Leptospiral Vaccine Research Articles

International regulatory requirements for Leptospira vaccine potency testing. Roundtable: Current requirements and opportunity for harmonization

Progress continues to be made in the ongoing efforts to replace, reduce, or refine the use of laboratory animals for Leptospira vaccine potency testing in certain markets/regions. Leptospira-containing vaccines, as with many veterinary vaccines, are manufactured and distributed both on a regional basis by local manufacturers and internationally by large multinational firms. Three general scenarios exist for the international testing and distribution of veterinary vaccines including: 1) the importing country recognizes the country of origin's testing and batch release data with no additional testing; 2) the importing country requires the manufacturer to conduct a specific potency assay based on the current importing market's regulations for the importing country or 3) the importing country requires retesting of the product in country prior to distribution. Scenarios 2 and 3 both have the potential to significantly increase the usage of laboratory animals for what may be considered redundant testing. Specific requirements for the importation of Leptospira vaccines in the United States, Europe, and Mexico were presented as well as efforts to reduce the use of laboratory animal testing through the availability of internationally recognized tests.

European regulatory framework and practices for veterinary Leptospira vaccine potency testing

In Europe, the legal basis for requirements for medicinal products is described in the European Pharmacopoeia (Ph. Eur.) In the European Union, the Ph. Eur. is supplemented by several guidelines issued by the European Medicines Agency. Immunological veterinary products must comply with the Ph. Eur. monograph on veterinary vaccines and the accompanying texts, as well as specific monographs. The Ph. Eur. includes monographs on canine leptospirosis and bovine leptospirosis vaccines (inactivated). Both monographs require that an immunogenicity test be performed once in the target species during the life of a vaccine. The hamster challenge test is applied for batch potency testing of canine vaccines. Alternatively, serological tests or suitable validated in vitro tests to determine the content of one or more antigenic components indicative of protection may be performed. Vaccines for use in cattle are tested in a serological test in guinea pigs. The acceptance criteria in alternative tests are set with reference to a batch of vaccine that has given satisfactory results in the immunogenicity test. At a January 2012 European workshop, the suitability of the hamster potency test was questioned and unanimous agreement was reached that moving toward complete in vitro testing is possible and should be promoted.

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.

Product-specific validation of a serological potency test for release of Leptospira vaccines in the European Union

Historically in the European Union, all Leptospira vaccines were released using the European Pharmacopoeia (Ph. Eur.) hamster potency assay. Recently, there has been a shift toward alternatives that offer either refinement of testing or replacement of animals for product release. This is being driven by animal welfare concerns but also by a drive to have more consistent, cheaper, and faster batch release tests. This publication discusses one such example of a multicomponent canine vaccine that includes three Leptospira serovars and has recently been registered in the European Union. The potency release test is a refinement because it uses rabbit serology rather than hamster challenge. This publication covers the principles of the test method, challenges faced during its development and registration, and discussion about benefits and limitations of this method. It concludes with a view of how the use of serology testing could fit into an overall strategy to move to fully in vitro testing by adopting a consistency approach.

Expansion of the in vitro assay for Leptospira potency testing to other serovars: Case study with Leptospira Hardjo

Evaluation of leptospiral vaccines for potency against Leptospira interrogans serovars Pomona, Icterohaemorrhagiae, Canicola, and Grippotyphosa is accomplished using the hamster potency test method described in 9 CFR 113.101-104. Applicability of this method to evaluation of bacterins developed for immunization against infection with L. interrogans serovar Hardjo or Leptospira borgpetersenii serovar Hardjo is complicated by several issues. Information from research on target host animal efficacy studies and evaluation of the immune response elicited using effective whole-cell bacterin formulations have revealed problems in relating these studies to either hamster-based or other potency testing methods. Future work on serovar Hardjo vaccines employing recombinant proteins will require preliminary testing methods in models other than the host animal. These models may also prove applicable to evaluation of potency for protein-based vaccines. Both an acute lethal infection model and a chronic infection model have been developed using two different strains of serovar Hardjo and will be described.

