Re-evaluating the LD50 requirements in the codified potency testing of veterinary vaccines containing Leptospira (L.) serogroup Icterohaemorrhagiae and L. serogroup Canicola in the United States.

Approximately one-third of the reportable USDA Category D and E laboratory animals in the United States are expended on the potency testing of leptospiral vaccines by the codified hamster vaccination-challenge assay. Valid tests require ≥80% of challenge controls to succumb to disease and an LD50 between 10 and 10,000. This work evaluates the risk associated with the removal of LD50 limits; thereby, eliminating back-titration hamsters from in vivo potency assays for Leptospira (L.) serogroups Canicola and Icterohaemorrhagiae. The impact was assessed through 1) retrospective analysis of industry and CVB serial release data from July 2011-April 2015 and 2) evaluation through vaccination-challenge assays. For the initial vaccination-challenge assays (n = 3/serogroup), one group received potent bacterin (PB) and six groups received subpotent bacterins (SB1-SB6). PB and SB1 were challenged with a single dilution of Leptospira between 10 and 10,000 LD50. SB2-SB6 received serial dilutions of more concentrated challenge. Based on the retrospective analysis and in vivo assays, 80% of the challenge controls succumbing to disease reasonably ensured the minimal LD50 was administered. Subpotent vaccines were not at increased risk for being deemed potent when challenged with >10,000 LD50, but potent vaccines were at risk of being deemed subpotent when challenged with >10,000 LD50.

Highly Pathogenic Leptospira Found in Urban Brown Rats (Rattus norvegicus) in the Largest Cities of Sweden.

Leptospirosis is an emerging zoonosis of global concern; however, its contemporary occurrence in Sweden, a European country partly located north of the Arctic Circle, is poorly known. Four out of 30 brown rats, captured within urban districts in Sweden, were found to be positive for antibodies to Leptospira interrogans serovar Icterohaemorrhagiae. This serovar causes Weil's disease in humans, a severe infection with jaundice, renal failure, and hemorrhage. Our study is the first finding of this highly pathogenic serovar in Swedish rats since the 1930s.

Role of 72 kDa protein of Leptospira interrogans as a diagnostic marker in acute leptospirosis.

BACKGROUND & OBJECTIVES: Leptospirosis is a widespread zoonotic disease and a public health problem, particularly in tropical and subtropical countries. Varied clinical manifestations of the disease frequently lead to misdiagnosis resulting in life-threatening multi-organ complications. Therefore, early laboratory investigation using an appropriate diagnostic approach is crucial. In the present study, a potential protein marker was identified and evaluated for its usefulness in the serodiagnosis of acute leptospirosis. METHODS: Leptospira interrogans serovar Icterohaemorrhagiae (L44), which represents a commonly prevalent serovar in Malaysia, was cultivated for preparation of sequential protein extract (SEQ). SDS-PAGE and immunoblotting were performed with a serum panel comprising confirmed cases of leptospirosis and controls (n=42 each). Identification and characterization of the highest scoring protein from the antigenic band was performed. Subsequently based on the nucleotide coding sequence of the protein, the corresponding recombinant protein was custom-produced. It was then evaluated for sensitivity and specificity by testing against 20 serum samples from leptospirosis patients and 32 from controls. RESULTS: Among the antigenic components, a 72 kDa protein band demonstrated significant sensitivity (83.3%) and specificity (95.2%) for the detection of specific anti-leptospiral IgM antibodies. The protein was identified by mass-spectrometry analysis as heat shock protein DnaK of L. interrogans. Recombinant form of the protein (r72SEQ) showed 85 per cent sensitivity and 81 per cent specificity for the detection of specific anti-leptospiral IgM antibodies. INTERPRETATION & CONCLUSIONS: The findings of our study indicate that a protein (72 kDa) of L. interrogans has the potential utility of being used for the diagnosis of acute leptospirosis. Further studies need to be done to confirm these findings.

