Experimental Leptospira borgpetersenii serovar hardjo infection of pregnant cattle

Objective To observe the effect upon the foetus of experimental infection of pregnant cattle with Leptospira borg-petersenii serovar hardjo .
Design A disease transmission study using pregnant cattle.
Procedure Fourteen heifers serologically negative to L hardjo were artificially inseminated and later challenged with a north-Queensland isolate of L hardjo by conjunctival inoculation. The heifers were serologically monitored and their urine examined for the presence of leptospires using culture and fluorescent-antibody tests at appropriate intervals. Elective caesarean sections were performed on pregnant heifers at 6.5 weeks after the challenge. Foetuses were examined using serological, histopathological, microbiological and fluorescent-antibody tests.
Results Ten of the heifers became pregnant, but three subsequently aborted before challenge. After challenge, all 14 heifers seroconverted and L hardjo was isolated from the urine of 6 of the 7 pregnant heifers. No evidence of foetal L hardjo infection was detected. Two of the foetuses had histopatho-logical lesions consistent with Neospora s p infection.
Conclusion It is likely that the isolate of L hardjo used in this study does not normally infect the foetus. Neospora s p may be a more significant cause of bovine reproductive wastage.     

Production and characterization of monoclonal antibodies specific for Leptospira borgpetersenii serovar hardjo type hardjobovis and Leptospira interrogans serovar hardjo type hardjoprajitno.

Murine monoclonal antibodies were produced by immunizing BALB/c mice with a killed whole-cell antigen prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis. Six of these antibodies recognized epitopes on the homologous antigen and on whole-cell antigen prepared from Leptospira interrogans serovar hardjo type hardjoprajitno. These antibodies did not cross-react with whole-cell antigens prepared from L. borgpetersenii serovar sejroe, 10 other pathogenic Leptospira serovars, or the saprophytic Leptospira biflexa serovar patoc. Three other monoclonal antibodies reacted with antigens prepared from the 2 hardjo serovars and serovar sejroe but not with antigens from the 10 other pathogenic serovars, or serovar patoc. The epitopes recognized by all of the hardjo-specific antibodies and 2 of the 3 hardjo/sejroe-specific antibodies were susceptible to sodium meta-periodate oxidation. All of the antibodies were characterized by Western blots with the hardjobovis whole-cell antigen. Each of the 9 monoclonal antibodies was inhibited from binding to the hardjobovis antigen by bovine sera which were obtained from cattle experimentally infected with hardjobovis and from field cattle, with anti-serovar hardjo microscopic agglutination test antibody titres ranging from 100 to 12800. Some of these antibodies may be suitable for incorporation into competitive enzyme immunoassays for the specific detection of antibodies to either of the hardjo serovars.     

Amoxycillin as an alternative to dihydrostreptomycin sulphate for treating cattle infected with Leptospira borgpetersenii serovar hardjo

Objective To assess the effect of amoxycillin treatment on urinary excretion of leptospires from cattle infected with Leptospira borgpetersenii serovar hardjo .
Design A chemotherapy trial with controls.
Procedure Fourteen heifers serologically negative to L hardjo were inoculated with L hardjo via the conjunctival route and assessed for evidence of infection by serological, fluorescent antibody and microbiological tests. Two injections (48 h apart) of amoxycillin at a dose of 15 mg/kg were administered intramuscularly to seven heifers 6.5 weeks after infection; the remaining heifers acted as untreated controls. Later, these seven control group heifers were treated with a single dose of amoxycillin (15 mg/kg). Samples of urine were collected before and after amoxycillin treatments; kidneys were collected at slaughter, and examined by fluorescent antibody test and microbiological culture.
Results Leptospires were isolated from the urine of 11 of 14 heifers inoculated with L hardjo . After treatment of six of these with two injections of amoxycillin, leptospires were not isolated. Of the controls, four of the five initially leptospiruric heifers continued to shed leptospires; after a single injection of amoxycillin, no leptospires were detected in the kidneys of these four.
Conclusion Amoxycillin may be an acceptable alternative to dihydrostreptomycin sulphate for the treatment of cattle infected with L hardjo .     

Evaluation of antibiotics for treatment of cattle infected with Leptospira borgpetersenii serovar hardjo