USDA regulatory guidelines and practices for veterinary Leptospira vaccine potency testing

Batch-release potency testing of leptospiral vaccines licensed by the United States Department of Agriculture (USDA) historically was conducted through animal vaccination-challenge models. The hamster vaccination-challenge assay was Codified in 1974 for bacterins containing Leptospira pomona, Leptospira icterohaemorrhagiae, and Leptospira canicola, and in 1975 for bacterins containing Leptospira grippotyphosa. In brief, 10 hamsters are vaccinated with a specified dilution of bacterin. After a holding period, the vaccinated hamsters, as well as nonvaccinated controls, are challenged with virulent Leptospira and observed for mortality. Eighty percent of vaccinated hamsters must survive in the face of a valid challenge. The high cost of the Codified tests, in terms of monetary expense and animal welfare, prompted the Center for Veterinary Biologics (CVB) to develop ELISA alternatives for them. Potency tests for other serogroups, such as Leptospira hardjo-bovis, that do not have Codified requirements for potency testing continue to be examined on a case-by-case basis.

Leptospirosis in horses

Leptospirosis in horses has been considered a relatively uncommon infection. However, recent data suggest that the infection is widespread, with the incidence and infecting serovars varying considerably in different geographical regions. The majority of infections remain asymptomatic. Clinical signs in equine leptospirosis resemble those seen in other animal species. However, leptospirosis as a cause of acute respiratory distress is becoming more frequently recognised. A particular feature of equine leptospirosis is post infection recurrent uveitis (moon blindness or periodic ophthalmia), which appears to be mediated by autoimmune mechanisms involving cross reactivity between ocular tissues and leptospiral membrane proteins. There are no leptospiral vaccines licensed for use in horses, with no prospect for any becoming available in the foreseeable future. Accordingly, prevention of equine leptospirosis must rely on good hygiene practices, minimisation of rodent contact, and vaccination of other species of production and companion animals.

A Conserved Region of Leptospiral Immunoglobulin-Like A and B Proteins as a DNA Vaccine Elicits a Prophylactic Immune Response against Leptospirosis

The leptospiral immunoglobulin-like (Lig) proteins LigA and LigB possess immunoglobulin-like domains with 90-amino-acid repeats and are adhesion molecules involved in pathogenicity. They are conserved in pathogenic Leptospira spp. and thus are of interest for use as serodiagnostic antigens and in recombinant vaccine formulations. The N-terminal amino acid sequences of the LigA and LigB proteins are identical, but the C-terminal sequences vary. In this study, we evaluated the protective potential of five truncated forms of LigA and LigB proteins from Leptospira interrogans serovar Canicola as DNA vaccines using the pTARGET mammalian expression vector. Hamsters immunized with the DNA vaccines were subjected to a heterologous challenge with L. interrogans serovar Copenhageni strain Spool via the intraperitoneal route. Immunization with a DNA vaccine encoding LigBrep resulted in the survival of 5/8 (62.5%) hamsters against lethal infection (P < 0.05). None of the control hamsters or animals immunized with the other vaccine preparations survived. The vaccine induced an IgG antibody response and, additionally, conferred sterilizing immunity in 80% of the surviving animals. Our results indicate that the LigBrep DNA vaccine is a promising candidate for inclusion in a protective leptospiral vaccine.

