Exploration of the potential utility of the luciferase immunoprecipitation system (LIPS) assay for the detection of anti-leptospira antibodies in dogs.

Canine leptospirosis represents a diagnostic challenge to veterinarians, due to the variability in presenting clinical signs and interpretation of serology test results in dogs that have been vaccinated previously. None of the commercially available serological assays, including the microscopic agglutination test (MAT), have been verified to be capable of differentiating infected from vaccinated animals (DIVA). Recent work identified that half of primary practice attending dogs were up to date with their leptospirosis vaccination and would be expected to have circulating anti-leptospira antibodies (Taylor et al., 2022), indicating that this is a relevant issue for suspected leptospirosis cases in dogs in the UK. This study aimed to explore the utility of three leptospiral outer membrane proteins (OMPs: LipL32, LipL21 and LipL41) as potential DIVA targets in the luciferase immunoprecipitation system (LIPS) assay. N and C terminal nanoluciferase tagged recombinant proteins were generated for each OMP. Differences in reactivity between serum samples from MAT positive dogs (n = 29) and paired samples (n = 6 dogs) taken pre and 21 days post leptospirosis vaccination were assessed against these six constructs. Reactivity was greater towards the N terminal than the C terminal recombinant proteins for all three OMPs. None of the constructs appeared to demonstrate DIVA capability, although two (pNLF1-N-FLAG/LipL32 and pNLF1-N-FLAG/LipL21) were able to detect vaccine seroconversion. The findings of this work suggest that these particular OMP targets do not offer DIVA ability, however LipL32 and LipL21 may be suitable for use in immunoassays for vaccine trials or for detection of infections in humans, where there is no requirement for DIVA capability.

A new tetravalent canine leptospirosis vaccine provides at least 12 months immunity against infection.

A key success factor in the vaccination of dogs against leptospirosis is long term protection against establishment of the renal carrier state, in order to protect other dogs, as well as humans, against this re-emerging zoonotic disease. In this paper, we describe the ability of a new European tetravalent vaccine containing antigen from Leptospira interrogans (sensu lato) serogroups Icterohaemorrhagiae, Canicola, Grippotyphosa and Australis to control infection and renal excretion in dogs at 12 months after vaccination. In order to demonstrate the efficacy of all four vaccine components, four separate challenge studies were performed. For each study two groups of dogs were used (a group receiving the leptospirosis vaccine and a control group). Twelve months after the second vaccination all dogs in the vaccine and control groups were challenged, both intraperitoneally and conjunctivally, using a pathogenic challenge strain from one of four serogroups. Parameters recorded post-challenge were: clinical signs of disease, change in body temperature, total leucocyte count, thrombocyte count, presence of challenge organisms in blood, urine and kidney tissue, and evidence of interstitial nephritis at necropsy four weeks after challenge. The vaccine was able to either prevent or significantly reduce infection following challenge with the strains of all four serogroups. The vaccine was also able to prevent or significantly reduce renal infection following Canicola and Icterohaemorrhagiae challenge, and there was a trend of reduction of renal infection with Australis (serovar Bratislava). In the case of the Grippotyphosa study, challenge led to no detectable renal infection in any dog of the control group. In conclusion, in this study significant protective immunity was achieved in dogs 12 months after a basic vaccination schedule of two doses against strains of serogroups Canicola, Icterohaemorrhagiae, Grippotyphosa and Australis.

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.

A novel tetravalent Leptospira bacterin protects against infection and shedding following challenge in dogs.

Recent evidence based on the current epidemiological situation suggests that vaccines against canine leptospirosis in Europe should be directed against infection with Leptospira interrogans (sensu lato) serogroups Canicola, Icterohaemorrhagiae, Grippotyphosa and Australis. In the eight studies presented here, dogs were vaccinated with Nobivac L4 (MSD Animal Health), a new tetravalent inactivated vaccine containing antigen from four strains representing these four serogroups. The dogs were then challenged, together with unvaccinated control dogs, using heterologous strains from the same four serogroups. In four of the studies, pups without agglutinating antibodies against the four serogroups were vaccinated with Nobivac L4 vaccine. In a further four studies, Nobivac L4 vaccine was given 48 hours after administration of antiserum from vaccinated dogs designed to mimic the serological status of pups with maternally derived antibodies against these serogroups. In all eight studies, vaccine efficacy was assessed in terms of antibody response, clinical signs, fever, thrombocyte count, frequency of positive isolation of challenge organisms from blood, urine and kidney and frequency of interstitial nephritis. The results demonstrate that Nobivac L4 vaccine induces sterile immunity against leptospiraemia and renal infection with strains of serogroups Canicola, Icterohaemorrhagiae and Grippotyphosa, and induces sterile immunity against leptospiraemia with a strain of serogroup Australis. Since sterile immunity was achieved in pups pretreated with antiserum as well, it can be concluded that this vaccine is also likely to be efficacious in the face of maternally derived antibodies in pups from the age of six weeks.

Comparison of the efficacy of a subunit and a live streptomycin-dependent porcine pleuropneumonia vaccine.

