Comparison of PCR versus culture for detection of Mycobacterium bovis after experimental inoculation of various matrices held under environmental conditions for extended periods.

The purpose of this study was to compare the performance of a molecular detection technique (nested PCR) with that of mycobacterial culture in the detection of Mycobacterium bovis DNA in a set of 687 samples of experimentally inoculated environmental substrates (hay, soil, corn, water) exposed to natural weather conditions in Michigan. Four replicates of each substrate were used; half were autoclaved for sterilization, all were inoculated with 50,000 CFU of M. bovis isolated from Michigan livestock, and all were placed in outdoor enclosures, with half under shade and the other half exposed to direct sunlight. Samples were tested for the presence of M. bovis during one 12-month period, with monthly sample testing and during three 12-week periods (winter, spring, summer) with weekly sample testing. Samples were subjected to mycobacterial culture for isolation of M. bovis and a nested PCR with two primer sets targeting IS6110 to detect M. bovis DNA. In 128 samples tested during the 12-month period, M. bovis was not detectable by culture after 2 months but M. bovis DNA was detectable by PCR for at least 7 months. Of the 559 samples tested during the 12-week periods, PCR detected M. bovis DNA for up to 88 days in all of the sample types. There were no significant differences in the detection of M. bovis between shade and sun samples or between sterile and unsterilized samples, regardless of the detection method (PCR or culture). For use in epidemiologic investigations, the PCR assay was more rapid than mycobacterial culture, was not hindered by contaminating organisms, and detected M. bovis DNA in environment samples much longer after initial contamination than mycobacterial culture did.

Changes in multidrug resistance of enteric bacteria following an intervention to reduce antimicrobial resistance in dairy calves.

An intervention study was conducted to determine whether discontinuing the feeding of milk replacer medicated with oxytetracycline and neomycin to preweaned calves reduced antimicrobial resistance in Salmonella, Campylobacter, and Escherichia coli bacteria. Results demonstrated that the intervention did reduce multidrug resistance in these bacteria but that other factors also influenced multidrug resistance.

Proliferation of Streptococcus zooepidemicus and Pseudomonas aeruginosa within a simulated subpalpebral lavage flushed with equine serum.

OBJECTIVE: To evaluate whether equine serum administered via a simulated subpalpebral lavage system (SPL) supports proliferation of Streptococcus zooepidemicus or Pseudomonas aeruginosa within the tubing. PROCEDURES: A sterile i.v. catheter with injection cap was inserted into sterilized silicone tubing (Mila). To mimic an SPL within the dorsal conjunctival fornix, the tubing was secured to an elevated platform. The tip of the tubing extended from the platform into a vial containing culture medium just inoculated with approximately 1.5 x 10(8) CFU/mL P. aeruginosa or S. zooepidemicus. To mimic administration of medication, the tubing was infused twice daily with equine serum, sterile saline (negative control), or culture medium (positive control) followed by air. Incubation was at 25 or 37 degrees C. At 24, 48, and 72 h postinoculation, samples were obtained for bacterial culture from one simulated SPL for each experimental variant. The following sections were cultured: (i) tubing tip previously submerged in the inoculated culture medium, (ii) tubing mid-section, and (iii) tip of the i.v. catheter. The experiment was performed in triplicate. RESULTS: Streptococcus zooepidemicus or P. aeruginosa were isolated from 100% of the tubing tips. Streptococcus zooepidemicus was isolated from one mid-section flushed with culture medium incubated at 37 degrees C. All other samples were negative for growth of the inoculated agents. CONCLUSIONS: Streptococcus zooepidemicus and P. aeruginosa did not proliferate within silicone tubing infused with equine serum. These data suggest that topical serum can be safely administered through a superiorly placed SPL in clinical cases.

Minimum inhibitory concentration and postantibiotic effect of amikacin for equine isolates of methicillin-resistant Staphylococcus aureus in vitro.

