Veterinaria Italiana, Volume 50 (2), April-June 2014 by Veterinaria Italiana

ISSN 0505-401X

Volume 50 (2) Aprile-Giugno April-June

2014

Rivista trimestrale di Sanità Pubblica Veterinaria, edita dall’Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” A quarterly journal devoted to veterinary public health, veterinary science and medicine, published by the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ in Teramo, Italy

Volume 50 (2), 2014

Pasquale Celommi ((Montepagano 1851 - Roseto degli Abruzzi 1928) Pastorella a Montepagano. Olio su tela/Oil on canvas, cm 34x44. Collezione privata/Private collection In un paesaggio campestre autunnale una pastorella regge con la destra una zucca e con la sinistra tenta di sottrarre all’avido tentativo di brucare le foglie di un tralcio di succosa uva con un atto che pare più simile ad un passo di danza che ad un repentino sottrarsi all’ingordigia dell’animale. In secondo piano pecore brucanti lungo una strada assolata e poco ricca di vegetazione. Sullo sfondo la collina lontanante e un agglomerato di case che parrebbero richiamare l’amata Montepagano. I colori dalle variazioni gialle sino al verde intenso del viticcio in secondo piano, schiariti nel vello delle pecore colpite dal sole sino al copioso e abbondante manto dell’animale in primo piano riccioluto e folto, si raccolgono in un vorticoso rincorrersi nel rosso corallo della veste della pastorella cui conferisce forte aggetto sottolineato dall’avvitamento repentino e grazioso del corpo, e dal volume della zucca dal giallo intenso. In a rural scenario a young shepherdess holds a pumpkin in her right hand and with the left hand prevents a sheep to browse on some grape, the gesture resembles more a dance step than subtle movement to avoid the sheep’s gluttony. In the background nibbling sheep are depicted along a sunny and gaunt path and a hill and a small village, which may recall the beloved Montepagano. The colours, from the yellow to the green offer a dynamic contrast to the red of the shepherdess’ dress. A cura di/By Polo Museale Città di Teramo

Questa rivista è nata nel 1950 con il nome di Croce Azzurra. Dal 1954 si chiamerà Veterinaria Italiana.

Comitato direttivo Managing Scientific Board Romano Marabelli Fernando Arnolfo

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Valerio Giaccone – Italy Ciriaco Ligios – Italy N. James MacLachlan – United States of America Paola Nicolussi – Italy Janusz Paweska – South Africa Giovanni Pezzotti – Italy Roberto Piro – Italy Giuseppe Ru – Italy Fabrizio Vitale – Italy Stéphan Zientara – France

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Segreteria di redazione Associate Editors Monica Bucciarelli, Guido Mosca, Mariarosaria Taddeo, Carlo Turilli Recensioni Book reviews Manuel Graziani Progetto grafico e web Graphic and web design Paola Di Giuseppe Amministrazione Administration Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” Campo Boario, 64100 Teramo, Italia veterinariaitaliana@izs.it Stampa Printer Giservice srl, Teramo, Italia http://www.izs.it/vet_italiana/index.html © 2014 Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” Campo Boario, 64100 Teramo, Italia

ISSN 0505-401X Formato elettronico Electronic format ISSN 1828-1427 Stampato su carta ecologica TCF Printed on 50% recycled, 100% chlorine- and acid-free environmentally friendly paper Aut. Trib. Teramo n. 299 del 16/05/1990 Sped. in Abb. Post. Art. 2 comma 20/c L. 66/96 DCB/DC Abruzzo Pescara

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The Steering Committee and the Scientific Committee are delighted to announce the IV International Conference on Bluetongue and related Orbiviruses, which will be held in Rome on November 5-7, 2014. Ten years after the III International Conference on Bluetongue, Orbivirus-related diseases are still priority and, in some cases, an emergency for official veterinary services and livestock industry.

The evolution of such diseases, their dissemination in different areas and the innovations introduced in laboratory diagnosis pose the need for a new meeting devoted to update the international scientific community on the current situation, compare different approaches, and define new strategies for disease control. Submissions of abstract for oral or poster presentations are welcome.

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Volume 50 (2), 2014 Carla Giansante, Annamaria Conte, Armando Giovannini, Luca Castriota, Franco Andaloro & Nicola Ferri Assessment of the effect of the climate variations of coastal surface water and study of Sepia officinalis spawing..................................................................... 87-97 Thomson Reuters Science Journal Citation Reports® database (JCR/Science Edition®) Journal impact factor 2012: 0.519 • National Library of Medicine’s MEDLINE/ PubMed system • Thomson Reuters Science Citation Index Expanded™ (SciSearch®) • CABI’s Full-Text Repository • Directory of Open Access Journals (DOAJ) • Elsevier’s SciVerse Scopus

Le pubblicazioni dell’Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise “G. Caporale” (IZSAM) sono protette dalla legge internazionale sul copyright. Gli estratti possono essere letti, scaricati, copiati, distribuiti, stampati, recuperati; è consentito inoltre il collegamento ai file pdf di Veterinaria Italiana. Informazioni per fini commerciali devono essere richieste all’IZSAM. Le traduzioni a stampa e gli adattamenti sono consentiti previa autorizzazione scritta da parte dell’IZSAM. Le opinioni espresse negli articoli pubblicati sono esclusivamente sotto la responsabilità degli autori. L’eventuale citazione di specifiche Ditte o prodotti, siano essi brevettati o meno, non implica che essi siano stati consigliati dall’IZSAM e vengano preferiti ad altri di simile natura non menzionati nei testi. Publications of the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ (IZSAM) are protected by international copyright law. Users are permitted to read, download, copy, distribute, print, search abstracts; besides they can link to Veterinaria Italiana full pdf files. Should information be required for commercial purposes, prior written permission must be sought from the IZSAM. Published translations and adaptations also require prior written approval from the IZSAM. The views expressed in signed articles are solely the responsibility of the authors. The mention of specific companies or products of manufacturers, whether or not patented, does not imply that these have been endorsed or recommended by the IZSAM in preference to others of a similar nature that are not mentioned.

Valutazione dell'incidenza delle variazioni climatiche nelle acque costiere e studio della produttività di Sepia officinalis in Abruzzo, Italia (riassunto)..............................................................................87

Silvia Vincenzetti, Laura Foghini, Stefania Pucciarelli, Valeria Polzonetti, Natalina Cammertoni, Daniela Beghelli & Paolo Polidori Hypoallergenic properties of donkey's milk: a preliminary study............................................................................... 99-107 Studio preliminare sulle proprietà ipoallergeniche del latte di asina (riassunto)...................................................................................................................99

Stefano Gavaudan, Anna Duranti, Francesca Barchiesi, Sara Ruschioni, Elisa Antognini, Erica Calandri, Paolo Mancini & Paola Riolo Seasonal monitoring of Aedes albopictus: practical applications and outcomes ................................................ 109-116 Descrizione di un sistema di monitoraggio stagionale di Aedes albopictus e dei risultati ottenuti (riassunto)............................................................... 109

John B. Muma, Kennedy K. Mwacalimba, Hetron M. Munang'andu, Gift Matope, Akinbowale Jenkins, Victor Siamudaala, Aaron S. Mweene & Tanguy Marcotty The contribution of veterinary medicine to public health and poverty reduction in developing countries .............................. 117-129 Il contributo della medicina veterinaria per la salute pubblica e la riduzione della povertà nei paesi in via di sviluppo (riassunto).......................................... 117

Aristarhos Seimenis & Darem Tabbaa Stray animal populations and public health in the South Mediterranean and the Middle East regions................... 131-136 Popolazioni di animali randagi e sanità pubblica nelle regioni del Sud Mediterraneo e del Medio Oriente (riassunto)................................................................. 131

Claudia Eleni, Valentina Panetta, Francesco Scholl & Paola Scaramozzino Causes of death in dogs in the province of Rome (Italy).................. 137-143 Cause di morte in cani provenienti dalla Provincia di Roma, Italia (riassunto)...................................................................................... 137

Volume 50 (2), 2014 SHORT COMMUNICATION Andrea Balboni, Patrizia Bassi, Mara Battilani, Roberta Biserni, Santino Prosperi & Francesco Dondi Severe, diffuse fibrinonecrotic pleuropneumonia in a cat affected by multiple viral infection....................................... 145-149 Grave pleuropolmonite fibronecrotica diffusa in un gatto con infezione virale multipla (riassunto)......................................................................................... 145

SHORT COMMUNICATION Alessio Lorusso & Giovanni Savini Old diseases for new nightmares: distemper strikes back in Italy ........................................................... 151-154 Vecchie malattie per nuovi incubi: il cimurro colpisce ancora (riassunto).............................. 151

LIBRI/Book reviews Rosario Fico, Simone Angelucci, Erika Ciarrocca Manuale delle attivitĂ investigative per i reati contro la fauna ..................155 Pietro Benazzi (a cura di) Cinzia Benazzi, Gabriella Martini Il Regolamento di Polizia Veterinaria .......................................................................157

Assessment of the effect of the climate variations of coastal surface water and study of Sepia officinalis spawing Carla Giansante1*, Annamaria Conte1, Armando Giovannini1, Luca Castriota2, Franco Andaloro2 & Nicola Ferri1 1 2

Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise 'G. Caporale', Campo Boario, 64100 Teramo, Italy Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA), Via Salvatore Puglisi 9, 90143 Palermo, Italy * Corresponding author at: Biologia delle Acque Interne, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise 'G. Caporale', Campo Boario, 64100 Teramo, Italy. Tel.: +39 0861 332631, e-mail: c.giansante@izs.it

Veterinaria Italiana 2014, 50 (2), 87-97. doi: 10.12834/VetIt.2004.52

Accepted: 24.09.2013 | Available on line: 23.05.2014

Keywords Abruzzo, Adriatic Sea, Climate change, Cuttlefish, Sepia officinalis, Sustainable fisheries, Water temperature.

Summary The aim of this study was to establish whether climate change affected migratory behaviour of Sepia officinalis (Linnaeus, 1758), which is an important resource for small-scale fishermen of Abruzzo region (Italy). Starting at the beginning of March until the end of April, the cuttlefish in this area migrates from deep cold water towards warmer coastal waters, where they spawn. Small-scale fishing of cuttlefish is permitted in costal waters from March to September. During the study period, between March and September 2008, both cuttlefish traps and trammel nets were used in 5 sampling areas along the Abruzzo coast to test their relative efficiency in catching cuttlefish. Trapped specimens were counted, weighed and measured, their gender and sexual maturity were also determined. The data obtained from the sampling were correlated to surface water temperature to assess possible changes in migration behaviours. The obtained data show that during the first months of migration (March and April), a greater percentage of large males was caught, while females and smaller males predominated later in the year. The study also showed that surface water temperature did not reveal any significant shifts from the trend over the last 10 years. As for the efficiency of the fishing methods, traps were found to be more effective than trammel nets.

Valutazione dell’incidenza delle variazioni climatiche nelle acque costiere e studio della produttività di Sepia officinalis in Abruzzo, Italia Parole chiave Abruzzo, Mare Adriatico, Cambiamenti climatici, Seppia, Sepia officinalis, Pesca sostenibile, Temperatura dell’acqua.

Riassunto La seppia (Sepia officinalis Linnaeus, 1758) costituisce una delle più importanti risorse per gli operatori della “piccola pesca” della regione Abruzzo, Italia. Lo studio ha avuto come obiettivo la valutazione della consistenza della popolazione di seppia nell’anno 2008 lungo la costa abruzzese (Mare Adriatico) e la verifica di eventuali alterazioni nei comportamenti migratori derivanti dai cambiamenti climatici. Annualmente nel Mare Adriatico, nei mesi di marzo‑aprile, la seppia migra dalle fredde acque profonde verso le acque costiere più calde dove si riproduce. In Abruzzo, la cattura della seppia è consentita da marzo a settembre. Lo studio effettuato si è svolto in questi stessi mesi del 2008, in 5 zone di campionamento, impiegando come attrezzi da pesca la nassa e il tramaglio, testando di questi ultimi anche l’efficienza. Gli esemplari pescati sono stati contati, pesati e misurati, determinando il sesso e lo stadio di maturazione sessuale. La nassa è risultata più efficace nelle catture rispetto al tramaglio. Durante i primi mesi di migrazione (marzo-aprile) è stata catturata una percentuale superiore di maschi di grandi dimensioni, seguita da catture di femmine e maschi di dimensioni ridotte nei mesi successivi. La nassa si è rivelata uno strumento di campionamento valido. L’elaborazione dei dati relativi alla temperatura superficiale dell’acqua negli ultimi 10 anni non ha mostrato scostamenti significativi dalla norma .

Climate change and productivity of Sepia officinalis Giansante et al.

Introduction Sepia officinalis belongs to the class of Cephalopoda (Linnaeus, 1758), it is a bentho-nectonic neritic species which can be found either in costal areas with sandy and muddy beds or in areas covered by algae and spermatophyte (Lucchetti 2004). This cuttlefish is widespread throughout the Mediterranean and West Atlantic areas; within the Mediterranean basin, it can be found in the Aegean Sea, Sea of Marmora and in the Sea of the East, whereas in the Atlantic Ocean its presence has been reported from the coastal water of Shetland Islands and Southern Norway to the 16° N, at the boundaries of Mauritania and Senegal (Pierce et al. 2008, Wolfram et al. 2006).

decreases. The migration process may depend on the water temperature, its salinity, the nature of the seabed and the marine current. Among these, however, the temperature of the coastal water is deemed to be the main factor affecting the migration behaviour of this species.This study aimed to correlate cuttlefish migration behaviour with surface water temperature. This hypotesis, if proved, would imply an early starting of the fishing season and a deployment of trammel nets and traps.

Materials and methods Surface seawater temperatures

Sepia officinalis is one of the most important resources for the local fishing activities1,2, particularly in the early spring, when the species’ reproductive season begins. Thanks to its swimming skills, it can easily face migrations. In fact, within the 2 year span of their life (Adamo et al. 2000, Dunn 1999, Wang et al. 2003), individuals of this species migrate often times from coastal to deep water depending on the season and in relation to their reproductive cycle (Denis et al. 2001, Mangold-Wirz 1963, Mangold 1989).

Surface seawater temperatures were measured using data collected in situ and data stored in the digital archive of the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ (IZSAM) for the period 1989-2007. Data were recorded at 500 m, 1,000 m, 2,000 m and 3,000 m from the outlet to the sea of the rivers Alento, Arielli, Cerrano, Feltrino, Foro, Moro, Pescara, Piomba, Saline, Salinello, Sangro, Sinello, Tordino, Trigno, Tronto, Vibrata e Vomano (Giansante 1998, Giansante 2001, Giansante 2008).

Like all cephalopods, cuttlefishes have distinct sexes and reproduce only once in their lifetime. Eggs are usually laid at water temperatures between 13°C and 15°C. The cuttlefish life cycle is 12-24 months, varying according to the environmental conditions. Growth is quite fast, the individuals born in summer from eggs laid in spring, usually spawn in the autumn of the following year, while those born in the autumn clutch spawn in the spring of their second year of life (Jereb and Roper 2005).

Surface temperatures around the Abruzzo coast between 1992 and 2008 were measured via satellite every 8 days within an area between 14° and 15°E and 42° and 43°N (Figure 1). Data from 1992 to 2007, originated from the SST (Sea Surface Temperatures) Pathfinder version 5 (developed by the University of Miami's Rosenstiel School of Marine and Atmospheric Science (RSMAS) and the National Oceanic and Atmospheric Administration (NOAA) National Oceanographic Data Center (NODC), United States Department of Commerce) were recorded by NOAA AVHRR (National Oceanic and Atmospheric Administration - Advanced Very-High-Resolution Radiometer; for the year 2008 (AVHRR data were not

During the early spring, S. officinalis leaves the seabed which it inhabits during the winter and moves toward the coastal waters where it begins its reproductive process (Wang et al. 2003). It should be noted that not all the specimens migrate in the same manner: the first individuals to leave the seabed are the bigger ones, usually these are male specimens which can begin to migrate up to a week before the females (Jereb and Roper 2005). In the Southern part of the Adriatic see, the bigger individuals start to move toward coastal waters between February and March, while smaller specimens migrate during the summer. The inverse migration path, from coastal water to seabed, can be observed with the approaching of the autumn when the temperature

L egge 13 marzo 1958, n. 250. Previdenze a favore dei pescatori della piccola pesca marittima e delle acque. Off J, 83, 5.4.1958. 2 E uropean Community (EC). 1999. Council Regulation (EC) No 2792/1999 of 17 December 1999 laying down the detailed rules and arrangements regarding Community structural assistance in the fisheries sector. Off J, L 337, 30.12.1999, 10-28. http://eur-lex.europa.eu/LexUriServ/LexUriServ. do?uri=OJ:L:1999:337:0010:0010:EN:PDF. 1

Figure 1. Selected sea area of the Abruzzo coast (Italy) for temperature monitoring by satellite.

Veterinaria Italiana 2014, 50 (2), 87-97. doi: 10.12834/VetIt.2004.52

Giansante et al.

Gaps in the satellite data were overcome using surface temperature values recorded by the Institute for Environmental Protection and Research (ISPRA) Marigraphic Service’s Ortona data buoy (42° 21´ 21” N, 014° 24´ 53” E). Measurements were taken at one-hour intervals every day from January 1998 to October 2008.

Scientific sampling of Sepia officinalis A sampling campaign was carried out from February 15 2008 to September 29 2008 in the areas around Martinsicuro, Giulianova, Montesilvano, Francavilla and Ortona, for a total of 55 sets of samples. Sampling was carried out using a 500 m long, 2 m high trammel net with a 40 mm mesh and 40 cuttlefish traps (nasse o bertovelli). Water and air temperatures were measured during each collection with a certified field thermometer (model THM912, Oregon Scientific, Portland, Oregon, USA). Trapped specimens were counted, transported to the laboratory under refrigeration for the determination of morphometric parameters, gender and maturity (Lipinski 1979, Mangold 1989, Mangold-Wirz 1963, Richard 1971, Richard et al. 1979). Specimens caught in the trammel net and any surplus animals caught in the traps were also measured (mantle length‑ML) from the anterior dorsal edge to the back and weighed in-field. Biometric measurements were made using a moulded fish ruler (Scubla Aquaculture, Udine, Italy) and a dynamometer (model PHS3000, MPIM, Chieti, Italy).

Statistical analysis The annual means of the surface seawater temperatures logged in the database of the IZSAM have been calculated. Monthly and annual means have also been calculated for data collected by the buoy. The significance of the regression line with a 95% confidence interval has been evaluated on the annual means. Finally, monthly means have been calculated for the data obtained from satellite measurements.

Results Surface temperatures Surface seawater temperatures measured by the IZSAM during the monitoring campaign carried out h ttp://gcmd.nasa.gov/records/GCMD_OBPG_MODIS_AQUA_L3_M4D_ SST.html. 4 http://noaasis.noaa.gov/NOAASIS/ml/avhrr.html. 3

Veterinaria Italiana 2014, 50 (2), 87-97. doi: 10.12834/VetIt.2004.52

Temperature (°C)

available), values were taken from the MODIS/Aqua (Moderate Resolution Imaging Spectroradiometer)3 developed by OCEAN ESIP (Ocean Earth Science Information Partners)4.

Climate change and productivity of Sepia officinalis

20 15 10 5 0 1985

1990

2000

1995

2005

2010

Year

Figure 2. Annual mean surface seawater temperature of the Abruzzo coast for the years 1989-2007. from 1989 to 2007 were extracted and processed (Figure 2). The mean daily temperatures were calculated, followed by the average monthly temperature, obtaining 120 temperature values. A standard decomposition analysis of the time series was conducted to identify the trend, seasonality and residue using the ‘decompose’ function of the Stats software package R 2.9.2™. The function first determines the trend of the time series using the rolling average of 13 values and weights (wi) of:

{

1/24, i={1;13} wi = 1/12 , i={2:12} The seasonal component of the detrended series is calculated as the mean of the values assumed for the same month in each year and the residual component is obtained on the basis of the selected model (in this case additive) by removing trend and seasonality from the original time series. The adequacy of the model is evaluated by analysis of the residues, testing the mean (t-test) and studying the distribution (Figure 3, Table I). The t-test demonstrated that the mean of the residues is not significantly different from 0. The trend analysis did not reveal any significant trends with respect to temperature rises or drops (slope: t = 0.748; p = 0.456). Satellite data showed frequent gaps, especially near the coast, probably due to cloud cover and signal noise causing sensor disorders. For this reason, it was not always possible to obtain temperature means representative of the entire area in question, and so no time series analysis was carried out. However, the monthly means for surface water temperature near the Abruzzo coast were analysed to show possible trend. The temperature value is averaged each month over the entire area taken into consideration. Figures 4a and 4b depict the surface temperature trend (monthly means) for 1992-2008 and show a sinusoidal trend over the seasons. With respect to the general temperature trend, there is a slight increase in the minimum temperature in 2007. Figure 5

Climate change and productivity of Sepia officinalis Giansante et al.

Average surface temperature (째C)

Table I. Regression analysis of trend to evaluate the adequacy of the model by analysis of the residues, testing the mean (by t-test) and studying the distribution.

Slope

Estimate

Std. Error

t value

p value

0.001056

0.001411

0.748

0.456

15 2000

2002

2004

2006

2008

Min.

1st Qu.

Median

Mean

3rd Qu.

Max.

-2.289

-0.574

0.0134

-0.000827

0.680

2.161

t-test for mean <>0: t = -0.009241469; p = 0.5036782.

Time

Figure 3. Fitting of a linear trend to the time series trend component.

a) 30 28

Temperature (째C)

26 24 22 20 18 16 14 12 January 2008

January 2007

January 2006

January 2005

January 2004

January 2003

January 2002

January 2001

January 2000

January 1999

January 1998

January 1997

January 1996

January 1995

January 1994

Gennaio 1992

January 1993

Year

b) 30 28 26

Temperature (째C)

24 22 20 18

min max

16 14 12

2008

2007

2006

2005

2004

2003

2002

2001

2000

1999

1998

1997

1996

1995

1994

1993

1992

Year

Figure 4. Sea surface temperature trends of the Abruzzo coast for the years 1992-2007 (a); maximums and minimums (b).

Veterinaria Italiana 2014, 50 (2), 87-97. doi: 10.12834/VetIt.2004.52

Giansante et al.

Climate change and productivity of Sepia officinalis

27-28 26-27

Sea Surface Temperature (째C)

24-25

December

October

November

September

July

August

June

May

April

March rch

Februaryy

2005 005

7 2007 January

2003

2004

2001 2002

1992 1993 1994 1995 1996 1997 1998 1999 2000

25-26 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10

23-24 22-23 21-22 20-21 19-20 18-19 17-18 16-17 15-16 14-15 13-14 12-13 11-12 10-11

January

27-28 26-27

February March April

25-26 24-25 23-24 22-23

May

21-22

June

20-21 19-20

July August September

18-19 17-18 16-17 15-16

October November

14-15 13-14 12-13

2007

2006

2005

2004

2003

2002

2001

2000

1999

1998

1997

1996

1995

1994

1993

1992

December

11-12 10-11

Figure 5. (a) and (b) SST (Sea Surface Temperature) gradient of the Abruzzo coast for 1992-2008. shows that the highest temperatures were recorded in August, whereas the lowest temperatures were observed in February-March. It is noteworthy that, starting in 2002, the temperatures recorded for July and August are increasingly similar. At the

Veterinaria Italiana 2014, 50 (2), 87-97. doi: 10.12834/VetIt.2004.52

same time, in 2007, data show an early increasing of the temperature in April and May, which is quite odd when compared with data concerning both, the previous and the subsequent year, when the temperature did not go above 15째C.

Climate change and productivity of Sepia officinalis Giansante et al.

Traps

Scientific sampling of Sepia officinalis

Trammel net

43.299

45 40

39.219

37.041

25.243

25 20

25.13 17.033

16.399

13.552

9.151

4.163

5 0

Ortona

Francavilla

Montesilvano

Giulianova

Martinsicuro

Sampling point

Figure 6. Total biomass of Sepia officinalis caught during monitoring runs by sampling point and tackle used.

Trap

25000

20000

Biomass (g)

40 30 20 10 0

Trammel net

15000 10000 5000

Sampling date

Figure 7. Total number of Sepia officinalis caught in traps and trammel net at all sampling points.

26/09/2008

12/09/2008

29/08/2008

15/08/2008

01/08/2008

18/07/2008

04/07/2008

20/06/2008

06/06/2008

23/05/2008

09/05/2008

25/04/2008

11/04/2008

28/03/2008

15/02/2008

26/09/2008

12/09/2008

29/08/2008

15/08/2008

01/08/2008

18/07/2008

04/07/2008

20/06/2008

06/06/2008

23/05/2008

09/05/2008

25/04/2008

11/04/2008

28/03/2008

14/03/2008

29/02/2008

15/02/2008

0 14/03/2008

N째

Trap

Trammel net

29/02/2008

Nine hundred ninty-two specimens were caught during the scientific sampling campaign: 720 were captured using traps, 272 using trammel nets. The total biomass was 230.23 kg, 143.032 kg in traps and 87.198 kg in trammel nets. The number of specimens caught, and consequently the biomass, was greater in April, May and June at all 5 sampling sites. Catches were poor in July, August and September (no more than 5 specimens captured with 40 traps), with the exeption of Ortona, where 12 cuttlefish were caught on August 20 and 7 on September 3 2008. Similar results were obtained using trammel nets: the most successful captures occurred in April, May and June, whereas in the following months only 2 individuals per run were caught with this tackle (Ortona sampling point).

Sampling date

Figure 8. Total biomass of Sepia officinalis caught in traps and trammel net at all sampling points.

800 700

N째 of specimens

600 500 400 300 200 100 0

Species

Figure 9. Number of specimens caught in traps over all sampling points on the Abruzzo coast between March and September 2008.

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Climate change and productivity of Sepia officinalis

With reference to the total biomass of cuttlefish caught at each sampling point, the data showed that the best catches occurred in Giulianova, probabily due to various factors including greater skill of the fisherman in positioning the tools, greater number of cuttlefish in the area, and/or weather and sea conditions. The data concerning the Giulianova site showed an inverse trend: the cuttlefish biomass caught in the trammel nets was greater than that caught in the traps. Also in this case the results might have depended on greater skill of the fisherman in positioning the tools, greater number of cuttlefish in the area, and/or weather and sea conditions.

the first 2 observations carried out for each sampling point. Until April, males were generally more numerous than females, however in May and June the male/famale ratio was inverted. Of all the speciments collected during the study, 300 out of 596 individuals were males, while 296 were female. Almost all the male specimens, 272 out of 300, were in the IV stage of gonad maturation, whereas 21 individuals were in the II or III stage and only 7 specimens had a completely vacant spermatophore capsule indicating that the reproductive phase was concluded. Almost all females, 291 out of 296, were in the III phase of the gonad maturation, which preceeds the reproduction stage; 4 specimens were in stage IV, while only 1 individual showed to have already layed its eggs. Most of the specimens were caught with a surface seawater temperature between 14.4° and 21.5°C.

