We would like to express several concerns regarding the study by Rinehart et al1 in which the authors evaluated the efficacy of the Leptospira interrogans serovar hardjo type hardjoprajitno component of a commercial, pentavalent, adjuvanted Leptospira bacterin against challenge with Leptospira borgpetersenii serovar hardjo type hardjobovis. Efficacy was determined on the basis of reduction of renal colonization and urinary shedding of the challenge strain. Although the results of the study meet current requirements for licensure and the authors mention potential reasons for differences between their results and results of previous studies2,3 involving multivalent bacterins containing L interrogans serovar hardjo type hardjoprajitno, we believe that the claim of 100% protection against challenge presented in the manuscript may overvalue the data in terms of vaccine efficacy.
Infection of cattle with serovar hardjo results in persistent renal infection, urinary shedding, and economic losses attributable to fetal infection and reproductive failure. Our chief concern with this study is the reliance solely on kidney and urine culture results to determine whether cattle were infected after challenge. In a vaccination and challenge study2 involving pregnant cattle, culture alone failed to identify 14 of 15 vaccinated animals that were subsequently determined to be infected and shedding leptospires after challenge with L borgpetersenii serovar hardjo. Thirteen of 15 calves born to these vaccinated animals were also shown to have leptospires in their tissues, clearly demonstrating that culture alone cannot be relied on to detect infection when evaluating vaccine efficacy. Other vaccination studies2–7 have also reported difficulties associated with isolation of L borgpetersenii serovar hardjo from either tissue or urine obtained from vaccinated cattle despite histopathologic, immunochemical, and molecular evidence that organisms were present. Results reported for these studies relied on application of additional methodologies available in diagnostic settings to demonstrate the presence of leptospiral infection and shedding (despite negative culture results). The rigor and reliability of vaccine evaluation should approach that applied to diagnosis of disease.8
Although the authors' discussion mentions studies correlating leptospirosis vaccine efficacy with CD4 and γδ T lymphocyte subsets and with induction of Th1-type immune responses, similar data evaluating host immune responses to the vaccine formulations were not presented in the manuscript. The only immunologic data collected in this study were apparently serologic response data, and these data were not shown. Using a seropositive cutoff of 1:100 in the microscopic agglutination test (MAT), the authors report that “A transient positive MAT serologic response to the L interrogans serovar hardjo was observed only on day 14 after second vaccination in < 24% of the vaccinates.” The authors discuss the lack of an anamnestic response in their study, which has been a common observation after challenge in L borgpetersenii serovar hardjo vaccine studies. However, the authors did not indicate that conclusions from other studies3,5 strongly suggest that the presence or absence of an anamnestic response is not a good indicator of exposure or infection status in vaccinated animals.
Finally, the authors report in error that the monovalent leptospiral vaccine reported to provide protection against challenge with L borgpetersenii serovar hardjo strain 203 was prepared from L borgpetersenii serovar hardjo strain 93U.5 The reference vaccine that failed to demonstrate efficacy in that study was indeed prepared from strain 93U, but the vaccine that provided protection was obtained directly from the manufacturer.
In summary, although the presented data are of interest to veterinarians and scientists working in the area of leptospirosis, we are concerned that procedures used in this study may not have been sufficiently rigorous to evaluate vaccine efficacy. In particular, data from other studies would suggest that strict reliance on culture of leptospiral organisms from blood or urine for assessment of vaccine efficacy for serovar hardjo is not optimal. Consideration should be given to incorporation of new standards for vaccine evaluation for protection against this important pathogen.
David P. Alt, DVM, PhD
Steven Olsen, DVM, PhD
National Animal Disease Center, Infectious Bacterial Diseases Research Unit, Ames, Iowa.
Carole Bolin, DVM, PhD
Diagnostic Center for Population & Animal Health, Lansing, Mich.
1. Rinehart CL, Zimmerman AD, Buterbaugh RE, et al. 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. Am J Vet Res 2012; 73:735–740.
2. Bolin CA, Thiermann AB, Handsaker AL, et al. Effect of vaccination with a pentavalent leptospiral vaccine on Leptospira interrogans serovar hardjo type hardjo-bovis infection of pregnant cattle. Am J Vet Res 1989; 50:161–165.
