Leptospirosis in domestic animals in France: serological results from 1988 to 2007.

Leptospirosis is a common infection in domestic animals. The microscopic agglutination test (MAT) is used for serological diagnosis. From 1988 to 2007, the Leptospira Medical and Molecular Bacteriology Laboratory at the Nantes National College of Veterinary Medicine, Food Science and Engineering used the MAT to test serum samples from more than 40,000 cattle, 40,000 pigs, 20,000 horses and 9,500 dogs. Five Leptospira serogroups were prominent, with specific variations within the four animal species: Icterohaemorrhagiae, Australis, Sejroë, Grippotyphosa and Autumnalis. The prevalence and incidence of each serogroup varied for each species over the 20-year period: some serogroups were emergent during some years but disappeared later. This study reports the complex epidemiological features of leptospirosis.

La leptospirose est une infection courante affectant les animaux domestiques. Le diagnostic sérologique est réalisé au moyen du test d'agglutination microscopique (MAT). De 1988 à 2007, le Laboratoire de l'Unité de recherche de bactériologie médicale et moléculaire des leptospires (École nationale vétérinaire, agroalimentaire et de l'alimentation de Nantes) a utilisé cette épreuve pour tester des échantillons de sérum prélevés sur plus de 40 000 bovins, 40 000 porcs, 20 000 chevaux et 9 500 chiens. Cinq sérogroupes de Leptospira étaient majoritairement présents, à savoir Icterohaemorrhagiae, Australis, Sejroë, Grippotyphosa et Autumnalis, avec des variations en fonction de l'espèce. La prévalence et l'incidence des différents sérogroupes dans chacune des quatre espèces ont présenté des fluctuations au cours des vingt années de l'étude, certains sérogroupes ayant émergé pendant quelques années, puis régressé. Cette étude fait état de la complexité des caractéristiques épidémiologiques de la leptospirose.

La leptospirosis es una infección frecuente en los animales domésticos, para cuyo diagnóstico serológico se utiliza la prueba de aglutinación microscópica. Entre 1988 y 2007, el Laboratorio de Bacteriología Médica y Molecular de las Leptospiras de la Facultad Nacional Veterinaria, Agroalimentaria y de Alimentación Nantes-Atlántico aplicó dicha prueba al análisis de muestras séricas de más de 40 000 vacunos, 40 000 cerdos, 20 000 caballos y 9 500 perros. Se observó que predominaban cinco serogrupos de Leptospira, con variaciones específicas dentro de las cuatro especies animales: Icterohaemorrhagiae, Australis, Sejroë, Grippotyphosa y Autumnalis. En cada especie, la prevalencia y la incidencia de uno u otro serogrupo fueron variando a lo largo de esos veinte años: algunos serogrupos aparecían durante unos años para desaparecer después. En este estudio, la autora da cuenta de las complejas características epidemiológicas de la leptospirosis.

Diagnosis algorithm for leptospirosis in dogs: disease and vaccination effects on the serological results.

Leptospirosis is a common disease in dogs, despite their current vaccination. Vet surgeons may use a serological test to verify their clinical observations. The gold standard is the Microscopic Agglutination Test (MAT). After infection, the dog produces agglutinating antibodies against the lipopolyosidic antigens shared by the infectious strain but also, after vaccination, against the lipopolyosidic antigens shared by the serovars used in the bacterins (Leptospira species serovars Icterohaemorrhagiae and Canicola in most countries). MATs were performed in a group of 102 healthy field dogs and a group of 6 Canicola-challenged dogs. A diagnosis algorithm was constructed based on age, previous vaccinations, kinetics of the agglutinating antibodies after infection or vaccination and the delay after onset of the disease. This algorithm was applied to 169 well-documented sera (clinical and vaccine data) from 272 sick dogs with suspected leptospirosis. Totally, 102 dogs were vaccinated according to the usual vaccination scheme and 30 were not vaccinated. Leptospirosis was confirmed by MAT in 37/102 (36.2 per cent) vaccinated dogs and remained probable in 14 others (13.7 per cent), thus indicating the permanent exposure of dogs and the weakness of the protection offered by the current vaccines to pathogenic Leptospira.

Synthetic peptide issued from Hap1/LipL32 for new early serodiagnosis of human leptospirosis.

