Gd update 1 2014

teaming up for animal health

Update

Rianne Buter:

Useful techniques for molecular typing of E. coli

Tailor-made programme for

healthier pigs

E. coli

Healthy udders, more milk

A pathogen with major economic impact

01 ApRiL 2014

GD Animal Health

Custom made animal health solutions GD Proficiency testing schemes R&D Veterinary laboratory GD Diagnostics Consultancy

www.gdanimalhealth.com

| COLUMN

| CONTENT 04 GD’s new module: Transfer

of Knowledge and Capacity Building

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06 Monitoring and consultancy in Finland: tailor-made programme for healthier pigs

08 E. coli: a pathogen with major economic impact

10 Healthy udders, more milk 12 News & Communications

08 10

Contact information GD international If you want to know more about what GD can do for you, please contact one of our staff members, responsible for international sales:

Gerard Wellenberg, PhD, BSc

Eveline Peereboom – de Haan, BSc

Head International and

Account manager

Special Accounts

Pharmacy

Annemiek Kolkman, BSc

Madelon Bolderman, MSc

Product sales manager

Account manager

­Diagnostics and PTS

Feed

phone In the Netherlands 0900-1770, Outside the Netherlands +31 (0)570-63 33 91 Fax + 31 (0)570-63 41 04 | E-mail info@gddeventer.com | Website www.gdanimalhealth.com | Mail address GD, P.O. Box 9, 7400 AA Deventer, the Netherlands | Delivery address for samples and post mortem ­material GD, CMD, Arnsbergstraat 7, 7418 EZ Deventer, the Netherlands.

Teaming up This new edition of the renewed GD Update focuses again on our motto “Teaming up for animal health”.

We would like to inform you on our new products related to the transfer of knowledge and capacity building – on “Healthy udders, more milk”, for example. An important focus of GD Animal Health is udder health and to assist farmers in achieving improvements in milk yields and milk quality. It has been proven that improvements in udder health result in more milk and thus higher earnings.

The GD Animal Health udder health approach (called UGA) is developed not only for Dutch farmers, but also can be transferred to farmers or dairy companies abroad. On pages 10 and 11 of this GD Update, you can read more about our new tailor-made projects to improve udder health and to increase milk production.

This GD Update again features four extra pages covering our activities in the field of Research and Development. We describe in more detail the different molecular typing methods on Escherichia coli – a pathogen with major impact. So which molecular typing method is used for which purpose.

We also focus on the transfer of knowledge and on our current consultancy activities related to monitoring programmes. In this edition, we focus on “Tailor-made programmes” for healthier pigs in Finland. GD Animal Health also started a collaborative project with the Veterinary Diagnostic Center in Kiev (Ukraine).

In addition to all the above-mentioned information, GD Animal Health launched a new Mycoplasma synovia (Ms) PCR to discriminate between infected and vaccinated animals (DIVA principle).

Enjoy reading this GD Update.

GERARD WELLENBERG, PhD, BSc

GD Update | April 2014 |

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TraiNiNG | Copy: GERARD WEllENBERG, PHD, BSC

GD’s new module:

Transfer of Knowledge and Capacity Building

After developing a successful module to establish veterinary diagnostic laboratories, GD Animal Health has now developed an additional module entitled “Transfer of Knowledge and Capacity Building”.

international partner GD Animal Health continues to partner many international projects related to animal health, such as disease-control programmes, monitoring programmes and lab capacity building projects. GD has been active in eastern and central Europe, Egypt, Indonesia, India, Ethiopia, Ireland, Finland and Russia. Successful projects include

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disease (mastitis) control programmes, risk-analysis studies, epidemiological studies, legal aspects of food safety related to animal products, IT infrastructure, capacity building of laboratories and training of veterinarians and laboratory staff.

Long-term support GD Animal Health has thus acquired a lot

of knowledge and now offers customized modules to transfer this knowledge and help other companies to increase their capacity. A custom-made module helps GD to meet the specific needs of different laboratories or countries. The aim of our collaboration or partnership is to ensure long-lasting support relationships (longterm contracts).

promoting veterinary know-how Support modules also focus on veterinary support, use of diagnostic test results (practical value of diagnostic data), field visits, advice on the registration of animals, the development of disease control, and on surveillance programmes. In the past, GD Animal Health has organised training sessions and courses for veterinarians on poultry, cattle and swine diseases. GD also organises workshops on location, such as on-site consultancy training sessions and field visits.