[Punt-driver with jaundice - case 1/2014]. / Stocherkahnfahrer mit Ikterus - Fall 1/2014.

HISTORY AND ADMISSION FINDINGS: A 30-year-old student, who worked part-time as a punt-driver, was admitted to the hospital with fever up to 39.5 °C, severe pain in in his lower extremity and the lower back, nausea and jaundice. INVESTIGATIONS: Physical examination showed jaundice of skin and sclera as well as conjunctivitis of both eyes. Blood examination results indicated high levels of bilirubin (mostly conjugated), C-reactive protein and creatinine. There were no pathological findings in the ultrasound examination except of discrete splenomegaly. Serology revealed Leptospira IgM antibodies. DIAGNOSIS, TREATMENT AND COURSE: The patient was diagnosed with leptospirosis and was treated with intravenous ceftriaxon, intravenous rehydration and symptomatic analgesia. Upon this treatment, the liver and kidney function recovered and the patient could be discharged from the hospital in a good general condition. CONCLUSIONS: Leptospirosis is a zoonosis which is mainly transmitted by urine of infected animals (predominantly rodents). In this case, the disease was presumably transmitted during the patients work as a professional punt-driver on the Neckar River. The course of the disease is mostly mild with flu-like symptoms, but there are also serious courses with live-threatening complications such as liver or kidney failure and an associated high mortality rate.

Development of Leptospira in vitro potency assays: EU/industry experience and perspectives.

Nobivac® Lepto (MSD Animal Health) is a non-adjuvanted canine leptospirosis vaccine containing inactivated whole cells of Leptospira interrogans serogroup Canicola serovar Portlandvere and L. interrogans serogroup Icterohaemorrhagiae serovar Copenhageni. The current standard in vivo potency test is a hamster challenge test associated with major drawbacks such as animal suffering and poor reproducibility. Here, the quantification of antigenic mass by ELISA as a new in vitro potency test is described, supporting the 3Rs concept (replacement, reduction, and refinement of animal tests) and in accordance with European Pharmacopoeia Monograph 0447 (Canine Leptospirosis Vaccine [Inactivated]). The two corresponding sandwich ELISAs are based on monoclonal antibodies specific for immunodominant leptospiral lipopolysaccharide epitopes. Protection in passive immunization experiments demonstrate that these monoclonal antibodies recognize key protective antigens in currently licensed human and veterinary whole cell Leptospira vaccines. The high precision and robustness renders the two ELISAs much more reliable correlates of potency in dogs than the hamster potency test. The recent approval of these assays for a new canine leptospirosis vaccine is an important contribution to the 3Rs in quality control testing of Leptospira vaccines.

Detection of reactive canines to Leptospira in Campeche City, Mexico.

Leptospira reactivity in stray and household dogs in Campeche as well as associated risk factors to the seropositivity in household dogs have been herein determined. The survey included 323 dogs, 142 of which were stray dogs and 181 household dogs. Nine Leptospira interrogans serovars were tested by the microagglutination test. Reactivity was 21.3 % (69/323), 17.2 % corresponded to household dogs and 26.7 % to stray dogs. Leptospira Canicola (29 %), Leptospira Hardjo (22.58 %), and Leptospira Icterohaemorrhagiae (16.12 %) were the most common serovars reacting against the serum of household animals, while Leptospira Canicola (15.78 %), Leptospira icterohaemorrhagiae (13.15 %), and Leptospira Pomona (7.89 %) were those reacting in stray dogs. Results showed that all dogs have been in contact with different Leptospira serovars and outdoor exposure is the main infection risk factor.

A prime-boost strategy using the novel vaccine candidate, LemA, protects hamsters against leptospirosis.