Figure 6 reports the data about the biomass originating from each type of tackle (traps and trammel nets) used at each monitoring point. Figures 7 and 8 compare the number and biomass of cuttlefishes caught in the traps and those caught in the trammel nets. The Mann-Whitney test identified a statistically significant difference with respect to both, number of specimens (U = 2048, p<0.01) and biomass (U = 1979.5, p<0.01). Both, the number of specimens and their biomass were significantly greater when using the traps.

Figures 10, 11, 12, 13, 14 the dashed lines indicate the opening of the fishing season according to current legislation, i.e. 15 March for traps, and 1 April for trammel nets. The data analysed in this study showed that:

The traps also caught 472 octopuses (Octopus vulgaris), not normally caught in trammel nets or using other fishing methods. The low number of other species caught by the traps confirms their high selectivity (Figure 9) .

• on February 15, the starting date of the scientific sampling in Montesilvano, no specimen was captured and the seawater temperature was 7.5°C;

Results showed that the first specimens to approach coastal water are males, as it was recorded during

Air temperature

Water temperature

• on March 1, in Giulianova during the second sampling, 12 individuals were captured with

Trap catches

Trammel net catches

Start fishing with trap (15 March)

Start fishing with trammel net (1 April)

90 80

60 50

40 10

N° of catches

Temperature (°C)

20 5 10 0 29/02/2008

24/09/2008

03/09/2008

25/08/2008

20/08/2008

31/07/2008

18/07/2008

30/06/2008

18/06/2008

05/06/2008

28/04/2008

17/03/2008

15/02/2008

Sampling date

Figure 10. Ortona: specimens of Sepia officinalis caught by tackle type in relation to air temperature and surface seawater temperature.

Veterinaria Italiana 2014, 50 (2), 87-97. doi: 10.12834/VetIt.2004.52

Climate change and productivity of Sepia officinalis Giansante et al.

traps and 23 using trammel nets, the seawater temperature was 10.4°C;

sampling, 1 specimen was captured using traps and 1 using trammel nets. The sea water temperature was 10.6°C;

• on March 13, in Martinsicuro, during the third sampling, no specimen was captured and the seawater temperature was 10.0°C;

• on April 2, in Francavilla, during the fifth sampling, 36 individual were captured with traps and 7 with trammel nets, the water temperature was 12.2°C.

• on March 17, in Ortona during the fourth

Air temperature

Water temperature

Trap catches

Trammel net catches

Start fishing with trap (15 March)

Start fishing with trammel net (1 April)

60 20

N° of catches

Temperature (°C)

70 25

20 5

29/09/2008

24/09/2008

10/09/2008

25/08/2008

20/08/2008

30/07/2008

18/07/2008

03/07/2008

17/06/2008

10/06/2008

09/05/2008

02/04/2008

0 15/02/2008

Sampling date

Figure 11. Francavilla: specimens of Sepia officinalis caught by tackle type in relation to air temperature and surface seawater temperature.

Air temperature

Water temperature

Trap catches

Trammel net catches

Start fishing with trap (15 March)

Start fishing with trammel net (1 April)

90 80

60 50

15 40 10

N° of catches

Temperature (°C)

20 5 10 0 29/09/2008

10/09/2008

04/09/2008

27/08/2008

21/08/2008

28/07/2008

07/07/2008

23/06/2008

09/06/2008

16/05/2008

14/04/2008

15/02/2008

Sampling date

Figure 12. Montesilvano: specimens of Sepia officinalis caught by tackle type in relation to air temperature and surface seawater temperature.

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Figures 15 and 16 show the linear regression of mantle length variations of the individuals caught during the sampling period. The length of the specimens variated over time and such variation was more evident in males than in females. Males captured during the first phase of the sampling could be up to 21 cm long.

Air temperature

Water temperature

The linear regression highlighted an important negative relationship between the sampling dates and the length of the specimens, with a significantly negative angular coefficient, especially in males (Table II).

Trap catches

Trammel net catches

Start fishing with trap (15 March)

Start fishing with trammel net (1 April)

80 70 60

40 30

N째 of catches

Temperature (째C)

20 5

29/09/2008

05/09/2008

01/08/2008

28/08/2008

28/07/2008

08/07/2008

24/06/2008

11/06/2008

22/05/2008

21/04/2008

01/03/2008

0 15/02/2008

Sampling date

Figure 13. Giulianova: specimens of Sepia officinalis caught by tackle type in relation to air temperature and surface seawater temperature.

Air temperature

Water temperature

Trap catches

Trammel net catches

Start fishing with trap (15 March)

Start fishing with trammel net (1 April)

90 80

60 50

15 40 10

N째 of catches

Temperature (째C)

20 5 10

29/09/2008

05/09/2008

27/08/2008

21/08/2008

29/07/2008

07/07/2008

25/06/2008

13/06/2008

28/05/2008

24/04/2008

13/03/2008

0 15/02/2008

Sampling date

Figure 14. Martinsicuro: specimens of Sepia officinalis caught by tackle type in relation to air temperature and surface seawater temperature.

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Climate change and productivity of Sepia officinalis Giansante et al.

Mantle lenght (cm)

16 14 12 10 8 6 4

20 15 10 5

2 0 3

Months

Figure 15. Regression analysis of mantle length and month of sampling (Sepia officinalis females).

Figure 16. Regression analysis of mantle length and month of sampling (Sepia officinalis males).

Table II. Regression analysis of mantle length variations in males and females Sepia officinalis caught during the sampling period. Group Females Males

Parameter

Coefficient

Standard error

t value

Significance

Constant

16.248

0.637

25.523

Time (months)

-0.504

0.114

Constant

18.805

Time (months)

-1.142

Lower limit

Upper limit

<0.0001

14.992

17.505

-4.436

<0.0001

-0.729

-0.28

0.558

33.712

<0.0001

17.705

19.905

0.11

-10.371

<0.0001

-1.359

-0.925

Discussion Data collected by the data buoy (1999-2008) did not show any significative trend concerning the increasing or decreasing of surface seawater, whereas satellite data concerning the same period showed an early increase of the seawater temperature occurring in April-May 2007. On the basis of these data it can be argued that during the last 10 years no significative variation of the surface seawater temperature has been recorded. Data collected via the scientific sampling highlighted that surface seawater temperature is likely to be a determining factor in the migration behaviour of the cuttlefish, which moves toward the coast only when the surface temparature is above 10 째C. The first evidence of the presence of S. officinalis in coastal water was obtained during the first days of March. During the following days it is likely that, given the worstening of the weather conditions, the cuttlefish returned offshore. Temperature seems to be the main factor limiting the presence of cuttlefish in the coastal area. Sudden drops in temperature resulted in cooler water and instigated cuttlefish to move away temporarily, as occurred in the first sampling at both, Giulianova (01.03.2008: 35 specimens caught, seawater temperature 10.4 째C) and Martinsicuro (13.03.2008: no specimens caught, seawater temperature 10.0째C). It is worthwhile stressing that rain and

95% confidence interval for coefficient

snow had fallen between the 2 samplings causing a decreasing of the surface seawater temperature. The length of caught specimens diminished from March to September and were consistent with the findings reported in literature (Mangold 1989). An analysis of the gonad maturation confirmed that the reproductive period corresponds to the most proficous period for the small fishery activities, stressing therefore the need to choose fishing tools and methods which woud have the least possible impact on the resource and on its reproductive cycle. Cuttlefish traps showed to be the most efficient tool to capture cuttlefish as well as other species such as octopus. Furthermore, cuttlefish would use the traps for egg deposition, so if the these tools are left in the sea until September, as it is currenly required by the existing regulations, they will foster the natural increasing of the resource. Hence, the number of traps that can be deployed by each fisherman (which is regulated by the local authority, Capitaneria di Porto) should be determined keeping in mind the presence of the resource in the area.

Conclusions On the basis of the data considered there were no significant shifts in surface water temperature with respect to the trend over the last 15 years.

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Trammel nets showed to be a less efficient tool for the capture of cuttlefish when compared to traps and, although there are not limitations set for their deployment, this tool has a high negative impact on several species (e.g. cetaceans and turtles, protected species).

Climate change and productivity of Sepia officinalis

We conclude that trammel nets should not be used for capturing cuttlefish; furthermore, in order to preserve this resource, traps should be the only tool allowed for the capture of this species and the opening of the fishing season should not be anticipated earlier.

References Adamo S.A., Brown W.M., King A.J., Mather J., Mather L., Shoemaker K.L. & Wood J.B. 2000. Agonistic and reproductive behaviours of the cuttlefish Sepia officinalis in a semi-natural environment. J Mollus Stud, 66, 417-419. Denis V. & Robin J.P. 2001. Present status of the French Atlantic fisheries for cuttlefish (Sepia officinalis). Fisheries Research, 52, 11-22. Dunn M.R. 1999. Aspects of the stock dynamics and exploitation of cuttlefish, Sepia officinalis (Linnaeus, 1758), in the English Channel. Fisheries Research, 40, 277-293. Giansante C. 1998. Programma di Ricerca e Sperimentazione Mare Adriatico (PRISMA Fase 1) anni 1995-1997. Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’- Regione Abruzzo, Teramo. Giansante C. 2001. Programma di monitoraggio delle acque marine costiere anni 1989-2000. Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ - Regione Abruzzo, Teramo. Giansante C. 2008. Programma di monitoraggio delle acque marine per la molluschicoltura anni 1996-2007. Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’ - Regione Abruzzo, Teramo. Jereb P. & Roper C.F.E. 2005. Cephalopods of the world. An annotated and illustrated catalogue of cephalopod species known to date. Volume 1. Chambered nautiluses and sepioids (Nautilidae, Sepiidae, Sepiolidae, Sepiadariidae, Idiosepiidae and Spirulidae). FAO Species Catalogue for Fishery Purposes. 4, 1, Rome, FAO. Lipinski M. 1979. Universal maturity scale for the commercially important squids. The results of maturity classification of the Illex illecebrosus (Lesueur, 1821) population for years 1973-1977. ICNAF Res. Doc. 79/11/38, Serial No. 5364, 40.

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Lucchetti A. 2004. La seppia comune. Biologia, pesca e consumo delle più importanti specie commerciali – Sepia officinalis (Linneo, 1758). Il Pesce, 2, 93-95. Mangold K. 1989. Reproduction, croissance et durée de vie. In Céphalopodes. Traité de zoologie. Anatomie, Systématique, Biologie. Grassé P.P., Masson, Paris, 5 (4), 493-552. Mangold-Wirz K. 1963. Biologie des céphalopodes benthiques et nectoniques de la Mer Catalane. Vie Milieu Paris, 13 (Suppl.), 285 pp. Pierce G.J., Valavanis V.D., Guerra A., Jereb P., Orsi-Relini L., Bellido J.M., Katara I., Piatkowski U., Pereira J., Balguerias E., Sobrino I., Lefkaditou E., Wang J., Santurtun M., Boyle P.R., Hastie L.C., MacLeod C.D., Smith J.M., Viana M., Gonzalez A.F. & Zuur A.F. 2008. A review of cephalopod-environment interactions in European Seas. Hydrobiologia, 612, 49-70. Richard A. 1971. Contribution à l’étude expérimentale de la croissance et de la maturation sexuelle de Sepia officinalis L. (mollusque cephalopode). PhD Natural Sciences. Université de Lille, Lille, 264 pp. Richard A., Van den Branden C. & Decleir W. 1979. The cycle of activity in the accessory nidamental glands from Cephalopods. Naylor E. & Hartno R.G., 2nd ed., 173-180. Wang J., Pierce G.J., Boyle P.R., Deni, V., Robin J.-P. & Bellido-Millan, J.M. 2003. Spatial and temporal patterns of cuttlefish (Sepia officinalis) abundance and environmental influences - a case study using trawl fishery data in French Atlantic coastal, English Channel and adjacent waters. ICES J Mar Sci, 60, 1149-1158. Wolfram K., Mark F.C., John U., Lucassen M. & Pörtner H.O. 2006. Microsatellite DNA variation indicates low levels of genetic differentiation among cuttlefish (Sepia officinalis L.) populations in the English Channel and the Bay of Biscay. Comp Biochem Physiol Part D Genomics Proteomics, 1(3), 375-383.

Hypoallergenic properties of donkey’s milk: a preliminary study Silvia Vincenzetti1*, Laura Foghini2, Stefania Pucciarelli2, Valeria Polzonetti2, Natalina Cammertoni1, Daniela Beghelli2 & Paolo Polidori3 1

School of Biosciences and Veterinary Medicine, section of Veterinary Medicine, University of Camerino, Via Circonvallazione 93/95, 62024 Matelica (MC), Italy 2 School of Biosciences and Veterinary Medicine, section of Biosciences, Via Gentile III da Varano, 62032 Camerino (MC), Italy 3 School of Pharmacy, University of Camerino, Via Circonvallazione 93/95, 62024 Matelica (MC), Italy

* Corresponding author at: School of Biosciences and Veterinary Medicine, section of Veterinary Medicine, University of Camerino, via circonvallazione 93/95, 62024 Matelica (MC), Italy. Tel.: +39 0737 403462, Fax: +39 0737 403402, e-mail: silvia.vincenzetti@unicam.it

Veterinaria Italiana 2014, 50 (2), 99-107. doi: 10.12834/VetIt.219.125.5 Accepted: 29.04.2014 | Available on line: 30.06.2014

LXVII Meeting of the Italian Society for Veterinary Sciences (SISVet). 17-19 September 2013, Brescia, Italy - Selected papers Keywords Cationic exchange chromatography, CMPA, Cow’s milk, Donkey’s milk, Goat’s milk, Immunoblotting, Hypoallergenicity, Reversed-phase chromatography.

Summary Cow’s milk protein allergy (CMPA) is an abnormal immunological response to cow milk proteins, which results in IgE-mediated reactions. The therapeutic strategy to respond to CMPA envisages the total elimination of milk or the administration of cow’s milk substitutes. For this reason the use of milk from other mammalian species was tested. Among them, donkey’s milk proved to be the best alternative in feeding infants affected by CMPA, since its chemical composition is comparable to human milk. In this work an in vitro study was performed in order to analyze the IgE reactivity to milk protein allergens from cow, donkey and goat. In particular, immunoblotting experiments using sera from milk-allergic and non-allergic adult volunteers were conducted with the aim of verifying the hypoallergenic property of donkey’s milk. This study provided a preliminary evidence of the hypoallergenicity of donkey’s milk when compared to bovine and goat milk. Considering the obtained results, it would be possible to develop a sensitive diagnostic method for CMPA detection, based on chromatographic and immunoblotting analysis.

Studio preliminare sulle proprietà ipoallergeniche del latte di asina Parole chiave APLV, Cromatografia a fase inversa, Cromatografia a scambio ionico, Immunoblotting, Ipoallergenicità, Latte di asina, Latte di capra, Latte vaccino.

Riassunto L’allergia alle proteine del latte vaccino (APLV) è una reazione immunologica IgE-mediata. La strategia terapeutica dell’APLV è basata sull’eliminazione totale del latte vaccino e sulla somministrazione di alcuni tipi di latte sostitutivi a quello vaccino. A tal proposito, il latte d'asina ha dimostrato di essere, per la sua composizione chimica molto simile a quella del latte umano, l’alternativa migliore nell’alimentazione di neonati affetti da APLV. In questo studio preliminare è stata analizzata la reattività delle proteine del latte di vacca, asina e capra con esperimenti di immunoblotting. Sono stati utilizzati sieri di soggetti adulti volontari allergici e non allergici (controlli) al latte vaccino. I risultati hanno permesso di evidenziare le proprietà ipoallergeniche del latte di asina rispetto al latte di capra ma, soprattutto, rispetto a quello vaccino. Lo studio può contribuire allo sviluppo di un metodo diagnostico veloce e sensibile per la rilevazione dell’APLV.

Vincenzetti et al.

Properties of donkey’s milk

Introduction Food allergy refers to an abnormal immunologic response to a food protein that occurs in a susceptible subject. This reaction occurs each time the food is ingested and it is often not dose dependent (Cianferoni and Spergel 2009). Food allergy affects around 11-26 million of the European population and, in general, is more frequent in the pediatric, rather than in the adult population (Fiocchi et al. 2010). The allergens responsible for more than 85% of food allergy are proteins contained in milk, egg, peanut, tree nuts, shellfish, wheat, sesame, seed and soy (Waserman and Watson 2011). The allergenic segments or ‘epitopes’ of these proteins tend to be small (from 10 to 70 kDa in size) watersoluble glycoproteins, which are generally resistant to denaturation by heat or acid and therefore can remain intact even after processing, storage, cooking and digestion (Waserman and Watson 2011). Examples of these glycoproteins include caseins in milk, vicilins in peanut, and ovomucoid in eggs. Based on the immunological mechanism involved, food allergies may be further classified in: IgE-mediated or mediated by IgE antibodies; cell-mediated, when the cell component of the immune system is responsible of the food allergy and mostly involves the gastrointestinal tract; mixed IgE mediated-cell mediate (Cianferoni and Seprgel 2009). Cow milk protein allergy (CMPA) is clinically an abnormal immunological reaction to cow milk proteins, which may be due to the interaction between one or more milk proteins and one or more immune mechanisms, and results in immediate IgE-mediated reactions. The clinical manifestations of CMPA include gastrointestinal, respiratory, cutaneous as well as systemic anaphylactic symptoms (Bahna and Gandhi 1983). Cow milk is one of the most common food allergies in children, occurring in between 0.3 and 7.5% of the infant worldwide population (Hill et al. 1986), although this allergy is considered transient, with a remission rate of 85% after 3 years of age, there are still some children who exhibit it at the age of 10 and in the adult age (Giner et al. 2012). The main allergens in cow’s milk are caseins (αs1- and β-caseins) followed by β-lactoglobulin and α-lactalbumin, although the latter occurs to a minor extent (Docena et al. 1996, Jarviner et al. 2001). The therapeutic strategy for CMPA envisages the total elimination of cow’s milk and all its derivatives. In the first 2 years of life, however, milk represents an important source of nutrients and as such it cannot be eliminated from the everyday diet, making therefore necessary to use substitutive milks. In addition, oral desensitization cannot be performed before 2 years of age (Meglio et al. 2004). Soy milk or hydrolysed formulas may be considered good substitutes to human milk and allow to the children to grow up well, nevertheless it

100

has been shown that 17-47% of milk allergic infants can have adverse reactions to soy. In order to find a good substitute to cow’s milk, the use of milk from other mammalian species like goat, sheep, mare and donkey was considered in all the cases in which breast feeding was not possible and when it is not possible to use soy milk or hydrolyzed formulas (Iacono et al. 1992, Dean et al. 1993, Carroccio et al. 2000, Muraro et al. 2002, Restani et al. 2002). In particular, donkey’s milk proved to be a good alternative for CMPA infants because of its similarity to human milk with regard to its composition in lipids, with particular regards to the triacylglycerol fraction and fatty acid profile (Chiofalo et al. 2011), protein fraction, mineral and lactose content (Monti et al. 2007, Vincenzetti et al. 2008). Furthermore, the high content of lactose confer to this milk a good palatability and optimize the intestinal absorption of calcium, essential for bone mineralization in infants. The low allergenicity of donkey’s milk is mainly due to the low casein content (Vincenzetti et al. 2007), which is very close to the casein content determined in human milk. In particular αs1- and β-caseins in different phosphorylated forms has been shown to be present in large amount in donkey’s milk, κ-casein and αs2-casein are also present although in very small amounts (Vincenzetti et al. 2008, Criscione et al. 2009, Bertino et al. 2010) differently from cow’s milk (Creamer 2003). In this work, an in vitro study was performed in order to analyze the IgE reactivity to milk protein allergens from cow, donkey and goat by immunoblotting experiments using sera from milk-allergic and non-allergic adult volunteers. The cross-reactivity between sera of children with CMPA and milk proteins from some mammalian species such as sheep, goat and buffalo has been addressed in several studies (Carroccio et al. 1999, Restani et al. 2002, Monti et al. 2007), the primary aim of this work was to verify the renowned hypoallergenic property of this milk. To achieve such a goal, a purification and characterization of the caseins and whey proteins present in donkey, goat and cow milk were performed and each separated protein was used as antigen in the immunoblotting experiments. However, due to the restricted number of sera available, the present work must be intended as preliminary study that could be considered as a starting point for further analysis on the allergenicity of different types of milk.

Materials and methods Milk samples Bulk milk in midstage of lactation from cow, goat and donkey was obtained from local farms (Umbria region, May 2013). Skimmed milk was

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Properties of donkey’s milk

obtained from 25 ml fresh milk by centrifugation at 3000g for 30 minutes at 15°C. Whole caseins were obtained from skimmed milk by adjusting the pH to 4.6 with 10% (v/v) acetic acid and centrifuged at 3000g for 30 minutes in order to obtain a supernatant of whey proteins and the isoelectrically precipitated caseins which were subsequently resuspended in 25 ml of buffer A (50 mM ammonium acetate, 8 M urea, pH 5.5). The protein concentration of both whey and casein fractions was determined by the Bradford protein assay method (Bradford 1976).

(controls). Sera were obtained by centrifugation at 3000 g for 10 minutes at 4°C. Allergic volunteers were selected according to a positive case history (i.e. gastrointestinal symptoms upon controlled ingestion of milk products), positive skin-prick reactions and determination of specific IgE to cow’s milk proteins. For the immunoblotting analysis, each purified casein and whey protein from cow, goat and donkey was firstly separated by SDS-PAGE and then transferred to a nitrocellulose membrane by a Mini Trans-Blot® electrophoretic transfer cell (BioRad Laboratories, Inc. Hercules, CA).

Casein and whey protein purification

The nitrocellulose sheets were washed in 50 mM Tris; 150 mM NaCl; 0.05% Tween 20, pH 7.6 (TBST) and soaked in blocking solution (TBST, 1% BSA) and incubated for 16 hours.

A reversed-phase (RP-HPLC) and an ion exchange chromatography (Vincenzetti et al. 2008) were performed in order to purify and characterize the whey protein and the casein fractions in cow, goat and donkey milk. The HPLC system used was an Äkta Purifier (GE-Healthcare, Uppsala, Sweden). One aliquot (500 µl) of whole caseins resuspended in buffer A was subjected to the cationic exchange chromatography on HPLC through a MONO S HR 5/5 column, (1.0 ml bed volume, GE Healthcare), equilibrated in buffer A (flow rate of 0.5 ml/min) and eluted by a linear gradient between buffer A and buffer B (1 M ammonium acetate, 8 M urea, pH 5.5). The gradient used was: %B = 0, time = 10 min; %B = 100, time = 100 min; %B = 100, time = 110 min. Whey proteins from bovine, donkey and goat were separated by a RP-HPLC using a C4 Prosphere (300 Å, 5 µm, 4.6 mm I.D., 150 mm., Alltech); 500 µL of whey proteins were added to 500 µl of CL buffer (0.1 M bistris, pH 8.0 containing 8 M urea, 1.3% trisodium citrate, 0.3% DTT) and 1 ml was loaded into the reversed phase column equilibrated in trifluoroacetic acid (TFA)/H2O 1:1000 v/v (buffer A). Elution was achieved by the following step gradient with TFA/H2O/ acetonitrile 1:100:900 v/v (buffer B): %B = 0, time = 10 min; %B = 20, time = 10 min; %B = 40, time = 0.1 min; %B = 60, time = 40 min. The flow rate was 1 ml/min. In all cases, the proteins eluted from the columns were detected at 280 nm by a UV 900 Monitor included in the HPLC system. Each chromatographic peak eluted from each chromatographic course was collected and, subsequently, analyzed by SDS-PAGE. After the chromatographic courses, each purified peak casein and whey protein was dialyzed against 50 mM ammonium acetate buffer (pH 5.5), and 20 mM Tris/HCl buffer, respectively. After dialysis the concentration of proteins was determined by the Bradford protein assay method (Bradford 1976).

Immunoblotting analysis Blood samples were obtained from a total of 6 volunteers: 3 milk-allergic and 3 non-allergic subjects

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After incubation with blocking solution, the nitrocellulose membranes were directly incubated with each whole serum diluted 1:500 in TBST for at least 3 hours, to allow IgE antibodies eventually present in serum to cross-react with a specific milk proteins. After a washing in TBST, the nitrocellulose sheets were incubated with mouse anti-human Ig-G secondary antibody conjugated with Alkaline Phosphatase (AP) diluted 1:20000 in TBST for another hour. The AP reaction was visualized by the Alkaline Phosphatase Conjugate chromogen/substrate kit (Bio-Rad) according to the manufacturer’s instructions.

Allergenicity evaluation of milk proteins Caseins and whey proteins separated from cow, goat and donkey milk by chromatography and electrophoresis were individually blotted onto a nitrocellulose membrane and incubated with each serum of subject affected by CMPA and controls. The volunteer subjects involved in this preliminary screening were: 1A: subject affected by CMPA, 22 years old, female; 2A: subject affected by CMPA, 25 years old, female; 3A: subject with suspect CMPA, 25 years old, female; C1: control, 48 years old, male; C2: control, 46 years old, female; C3: control, 76 years old, male.

Results and discussion In this study, casein and whey protein fractions from cow, goat and donkey milk were separated using the different chromatographic procedures described in the Materials and methods section. The total whey proteins content was 1.7 mg/ml for bovine milk, 1.91 mg/ml for donkey milk and 1.77 mg/ml for goat

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Figure 1. (a) RP-HPLC of whey proteins from bovine milk. (b) 15% SDS-PAGE of peaks A, B, C and D eluted from RP-HPLC. St: Bio-Rad low molecular weight standard (97.4 kDa, phosphorylase b; 66.2 kDa, bovine serum albumin; 45.0 kDa ovalbumin; 31.0 kDa carbonic anhydrase; 21.5 kDa, soybean trypsin inhibitor; 14.4 lysozyme).

Figure 2. (a) Cationic-exchange chromatography on HPLC on casein from bovine milk. (b) 13% SDSPAGE of the peaks eluted from the MONO S HR 5/5 column. St: Bio-Rad low molecular weight standard.