OBJECTIVE: To report the minimum inhibitory concentration (MIC) of amikacin sulfate for equine clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and characterize the initial kill and duration of the postantibiotic effect (PAE) for selected strains. STUDY DESIGN: Experimental study. METHODS: Isolates of MRSA (n=35) had their amikacin MIC determined using the E-test agar diffusion method. Two isolates with MICs>256 microg/mL limit were further characterized using broth macrodilution. Six distinct isolates with amikacin MICs of 32, 48, 128 (2 isolates) and 500 (2 isolates) microg/mL had PAE determinations made over a range of amikacin concentrations from 31.25-1000 microg/mL using standard culture-based techniques. RESULTS: Median MIC of the 35 isolates was 32 microg/mL (range 2 to >256 microg/mL). Mean PAE of selected MRSA strains had an overall mean (all amikacin doses) of 3.43 hours (range 0.10-9.57 hours). PAE for MRSA exposed to amikacin at 1000 microg/mL was 6.18 hours (range 3.30-9.57 hours), significantly longer than that for all other concentrations (P<.0001). There was no statistically significant effect of isolate MIC on PAE. CONCLUSIONS: Isolates had a wide range of MIC; however, growth of all 6 selected strains were inhibited within the range of concentrations tested, including 2 strains with MICs of 500 microg/mL. PAE duration was not influenced by the MIC of amikacin but was significantly longer with treatment at 1000 microg/mL than at lower concentrations. CLINICAL RELEVANCE: Clinical isolates of MRSA are susceptible to amikacin at concentrations achieved by regional perfusion: however, the modest duration of PAE observed suggest that further laboratory and in vivo evaluation be conducted before recommending the technique for clinical use.

Changes in tetracycline susceptibility of enteric bacteria following switching to nonmedicated milk replacer for dairy calves.

A randomized intervention study was conducted to determine if discontinuing use of calf milk replacer medicated with oxytetracycline results in increased tetracycline susceptibility in Salmonella and Campylobacter spp. and Escherichia coli in dairy calves over a 12-month period. Dairy herds with enteric bacteria with known low tetracycline susceptibility were enrolled for the study. Fecal samples from preweaned calves and environmental samples were collected from eight dairy herds in Michigan and New York State. Samples were collected monthly for 3 months prior to and 12 months after four of the eight herds discontinued medicated milk replacer feeding. Salmonella and Campylobacter spp. and E. coli were isolated, and antimicrobial susceptibility testing was conducted using automated broth microdilution. A total of 804 intervention and 1,026 control calf fecal samples and 122 intervention and 136 control environmental samples were collected for testing. No differences in owner-reported morbidity and mortality between treatment groups were seen. The intervention was significantly associated with increasing tetracycline susceptibility in E. coli and Salmonella. Tetracycline susceptibility increased in intervention herds for the first 3 months after switching to nonmedicated milk replacer but declined in subsequent months. Discontinuing the practice of feeding medicated milk replacers to calves increased tetracycline susceptibility in E. coli and Salmonella on dairy farms, without increasing cattle disease, but declines in effectiveness after 3 months suggest that other factors contribute to decreasing susceptibility on the farm.

Experimental inoculation of meadow voles (Microtus pennsylvanicus), house mice (Mus musculus), and Norway rats (Rattus norvegicus) with Mycobacterium bovis.