Do you want more information about the customized modules GD can offer you?

Then please contact us at info@gddeventer.com.

GD provides trainings for the veterinary Diagnostic Center in Kiev GD Animal Health and the Veterinary Diagnostic Center of the BioTestlab group companies in Kiev, Ukraine, agreed to enter a collaborative venture focussing on the transfer of knowledge and capacity building.

Each module is based on different subjects or specific customer demands.

These might be related to the establishment of a veterinary diagnostic lab, but could also involve the transfer of lab knowledge (technical support) in terms of: • the selection and use of diagnostics and reagents, • the transfer of test methods related to bacteriology, (clinical) chemistry, immunology, molecular biology, pathology, histopathology and virology, as well as trouble shooting and how to interpret diagnostic data.

supporting Quality systems Transfer of knowledge could also be based on the implementation of a Quality System (ISO17025), and on the implementation of Quality Control and Quality Management.

This kind of support relates to: • the validation strategy of test methods, and • the use of internal test control samples and ring test (proficiency testing schemes), etc.

Training courses could be given on-site but also at the GD Animal Health laboratory.

The Veterinary Diagnostic Center was funded in 2001 and has official approval from the State Veterinary Department in Ukraine for performing diagnosis on animal diseases, and feed and water analyses. The lab is certified according to ISO 9001 by local and foreign accreditation bodies.

The Veterinary Diagnostic Center performs more than 110,000 analyses a year, and has built up strong and close relationships with more than 500 poultry and livestock farms. This private and independent diagnostic company already enjoyed a long-lasting relationship with GD Animal Health and this relationship will now be intensified.

GD will provide training to support the high-quality laboratory tests performed by the Veterinary Diagnostic Center and the veterinary services.

GD Update | April 2014 |

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research & development | Copy: Theo Geudeke, DVM PhD and Paul Franssen, DVM

Monitoring and consultancy in Finland:

Tailor-made programme for healthier pigs GD Animal Health has the unique opportunity to combine advanced laboratory testing, specialized pathology and practical veterinary knowledge. A good example of what this means in practice is GD’s monitoring and consultancy project together with the large Finnish cooperative for pig farmers, HK Agri. This has already resulted in greater insight and more practical advice for pig farmers.

A group of 21 large pig farms representing about 18,000 sows and over 100,000 finishers started cooperating with GD at the initiative of their veterinary advisor.

Once a year, the farmers submit serum samples of sows, weaned piglets and

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finishers (or rearing gilts) to GD. A few years ago GD also introduced the testing of drinking water samples. Furthermore, the veterinarian consults to the Finnish farms fills in meticulous checklists on management, housing, feeding

and health care on every farm. This information is completed with technical results in the sow herd and finishing pigs. Finally, slaughterhouse information, such as pathological findings, is also collected.

Serology & biochemistry

Technical results & management

The serum samples are tested for antibodies against APP, Influenza (type H1N1, H1N2 and H3N2), PIA / Ileitis and PCV2. In each age group, blood samples are collected from 12 animals. Since PRRS and Mycoplasma are of no concern in Finland, the animals are not tested for these pathogens. Every time, the necessity of testing certain pathogens is debated with the participants. For instance, if a certain age group is vaccinated or if all tested animals appear to be seropositive in many consecutive rounds of testing, tests may be skipped.

The results of the serological tests are presented in clear graphs and compared to previous results in order to assess the progress or changes.

The technical results of the sow farms are converted to financial figures in comparison with the results in the top 25% of the HK-Agri farms and in comparison with the results of the previous period. Figures on litter size, pregnancy rate, piglet mortality and sow replacement are converted to euros to demonstrate more clearly the weak and strong aspects of a farm and whether or not progress is being realized.