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milk, whereas the total casein protein content for cow, donkey and goat milk was 5.08, 2.42 and 5.37 mg/ml, respectively.

By a cationic-exchange chromatography (MONO S HR 5/5) caseins from bovine milk were separated into 8 peaks named E, F, G, H, I, L, M, N as shown in Figure 2a.

By RP-HPLC whey proteins from bovine milk were separated into 4 peaks, A, B, C and D as shown in Figure 1a, 15% SDS-PAGE analysis revealed that the peak C corresponds to α-lactalbumin with a molecular weight of 13.1 kDa and peak D correspond to β-lactoglobulin having a molecular weight of 18.97 kDa. For peak A and B, no proteins were identified. In according to data reported in literature, we did not find the presence of lysozyme in bovine milk (Figure 1b).

Each peak subjected to 13% SDS-PAGE showed a pattern similar to the one reported in the literature for bovine milk (Rasmussen 1994). By comparing protein migration patterns with those previously published for bovine milk, the chromatographic peaks were identified as follows: peaks E, F were assigned to the β-casein with a molecular weight of 32.56 kDa, the peaks H-I to a mixture of αs1 with a molecular weight of 33.52 kDa and κ-casein with a molecular weight of 26.55 kDa. The peaks L-M

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Figure 3. (a) RP-HPLC of whey proteins from donkey milk. (b) 15% SDS-PAGE of peaks O, P and Q eluted from the reversed phase. St: Bio-Rad low molecular weight standard.

Figure 4. (a) Cationic-exchange chromatography of casein from donkey’s milk. (b) 13% SDSPAGE of the peaks eluted from the MONO S HR 5/5. St: Bio-Rad low molecular weight standard. were assigned to a mixture of αs1-, αs2- (molecular weight 32.03 kDa) and κ-casein whereas the peak N to the αs2 casein. By RP-HPLC, whey proteins from donkey milk were separated into 3 peaks named O, P and Q as shown in Figure 3a, 15% SDS-PAGE analysis (Figure 3b) revealed that the peak O corresponds to lysozyme with a molecular weight of 14.6 kDa, peak P corresponds to α-lactalbumin with a molecular weight of 14.1 kDa and peak Q corresponds to β-lactoglobulin with a molecular weight of 22.4 kDa, as previously reported (Vincenzetti et al. 2008).

chromatography (MONO S HR 5/5) as shown in Figure 4a. 13% SDS-PAGE analysis (Figure 4b) identifies mainly αs1 (32.3 kDa) and β-caseins (36.0 kDa): peak S was identified as β-casein, whereas peaks T and U were identified as αs1 casein. Peak R presents a weak band of 29.7 kDa presumably corresponding to an isoforms of αs1 casein (Vincenzetti et al. 2008). It was not possible to determine, in this experimental conditions, the presence of other types of caseins, such as αs2-, κ- and γ- , however other authors detected them in donkey’s milk but in very small amount (Chianese et al. 2010).

Caseins from donkey’s milk were separated into 4 peaks, named R, S, T and U, by cationic-exchange

Whey proteins from goat’s milk were separated into 3 peaks named V, W and X by reversed-phase

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Figure 5. (a) RP-HPLC of whey proteins from goat’s milk. (b) 15% SDS-PAGE of peaks V, W and X.

Figure 6. (a) Cationic-exchange chromatography analysis of casein from goat’s milk. (b) 13% SDSPAGE of the eluted peaks. St: Bio-Rad low molecular weight standard. chromatography (Figure 5a), 15% SDS-PAGE analysis (Figure 5b) revealed that the peak W corresponds to α-lactalbumin with a molecular weight of 12.7 kDa, peak X corresponds to β-lactoglobulin with a molecular weight of 18.27 kDa. Similarly to bovine milk, lysozyme was not present in goat’s milk. The goat milk’s whole casein was separated into 6 peaks, named Y, Z, K, J1, J2 and J3 by cationic‑exchange chromatography (MONO S HR 5/5), as shown in Figure 6a, 13% SDS-PAGE analysis on the peaks eluted from the cationic‑exchange chromatography, showed a pattern similar to that reported in the literature for goat’s milk (Greppi et al. 2008). By comparing protein

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migration patterns with those previously published for goat’s milk, the chromatographic peaks were identified as follows (Figure 6b): the peaks Y-Z were assigned to the β-casein (molecular weight of 29.70 kDa), the peaks K-J1 correspond to the κ-casein (molecular weight of 26.42 kDa), the peak J2 resulted to be a mixture of αs2-casein (molecular weight of 32.66 kDa), κ-casein (predominant) and αs1-casein (molecular weight of 23.19 kDa). The serum of the allergic subject 1A showed a weak cross-reactivity with bovine β-lactoglobulin (Figure 7, lane D), but a strong cross-reactivity was obtained towards the bovine αs1- and αs2- caseins (Figure 8a, lanes H-I, L and M), no cross‑reactivity

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in correspondence to the β-casein was observed (Figure 8a, lane G). In addition, the 1A subject showed cross-reactivity with goat αs1- and αs2- casein (Figure 8b), as well. No positivity was observed in correspondence of the donkey’s milk proteins (Figure 7, lanes O-P-Q and Figure 8c).

also with the αs2-casein from goat’s milk (Figure 9b) but did not show cross-reactivity with the donkey’s milk proteins (Figure 9c). The serum of subject 3A with suspected CMPA did not show any cross-reactivity with any of the blotted whey proteins (data not shown), while a weak crossreactivity towards the bovine casein fractions was observed, but not towards the goat and the donkey’s milk proteins (Figure 10).

The serum of allergic subject 2A did not show cross reactivity with any of the blotted whey proteins, indicating that its allergy is probably only due to casein fraction (data not shown). This subject showed in fact a very strong cross-reactivity towards all bovine casein fractions (Figure 9a), cross-reacted

Control subjects C1, C2 and C3 did not show crossreactivity with any of the blotted whey proteins and caseins from bovine, goat, donkey’s milk.

Conclusions The primary aim of this work was to verify the renowned hypoallergenic property of donkey’s milk, given the common knowledge and the recent evidences of the importance of this milk as substitute of human milk in CMPA infants. . The immunoblotting was performed with the serum of 6 volunteers, 2 of them (1A and 2A) were affected by CMPA, 1 (3A) was suspected to be affected by CMPA and 3 were healthy controls (C1, C2, C3). The serum of subject 1A showed a strong cross‑reactivity with the bovine caseins, a weak cross‑reactivity with the goat caseins, but no reactivity with the donkey caseins. Moreover, a very weak cross‑reactivity with 2 isoforms of bovine β-lactoglobulin was determined.

Figure 7. Immunoblotting analysis of the serum of the subject 1A affected by CMPA towards: α-lactalbumin from bovine (C), goat (W), donkey (P); β-lactoglobulin from bovine (D), goat (X), donkey (Q) and lysozyme from donkey milk (O).

The serum of subject 2A showed a very strong cross‑reactivity with the bovine casein fractions, a cross‑reactivity (but lesser than bovine) with the

Figure 8. Immunoblotting analysis of the serum of the subject 1A affected by CMPA towards bovine (a), goat (b) and donkey whole caseins (c). Lane G, bovine β-casein; lane H-I, bovine αs1- and κ-casein; lane L-M, bovine αs1 and αs2-caseins; lane Y, goat β-casein; lane J1-2-3, goat αs1 and αs2-caseins.

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Figure 9. Immunoblotting analysis of the serum of the subject 2A affected by CMPA towards bovine (a), goat (b) and donkey whole caseins (c). Lane G, bovine β-casein; lane H-I, bovine αs1- and κ-casein; lane L-M, bovine αs1 and αs2-caseins; lane Y, goat β-casein; lane J1-2-3, goat αs1 and αs2-caseins. The weaker serum cross-reactivity with goat milk proteins and the absence of serum cross-reactivity with donkey milk proteins in 2 of the 3 allergic volunteers included in this study confirm the lower allergenic power of goat and donkey milk proteins compared to those of bovine milk.

Figure 10. Immunoblotting analysis of the serum of the subject 3A with suspect CMPA towards bovine, goat and donkey whole caseins. goat caseins, and no reactivity with donkey caseins. The serum of A2 did not show cross-reactivity with any of the blotted whey proteins.

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The less allergenicity of goat’s milk has been ascribed to its lower α-casein content. For this reason goat milk is one of the most frequently suggested alternative to cow milk, although evidence of its tolerability is reported only by few clinical studies (Fiocchi et al. 2010). Some studies (Carroccio et al. 2000, Vita et al. 2007) indicated donkey milk as a valid substitute to cow milk; it has been demonstrated to be more tolerated than cow and goat milk and also than hydrolyzed milks in CMPA patients. In this preliminary study donkey’s milk proteins did not show any cross-reactivity with allergic sera, giving an important in vitro proof of its hypoallergenicity if compared to bovine and goat milk. However, due to the limited number of cases examined, further studies are needed to confirm these data. Considering the results obtained in this study, it would be possible to develop a sensitive diagnostic method for CMPA detection, based on chromatographic and immunoblotting analysis.

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References Bahna S.L. & Gandhi M.D. 1983. Milk hypersensitivity. I. Pathogenesis and symptomology. Ann Allergy, 50, 218-224. Bertino E., Gastaldi D., Monti G., Baro C., Fortunato D., Perono Garoffo L., Coscia A., Fabris C., Mussap M. & Conti A. 2010. Detailed proteomic analysis on DM: insight into its hypoallergenicity. Front Biosci, 2, 526-536. Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 249-254. Carroccio A., Cavataio F. & Iacono G. 1999. Cross-reactivity between milk portions of different animals. Clin Exp Allergy, 29, 1014-1016. Carroccio A., Cavataio F., Montalto G., D’Amico D. & Alabrese L. 2000. Intolerance to hydrolysated cow’s milk proteins in infants: Characteristics and dietary treatment. Clin Exp Allergy, 18, 1597-1603. Chianese L., Calabrese M.G., Ferranti P., Mauriello R., Garro G., De Simone C., Quarto M., Addeo F., Cosenza G. & Ramunno L. 2010. Proteomic characterization of donkey milk “caseome”. J Chromatogr A, 1217, 4834-4840. Chiofalo B., Dugo P., Bonaccorsi I.L. & Mondello L. 2011. Comparison of major lipid components in human and donkey milk: new perspectives for a hypoallergenic diet in humans. Immunopharmacol Immunotoxicol, 33, 633-644. Cianferoni A. & Spergel J.M. 2009. Food allergy: Review, Classification and Diagnosis. Allergol Int, 58, 457-466. Creamer L.K. 2003. Milk Proteins / Casein Nomenclature, structure and association properties. In Enciclopedia of Dairy Science (H. Roginski, J.W. Fuquay & P.F. Fox, eds). Academic Press, Amsterdam, 1895-1902. Criscione A., Cunsolo V., Bordonaro S., Guastella A.M., Saletti R., Zuccaro A., D’Urso G. & Marletta D. 2009. Donkey’s milk protein fraction investigated by electrophoretic methods and mass spectrometry analysis. Intern Dairy J, 19, 190-197. Dean T.P., Adler B.R., Ruge F. & Warner J.O. 1993. In vitro allergenicity of cow’s milk substitutes. Clin Exp Allergy, 23, 205-210. Docena G.H., Fernandez R., Chirdo F.G. & Fossati C.A. 1996. Identification of casein as the major allergenic and antigenic protein of cow’s milk. Allergy, 51, 412-416. Fiocchi A., Brozek J., Schünemann H., Bahna S.L., von Berg A., Beyer K., Bozzola M., Bradsher J., Compalati E., Ebisawa M., Guzmán M.A., Li H., Heine R.G., Keith P., Lack G., Landi M., Martelli A., Rancé F., Sampson H., Stein A., Terracciano L., Vieths S. & World Allergy Organization (WAO) Special Committee on Food Allergy. 2010. World Allergy Organization (WAO) Diagnosis and Rationale for Action against Cow's Milk

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Allergy (DRACMA) Guidelines. Pediatr Allergy Immunol, 21, 1-125. Giner M.T., Vasquez M., Dominiguez O. 2012. Specific oral desensitization in children with IgE-mediated cow’s milk allergy. Evolution in one year. Eur J Pediatr, 171, 1389-1395. Hill D.J., Firer M.A., Shelton M.J. & Hosking C.S. 1986. Manifestations of milk allergy in infancy: clinical and immunologic findings. J Pediatr, 109, 270-276. Greppi, G.F., Roncada P. & Fortin R. 2008. Protein Components of Goat’s Milk. In Dairy Goats Feeding and Nutrition (A. Cannas and G. Pulina eds). CAB International, Oxfordshire, UK. Iacono G., Carroccio A., Cavataio F., Montalto G., Soresi M. & Balsamo V. 1992. Use of ass’s milk in multiple food allergy. J Pediatr Gastr Nutr, 14, 177-181. Järvinen K.M., Chatchatee P., Bardina L., Beyer K. & Sampson H.A. 2001. IgE and IgG binding epitopes on alpha-lactalbumin and betalactoglobulin in cow’s milk allergy. Int Arch Allergy Immunol, 126, 111-118. Meglio P., Bartone E., Plantamura M., Arabito E. & Giampietro P.G. 2004. A protocol for oral desensitization in children with IgE-mediated cow’s milk allergy. Allergy, 59, 980-998. Monti G., Bertino E., Muratore M.C., Coscia A., Cresi F., Silvestro L., Fabris C., Fortunato D., Giuffrida M.G. & Conti A. 2007. Efficacy of donkey's milk in treating highly problematic cow's milk allergic children: an in vivo and in vitro study. Pediatr Allergy Immunol, 18, 258-264. Muraro M.A., Giampietro P.G. & Galli E. 2002. Soy formulas and nonbovine milk. Ann Allergy Asthma Immunol, 89, 97-101. Rasmussen L.K., Højrup P. & Petersen T.E. 1994. Disulphide bridges in bovine caseins: localization of intrachain disulphide bridges in monomers of κ– and αs2-casein from bovine milk. J Dairy Res, 6, 485-493. Restani P., Beretta B., Fiocchi A., Ballabio C. & Galli C.L. 2002. Cross-reactivity between mammalian proteins. Ann Allergy Asthma Immunol, 89, 11-15. Vincenzetti S., Polidori P. & Vita A. 2007. Nutritional characteristics of donkey’s milk protein fraction. In Dietary protein research trends (J.R. Ling ed). Nova Science Publisher, Inc., New York, USA, 207-225. Vincenzetti S., Polidori P., Mariani P., Cammertoni N., Fantuz F. & Vita A. 2008. Donkey’s milk protein fractions characterization. Food Chem, 106, 640-649. Vita D., Passalacqua G., Di Pasquale G., Caminiti L., Crisafulli G., Rulli I. & Pajno G.B. 2007. Ass’s milk in children with atopic dermatitis and cow’s milk allergy: Crossover comparison with goat’s milk. Pediatr Allergy Immunol, 18, 594-598. Waserman S. & Watson W. 2011. Food allergy. Allergy Asthma. Clin Immunol, 7, Suppl 1, S7.

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Seasonal monitoring of Aedes albopictus: practical applications and outcomes Stefano Gavaudan1*, Anna Duranti1, Francesca Barchiesi1, Sara Ruschioni2, Elisa Antognini1, Enrica Calandri1, Paolo Mancini1 & Paola Riolo2 2

1 Istituto Zooprofilattico Sperimentale Umbria e Marche, Perugia, Italy Dipartimento di Scienze Agrarie, Alimentari e Ambientali, Università Politecnica delle Marche, Ancona, Italy

* Corresponding author at: Istituto Zooprofilattico Sperimentale Umbria e Marche, Via Cupa di Posatora 3, 60100 Ancona, Italy. Tel.: +39 071 41760, fax: +39 071 42758, e-mail: s.gavaudan@izsum.it

Veterinaria Italiana 2014, 50 (2), 109-116. doi: 10.12834/VetIt.1009.05

Accepted: 22.07.2013 | Available on line: 18.10.2013

Pest Management e salute pubblica. Veterinaria Italiana, Collana di monografie, Monografia 23, 2011 - Reprint Keywords Aedes albopictus, Arbovirus, Mosquito, Ovitrap, Pest control, Surveillance system, Vector monitoring.

Summary The introduction of the Asian tiger mosquito Aedes (Stegomya) albopictus (Diptera: Culicidae) into temperate regions poses serious concerns for the risk of the spreading of arboviral epidemics, as confirmed by the Chikungunya fever outbreak in Italy. This article describes the implementation and the results of a strategy for the pest management implemented over 4 years in Pesaro (a city in the Marche region, Italy). The strategy used 60 integrated wide‑sized ovitraps for monitoring purposes. Twenty-day larvicide-based treatment cycles were implemented for the manholes of the urban area and also the inhabitants were involved in pest control relating to their own properties. It was observed that the weekly median of eggs laid decreased consistently from 2008 to 2011, indicating the good performance of the vector control and a reduction in the related epidemics risk.

Descrizione di un sistema di monitoraggio stagionale di Aedes albopictus e dei risultati ottenuti Parole chiave Aedes albopictus, Arbovirus, Controllo dei parassiti, Monitoraggio, Sorveglianza, Trappole, Zanzara.

Riassunto La diffusione sul territorio italiano della cosiddetta zanzara tigre Aedes (Stegomya) albopictus ha aumentato il rischio della propagazione di epidemie arbovirali, quali ad esempio i casi di Chikungunya registrati nel Nord Italia. Questo articolo descrive il monitoraggio effettuato nella città di Pesaro (Marche, Italy) per un periodo di 4 anni sulla popolazione di Ae. albopictus. Il monitoraggio è stato eseguito usando 60 trappole, uno strumento sempre utile nell’ambito di strategie di controllo degli insetti. Nel presente studio sono stati effettuati cicli larvicidi della durata di 20 giorni che hanno interessato i sistemi fognari della città; anche i cittadini sono stati coinvolti nello studio per quanto concerne il controllo nelle proprietà private. Tra il 2008 e il 2011 è stata osservata una diminuzione del numero di uova deposte settimanalmente. Quest’ultimo dato evidenzia l’efficacia della strategia di controllo di Ae. albopictus e indica, quindi, una considerevole riduzione del rischio epidemico correlato al vettore.

Introduction The diffusion of the Asian tiger mosquito Aedes (Stegomya) albopictus (Skuse, 1897) (Diptera: Culicidae) is increasing across Europe, as well as on other continents. Over the last two decades, its presence in Mediterranean areas has increased, as has its spread into the northern countries of the European Union, with new exotic species of mosquito also being introduced (Medlock

et al. 2006, Werner et al. 2012, Eritja et al. 2005, Alto & Juliano 2001, Capelli et al. 2011). In some countries, Ae. albopictus has displaced Ae. aegypti (Linnaeus, 1862). This poses concern for dengue and chikungunya epidemics, for the increased involvement of Ae. albopictus as a vector, and for the spread of arboviruses outside endemic areas (Carrieri et al. 2011, ECDC 2007, Vazeille et al. 2008, Hawley 1988, Gratz 2004). This situation has been

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confirmed by the recent outbreaks of dengue fever in France (La Ruche et al. 2010) and Croatia (Gjenero-Margan et al. 2011), and of chikungunya fever in Italy (Rezza et al. 2007). These outbreaks also suggest the need to improve the surveillance and control of this arbovirus vector and pest, Ae. albopictus (Carrieri et al. 2011). Indeed, it is known that a lack of mosquito control and related public health strategies can enhance the population density of Ae. albopictus, which can thus increase the risk of further epidemics (ECDC 2007, Eritja et al. 2005, Mitchell 1995, Dana et al. 2000, Gratz 2004). In Italy, the Ministry of Health has recommended that the Public Health Services improve arbovirus surveillance. However, mosquito surveillance and control are often not very well practised and are based on little experience, because of the lack of national schemes for these activities (Ministero della Salute 2011). Consequently, every municipality is responsible for its own territory, as well as the population being responsible for their own properties, with only a few regions carrying out wellcoordinated surveillance (ECDC 2007, Romi & Majori 2009, Ministero della Salute 2007). The distribution of Ae. albopictus is determined by several environmental variables (Medlock et al. 2006, Alto & Juliano 2001, Roiz et al. 2011), with the main parameters arising from previous studies being the mean temperature in January (JanTmean), the annual mean temperature (AnnTmean), and the annual precipitation. A JanTmean <0 °C affects the survival rate of the diapausing eggs during the winter period (IZSUM, Knudsen et al. 1996). When the AnnTmean is >11 °C, this determines the areas that are suitable for adult survival (Medlock et al. 2006, Knudsen et al. 1996, ECDC 2009, Kobayashi et al. 2002). Finally, an annual rainfall >500 mm is the threshold for habitats where mosquito populations can thrive (Roiz et al. 2010). In the regions along the Adriatic coast in Italy, the Ae. albopictus population trend is characterised by a peak in summer and the disappearance of the adults in winter (with the exceptions of most of the more southern latitudes). This trend helps to control the mosquito population early in spring (at the initial stage of infestation), which will also benefit from focused surveillance of human cases of arboviruses during the higher risk periods of epidemics (Carrieri et al. 2008, Ministero della Salute 2011, Romi & Majori 2009). To achieve this goal, a regional scheme for chikungunya and dengue surveillance and Ae. albopictus monitoring was set-up in the Marche and Emilia Romagna regions of central-eastern Italy, with the involvement of the larger municipalities in both of these regions (Carrieri et al. 2011, Ministero della Salute 2007). This scheme was carried out in the Marche region

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with very limited budget and resources. The monitoring system that was followed, which was not mandatory for all municipalities in the region, also included staff training (community workers, environmental officers, cleaners, volunteers of the regional Civil Protection Authorities). Information and practices were shared with the stakeholders (the local population, politicians and general practitioners). This report describes the monitoring system adopted in the town of Pesaro (Marche, Italy), and it discusses the results of the survey through the years of 2008 to 2011.

Materials and methods The monitoring system presented here was carried out in the Pesaro municipality (43°91’20”N; 23°91’57”E), in the north of the Marche region (central-eastern Italy), at 11 m a.s.l., with a population of 95,011 inhabitants, and a total urban area of ca. 12,658 ha (ISTAT 2010). The monitoring was achieved through the use of ovitraps. Each ovitrap consisted of a black plastic pot of 400 ml that was filled with dechlorinated water that contained a strip of masonite (12.5 × 2.5 cm in size), where the Ae. albopictus laid their eggs. A total of 60 ovitraps were distributed homogeneously in the urban area. Each trap was placed at a minimum distance of 500 m from the next one. The positions of ovitraps were previously planned using the geographic information system. The ovitrap density was decided upon according to the Emilia Romagna guidelines, as reported for municipalities with urbanised areas between 3,001 ha and 5,000 ha and without previous monitoring data (Ministero della Salute 2007, Regione Emilia Romagna 2008). The ovitraps were placed at ground level by trained technicians, preferably on grass, hidden and in the shade, so the Ae. albopictus can rest and lay their eggs. The ovitraps were left in these same positions through the whole period of the survey. On a weekly basis, and usually on Wednesdays, the masonite strips were collected and delivered to the laboratory. The ovitraps were then filled up again, with the masonite strip was replaced (Carrieri et al. 2011). Identification and counting of the eggs were performed in the laboratory, under stereomicroscopy at 40× magnification (Olympus SZX7, Japan). Over the four years of the survey (2008-2011), the ovitraps were activated for a period of 20 weeks each year, during the vectorial season. This occurred from week 20 to week 40 of each year (late May to early October). The total number of eggs per ovitrap was published on-line in a weekly bulletin (IZSUM). The median number of total eggs harvested weekly from the ovitraps was used for the descriptive analysis.

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The median was used because the data do not fit a normal distribution. Analysis of the seasonal population dynamics of Ae. albopictus was performed using the ovitrap index, as the number of positive ovitraps/ the number of active ovitraps ×100. These are reported as weekly box-and-whisker plots. The presence of annual trends was tested for by using the Cuzick test, a non-parametric test for trends across ordered groups, which is an extension of the Wilcoxon rank-sum test (Cuzick 1985, Altman 1991, Dana et al. 2000). The Cuzick test was used to evaluate the trends in the median levels of eggs laid across the four years considered. The statistical analyses were performed with the Stata 11.1 software, and the data have also been reported using the Map Info Professional software, version 7.5 (data not shown). Each ovitrap was identified by its own barcode,

which also coded for the coordinates and elevation of each site monitored. The week of sampling and the number of eggs were the data produced by the laboratory, and this information was automatically included in a database. Excel files of the weekly monitoring data were sent to the municipalities. The meteorological data were obtained from a local weather station: the Meteorological Observatory “Valerio”, Pesaro. These data included the daily temperatures (Tmin, Tmax) and the rainfall through the four years of 2008 to 2011.

Results The percentage of active ovitraps over the four years of monitoring ranged from a minimum of 92% to a maximum of 100%. The median numbers of eggs and the selected percentiles per year are detailed in Table I.

Table I. Median egg numbers per year, with selected percentiles (P). Year

Maximum 980 547 330 320

2008 2009 2010 2011

Minimum 0 0 0 0

Eggs per year, with percentiles (P) P25 P50 P75 0 40 107 0 8 51 0 0 4 0 0 3

Eggs/Ovitrap/Week

2008

P98 407 289 75 145

P99 535 325 150 205

2009

800

600

400

200

0 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Week

2010

2011

800

600

400

200

0 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Week

Figure 1. Box-and-whisker plots of the seasonal population dynamic based on egg density for the years 2008 to 2011.

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Ovitrap index

2008

2009

100

0 20

Week

2010

2011

100

Week

0 20

Week

Figure 2. Ovitrap index trends for the years 2008 to 2011.