Mycobacterium bovis has a wide host range that includes several wildlife species, and this can hamper attempts to eradicate bovine tuberculosis from livestock. The purpose of this study was to determine if common rodent species, namely meadow voles (Microtus pennsylvanicus), house mice (Mus musculus), and Norway rats (Rattus norvegicus), that inhabit the bovine tuberculosis endemic area of Michigan, can be experimentally infected with M. bovis. The objectives of the study were: 1) to determine if these rodent species can be infected, and if so, to document attendant pathologic processes/pathogenesis; 2) to detect any fecal shedding of M. bovis; and 3) to evaluate the relative susceptibility of the three species to M. bovis infection. For each species (n=36) there were two treatment (n=12/group) and one or two control groups depending on species (n=6-12/group); the maximum study duration was 60 days. The meadow vole treatments consisted of high dose inocula that were given by oral or intranasal routes, whereas the house mice and Norway rats were given only oral inocula at either a high or low dose. Of the three species, meadow voles were most susceptible to M. bovis infection. Upon intranasal inoculation, all 12 voles were infected as determined by gross and microscopic lesions and culture of M. bovis from tissue and feces. Seven of the 12 meadow voles inoculated orally were infected. House mice also were susceptible; M. bovis was isolated from 14 of 24 animals. Only one Norway rat in the high dose treatment group was positive by culture and this was the only animal from which minimal attendant lesions were observed. Results of this study indicate that meadow voles and house mice can be infected with M. bovis and might serve as spillover hosts. Concerted efforts should, therefore, be made to reduce or eliminate these rodents on premises where M. bovis-infected livestock are present.

Prevalence of serum antibodies against six Leptospira serovars in healthy dogs.

OBJECTIVE: To determine the prevalence of antibodies against 6 Leptospira serovars and determine risk factors associated with positive Leptospira titers in healthy client-owned dogs in Michigan. DESIGN: Cross-sectional study. ANIMALS: 1,241 healthy dogs at least 4 months of age. PROCEDURES: Dogs were examined by veterinarians at private practices. Vaccinated and unvaccinated dogs were enrolled in the study, which occurred prior to the availability of a 4-serovar (Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona) Leptospira vaccine. Sera were tested by use of the microscopic agglutination test to determine antibody titers against Leptospira serovars Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona. A questionnaire was used to collect demographic information about each dog to identify risk factors associated with seropositive status. RESULTS: 309 of 1,241 (24.9%) dogs had antibody titers against at least 1 of the 6 Leptospira serovars, which suggested exposure to Leptospira spp. Prevalence of antibodies was highest to serovar Grippotyphosa, followed by Bratislava, Canicola, Icterohaemorrhagiae, and Pomona. Age, travel outside Michigan, exercise outside fenced yards, and exposure to livestock and wildlife were significant risk factors for positive titers. CONCLUSIONS AND CLINICAL RELEVANCE: Among healthy dogs from the lower peninsula of Michigan, > 20% have antibodies against leptospiral serovars historically considered uncommon but more recently incriminated as causing clinical canine leptospirosis. Wildlife and livestock may be of increasing importance as reservoirs for canine leptospirosis as urbanization continues to occur. Expanded vaccination strategies may partially mitigate these trends.

Survivability of Mycobacterium bovis on salt and salt-mineral blocks fed to cattle.

OBJECTIVE To determine the survivability of Mycobacterium bovis on salt and salt-mineral blocks in typical weather conditions in Michigan over two 12-day periods at the height of summer and winter. SAMPLE 4 salt (NaCl) and 4 salt-mineral blocks inoculated with pure cultures of a strain of M bovis currently circulating in Michigan livestock and wildlife. PROCEDURES In the summer and again in the winter, inoculated blocks were placed in secured outdoor facilities where equal numbers of each block type (2/type/season) were exposed to shade or sunlight. Samples were collected from randomly selected areas on the surface of each block beginning within 1 hour after placement (day 0) twice a day for the first 4 days and once a day from days 7 through 11. Bacterial culture of samples was performed to detect viable M bovis. RESULTS Depending on the exposure conditions, salt blocks yielded viable M bovis for up to 2 days after inoculation and salt-mineral blocks yielded viable M bovis for > 3 days. Survival time was greatest on salt-mineral blocks kept outdoors in the shade during the winter. The odds of recovering viable M bovis from salt-mineral block samples were 4.9 times as great during the winter (vs the summer) and 3.0 times as great with exposure to shade (vs sunlight). CONCLUSIONS AND CLINICAL RELEVANCE Results from this study indicated that salt and salt-mineral blocks should be considered potential sources of bovine tuberculosis when designing risk mitigation programs for cattle herds in areas with wildlife reservoirs of M bovis.