Immunomodulatory cytokine gene expression profiles and lymphoprolifration response in hamsters vaccinated by leptospiral vaccine with several adjuvants

Event Abstract Back to Event Immunomodulatory cytokine gene expression profiles and lymphoprolifration response in hamsters vaccinated by leptospiral vaccine with several adjuvants Reza Banihashemi1, 2* 1 Razi vaccine serum research inistitute, bacterial vaccine, Iran 2 Tarbiyat modares univercity, medical scinces, Iran Abstract: Introduction Leptospirosis is an acute infectious disease caused by Leptospira spp. that produces a broad range of health problems mainly in developing world. The present work aimed to quantify the mRNAs from cytokines ex vivo and its relation with the lymphoprolifration response of vaccine-sensitized spleenocytes as major elements in protection against leptospirosis. Materials and Methods 64 Four-week-old male hamsters separated in eight equal groups were subcutaneously inoculated with four-leptospiral vaccine and four-control adjuvant through a triple-injection regiment at fortnightly intervals. Animals in group one only received the plain vaccine preparation with no adjuvant while other groups were immunized with the same vaccine preparation but different adjuvants. Two weeks after the last injections, all animals were euthanized and subjected to splenectomy. After preparation of splenocytes, cytokine profiles and lymphoprolifration response has been evaluated against the recall antigen. Results Vaccination of hamsters led to significant lymphoprolifration response in all of the animals although differences were noticed in reaction levels between groups. The induction of Th1 (IL12p40, IFNү and TNFα), Th2 (IL4, IL10) and Treg (TGFβ ) type cytokine were evaluated from tested animals lymphocyte stimulated by ex vivo antigen utilizing real time PCR Conclusion We have demonstrated that adjuvant plays a fundamental role in the type and extent of the immune responses raised by leptospiral vaccination. In our hands, alum triggered both type 1 and type 2 immune reactions. Giving the fact that both Th1 and Th2 responses were stimulated, we strongly recommend selection of alum in any formulation of leptospiral vaccine. Acknowledgements This work was completely financed with the state funds from Razi Vaccine and Serum Research Institute under grant no. 2-18-18-90012. All staff at the Aerobic Bacterial Research and Vaccine Production Department of Razi Institute are thanked for their contribution to conduction of this research. Dr Moazeni jula are much thanked for critically reading and excellent editing the manuscript. Dr j majidi, a highly respected academician at the Tabriz medical University, He will be missed by all his colleagues and friends. This work was performed under the auspices of the Razi Vaccine and Serum Research Institute (RVSRI) of Iran Keywords: Vaccine, Leptospirosis, adjuvant, real time PCR, Lymphoprolifration Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Banihashemi R (2013). Immunomodulatory cytokine gene expression profiles and lymphoprolifration response in hamsters vaccinated by leptospiral vaccine with several adjuvants. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00175 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 10 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Reza Banihashemi, Razi vaccine serum research inistitute, bacterial vaccine, karaj-Iran, Alborz, 3197619751, Iran, reza7471@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Reza Banihashemi Google Reza Banihashemi Google Scholar Reza Banihashemi PubMed Reza Banihashemi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Development of Leptospira Killed Whole Culture Vaccine Using Different Adjuvants and Evaluation of Humoral Immune Response in Hamsters

Leptospirosis is an acute infectious disease caused by Leptospira spp. that produces a broad range of health problems mainly in developing world. In several affected countries, a major attempt has been begun to find a potent and effective vaccine. This is while; the available vaccines neither perused immunological memory nor confer cross-protection. Four-week-old male hamsters were subcutaneously inoculated with four-leptospiral vaccine and four-control adjuvant through a triple-injection regiment at fortnightly intervals. Animals were bled through the saphenous vein weekly until the seventh week. We have demonstrated that adjuvant plays a fundamental role in the type and extent of the immune responses raised by leptospiral vaccination. We strongly recommend selection of alum in any formulation of leptospiral vaccine.