Also the slaughterhouse information on pathological findings, such as percentage of finishers with pleurisy, pneumonia, arthritis, abscesses or condemned livers, is converted to economic figures based upon scientific sources. This reveals the possible revenues in hard currency of a healthier pig herd.

Apart from the organoleptic examination (smell, colour, clarity and residues of iron or hydrogen sulphide), the drinking water quality is tested for ammonium, nitrite, nitrate, iron, manganese, sodium, chlorine, sulphate, hardness and acidity (pH). The bacteriological water quality is also investigated for overall bacterial and specific E. coli count. Just like in Dutch farms, the water quality is frequently disappointing and sometimes this essential substrate for living creatures is basically not suitable for drinking.

Finally, on each farm an extended checklist is used to assess housing, indoor climate, feeding and health care. Also clinical symptoms like diarrhoea, coughing and wasting are recorded. These checklists are also submitted to GD Animal Health.

Integration of data All information from the farms, such as serology, water quality tests, slaughterhouse information, technical results and the completed checklist, is aggregated by a veterinarian of GD’s pig-health department. The integrated information is then

summarized and converted into practical recommendations in consultation with the veterinary advisor of the Finnish farms.

The recommendations given may include treatments or preventive measures like vaccinations. But often advice is related to housing (indoor climate or pen design) or drinking water disinfection. Another issue might be the feeding of sows in relation to reproduction. In conclusion, the recommendations are not limited to typical health topics like antimicrobial treatments or vaccinations, but extend to all sorts of zootechnical advice concerning reproduction in sows, growth rate in finishers, housing, hygiene and feeding.

Additional activities Incidentally a GD pig-health veterinarian visits some of the farms at the request of and in consultation with the local advisors. Furthermore, every now and then workshops are organized on specific topics like farrowing and colostrum management or feeding sows during gestation. GD’s pig-health department has thus designed a tailor-made monitoring and consultancy programme with the group of Finnish pig producers. In doing so it uses different kinds of expertise from within GD, like in this case the practical knowledge of our pig veterinarians in collaboration with the serology and biochemistry sections of the GD-laboratory.

A veterinary consultant is performing pregnancy testing.

GD Update | April 2014 |

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Research & Development | Copy: Margriet Brus, MA

E. coli:

a pathogen with major economic impact What molecular typing techniques for typing E. coli have sufficient discriminatory power? And to what degree is E. coli genetically diverse? These questions were the aims of the study of GD Animal Health research technician Rianne Buter as part of her graduation project from the University of Applied Sciences. Not only did Buter find the answers she was looking for, the results are also promising because most typing techniques can also be used for the molecular typing of other pathogens than E. coli.

Introducing new tests Rianne Buter works as research technician at the Research & Development (R&D) Department of GD Animal Health. She carried out her study together with colleague and supervisor Remco Dijkman and others.

With her research into E. coli Buter aimed to introduce to GD new, cheaper and faster molecular typing methods with sufficient discriminatory power. To this end, she examined 500 E. coli isolates – primarily from cattle, poultry and pigs – using five different typing techniques. “The isolates were already classified beforehand as either pathogenic or commensal based on post mortem results and bacteriology. The five techniques were then applied for molecular typing of the isolates”, Buter says.

“Four techniques were new to GD, but we already were familiar with one technique, Pulsed Field Gel Electrophoresis (PFGE).

MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization, Time-of-flight Mass Spectrometry) was already used in our routine laboratory for identification of bacteria, but there was no experience with this technique in typing bacteria at the subspecies level.”

Searching for a rapid test

Remco Dijkman and Rianne Buter at work in the GD laboratory.

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The five techniques used in the study were PFGE, MLVA (Multiple-locus Variable number of tandem repeat Analysis), MLST (Multi Locus Sequence Typing), ECOR (Escherichia Coli Reference Collection) phylotyping and MALDI-TOF MS. PFGE was used as reference technique for the study. Buter: “Together with the MLVA, this technique proved to have the most discriminatory power. With MLVA it also seemed possible to discriminate E. coli

pathovars (pathogenic genotypes). MLST is primarily suited for epidemiological and evolutionary studies.” Molecular typing using MALDI-TOF MS is still in its infancy, but could be very interesting. However, more research needs to be done. One of the things that Buter and her colleagues had hoped for was that the ECOR technique would be suitable as rapid test. “A test that would demonstrate directly: this E. coli is a pathogenic strain that causes disease”, says Buter. “Sadly we had to reject this idea. However, this technique proved to be interesting in gathering more information about evolutionary history of E. coli isolates. Also the correlation of ECOR phylotypes and MLST genotypes was very good. This makes this technique useful in future research projects for E. coli.”