3. Bolin CA, Zuerner RL, Trueba G. Effect of vaccination with a pentavalent leptospiral vaccine containing Leptospira interrogans serovar hardjo type hardjo-bovis on type hardjo-bovis infection of cattle. Am J Vet Res 1989; 50:2004–2008.
4. Bolin CA, Cassells JA, Zuerner RL, et al. Effect of vaccination with a monovalent Leptospira interrogans serovar hardjo type hardjo-bovis vaccine on type hardjo-bovis infection of cattle. Am J Vet Res 1991; 52:1639–1643.
5. Bolin CA, Alt DP. Use of a monovalent leptopsiral vaccine to prevent renal colonization and urinary shedding in cattle exposed to Leptospria borpetersenii serovar hardjo. Am J Vet Res 2001; 62:995–1000.
6. Zuerner RL, Alt DP, Palmer MV, et al. A Leptospira borgpetersenii serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. Clin Vaccine Immunol 2011; 18:684–691.
7. Naimann BM, Blumerman S, Alt D, et al. Evaluation of type 1 immune response in naive and vaccinated animals following challenge with Leptospira borgpetersenii serovar hardjo: involvement of WC1+ and CD4 T cells. Infect Immun 2002; 70:6147–6157.
8. Smith CR, Ketterer PJ, McGowan MR, et al. A review of laboratory techniques and their use in the diagnosis of Leptospira interrogans serovar hardjo infection in cattle. Aust Vet J 1994; 71:290–294.
The authors respond:
We appreciate the opportunity to respond to the letter from Drs. Alt, Olsen, and Bolin. Their primary concern appears to be that the level of protection provided by the vaccine is overstated. Although their letter refers to a 100% protection claim, our report does not specifically imply that. The true problem lies in assessing a correlate for vaccine protection when dealing with an infectious organism, such as Leptospira interrogans, that is host adapted, results in chronic infection, is difficult to culture, and causes little obvious clinical disease. The studies referenced by by Drs. Alt, Olsen, and Bolin demonstrate that no Leptospira vaccines have established complete protection against infection. Therefore, we agree that it is likely infection occurred in vaccinated animals in our study. The Center for Veterinary Biologics sets forth the requirements for licensure of Leptospira vaccines, which were followed in this study. The actual label claim issued by the Center for Veterinary Biologics regarding the vaccine in question was protection against urinary shedding, not protection against infection. The sticking point comes with regard to detection of Leptospira infection and the biological importance of the results of various detection methods. Finding viable, replicating Leptospira organisms in cultures of urine or kidney samples is truly the gold standard to indicate whether animals are actively infected or potential reservoirs. This becomes problematic with Leptospira serovars that are difficult to isolate from tissue samples and are intermittently shed. On the other hand, detection of Leptospira DNA (eg, by use of a PCR assay) or antigen (ie, by use of a fluorescence antibody test) indicates the presence of the organism but does not indicate whether the organism is alive or dead, making it unclear whether the vaccine may have protected against infection by killing the organism. In a perfect world with a perfect vaccine that induced sterilizing immunity, there would be no evidence whether by culture, histologic testing, or DNA- or antigen-based testing of the presence of Leptospira organisms. That world doesn't exist, so we are left with the problem of trying to determine what is the best correlate of protection against Leptospira infection. With the uncertainty surrounding what defines protection against Leptospira infection, the last statement by Drs. Alt, Olsen, and Bolin that “Consideration should be given to incorporation of new standards for vaccine evaluation for protection against this important pathogen” merits further investigation. The question in front of us is what should those new standards be to prove a Leptospira vaccine protects against infection.
Carol L. Rinehart, PhD
Rika A. Jolie, DVM, PhD, MBA
Boehringer Ingelheim Vetmedica Inc, St Joseph, Mo.
Alicia D. Zimmerman, DVM
Robin E. Buterbaugh, MS
Rural Technologies Inc, Brookings, SD.
Christopher C. L. Chase, DVM, PhD
Department of Veterinary and Biomedical Science, College of Agriculture and Biological Sciences, South Dakota State University, Brookings, SD.