Median egg density

Fitted values

200

150

100

Table II. Mean temperatures and rainfall for the years 2008 to 2011. Year

JanTmean (°C)

AnnTmean (°C)

Rainfall (mm)

2008

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0 01July2008

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Figure 3. Trend in the weekly median number of eggs for the years 2008 to 2011. Figure 1 illustrates the weekly box-and-whisker plots over the whole of the monitoring season through each of the four years (as weeks 20 to 44). Figure 2 shows the ovitrap indices calculated for the same periods. In the first year of monitoring (2008) the seasonal dynamics analysis (Figures 1, 2) showed three peaks of egg density: the first during the week 28, in mid-July (median number of eggs/ ovitrap/ week, 91; ovitrap index, 87%); the second for week 32, in mid‑August (median number of eggs/ ovitrap/

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week, 195; ovitrap index, 93%); and the third for week 37, in mid‑September (median number of eggs/ ovitrap/ week, 155; ovitrap index, 96%). Considering the whole observation period, the weekly ovitrap index was >50% and the median number of eggs exceeded 50 per week from week 26 to week 37 (Figures 1, 2). In the second year of monitoring (2009), there were again three peaks seen: the first peak of eggs was detected during week 25, in mid-to-late June (median number of eggs/ ovitrap/ week, 31; ovitrap index, 72%); the second peak for week 28, in mid‑July (median number of eggs/ ovitrap/ week, 52; ovitrap index, 77%), and the third peak for week 31, in early August (median number of eggs/ ovitrap/ week, 93; ovitrap index, 91%) (Figures 1, 2). In the last two years of monitoring (2010, 2011), the

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20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

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Tmean

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Figure 4. Mean weekly temperatures (line graph) and rainfall (histogram) from week 20 to week 40 for the years 2008 to 2011. data showed greatly lowered seasonal trends for the median numbers of eggs (Figures 1, 2). In 2010, three small peaks were again detected: the first in week 25, in mid-to-late June (median number of eggs/ ovitrap/ week, 4; ovitrap index, 54%); the second in week 29, in mid-to-late July (median number of eggs/ ovitrap/ week, 16; ovitrap index, 79%), and the third in week 34, towards the end of August (median number of eggs/ ovitrap/ week, 7; ovitrap index, 68%). Finally, in 2011, the single peak for the median numbers of eggs was seen for week 35, early in September (median number of eggs/ ovitrap/ week, 37; ovitrap index, 75%). Although they showed low median numbers of eggs, the ovitrap indices for 2010 and 2011 remained at relatively high levels (as ovitrap index >50%) for weeks 34 and 35, and for weeks 31 to 35, respectively (Figures 1, 2). The seasonal population trends of the Ae. albopictus dynamics from 2008 to 2011 are shown in Figure 3. The significant of the progressive reduction in the number of eggs from 2008 to 2011 was verified by the Cuzick test for the trend (p <0.0001). Meteorological data were collected and analysed to provide the means for the January and annual temperatures and the total annual rainfall (Table II). The JanTmean through these four years was always >0 Veterinaria Italiana 2013, 50 (2), 109-116. doi: 10.12834/VetIt.1009.05

°C, with JanTmean from 3.1 °C (2010) to 5.8 °C (2008). Similarly, the AnnTmean are considerably higher than 11 °C, varying from 17.8 °C (2010) to 19.1 °C (2008). The rainfall from 2008 to 2010 was also a lot higher than the minimum of 500 mm: from 664 mm (2011) to 1,203 mm (2010). The mean temperatures and the rainfall measured during the monitoring period are briefly reported in Figure 4.

Discussion As indicated above, the median number of egg per week showed a significant decreasing trend over the years from 2008 to 2011. During the second year of monitoring, the median number of eggs remained at lower levels than for the previous year, 2008, and especially during the period of the maximum population density. However, although in the second year of monitoring the peaks were lower, the ovitrap index and median number of eggs remained high from week 25 to week 32 (ovitrap index >50%; median number of eggs >50). In the other two years (2010 and 2011) very low egg densities were found, with the median number of eggs rarely exceeding 50. The ovitrap index is related to the levels of diffusion of Ae. albopictus in the period of maximum

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population development, and not to the numbers of eggs harvested in the ovitraps, which are instead related to the population abundance. This pattern is due to the high sensitivity of these ovitraps, as it is known that each ovitrap can capture at least one egg even if only a few females are present. Nevertheless, there was a decreasing trend in median eggs per week from 2008 to 2011 (although the ovitrap indices remained high in the period of maximum development). Furthermore, on the basis of preliminary assessments, there were no significant differences in the climatic parameters to justify such a massive reduction in the infestation in the years of surveillance. These data suggest excellent control of this pest. This mosquito control was achieved using a diflubenzuron-based larvicide product over 8 cycles, of about 20 days each, over the 24 weeks each year during the vectorial season, so from May to October. Every week, the treatment of one district of the town of Pesaro was carried out, covering the whole total of 18,000 manholes for each cycle. Treatments to kill the adult mosquitoes with a pyriproxyfen-based product were suggested only if social events were planned that were close to the population communities, and only if they could be confirmed to be a particular nuisance for the population by an inspection. The Public Health Office authorized all of these treatments. The pest control strategy also included education of the population, particularly providing information to prevent, discover and eliminate mosquito breeding sites. Larvicide products for the treatment of inextinguishable breeding sites were also distributed to the population through reduced prices in local pharmacies (about 1,200 tablets/year of Bacillus thuringensis israelensis spores); these were also free to schools, agencies, and other partners. Information was set up throughout all meetings, with fact sheets and a toll free number established. Finally, a staff of technicians was available for visits upon request, and an entomologist provided sampling for larvicidal efficacy in manholes.

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Conclusions As suggested in the literature, the January and annual temperatures and the annual precipitation are the most important limiting climatic factors for the mosquito breeding cycles. Over these four years of investigation, the JanTmean, AnnTmean and annual precipitation were always above the thresholds: >0 째C, >11 째C, >500 mm, respectively. Therefore, we can assume that in the Pesaro area the survival rate of the diapausing eggs during the winter period was not influenced by the winter temperatures. Moreover, looking at the AnnTmean and the rainfall, we can state that this area is very suitable for Ae. albopictus to thrive. Thus, the climatic factors were not significant in limiting potential seasonal increase and spread of the local Pesaro Ae. albopictus population. In our experience, monitoring is a fundamental tool for effective mosquito control. This system based on ovitraps, as described herein, is economic and effective, and allowed the measuring of the main factors relating to the Ae. albopictus population: the abundance and diffusion. These parameters are very important for the evaluation of seasonal activities during any effective pest control system. To ensure the reliability of pest control systems, control and monitoring should be performed by different interested parties. If both monitoring and pest control are well performed and managed, the risk of arbovirosis is also reduced and improved responses are possible in the case of any outbreak. However, it is still necessary to establish this level of organization in the major urban areas of the Italian peninsula, where mosquito infestation is often largely out of control, with the consequent high risk of epidemics. This concern is probably due to a lack of knowledge of the risks associated with emerging arboviruses, and the lack of specific expertise among municipal technicians. The task of public health scientists (e.g. entomologists, surgeons, epidemiologists) is therefore to accelerate the timetable for the implementation of existing techniques and to design innovative techniques for mosquito monitoring and control. They also need to disclose the use of and spread the knowledge of such methods among politicians and administrators.

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The contribution of veterinary medicine to public health and poverty reduction in developing countries John B. Muma1, Kennedy K. Mwacalimba2*, Hetron M. Munang’andu3, Gift Matope4, Akinbowale Jenkins5, Victor Siamudaala6, Aaron S. Mweene1 & Tanguy Marcotty5,7 Department of Disease Control, University of Zambia, School of Veterinary Medicine, P.O. Box 32379 Lusaka, Zambia. 2 Independent Health Policy Researcher, 4639D Santa Cruz Dr, Indianapolis, Indiana 46268, USA. 3 Section of Aquatic Medicine and Nutrition, Department of Basic Sciences and Aquatic Medicine, Ullevålsveien 72, P.O. Box 8146 Dep., 0033 Oslo, Norway. 4 Department of Paraclinical Veterinary Studies, University of Zimbabwe, Faculty of Veterinary Science, P.O. Box MP 167, Mount Pleasant, Harare, Zimbabwe. 5 Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Onderstepoort, South-Africa. 6 KAZA TFCA Secretariat, Plot 2951, Madiba Shopping Complex, P.O BOX 821 Kasane, Botswana. 7 Department of Animal Health, Institute of Tropical Medicine, Antwerp, Belgium. 1

* Corresponding author at: Independent Health Policy Researcher, 4639D Santa Cruz Dr, Indianapolis, Indiana 46268, USA. Tel.: +1317 735 2920, e-mail: kapala.mwacalimba@gmail.com

Veterinaria Italiana 2014, 50 (2), 117-129. doi: 10.12834/VetIt.1405.323 Accepted: 14.05.2014 | Available on line: 30.06.2014

Keywords Africa, Diseases, Livelihoods, Livestock Production, One Health, Poverty alleviation.

Summary Few studies have explicitly examined the linkages between human health, animal disease control and poverty alleviation. This paper reviews the contribution that veterinary medicine can make to poverty alleviation in sub-Saharan Africa. Our analysis attempts to explore aspects of this contribution under five themes: food production; food safety; impact and control of zoonotic infections; promotion of ecotourism; and environmental protection. While these areas of human activity have, more or less, fallen under the influence of the veterinary profession to varying degrees, we attempt to unify this mandate using a ‘One Health’ narrative, for the purpose of providing clarity on the linkages between the veterinary and other professions, livestock production and poverty alleviation. Future opportunities for improving health and reducing poverty in the context of developing African countries are also discussed. We conclude that veterinary science is uniquely positioned to play a key role in both poverty reduction and the promotion of health, a role that can be enhanced through the reorientation of the profession’s goals and the creation of synergies with allied and related professions.

Il contributo della medicina veterinaria per la salute pubblica e la riduzione della povertà nei paesi in via di sviluppo Parole chiave Africa, Allevamento, Animale, One Health, Patologia, Povertà, Scienze veterinarie.

Riassunto Le relazioni tra salute umana, controllo delle patologie animali e programmi di riduzione della povertà raramente sono state oggetto di analisi. Questo articolo analizza il contributo che la medicina veterinaria può fornire ai processi di riduzione della povertà nell’Africa sub‑sahariana. In particolare, vengono analizzate le implicazioni della medicina veterinaria su: produzione di alimenti, igiene alimentare, impatto e controllo delle zoonosi, promozione di ecoturismo e protezione dell’ambiente. Lo studio ha l’obiettivo di riconsiderare questi aspetti sulla base dell’approccio “One Health” e di chiarire le relazioni che la professione veterinaria ha con le altre professioni, gli allevamenti animali e i programmi di riduzione della povertà. L’articolo esamina le opportunità future per migliorare le condizioni di salute e ridurre il sottosviluppo nei paesi africani, evidenziando il ruolo determinante delle scienze veterinarie. Ruolo che può essere ancor più potenziato attraverso la ridefinizione degli obiettivi professionali e la creazione di sinergie con le altre professioni.

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Introduction The ‘One World, One Health’ framework supports an integrated approach for addressing the surveillance of, and response to, human, animal and environmental health concerns. First articulated by William Osler and Rudolf Virchow over a century ago (Kahn et al., 2007), ‘One Health’ was re-introduced to the world in Schwabe’s ‘Veterinary Medicine and Human Health’ (AVMA 2008; Battelli and Mantovani, 2011). Its contemporary precepts were articulated at a symposium organised by the Wildlife Conservation Society (WCS) in New York in 2004 (WCS 2013). While the inextricable linkages between human and animal health and their shared environment have been outlined in the literature, context-specific illustrations of the need to maximise the benefits of a holistic approach to animal and human health in the Sub-Saharan African context are still required. In the war against infectious diseases, physicians in Sub-Saharan Africa often face what seem to be insurmountable odds; odds which could be improved by a partnership between the veterinary and environmental professions to explore and address Africa’s social determinants of health. At the same time, the veterinary profession in Sub-Saharan Africa, however, faces unique context-specific challenges. Following the International Monetary Fund Structural Adjustment Programmes of the late 80s and 90s, animal health programmes, which used to be government run, were relegated to the private sector so to scale down the role of public administration involved in the management of veterinary service (Cheneau et al. 2004). However, in many contexts, the disincentives of working in rural or low input areas led to detrimental consequences for overall animal health and production. On the one hand, the vulnerability of rural areas makes them a key source of both human and animal infectious diseases; a situation worsened by the paucity of rural veterinary support. On the other hand, under commercial settings of Sub‑Saharan Africa, the information technology boom of the 21st century allowed farmers to become much more educated than they used to be, having access to specialised information through formal and informal educational resources, making the farmers less likely to consult veterinarians on areas such as cattle, pig and poultry production and other husbandry practices. The veterinary profession in Sub-Saharan Africa has therefore suffered severe setbacks as the need for specialist veterinary care in animal production has decreased. That said, the public good aspect of the veterinary profession has special relevance for the SubSaharan African context, particularly in those nations in which the vast majority of the population

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is dependent on livestock and agriculture. These populations face a lack of knowledge concerning the veterinary public health risks of uncontrolled zoonoses, the importance of food safety, the need for environmental protection and the negative impacts of limited investments in disease control and quality assurance systems. With international development discourse now shifting towards propoor initiatives (e.g. the millennium development goals), whose core objective is the alleviation of poverty, the veterinary profession in Sub-Saharan Africa is presented with an opportunity to refocus its efforts on livelihood advancement through contributions to areas such as sustainable animal health, tourism, trade and advancing the cause of environmental protection. Therefore, veterinarians operating in these countries need to propose and develop control strategies within their mandate that are both effective and context appropriate, taking into account economic, cultural and sociological considerations (Marcotty et al. 2009). To achieve this, we suggest using the ‘One Health’ framework in a livelihoods approach (Scoones 2010). This involves understanding the context and needs of rural communities to define control and response strategies that are equitable, accessible and appropriate. In this paper, we examine the role of veterinary profession in poverty reduction and public health in resource-poor communities of Sub-Saharan Africa by focusing on five thematic areas: food production and food security; food safety; control of zoonotic infections, environmental protection and the promotion of ecotourism. The overarching question through which we explore these thematic areas is: ‘how can the adoption of pro-One Health strategies aid the veterinary profession in effectively contributing to poverty alleviation and rural livelihoods?’ Addressing this question requires to consider two preliminary and interlinked questions, whose analysis constitute the conceptual framework for this paper. 1. How does veterinary medicine fit into the complex arena of rural livelihood advancement, animal production, food safety and environmental protection? 2. How can the profession go beyond its animal health limits to positively impact rural livelihoods and poverty alleviation?

Background Among rural livestock-keeping communities, livestock carry enormous currency, what Smith and colleagues (Smith et al. 2001) termed “multifunctionality”. Multifunctionality describes the myriad roles that livestock play in the maintenance

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of rural livelihoods, as sources of nutrition and assurance of domestic food through the provision of meat and milk; traction power and manure for crops; the maintenance of distinctive rural cultures in their role in marriages (the quoting of dowry in livestock) and traditional ceremonies; and their role in providing social security by offsetting crop failure and acting as banks (Ilemobade 2009; Maudlin et al. 2009; Perry et al. 1984; Mwacalimba et al. 2013). Furthermore, low-scale free range poultry rearing and small ruminant production has been identified both as major sources of protein and alternative income for the poor since they are readily marketable (Dolberg 2003). The multifunctionality of livestock implies that for most rural livestock keepers in Sub-Saharan Africa, human livelihoods are inextricably linked to the welfare of their stock. Emotional distress is frequent in times of high cattle mortalities, owing to the helplessness farmers feel in the absence of veterinary intervention during high impact disease outbreaks. This is a phenomenon experienced also in high income countries, for example it has been reported that that life after the UK Foot and Mouth (FMD) crisis of 2001 “was accompanied by distress, feelings of bereavement, fear of a new disaster, loss of trust in authority and systems of control, and the undermining of the value of local knowledge” (Mort et al. 2008). The authors further noted that “such distress remained largely invisible to the range of ‘official’ inquiries into the disaster” (Mort et al. 2008). Such emotional traumas are not easily quantifiable and, thus, do not attract the sympathy of policymakers who focus only on animal morbidity and mortality without weighing the emotional investment made by livestock owners. Livestock and livestock production are critical to the welfare and livelihoods of many people living in pastoral and mixed crop-livestock farming communities in developing countries (FAO 2004; WHO 2006). Animals owned by poor farmers in such contexts remain susceptible to a broad spectrum of diseases due to their owners’ inability to meet the cost of disease prevention and production inputs (FAO 2002). Animal health status is a particularly important constraint to poverty alleviation. It can thus be argued that maintaining livestock health, adequate veterinary services can actually bolsters crop production, rural livelihoods and the social and emotional wellbeing of livestock owners (Muma et al. 2012). We argue that this can be strengthened via veterinary input in animal production, disease control, food safety and environmental protection. The following sections offer an overview of each of these thematic areas, followed by a description of the role the veterinary profession can play in each of such areas.

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Veterinary medicine and poverty alleviation Contribution to food production Food production and food security are critical components of rural livelihoods in Sub-Saharan Africa. In crop-livestock production systems, animal traction power is a vital input in the production cycle (Perry et al. 1984). It has been demonstrated that when oxen are available for cultivation, maize production is increased 4 to 5 times (Connor 1989). However, cattle kept by rural populations remain vulnerable to diseases and adverse climatic conditions, which all tend to impact negatively on rural welfare and food security (WHO 2006). Serious livestock epidemics have the potential to threaten entire crop-livestock production systems by adversely affecting those communities whose livelihoods actively depend on animal draft power. The impact of the now eradicated Rinderpest virus on human communities is an example of how a livestock disease can change the course of nations. The broad impact of infectious diseases is not unique to Africa. The 2001 Foot and Mouth Disease (FMD) outbreak in the UK provides another example of how a nation’s economy can be severely affected by livestock diseases. Foot and Mouth Disease is reported to have brought about losses to agriculture and the food chain amounting to £ 3.1 billion. The majority of costs went towards compensation for slaughtered livestock, waste disposal and cleanup, while agricultural producers were expected to suffer losses estimated at £ 355 million, representing about 20% of the estimated total income from UK farming in 2001 (Thompson et al. 2002). A 2002 study conducted by the National Audit Office estimated the direct costs of the outbreak at £ 3 billion and the indirect costs at £ 5 billion (Oxford Analytica 2012). In Ethiopia, when oxen numbers were halved by trypanosomosis, production of cereals also fell. Further losses of oxen forced cultivators to abandon fertile areas for higher ground (Slingenberg 1992). As an alternative, small ruminant keeping has been suggested for such communities, due to their higher disease tolerance and resilience to drought and other adverse climatic conditions (Omondi et al. 2008). However, in many parts of Sub-Saharan Africa, cattle remain central to the livelihoods and identity of crop keeping rural communities. They are also valued for their input in crop production, higher sale price and milk production potential compared to sheep and goats. Livestock movement bans are usually instituted during severe disease outbreaks of conditions such as FMD or Contagious Bovine Pleuropneumonia (CBPP)

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(Pineda-Krch et al. 2010; Woolhouse 2003). These movement restrictions incapacitate subsistence farmers by denying them the opportunity to obtain returns on livestock and their products, which are critical sources of income (Pineda-Krch et al. 2010; Woolhouse 2003). Movement bans also restrict access to pasture and water, often located long distances from households that practice transhumant grazing and nomadic pastoralism systems. When livestock movement bans are implemented just before the crop planting season, farmers often fail to raise funds needed to obtain farming inputs such as seed and fertilizer. Funds are usually acquired through the sale of livestock at markets far from their homes. Movement bans also lead to problems in food security. In West Africa for instance, an interstate ban on the movement of poultry and poultry products instituted following an outbreak of avian influenza ultimately led to regions with low poultry production unable to obtain poultry and poultry meat from the high poultry producing areas (Dolberg 2003). The result was the reduced availability of the cheapest and commonest source of protein for low-income consumers (Dolberg 2003). Often, outbreaks which occur in Sub-Saharan Africa are not due to a failure to detect disease occurrences, they rather follow a lack of appropriate response tools for early detection by veterinarians or qualified animal health technicians. There is a direct correlation between consistent up-scaled veterinary service delivery and food availability. This has been seen in developed economies in which there is a rapid response to disease outbreaks and effective monitoring and surveillance systems. An example is the control of FMD in Japan where outbreaks are rapidly quelled because of financial investments in the veterinary services (Muroga et al. 2012; Sugiura et al. 2006). Well-established interventions such as vaccination of cattle against diseases of economic importance like FMD, Contagious Bovine Pleuropneumonia (CBPP), brucellosis and avian influenza, as well as the promotion of good husbandry practices and community orientated bio-sanitation are among the most effective tools of controlling livestock diseases and thus provide an important adjuvant to poverty alleviation in rural communities.

Contribution to food safety With the increase of the worldâ&#x20AC;&#x2122;s population, a global food crisis has been foreseen, with developing nations identified as future significant contributors to food production, particularly from livestock and livestock products (Delgado et al. 1999). There is now an increased global awareness of food borne diseases and the importance of food safety in

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many parts of the world (Knight et al. 2003). The importance of food safety cannot be overstated. Milk and other animal products must be safe, sound and wholesome if they are to contribute to the creation of healthy societies, which is essential for national productivity (Choudhury et al. 2013). Most developing countries suffer chronic food safety problems, mainly attributed to poor food safety governances systems; insufficient food hygiene education; poor and non-existent waste disposal systems; abundant insect and animal disease reservoirs and vectors of disease agents; and inadequate human and financial resources to invest in food safety. Furthermore, in many developing countries, veterinary drug controls are inadequate and food safety assurance systems such as the Hazard Analysis Critical Control Point (HACCP) or the farm-to-fork concept of food safety have not been implemented along many food supply chains, making it difficult to assure food safety. In order for communities to reap the benefits of increased livestock production, there is need to structure veterinary support in rural Africa towards the prevention of the introduction of biological (disease agents), chemical (antibiotic residues and pesticides) and physical (radioactive materials) hazards along the food chain. The safety of food of animal origin, however, begins with production (Wood et al. 1998). Increased livestock production and food safety must therefore be seen as the core objectives of the veterinary professionâ&#x20AC;&#x2122;s contribution to food security. In the 21st century, no livestock veterinarian should feel comfortable only in controlling animal diseases in rural areas. In the execution of their duties, they should also take keen interest in the viability, sustainability and growth of livestock production, and ultimately in the protection of humans from hazards and the promotion of human advancement. The inspection of animals at slaughter provides a valuable contribution to surveillance for diseases of animal and public health importance (OIE 2010). By applying their skills and knowledge in abattoirs, veterinarians contribute to public health mainly through the control of zoonoses and diseases transmissible to humans through food and preventing human exposure to antibiotics and other chemical pollutants that are likely to enter the food chain. There is also need for veterinarians to be actively involved in the regulation of antibiotic and anthelmintic drug used in animals to reduce the risk of the development of drug resistant organisms potentially harmful to human health (Geerts and Gryseels 2000; WHO 2006). This requires a shift from the classical approach of controlling animal diseases to full engagement in the production of food that is safe, sound and wholesome. Food of animal origin often serves as a vehicle for many food-borne diseases. The education and

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training of veterinarians in animal health and food hygiene means that they are uniquely equipped to play a central role in ensuring food safety, especially the safety of foods of animal origin (OIE 2010). However, in many African countries, the mandate to assure the safety of food of animal origin falls under multiple jurisdictions: Ministries of Health, Ministries of Local Government and Ministries of Agriculture. This can result in professional rivalries that impact negatively the operationalization of food safety governance. To ensure that foodborne hazards can be managed, veterinarians in developing nations need therefore to seek legislative reform to realign mandates and competencies towards cooperation and synergism among stakeholders along the food production value chain.

Contribution to zoonoses prevention and control The global public health community now recognizes that control of diseases in animals is the principal means of reducing human exposure to the majority of emerging infectious diseases (EIDs) (Levings 2012). A recent study involving 1415 human pathogens, observed that 61% of these were of zoonotic origin (Taylor et al. 2001). A large proportion of the zoonotic EIDs are believed to be in the developing world (Cleaveland et al. 2007; Faye and Lancelot 2006; Palmer 2007; Pappas et al. 2008). The emergence of zoonotic EIDs such as Severe Acute Respiratory Syndrome (SARS), highly pathogenic avian influenza and West Nile Virus, have brought the world’s attention to the need for effective and efficient veterinary services to avert the global economic losses associated with pandemic-scale infectious disease threats, as well reducing human morbidity and mortality (Bengis et al. 2004; Kahn et al. 2009; Mazet et al. 2009). Although it is understood that many animal diseases and zoonoses negatively affect people’s livelihoods, the impacts of these diseases may, in some circumstances, be largely underreported, thus underestimating their incidence and artificially downgrading their importance on the policy agendas of both governments and funding agencies (Maudlin et al. 2009; Dorny et al. 2009; Welburn et al. 2009). Many zoonotic diseases that were once thought to be insignificant and generally neglected are re-emerging and are likely to become increasingly important (Cleaveland et al. 2007; Aluwong and Bello 2010). For instance, parasitic zoonoses remain a major health problem in poor communities in unsanitary environments (Dorny et al. 2009; Aramayo et al. 2009; Casapia 2006). There is also an increase in the incidence of non-typhoidal Salmonellosis as well as other enteric infections whose source of transmission are largely

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of animal origin in rural communities in developing countries (see Morpeth et al. 2009). The risk groups most susceptible, such as HIV-infected individuals, the elderly and children below the age of 3, are all increasing. The symbiotic relationship between rural communities and their livestock in developing countries, evinced by the inevitable proximity of the 2 species, is a favourable environment for the transmission of zoonoses (Perry 1984; Mfinanga et al. 2003). The risk of contracting zoonoses from wildlife is higher in these poor communities whose people and livestock interact with wildlife, commonly referred to as wildlife-livestock interface areas (Karesh et al. 2005). Wildlife-livestock interfaces pose a challenge to human, animal and environmental health practitioners due to the complex and continuous cycle of disease transmission that such areas foster (Malama et al. 2013). In the Southern Province of Zambia, for instance, livestock owners trek their cattle to water sources located mostly in and around wildlife sanctuaries during the dry season or along nomadic tracks with year round interaction between livestock and wildlife. This provides opportunities for livestock exposure to parasites, diseases and their vectors while sharing pasture and water sources with wildlife (Muma et al. 2007; Munyeme et al. 2008; Muma et al. 2006). In Nigeria, a well characterized Mycobacterium bovis isolate was identified in humans constantly exposed to cattle, and similarly, a well characterized Mycobacterium tuberculosis isolate was isolated from a goat (Jenkins et al. 2011). In Zambia, two studies (Muma et al. 2007 and Munyeme et al. 2008) demonstrated that cattle interaction with the Kafue lechwe antelope (Kobus leche kafuensis) was an important risk factor associated with increased Brucella spp. and M. bovis infections respectively, in cattle on the Kafue flats. In this same area, an association was earlier observed between keeping cattle and the having a tuberculosis infected person in a household (Cook et al. 1996). It is probable that diseases in both wildlife and cattle, if unchecked, could have serious repercussions for human health (Oloya et al. 2008; Kazwala et al. 1998; Cleaveland et al. 2007; Mwacalimba et al. 2013). The cost‑benefit analysis performed by Mwacalimba and colleagues (Mwacalimba et al. 2013) suggests that, albeit difficult, a combined control of tuberculosis in lechwe and cattle in this interface would have the added benefit of controlling its zoonotic impact and overall disease prevalence in the area. Another example is rabies, which is estimated to kill approximately 50,000 people around the globe annually (Meslin et al. 2000). In a human rabies survey conducted in Zimbabwe, over 90% of the cases were due to dog bites, with jackals (Canis adustus and C. mesomelas) and honey

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badgers (Mellivora capensis) contributing less to the positive cases (Pfukenyi et al. 2007). While the condition has both sylvatic and domestic cycles, the control of this disease in domestic animals is likely to reduce its incidence in humans. The repercussions of these and similar zoonoses include increased health costs for already impoverished communities; reduced labour outputs from infirm members of the community, reduced output from diseased livestock and increased animal health costs. Most infectious diseases are poorly controlled in both animals and humans in many sub-Saharan states. Some of the factors responsible include limited veterinary resources due to low budgetary allocations from central treasuries, insufficient diagnostic tools for rapid field detection of animal diseases; dysfunctional laboratories; poor surveillance systems as well as inefficient disease reporting systems and poor attitudes among public service providers (Muuka et al. 2013). One Health approaches may ensure a speedy response to such threats, forestalling the spread of zoonoses to in‑contact communities and beyond. Because poor health delivery systems attract community attention and threaten political fortunes, human health enjoys strong political will over a number of other sectors in most sub-Saharan countries. Unless policy-makers begin to see the benefits of holistic preventive medicine, there will always be an attraction to curative therapy in humans due to its immediate and visible impact on the affected communities and countries. The implementation of holistic preventive medicine therefore requires unwavering political will and the sound implementation of integrated health programmes. Despite continued resource challenges and minimal government attention to veterinary services and research in recent years, the veterinary profession in many African countries has made major contributions to the body of knowledge on zoonoses through research in various aspects of disease epidemiology (Cadmus et al. 2004; Muma et al. 2006, Oloya et al. 2006; Munyeme et al. 2009; Matope et al. 2010), such as risk factors for transmission in domestic animals and wildlife (Munyeme et al. 2008; Matope et al. 2010; Kabagambe et al. 2001; Kazwala et al. 2001); phenotypic and molecular characteristics of zoonotic agents (Cadmus et al. 2006; Hilty et al. 2005; Kazwala et al. 2006; Oloya et al. 2008; Michel et al. 2008); and cost-benefit analysis of zoonoses control (Mwacalimba et al. 2013). Such research is useful for informing the development of disease control policies tailored to the primary healthcare needs of rural Sub-Saharan Africa. By embracing a ‘One Health’ approach, it is envisaged that the collaboration between veterinarians, medical and paramedical professionals will strengthen national, regional and international strategies for zoonoses

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control (Kahn et al. 2009; Mazet et al. 2009). Indeed, because of the convergence of human and animal diseases, a common approach to biosecurity has become essential (Kahn et al. 2009; Muma et al. 2012). But often, there is little utilisation of knowledge generated by researchers due to non‑existent knowledge exchange platforms between the knowledge generators (Universities and research institutions) and the end users (policy-makers and the general public).