Toward developing an effective vaccine capable of conferring heterologous protection, the putative lipoprotein LemA, which presents an M3 epitope similar to that of Listeria, was evaluated as a vaccine candidate in the hamster model of leptospirosis. LemA is conserved (>70% pairwise identity) among the pathogenic Leptospira spp., indicating its potential in stimulating a cross-protective immune response. Using different vaccination strategies, including prime-boost, DNA vaccine, and a subunit preparation, recombinant LemA conferred different levels of protection in hamsters. Significant protection against mortality was observed for the prime-boost and the DNA vaccine strategies, which showed 87.5% (P < 0.01) and 62.5% (P < 0.05) efficacy, respectively. Although the subunit vaccine preparation protected 50.0% of immunized hamsters, the level of protection was not significant. None of the hamsters in the control groups survived challenge with a virulent strain of Leptospira interrogans serogroup Icterohaemorrhagiae. Characterization of the immune response found that the strongest antibody response was stimulated by the subunit vaccine preparation, followed by the prime-boost strategy. The DNA vaccine failed to elicit an antibody response in immunized hamsters.

Detection of reactive canines to Leptospira in Campeche City, Mexico / Detection of reactive canines to Leptospira in Campeche City, Mexico.

Leptospira reactivity in stray and household dogs in Campeche as well as associated risk factors to the seropositivity in household dogs have been herein determined. The survey included 323 dogs, 142 of which were stray dogs and 181 household dogs. Nine Leptospira interrogans serovars were tested by the microagglutination test. Reactivity was 21.3

corresponded to household dogs and 26.7

), and Leptospira Icterohaemorrhagiae (16.12

) were the most common serovars reacting against the serum of household animals, while Leptospira Canicola (15.78

), and Leptospira Pomona (7.89

) were those reacting in stray dogs. Results showed that all dogs have been in contact with different Leptospira serovars and outdoor exposure is the main infection risk factor.

Detection of reactive canines to Leptospira in Campeche City, Mexico. / Detection of reactive canines to Leptospira in Campeche City, Mexico.

Leptospira reactivity in stray and household dogs in Campeche as well as associated risk factors to the seropositivity in household dogs have been herein determined. The survey included 323 dogs, 142 of which were stray dogs and 181 household dogs. Nine Leptospira interrogans serovars were tested by the microagglutination test. Reactivity was 21.3

(69/323), 17.2

corresponded to household dogs and 26.7

to stray dogs. Leptospira Canicola (29

), Leptospira Hardjo (22.58

), and Leptospira Icterohaemorrhagiae (16.12

) were the most common serovars reacting against the serum of household animals, while Leptospira Canicola (15.78

), Leptospira icterohaemorrhagiae (13.15

), and Leptospira Pomona (7.89

) were those reacting in stray dogs. Results showed that all dogs have been in contact with different Leptospira serovars and outdoor exposure is the main infection risk factor.

Seroprevalence of major bovine-associated zoonotic infectious diseases in the Lao People's Democratic Republic.

Bovine-associated zoonotic infectious diseases pose a significant threat to human health in the Lao People's Democratic Republic (Lao PDR). In all, 905 cattle and buffalo serum samples collected in northern Lao PDR in 2006 were used to determine seroprevalence of five major bovine zoonotic infectious diseases that included Taenia saginata cysticercosis, bovine tuberculosis, Q-fever, bovine brucellosis, and bovine leptospirosis. Five enzyme-linked immunosorbent assays (ELISAs) were used to test for the presence of antibodies to the diseases, except Taenia saginata, for which we tested for the presence of Taenia metacestode circulating antigens. The overall highest prevalence was for T. saginata (46.4%), with lower prevalence for Q-fever (4%), leptospirosis (3%), tuberculosis (1%), and brucellosis (0.2%). Although there were no significant differences in the proportion of seroprevalence between sex and age of the animals sampled, there were significant differences between the provincial distributions. Further studies are required to determine the seroprevalence of these infections in other locations in Lao PDR, as well as other animal species including humans, in order to develop effective prevention and control strategies. This is the first study to investigate the prevalence of bovine zoonotic infectious agents in the Lao PDR. Positivity was demonstrated for all diseases investigated, with the highest prevalence for T. saginata antigen and Coxiella burnetti antibodies. For T. saginata, there were significant differences in the provincial distribution. Approximately 16% seroprevalence of Coxiella burnetti was noted in Xayabuly Province; however, there are no clear reasons why this was the case, and further studies are required to determine risk factors associated with this observation.