Sequence of Leptospira santarosai serovar Shermani genome and prediction of virulence-associated genes

Leptospirosis, a widespread zoonosis, is a re-emerging infectious disease caused by pathogenic Leptospira species. In Taiwan, Leptospira santarosai serovar Shermani is the most frequently isolated serovar, causing both renal and systemic infections. This study aimed to generate a L. santarosai serovar Shermani genome sequence and categorize its hypothetical genes, particularly those associated with virulence. The genome sequence consists of 3,936,333 nucleotides and 4033 predicted genes. Additionally, 2244 coding sequences could be placed into clusters of orthologous groups and the number of genes involving cell wall/membrane/envelope biogenesis and defense mechanisms was higher than that of other Leptospira spp. Comparative genetic analysis based on BLASTX data revealed that about 73% and 68.8% of all coding sequences have matches to pathogenic L. interrogans and L. borgpetersenii, respectively, and about 57.6% to saprophyte L. biflexa. Among the hypothetical proteins, 421 have a transmembrane region, 172 have a signal peptide and 17 possess a lipoprotein signature. According to PFAM prediction, 32 hypothetical proteins have properties of toxins and surface proteins mediated bacterial attachment, suggesting they may have roles associated with virulence. The availability of the genome sequence of L. santarosai serovar Shermani and the bioinformatics re-annotation of leptospiral hypothetical proteins will facilitate further functional genomic studies to elucidate the pathogenesis of leptospirosis and develop leptospiral vaccines.

Concerns about Leptospira vaccine efficacy study

Concerns about Leptospira vaccine efficacy study

Efficacy of vaccination of cattle with the Leptospira interrogans serovar hardjo type hardjoprajitno component of a pentavalent Leptospira bacterin against experimental challenge with Leptospira borgpetersenii serovar hardjo type hardjo-bovis

To evaluate the efficacy of vaccination with the Leptospira interrogans serovar hardjo type hardjoprajitno component of a pentavalent Leptospira bacterin against a virulent experimental challenge with Leptospira borgpetersenii serovar hardjo type hardjo-bovis strain 203 in cattle. Fifty-five 6-month-old Holstein heifers. Heifers that were negative for persistent infection with bovine viral diarrhea virus determined via immunohistochemical testing and negative for Leptospira interrogans serovar pomona, Leptospira interrogans serovar hardjo, Leptospira interrogans serovar grippotyphosa, Leptospira interrogans serovar bratislava, Leptospira interrogans serovar canicola, and Leptospira interrogans serovar icterohaemorrhagiae determined via microscopic agglutination assay were enrolled in the study. Two heifers were separated and used for the challenge passage. The remaining heifers were vaccinated twice with a commercial pentavalent bacterin or a sham vaccine 21 days apart and subsequently challenged with L borgpetersenii serovar hardjo type hardjo-bovis strain 203. Urinary shedding, antibody titers, and clinical signs of leptospirosis infection were recorded for 8 weeks after challenge. Heifers that received the pentavalent bacterin did not shed the organism in urine after challenge and did not have renal colonization at necropsy. Heifers that were sham vaccinated shed the organism in urine and had renal colonization. Results provided evidence that a pentavalent Leptospira vaccine containing L interrogans serovar hardjo type hardjoprajitno can provide protection against challenge with L borgpetersenii serovar hardjo type hardjo-bovis strain 203. It is important to demonstrate cross-protection that is vaccine specific against disease-causing strains of organisms that are prevalent under field conditions.

Longitudinal serological survey and herd-level risk factors for Leptospira spp. serovars Hardjo-bovis and Pomona on deer farms with sheep and/or beef cattle