Experimental canine leptospirosis caused by Leptospira interrogans serovars pomona and bratislava.

OBJECTIVE: To evaluate gross, histopathologic, and serum biochemical findings caused by Leptospira interrogans serovars pomona and bratislava inoculated in dogs. ANIMALS: Twenty-seven 8-week-old female Beagles. PROCEDURE: Dogs were randomly assigned to challenge or control groups. Challenge groups were conjunctivally inoculated on 3 successive days with 5 x 10(7) L interrogans serovar pomona (n = 12) or serovar bratislava (11). Clinical signs were recorded throughout the experiment, and clinical pathology assays, bacteriologic culture, and necropsies (6 or 7 dogs necropsied at each time point) were done on postinoculation day (PID) 7, 10, 14, and 20. RESULTS: Infection could not be confirmed in any serovar bratislava-inoculated dog, and control dogs remained healthy throughout the experiment. Positive culture and fluorescent antibody test results were confirmed in 11 of 12 serovar pomona-inoculated dogs. Fever and lethargy starting at PID 7 were the most common clinical signs in serovar pomona-infected dogs. On day 10, gross lesions included multifocal renal and pulmonary hemorrhage and perirenal edema. Serovar pomona-inoculated dogs had histopathologic lesions including hepatitis, interstitial nephritis, and pneumonia at PID 7, 10, 14, and 20. Increases in BUN, anion gap, and bilirubin concentration occurred on PID 10, 14, and 20. Platelet counts in dogs with positive results of bacteriologic culture were decreased from baseline values on PID 10, 12, and 14. CONCLUSIONS AND CLINICAL RELEVANCE: Conjunctival inoculation with L interrogans serovar pomona resulted in a high rate of infection with concomitant hemorrhagic and inflammatory lesions of the kidneys, liver, and lungs.

Effects of positive results for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA on results of caudal fold tuberculin test and interferon-gamma assay for tuberculosis in cattle.

OBJECTIVE: To determine whether cattle testing positive for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA were more likely to have false-positive responses on the caudal fold tuberculin (CFT) test or interferon-gamma (IFN-gamma) assay for Mycobacterium bovis than cattle testing negative for M paratuberculosis. ANIMALS: 1043 cattle from 10 herds in Michigan. PROCEDURE: Feces and blood samples for plasma were collected from cattle > or =24 months old on the day the CFT test was read. Fecal samples were submitted for microbial culture for M paratuberculosis. Plasma samples were tested for antibody against M paratuberculosis, and IFN-gamma after stimulation with purified protein derivative tuberculin from M bovis or M avium. RESULTS: Of 1043 cattle, 180 (17.3%) had positive CFT test results (suspects) and 8 (0.8%) had positive IFN-gamma assay results after stimulation with purified protein derivative tuberculin from M bovis. Forty-five (4.3%) and 115 (11.0%) cattle tested positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA, respectively. Cattle with positive responses for M paratuberculosis appeared to have an increased likelihood of false-positive results on the CFT test, although this association was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: No significant association was detected among cattle testing positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA and positive CFT test and IFN-gamma assay results for M bovis.

Outbreak of leptospirosis among triathlon participants and community residents in Springfield, Illinois, 1998.

We investigated an outbreak of leptospirosis among athletes and community residents after a triathlon was held in Springfield, Illinois. A telephone survey was conducted to collect clinical information and data on possible risk factors, community surveillance was established, and animal specimens and lake water samples were collected to determine the source of the leptospiral contamination. A total of 834 of 876 triathletes were contacted; 98 (12%) reported being ill. Serum samples obtained from 474 athletes were tested; 52 of these samples (11%) tested positive for leptospirosis. Fourteen (6%) of 248 symptomatic community residents tested positive for leptospirosis. Heavy rains that preceded the triathlon are likely to have increased leptospiral contamination of Lake Springfield. Among athletes, ingestion of 1 or more swallows of lake water was a predominant risk factor for illness. This is the largest outbreak of leptospirosis that has been reported in the United States. Health care providers and occupational and recreational users of bodies of freshwater in the United States should be aware of the risk of contracting leptospirosis, particularly after heavy rains.