Gene expression profiles of immune mediators and histopathological findings in animal models of leptospirosis: comparison between susceptible hamsters and resistant mice.

Leptospirosis is a widespread zoonosis characterized by multiple organ failure and variable host susceptibility toward pathogenic Leptospira strains. In this study, we put the role of inflammatory mediators in parallel with bacterial burdens and organ lesions by comparing a susceptible animal model, the hamster, and a resistant one, the Oncins France 1 (OF1) mouse, both infected with virulent Leptospira interrogans serovar Icterohaemorrhagiae strain Verdun. Histological observations evidenced edema, congestion, hemorrhage, and inflammatory infiltration in the organs of hamsters, in contrast to limited changes in mice. Using reverse transcription-quantitative PCR techniques, we showed that the relative Leptospira burden progressively increased in hamster tissues, while a rapid clearance was observed in mouse tissues. The early regulation of the proinflammatory mediators interleukin-1ß (IL-1ß), IL-6, tumor necrosis factor alpha, and cyclo-oxygenase-2 and the chemokines gamma interferon-inducible protein 10 kDa/CXCL10 and macrophage inflammatory protein-1α/CCL3 in mouse tissues contrasted with their delayed and massive overexpression in hamster tissues. Conversely, the induction of the anti-inflammatory cytokine IL-10 was faster in the resistant than in the susceptible animal model. The role of these cytokines in the pathophysiology of leptospirosis and the implications of their differential regulation in the development of this disease are discussed.

[Epidemiological surveillance of leptospirosis on Reunion Island in 2004-2008: possible impact of Chikungunya infection on the case fatality rate of leptospirosis]. / Surveillance épidémiologique de la leptospirose à la Réunion, 2004-2008: possible impact de l'épidémie de chikungunya sur la létalité de la leptospirose.

In 2006, increased mortality due to leptospirosis in Reunion Island had alerted the authorities and justified the conduct of this study in order to update knowledge on the epidemiology of leptospirosis, whereas the latest epidemiological data published on the disease dated back to 2003. This study followed the scheme of a descriptive retrospective survey based on data from reporting and investigation of hospitalized cases of leptospirosis that occurred in Reunion between the 1st January 2004 and 31st December 2008. Data from the National Reference Center (NRC) have also been used. The annual number of reported cases (40 to 50) was stable over the period, which contrasted with the historical decreasing trend of incidence reported by the NRC. The circumstances of exposure were those usually associated with the disease on the island: about 80% of cases were infected between January and June, during the wet season; the main exposure factor identified was the practice of agriculture, declared or not; Leptospira icterohaemorrhagiae remained the most frequently isolated serovar, although regressing. Finally, our study has shown that excess mortality observed in 2006 did not result from an increased incidence but from a rise in the case fatality rate. This could be linked to the outbreak of chikungunya, which peaked in February 2006. In endemic areas of leptospirosis, health professionals should remain aware of the risk of occurrence of fatal cases during arbovirosis outbreaks.

Onset and duration of protective immunity against clinical disease and renal carriage in dogs provided by a bi-valent inactivated leptospirosis vaccine.