Widely applicable Colleague Remco Dijkman adds: “Most of these techniques are interesting because they can be used more widely, on other bacteria or even viruses. We recently used the PFGE technique, for instance, for typing Listeria monocytogenes. During hygiene monitoring of a meat processing company this bacteria was detected and isolates from the entire production line were examined using PFGE. In the end, just one contaminated machine was re-

poultry cattle poultry pigcattle human

pig

goat

human

horse

goat

duck

horse sheep catduck

sheep cat

Minimum Spanning Tree for 420 E. coli isolates analyzed with MLST. Each isolate is represented by a dot. Individual isolates are represented by a small dot and larger dots represent more than one isolate with identical genotype.

Dots are colored by animal type as shown in the legend. Dots connected with a line are related. Groups of dots with identical shading are very closely related isolates belonging to the same clonal complex.

sponsible for the persisting contamination of ready to eat meat products. After the machine was subjected to improved cleaning and disinfection, the problem was solved. A great example where molecular typing techniques were used to pinpoint the source of the contamination.”

351 E. coli types In addition to greater knowledge and experience with typing techniques, Buter also wanted to acquire insight into the genetic diversity of E. coli and their relation to pathogenicity. This genetic diversity turned out to be very high. Buter: “We have detected 351 genotypes with PFGE, and that is a lot. The discriminatory power of the PFGE test thus proved very high.”

Furthermore, Buter found genetic differences between E. coli isolates of mammals and those of poultry with MLST (see figure). E.g. some genotypes belonged mainly to extra-intestinal E. coli. Other genotypes were very divers and contained suspect commensal as well as pathogenic E. coli from different origin. “The advantage of MLST is that we are able to compare the genotypes we find with results found by other institutes all over the world using a public database”, says Buter.

E. coli E. coli are bacteria that occur frequently in poultry, reptiles and mammals, including humans. Not all types of the bacteria cause disease, but certain types may lead to problems in livestock farming. ‘Intra-intestinal’ (found inside the gut) E. coli may cause diarrhoea and severe dysentery.

It is a particularly acute problem in poultry. In other species, especially in young animals, diarrhoea caused by E. coli is a problem. ‘Extra-intestinal’ (found outside the gut) E. coli may lead to inflammation in organs outside the intestines, for instance in the human urinary tract and for poultry leasions in the peritoneum, oviduct and air sacs e.g. occur frequently.

E. coli can cause significant losses and is therefore of economic relevance. To deal with this problem effectively, further typing of the bacteria is required.

Wide diversity “The diversity between farms is very high”, Buter continues. “Only at three farms identical E. coli genotyes were found with PFGE, so almost every farm had its ‘own’ pathogen. And that makes it easier to trace back problems caused by E. coli.” When using a farm-specific E. coli vaccine (autogenous vaccines) it may be very important to add more than one genotype if detected at a farm. “You can add up to four different isolates in a single auto­genous vaccine,” says Dijkman. Auto­genous vaccines are now used primarily at poultry farms. GD makes these vaccines, but it is essential to first analyse which genotypes are present on a farm by testing a representative number of affected animals from a flock.

Practical use Dijkman: “The expertise we have gained on typing E. coli could now be used for typing other pathogens. And that may be of interest to the food and feed industry, companies involved in commercial livestock farming and the Dutch Commodity Boards.” Buter adds: “The data we collected on ESBL producing [enzymes that can inactivate antibiotics, ed.] E. coli was also interesting. These data were as yet insufficient to draw any conclusions, but we will be expanding research on ESBL typing. We are due shortly to launch a number of major ESBL projects.”