AIM: To investigate the seroprevalence of Leptospira spp. serovars Hardjo-bovis and Pomona on deer and mixed deer, sheep and/or beef cattle farms in the lower North Island of New Zealand and to examine associations between putative risk factors for seropositive deer herds. METHODS: Serological screening was conducted on 19 commercial deer farms, 16 with sheep and/or beef cattle, between August and October each year between 2006 and 2008. No leptospiral vaccination had been conducted on the farms. On each farm every year, serum samples were collected from a random sample of 20 or more rising 2-year-old replacement animals from each species. The microscopic agglutination test (MAT) was used to detect leptospiral antibodies against Leptospira borgpetersenii serovar Hardjo-bovis and Leptospira interrogans serovar Pomona. For both serovars, a titre of ≥1:48 was considered positive and a herd was considered seropositive if >3 of 20 serum samples were positive. Information on potential herd-level risk factors for deer herds being seropositive was obtained from a questionnaire completed by the farm owner or manager. RESULTS: The mean percentage of deer, cattle and sheep herds seropositive for Hardjo-bovis alone over 3 years was 42%, 53% and 54%, respectively, and for serovar Pomona was 7%, 5% and 0%, respectively. Antibodies to both serovars were found in 23%, 16% and 10% of deer, cattle and sheep herds, respectively. At the individual animal level, 228/1,107 (21%) deer, 308/767 (40%) cattle and 369/1,244 (30%) sheep were seropositive for Hardjo-bovis, 102 (9%) deer, 51 (7%) cattle and 23 (2%) sheep were seropositive for Pomona, and 49 (4%) deer, 28 (4%) cattle and 18 (1%) sheep were seropositive for both serovars. Deer herds were more likely to be seropositive for Hardjo-bovis in 2006 than 2008 (p=0.008), when seropositive in the preceding year (p=0.016) and on hilly compared with flat topography (p<0.001). Deer herds were more likely to be seropositive for Pomona when seropositive in the preceding year (p=0.016), when co-grazing with sheep flocks that were seropositive for Pomona (p<0.001), and when herds had a closed- compared with open-herd replacement policy (p<0.001). CONCLUSIONS: Exposure to Leptospira spp. was widely distributed in deer, sheep and beef cattle in the lower North Island of New Zealand. Co-grazing of deer with sheep that were seropositive was a potential risk factor for deer herds to be seropositive.

Humoral Immune Response of Inactivated Bivalent &amp;lt;i&amp;gt;Leptospira&amp;lt;/i&amp;gt; Vaccine among Dogs in Tiruchirappalli, Tamilnadu, India

Leptospirosis has been recognized as the disease of the dogs. The prevention of canine leptospirosis can block the transmission of the etiological agent to humans. Commercial vaccines prevent not only clinical leptospirosis but also the renal carrier state in dogs. Thus in this present investigation, the humoral immune response of the vaccinated dogs with the multivalent vaccine (Megavac-6) was analyzed. Enzyme linked immunosorbent assay (ELISA) with preparations of 2 whole cell lysate, 2 leptospiral LPS, 4 purified recombinant proteins were used to test sera from 30 dogs vaccinated with MEGAVAC-6, a commercial vaccine practiced by the animal husbandary Departments in Tamilnadu. All 30 sera were positive by ELISA with whole cell lysate of Canicola and Icterohaemorrhagiae, and ELISA with Canicola LPS, Icterohaemorrhagiae LPS; antibody titres ranged from 20 to ≥10,240. Although there was less frequent reactivity of sera with recombinant antigens, antibodies to LipL32 and LigA were detected in 25 (83.3%) serum samples. Less frequent reactivity was noted when recombinant GroEl, LK73.5 antigens were included separately in ELISAs. The highest MAT titre was observed against the serovar Canicola and Icterohaemorrhagiae. The more reactivity against LPS may be due to the dominance of serovar specific leptospiral LPS, which may be the dominant immunogenic antigen in inactivated bivalent leptospiral vaccines. Eventhough LPS is the dominant antigen. It is serovars specific and hence recommends the incorporation of the locally circulating serovars in vaccine for its efficient use. The efficiency at this point can be increased by the use of subunit vaccines rather than recombinant proteins as such. The present study has proposed certain epitopes with increased antigenic potency.