An improved immunohistochemical diagnostic technique for canine leptospirosis using antileptospiral antibodies on renal tissue.

The purpose of this study was to compare the immunoreactivity in canine renal tissues stained with antisera specific for 3 leptospiral antigens and those processed with traditional staining methods. In addition, immunoglobulin staining was done on tissues with immunoreactivity to leptospiral antigens. Formalin-fixed renal sections from 12 dogs with chronic interstitial nephritis suspected or proven to have leptospirosis (6 dogs with silver-stained leptospires and 6 dogs in which silver-stained leptospires were not detected) were used. Antibodies consisted of a monoclonal antibody to Leptospira kirschneri serovar grippotyphosa lipopolysaccharide (LPS) and 2 polyclonal antibodies to outer membrane proteins, including OmpL1, a leptospiral porin, and LipL41, an outer membrane lipoprotein. The murine monoclonal antisera against LPS (F71C2-1) had the most abundant and consistent immunoreactivity. Immunoreactive areas were present in 6 of 6 sections positive by silver staining and included extracellular granular debris in intertubular areas, debris in macrophages, organisms in tubular lumina, and cytoplasmic granules in tubular epithelia. Antisera with specificity for the outer membrane proteins OmpL1 and LipL41 detected only intact organisms in tubular lumina. Immunoreactivity to OmpL1 (polyclonal 338) occurred in 4 of 5 sections positive by silver staining, but immunoreactivity to LipL41 (polyclonal 813) occurred in only 1 of 6 silver-positive sections. Each of the kidney sections in which leptospiral antigens were detected by immunohistochemistry also was positive by silver staining. Sections negative by silver staining were also negative by immunostaining. Although immunohistochemistry did not enhance sensitivity, amplification of signal by secondary antibody and hematoxylin counterstaining improved the ease of diagnosis and allowed better evaluation of tissue morphology than did silver staining methods. IgG was the most abundant immunoglobulin. IgG immunoreactivity occurred predominantly in plasma cells within interstitial infiltrates. Interstitial infiltrates contained abundant immunoreactivity to LPS, but immunoreactivity to OmpL1 and LipL41 was not noted.

Clinical and pathologic comparison of acute leptospirosis in dogs caused by two strains of Leptospira kirschneri serovar grippotyphosa.

OBJECTIVE: To develop a method for inducing acute leptospirosis in dogs. ANIMALS: 31 nine-week-old female Beagles. PROCEDURE: Beagles were randomly assigned to 2 inoculation groups or a control group. Dogs were inoculated on 3 successive days by conjunctival instillation of 5 x 10(7) cells of Leptospira kirschneri serovar grippotyphosa strain 82 (12 dogs) or strain RM 52 (14 dogs). Control dogs (n = 5) were similarly inoculated with sterile leptospiral culture media. Clinical signs, clinicopathologic variables, anti-leptospiral antibody titers, and evidence of leptospires in tissues and body fluids were evaluated. Dogs were euthanatized and necropsied on days 7, 14, 22, or 28 after inoculation or as required because of severe illness. RESULTS: Clinical signs in infected dogs included conjunctivitis, lethargy, diarrhea, dehydration, vomiting, and icterus. Consistent clinicopathologic alterations included azotemia, hyperphosphatemia, increased anion gap, hyperbilirubinemia, and an increase in alkaline phosphatase activity. Leptospires were cultured from the kidneys (11/12), urine (6/9), aqueous humor (9/12), blood (12/12), and liver (12/12) of dogs inoculated with strain 82. Only 3 of 14 dogs became infected after inoculation with strain RM 52. Histopathologic lesions in infected dogs included interstitial nephritis, renal tubular degeneration and necrosis, pulmonary hemorrhage, and hepatic edema and perivasculitis. CONCLUSIONS AND CLINICAL RELEVANCE: Conjunctival exposure to L kirschneri serovar grippotyphosa strain 82 resulted in acute leptospirosis in all inoculated dogs, but only 3 of 14 dogs inoculated with strain RM 52 became infected. This method of infection by serovar grippotyphosa can be used to study the pathogenesis and prevention of leptospirosis in dogs.