OBJECTIVE: To evaluate antibiotics for treatment of cattle with leptospirosis caused by Leptospira borgpetersenii serovar hardjo. DESIGN: Randomized controlled trial. ANIMALS: 42 healthy mixed-breed cattle. PROCEDURE: Cattle were inoculated via conjunctival instillation with L. borgpetersenii serovar hardjo. After infection and urinary shedding of L. borgpetersenii were confirmed, cattle were treated with various antibiotics. To determine effectiveness of antibiotic treatment, urinary shedding of L. borgpetersenii was monitored for 4 to 6 weeks after administration of antibiotics, using darkfield microscopic examination, microbial culture, immunofluorescence testing, and a polymerase chain reaction assay. RESULTS: All inoculated cattle developed leptospirosis and shed leptospires in their urine. The following antibiotic treatments resulted in elimination of urinary shedding of leptospires: a single injection of oxytetracycline (20 mg/kg 19 mg/lb] of body weight, IM), tilmicosin (10 mg/kg [4.5 mg/lb], SC), or a combination product that contained dihydrostreptomycin-penicillin G (25 mg/kg [11.4 mg/lb], IM) or multiple injections of ceftiofur sodium (2.2 or 5 mg/kg [1 or 2.3 mg/lb], IM, once daily for 5 days, or 20 mg/kg, IM, once daily for 3 days). CONCLUSIONS AND CLINICAL RELEVANCE: Successful resolution of leptospirosis in cattle by administration of dihydrostreptomycin-penicillin G confirms results obtained by other investigators. Three other antibiotics (oxytetracycline, tilmicosin, and ceftiofur) also were effective for resolving leptospirosis and may be useful substitutes for dihydrostreptomycin, an antibiotic that is no longer available for use in food-producing animals in the United States. Cost, safety, and withdrawal times of these various treatment options need to be considered.     

The efficacy of a Leptospira interrogans serovars pomona and copenhageni and L. borgpetersenii serovar hardjo vaccine in cattle

An experimental, trivalent, bovine, leptospiral vaccine, containing inactivated Leptospira interrogans serovars pomona and copenhageni and L. borgpetersenii serovar hardjo Hardjobovis, was developed. The experimental vaccine was shown to protect hamsters against virulent challenge with each of the component serovars. In a serological efficacy test in cattle, the experimental vaccine was compared for bioequivalence with a similar product, registered in New Zealand for veterinary use. The experimental vaccine induced higher titres in cattle than the latter mentioned product.     

Leptospira borgpetersenii serovar hardjo type hardjobovis in bovine embryos fertilized in vitro.

The association of Leptospira borgpetersenii serovar hardjo type hardjobovis with bovine embryos produced by in vitro fertilization was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Morula stage embryos with an intact zona pellucida (ZP) were exposed to this spirochete for 24 h in culture medium, washed by the standard washing procedure as recommended by the International Embryo Transfer Society, and then examined. SEM showed typical helicoid leptospires on the surface and in the pores of the ZP. TEM showed cross and longitudinal sections of leptospires in the matrix and channels of the ZP, in the perivitelline and intercellular spaces, on the vitellus and in the embryonic cells. Some of the embryos that were penetrated showed damage to the membranes and the cytoplasm. The ineffectiveness of the washing procedure, for the removal of hardjobovis from exposed embryos may be of importance to the industry.     

Use of a monovalent leptospiral vaccine to prevent renal colonization and urinary shedding in cattle exposed to Leptospira borgpetersenii serovar hardjo.

OBJECTIVE: To determine whether a monovalent Leptospira borgpetersenii serovar hardjo (type hardjobovis) vaccine commercially available in Australia, New Zealand, Ireland, and the United Kingdom would protect cattle from renal colonization and urinary shedding when exposed to a US strain of Leptospira borgpetersenii serovar hardjo. ANIMALS: 24 Hereford heifers that lacked detectable antibodies against serovar hardjo. PROCEDURE: Heifers received 2 doses, 4 weeks apart, of the commercial hardjo vaccine (n = 8) or a monovalent US reference hardjo vaccine (8) or were not vaccinated (controls; 8). Heifers were challenged 16 weeks later by intraperitoneal inoculation or conjunctival instillation. Serum antibody titers were measured weekly, and urine samples were examined for leptospires. Heifers were euthanatized 11 to 14 weeks after challenge, and kidney tissue was examined for evidence of colonization. RESULTS: All 8 heifers vaccinated with the reference vaccine were found to be shedding leptospires in their urine and had evidence of renal colonization. All 4 control heifers challenged by conjunctival instillation and 2 of 4 control heifers challenged by intraperitoneal inoculation shed leptospires in their urine, and all 8 had evidence of renal colonization. In contrast, leptospires were not detected in the urine or tissues of any of the 8 heifers that received the commercial hardjo vaccine. Heifers that received the commercial hardjo vaccine had significantly higher antibody titers than did heifers that received the reference vaccine. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cattle that received 2 doses of the commercial hardjo vaccine were protected against renal colonization and urinary shedding when challenged with L borgpetersenii serovar hardjo strain 203 four months after vaccination.     

Evaluation of two antimicrobial therapies in the treatment of Leptospira borgpetersenii serovar hardjo infection in experimentally infected cattle

This study demonstrated the ability of the antimicrobials tulathromycin (Draxxin) and ceftiofur crystalline free acid sterile suspension (Excede) to clear the spirochete Leptospira borgpetersenii serovar hardjo type hardjo-bovis (L. hardjo-bovis) from experimentally infected cattle. Treatment with tulathromycin resulted in clearance of L. hardjo-bovis organisms from the urine and kidney tissue of all animals (9 of 9), and treatment with ceftiofur crystalline free acid resulted in clearance of the organisms from the urine of 8 of 10 heifers and the kidney tissue of all 10 animals. In contrast, 10 of 10 placebo-treated cattle had L. hardjo-bovis organisms in their urine and 8 of 10 had the organisms in kidney tissue.     