Leptospirosis is a worldwide zoonosis. Today, serological diagnosis is generally assessed by MAT. We performed ELISA with a synthetic peptide derived from Hap1/lipL32 which is a protein expressed only by pathogenic Leptospira. Repeatability and thresholds were defined with 85 controls sera and 119 hospitalized leptospirosis. The PP-ELISA repeatability and IgM/IgG cut-off values were based on control sera. For these cut-off values, we observed the IgM-PP-ELISA specificity of 89%, whereas it was 100% for the IgG. Then, we compared PP-ELISA and standard MAT results for leptospirosis patients. The concordance rate for IgM-PP-ELISA and MAT was low (43%), whereas it was 85% for IgG-PP-ELISA and MAT. During the first 5 days after hospitalization, PP-ELISA gave positive results in 13 out of 16 patients (81%) whereas 8 out of 14 patients (57%) were positive to MAT. ELISA using Hap1/lipL 32-derived synthetic peptide PP is an earlier serological diagnosis of human leptospirosis than MAT.

Leptospira exposure in the human environment in France: A survey in feral rodents and in fresh water.

This paper confirms the important role of rodents to be maintenance hosts of leptospires. Their role is related to renal carriage and shedding of leptospires into urine, thus contaminating fresh water. Serological and carriage of feral rodents trapped in France were determined by MAT and hap1PCR specific for pathogenic leptospires. In same areas, fresh water samples were analyzed by hap1PCR. The overall seroprevalence was 44% in 649 rodents and was similar regardless of the species. Seroprevalence for leptospirosis is about 20-53% according to species. hap1PCR (516 kidneys) showed that renal carriage was higher in brown rats (34.7%) and muskrats (15.8%) than in coypus (3.3%). hap1PCR demonstrates a significative difference (P-value > 10(-12)) for the renal carriage between the different species: muskrats and rats are more efficient maintenance hosts than coypu but all infect water. Moreover 5/38 water samples associated with human cases were hap1PCR positive and 1/113 in controlled waters.

[Seroprevalence of Lyme Borreliosis and tick-borne encephalitis in workers at risk, in eastern France]. / Séroprévalence de la borréliose de Lyme et de l'encéphalite à tiques chez des professionnels exposés dans le Grand est de la France.

OBJECTIVE: The aim of this article was to assess the seroprevalence of Lyme Borreliosis and tick-borne encephalitis (TBE) among occupationally exposed forest workers. METHODS: Workers exposed to tick bites in Eastern France were interviewed by occupational health physicians of the mutualité sociale agricole (MSA) on their sociodemographic features, their occupational activity, their last tick bite, their clinical history, and their means of prevention. Blood sampling was carried out for antibody detection. RESULTS: Among the 2975 subjects included in the study, the observed seroprevalence was 14.1% for Lyme borreliosis and 3.4% for TBE. Age, occupational activity, and place of residence significantly influenced the serological status of Lyme borreliosis. The seroprevalence was significantly higher among woodcutters (17.5%) than among other occupational categories (p<.001). Seroprevalence in Alsace (26.9%) and Lorraine (16.5%) were significantly higher than in other regions (p<0.001 and p<0.01, respectively). The seroprevalence of TBE was significantly higher in Alsace (5.5%; p<0.001). The rates of seroprevalence for both infections varied according to forest areas. The multifactorial analysis of prevention practices revealed three types of behaviors as far as protection was concerned: "rigorous", "partial", or "insufficient". CONCLUSION: These results do not change the present French indications for use of TBE vaccine. They highlight the importance of information on these diseases and the need for further studies on microbial ecology and risk-factors identification.

Protection against Leptospira interrogans sensu lato challenge by DNA immunization with the gene encoding hemolysin-associated protein 1.

The use of DNA constructs encoding leptospiral proteins is a promising new approach for vaccination against leptospirosis. In previous work we determined that immunization with hemolysis-associated protein 1 (Hap1) (LipL32) expressed by adenovirus induced significant protection against a virulent Leptospira challenge in gerbils. To avoid the use of the adenovirus vector, we checked for clinical protection against lethal challenge by DNA vaccination. A DNA vaccine expressing Hap1 was designed to enhance the direct gene transfer of this protein into gerbils. A challenge was performed 3 weeks after the last immunization with a virulent strain of serovar canicola. Our results show that the cross-protective effect with pathogenic strains of Leptospira, shared by Hap1, could be mediated by the DNA plasmid vector. This finding should facilitate the design and development of a new generation of vaccines against bacteria, particularly Leptospira interrogans sensu lato.