Contribution of veterinarians to environmental protection and improved land utilization It is estimated that an area of approximately 10 million square kilometres in Africa is infested with tsetse flies which transmit trypanosomes (Connor 1989; Slingenbergh 1992). These vast tracts of land are generally unsuitable for human habitation or the rearing cattle and other livestock. Veterinarians have developed methods to control tsetse flies, especially the savannah species, Glossina morsitans. Successful control programmes have been mounted in Southern Africa and large tracts of land have been reclaimed for people to live and farm (Masiga 1998). With the increasing levels of pollution in African rivers, the use of animals living in close proximity to, or in, these rivers as sentinels of contamination is becoming increasingly important (Myburgh et al. 2011; Mariano et al. 2009). Significant contributions have been made by veterinarians and biologists in the area of eco-toxicology by monitoring the effects of industrial pollutants and pesticides on humans and animals. Veterinarians, in a bid to save animal populations, have been pivotal in developing protocols for evaluating the levels of environmental pollution. For instance, the effect of mining activities in Zambia on the water population and aquatic life has been a subject of investigation by veterinary researchers (M'kandawire, et al. 2012; Mwase et al. 2002; Almli et al. 2005). As developing economies pursue industrial advancement, there is the inevitable but poorly researched challenge of waste management. The increased demand for animal protein caused by increasing incomes in developing nations (Delgado et al. 1999) has resulted in the growth of urban animal keeping, commonly known as ‘backyard’ production. With the world’s growing population competing for the same geo-physical resources such as land and water, backyard animal production is likely to increase to meet the growing demand for animal protein. However, this animal production model has created novel challenges in waste disposal as the conventional municipal sewage systems are not designed to deal with the effluent

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from livestock production in human residential areas. Veterinarians, through the application of their training and resources, can significantly contribute to research into the design of innovative waste management systems and surveillance tools for monitoring environmental pollution and gauging the impacts of mitigation measures (Sim and Wu 2010; Cabaret et al. 2002). However, this can only be feasible if veterinarians work in close collaboration with environmental health professionals in the development of new innovations for mitigating the public health risks associated with animal waste management. Furthermore, all animals, including domestic livestock and wildlife, can potentially cause environmental degradation through overgrazing if their stock densities exceed land carrying capacities, especially in the semi-arid savannah during, and subsequent to, drought years (Fynn and O'Connor 2000). Veterinarians can contribute to environmental protection by designing efficient livestock stocking and management strategies. They also possess the expertise to do the same in wildlife management, through informing controlled game hunting programmes, culling procedures and the protection of vulnerable species.

Contribution to ecotourism Like many other countries on the African continent, Zambia provides an example of the ecological diversity that makes Sub-Saharan Africa a competitive candidate for state, private and foreign investment in ecotourism. In-situ conservation in Zambia includes protected National Parks (NP) and Game Management Areas (GMAs) (Saiwana 1995). There are 19 NPs and 32 GMAs covering 8.6% and 22% of Zambiaâ&#x20AC;&#x2122;s total landmass respectively. Some of the fauna in the countryâ&#x20AC;&#x2122;s conservancies include species like the African wild dog (Lycaon pinctus), Shoebill stork (Balaeniceps rex), Kafue lechwe (Kobus leche kafuensis), Black lechwe (Kobus leche smithemani) and Ansellâ&#x20AC;&#x2122;s shrew (Crocidura ansellorum), which have been listed as endangered species by the International Union for Conservation of Nature (IUCN) (IUCN 2009). GMAs act as buffer zones between NPs and communal lands. Livestock ownership and consumptive utilization of wildlife are permitted in the GMAs (Saiwana 1995). In recent years, wildlife populations in the NPs and GMAs have significantly reduced due to poaching, infectious disease outbreaks and the over-exploitation of natural resources by expanding human populations (Siamudaala et al. 2004). In order to save wildlife, governments in many Sub-Saharan African states are promoting ex-situ conservation which involves private rearing of wildlife outside

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state-owned protected areas (Siamudaala et al. 2004). Currently, Zambia has more than 100 game ranches and over 10 large crocodile farms. The number of aviaries is also slowly increasing (Siamudaala 2004). The negative impact of tick-borne diseases and trypanosomosis (transmitted by tsetse flies that cover over 30% of the landmass in Zambia) on livestock production have accelerated the shift from cattle ranching to game ranching, because game are trypano-tolerant. The involvement of the private sector in the rearing of endangered species such as the Kafue and Black lechwe serves as a back-up, increasing the declining populations of these Kobus leche sub-species. Wildlife, however, serve as important disease reservoirs. Examples include FMD and theileriosis in buffalo; malignant catarrhal fever in wildebeests and African swine fever in warthogs (Masiga 1998). These diseases constrain the expansion of the wildlife industry in many sub-Saharan countries (Munang'andu et al. 2006; Siamudaala et al. 2003). In one instance, an outbreak of anthrax in Malilangwe conservancy in Zimbabwe in 2004 nearly wiped out the entire population (n=500) of kudu (Tragelopus strepsiceros) and a number of other antelopes such as the nyala (Tragelaphus angasi), bushbuck (Tragelaphus scriptus), water buck (Kobus ellipsprymus) and roan antelope (Hippotragegus equinus), suffering losses of approximately 68%, 48%, 44% and 42% of their populations, respectively (Clegg et al. 2007). The disease further killed livestock in adjacent wildlife-livestock interface areas (Clegg et al. 2007). Other examples include the canine distemper epidemics in East Africa which are believed to have resulted in disturbed wild animal population dynamics with negative environmental repercussions (Roelke-Parker et al. 1996). Ex-situ conservation also presents infectious disease threats such as the report of tuberculosis in Kafue lechwe and in a bushbuck (Tragelaphus scriptus) on a game ranch in the Central Province of Zambia (Zieger et al. 1998). Wildlife conservation not only brings the threat of wildlife diseases to livestock, but also to humans, making the multi-species transmission infections highly probable. The resultant complex cycle of infectious disease transmission can have catastrophic effects on all species involved. For instance, the introduction of bovine tuberculosis from cattle to the Kafue lechwe, as well as the introduction of rabies and canine distemper from domestic dogs to wild dogs has contributed to the significant reduction of these species, subsequently leading to their inclusion on the IUCN red list of threatened species (IUCN 2009). A case of transmission of human tuberculosis to monkeys has been reported in South Africa (Michel 2003). Anthrax outbreaks in the Luangwa National Park of Zambia have contributed

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to the decline of several wildlife species, including wild dogs (Turnbull et al. 1991). A significant problem is that wildlife conservation usually occurs in areas remote to veterinary services (Siamudaala 2004). Consequently, game is consumed and wildlife trophies are handled without veterinary clearance. For instance, in 2011, an outbreak of anthrax in hippos (Hippopotamus amphibious) in the Luangwa National Park resulted in human infections and environmental contamination in Chama district of Western province in Zambia (Hangombe et al. 2012). It was reported that all the patients involved in the outbreak had a history of having consumed or touched anthrax-infected hippo meat. A combined team of veterinary and medical professionals investigated the outbreak and implemented control measures offering an example of ‘One Health’ approach to the control of zoonoses. Zoonoses such as anthrax not only threaten the existence of wildlife resources, they also threaten tourism. These concerns gain an international dimension in the implementation of Trans-frontier Conservational Areas (TFCAs) currently being advocated and developed within the Southern African Development Community (SADC). Trans-frontier Conservation Areas are large natural systems of one or more protected areas straddling 2 or more countries. The concept of creating TFCAs is recognised as an important tool for promoting the conservation of biodiversity and protection of endangered animal species. This conservation model also presents the risk of sharing of infectious diseases between humans, livestock and wildlife. The mitigation of this risk requires significant veterinary input in TFCA implementation. Ecotourism is defined as “responsible travel to natural areas that conserves the environment and improves the well-being of local people” (The International Ecotourism Society, TIES 1990). Tourism is not only important for state revenue, since facilities for game viewing and hunting safaris tend to be located in rural areas, it also offers rural communities opportunities for gainful employment, and a market for their agricultural produce and crafts. Such investments greatly contribute to poverty reduction. How do considerations of wildlife conservation, ecotourism and hunting add to this discussion on livelihoods, health and the role that veterinary medicine could play in poverty reduction? It is in the mêlée of infectious disease threats that the veterinary profession must reorient itself to proactively support ecotourism. Siamudaala (2004) has argued that veterinary services play a pivotal role in poverty alleviation among local communities in and around wildlife protected areas. Under such circumstances, the challenge

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for the veterinary fraternity is to design disease control and conservation strategies that allow for the co-existence of wildlife and livestock not only in interface areas, but also in game‑ranches where wildlife exists in close proximity to livestock (Haigh 1996). Veterinarians and para-veterinarians are also involved in the establishment and maintenance of ex-situ conservancies, thus contributing to wealth creation. They are involved in carrying out ecological audits to determine the carrying capacity and type of fauna and area chosen to be a game ranch is able to support the wildlife species to be introduced. They also carry out veterinary audits to determine the disease profile and vector species found in ecosystems chosen for the establishment of ex-situ conservancies (Munang’andu 2000). Veterinarians are responsible for the translocation of animals from NPs, GMAs or other private properties to the new conservancies. On established game ranches, disease surveillance and monitoring is carried out during safari hunting, capture operations, or cropping of excess stock (Munang’andu et al. 2006). Therefore, veterinarians are well placed to implement the prevention and control measures necessary to avert the negative impacts of emergence of diseases in and from wildlife. However, additional skills are required in risk analysis for veterinarians to help stakeholders in wildlife conservation to take into account the sanitary and phytosanitary measures necessary for safe animal translocation and health management.

Discussion: veterinary activities in the 21st century We can now summarise how the veterinary profession can go beyond its ‘animal treatment’ role to positively impact production, rural livelihoods and poverty alleviation. It is important that veterinarians in Sub-Saharan Africa are equipped with novel and interdisciplinary skill sets that appropriately meet the contemporary challenges of holistic animal, human and environmental health. A review of veterinary curricula to include ‘One Health’ precepts within a livelihoods framework should be an urgent undertaking, if Sub-Saharan African veterinarians are to remain relevant to the context in which they live and work. This should also include competence in analysing the costs and benefits of the control strategies they institute, to assure the best response to the needs of the society (Swan and Kriek 2009; Kock 1996). Many disease control campaigns have been conducted without a full assessment of their economic impacts both in terms of the cost of conducting the exercise, disposal, and the effects of other related non-agriculture industries such as tourism (Sugiura et al. 2001; Thompson et al. 2002).

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Wildlife and nature conservation are important in the conservation of the world’s biodiversity. Veterinarians can be particularly instrumental in monitoring wildlife diseases and designing disease control strategies (Kock 1996). However, the challenges of infectious disease control and eco-conservation require that veterinarians no longer remain isolated as a profession but, instead, engage and collaborate with other professionals in allied fields. In the 21st century, animal health and production, in its various forms, remains an important asset in Sub-Saharan Africa. Despite the differences in approaches and mandates among the professions dealing with human, animal and environmental health, cooperation between the disciplines that link this triad of people, animals and their shared environment is critical in assuring sustainable policies for health and poverty reduction. The burden of veterinary responsibility will only grow with the increasing incidence of emerging and re-emerging diseases. The strengthening of national veterinary services should thus be viewed as fundamental to the surveillance and control of domestic, wild animal and zoonotic diseases. Policy-makers in Sub-Saharan Africa should not ignore the various sociological roles that livestock play in developing countries. For instance, understanding community perceptions and interpretations of animal diseases in rural

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African settings provides a significant tool for disease surveillance and the institution of control interventions (Muuka et al. 2012). Therefore, in order to change policymaker perceptions towards livestock, veterinarians cannot take the view that the control of animal diseases in African countries is solely an exercise of laboratory science. It should also include an application of human anthropology (Marcotty et al. 2009; Muuka et al. 2012) and politics (Green 2012; Mwacalimba 2012; Mwacalimba and Green 2014). In conclusion, in adapting the ‘One Health’ approach to the Sub-Saharan African context, veterinarians need to be cognizant of the fact that animal health is one, albeit important, pathway for the improvement to human health and welfare. The veterinary profession should therefore refocus its energies on adopting new knowledge and new partnerships. It should identify and understand the various economic constraints and challenges that the veterinary profession faces in contributing efficiently to human development, public health, animal production, draught power and wildlife conservation (Swan et al. 2009). Veterinary science is uniquely positioned to play a key role in both poverty reduction and the promotion of global health. This role can be enhanced through the reorientation of the profession’s goals and the creation of synergies with allied and related professions.

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Stray animal populations and public health in the South Mediterranean and the Middle East regions Aristarhos Seimenis1 & Darem Tabbaa2* WHO-Mediterranean Zoonoses Control Centre, 25 Neapoleosst, 15310 Athens, Greece. Department of Public Health, Faculty of Veterinary Medicine, Al Baath University, Hama, Syria. 1

* Corresponding author at: Department of Public Health, Faculty of Veterinary Medicine, Al Baath University, Hama, Syria. Tel.: +963 944 233 966, e-mail: daremtabbaa@yahoo.com

Veterinaria Italiana 2014, 50 (2), 131-136. doi: 10.12834/VetIt.48.134.3 Accepted: 09.04.2014 | Available on line: 30.06.2014

Keywords Environment, Overpopulation, Public health, Stray animals, Veterinary public health, Zoonoses.

Summary Uncontrolled urban growth in South Mediterranean and the Middle East regions involves city dwellers and stray animals (mainly dogs and cats) creating a dense and downgraded environment, in which irregular street garbage collection disposes sufficient food for survival and proliferation of stray animals. Under such conditions serious public health hazards are expected due to the increase of animal bites, the multiplication of insects and rodents vectors of different viral, bacterial, fungal and parasitic agents to which humans are exposed. Traditional national stray animal eradication programs and occasional small animals’ humane elimination campaigns are insufficient to avert human and veterinary health risks when not coupled with modern technologies. In such environments, multiple foci of emerging and re‑emerging zoonoses easily spread, i.e. rabies, hydatidosis, leishmaniasis and toxoplasmosis. Upgrading urban and peri-urban situations requires integrated/coordinated management programmes, in which public and animal health services as well as municipalities have a crucial role. Control and upgrading programmes should be flexible and able to adapt to the specific conditions of the given country/region. In this context, intersectoral/interprofessional collaborations and community participation are crucial for any national and regional development strategies. In this respect, a global approach considering both public health and socio-economic problems shows to be extremely adequate and effective.

Popolazioni di animali randagi e sanità pubblica nelle regioni del Sud Mediterraneo e del Medio Oriente Parole chiave Ambiente, Animali randagi, Sanità pubblica, Sanità pubblica veterinaria, Sovraffollamento, Zoonosi.

Riassunto Lo sviluppo urbano non controllato, rilevabile in alcuni paesi del Sud-Mediterraneo e Medio Oriente, coinvolge abitanti e animali randagi (soprattutto cani e gatti). L’ambiente risulta saturo e degradato visto che esseri umani e animali coesistono in situazioni di sovraffollamento. La raccolta irregolare dei rifiuti urbani offre la disponibilità di alimenti sufficienti per la sopravvivenza e proliferazione degli animali randagi. Questa condizione rappresenta un serio pericolo per la salute pubblica, in quanto si moltiplicano le morsicature da animali randagi e aumenta la proliferazione di insetti e roditori trasmettitori di agenti virali, batterici, fungini e parassitari. I vecchi programmi nazionali di lotta al randagismo, così come le campagne di eliminazione dei piccoli animali, non sono sufficienti se non sono accompagnate da tecnologie moderne e risorse adeguate. In questi contesti, è facilitata la diffusione di zoonosi emergenti e riemergenti, quali per esempio rabbia, echinococcosi, leishmaniosi, toxoplasmosi. Il miglioramento degli ambienti urbani e sub-urbani richiede programmi di gestione coordinati e integrati. A tal fine, i servizi di sanità pubblica, sanità animale e le amministrazioni locali rivestono un ruolo cruciale. I programmi di controllo e sviluppo devono essere flessibili e capaci di adattarsi alle condizioni del territorio. La collaborazione intersettoriale e interprofessionale, con la partecipazione della comunità, sono fattori di fondamentale importanza nonché componenti essenziali delle strategie di sviluppo a livello nazionale e regionale. Questo articolo descrive un tentativo di approccio globale ai problemi di sanità pubblica e socio-economici legati al randagismo nei paesi del Sud‑Mediterraneo e Medio Oriente, fornendo alcune indicazioni generali sulle possibili soluzioni.

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Introduction In the epidemiology of the most important zoonoses such as cystic echinococcosis (hydatidosis), leishmaniasis and rabies, South Mediterranean and Middle East regions (SMMER) present many similar climatic, geophysical and socio‑cultural characteristics. The endemicity and/ or the gradual spread of such diseases in these neighbouring regions create serious public health and socio‑economic hazards (Mantovani 2005). Several interconnected factors are prevailing in characterising such situation. The most significant among them may include: • the increasing human population density and displacement in urban areas; • close and intensive coexistence between humans and animals; • high number of stray animals in urban settings; • intensification of animal production and trade; • illegal and unsafe production, processing and use of food of animal origin; • traditional socio-cultural characteristics; • lack of awareness of most of community concerning health hazards; • weak intersectoral collaboration; • strong financial limitations and shortages of resources; • badly informed decision-makers; • extreme difficulty in evaluating the phenomenon of stray animals in terms of numbers and then epidemiological geographic areas. In this context, dog and cat roaming is recognized as an important public health and socio‑economic hazard. Small animals may be either stray or owned but not adequately controlled, may be formerly owned but have become feral or may have always been free‑living (Abdou 1999, Baldelli et al. 2000, Boegel 2001). These aspects depict a quite different scenario than the one characterising coastal countries of the Northern area of the Mediterranean basin. In the SMMER canine and feline populations play their own roles in the epidemiological status of zoonotic diseases, where animals are passively supported by the considerable amounts and the growing quantities of feed available in streets’ garbage. While stray animals are not allowed in houses due to the hygienic and health hazard they represent, they are not chased. In urban areas few domestic cats’ owners permit their cats to wander around to collect their feed. Free-roaming cats create additional social hazards. They can be a nuisance, feed on garbage,

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defecate in places they are digging and are an additional factor to overpopulation of stray animals. The large number of stray animals, with or without owners, poses many problems, among which: • the spread of zoonoses to humans; • the transmission of disease to other animals; • bites, scratches and related problems; • economic problems, mainly connected with attacks on domestic animals; • pollution of soil and water; • problems connected with public security (road accidents, attacks by single or packs of dogs); • attraction of wild animals. Overpopulation in developing countries includes also the city dwellers living in proximity with animal populations, generating large volumes waste. The management of urban waste in these circumstances becomes a difficult issue for city administrations, which should contribute to properly approaching financial and public health problems created under such undesirable conditions (Baldelli et al. 2000, Poglayen 2003).

Stray Animals and Public Health Hundreds of thousands of stray dogs are eliminated every year in SMMER countries, without significant results given the ‘holding capacity’ of a territory, directly related to the amount of waste food available. Animals surviving mass elimination can easily reproduce, and together with newly abandoned animals, can create new stray animal populations (Mantovani 2003, Vos 2000). Public health impact of the phenomenon of roaming animals in urban and peri-urban areas includes the emergence and endemicity of a lot of zoonoses (Calum et al. 2000). Without forgetting brucellosis, campylobacteriosis, cat scratch disease, cheyletelliosis, cryptosporidiosis, dermatophytozoonoses, dirofilariasis, giardiasis, leptospirosis, Mediterranean spotted fever, opistorchiasis, pasteurellosis, visceral and cutaneous larva migrans, and many other, which all fall outside the scope of this article. The most important zoonoses in dogs and cats in SMMER may be considered the following: rabies, cystic echinococcosis, zoonotic visceral leishmaniasis and toxoplasmosis.

Urban and peri-urban rabies The World Health Organization (WHO) estimates that there are more than 200 million stray dogs worldwide and that every year, 55 thousand people die from rabies, while another 15 million receive

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post exposure treatment to avert the deadly disease; 95% of these cases occur in Asia and Africa, and 99% of the fatalities are caused by dogs (WHO 2013). Urban Rabies is known to be an important public health hazard. It is maintained and propagated primarily by dogs; their bites are the main route of rabies among humans. Cats constitute the second most important group of human infection followed by other domestic and wild animal species occasionally found in cities or peri-urban settlements (Meslin et al. 2002). The reported cases in the SMMER are supposed to range from 800 to 1000 annually, while post-exposure treatments reach several tens of thousands in each country. Children account for the largest group receiving such treatment. At the same time, there is an increasing role of wildlife in rabies epidemiology in the same area (Seimenis 2004, Seimenis 2009). Only a part of the infected animals is recognized and recorded by the veterinary services, mainly in connection with human exposure to animals, while only part of the rabies reported cases are laboratory confirmed (Seimenis 2004).

Cystic echinococcosis The increasing interaction between humans and animals is a well-known factor influencing the occurrence and endemicity of other zoonoses such as Cystic echinococcosis (CE) by Echinococcus granulosus. It involves mainly dogs and rural families, together with urban dwellers, which may be (and are) endangered by close contact with these animals. (Mantovani 2003, Mantovani and Seimenis 2003). There are many possible sources providing infected food for stray dogs. For example, human habits of feeding dogs of slaughter and/or butcher waste in specific situations are responsible for canine infection; also the parasite may be spread in urban and sub-urban districts through owned and stray dogs roaming in the country or peri-urban areas (Mantovani 2003, Mantovani and Seimenis 2003, Mantovani 2005). Slaughterhouses may be insufficiently attended in developing countries, being so accessible to dogs; they may also lack facilities for the proper destruction of infected material. Illegal and noninspected slaughtering represents an important infection source for dogs by discarded offal. Therefore, measures to be taken should aim, besides the control of free roaming dogs and cats, to impede their access to the sources of infection, particularly slaughterhouses and butcheries (Abdou 1999, Baldelli et al. 2000, Mantovani 2003, Mantovani and Seimenis 2003).

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Zoonotic visceral leishmaniasis There is no doubt that dogs are the reservoir of leishmaniasis caused by viscerotropic and dermotropic strains of Leishmania infantum (Gramiccia and Gradoni 2005), while its role in the epidemiology of cutaneous leishmaniasis due to dermotropic Leishmania tropica is still to be proved. Canine leishmaniasis is widespread in the Mediterranean area showing typical focal distribution and a broad spectrum of infection prevalence from 2 to 40%, in countries such as Afghanistan, Egypt, Iran, Iraq, Jordan, Lebanon, Libya, Morocco, Saudi Arabia, Syria, Tunisia and Yemen. Dogs in general, but most particularly stray dogs, are exposed to infected sandflies, which then act as domestic reservoirs. Once the parasite is introduced into a community it is maintained in a dog-insect-dog transmission cycle. The uncontrolled overpopulation in a deteriorated urban environment constitutes the appropriate background for the multiplication of hosts and vectors. Infected sandflies bite people causing visceral and cutaneous leishmaniasis. If appropriate measures are not timely undertaken towards urban and peri-urban environmental management, the disease becomes endemic affecting an increasing number of individuals, among which children are prevailing. (Postigo 2010, Seimenis 2010, Tesh 1998, World Health Organization-Eastern Mediterranean Regional Office 2009).