Clinical features and risk factors for development of urinary tract infections in cats.

The clinical and diagnostic features of 155 cats with urinary tract infection (UTI) and 186 controls with negative urine culture/s were characterized retrospectively (signalment, clinical signs, urinalysis, urine culture, concurrent diseases, lower urinary tract diagnostic/therapeutic procedures). Multivariable logistic regression was used to identify risk factors associated with UTI. Cats of all ages were affected by UTI with no sex/breed predisposition. Lower urinary tract signs were absent in 35.5% of cats with UTI. Pyuria and bacteriuria had sensitivities of 52.9% and 72.9%, and specificities of 85.5% and 67.7% for detection of UTI, respectively. Risk factors significantly associated with increased odds of UTI were urinary incontinence [odds ratio (OR)=10.78, P=0.0331], transurethral procedures (OR=8.37, P<0.0001), urogenital surgery (OR=6.03, P=0.0385), gastrointestinal disease (OR=2.62, P=0.0331), decreased body weight (OR=0.81, P=0.0259) and decreased urine specific gravity (OR=0.78, P=0.0055). Whilst not independently significant, renal disease and lower urinary tract anatomic abnormalities improved statistical model performance and contributed to UTI.

Use of the intradermal tuberculin test in a herd of captive elk (Cervus elaphus nelsoni) naturally infected with Mycobacterium bovis.

In the United States, tuberculosis of captive cervids, caused by Mycobacterium bovis, attracted attention in 1991 when investigations, prompted by the identification of a tuberculous elk (Cervus elaphus nelsoni) of U.S. origin exported to Canada, revealed tuberculosis in 10 different elk herds in 8 different states. Based on methods used in cattle, official regulations pertaining to testing and eradication of tuberculosis in captive cervids were added to the U.S. Department of Agriculture bovine tuberculosis eradication effort in 1994. However, little published information exists on the accuracy of intradermal tuberculin testing in naturally infected cervids. Evaluation of a captive herd of 71 animals in Wisconsin included postmortem examination and tissue sample collection from both tuberculin test responders and nonresponders. Within this captive herd, of admittedly small size, results showed the single cervical test to have a sensitivity of 88% and a specificity of 69%. Evaluation of diagnostic tests in the species of interest is important, as extrapolation of data obtained from other species may not be appropriate.

A Study of the Persistence of Mycobacterium bovis in the Environment under Natural Weather Conditions in Michigan, USA.

Reisolation of Mycobacterium bovis from inoculated substrates was used to follow the persistence of viable M. bovis bacteria exposed to natural weather conditions over a 12-month period. Environmental factors were recorded continuously, and factors affecting M. bovis persistence (i.e., temperature, season, and substrate) were studied using survival analysis and Cox's proportional hazards regression. Persistence of M. bovis in the environment was significantly shorter in the spring/summer season, characterized by the highest average daily temperatures over the 12-month period. M. bovis persisted up to 88 days in soil, 58 days in water and hay, and 43 days on corn. These studies demonstrate that M. bovis bacteria persist long enough to represent a risk of exposure for cattle and/or wildlife and strengthen evidence that suggests cattle farm biosecurity and efforts to eliminate supplemental feeding of white-tailed deer will decrease the risk of bovine TB transmission among and between cattle and deer populations.