[Efficacy of Spirolept vaccine against human leptospirosis as estimated by passive protection of laboratory rodents]. / Etude de l'efficacité du vaccin Spirolept contre la leptospirose par la protection passive de rongeurs de laboratoire.

OBJECTIVE: This study had for aim to assess the serological response induced by the Spirolept vaccine against human leptospirosis. METHOD: A serological follow-up was made on 31 patients at a risk of occupational exposure. The antibody titers of vaccinated patients were assessed by MAT and ELISA. In a second step, vaccinal protection was studied in vivo by checking the seroprotective effect of the human sera injected in an animal model (Meriones unguiculatus) naturally susceptible to the disease. The passive protection was studied by comparing the death rate on five batches of animals to which the bacterium was inoculated. Thus, four batches of animals were injected subcutaneously with a pooled sera of vaccinated people sampled at D0, D15, D135, and D320 after Spirolept vaccination. One control batch was given PBS. One day after injection, the latter batch was inoculated with the homologous strain Verdun of Leptospira interrogans ss icterohemorrhagiae (serogroup Icterohemorrhagiae) used to make the vaccine. RESULTS: The death rate was significantly decreased as soon as D15 after the first injection, even with pooled sera of vaccinated people negative for the MAT. COMMENTS: The Spirolept vaccine induces a protective response against icterohemorrhagiae, which can be transmitted to the animal model and thus is linked to a humoral response.

Comparison of the efficacy of three commercial bacterins in preventing canine leptospirosis.

Twenty-four specific pathogen-free beagles were randomly allocated into four groups (three vaccinated groups and one control group) and inoculated at nine and 12 weeks of age with one of three commercial inactivated Leptospira vaccines: A (Vanguard 7; Pfizer Santé Animale), B (Dohyvac 7L; Fort Dodge), and C (Nobivac DHPPi + Lepto; Intervet International); the control group received Nobivac DHPPi (Intervet International). Seven weeks after the second vaccination all the dogs were challenged with Leptospira interrogans serogroup canicola. All the vaccinated dogs developed a mild serological response (microscopic agglutination titres) after the booster vaccination. A significant serological response after the challenge was observed, particularly in the controls. The challenge induced fever and clinical disorders in the control group, whereas in the vaccinated groups the clinical signs were mild. Blood cultures became positive in all control dogs, and in one of six dogs vaccinated with vaccine A and two of four dogs vaccinated with vaccine B; none of the six dogs vaccinated with vaccine C was leptospiraemic at any stage of the experiment. Urine cultures were positive in all the control dogs two weeks after the challenge. One of six dogs vaccinated with vaccine A and two of four dogs vaccinated with vaccine B shed bacteria in their urine after the challenge, but none of the dogs vaccinated with vaccine C shed bacteria in their urine at any time during the experiment.

Clustered cases of leptospirosis in Rochefort, France, June 2001.

Five clustered cases of leptospirosis were diagnosed in the area of Rochefort, France, in June 2001, among teenagers who had swum in the Genouillé canal. The symptoms included fever, headache, abdominal pain and vomiting, chills and myalgia. Three cases were confirmed by PCR and serology. The mean cumulative duration of bathing was significantly higher in cases (23.8 hours) compared to controls (14.4 hours). No other particular risk factor was observed. The environmental investigation revealed the presence of rodents excreting of leptospires near the bathing area. For all antigens considered, the occurence of seropositive rodents was 30.8%, L. icterohaemorrhagiae being the predominant serogroup (23,1%).

Role of the coypu (Myocastor coypus) in the epidemiology of leptospirosis in domestic animals and humans in France.

The coypu (Myocastor coypus), a rodent whose natural habitat is stagnant freshwater, has become a widespread pest in France within the last decade. This study investigated the prevalence of seropositivity and the renal carriage of leptospires in coypus in order to evaluate their role in terms of the risk of infection by Leptospira interrogans in domestic animals and humans. The study involved the application of serological and bacteriological methods to identify leptospires infection and/or carriage in 738 coypus trapped from 1996 to 1999 in six areas of France. Seroprevalence in samples ranged from 16.5 to 66%, and three field strains were isolated (two L. interrogans Icterohaemorrhagiae and one L. interrogans Sejroe). This first report on the isolation of leptospires from coypus in France emphasises the role of this animal in the epidemiology of leptospirosis.

