PRESENTATION
BROCHURE
ESSENTIAL GUIDES ON SWIN HEALTH AND PRODUCTION
L ptospirosis Francisco Javier García Peña
ESSENTIAL GUIDES ON SWINE HEALTH AND PRODUCTION
Leptospirosis
ESSENTIAL GUIDES ON SWIN HEALTH AND PRODUCTION
L ptospirosis Francisco Javier García Peña
AUTHORS: Francisco Javier García Peña. FORMAT: 17 × 11 cm. NUMBER OF PAGES: 88. NUMBER OF IMAGES: 40. BINDING: paperback, wire-o.
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A visual and practical guide on swine leptospirosis aimed at field practitioners to assess and review the current situation of this disease. This book, written by an author with a wide experience, describes the most relevant aspects of the aetiology, epidemiology (with a special emphasis on concepts such as “natural nidi”, maintenance host and accidental host), pathogenesis, clinical picture, lesions, diagnosis (particularly the interpretation of the results of the diagnostic tests performed), treatment and control of the disease. The book includes numerous visual resources that facilitate the understanding of the text.
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Leptospirosis
Presentation of the book Most of the studies carried in different countries indicate a high prevalence of leptospirosis in pigs. However, diagnosing this infection is difficult and few laboratories can perform this task, which is probably why it can be said that the disease is underdiagnosed and often forgotten as a possible cause of reproductive disorders. Furthermore, there is a school of thought that believes the changes made to the production systems as a consequence of new animal welfare regulations and restrictions on the use of medicated feed and other preventive treatments may give rise to an increase in leptospirosis-related reproductive disorders. That is why we believe it could be useful for field practitioners to assess and review the current situation of the disease. The purpose of this book is to gather the existing knowledge on swine leptospirosis in a visually attractive and practical manner. The book first includes a review of the aetiology of the disease, since there have been major taxonomic changes affecting the genus Leptospira in the past few years. It then reviews the concepts of “natural nidi”, maintenance host and accidental host, which are key elements to understand the epidemiology of the disease. It also explains the differences and most important aspects of the pathogenesis, clinical picture, interpretation of the diagnostic test results, and control strategies of the disease, depending on whether the infection is caused by adapted or non-adapted serovars. Francisco Javier García Peña
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Leptospirosis
The author Francisco Javier García Peña Francisco Javier García Peña is currently working as a public health inspector (products for human consumption) at the Barajas Airport Border Inspection Post, Madrid. He graduated in biological sciences in 1982 and in veterinary medicine in 1986 from the Complutense University of Madrid (UCM). He worked at the Department of Bacteriology 2 at the Central Veterinary-Animal Health Laboratory (LCV) of Algete (Spanish Ministry of Agriculture and Fisheries, Food and Environment), Madrid, from 1992 to 2017. Before working at the LCV of Algete, he was professor at the Department of Animal Health (Infectious Pathology) of the Faculty of Veterinary Medicine, UCM for five years. During this period, his research focused mainly on the epidemiology and diagnosis of pleuropneumonia and streptococcal infections in pigs. At the LCV of Algete, the Department of Bacteriology 2 houses the national research centres on several zoonoses and animal diseases such as campylobacteriosis, leptospirosis, botulism, verotoxigenic Escherichia coli infections, contagious equine metritis, vibriosis and bacterial diseases of fish and bees. His main activity there has therefore been the development and validation of diagnostic techniques for the previously mentioned diseases. In addition, he was the first to describe several bacterial diseases in Spain, such as tularaemia in lagomorphs and rodents, contagious equine metritis or haemorrohagic septicaemia in the deer. After a brief visit at the Leptospirosis Reference Laboratory of the World Organisation for Animal Health (AFBI Veterinary Sciences Division, Stormont, Belfast), he started conducting research on this disease in several animal species, both domestic and wild. In collaboration with the Faculty of Veterinary Medicine of the UCM, NEIKER (Basque Institute of Agricultural Research and Development), CENSYRA (Centre for Animal Selection and Reproduction) in the Spanish region of Asturias, and other companies specialised in animal health, he has conducted prevalence studies in cattle, pigs, deer and wild boars, and studied outbreaks in production animals with the isolation of the strains involved and the determination of the serovars maintained by wild rodents. His current work mainly focuses on his participation in the Group for the Study of Wild Animal Medicine and Preservation (GEMAS), whose purpose is to study the health status of the Spanish native fauna and the interrelationships of some diseases between wild and domestic animals and human beings.