OBJECTIVE: To evaluate the efficacy of two new-generation porcine pleuropneumonia vaccines when challenged with Australian isolates of Actinobacillus pleuropneumoniae of serovars 1 and 15. DESIGN: The Porcilis APP vaccine and an experimental streptomycin-dependent strain of A pleuropneumoniae were evaluated in a standardised pen trial. Each vaccine/challenge group consisted of 10 pigs. RESULTS: With the serovar 1 challenge, the Porcilis APP vaccine and the live vaccine, compared with the control group, gave significant protection in terms of clinical signs, lung lesions, re-isolation scores and average daily gain (ADG) postchallenge. Only the Porcilis APP vaccine provided significant protection against mortality. In the serovar 15 challenged pigs, the only significant difference detected was that the Porcilis APP vaccinated pigs had a better postchallenge ADG than the controls. None of the Porcilis APP vaccinated pigs showed signs of depression postvaccination and none were euthanased after challenge with either serovar 1 or 15. The pigs vaccinated with the live vaccine showed obvious depression after each vaccination and a total of 3 pigs were euthanased after challenge (one with serovar 1 and two with serovar 15). CONCLUSIONS: Both of the vaccines provided significant protection against a severe challenge with serovar 1 A pleuropneumoniae. Neither vaccine was effective against a serovar 15 A pleuropneumoniae challenge. There was evidence that the Porcilis APP vaccine did provide some protection against the serovar 15 challenge because the ADG, after challenge of pigs given this vaccine, was greater than the control pigs.

An evaluation of the role of antibodies to Actinobacillus pleuropneumoniae serovar 1 and 15 in the protection provided by sub-unit and live streptomycin-dependent pleuropneumonia vaccines.

OBJECTIVE: To evaluate the serological response of pigs receiving either the Porcilis APP vaccine or a modified live vaccine based on a streptomycin-dependent (SD) strain of Actinobacillus pleuropneumoniae, and then challenged with an Australian isolate of A. pleuropneumoniae of either serovar 1 or 15 as a means of understanding the protection provided by both vaccines against serovar 1 but not against serovar 15. DESIGN: The serological tests evaluated were serovar-specific polysaccharide ELISA tests (for serovar 1 and 15), ELISA tests for antibodies to three A. pleuropneumoniae toxins (ApxI, ApxII and ApxIII) as well as to a 42 kDa outer membrane protein (OMP), a haemolysin neutralisation (HN) assay and immunoblotting. The tests were used to detect antibodies in vaccinated pigs that had been shown to be protected against serovar 1 but not serovar 15. RESULTS: In the polysaccharide antigen ELISA assays, both vaccines resulted in a significant rise in the titre in the serovar 1 ELISA but not the serovar 15 ELISA. The Porcilis APP vaccinated pigs showed a significant response in the ApxI, ApxIII and 42 kDa OMP ELISA. In the ApxII ELISA, all pigs tested (the Porcilis APP vaccinates and the controls) were positive on entry to the trial. In the HN assay, the Porcilis APP vaccinated pigs showed a significant response after one dose while the SD vaccinated pigs required two doses of vaccine before a marked rise in titre was induced. Immunoblotting revealed that neither vaccine generated antibodies that recognised the ApxIII produced by serovar 15. CONCLUSIONS: The failure of these vaccines to provide protection against serovar 15 may be due to novel virulence factors possessed by serovar 15, significant differences between the ApxIII toxin of serovar 15 and those present in the Porcilis APP vaccine or failure by both vaccines to induce antibodies to the serovar 15 specific polysaccharide.

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.

Duration of immunity in dogs vaccinated against leptospirosis with a bivalent inactivated vaccine.

Duration of immunity in dogs induced with current commercial inactivated leptospirosis vaccines and evaluated against experimental infection, to date, has hardly been documented. The purpose of the present work was to assess the duration of immunity in dogs that is attainable with a commercial inactivated bivalent leptospirosis vaccine. For this purpose, young dogs were vaccinated twice followed by challenge with either Leptospira interrogans serovar canicola or L. interrogans serovar icterohaemorrhagiae 5 weeks, 27 weeks or 56 weeks after the second vaccination. For assessment of the duration of immunity, titres of agglutinating serum antibodies were measured before and after challenge, and the effects of challenge on a variety of parameters were determined including reisolation of challenge organisms from blood, urine and kidney. Both challenge strains induced a generalised infection in control dogs, the canicola strain being most virulent. From the results with different parameters it appeared that the two vaccinations induced a high rate of protection from generalised infection with canicola and icterohaemorrhagiae at 5, 27 and 56 weeks after the second vaccination. In addition, after 56 weeks, still a high level of immunity against renal infection with sv. canicola and, as a consequence, urinary shedding of sv. canicola bacteria, was demonstrated. It was, therefore, concluded that with this vaccine, using this vaccination schedule, a duration of immunity of 1 year can be attained against infection with both serovars.

Proposal of a new serovar of Actinobacillus pleuropneumoniae: serovar 15.

We report on the re-examination of nine Australian isolates of Actinobacillus pleuropneumoniae that have been previously assigned to serovar 12. In the ring precipitation test, none of the nine isolates reacted with antisera to serovars 1-14 of A. pleuropneumoniae. Antiserum prepared against one of the Australian isolates gave no reaction with any of the 14 recognised serovar reference strains, except serovar 7. This reaction of the HS143 antiserum with serovar 7 antigen could be removed by adsorption with serovar 7 antigen. The adsorbed antiserum remained reactive with HS143 and the other eight Australian isolates. The nine Australian isolates were all shown to express ApxII and ApxIII, found in serovars 2, 4, 6 and 8, as well as the 42kDa outer membrane protein found in all serovars of A. pleuropneumoniae. The nine Australian isolates were found to possess the following toxin associated genes apxIBD, apxIICA, apxIIICA, apxIIIBD and apxIVA. The toxin gene profile of the Australian isolates is typical of A. pleuropneumoniae serovars 2, 4, 6 and 8. On the basis of the serological characterisation results and the toxin gene profiles, we propose that these isolates represent a new serovar of A. pleuropneumoniae--serovar 15--with HS143 being the reference strain for the new serovar.