Efficacy of Leptospiral vaccine vax-SPIRAL against challenge with strains isolated from leptospirosis epidemic in Nicaragua using the hamster as biomodel

A hamster model was used to determine the protector capacity of vax-SPIRAL against the lethal infection after challenge with 10,000 LD of four strains isolated of a leptospirosis outbreaks in Nicaragua. These strains were 50 selected from a primary group of 16 belonging to the serogroups strains Ballum, Icterohemorragiae, Pomona, Serjoe and Pyrogenes. Most of these, except Pyrogenes serogroup, reproduced the infection in animal model and the most virulent representatives of each serogroup were selected for the challenge. The results of this study

Growth response and shedding of Leptospira spp. in urine following vaccination for leptospirosis in young farmed deer

AIM: To investigate the effect of vaccination against Leptospira serovars Hardjo-bovis and Pomona on growth rate and shedding of leptospires in urine in rising 1-year-old farmed red deer. METHODS: In early March 2007, 230 female and 205 male, 3-month-old deer on five farms were treated with streptomycin then were randomly allocated to a control group (n = 218), or were vaccinated (n = 217) with a bivalent whole-cell killed leptospiral vaccine (Leptavoid-2) followed by a booster 4 weeks later. These animals were isolated from other 3-month-old deer on each property until May, when all vaccinated and control deer were combined with deer not treated with streptomycin, for maximum exposure to natural leptospiral challenge. Seroconversion was monitored in control deer against Leptospira borgpetersenii serovar Hardjo-bovis and Leptospira interrogans serovar Pomona using the microscopic agglutination test (MAT). A cut-off of ≥ 1:48 was used to identify a seropositive response to infection, and ≥1:24 to indicate a response to vaccination. Urine was collected from females in all groups and tested for shedding of leptospires using bacterial culture and real-time PCR. Male deer from vaccinated and control groups were weighed every 2–3 months to measure growth rate. RESULTS: In vaccinated female deer, antibody was present to Hardjo-bovis in 39–73% and to Pomona in 78–100% of animals in May, with titres ranging between 1:24–1:96 and 1:24–1:1536, respectively; control female deer were all serologically negative. In November, female control deer were seropositive to Hardjo-bovis on 4/5 farms (seroprevalence range 4–78%) but seronegative to Pomona on all farms. Between March and November mean growth rate was higher in vaccinated than control deer (26 g/day) on one farm (p = 0.049) that had the highest prevalence of seroconversion (78–88%) and urinary shedding (56%). Between August and November on 4/5 farms, when almost all seroconversions occurred, growth rates were higher in vaccinated than control deer (31 g/day; p = 0.011). Urinary shedding of leptospires was detected by culture and/or PCR in 8/34 (24%) unvaccinated control and 20/38 (53%) animals not treated with streptomycin on 2/5 farms; no shedding was detected in vaccinated deer. The average risk difference for shedding between vaccinated and control deer was –11% (95%CI = –18 to –4) (p = 0.001). CONCLUSION: Vaccination against Leptospira serovars Hardjo-bovis and Pomona was associated with higher liveweight gain and reduced urinary shedding of leptospires in young deer naturally challenged by Hardjo-bovis. The association with liveweight gain was evident on some but not all farms.

Effects of Recent Leptospira Vaccination on Whole Blood Real‐Time PCR Testing in Healthy Client‐Owned Dogs

Bacterin-based canine Leptospira vaccines could present a challenge for the use of whole blood real-time polymerase chain reaction (PCR) as a diagnostic tool. Recent vaccination could induce positive results if the targeted DNA fragment is present within the vaccine and in the blood of the recently vaccinated dog. The objective of this study was to assess whether 2 available 4-serovar vaccines induce a positive real-time PCR reaction in the blood of healthy recently vaccinated dogs. Twenty healthy dogs. This was a prospective study. Dogs were assigned to 1 of 2 vaccine groups. Both vaccines were culture-based and include Leptospira interrogans serovars Pomona, Canicola, and Icterohaemorrhagiae and Leptospira kirschneri serovar Grippotyphosa. Whole blood for real-time PCR and serum for the microscopic agglutination test (MAT) were collected prior to and 3 and 7 days after vaccination and weekly thereafter for 8 weeks. Two real-time PCR tests targeting 2 different genes were performed independently in a blinded fashion. Both Leptospira vaccines produced positive real-time PCR reactions when assayed undiluted or diluted 1 : 100 in canine blood. However, blood samples drawn from all dogs at all time points after vaccination were negative on PCR. All dogs developed MAT titers. Recent vaccination with 2 commercially available vaccines does not interfere with the use of real-time PCR for the identification of acute Leptospira infection in dogs.