Toxoplasmosis It is of particular health significance because of long-term contamination of soil, water and dust by oocysts, through faeces. Contact with oocyst‑contaminated soil and water is probably the major means through which different species (rodents, ground-feeding birds, sheep, goats, pigs, and cattle, as well as humans living in the SMMER) are exposed to Toxoplasma gondii. The uncooked, raw meat is another important source of infection for humans and also for cats. Cats are the main host species contaminating the environment near human settlements. Stray and feral cats are much more implicated in this role than pet ones (Baldelli et al. 2000, Boegel 2001, Poglayen 2003).

General measures for the control of stray animals to enhance public health in the SMMER Upgrading urban general public and veterinary hygienic conditions and infrastructure, community’s educational level, as well as human and animal populations’ density rationalization, are interconnected factors to be managed in an

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integrated way in order to achieve an acceptable level of veterinary public health in urban and peri-urban environments. Interventions of urban veterinary hygiene in order to be effective should cover simultaneously at least the most important among the factors creating and maintaining an existing stagnatory epidemiology. This is more than a hard and difficult task (Abdou 1999, Baldelli et al. 2000, Wisner and Adams 2002). Public and animal health services and councils have a crucial role in the improvement of urban hygiene conditions. The main measures expected to be taken concern: • the establishing of an effective and comprehensive policy for roaming animal populations management, and provide resources for implementation; • providing adequate and regular garbage collection systems in urban and peri-urban areas, and monitor their management; • activating an efficient system for collection, rendering and/or destruction of dead animals and infected viscera; • protecting slaughterhouses, butcheries, dumps and other attraction places from dog and cat access; • promoting adequate legislation on management of animal populations in urban settlements, based on their welfare; • undertaking public health educational campaigns for the community focusing, among other things, on the human – animal interaction and ‘responsible animal ownership’. With regard to the dog population management, the systematic or incidental removal campaigns are still a much-applied ‘tool’ in many developing countries. However, as previously referred, this offers only limited spatial and temporal success, as there is rapid repopulation of the evacuated area (Vos 2000, World Health Organization-Eastern Mediterranean Regional Office 2009). The most appropriate method for the elimination of the animal stray population and for avoiding its reproduction remains open for discussion. In this context it is worth recalling the International Companion Animal Management Coalition (ICAMC), which was formed in 2006 from representatives of the World Society for Protection of Animals (WSPA), the World Small Animal Veterinary Association, the Alliance for Rabies Control and other parent organizations. Based on the original 1990 WHO/ WSPA document, the Coalition published in 2008 a revised set of guidelines to manage roaming dog populations and the risks these may present, including population size reduction when this

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is considered necessary (ICAM Coalition 2008). Coordinated activities together with the community involvement could offer approaches appropriate for each country. Basic rule of cardinal importance remains the implementation of integrated management schemes either in normal or in emergency situations (Abdou 1999, Baldelli et al. 2000, Massei 2009, Aidaros 2009). Decision-makers at central and local levels should be well informed of the environmental health hazards and the inter-relationships between population, health and sustainable development. There is a need for political commitment to the development of environmental health activities including well organized governmental services and community participation, in order to ensure their incorporation in urban and peri-urban development plans. Such plans should provide strengthening of local authorities and enabling them to tackle health and environmental problems including those due to roaming animals (Wisner and Adams 2002). In order to achieve the best possible effect, all programmes and activities concerned with the management of stray animal population in urban and peri-urban areas should be based on the principles of the intersectoral cooperation. The lack of such cooperation undermines any kind of efforts aiming at improving the situation. Multi-disciplinary and cross-sectoral structures associated with an appropriate coordinating administrative mechanism are absolutely essential. The combination of financial constraints, weak cooperation/coordination and lack of community involvement maintain important problems, becoming very difficult to address. Such situation is an additional characteristic in most SMME countries. Therefore, this is one more occasion to emphasize the importance of attitudes changing and the continuous strengthening of a global social and technical involvement aiming to the development of public health (Busani et al. 2006, Vos 2000).

Conclusion The task of establishing a step-by-step consistent improvement of urban hygiene conditions (in developing world in general but most particularly in the SMMER) is extremely complex and difficult, but it deserves the challenge. It is the only way which may lead to ensuring acceptable living conditions, alleviating human suffering as well as maintain socio-economic development, while combating the impact to public health coming from the uncontrolled human and animal co-existence. Public health impact and the associated social and financial damages of free-roaming animals in developing countries require action. These actions

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seem to collide with animal welfare and especially stray dogs and cats are at the centre of these collisions and admittedly, the method for managing stray animals population in poor societies is still to be improved. At the same time, financial limitations, often ineffective legislative implementation and enforcement, the lack of intersectoral coordination and the insufficient awareness of the urban and peri‑urban communities in the SMMER, create difficulties on how best such problems could be managed (Battelli 2003, Vos 2000, Massei 2009, Aidaros 2009). This is true even when the resources come from supranational organizations (see for example the case of Ukraine and World Cup).

method to the traditional parenteral vaccination of dogs, could mean a breakthrough in this aspect (Vos 2000, Seimenis 2009).

The challenge is to identify and implement, as best as possible, an integrated, effective, comprehensive and affordable management program for roaming animal population, tailored for countries sharing conditions such as those characterising the SMMER.

Preparedness on contingency programmes to face emergencies of different kinds should be elaborated and inter-professional teams have to be trained for intervention in case of necessity.

The mass vaccination of dogs against rabies put in place in the SMMER will not achieve its target and same results it has achieved in developed countries, just because many dogs cannot be handled and therefore cannot be vaccinated. The possibility of oral vaccination campaigns, as a supplementary

Specialized international organizations and NGOs are offering their expertise and technical assistance, including training of inter-professional staff, so that all relevant programmes could be actively managed, therefore, their contribution is always essential. Public health education regarding hygienic sanitation of the environment and responsible ownership of dogs and cats should become among the most important working tools. Schools and mass media play an important educational role for the community in this domain.

Difficult and complex situations can only be afforded through inter-sectoral/inter-professional collaboration and community involvement. It is the only strategy, leaving aside local cultures and beliefs entrenchments, could contribute to address the socio-economic development and human suffering alleviation in the SMMER.

References Abdou A.E. 1999. Survey on animal straying in urban and rural environments. Possible approaches to the problem in the Southern and Eastern Mediterranean region. Ιn Proceedings of MZCP Workshop on Dog and Cat Populations Management in Normal and Disaster Situations. Repubblica di San Marino, 18-20 March 1999, 39-45. Aidaros H. 2009. Combating stray dog as a mean of controlling rabies. In World Health Organization (Head Quarters – Eastern Mediterranean Regional Office – Mediterranean Zoonoses Control Programme)/ OIE Inter-country. Report of an Inter-country Workshop on Protecting Humans from Domestic and Wildlife Rabies in the Middle East, Amman, Jordan, 23-25 June 2008, 43-46. Baldelli R., Battelli G. & Poglayen G. 2000. Zoonoses and other health problems connected with the coexistence of man-dog-cat in normal situations and in emergencies. Information Circular WHO Mediterranean Zoonoses Control Center, 49, 2-5. Battelli G. 2003. Cystic echinococcosis: control problems in normal and emergency situations. Information Circular WHO Mediterranean Zoonoses Control Center, 57, 10-12.

& Seimenis A. 2006. Multidisciplinary collaboration in veterinary public health. Ann Ist Sup Sanita, 42(4), 397-406. Gramiccia M. & Gradoni L. 2005. The current status of zoonotic leishmaniases and approaches to disease control. Int J Parasitol, 35, 1169-1180. International Companion Animal Management Coalition (ICAM Coalition). 2008. Human dog population management guidance (http://www.icam-coalition. org/downloads/Humane_Dog_Population_ Management_Guidance_English.pdf accessed on 3 June 2014). MacPherson C.N.L., Meslin F.-X. & Wandeler A.I. 2000. Dog zoonoses and Public Health. CABI Publishing, Wallingford, 382 p. Mantovani A. 2003 The role of dogs in the epidemiology of cystic echinococcosis. In Cystic Echinococcosis and the Mediterranean. Information Circular WHO Mediterranean Zoonoses Control Center, 57, 3-4.

Boegel K. 2001. Cats as reservoir of infectious diseases to humans. Information Circular WHO Mediterranean Zoonoses Control Center, 52, 8-10.

Mantovani A. 2005. Mediterranean and zoonoses: a relationship. In Proc. 35th International Congress World Association History Veterinary Medicine (Veggetti A., Zoccarato I. & Lasagna E., eds). Grugliasco, Torino, 8-11 September 2004. Fondazione Iniziative Zooprofilattiche e Zootechniche, Brescia, 59-62.

Busani L., Caprioli A., Macri A., Mantovani A., Scavia G.

Mantovani A. & Seimenis A. 2003. Factors affecting

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the maintenance of cystic echinococcosis in the Mediterranean region. Information Circular WHO Mediterranean Zoonoses Control Center, 57, 2.

Tesh R.B. 1998. Control of zoonotic visceral leishmaniasis: is time to change strategies? Information Circular WHO Mediterranean Zoonoses Control Center, 46, 7-10.

Massei G. 2009. Immunocontraception for dogs. In World Health Organization (Head Quarters – Eastern Mediterranean Regional Office – Mediterranean Zoonoses Control Programme)/ OIE Inter-country. 2009. Report of an Inter-country Workshop on Protecting Humans from Domestic and Wildlife Rabies in the Middle East. Amman, Jordan, 23-25 June 2008, 35-37.

Vos A. 2000. Dog population management: a misconception. WHO Mediterranean Zoonoses Control Center Information Circular, 50, 12-14.

Meslin F.-X., Miles M.A., Vaxenat J.A. & Gemmel M.A. 2002. Zoonoses control in Dogs. In Dogs, Zoonoses and Public Health (MacPherson C.N.L., Meslin F.-X. & Wandeler A.I., eds). CABI Publishing, Wallingford, 331-361. Poglayen G. 2003. Zoonoses from faeces in urban environment. In Urban Parassitology (Puccini V. & Tarsitano E., eds), Edagricole, Bologna, 37-47. Postigo J.A.R. 2010. Leishmaniasis in the World Health Organization Eastern Mediterranean Region. Int J Antimicrob Agents, 365, 562-505. Seimenis A. 2004. Rabies in Southern, Eastern Mediterranean and Middle East Countries. Information Circular WHO Mediterranean Zoonoses Control Center, 58, 7-8. Seimenis A. 2009. General principles on planning rabies control programmes. In World Health Organization (Head Quarters – Eastern Mediterranean Regional Office – Mediterranean Zoonoses Control Programme)/ OIE Inter-country. 2009. Report of an Inter-country Workshop on Protecting Humans from Domestic and Wildlife Rabies in the Middle East. Amman, Jordan, 2325 June 2008, 28-30.

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Wandeler A.I. & Bingham J. 2002. Dogs and Rabies. In Dogs, Zoonoses and Public Health (MacPherson C.N.L., Meslin F.-X. & Wandeler A.I., eds). CABI Publishing, Wallingford, 63-90. Wisner B. & Adams J. 2002. Environmental health measures in the emergency phase. In Environmental health in emergencies and disasters. World Health Organization, Geneva, 48-51. World Health Organization. 2013. Rabies Fact Sheet n 99 (http://www.who.int/mediacentre/factsheets/fs099/ en/index.html accessed on 3 June 2014). World Health Organization (Head Quarters – Eastern Mediterranean Regional Office – Mediterranean Zoonoses Control Programme)/ OIE Inter-country. 2009. Report of an Inter-country Workshop on Protecting Humans from Domestic and Wildlife Rabies in the Middle East. Amman, Jordan, 23-25 June 2008. World Health Organization-Eastern Mediterranean Regional Office. 2009. Report of the intercountry meeting on leishmaniasis control strategies in the Eastern Mediterranean Region. Aleppo, Syrian Arab Republic, 27-30 October 2008. http://applications. emro.who.int/docs/intercountr y_meeting_ ctd_053_14277.pdf.

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Causes of death in dogs in the province of Rome (Italy) Claudia Eleni1, Valentina Panetta2, Francesco Scholl1 & Paola Scaramozzino1* 1

Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova 1411, 00178 Rome, Italy 2 L’altrastatistica S.r.l, via Erminio 16, 00174 Rome, Italy Corresponding author at: Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Via Appia Nuova 1411, 00178 Rome, Italy. Tel.: +39 06 79099472, e-mail: paola.scaramozzino@izslt.it

Veterinaria Italiana 2014, 50 (2), 137-143. doi: 10.12834/VetIt.13.07.01 Accepted: 09.04.2014 | Available on line: 30.06.2014

Keywords Cause of death, Dog, Necropsy, Infectious diseases, Poisoning, Province of Rome, Italy.

Summary Dogs share with humans several zoonotic diseases as well as some important determinants of degenerative syndromes and tumours. For this reason, systematic surveillance on small animal disease carried out through the collection and analysis of necropsy records could be helpful to public health. To describe the causes of death in dogs from the province of Rome (Italy) submitted to the Istituto Zooprofilattico Sperimentale del Lazio e della Toscana for necropsy during 2003–2007, a retrospective study was conducted on diagnostic data of 870 dogs. The final diagnosis was established by anatomo-histopathological examinations and, when needed, by ancillary laboratory tests. The most common causes of death were ‘infectious disease’ (23%) and ‘poisoning’ (17%). In 5% of the cases, the cause remained undetermined. The frequency of ‘poisoning’ was higher (39%) in stray dogs, while ‘infectious disease’ was more frequent (49%) in dogs from breeding farms. Parvovirosis was the most frequent infectious disease (33%) while anticoagulants accounted for 30% of the cases involving toxicity. Death by neoplastic lesions was quite infrequent (7%). Findings from this study provide veterinarians with an overview of the causes of death in dogs and it could provide public health authorities with new data about both novel and re-emerging threats.

Cause di morte in cani provenienti dalla Provincia di Roma, Italia Parole chiave Autopsia, Avvelenamento, Cane, Causa di morte, Malattia infettiva, Provincia di Roma, Italia.

Riassunto I cani condividono con l’uomo diverse zoonosi e alcuni importanti determinanti di malattie degenerative e tumori. Pertanto, una sorveglianza sanitaria sistematica sui cani, condotta anche tramite raccolta e analisi dei dati autoptici, può essere di grande utilità in sanità pubblica. A questo scopo è stato condotto, tra il 2003 e il 2007, uno studio retrospettivo sui dati diagnostici di 870 cani della provincia di Roma sottoposti ad autopsia all’Istituto Zooprofilattico Sperimentale del Lazio e della Toscana. La diagnosi conclusiva per ogni reperto autoptico è stata ottenuta tramite esame anatomo-istopatologico e test di laboratorio complementari. Le cause di morte più frequenti sono risultate: “malattia infettiva” (23%) e “avvelenamento” (17%), nel 5% dei casi la causa non è stata determinata. Le morti per malattie infettive sono risultate più frequenti in cani provenienti da allevamenti (49%), la parvovirosi è risultata la patologia infettiva più rilevata (33%). La frequenza di “avvelenamento” è risultata più alta nei cani randagi (39%), gli anticoagulanti sono stati usati nel 30% di questi casi. Le morti per neoplasia sono risultate poco frequenti (7%). I risultati di questo studio forniscono ai veterinari clinici un quadro generale sulle cause di morte della popolazione canina e possono costituire una fonte di dati per le autorità di sanità pubblica per eventuali allerte su malattie emergenti e ri-emergenti.

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Causes of death in dogs in the province of Rome (Italy)

Introduction The role of companion animals as a sentinel for human health disorders is widely recognised. As a matter of fact, pets share with their owners not only the domestic habitat, but also other environmental risk factors (Backer et al. 2001). Moreover, unlike humans, animals are not affected by common confounders related to individual behaviours (Schmidt 2009). In this context, data collection by veterinary clinics (Ward & Kelman 2012) and laboratory records could be useful for outlining potential risks to public health. In a recent paper, Doera et al. (Doera et al. 2011) reviewed several examples of syndromic surveillance in small animal practices, aimed to empower public health awareness towards new and emerging risks. Cause-specific mortality rates are considered to be among the most powerful and informative epidemiological indicators, even though in veterinary medicine, and in particular in small animals, they are not widely used (Dohoo et al. 2009). One of the reasons for the lack of this kind of studies is the difficulty to clearly define, in most cases, the cause of death in the context of a complex pathogenetic process. Studies on the causes of death in dogs have been carried out using autopsy files (Bronson 1982, Craig 2001, Eichelberg & Seine 1996, Moore et al. 2001, Olsen & Allen 2000, Schmidt 2009), especially through designed questionnaires (Adams et al. 2010, Michell 1999, Proschowsky et al. 2003), data from animal insurance databases (Bonnett et al. 1997, Egenvall et al. 2000), and animal cemetery records (Hayashidani et al. 1988). No similar surveys had been previously carried out in Italy, mostly because of the lack of information on the number and characteristics of the canine population. The Istituto Zooprofilattico Sperimentale del Lazio e della Toscana (IZSLT) conducts extensive investigations regarding animal diseases, which include not only anatomical and histopathological examinations, but also virological and bacteriological tests and chemical analyses for toxic substances. Dogs, cats, and other pets are submitted to the laboratory by veterinarians and owners whenever there is the need to know the cause of death. In fact, the post-mortem examination (PM) is not a routine procedure, but is usually carried out in the event of sudden death or when the cause leading to euthanasia is not known. Sometimes, as in the case of stray dogs and in all the cases for which a crime is suspected, the request for a PM is routinely carried out by official veterinarians. The aim of this study was to estimate the proportional mortality of the causes of death in dogs from the province of Rome, a

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mainly urbanized area, delivered to the Pathology Department of the IZSLT for PM over the period of 2003-2007.

Materials and methods Data collection An observational retrospective study of dogs from the province of Rome submitted by about 90 veterinary practitioners and official veterinarians was carried out over a 5-year period. For the necropsy of dogs submitted by practitioners, the owners were usually charged with a service fee, while the necropsy of dogs sent by official vets was free of charge. The original submission forms were collected in order to gather information about the identification, sex, age and, when available, the medical history of each dog. Eventually, the data were transferred to Microsoft Excel®. Regarding breed, age, and type of property, dogs were grouped into the following classes: purebred and crossbred; 0-1 month, 2-6 month, 7-12 month, 13-60 month, 61-119 month, > 120 month; and kennel (public or private), breeding farm, stray dog or free range, and owned dogs. Information on the specific breed was recorded and used when it was considered of interest. Military and police dogs were considered in the same category as those in breeding establishments, commercial puppy farms and pet shops because of the similarity in lifestyle and number of animals living in confined spaces. Each dog was subjected to a complete PM, independently from the reason for the submission. After the PM, specific diagnostic investigation was carried out according to: • specific request from the veterinary practitioner; • medical history, when available; • anatomo-pathological findings; and • state of conservation of the carcass.

Diagnosis and classification of causes of death Samples were submitted for one or more tests, such as virological, bacteriological, parasitological, histological, and toxicological tests in order to confirm or exclude specific causes of death. Test results and final diagnosis were all extracted from the information system of the IZSLT. The cause of death was unambiguously specified for each record and then all of the cases were grouped into 12 macro-categories accordingly. The macrocategories, intended as the main causes of death, were: ‘infectious disease’; ‘poisoning’; ‘inflammation of unknown origin’; ‘trauma’; ‘organ displacement’;

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‘neoplasia’; ‘cardiovascular disease’; ‘degenerative disease’; ‘combined causes’; ‘congenital anomalies’; ‘other cause’; and ‘not determined cause’. These categories include effective causes of death, i.e., diseases or injuries that are sufficient to lead to death as well as conditions characterized by severe structural or functional disorders that are considered incompatible with life by pathologists. When the PM was inconclusive and the results of laboratory tests were negative, the case fell into the latter category.

Statistical analysis Frequencies were calculated for each categorical variable included in the study. The information about the medical history was used only to address the diagnosis and not for data analysis. The proportion of each cause of death and its 95% binomial exact confidence interval (95% CI) were calculated. The χ2 test was used to compare proportions of causes of death in dogs of different types of property, breeds, and classes of age. Two‑sided tests were conducted and a p-value of less than 0.05 was considered statistically significant. The statistical analysis was performed using SPSS® (ver. 13) and STATA® 12.0.

Results The study included 870 dogs whose characteristics are summarized in Table I. The sample was built as follows: 5% of dogs were euthanized before being submitted to the laboratory; 20% died after a disease course longer than a few days; and 75% were found dead or died immediately afterwards. Fifty-eight carcasses (7%) were in the initial or advanced putrefaction stage. The majority of the dogs were male (54%), adult, and elderly (60%). Fifty-one percent were owned dogs and about 50% were purebred, the most frequent breed being the German Shepherd. In 95% of the examined cases, it was possible to establish at least 1 possible cause of death. In 47 cases (5.4%, 95% CI 4.0–7.1), where no significant lesions were observed, it was not possible to define the diagnosis even when minimum standards tests, i.e. bacteriological and histological tests, were performed. The most frequent main causes of death were ‘infectious disease’ (n=204, 23.4%, 95% CI 20.7-26.4) and ‘poisoning’ (n=149, 17.1%, 95% CI 14.7-19.8; Figure 1). In 122 cases (14.0%, 95% CI 11.8-16.5), the observed inflammatory changes could not be attributed to any infectious agent or specific cause. In 23 cases (2.6%, 95% CI 1.7-3.9), the degenerative changes were so severe that they were considered to be the cause of death, although the primary cause of death remained unknown.

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Causes of death in dogs in the province of Rome (Italy)

Table I. Characteristics of dogs in the study population (N=870) examined in Rome province between 2003-2007. Variable

N° of dogs (%) Sex

Female Male Not reported

323 (37.1) 470 (54.0) 77 (8.9) Age classes (months)

0-1 2-6 7-12 13-60 61-119 > 120 Not reported

50 (5.7) 139 (16.0) 67 (7.7) 199 (22.9) 169 (19.4) 152 (17.5) 94 (10.8) Property/Breeding

Kennel Breeding farm Stray dog Owned dog Not reported

297 (34.1) 35 (4.0) 84 (9.7) 440 (50.6) 14 (1.6) Breed

Crossbred Purebred Not reported Total

396 (45.5) 435 (50.0) 39 (4.5) 870 (100.0)

poisoning

17.1%

inflammation unknown origin

14.0%

infectious disease

23.4%

trauma

12.6% not determined cause

5.4%

congenital anomalies

neoplasia

0.5%

7.2%

combined cause 0.9% other cause

2.2%

degenerative disease

2.6%

organ displacement

9.1%

cardiovascular disease

4.8%

Figure 1. Main causes of death in dogs (N=870) from Rome province examined between 2003-2007. No differences were detected in the frequency of causes between sexes. Categorising by type of property, the proportion of poisoning in stray dogs (n=33, 39.3%) was significantly higher than in owned (n=100, 22.7%), kennelled (n=15, 5.1%) and

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Table II. Specific causes of death according to the macro-categories of dogs (N=870) from Rome province examined between 2003-2007. Macro-categories/ specific causes

N (%)

Infectious disease parvovirosis 68 (33.3) Gram + septicaemia 57 (27.9) Gram - septicaemia 37 (18.2) others 42 (20.6) Total 204 (100.0) Poisoning anticoagulants 45 (30.2) strychnine 33 (22.1) zinc phosphide 28 (18.8) metaldehyde 22 (14.8) others 21 (14.1) Total 149 (100.0) Inflammation of unknown origin haemorrhagic enteritis 31 (25.4) nephritis 30 (24.6) pneumonia 20 (16.4) others 41 (33.6) Total 122 (100.0) Trauma generic trauma 44 (40.0) dog bite 23 (20.9) car accident 12 (10.9) post-surgery haemorrhages 9 (8.2) others 22 (20.0) Total 110 (100.0) Not determined cause Total 47 (100.0)

95% CI 26.9–40.3 21.9–34.8 13.1–24.1 15.2–26.8 ---23.0–38.3 15.8–29.7 12.9–26.0 9.5–21.5 8.9–20.7 ---18.0–34.1 17.2–33.2 10.3–24.2 25.3–42.7 ---30.8–49.8 13.7–29.7 5.8–18.3 3.8–15.0 13.0–28.7 -------

farming dogs (n=1, 2.9%, p<0.001). Dogs housed in kennels seemed to die of ‘inflammation of unknown origin’ (n=58, 20%), ‘neoplasia’ (n=33, 11%), and ‘degenerative disease’ (n=15, 5%) more often than owned dogs (n=51, 12%, p=0.014; n=26, 6%, p=0.005; n=7, 2%, p=0.012, respectively). Proportional mortality due to ‘infectious disease’ was comparable between owned and kennelled dogs (n=110, 25% and n=68, 23%, respectively), but was significantly higher in dogs from breeding farms (n=17, 49%, p<0.001). With regard to the cause of death by age, the proportion of deaths by ‘infectious disease’ was higher in the first 2 age classes (n=36, 72% and n=83, 60%, respectively) than in all the others (p<0.001). The highest proportion of death by ‘neoplasia’ was detected in the class ‘> 120 month’ (n=36, 24%). After stratifying the sample by breeds, purebred

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Macro-categories/ specific causes

N (%)

Neoplasia haemangiosarcoma 17 (27.0) lymphoma 9 (14.3) others 37 (58.7) Total 63 (100.0) Cardiovascular disease cardiomyopathy 16 (38.1) heat stroke 8 (19.0) vascular diseases 7 (16.7) others 11 (26.2) Total 42 (100.0) Degenerative disease hepathosis 13 (56.5) myocardosis 6 (26.1) others 4 (17.4) Total 23 (100.0) Combined causes Total 8 (100.0) Congenital anomalies Total 4 (100.0) Organ displacement gastric dilatation and volvolus 62 (78.5) others 17 (21.5) Total 79 (100.0) Other cause organ laceration 11 (57.9) others 8 (42.1) Total 19 (100.0)

95% CI 16.6–34.0 6.7–25.4 45.6–71.0 ---23.6–54.4 8.6–34.1 7.0–31.4 13.9–42.0 ---34.5–76.8 10.2–48.4 4.9–38.8 ---------67.8–86.9 13.1–32.2

33.5–79.7 20.2–66.5 ----

dogs seem to die more frequently of ‘infectious disease’ (n=121, 28%) and ‘organ displacement’ (n=50, 12%) than crossbreds (n=66, 17%, p<0.001 and n=26, 7%, p=0.014, respectively), while the frequency of ‘inflammation of unknown origin’ was higher in crossbreds (n=45, 10%, p=0.001). Amongst German Shepherd dogs, gastric dilatation volvolus syndrome accounted for 25% (n=24) of the observed causes of death. Within each macro-category, the most frequent and specific cause of death was recorded (Table II). For instance, within ‘infectious disease’, parvovirosis was the most frequent diagnosis (n=68, 33.3%, 95% CI 26.9-40.3), while salmonellosis, the only important zoonosis detected, was diagnosed in 5 dogs out of 870 and was considered the primary cause of death in only 2 of them. Between the 2 classes of bacteria generating septicaemia, Gram + were more frequent (n=57, 27.9%). In particular,

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the proportion of Gram + septicaemia in puppies up to 30 days old was 50% (25/50). Among toxic substances, anticoagulants (especially difenacoum) were detected in 45 cases (30.2%, 95% CI 23.0-38.3).

of microbiological tests. Furthermore, the available tests may be not sufficiently sensitive or are, as in the case of toxicological testing, too expensive to be performed for every possible substance.