Identification of the hemolysis-associated protein 1 as a cross-protective immunogen of Leptospira interrogans by adenovirus-mediated vaccination.

New vaccine strategies are needed for the prevention of leptospirosis, a widespread human and animal disease caused by pathogenic leptospires. Our previous work determined that a protein leptospiral extract conferred cross-protection in a gerbil model of leptospirosis. The 31- to 34-kDa protein fraction of Leptospira interrogans serovar autumnalis was shown sufficient for this purpose. In the present study, N-terminal sequencing of a 32-kDa fraction and Southern blotting of genomic DNA with corresponding degenerated oligonucleotide probes identified two of its constituents: hemolysis-associated protein 1 (Hap1) and the outer membrane Leptospira protein 1 (OmpL1). Adenovirus-mediated Hap1 vaccination induces significant protection against a virulent heterologous Leptospira challenge in gerbils, whereas a similar OmpL1 construct failed to protect the animals. These data indicate that Hap1 could be a good candidate for developing a new generation of vaccines able to induce broad protection against leptospirosis disease.

Antimicrobial activity of enrofloxacin against Staphylococcus intermedius strains isolated from canine pyodermas.

This study examined and compared the minimal inhibition concentrations (MICs) of enrofloxacin against 393 Staphylococcus intermedius strains isolated in France from canine pyodermas during three different years, 1995 (174 isolates), 1997 (101 isolates) and 1999 (118 isolates). The MICs of enrofloxacin against these strains ranged from 0.063 to 64 mg L-1, with MIC50 and MIC90 equal to 0.125 and 0.25 mg L-1, respectively. Two resistant strains were found, but only among isolates collected in 1999. The data show that resistance to enrofloxacin among S. intermedius strains is still rare in dogs, but the selection in vitro of variants in which the MICs were increased 4-16-fold after 10 serial passages in subinhibitory concentrations of enrofloxacin suggests that inappropriate use might favour the development of resistant strains in vivo.

Binding of rabbit hemorrhagic disease virus to antigens of the ABH histo-blood group family.

The ability of rabbit hemorrhagic disease virus to agglutinate human erythrocytes and to attach to rabbit epithelial cells of the upper respiratory and digestive tracts was shown to depend on the presence of ABH blood group antigens. Indeed, agglutination was inhibited by saliva from secretor individuals but not from nonsecretors, the latter being devoid of H antigen. In addition, erythrocytes of the rare Bombay phenotype, which completely lack ABH antigens, were not agglutinated. Native viral particles from extracts of infected rabbit liver as well as virus-like particles from the recombinant virus capsid protein specifically bound to synthetic A and H type 2 blood group oligosaccharides. Both types of particles could attach to adult rabbit epithelial cells of the upper respiratory and digestive tracts. This binding paralleled that of anti-H type 2 blood group reagents and was inhibited by the H type 2-specific lectin UEA-I and polyacrylamide-conjugated H type 2 trisaccharide. Young rabbit tissues were almost devoid of A and H type 2 antigens, and only very weak binding of virus particles could be obtained on these tissues.

Evidence of cross-protection within Leptospira interrogans in an experimental model.

Killed whole-cell preparations were used as bacterins against leptospirosis. As this type of protection is considered to be serogroup-specific, several serogroups were added to the usual vaccines, and the most pathogenic serovar was chosen for each group. Different leptospire extracts were evaluated for their protective capacity against acute lethal leptospirosis in gerbils (Meriones unguiculatus). Total extracts induced complete protection against homologous challenges and partial protection against heterologous challenges. LPS fractions protected against homologous but not heterologous challenges, whereas protein extract induced significant protection against both types of challenge. Thus, cross-protection within L. interrogans was related to the protein extract.

Chronic hepatitis associated with leptospiral infection in vaccinated beagles.

Sixteen juvenile Beagle dogs originating from a single breeding colony and regularly vaccinated against Leptospira interrogans (serogroups Canicola and Icterohaemorrhagiae) developed a clinical syndrome characterized by retarded growth, weight loss and often ascites. Over a 10-month period, post-mortem examinations were performed on all affected dogs. Gross lesions were confined to the liver which was often firm, tan-coloured and mottled. Microscopically, hepatic lesions ranged from those of severe chronic hepatitis to mild diffuse hepatocellular vacuolation, with bile stasis, occasional scattered lymphocytic aggregates and haemosiderin granulomas. Special stains and electron microscopy revealed spirochaetes within bile canaliculi. The genus Leptospira was recognized by immunohistochemical methods in nine dogs. Leptospires were isolated from six dogs, but serological tests failed to detect significant titres of antibody to L. interrogans in these animals. A serological survey of 37 kennelmates demonstrated that 20 dogs had high titres of serogroup Australis leptospiral antibody, which could not have resulted from vaccination. These findings strongly suggest a connection between the presence of leptospires and the hepatic lesions.