Protection against clinical disease and prevention of the renal carrier state remain the key objectives of vaccination against leptospirosis in the dog. In the present paper, groups of dogs were vaccinated twice with a commercial bacterin (EURICAN L) containing Leptospira interrogans serovars icterohaemorrhagiae and canicola and challenged with heterologous representatives of both serovars at 2 weeks (onset of immunity) or 14 months (duration of immunity) after the second vaccination. Control dogs were not vaccinated against leptospirosis and kept with the vaccinated dogs. The challenges, irrespective of the serovar, reliably produced clinical signs consistent with Leptospira infection in the control pups with up to 60% mortality. As expected clinical disease in the adult controls was less severe, but we were able to induce morbidity and mortality as well. Under these extreme challenge conditions, clinical signs in the vaccinated dogs were rare, and when observed, mild and transient in nature. Following experimental infection, 100% of the control pups and 83% of the adult controls became renal carriers. Despite the heavy challenges, none of the 18 vaccinated puppies (onset of immunity studies) and only 2 out of the 16 vaccinated adult dogs (duration of immunity studies) developed a renal carrier state. These results show that a primary course of two doses of EURICAN L provided quick onset and long-term protection against both clinical leptospirosis and the renal carrier stage. This vaccine should provide veterinarians with a powerful tool to prevent clinical disease in dogs and zoonotic transmission of leptospirosis to humans.

Severe course of rat bite-associated Weil's disease in a patient diagnosed with a new Leptospira-specific real-time quantitative LUX-PCR.

Leptospirosis is a zoonotic disease with global distribution, caused by spirochaetes of the genus Leptospira. Transmission of Leptospira interrogans serovar Icterohaemorrhagiae, the causative agent of Weil's disease, to humans usually results from exposure to the urine of infected, but mostly asymptomatic, rodents, either by direct contact or indirectly through contaminated soil or water. Although regarded as a re-emerging infectious disease, human leptospirosis is probably underdiagnosed due to its often unspecific clinical appearance and difficulties in culturing leptospires. Therefore, more rapid and specific diagnostic procedures are needed. Here we describe a novel real-time quantitative PCR system developed for the accurate and fast diagnosis of pathogenic Leptospira spp. Its usefulness in the management of a patient with rat bite-associated multiorgan failure is demonstrated.

Rapid serodiagnosis of leptospirosis by latex agglutination test and flow-through assay.

PURPOSE: Diagnosis of leptospirosis facilitates patient management and initiation of therapy. The microscopic agglutination test (MAT) is the serological test used in reference laboratories because of its high degree of sensitivity and specificity. But the results are not available quickly for patient management. In the present study, in order to develop a simple, rapid immunodiagnostic assay, one of the outer membrane proteins (OMPs), recombinant LipL41 (rLipL41) has been utilised in latex agglutination test (LAT) and flow-through assay. METHODS: Part of LipL41 gene was expressed in Escherichia coli system and purified. The rLipL41 antigen of pathogenic Leptospira interrogans serovar Icterohaemorrhagiae, which is conserved in all pathogenic Leptospira spp. was used as capture antigen in the LAT and flow-through test. Both tests are very rapid and could be completed within 5 minutes. The sensitivity and specificity of rLipL41 was assessed and evaluated in LAT and flow-through assay in comparison with standard MAT. RESULTS: The sensitivity and specificity of the LAT were 89.70 and 90.45% and flow-through assay were 89.09 and 77.70%, respectively. CONCLUSIONS: The developed LAT and flow-through assays were simple, rapid and economical for the detection of leptospira infection and suitable for large-scale screening of samples in endemic areas without any sophisticated equipment.

Leptospirosis pulmonary haemorrhage: a diagnostic challenge.

Leptospirosis is a re-emerging zoonosis with worldwide distribution. Antibiotic treatment is available, but leptospirosis is rarely suspected due to the non-specific clinical presentation, and is also underreported due to the difficulty in confirming the diagnosis. The syndrome of leptospirosis pulmonary haemorrhage is increasingly described; it is severe and can present without the classical manifestations of Weil's disease. We discuss two cases of leptospirosis with pulmonary haemorrhage; both our patients experienced a dramatic deterioration hours after their admission, and required emergency intubation and intensive therapy unit support. Massive haemoptysis is a life threatening complication of leptospirosis, even outside tropical areas.