Development of a monoclonal antibody-based competitive enzyme-linked immunosorbent assay for the detection of Leptospira borgpetersenii serovar hardjo type hardjobovis antibodies in bovine sera.

A murine monoclonal antibody (designated M553) that binds to an epitope on whole cell antigens prepared from Leptospira borgpetersenii serovar hardjo type hardjobovis and Leptospira interrogans serovar hardjo type hardjoprajitno, was produced and incorporated into a competitive enzyme-linked immunosorbent assay for the detection of bovine antibodies to serovar hardjo. The epitope recognized by M553 was susceptible to periodate oxidation. The M553 antibody was characterized by western blot with hardjobovis whole cell antigen. This antibody does not cross-react with whole cell antigens prepared from 11 other pathogenic Leptospira serovars, or, Leptospira biflexa serovar patoc. The sensitivity estimate of the competitive ELISA was 100% with field sera (n = 165) with serovar hardjo microscopic agglutination test (MAT) titres of > or = 100. The specificity estimate was 100% with sera (n = 128) obtained from a specific pathogen free herd of cattle that were negative in the MAT at a dilution of 1:100 for serovars hardjo, pomona, sejroe, copenhageni, canicola, and grippotyphosa. The specificity estimate with field sera (n = 301) with serovar hardjo MAT titres of < 100, was 98% (95% confidence interval = +/- 1.58%). There was no cross-reactivity with field sera (n = 306) with serovar pomona titres > or = 100 and serovar hardjo titres < 100. The specificity estimate with the combined populations of sera with serovar hardjo MAT titres of < 100 (n = 735) was 99.18% (95% confidence interval = +/- 0.65%). There was a high level of agreement (kappa = 0.977) between the results of the competitive ELISA and those of the MAT.     

Antimicrobial drug susceptibility of Leptospira interrogans serovar hardjo isolated from cattle.

The susceptibility to commonly used drugs of 18 isolates of Leptospira hardjo from the kidneys of feedlot cattle from different sources was determined quantitatively. All isolates were susceptible to penicillin G, ampicillin, tetracycline, erythromycin and streptomycin. Susceptibility to sulphamethazine was ambiguous. No drug resistance was detected and the results were similar to those described for other serovars.     

Restriction endonuclease analysis of Leptospira interrogans serovar hardjo isolates from cattle

The genomes of 253 strains of Leptospira interrogans serovar hardjo were examined by restriction endonuclease analysis of their DNA. The strains had been isolated from cattle at an abattoir (190), milk of agalactic cows (seven) and from aborted bovine fetuses (56). Two distinct genome types, Hardjoprajitno and Hardjobovis, were detected. The majority (91 per cent) of isolates from abattoir cattle were of the Hardjobovis type while most (76 per cent) of the isolates from clinical/pathological material were similar to Hardjoprajitno.     

Comparison of three techniques to detect Leptospira interrogans serovar hardjo type hardjo-bovis in bovine urine

Nucleic acid hybridization, bacteriologic culture, and a fluorescent antibody test were compared for detection of Leptospira interrogans serovar hardjo type hardjo-bovis in bovine urine. Seventy-five urine samples were collected from pregnant cows challenge exposed with type hardjo-bovis. Twenty samples were collected from steers not exposed to hardjo-bovis. Sediments from each sample were examined, using fluorescent antibodies and a repetitive sequence element nucleic acid probe, to detect the presence of leptospires. Urine samples were processed for bacteriologic culture, using standard techniques. Under laboratory conditions typically used for these techniques, leptospires were detected in 60 of 75 urine samples from challenge exposed cows by nucleic acid hybridization, in 24 samples by fluorescent antibody test, and in 13 samples by bacteriologic culture. Leptospires were not detected in the urine of steers not exposed to hardjo-bovis.     

Prevalence of Leptospira interrogans serovar hardjo antibodies in milk in Belgian dairy herds

Pooled milk samples were collected from 2000 Belgian dairy herds in the autumn of 1989. Antibodies against Leptospira interrogans serovar hardjo were detected in 9.2% of the herds, with the incidence being higher in the southern part of the country.     

Protection of cattle against natural challenge with Leptospira interrogans serovar hardjo using a hardjo-pomona vaccine

The hardjo component of a bivalent hardjo/pomona vaccine prepared from cultures grown in a protein-free medium was tested in calves which experienced a natural challenge about 7 months after vaccination. A significant degree of protection was obtained using either seroconversion or positive urine cultures as in-dicators of infection. Seroconversion occurred in 10/10 non-vaccinates and 2/9 vaccinates (p <0.01), Leptospires of serovar hardjo were isolated from the urine of 6/10 non-vaccinated calves and 0/9 vaccinated animals (p <0.05).