GD Update | April 2014 |

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Udder health | Copy: Christian Scherpenzeel, DVM

GD’s new udder health approach explained:

“Healthy udders, more milk” GD Animal Health has a leading role in animal health and food production in the Netherlands. Over ninety years, GD has provided animal health programmes and laboratory diagnostic services, supporting industrial customers, governments, veterinary practitioners and farmers. GD is unique in combining practical expertise in the field of animal health management with expertise in laboratory diagnostic services. An important focus of GD is udder health.

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Scientific and practical expertise To help farmers improve udder health on their farms, GD Animal Health recently introduced a new ‘Udder Health Approach’ [In Dutch: ‘Uiergezondheidsaanpak’ or UGA, red.] aiming for healthy udders and, as a result, more milk and fewer financial loses.

UGA integrates state-of-the-art scientific udder-health expertise with the implementation of practical expertise, working closely together with dairy farmers and associated veterinarians. A true example of GD’s motto ‘Teaming up for animal health’.

Overall concept The Udder Health Approach is an overall concept for the improvement and optimization of udder health and milk quality on (large) dairy farms by offering laboratory products, services, monitoring and training or consultancy. The extended know-how regarding the most optimal and reliable diagnostic tests for udder-health parameters, together with our practical expertise and network is of great value for both farmers and veterinarians. Our team is available to supply all the information needed. Two dairy veterinarians and three specialized extension workers operate close together in the field with practitioners and farmers and bring independent scientific know-how into practice.

Laboratory tests In 2013, over 15,000 individual cow milk samples and 27,000 bulk tank samples were analyzed for classic bacteriological culturing, with individual samples subject to optional antibiotic sensitivity testing.

Over 4,000 sensitivity tests were conducted. In addition to classical cow milk cultures, UGA provides tests for molecular biology (PCR) as well as tests for bedding and on-farm udder-health consultancy.

Training and consultancy Besides diagnostic testing, UGA performs over 100 veterinary dairy farm visits, 200 dynamic tests and more than 400 training sessions and workshops each year. We generate a lot of experience and knowledge by participating in at least two large (clinical) trials annually for the (pharmaceutical) industry under GLP and GCP conditions.

Monitoring The UGA Team monitors udder health and antimicrobial resistance in the Netherlands and other countries 365 days a year. With a national network of 500 dairy veterinarians and 18,000 dairy farmers, our customers provide a lot of useful information that is used for early warning of emerging

diseases. The UGA Team has over 5,000 consultations by phone each year, triggering more than 1,000 farm visits each year.

Partner for the industry UGA is a reliable partner for scientific research, product development and training of employees for companies in the dairy industry. For the pharmaceutical industry, we perform at least two large marketing or registration (clinical) trials each year, under GLP and GCP conditions, if necessary.

We also collaborate and cooperate with other companies in the dairy industry (i.e. dairy plants, milking-machine manufacturers, etc.). UGA is the authority for stateof-the-art udder-health knowledge and experience and an excellent partner for the industry, with a focus on improving udder health. After all, healthy udders, more milk.

Curious about what our UGA Team can do for you? Please do not hesitate to contact GD Animal Health.

The UGA Team: Jantijn Swinkels, Anton Gosselink, Sabine Tijs, Hans Miltenburg and Christian Scherpenzeel

GD Update | April 2014 |

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News & communications New differentiating Ms pCr test available

Naomi de Bruijn, specialist ECvp

The increasing clinical and economic relevance of Mycoplasma synoviae (Ms) prompted the Dutch poultry industry to launch a control and eradication programme for this mycoplasma species in 2013. Awareness of the relevance of Ms is also increasing in many other countries. live Ms vaccines are used in the field for the prevention of clinical problems. However, they may interfere with the control and eradication of Ms unless diagnostic tests able to differentiate vaccinated infected from vaccinated noninfected flocks are available.

Therefore GD Animal Health has developed a differentiating Ms PCR which can distinguish Ms field strains from vaccine strain MS-H (AH product number W1434). Possible test results of this PCR in a (vaccinated) flock are presented in the table below.

For more information contact Dr. Anneke Feberwee on +31 (0)570 660 384 or at a.feberwee@gddiergezondheid.nl.