Communication services Website Online visualisation of the sample chapter. Presentation brochure in PDF format. Author’s CV. Sample chapter compatible with iPad.
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ESSENTIAL GUIDES ON SWIN HEALTH AND PRODUCTION
L ptospirosis Francisco Javier García Peña
Table of contents 1. Aetiology
4. Diagnosis
Main characteristics of the genus Leptospira
When should leptospirosis be suspected?
Taxonomy
Samples sent to the laboratory
2. Epidemiology Pigs as hosts for Leptospira spp. Pigs as maintenance hosts Pigs as accidental hosts
Geographical distribution and prevalence Geographical distribution Prevalence
Sources of infection, transmission and risk factors Health implications
3. Pathogenesis, clinical presentation and lesions Pathogenesis Clinical presentation Acute leptospirosis Chronic leptospirosis
Lesions Financial impact
Selection of animals to be tested and suitable samples Sending the samples
Laboratory diagnostic methods Objective of diagnosis Diagnosis of acute leptospirosis Diagnosis of abortions, stillbirths, and non-viable piglets at birth Diagnosis of infertility.Do I have leptospirosis on my farm? If I do, is it active?
Interpreting the results
5. Treatment and control Treatment Control Health–hygiene prophylaxis Antibiotic metaphylaxis Vaccination
References
4
Diagnosis
Leptospirosis
When should leptospirosis be suspected? One of the most problematic aspects of leptospirosis is its diagnosis, as the clinical signs are not usually very specific. Leptospirosis should be suspected in the event of: ◗ Fever, jaundice, and/or nervous signs in piglets <3 months old.
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◗ Piglets that are aborted, mummified, stillborn, or weak and non-viable. ◗ Endometritis and poor fertility in the sow with cyclic or acyclic returns (28–35 days). Most infections are subclinical, and the only clinical signs observed are usually reproductive. This makes laboratory diagnosis necessary, as other diseases, such as PRSS, brucellosis, parvovirus, and SMEDI, can develop with similar clinical signs.
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DIAGNOSIS
Samples sent to the laboratory Selection of animals to be tested and suitable samples ◗ Animals with acute signs: jaundice, haemoglobinuria, etc.
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◗ Animals with reproductive problems that have not been treated with antibiotics. ◗ Fresh foetuses which have not yet started to decay. Include foetal stomach contents for polymerase chain reaction (PCR).
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4
Diagnosis
Leptospirosis
Disease phase
Humoral immune response: antibody titre
Leptospiraemia
Acute clinical disease
Chronic clinical disease
Samples for direct diagnosis Blood and cerebrospinal fluid
Kidneys, liver, lungs, brain and body fluids
Urine, kidneys, and samples from the genital tract (oviducts, uterus, prostate gland, testicles, etc.) Vaginal discharge, placenta, and foetal organs
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Samples for serological diagnosis Paired serum samples: seroconversion
1
2
A single serum sample
6–12
3 Weeks post infection
Figure 1. Samples sent to the laboratory according to the clinical signs observed and development of the immune response.
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DIAGNOSIS
Sending the samples It is advisable to contact the laboratory where the tests are to be conducted before sending specimens, so that they can advise on the samples to be taken and how to collect, preserve, and transport them, as these requirements depend on the diagnostic tests to be used. The least time possible should elapse between sample collection and arrival at the laboratory.