Among the inflammatory syndromes, where no specific causes were detected, the proportions of haemorrhagic enteritis (n=31, 24.6%, 95% CI 17.2‑33.2) and nephritis (n=30, 25.4%, 95% CI 18.0‑34.1) were similar. Within the “trauma” main category, generic trauma was the most frequent cause of death (n=44), followed by dog bite (n=23). Haemangiosarcoma was the most frequent neoplasia observed (n=17, 27.0%, 95% CI 16.6-34.0), most often in the spleen (8 out of 17 cases).

In the present study, ‘infectious disease’ was the most frequent cause of death. Among these, zoonoses were not common, since Salmonella spp. were found only in the 0.6% of cases. Due to the high prevalence of leishmaniasis in the area of study (Scarpulla et al. 2010), it is possible that some cases of observed nephritis were attributable to this disease, but unfortunately no specific tests were performed.

Among the combinations of causes, various infectious diseases were observed, such as canine distemper or salmonellosis, which were often associated with degenerative syndromes like hepathosis or nephrosis.

Discussion The present study is the first attempt in Italy to investigate the causes of death in pets with the main purpose of obtaining information useful for epidemiological research and surveillance. Unlike similar studies (Fleming et al. 2011, Lord et al. 2007), it did not only include a set of specific breeds or certain age classes, but the general dog population living in the province of Rome. Some results, i.e. the proportion and typology of poisoning or the identification of the prevalent infective agents in kennels, could be used by regional official veterinarians to better address control and prevention measures. According to Olsen and Allen (Olsen & Allen 2000), the cause-specific proportional mortality may be influenced by the category of dead dogs received at the laboratory for post-mortem diagnosis. Indeed, for most of these dogs, death was characterised by sudden and unexpected events, such as hyperacute infectious disease and poisoning, and the owner was not aware of any previous diseases. Since the service is not free, only owners who are motivated and with adeguate means, as well as veterinarians, sometimes for legal reasons, apply for the diagnostic service. Due to this selection bias, it is not possible to extend the results to the general dog population. The low percentage of undetermined cause of death compared with reports by other authors (Graig 2000, Olsen & Allen 2000) is likely to be a consequence of the access to multiple tests at the IZSLT. Nevertheless, a small proportion of examined cases remained undetermined because of the poor condition of the carcass, which did not allow the identification of specific lesions and compromised the accuracy

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As expected, the proportional mortality for poisoning in stray dogs was higher than in dogs of other categories. Nevertheless, the frequency of poisoning in owned dogs might be due to a low level of social tolerance to neighbours’ dogs in overcrowded urban environments, in the city of Rome and its surrounding areas where it is not uncommon to find intentionally located poisoned baits. To confront this illegal and dangerous phenomenon (well known to the authorities), the Italian Ministry of Health issued legislation that made the toxicological analysis compulsory and free of charge on any suspected bait found within the whole national territory1. The easy access to the purchase of anticoagulants, used primarily as a means for pest control, results in their frequent use for intentional poisoning, although accidental ingestion by dogs is also likely. However, it is notable that strychnine, forbidden by law since 1977 for commercial sale and use, was detected, even if its occurrence is progressively decreasing. Infectious diseases, especially parvovirosis, are obviously more common in young animals, as already reported by Walter and Kirchhoff (Walter & Kirchhoff 1995), either in dogs housed in domestic environments or in those from shops or puppy farms. In this respect, one possible risk factor that must be mentioned is the incorrect implementation of vaccination programmes. Strains of parvovirus not completely matching the commercial vaccines were indeed found in Italy (Decaro et al. 2011). In the case of pet shop puppies, a legal certificate of the cause of death is often required by the owners. This could justify the frequent recourse to PM investigation in cases of clinically evident gastroenteritis. In rehoming kennels, dogs seem to die quite often because of inflammation of unknown origin. This may be due to 2 primary factors: first, the high frequency of old animals in kennels with a consequent high prevalence of chronic syndromes; and the negative results in bacteriological analysis because of the prompt use of antibiotics in those premises when an infection occurs.

M inisterial Order of 18 December 2008.

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The high susceptibility of some breeds to infectious diseases has already been reported by other authors (Houston et al. 1996, Walter & Kirchhoff 1995). Also, the susceptibility of the German Shepherd dog and other large and giant breed dogs to gastric volvolus is well known to be due to the large thoracic depthâ&#x20AC;&#x2018;to-width ratio (Monnet 2003). Old age (64.7% were more than 60 months old) was also the most probable reason for the relatively high frequency of neoplasia in kennels, although the present estimate is lower than that of other reports (Adams et al. 2010, Bronson 1982, Craig 2001, Eichelberg & Seine 1996, Proschowsky et al. 1993) because of the selection bias mentioned above. In the neoplasia macro-category, haemangiosarcoma might have been diagnosed more frequently because it can easily cause sudden death following internal haemorrhage. A high proportional mortality for this neoplasia was also reported by Olsen and Allen (Olsen & Allen 2000) and Craig (Craig 2001). Conversely, very common tumours like skin and mammary carcinoma were only

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sporadically found in this study, probably because they are easily diagnosed by clinical examination and do not predispose to sudden death. In conclusion, findings from this study provide veterinarians and health professionals with an overview of the causes of death in dogs, especially for young ones and for sudden death events. This study could be considered a first step to building a more comprehensive and representative surveillance system for small animal diseases aimed at supplying data to public health authorities about known diseases and emerging threats.

Acknowledgments The authors wish to thank Mrs Arianna Miconi for her help in drawing graphs and tables and Mr Antonino Caminiti, Mrs Laura Weinstein, Mrs Alessandra Di Egidio and Mr Andrea Carvelli for revising the text.

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References Adams V.J., Evans K.M., Sampson J. & Wood J.L.N. 2010. Methods and mortality results of a health survey of purebred dogs in the UK. J Small Anim Pract, 51, 512-524. Backer L.C., Grindem C.B., Corbett W.T., Cullins L. & Hunter J.L. 2001. Pet dogs as sentinels for environmental contamination. Sci Total Environ, 274, 161-169. Bonnett B.N., Egenvall A., Olson P. & Hedhammar A. 1997. Mortality in insured dogs: rates and cause of death in various breeds. Vet Rec, 141, 40-44. Bronson R.T. 1982. Variation in age at death of dogs of different sexes and breeds. Am J Vet Res, 43, 2057-2059. Craig L.E. 2001. Cause of death in dogs according to breed: a necropsy survey of five breeds. J Am Anim Hosp Assoc, 37, 438-443. Decaro N., Desario C., Billi M., Mari V., Elia G., Cavalli A., Martella V. & Buonavoglia C. 2011. Western European epidemiological survey for Parvovirus and Coronavirus infections in dogs. Vet J, 187, 195-199. Dohoo I., Martin W. & Stryhn H. 2009. Measures of diseases frequency. In Veterinary epidemiological research, 2nd ed. VER Inc, Charlottetown, Canada, 73-85. Dorea F.C., Sanchez J. & Revie C.W. 2011. Veterinary syndromic surveillance: Current initiatives and potential for development. Prev Vet Med, 101, 1-17. Egenvall A., Bonnett B.N., Olson P. & Hedhammar A. 2000. Gender, age, breed and distribution of morbidity and mortality in insured dogs during 1995 and 1996. Vet Rec, 143, 519-525. Eichelberg H. & Seine R. 1996. Life expectancy and cause of death in dogs. I. The situation in mixed breeds and various dog breeds. Berl Münch Tierärztl Wochenschr, 109, 292-303. Fleming J.M., Creevy K.E. & Promislow D.E.L. 2011. Mortality in north american dogs from 1984 to 2004: an investigation into age-, size-, and breed-related causes of death. J Vet Intern Med, 25, 187-198. Hayashidani H., Omi Y., Ogawa M. & Fukotomi K. 1988. Epidemiological studies on the expectation of life for

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dogs computed from animal cemetery records. Jpn J Vet Sci, 50, 1003-1008. Houston D.M., Ribble C.S. & Head L.L. 1996. Risk factors associated with parvovirus enteritis in dogs: 283 cases (1982-1991). J Am Vet Med Assoc, 208, 542-546. Lord L.K, Yaissle J.E, Marin L. & Couto C.G. 2007. Results of a web-based health survey of retired racing greyhounds. J Vet Intern Med, 21, 1243-1250. Michell A.R. 1999. Longevity of British breeds of dog and its relationship with sex, size, cardiovascular variables and disease. Vet Rec, 145, 625-629. Monnet E. 2003. Gastric dilatation-volvulus syndrome in dogs. Vet Clin N Am-Small, 33, 987-1005. Moore G.E., Burkman K.D., Carter M.N. & Peterson M.R. 2001. Causes of death or reason for euthanasia in military working dogs: 927 cases (1993-1996). J Am Vet Med Assoc, 219, 209-214. Olsen T.F. & Allen A.L. 2000. Causes of sudden and unexpected death in dogs: a 10-year retrospective study. Can Vet J, 41, 873-875. Proschowsky H.F., Rugbjerg H. & Ersboll A.K. 2003. Mortality of purebred and mixed-breed dogs in Denmark. Prev Vet Med, 58, 63-74. Scarpulla M., Macrì G., Salvato L., Spallucci V., Aquilini E. & Rombolà P. 2010. Prevalenza di campioni sieropositivi per leishmaniosi canina nel Lazio (2005-2008). In Volume degli atti. Proc. 12th Congresso SIDILV, Genova, 27-29 October 2010, Litografia La Ducale, Parma, 366-367. Schmidt P.L. 2009. Companion animals as sentinel for public health. Vet Clin N Am-Small, 39, 241-250. Walter J.H. & Kirchhoff A. 1995. Causes of death in young dogs according to the autopsy files (1980-1993). Berl Münch Tierärztl Wochenschr, 108, 121-126. Ward M.P. & Kelman M. 2012. Disease surveillance in dogs and cats: a practitioner-based system. In Book of Abstracts. Proc. of the 13th International Symposium on Veterinary Epidemiology and Economics, Wageningen, 20-24 August 2012, Academic Publishers, Maastricht, 57.

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SHORT COMMUNICATION Severe, diffuse fibrinonecrotic pleuropneumonia in a cat affected by multiple viral infection Andrea Balboni*, Patrizia Bassi*, Mara Battilani, Roberta Biserni, Santino Prosperi & Francesco Dondi Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano dell’Emilia (BO), Italy * Andrea Balboni and Patrizia Bassi contributed equally to this short communication. Corresponding author at: Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano dell’Emilia (BO), Italy. Tel.: +39 051 2097084, e-mail: a.balboni@unibo.it

Veterinaria Italiana 2014, 50 (2), 145-149. doi: 10.12834/VetIt.49.144.2 Accepted: 09.04.2014 | Available on line: 08.05.2014

Keywords Cat, Coinfection, Feline calicivirus, Feline coronavirus, Feline herpesvirus, Feline panleukopenia virus, Histopathology, Pneumonia.

Summary This communication describes the coinfection with feline panleukopenia virus (FPV), feline herpesvirus 1 (FeHV-1), feline calicivirus (FCV) and feline coronavirus (FCoV) in a 1 year‑old domestic cat living in a feline shelter. The cat was referred to veterinary hospital with clinical signs related to diffuse gastro-intestinal inflammation, it had developed a severe pneumopathy with fibrinous exudation in all body cavities and died 8 days after initial presentation. Pathological findings and biomolecular diagnostic test results were compatible with an initial FPV infection that, in consequence of the lymphoid depletion, has fostered coinfection or reactivation of chronic-latent infections with FeHV-1, FCV, and FCoV. In the reported case, the simultaneous presence of different viruses exacerbated the clinical status of the host, resulting in multiple organ damage and leading it to its death.

Grave pleuropolmonite fibronecrotica diffusa in un gatto con infezione virale multipla Parole chiave Coinfezione, Feline calicivirus (FCV), Feline coronavirus (FCoV), Feline herpesvirus-1 (FeHV-1), Feline panleukopenia virus (FPV), Gatto, Istopatologia, Polmonite.

Riassunto Questa comunicazione descrive la coinfezione con feline panleukopenia virus (FPV), feline herpesvirus-1 (FeHV-1), feline calicivirus (FCV) e feline coronavirus (FCoV) in un gatto di un anno proveniente da un gattile. L’animale, pervenuto all’ospedale veterinario con segni clinici di gastro-enterite, ha successivamente sviluppato una grave pneumopatia accompagnata da essudato fibrinoso in tutte le cavità corporee, decedendo dopo 8 giorni. I reperti anatomopatologici e i risultati dei test biomolecolari sono risultati compatibili con un’infezione da FPV allo stadio iniziale che, in conseguenza della deplezione linfoide, ha favorito la coinfezione o la riattivazione di infezioni croniche o latenti da FeHV-1, FCV e FCoV. Nel caso riportato, la simultanea infezione con diversi virus ha esacerbato lo stato clinico dell’ospite determinando un danno multiorganico che ha portato a morte l’animale.

Episodes of viral coinfection have been frequently reported in cats. The simultaneous presence of feline herpesvirus 1 (FeHV-1) and feline calicivirus (FCV), as well as the presence of other viral and bacterial agents, is recognized as the leading cause of Feline Respiratory Disease Complex (FRDC), characterized by injury to the upper respiratory tract and by ocular and oral lesions (Cohn 2011). Furthermore, multiple infections sustained by feline panleukopenia virus (FPV) and canine parvovirus (CPV) variants (2a/2b/2c) or by FPV and other viruses

have already been reported and systemic infection of feline coronavirus (FCoV) have been frequently associated with immunosuppressive viruses, as feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV), which act as predisposing factor for the onset of infectious peritonitis (Battilani et al. 2013, Lutz et al. 1995, Moschidou et al. 2011, Pedersen 2009). In this study was reported a case of multiple infection with FPV, FeHV-1, FCV and FCoV, which all together caused enteritis, lymphoid depletion and severe lung injury in a cat.

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A 1 year-old domestic shorthair cat living in a rescue shelter in a residential area of Bologna (Emilia‑Romagna region, Northern Italy) and vaccinated upon entry into the cattery with a trivalent modified live vaccine composed by attenuated FCV, FeHV‑1 and FPV strains (Feligen CRP, Virbac Srl, Carros, France), was referred to the Veterinary Teaching Hospital of the Department of Veterinary Medical Sciences, University of Bologna, for the acute onset of vomiting, anorexia and moderate depression. Physical examination revealed mild weight loss and moderate dehydration; a blood sample was collected for a clinicopathological evaluation and stored at -20°C. Complete blood count (CBC) showed increased red blood cell count (13.45 × 109/l; reference interval 5-10 × 109/l), hemoglobin concentration 15.7 g% (reference interval 8-15 g%), hematocrit value 49.3% (reference interval 24‑45%), and severe lymphopenia (250/µl; reference interval 1,500-7,000/µl). Biochemistry results were characterized by increased concentration of total proteins 8.84 g/dl (reference interval 6-8 g/dl), albumin 3.77 g/dl (reference interval 2.10-3.30 g/ dl), urea 197 mg/dl (reference interval 15-60 mg/dl), phosphorus 9.0 mg/dl (reference interval 2.9-8.3 mg/ dl), and hypocloremia 112 mEq/l (reference interval 119-132 mEq/l). Serum Amyloid A (SAA) tested with immunoturbidometric method (LZ test SAA, Eiken Chemical Co, Tokyo, Japan) was also increased (150 mg/l; reference interval 0-10 mg/l). Serum protein electrophoresis revealed an increase in α-2 globulin fraction (2.40 g/dl; reference interval 0.58-1.26 g/dl). Fecal flotation was negative. Thoracic radiographs upon admission were unremarkable, while abdominal ultrasound findings were consistent with diffuse gastro-intestinal inflammation. The cat was treated with intravenous (IV) fluids, amoxicillin/ clavulanate (20 mg/Kg IV TID) and ranitidine (1 mg/ Kg IV BID). Clinical signs slightly improved during the following days, however 5 days after the hospital admission the patient developed an acute dyspnea. Thoracic radiographs showed a severe pneumopathy, particularly broncopneumonia (Figure 1). The cat died 8 days after initial presentation, despite intensive care treatment, and it was subsequentely submitted to necropsy. A complete post mortem examination was performed and samples from different tissues (lung, heart, liver, kidney, small intestine, mesenteric lymphnodes, brain and eyes) were collected and fixed in 10% neutral buffered formalin. After fixation, selected tissues were embedded in paraffin, sectioned at 4 μm and stained with hematoxylin and eosin. Corneal and oropharyngeal swabs and aseptically taken specimens of tongue, intestine, lung, thoracic lymph node and pleural effusion were also collected for virological investigations. Viral DNA and RNA were extracted from biological samples using commercial kits

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Figure 1. Right lateral thoracic radiograph of a 1 year-old domestic cat coinfected with FPV, FeHV-1, FCV, FCoV. Note the severe ventral located alveolar pattern consistent with bronchopneumonia. (NucleoSpin Tissue Mini Kit, Macherey-Nagel, Düren, Germany; QIAamp Viral RNA and RNeasy Mini Kit, Qiagen, Hilden, Germany). Polymerase chain reaction (PCR) assay was conducted to detect feline and canine parvovirus (FPV and CPV‑2a/2b/2c respectively) and FeHV-1. The reactions were carried out using the sets of primers reported in Table I and a commercial Taq‑polymerase (Taq DNA Polymerase Kit, Qiagen, Hilden, Germany). FPV/CPV detection was done on DNA extracted from intestine and tongue, while FeHV1 detection was done on DNA extracted from corneal swab, lung and thoracic lymph node. The FPV strain 1033/09 (Battilani et al. 2011) and a FeHV‑1 vaccine strain (Feligen CRP/R, Virbac Srl, Carros, France) were used as positive controls for parvovirus and FeHV‑1 detection respectively. One-tube real‑time reverse transcription-PCR (qRT-PCR) assay was carried out to detect FCV and feline coronavirus (FCoV) using the sets of primers reported in Table I and a one‑step SYBR Green system (EXPRESS One‑step SYBR GreenER Kit, Invitrogen, Carlsbad, CA, USA). Feline calicivirus detection was done on RNA extracted from oropharyngeal swab and lung while FCoV detection was done on RNA extracted from thoracic lymph node and pleural effusion. A plasmid (pCR4 plasmid, Invitrogen, Carlsbad, CA, USA) containing 1 copy of the target sequence of a FCV vaccine strain (Feligen CRP/R) and a plasmid (pCR4 plasmid) containing 1 copy of the target sequence of FCoV strain TN/420/00 (Battilani et al. 2010) were used as external standards for the construction of the FCV and FCoV assay standard curves respectively. Feline immunodeficiency virus and FeLV rapid immunoenzimatic test (SNAP FIV/FeLV Combo Plus Test, IDEXX Laboratories, Westbrook, ME, USA) and Toxoplasma gondii immunofluorescent antibodies titer (IFAT) for IgG and IgM (MegaScreen FLUOTOXOPLASMA, MegaCor Diagnostik, Hoerbranz, Austria) were performed on a stored serum sample. A titer of 1:40 was used as first dilution for both IgG and

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Table I. Primers used for molecular detection of RNA and DNA viruses in a 1 year-old domestic cat coinfected with FPV, FeHV-1, FCV, FCoV. Virus FPV/CPV FeHV-1 FCoV FCV

Primer name P1 for VP rev gB for gB rev FCoV1128 for FCoV1229 rev qFCV for qFCV rev

Primer sequence 5'-ATGAGTGATGGAGCAGTTC-3' 5'-TTCTAGGTGCTAGTTGAG-3' 5’-GCACACGACCGGCTAATACAGG-3’ 5’-CAGCTTTCGAGAGGCACATACCC-3’ 5’-GATTTGATTTGGCAATGCTAGATTT-3’ 5’-AACAATCACTAGATCCAGACGTTAGCT-3’ 5’-TAATTCGGTGTTTGATTTGGCCTGGGCT-3’ 5’-CATATGCGGCTCTGATGGCTTGAAACTG-3’

Fragment amplified Gene VP2 1745pb Gene gB 737bp ORF 7b 102bp ORF 1 83bp

Reference Battilani et al. 2006 Vögtlin et al. 2002 Gut et al. 1999 Battilani et al. 2013

IgM. Titers were considered as indicative of infection if >1:160 for IgG and ≥ 1:40 for IgM. Post mortem external examination showed dehydration, loss of body conditions and a small amount of white fluid exudate in both eyes. The most consistent gross findings were located in the chest. Lungs were diffusely covered by a thin layer of fibrin. All pulmonary lobes were adherent each other; the caudal lobes were completely firmly adherent to the diaphragm (Figure 2) and rubbery in consistence. Moreover, there was a small amount of yellowish fluid admixed with fibrin in all the body cavities (thorax, abdomen and pericardium), the bowel was dilated with (mostly) yellow-brown fluid content and hyperemic mucosa. Mesenteric lymph nodes were prominent and edematous. Histological sections of the lungs showed a severe, diffuse fibrinonecrotic pleuropneumonia (Figure 3). Alveolar walls were moderately thickened with microthrombi and fibrin; alveoli were filled by neutrophils, macrophages, erythrocytes and fibrin. Occasionally, type II pneumocytes were present. Multifocally, bronchial epithelial necrosis was present with lumina filled by cell debris, neutrophils, macrophages and mucus. Marked villous stunting, crypt distortion with disseminated crypt abscesses, and mild submucosal infiltration of lymphocytes, macrophages and scattered neutrophils were seen in the ileum. Ileal crypts were lineated by cuboidal or flattened cells but scattered bizarre large cells with swollen nuclei and prominent nucleoli were evident (Figure 4). Mesenteric lymph nodes were enlarged with cortical atrophy, follicular hyalinosis, mild sinus histiocytosis and occasional erythrophagocytosis. Sections from the brain showed disseminated Alzheimer type II cells, often arranged in pairs. Sections from the liver showed only mild diffuse congestion of sinusoids. Focal necrosis of the superficial epithelium and mild focal subepithelial infiltration of lymphocytes and plasma cells were detected in the nictitating membrane. Inclusion bodies were not observed in all examined sections.

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Figure 2. Gross findings: lung of a 1 year-old domestic cat coinfected with FPV, FeHV-1, FCV, FCoV. Note the thin layer of fibrin covering part of the cranial and caudal right lobes and the adherences between the caudal lobes and the diaphragm. Biomolecular investigations showed positivity for Parvovirus, FeHV-1, FCV and FCoV infections in all tested samples. In particular, quantitative real‑time RT-PCR allowed to detect the following concentrations of FCV target RNA: 5.7 x 109 copies/g of lung and 1.8 x 105 copies/µl of oro-pharingeal swab RNA extract (RNA extracted to oro-pharingeal swab was eluted in 60µl of RNasi-free water). Instead, concentrations of 7.6 x 107 copies/g of thoracic lymph node and 7.8 x 102 copies/ml of pleural effusion have been detected for FCoV. In order to distinguish FPV from CPV, the amplicon of VP2 gene was directly sequenced, the analysis of the deduced amino acid residues at critical positions allowed for the identification of the FPV parvovirus species. Feline herpesvirus gB gene amplification was confirmed by sequencing of PCR products obtained to corneal swab and lung DNA extracts. The cat was negative for FIV and FeLV while IFAT for Toxoplasma gondii revealed a titer of 1:80 for IgG and was negative for IgM. The severe histological characters previously described in ileum, mainly collapsed villi, crypts lineated by cuboidal cells and scattered bizarre

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Figure 3. Lung, caudal lobe of a 1 year-old domestic cat coinfected with FPV, FeHV-1, FCV, FCoV. Note the adherence, characterized by fibrin and mixed inflammatory cells, between the caudal lobe (arrow) and the diaphragm (arrowhead). HE. Bar = 100 μm.

Figure 4. Ileum of a 1 year-old domestic cat coinfected with FPV, FeHV1, FCV, FCoV. Villous atrophy, dilatation of some crypts with cellular debris in the lumen, and other crypts lined by large cells with swollen nuclei and prominent nucleoli. HE. Bar = 50 μm.

cells with swollen nuclei and prominent nucleoli are reported in the latest stage of FPV infection and was confirmed by detection of parvovirus genome in intestine and tongue DNA extracts. The lymphoid depletion with follicular hyalinosis detected in mesenteric lymph nodes is also described in parvoviral infection in cats, often associated with erytrophagocytosis (Brown et al. 2007) that was only occasional in the present case.

and corneal ulcerations. In the described case, neither oral nor ocular injury were present; however, the histological lung lesions and the detection of both viral genomes are compatible with a concomitant FeHV-1 and FCV infection. In particular, the absence of inclusion bodies in the histological lung sections is compatible with a FeHV-1 pulmonary infection occurred from more than 7 days.