GD researcher Naomi de Bruijn recently passed the European College of Veterinary Pathologists (ECVP) exam. After an intensive study of several years, Naomi can now call herself a “Specialist in Veterinary Pathology”. GD Animal Health – specifically the Poultry Department – is very proud to announce that poultry pathology in GD is now performed by an ECVP-certified pathologist. GD congratulates Naomi with this brilliant result.

Differentiating Ms PCR MS-H vaccine strain

Ms field strain

Infection status

Result 1

detected

detected

infected

Result 2

detected

not detected

free

Result 3 not detected

detected

infected

Result 4 not detected

not detected

free

MOrE iNfOrMaTiON? on our website www.gdanimalhealth. com you will find information about our products, services and various GD projects as well as the latest information about submitting samples to GD. Furthermore, we have an e-letter to keep you informed about new tests, recent publications by GD employees, proficiency testing schemes and much more. you can register for our e-letter on the website.

GD, po Box 9, 7400 AA Deventer, the Netherlands, t. +31 570 63 33 91, F. +31 570 63 41 04 www.gdanimalhealth.com info@gddeventer.com

pigMatch: new opportunities PigMatch, the international and independent web portal on pig-health, has expanded. Participating Dutch and German pig farms now have even more options for indicating their health status to potential customers in a more flexible way.

One and a half years ago, PigMatch was introduced in Germany and the Netherlands to provide pig farmers the opportunity to exchange information on the health status of their herds. Thus potential purchasers can gather data on pathogens like PRRS, APP, Mycoplasma hyopneumoniae, Brachyspira hyodysenteriae and B. pilosicoli, but also on diseases like Atrophic Rhinitis, mange and Salmonellosis. The sample size is fixed, but different levels of monitoring may be selected (3, 6 or 12 times a year).

A new option has recently been added to differentiate the level per pathogen or to adapt the interval of sampling to the farm visit routine of the practicing vet. More detailed information about PigMatch can be found at www.PigMatch.eu.

GD present at EavLD GD Animal Health will be present as Gold Sponsor at the 3rd EAVlD Congress on veterinary diagnostics, held on 12–15 October 2014 in Pisa, Italy. We look forward to meeting you there. If you would like to make an appointment, please send an email to Annemiek Kolkman at a.kolkman@gddeventer.com.

pTs 2014 AUGUst PTS for Avian Metapneumovirus (aMPV)/Turkey Rhinotracheitis (TRT) antibody detection in serum

VlDIA255*

PTS for Avian REO Virus (ARV/REO) antibody detection in serum

VlDIA277*

PTS for Infectious laryngotracheitis (IlT) antibody detection in serum

VlDIA292*

oCtoBER PTS for Avian Infl uenza Virus (AI) antibody detection in serum

VlDIA232*

PTS for Salmonella (SA) antibody detection in chicken serum

VlDIA233*

PTS for Salmonella (SA) antibody detection in porcine serum

VlDIA171*

PTS for Swine Infl uenza Virus (SIV) antibody detection in serum

VlDIA295

* officially accredited according to the international guideline ISO 17043:2010

parr test for canine lymphoproliferative disorders lymphoproliferative disorders represent a significant clinical problem in dogs. It is crucial to differentiate neoplastic lymphoid disease – i.e. lymphomas and leukaemias – from reactive, hyperplastic lymphoid conditions. GD offers a PARR test which makes this possible.

While the combination of histopathology and immunohistochemistry is a powerful tool to accurately assess the neoplastic or reactive nature of a lymphoproliferative condition in a significant percentage of such cases, the essence of lymphoid neoplasia – i.e. the clonal expansion of neoplastic lymphoid cells – cannot be proved by this microscopic approach. GD’s PARR test for formalin-fixed and paraffin-embedded tissues and also smears will supply additional information. The PARR has been tested and validated and the overall specificity is 90% and sensitivity 92%. It will generate results even when small lymph node trucut biopsies are used as DNA source. Inherent to this, it is therefore always prudent to interpret PARR data in combination with other findings, such as history, histopathology and immunohistochemistry. GD also offers a service for feline PARR testing.