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Table 1. Sample type and transport conditions according to the diagnostic technique used. Diagnostic technique Culture Histology Immunofluorescence (IF) or immunoperoxidase (IP) PCR Serology by microagglutination (MAT) Serology by enzymelinked immunosorbent assay (ELISA)
Storage temperature
Transport medium with serum albumin
Use of swabs
✔ ✘
✘ ✘
✔ ✔
✘ ✘
✘ ✔
✘
✘
✔
✔
±
✘
✔
✔
✔
✔
✘
✘
✔
±
✘
✘
✘
✔
✔
✘
25 °C -20 °C
Immersion in formalin
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Diagnosis
Leptospirosis
Laboratory diagnostic methods Diagnostic methods are classified into 2 broad groups: ◗ Direct: based on detecting the bacteria themselves, or bacterial antigens, or their nucleic acid. ◗ Indirect: based on detecting antibodies to infection. Table 2. Characteristics of the main methods of laboratory diagnosis for leptospirosis. Characteristic
Direct methods
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Serological tests
Direct observation
Culture
IF or IP
PCR
MAT
ELISA
+++ ++
+++ +++ +++
+++ + ++ +
+++ ++ ++ -
+ ++ +++ ++
+++ + ++ -
-
+++
-
-
++
+
Speed Sensitivity Specificity Difficulty Identification of the serogroup involved
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DIAGNOSIS
Table 3. Advantages and disadvantages of diagnostic techniques for leptospirosis. Test
Advantages
Disadvantages Direct
◗
Culture
◗ ◗
Immunohistochemical tests (IF and IP)
◗ ◗ ◗ ◗
PCR
◗ ◗
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Definitive diagnosis: the only technique that enables the serovar involved to be identified. Identifies the serovars circulating in a country or region. Creates a collection of strains for epidemiological and antibiotic resistance studies. Speed Useful to diagnose foetal infection and acute cases. Do not require viable leptospires. Distinguishes between pathogenic and saprophytic leptospires. Does not require viable or complete leptospires. Speed, especially real-time PCR (RT-PCR).
◗ ◗ ◗
◗ ◗ ◗ ◗ ◗ ◗ ◗
Very dependent on sample quality. Tedious: up to 6 months for a negative result. Difficult: contamination, addition of fresh culture medium, etc.
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Lack of commercial reagents. Not very effective in detecting chronic carriers. Require leptospires that have retained their antigenic integrity. Lack of validation for animal tissues. Proven specificity compared with MAT, but not with culture. Inhibitors present in the sample, especially in foetal samples. Does not distinguish between serogroups.
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Diagnosis
Leptospirosis
Test
Advantages
Disadvantages
Indirect or serological ◗
Possible to identify the serogroup involved. Method of reference for international trade. Useful for individual diagnosis of acute cases, herd diagnosis, and epidemiological studies.
MAT
◗
Outer membrane proteins (OMPs) ELISA Outer membrane lipopolysaccharides (LPS)
◗
Reacts to all pathogenic leptospires: useful to diagnose acute infections.
◗
Specific to the serogroup: useful in epidemiological studies and monitoring programmes.
◗
◗ ◗ ◗
◗ ◗
Storage and testing of strains of live organisms. Sensitivity depends on the spectrum of serogroups selected. Individual diagnosis: useful in chronic infections, but of limited use if the disease is caused by strains from the Australis serogroup (Bratislava serovar).
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Validated against MAT in presence of titres of ≥1/100: poor sensitivity for chronic infections. Does not distinguish between infected and vaccinated animals.
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DIAGNOSIS
Cell culture is the only test that allows the serovar involved to be identified, but it is a complicated, slow, and tedious procedure. For this reason, it is advisable to: ◗ Use it in combination with a quick test, immunofluorescence (IF) or PCR, to reach a positive or negative diagnosis of leptospirosis. ◗ Send serum samples for a microscopic agglutination test (MAT), to determine the serogroup involved. The serogroup will usually be the one with the highest titres. The spectrum of strains that should be selected for MAT are representatives of strains that are adapted to the pig (Bratislava, Pomona, and Tarassovi), and strains that are not adapted but that circulate in the country or region (e.g. the Castellonis, Icterohaemorrhagiae, Canicola, and Hardjo serovars in Spain).
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Figure 2. Positive IF for homogenised foetal viscera.
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Diagnosis
Leptospirosis
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Figure 3. Different degrees of agglutination in a culture of the Pomona serogroup, using MAT.
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DIAGNOSIS
Objective of diagnosis Animals with fever, jaundice, haemoglobinuria
Observation of the herd
No Vaginal discharge and infertility
Yes
No Abortions, increase in mummified piglets, stillbirths, and weak, non-viable piglets
Yes
Paired serum samples for MAT Seroconversion: 4-fold increase in titre of a particular serogroup between the first and second sample 53
Serum for MAT or ELISA testing for the Bratislava serovar Genital swabs for PCR or IF
Serum samples for MAT Placenta, viscera from 3–4 foetuses for PCR or IF
Acute leptospirosis caused by the serogroup with the highest titres
–/–: no leptospirosis +/+: active infection with the Bratislava or Muenchen serovars and sows carrying the infection in the genital tract; active foetal infection and chronic infection of the sow by the leptospirosis serogroup with the highest titres +/–: possible old contact. See titres and review PCR –/+: suspect. Review antigens included in the MAT Figure 4. Leptospirosis: diagnosis of the different clinical pictures.