Proteome and immunome of pathogenic Leptospira spp. revealed by 2DE and 2DE-immunoblotting with immune serum.

In this study, proteomes of two pathogenic Leptospira spp., namely L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni and L. borgpetersenii, serogroup Tarassovi, serovar Tarassovi, were revealed by using two dimensional gel electrophoresis (2DE)-based-proteomics. Bacterial cells were disrupted in a lysis buffer containing 30 mM Tris, 2 M thiourea, 7 M urea, 4% CHAPS, 2% IPG buffer pH 3-10 and protease inhibitors and then subjected to sonication in order to solubilize as much as possible the bacterial proteins. The 2DE-separated components of both Leptospira homogenates were blotted individually onto membranes and antigenic components (immunomes) were revealed by probing the blots with immune serum of a mouse readily immunized with the homogenate of L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni. The immunogenic proteins of the two pathogenic Leptospira spp. could be grouped into 10 groups. These are: 1) proteins involved in the bacterial transcription and translation including beta subunit transcription anti-termination protein of DNA polymerase III, elongation factors Tu and Ts, and tRNA (guanine-N1)-methyltransferase; 2) proteins functioning as enzymes for metabolisms and nutrient acquisition including acetyl-Co-A acetyltransferase, putative glutamine synthetase, glyceraldehyde-3-phospahte dehydrogenase, NifU-like protein, 3-oxoacyl-(acyl-carrier-protein) reductase, oxidoreductase, sphingomyelinase C precursor, spermidine synthase, beta subunit of succinyl-CoA synthetase, and succinate dehydrogenase iron-sulfur subunit; 3) proteins/enzymes necessary for energy and electron transfer, i.e. electron transfer flavoprotein, and proton-translocating transhydrogenase; 4) enzymes for degradation of misfolded proteins, i.e. ATP-dependent Clp protease; 5) molecular chaperone, i.e. 60 kDa chaperonin; 6) signal transduction system, i.e. response regulator; 7) protein involved in immune evasion in host, i.e. peroxiredoxin; 8) cell structure proteins including MreB (cytoskeletal) and flagellin/ periplasmic flagellin; 9) lipoproteins/outer membrane proteins: LipL32, LipL41, LipL45 and OmpL1; and 10) various hypothetical proteins. Many immunogenic proteins are common to both Leptospira spp. These proteins not only are the diagnostic targets but also have potential as candidates of a broad spectrum leptospirosis vaccine especially the surface exposed components which should be vulnerable to the host immune effector factors.

OmpL1 DNA vaccine cross-protects against heterologous Leptospira spp. challenge.

Available leptospirosis vaccines made up of inactivated bacteria or their membrane components elicit immunity which is serovar specific and unsatisfactory immunological memory. A vaccine that protects across Leptospira serogroups/serovars, i.e. broad spectrum, and induces long-lasting memory is needed for both human and veterinary uses. In this study, a plasmid DNA vaccine was constructed from cloning gene encoding a transmembrane porin protein, OmpL1, of pathogenic Leptospira interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni into a mammalian expression vector pcDNA3.1(+). The protective efficacy of the ompL1-pcDNA3.1(+) plasmid DNA vaccine was studied by immunizing hamsters intramuscularly with three doses of the vaccine (100 microg per dose) at two week intervals. The empty pcDNA3.1(+) and PBS were used as mock as negative vaccine controls, respectively. All animals were challenged with the heterologous Leptospira interrogans, serogroup Pomona, serovar Pomona (10 LD50), at one week after the last vaccine booster. The ompL1-pcDNA3.1(+) plasmid DNA vaccine rescued some vaccinated animals from the lethal challenge and delayed death time, reduced morbidity, e.g. fever, and/or the numbers of Leptospira in the tissues of the vaccinated animals. While the results are encouraging, further studies are needed to optimize the immunization schedule, vaccine dosage and formulation in order to maximize the efficacy of the vaccine.