Vaccination for leptospirosis improved the weaning percentage of 2-year-old farmed red deer hinds in New Zealand

AIM: To investigate the effect of leptospiral vaccination against serovars Hardjo-bovis and Pomona on fetal loss and weaning percentage in rising 2-year-old farmed red deer hinds. METHODS: In mid-February 2007, 252 rising 2-year-old hinds on four farms received a single dose of streptomycin (25 mg/kg), to minimise leptospiral infection. They were randomly allocated to vaccinated and control groups. Vaccinated hinds (n=125) received a 2-ml S/C injection of a bivalent whole-cell killed leptospiral vaccine (Leptavoid-2) followed by a booster 4–6 weeks later, and were grazed with control hinds (n=127). These animals were isolated from other hinds on each property, until after mating (June 2007), when all vaccinated and control hinds were combined with hinds not treated with streptomycin, for maximum exposure to natural leptospiral challenge. Evidence of natural challenge by Leptospira spp. was assessed in blood samples from control hinds by serology against L. borgpetersenii serovar Hardjo-bovis and L. interrogans serovar Pomona, using the microscopic agglutination test (MAT), and in hinds not treated with streptomycin by detection of shedding of organisms in urine, using bacterial culture and real-time PCR. Pregnancy diagnosis was carried out in May/June 2007, using transrectal ultrasonography, to determine conception. In late October, prior to calving, the pregnant vaccinated and control hinds were examined by palpation of the abdomen and udder, to determine the percentage of hinds pregnant at term and assess fetal loss. In March 2008, at weaning, vaccinated and control hinds were examined for lactation status, using observation and palpation of the udder. The differences between the groups were evaluated using matched logistic regression analysis. RESULTS: After mating, pregnancy was diagnosed in 97/125 (77.6%) vaccinated and 106/127 (83.5%) control hinds. All four farms had serological evidence of Hardjo-bovis infection, and a single hind was serologically positive for Pomona between October and March. Real-time PCR confirmed urinary shedding on two farms. The mean percentage of hinds pregnant at term, for those animals confirmed pregnant after mating, in the vaccinated and control groups was 95/97 (98%) (range 95–100%) and 103/106 (97%) (range 94–100%), respectively (p>0.05). The mean weaning percentage for vaccinated and control groups was 86/97 (89%) (range 78–95%) and 88/106 (83%) (range 76–88%), respectively (p=0.015). CONCLUSION: Vaccination for leptospirosis resulted in no difference in the percentage of hinds pregnant at term, but a higher weaning percentage compared with unvaccinated controls suggesting that vaccination reduced perinatal and/or pre-weaning mortality.

Improvement of trivalent leptospira vaccine by removal of anaphylactic agents

The anaphylactic reactions in cattle following leptospira vaccination mostly booster dose in different parts of Iran have been reported. The serum proteins as allergic substances are components of liquid phase of the vaccine. Therefore, the vaccine was modified by washing the whole cultures by centrifugations. The modified vaccine was safe in laboratory animals and cattle as well as under field conditions. Microagglutination test revealed a similar pattern of antibody response to the three Leptospira interrogans serovars (Canicola, Grippotyphosa, and Sejro hardjo) in all vaccinated cattle groups while was higher than the response of control animals. The results of the present investigation revealed that we can minimize postvaccination shock in vaccinated cattle populations with removing the shock proteins.