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Diagnosis
Leptospirosis
Diagnosis of acute leptospirosis Acute leptospirosis is caused by infection with some serovars of the Pomona serogroup and accidental serovars (adapted to other species). Diagnosis is relatively straightforward because a first serum sample can be taken from the affected animals as soon as signs are first seen, and a second at 14â&#x20AC;&#x201C;20 days. These paired samples are examined using MAT to detect any seroconversion. The serogroup with the highest titres and most seroconversion is usually the serogroup responsible. If the affected animals have died, there is also usually a large number of leptospires in almost all tissues, and these are easily detected by IF, PCR, and even by cell culture.
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However, even if serology is negative for the most common serogroups (Icterohaemorrhagiae, Canicola, Grippotyphosa, Pomona, etc.), leptospirosis disease still cannot be ruled out. Representative strains of all the serogroups would have to be included in the selection of MAT antigens in order to rule out leptospirosis.
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DIAGNOSIS
Diagnosis of abortions, stillbirths, and non-viable piglets at birth Samples of the placenta and foetal viscera should be sent to the laboratory. This should be from several foetuses as foetal infection is sequential and not all foetuses may be infected when abortion occurs. Serum from animals that abort, or give birth to small litters or weak, non-viable piglets, should also be submitted. Diagnosis of accidental serovars is relatively simple: there is a high level of leptospires in foetal tissue, and this is easily detected by IF or PCR. MAT does not detect seroconversion, as the antibody titre has stabilised or may be decreasing when abortion occurs. Despite this, the antibody titre is normally still high (>1/300) when abortion takes place.
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Diagnosis is more difficult if reproductive problems are caused by adapted serovars, because there are fewer leptospires in the tissues. However, IF and PCR are still useful for detecting leptospires in foetal tissues. The usefulness of MAT depends on the serogroup/serovar involved: â&#x2014;&#x2014; Pomona: a high antibody titre is usually detected at the time of abortion. â&#x2014;&#x2014; Bratislava: MAT is capable of detecting low titres if present. However, no antibodies at all may be found in a third of the animals affected at the time of abortion.
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Diagnosis
Leptospirosis
Diagnosis of infertility Do I have leptospirosis on my farm? If I do, is it active? Infertility is caused by infection with strains of the Australis serogroup (Bratislava and Muenchen serovars). The main problem in these cases is that immune response to these serovars is poor and titres can drop quickly. It is advisable to consider MAT titres as low as 1/10 to be potential positives in these cases. Serum from 10 % of the farm’s sows, or a minimum of 10 serum samples from farms with <100 animals, should be sent for testing. An equal number of samples should be taken from each group of sows. Serum samples should also be sent from all boars.
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The results should be evaluated to determine the serological profile. Infection is considered active if ≥10 % of the animals present with titres ≥1/100. If animals with any detectable antibody titre (≥1/10) are taken to be seropositive, various patterns of infection can be envisaged. Depending on the results, swabs of any vaginal discharge from repeating sows should be sent to the laboratory, with no transport medium, for PCR.
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2nd
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≥5th
% Seropositive with titre ≥1/100
% Seropositive
DIAGNOSIS
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0
Figure 5. Recent active infection. High percentage of seropositive animals, regardless of parity, a high proportion of which has titres ≥1/100.
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Leptospirosis
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Diagnosis
% Seropositive
4
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0
Figure 6. Endemic active infection. High percentage of seropositive animals in first and second parity sows, a high proportion of which has titres ≥1/100.
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≥5th
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% Seropositive with titre ≥1/100
% Seropositive
DIAGNOSIS
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Figure 7. Recent inactive infection. (a) Relatively high percentage of new seropositive sows in the first and second parity groups, while the percentage of animals with titres ≥1/100 is very low. There is a low percentage of seropositive animals in the higher parity groups, and the percentage of animals with titres ≥1/100 is also very low in these groups. (b) Very low percentage of seropositive first and second parity sows, although a high percentage of these has titres ≥1/100. There is a low percentage of seropositive animals in the higher parity groups, and the percentage of animals with titres ≥1/100 is also very low in these groups.
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