Detection of antibodies to rabbit haemorrhagic disease virus: an immunoblotting method using virus-coated human erythrocyte membranes.

The virus of rabbit haemorrhagic disease (RHDV) was purified from infected rabbit liver homogenate by using its property to bind to human red blood cells. Lysates from virus coated cells contained a 60 kDa protein identified as the major viral protein. Immunoblots prepared with that preparation were proved to be useful for immunochemical analysis since the 60 kDa component was intensively stained by subsequent incubation with rabbit sera from infected rabbits and with a secondary labelled antibody. The sera from 114 rabbits were analysed with this test and the data were compared with those obtained by using the haemagglutination inhibition test (HIT). Among the 114 field sera tested by Western blot, 86 contained antibodies to the 60 kDa RHDV antigen whereas only 76 showed positive reaction by HIT. The sensitivity and the specificity of the Western blot were 0.85 and 0.45, respectively, with a concordance between the two techniques of 0.72. Additionally, the European brown hare syndrome virus antibodies reacted with the 60 kDa RHDV protein on immunoblots.

Characterization of group EF-4 bacteria from the oral cavity of dogs.

Samples from gingival scrapings of dogs were examined for the presence of CDC Groups EF-4 bacteria. Isolation procedures were performed in 5% sheep blood agar supplemented with thiostrepton and trimethoprim (10 mg/l). Fifty nine EF-4 strains were isolated from 92% of 49 dogs. Among the Group EF-4 bacteria, the majority of isolates belonged to the arginine-negative (biovar "b") Group EF-4 (42 strains recovered in 82% of dogs). Seventeen arginine-positive strains (biovar "a") were recovered only from 35% of dogs. The strains were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The analysis of electrophoretic protein pattern of these bacteria supported the results of conventional testing, confirmed the distinction between the biovars "a" and "b" of Group EF-4 and supported the division of biovar EF-4b into two subgroups of either producing or non-producing acid from gluconate.

Partial characterization of the human erythrocyte receptor for rabbit haemorrhagic disease virus.

An important, well known property of the rabbit haemorrhagic disease virus is its ability to agglutinate human red blood cells. Accordingly, red cells from human adult donors were agglutinated despite their blood group ABO status, and treatments with proteases or glycosidases did not prevent agglutination. However, we discovered that the cells from human umbilical cords or foetuses were not agglutinated. In order to identify the viral receptor on human erythrocytes, glycolipids and glycoproteins from adult red cells were separated and tested for their potency in inhibiting agglutination. The bulk of the biological activity was associated with the highly glycosylated glycolipids (polyglycosylceramides), whereas a lower but significant activity was also associated with neutral glycolipids. No activity was found in the lipid-free sialoglycoprotein fractions. All these data strongly suggest that the RHDV receptor on human red cells corresponds to a development antigen which is not expressed on foetal cells and is mainly carried by glycolipids. Faint activity was also found in membranes from sheep red cells, suggesting that a similar glycolipid component is carried by these animal cells.

Fatal disease mimicking leptospirosis in a dog, caused by Aeromonas hydrophila.

A dog was treated for leptospirosis on clinical and epidemiological arguments. The amoxicillin treatment was not successful. Pure culture of Aeromonas hydrophila was then obtained from liver and kidney, indicating that the septicemia was due to this bacteria commonly found in waters.

Recognition of Leptospira interrogans antigens by vaccinated or infected dogs.

Antigenic recognition of leptospiral antigens by vaccinated or infected dogs was studied by microagglutination test (MAT) and by western blots. In western blots, serovar specific antigens detected by MAT migrated in the 18-31 kDa zone. The 25-31 zone seemed to be linked to antigens indicating virulence of the strain. These antigens are LPS. The first antibodies made after infection are produced against LPS migrating in the 14 kDa zone. Many protein antigens are common in leptospires belonging to different serogroups. Virulent strains exhibited specific antigens in the 45 and 32-34 kDa zones.