Novel 45-kilodalton leptospiral protein that is processed to a 31-kilodalton growth-phase-regulated peripheral membrane protein.

Leptospiral protein antigens are of interest as potential virulence factors and as candidate serodiagnostic and immunoprotective reagents. We identified leptospiral protein antigens by screening a genomic expression library with serum from a rabbit hyperimmunized with formalin-killed, virulent Leptospira kirschneri serovar grippotyphosa. Genes expressing known outer membrane lipoproteins LipL32 and LipL41, the heat shock protein GroEL, and the alpha, beta, and beta' subunits of RNA polymerase were isolated from the library. In addition, a new leptospiral gene that in Escherichia coli expressed a 45-kDa antigen with an amino-terminal signal peptide followed by the spirochetal lipobox Val(-4)-Phe(-3)-Asn(-2)-Ala(-1) (downward arrow)Cys(+1) was isolated. We designated this putative lipoprotein LipL45. Immunoblot analysis of a panel of Leptospira strains probed with LipL45 antiserum demonstrated that many low-passage strains expressed LipL45. In contrast, LipL45 was not detected in high-passage, culture-attenuated strains, suggesting that LipL45 is a virulence-associated protein. In addition, all leptospiral strains tested, irrespective of culture passage, expressed a 31-kDa antigen that was recognized by LipL45 antiserum. Southern blot and peptide mapping studies indicated that this 31-kDa antigen was derived from the carboxy terminus of LipL45; therefore, it was designated P31(LipL45). Membrane fractionation studies demonstrated that P31(LipL45) is a peripheral membrane protein. Finally, we found that P31(LipL45) levels increased as Leptospira entered the stationary phase, indicating that P31(LipL45) levels were regulated. Hamsters infected with L. kirschneri formed an antibody response to LipL45, indicating that LipL45 was expressed during infection. Furthermore, the immunohistochemistry of kidneys from infected hamsters indicated that LipL45 was expressed by L. kirschneri that colonized the renal tubule. These observations suggest that expression of LipL45 responds to environmental cues, including those encountered during infection of a mammalian host.

Evaluation of type 1 immune response in naïve and vaccinated animals following challenge with Leptospira borgpetersenii serovar Hardjo: involvement of WC1(+) gammadelta and CD4 T cells.

Organisms within the Hardjo serovar of Leptospira species are harbored in cattle throughout the world, causing abortion in pregnant animals as well as being shed in the urine, thereby providing sources of zoonotic infection for humans. We recently showed that sterile immunity in vaccinated cattle is associated with induction of a type 1 (Th1) cell-mediated immune response. Here naïve and previously vaccinated pregnant cattle were challenged with a virulent strain of serovar Hardjo and subsequently evaluated for expression of a type 1 immune response. Lymphocytes that responded in a recall response to antigen by undergoing blast transformation were evident in cultures of peripheral blood mononuclear cells (PBMC) from vaccinated cattle throughout the postchallenge test period while those from naïve cattle were evident at one time point only. Nevertheless, beginning at 2 weeks after challenge, gamma interferon (IFN-gamma) was measured in supernatants of antigen-stimulated PBMC cultures from nonvaccinated animals although the amount produced was always less than that in cultures of PBMC from vaccinated animals. IFN-gamma(+) cells were also evident in antigen-stimulated cultures of PBMC from vaccinated but not from nonvaccinated animals throughout the postchallenge period. The IFN-gamma(+) cells included CD4(+) and WC1(+) gammadelta T cells, and a similar proportion of these two subpopulations were found among the dividing cells in antigen-stimulated cultures as ascertained by carboxyfluorescein succinimidyl ester loading. Finally, while naïve and vaccinated animals had similar levels of antigen-specific immunoglobulin G1 (IgG1) following challenge, vaccinated animals had twofold-more IgG2. In conclusion, while infection may induce a type 1 response we suggest that it is too weak to prevent establishment of chronic infection.