Usually, both clinical and pathological findings in FeHV-1 and FCV infections involve upper respiratory tract and are quite similar, so differential diagnosis is often difficult. However, simultaneous viral infections are possible and can complicate the clinical and pathological presentation (Cohn 2011). In the reported clinical case there were no typical FCV-induced skin and oral ulcerations. Nonetheless, lower respiratory lesions as microthrombi, occasional type II pneumocytes, hyperplasia and accumulation of foamy histiocytes, cells debris and fibrin in alveoli, have also been described in virulent systemic FCV infections in cats (Pesavento et al. 2004). Moreover, severe fibrinonecrotic pneumonia with necrosis of bronchial and bronchiolar epithelium, has been reported also in FeHV-1 induced pneumonia in both kitten and adult cats (Chvala-Mannsberger et al. 2009). Large eosinophilic intranuclear inclusion bodies can be evident in bronchial epithelium infected by FeHV-1 up to 7 days after infection, but they are rarely detected after that time (Caswell et al. 2007). Detection of inclusion bodies can commonly differentiate FeHV-1 from FCV infection, because the latest is not associated with inclusion bodies, but their lack does not allow for excluding FeHV‑1 infection. Feline calicivirus and FeHV-1 can be responsible for symptomatic or asymptomatic infections and, in symptomatic infections, FCV and FeHV-1 are respectively associated to oral ulcerations, keratitis

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The involvement of the lower respiratory tract in FeHV-1 infection has been related to concomitant immunosuppressive viruses infections, such as FIV and FeLV (Chvala-Mannsberger et al. 2009). In the present case neither FIV nor FeLV infection was detected but immunosuppression, supported by lymphopenia and histological findings in mesenteric lymph nodes, is probably due to FPV. Immunosuppression and stress are predisposing factors also for the development of feline infectious peritonitis (FIP) and the detection of FCoV in thoracic lymph nodes and in pleural effusion may be compatible with the wet form of FIP. These findings could be also congruent with the systemic spread of an enteric non-virulent FCoV variant (FECV) in consequence of injury to the intestinal mucosa due to FPV infection, but the presence of fibrinous exudate in all body cavities could be indicative for the development of FIP. In view of abnormal physical examination, pathology and diagnostic test results, compatible with an initial viral gastroenteritis and a subsequent pneumonia with fibrinous exudation in all body cavities, it is possible to assume that the lymphoid depletion resulting from initial FPV infection has fostered coinfection or reactivation of chronic‑latent infections with FeHV-1, FCV, and FCoV, viruses widespread in feline communities. On the basis of the results of serological and immunoenzimatic test, the role of other common

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feline pathogens such as toxoplasma or retrovirus appeared negligible in this case. The viruses that affected the cats have acquired a particularly opportunistic character and are often associated with persistent, chronic and/ or asymptomatic infections (Clegg et al. 2012, Povey 1986, Vogel et al. 2010); severe clinical forms with acute course occur only under predisposing environmental conditions or when cats are subjected to stressors triggers. In the reported case, the pathogenic action exerted by each virus predisposes to infection of other viral agents and the simultaneous presence of different viruses

Fibrinonecrotic pleuropneumonia in a cat

exacerbates clinical status of the host, resulting in severe systemic inflammatory response and multiple organ damage, and leading to its death. The most common viruses that infect cats are widespread in the feline communities where the risk to contract multiple viral infections is higher also due to close contact between animals. For this reason, it is important to apply the rules of biosecurity to prevent the spread of infectious diseases in cat shelters (Möstl et al. 2013) and to avoid multiple infections that under conditions of stress can lead to the onset of severe diseases.

References Battilani M., Balboni A., Bassani M., Scagliarini A., Paltrinieri S. & Prosperi S. 2010. Sequence analysis of the nucleocapsid gene of feline coronaviruses circulating in Italy. New Microbiol, 33 (4), 387-392. Battilani M., Balboni A., Giunti M. & Prosperi S. 2013. Co‑infection with feline and canine parvovirus in a cat. Vet Ital, 49 (1), 127-129. Battilani M., Balboni A., Ustulin M., Giunti M., Scagliarini A. & Prosperi S. 2011. Genetic complexity and multiple infections with more Parvovirus species in naturally infected cats. Vet Res, 42 (1), 43. Battilani M., Scagliarini A., Ciulli S., Morganti L. & Prosperi S. 2006. High genetic diversity of the VP2 of a canine parvovirus strain detected in a domestic cat. Virology, 352 (1), 22-26. Battilani M., Vaccari F., Carelle M.S., Morandi F., Benazzi C., Kipar A., Dondi F. & Scagliarini A. 2013. Virulent feline calicivirus disease in a shelter in Italy: a case description. Res Vet Sci, 95 (1), 283-290. Brown C.C., Baker D.C. & Barker I.K. 2007. Alimentary System. In Jubb, Kennedy and Palmer’s pathology of domestic animals, 5th Eds (M.G. Maxie, ed). Elsevier Inc., St. Louis, 2, 1-296. Caswell J.L. & Williams K.J. 2007. Respiratory system. In Jubb, Kennedy and Palmer’s pathology of domestic animals, 5th Eds (M.G. Maxie, ed). Elsevier Inc., St. Louis, 2, 523-653. Chvala-Mannsberger S., Bago Z. & Weissenböck H. 2009. Occurrence, morphological characterization and antigen localization of felid herpesvirus-induced pneumonia in cats: a retrospective study (2001-2006). J Comp Pathol, 141 (2-3), 163-169. Clegg S.R., Coyne K.P., Dawson S., Spibey N., Gaskell R.M. & Radford A.D. 2012. Canine parvovirus in asymptomatic feline carriers. Vet Microbiol, 157 (1-2), 78-85. Cohn L.A. 2011. Feline respiratory disease complex. Vet Clin North Am Small Anim Pract, 41 (6), 1273-1289. Gut M., Leutenegger C.M., Huder J.B., Pedersen N.C. & Lutz H. 1999. One-tube fluorogenic reverse transcription-

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polymerase chain reaction for the quantitation of feline coronaviruses. J Virol Methods, 77 (1), 37-46. Lutz H., Castelli I., Ehrensperger F., Pospischil A., Rosskopf M., Siegl G., Grob M. & Martinod S. 1995. Panleukopenialike syndrome of FeLV caused by co-infection with FeLV and feline panleukopenia virus. Vet Immunol Immunopathol, 46 (1-2), 21-33. Moschidou P., Martella V., Lorusso E., Desario C., Pinto P., Losurdo M., Catella C., Parisi A., Bányai K. & Buonavoglia C. 2011. Mixed infection by Feline astrovirus and Feline panleukopenia virus in a domestic cat with gastroenteritis and panleukopenia. J Vet Diagn Invest, 23 (3), 581-584. Möstl K., Egberink H., Addie D., Frymus T., Boucraut-Baralon C., Truyen U., Hartmann K., Lutz H., Gruffydd-Jones T., Radford A.D., Lloret A., Pennisi M.G., Hosie M.J., Marsilio F., Thiry E., Belák S. & Horzinek M.C. 2013. Prevention of infectious diseases in cat shelters: ABCD guidelines. J Feline Med Surg, 15 (7), 546-554. Pedersen N.C. 2009. A review of feline infectious peritonitis virus infection: 1963-2008. J Feline Med Surg, 11 (4), 225-258. Pesavento P.A., MacLachlan N.J., Dillard-Telm L., Grant C.K. & Hurley K.F. 2004. Pathologic, immunohistochemical, and electron microscopic findings in naturally occurring virulent systemic feline calicivirus infection in cats. Vet Pathol, 41 (3), 257-263. Povey R.C. 1986. Persistent viral infection. The carrier state. Vet Clin North Am Small Anim Pract, 16 (6), 1075-1095. Vögtlin A., Fraefel C., Albini S., Leutenegger C.M., Schraner E., Spiess B., Lutz H. & Ackermann M. 2002. Quantification of feline herpesvirus 1 DNA in ocular fluid samples of clinically diseased cats by real-time TaqMan PCR. J Clin Microbiol, 40 (2), 519-523. Vogel L., Van der Lubben M., te Lintelo E.G., Bekker C.P., Geerts T., Schuijff L.S., Grinwis G.C., Egberink H.F. & Rottier P.J. 2010. Pathogenic characteristics of persistent feline enteric coronavirus infection in cats. Vet Res, 41 (5), 71.

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SHORT COMMUNICATION Old diseases for new nightmares: distemper strikes back in Italy Alessio Lorusso* & Giovanni Savini Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Campo Boario, 64100 Teramo, Italy * Corresponding author at: Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise ‘G. Caporale’, Campo Boario, 64100 Teramo, Italy. Tel.: +39 0861 332440, e-mail: a.lorusso@izs.it.

Veterinaria Italiana 2014, 50 (2), 151-154. doi: 10.12834/VetIt.66.191.2 Accepted: 14.02.2014 | Available on line: 30.04.2014

Keywords Abruzzo region, Canine distemper virus, Dogs, Italy, Vaccination, Wolves.

Summary This article analyses the distemper outbreak that affected the population of Apennine wolves (Canis lupus) in Italy during 2013. Distemper, as rabies, is a well-known viral infectious disease that concerns the canine population worldwide and represents a threat for wild species too. Implementation of vaccination and legislation for compulsory vaccination strategies should be achieved in areas with endangered wild species.

Vecchie malattie per nuovi incubi: il cimurro colpisce ancora Parole chiave Abruzzo, Cane, Cimurro, Italia, Lupo appenninico, Vaccinazione.

Riassunto L’articolo descrive il focolaio di cimurro che, nel corso del 2013, ha coinvolto la popolazione di lupo appenninico (Canis lupus) presente principalmente nel Parco Nazionale d’Abruzzo, Lazio e Molise. Il cimurro, al pari della rabbia, è una malattia virale estremamente nota per la sua patogenicità nei confronti della popolazione canina mondiale e rappresenta una seria minaccia per i carnivori selvatici a rischio di estinzione. In relazione a quanto riportato è auspicabile un miglioramento delle strategie vaccinali attualmente in vigore che preveda l’obbligatorietà della vaccinazione dei cani domestici presenti in prossimità di aree con animali selvatici suscettibili alla malattia.

Throughout its history mankind has been fascinated and scared at the same time by novelties. The printing revolution, nuclear power, television, space race and the Internet offer all good examples of novel tools or achievements that definitely improved our life style and which, all at once, posed the need for new forms of control and management. Similarly, the emergence of novel pathogens and diseases such as avian and swine flu, severe acute respiratory syndrome (SARS), mad cow disease (BSE), West Nile disease (WND), Middle east respiratory syndrome (Mers) and the entire complex of microorganisms involved in foodborne illnesses, attracted the global attention upon new biological threats and upon the pursuing of innovative prevention strategies. A vast amount of money has been invested worldwide to study and face novel diseases that in the majority of cases have their biological reservoir in wild and domestic animals. The One World One Health program, whose philosophy promotes collaboration and sharing expertise between the several actors operating at

the human-animal-environment interface, offers in this case a paradigmatic example. Although the research on these emerging pathogens gave brilliant outcomes in terms of control and prevention, ancient diseases affecting humans and animals still do exist. We should keep in mind that only 2 (viral) diseases have been so far eradicated from earth: smallpox in humans and rinderpest in cattle (Mariner et al. 2012), while several other well-known pathogens still circulate. Rabies, for instance, still kills humans. This is an ancient disease well-known to man since ever, it is probably one of the oldest disease ever recorded. The word rabies comes from the Sanskrit ‘rabbhas’ which means ‘acting ferociously’. The Greek used to call this tremendous infection Lyssa which stays for ‘mad, furious’. Preventive strategies were taken already at the time of Babylon: biting dog responsible for rabies transmission to a man resulted into a heavy fine for its owner (Dunlop and Williams 1996). To these days rabies is still a major threat for public health, and governments should

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implement measures to prevent its diffusion. In fact, rabies occurs in more than 150 countries and nearly 60,000 people die every year because of it, mostly in Asia and Africa (Knobel et al. 2005). According to the World Health Organization (WHO)1, 40% of people who are bitten by suspect rabid animals are children under 15 years of age and dogs represent the source of the vast majority of human rabies deaths. Fortunately, an effective vaccine to prevent rabies does exist for both humans and dogs. As stray and unvaccinated dogs are the primarily source of rabies for man, strong intervention to fight feral dogs, mass vaccination of both dogs and humans and upgrading the availability of post-exposure prophylaxis (PEP) must be implemented and pursued in developing countries. Besides rabies, many other infectious diseases of domestic animals keep causing headaches to veterinarian and diagnosticians. One of these is a disease called distemper, which affects primarily dogs. Rabies and distemper share some common grounds: dogs are recognized as the main reservoir of both diseases and they are both related to wildlife. However, the impact on public opinion is quite different, as the global attention usually devoted to rabies becomes worthless when it comes to distemper, whose importance remains confined on a regional scale and to animal health. However the 2 diseases might be connected when considered from the preventive approach point of view. Besides dogs, distemper affects a large number of carnivores as well, both in captivity and the wild, and it is transmitted mainly through direct contact. Distemper is a well-known disease too, in the past it was known as Distemper in the UK, morve or maladie des chains in France and Hundestaupe or Hundeseuche in Germany. It is not documented from ancient times as rabies, as it is believed to have invaded Europe much more recently, being it imported from Peru into Spain during the 17th century (Blancou 2004). In 1764, distemper made the first appearance in Italy and since then it has been responsible for epidemics within the domestic dog population. This is a highly contagious and severe systemic disease frequently involving the respiratory, gastrointestinal and nervous systems caused by canine distemper virus (CDV). It is very similar to measles virus of humans and rinderpest virus of ruminants; indeed they all belong to the Morbillivirus genus that forms a serologically closely related group within the family Paramixoviridae (Appel 1987, Barrett 1999, Martella et al. 2008). To better represent the evolution of the currently circulating CDV strains, a cluster classification has 1

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W HO. 2013. WHO Expert Consultation on Rabies. Second report. World Health Organ Tech Rep Ser, 982, 1-139, http://www.fao.org/fileadmin/ templates/faovn/files/Administration/ECTAD/Rabbies_report.pdf.

Lorusso & Savini

been proposed based upon the genetic relatedness of the haemagglutinin (H) encoding gene. When an unprotected dog becomes infected with distemper virus, it is doomed to succumb in the large majority of cases. It is therefore an important pathogen to deal with, especially if we consider that during the last 3 decades, a large number of CDV detection in free-living and captive wild animals has been reported. Thus, CDV represents an important conservation threat too. Renovate attention upon CDV arose in Italy during winter/spring 2013, as an epizootic occurred in dogs in the Abruzzo and Apulia regions (Di Sabatino et al. 2014). Interestingly, the novel CDV outbreak caused for the first time an epizootic among the population of Apennine wolves (Canis lupus) living mainly in the area of the National Park of Abruzzo, Lazio and Molise (PNALM) and in other wild species including foxes (Vulpes vulpes) and badgers (Meles meles). Phylogenetic analysis conducted using H gene sequences revealed that the strains involved in the distemper outbreak of Apennine wolves cluster in the Arctic lineage of CDV. Strains belonging to this lineage were demonstrated for the first time to cause disease in wildlife animals. Previous studies proved that at least 3 CDV lineages were circulating in Italy including Europe-1, Europe Wildlife and Arctic lineages. Europe-1 lineage is commonly found in domestic dogs all-over European countries; Europe Wildlife lineage encloses CDV strains detected in wild animals and domestic dogs in Hungary. Unlike Europe-1 and Europe Wildlife, CDV strains of the Arctic lineage were detected only in domestic dogs in Italy, Hungary and North America. They are called Arctic as they were firstly identified in the susceptible population of the Arctic ecosystem. Therefore, the presence of this lineage in wolves definitely represents a novelty. Though, which was the source of infection for wolves and why CDV spread so rapidly affecting a large number of animals? To answer this question we should consider a plethora of factors including: a. several cases of clinical distemper in unvaccinated domestic dogs were reported to the veterinary authorities; b. the number of naïve dogs was extremely high meaning that vaccination was not carried out properly in the previous years; c. the presence of wolves nearby villages, urban roads and farms is a common finding in the Abruzzo region; in addition transhumance with shepherd dogs is commonly practiced in proximity of places where wildlife exists, scenario that likely enhances the risk of direct contact between dogs and wolves; d. exceptional delay of the spring season.

Veterinaria Italiana 2014, 50 (2), 151-154. doi: 10.12834/VetIt.66.191.2

Lorusso & Savini

We were able to trace distemper back to summer 2012 in dogs, prior to the epizootics in wolves, and molecular investigation revealed that CDV strains of the Arctic lineage were behind it. Thus, this scenario suggests a spillover of virus from dogs to wolves. The virus spread extensively as the population immunity was very low, while this can be reasonable and acceptable within the wildlife population, this is not a suitable explanation for the domestic dogs, which need to be vaccinated and protected. Only in this way the population immunity is high enough to keep CDV infection under control, so that only sporadic cases may occur. Although the high genetic variation of the H gene raises interrogatives regarding the efficacy of the CDV strains currently used for the attenuated vaccines production (Martella et al. 2006), vaccination remains the compulsory preventive measure for dogs. As for rabies, vaccination has been shown to be highly effective, and for CDV, long lasting if carried out properly. Thus, in order to prevent future and potential devastating distemper outbreaks in the wild population, vaccination strategies should be implemented particularly among the dogs living in rural areas and dogs housed in local kennels. The main concern during the 2013 distemper outbreak was the possibility of CDV transmission to the Marsican brown bear (Ursus arctos marsicanus). Approximately, 40 bears (95% CI: 37-52%) live in the PNALM and CDV was previously shown serologically to circulate in the Marsican brown bear of the park (Marsilio et al. 1997). Since the Arctic lineage represents a new viral introduction into a naïve ecosystem, it is not possible to rule out that the virus may mutate and potentially increase the chances of expanding its host range to include the bear and cause clinical disease. In this respect, fighting the uncontrolled trading of low cost pets from Eastern Europe and to feral dogs will be

Veterinaria Italiana 2014, 50 (2), 151-154. doi: 10.12834/VetIt.66.191.2

Distemper strikes back in Italy

instrumental to control the spread of the disease. Furthermore the implementation of surveillance/ vaccination strategies and further studies, regarding antigenic mapping of CDV strains circulating in the susceptible population, are warranted in order to prevent the loss of endangered wild species and vaccine breakthrough events. In conclusion, in the era of genome manipulation by reverse genetics and next generation sequencing, in the era in which we are able to identify single amino acids to be crucial for pathogenesis and virulence, in the era of recombinant vaccines, humans and animals still suffer from old diseases which cause awful deaths in the case of rabies or conservation threat in distemper infection. Effective vaccines exist, nonetheless these diseases still keep killing. Why is this happening? There is only one simple answer: human habits. Human habits including poor planning strategies are responsible for it. Massive preventive strategies against rabies must be implemented and sustained in poor countries and compulsive vaccination in domestic and shepherd dogs against distemper must be made compelling by law in areas where endangered wild species live. While indigent population cannot afford the vaccine cost (from 7–20 USD in most low-income countries) or PEP (in high throughput clinics use of intradermic regimens can reduce costs to just 60 USD/death averted), the international community must implement strategies aimed to reduce the excessive number of children infected with rabies (Hampson et al. 2008). Rabies can only be eliminated through intervention in the animal reservoir (Hampson et al. 2009) and this is likely to be the most cost‑effective way of averting human rabies deaths in the long‑term (Zinsstag et al. 2009). It is then clear that veterinarians must occupy a central role in the management of such important infectious diseases with an obvious animal origin.

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References Appel M.J. 1987. Canine distemper virus in virus infections of carnivores. Elsevier Science Publishers BV, Amsterdam, The Netherlands, 133-159. Barrett T. 1999. Morbillivirus infections, with special emphasis on morbilliviruses of carnivores. Vet Microbiol, 69, 3-13. Blancou J. 2004. Dog distemper: imported into Europe from South America? Hist Med Vet, 29, 35-41. Dunlop R.H. & Williams D.J. 1996. Veterinary Medicine: An Illustrated History. R.R. Donnelley & Sons Company Publishers, Saint Louis, MO-USA. Di Sabatino D., Lorusso A., Di Francesco C.E., Gentile L., Di Pirro V., Bellacicco A.L., Giovannini A., Di Francesco G., Marruchella G., Marsilio F. & Savini G. 2014. Arctic lineage-canine distemper virus as a cause of death in Apennine wolves (Canis lupus) in Italy. Plos ONE, 9(1), e82356. doi: 10.1371/journal.pone.0082356. Hampson K., Dobson A., Kaare M., Dushoff J., Magoto M., Sindoya E. & Cleaveland S. 2008. Rabies exposures, post-exposure prophylaxis and deaths in a region of endemic canine rabies. PLoS Negl Trop Dis, 2(11), e339. doi: 10.1371/journal.pntd.0000339. Hampson K., Dushoff J., Cleaveland S., Haydon D.T., Kaare M., Packer C. & Dobson A. 2009. Transmission Dynamics and Prospects for the Elimination of Canine Rabies. PLoS Biol, 7(3), e1000053. doi:10.1371/journal.pbio.1000053.

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Knobel D.L., Cleaveland S., Coleman P.G., Fèvre E.M., Meltzer M.I., Miranda M.E.G., Shaw A., Zinsstag J. & Meslin F.X. 2005. Re-evaluating the burden of rabies in Africa and Asia. Bulletin of the World Health Organization, 83, 360‑368. doi: /S0042-96862005000500012. Mariner J.C., House J.A., Mebus C.A., Sollod A.E., Chibeu D., Jones B.A., Roeder P.L., Admassu B. & van 't Klooster G.G. 2012. Rinderpest eradication: appropriate technology and social innovations. Science, 337, 1309-1312. Martella V., Elia G. & Buonavoglia C. 2008. Canine distemper virus. Vet Clin N Am-Small, 38, 787-797. Martella V., Cirone F., Elia G., Lorusso E., Decaro N., Campolo M., Desario C., Lucente M.S., Bellacicco A.L., Blixenkrone-Møller M., Carmichael L.E. & Buonavoglia C. 2006. Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy. Vet Microbiol, 116, 301-309. Marsilio F., Tiscar P.G., Gentile L., Roth H.U., Boscagli G., Tempesta M. & Gatti A. 1997. Serologic survey for selected viral pathogens in brown bears from Italy. J Wild Dis, 33, 304-307. Zinsstag J., Dürr S., Penny M.A., Mindekem R., Roth F., Menendez Gonzalez S., Naissengar S. & Hattendorf J.. 2009. Transmission dynamics and economics of rabies control in dogs and humans in an African city. Proc Natl Acad Sci USA, 106, 14996–15001. doi: 10.1073/ pnas.0904740106.

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a cura di Manuel Graziani

LIBRI/Book reviews

Rosario Fico, Simone Angelucci, Erika Ciarrocca

Manuale delle attività investigative per i reati contro la fauna (Majambiente Edizioni, pp. 198) www.lifewolf.net

Il manuale realizzato dal Parco Nazionale della Majella, IZS di Lazio e Toscana, Corpo Forestale dello Stato e Legambiente, è il primo tentativo di tracciare un percorso condiviso nell’ambito delle investigazioni per i reati contro gli animali. Le attività di bracconaggio o di uccisione illegale di animali, infatti, rappresentano ancora oggi un fenomeno tristemente attuale nel nostro Paese; si pensi ai tanti lupi uccisi per avvelenamento o attraverso lacci e armi da fuoco. Gli autori del manuale, realizzato con il cofinanziamento della CE nell’ambito del progetto Life Wolfnet “Development of coordinated protection measures for Wolf in Apennines”, sono medici veterinari che ben conoscono la materia, a partire dal dott. Rosario Fico che dirige il Centro di Referenza Nazionale per la Medicina Forense Veterinaria dell’IZS di Lazio e Toscana. La pubblicazione nasce da un progetto, teso alla conservazione e gestione coordinata del Lupo nell’Appennino, che in primis ha messo in atto azioni dirette a ridurre il conflitto verso il lupo conseguente a sistemi normativi e procedurali di indennizzo del danno insufficienti, per poi combattere l’ostilità nei suoi confronti da parte degli allevatori e delle comunità locali. Sono state inoltre intraprese azioni per contrastare il fenomeno della mortalità illegale, ridurre la persistenza di rischi o lo sviluppo di nuove criticità sanitarie per la popolazione di lupo. Il progetto ha infine avviato attività finalizzate alla riduzione del disturbo diretto o indiretto al lupo e l’attivazione di un Network istituzionale. Il manuale è strutturato in 8 capitoli: Normativa essenziale; Ruoli e competenze; La scena del crimine; Dopo la scena del crimine; Il video-foto-trappolaggio come strumento di prevenzione e di indagine; Casi studio; Procedura operativa per la necroscopia del lupo; Modulistica. Da segnalare i contributi di Rita Lorenzini e Antonio Antonucci che si aggiungono ai tre autori principali.

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a cura di Manuel Graziani

LIBRI/Book reviews

Pietro Benazzi (a cura di) Cinzia Benazzi, Gabriella Martini

Il Regolamento di Polizia Veterinaria

(Società Editrice Esculapio, Opera in 3 volumi, pp. 2.300, € 148,00) www.editrice-esculapio.com

La normativa in materia di Polizia veterinaria stabilisce un quadro generale per la prevenzione, la lotta e l’eradicazione delle malattie infettive degli animali. Dalla prima pubblicazione del Regolamento (con D.P.R. n. 320 dell’8 febbraio 1954) ad oggi, come facilmente immaginabile, il panorama di riferimento è estremamente cambiato per vari fattori: le mutate modalità di allevamento degli animali, la comparsa di malattie fino a poco tempo fa sconosciute, la periodica ricomparsa di malattie “storiche” come l’afta epizootica, la febbre catarrale degli ovini, l’influenza aviaria; senza dimenticare l’aumentata consapevolezza e l’interesse crescente dei cittadini europei per gli aspetti di sanità pubblica e sicurezza degli alimenti. Oggi le maggiori preoccupazioni dei consumatori riguardano, inoltre, l’approvvigionamento alimentare connesso alla sanità animale, i costi economici derivanti dall’insorgenza di malattie negli animali e le considerazioni sul benessere degli animali, comprese le relative implicazioni determinate dalle misure di lotta alle malattie stesse. Ci si aggiunga che non è da sottovalutare il notevole aumento degli scambi commerciali di animali e prodotti di origine animale, spesso fuori controllo, sia all’interno dell’Unione Europea che con i Paesi terzi. Questo complesso insieme di cose ha dato origine ad una proliferazione di norme, prima nazionali, poi in recepimento di direttive europee, infine regolamenti comunitari immediatamente applicabili in tutti gli Stati membri. La nuova edizione del testo di Pietro Benazzi, che quest’anno compie 60 anni, aggiornata al 28 febbraio 2014 e da poco data alle stampe in 3 volumi dall’Editore Esculapio, integra le norme attualmente vigenti con il testo originario del Regolamento di Polizia Veterinaria. I curatori hanno posto una particolare attenzione alla fruibilità del testo che, peraltro, è stato arricchito di argomenti non trattati dal Regolamento originale come la biosicurezza, l’identificazione e registrazione degli animali e il benessere.

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Pasquale Celommi (Montepagano, Roseto degli Abruzzi, 1851 â&#x20AC;&#x201C; Teramo, Roseto degli Abruzzi, 1928), Riposo. Olio su tela, 63,6x114 cm. Collezione privata.

Prorogata fino al

31 AGOSTO