Phage typing of staphylococci isolated from dairy cows in New Zealand

Four red deer calves (Cervus elaphus) died with severe nephritis apparently associated with infection by Leptospira interrogans serovar pomona. The sera of 12 in-contact red deer calves were examined for leptospiral agglutinins and nine showed titres to pomona consistent with recent infection. Two also showed titres of 1:100 to serovar hardjo. The urine of five of these in-contact calves was examined periodically over a period of nine months. All were initially leptospiruric, four being infected with pomona and one with hardjo. In four animals leptospiruria could only be detected for up to six months, but one animal infected with pomona was leptospiruric for at least eight months.

The apparent source of infection was from infected cattle, and it is suggested that deer are unlikely to act as maintenance hosts for serovar pomona.     

Haemolytic disease associated with Leptospira interrogans serovar pomona in red deer calves (Cervus elaphus)

Three red deer calves (Cervus elaphus) died with a haemolytic disease associated with infection by Leptospira interrogans serovar pomona. Infection within the herd was more prevalent than disease. Sera from 16 herd mates were tested by the microscopic agglutination test (MAT) and 12 had leptospiral titres, the majority to serovar pomona. A few calves had titres to balcunica and hardjo. Urine was obtained for culture from six of these calves and serovar pomona was isolated from five with titres to pomona, and hardjo from one with a titre to hardjo but not pomona. A fourth calf died with severe nephritis but a diagnosis of leptospirosis was not confirmed in this case.     

Serological responses of farmed red deer to a vaccine against Leptospira interrogans serovars hardjo and pomona

Six adult, female, red deer were vaccinated with a hardjo-pomona vaccine followed by a second vaccination four weeks later and thereafter, at yearly intervals for two years. Serological responses were determined at intervals using a standard microscopic agglutination test. Increased responses to hardjo were observed in two deer which were seropositive to this serovar before vaccination compared to those which were initially seronegative. Some initially seronegative deer developed titres to hardjo and pomona ranging from 1:24 to 1:48 following the first dose of vaccine, and all deer produced a serological response to both serovars ranging from 124 to 1:96 following the second vaccination except one animal which failed to produce a pomona titre of 124 or greater. Responses to annual revaccinations were more variable both within and between years. Most deer produced titres which ranged from 1:24 to 1:96 for three to live months, although some deer failed to seroconvert following the annual revaccinations. Peak responses were similar to those recorded in cattle following an identical vaccination programme with the same vaccine but titres fell to undetectable levels after three to live months whereas titres reported in cattle generally persisted for at least 12 months.     

Observations of leptospirosis in farmed deer

AIMS: Slaughterhouse and on-farm surveys were undertaken to investigate some aspects of leptospirosis (Leptospira interrogans) in farmed deer in the lower North Island of New Zealand. METHODS: Blood samples and kidneys were collected at slaughter from 601 l-year and older red and red X Wapiti stags and 21 adult hinds from 53 farms (10 or 12 deer per farm). Serum samples were analysed for up to seven Leptospiral serovars. Gross and histological examinations of kidneys were undertaken. Kidneys from 202 deer were cultured for leptospires. A follow-up postal questionnaire (68% response) indicated one herd had been vaccinated prior to the survey. Serological analyses were also carried out on serum bank samples from a previous on-farm survey involving male and female weaner, yearling and adult red deer from 16 commercial deer farms in March and November. RESULTS: Serological reactions at titres > or = 96 to serovar hardjo were present in 73.6%, pomona in 41.5%, copenhageni in 11.3% and tarassovi in 15.1% of farms from the slaughterhouse survey. Antibodies to serovars australis, ballam and balanica were present in three, one and four of six herds studied, respectively. Titre prevalence to hardjo was higher than that of pomona and other serovars within farms. Cultures for Leptospira were positive in 10 stags from six lines with similar prevalence across age groups. Histological examination showed many gross lesions were associated with mild interstitial cellular infiltration characteristic of subclinical Leptospiral infections. Some sections from culture-positive kidneys contained spirochetes in renal tubules. The on-farm survey showed a 10-30% within-herd prevalence of pomona and hardjo titres in 56% of 3-month-old deer herds, but by 11 months of age, 100% of herds were titre-positive with high prevalences to one or both serovars. Concurrently, herds of 1-year-old and adult deer on the same farms were all seropositive. CONCLUSION: This study has shown that Leptospiral infections are common in farmed deer in the survey area.     

An unusual serological response in cattle after use of a commercial leptospiral combined hardjo-pomona vaccine

Vaccination of four calves with Leptavoid (Wellcome New Zealand Limited) gave rise to Leptospira interrogans serovars hardjo and pomona microscopic agglutination test titres that could not he distinguished in magnitude from post-infection titres. Vaccination of four calves with Lepto-3 (ICI Tasman Limited) gave rise to much lower titres. Revaccination of cows with Leptavoid caused a rise in hardjo titres which was significantly greater than after use of Lepto-3. The possibility that titres were due to the simultaneous infection with serovars pomona and hardjo of only the animals vaccinated with Leptavoid must be discounted.     

A serological study of bovine leptospirosis in the district of Taranaki

A cross-sectional serological survey of dairy cattle in Taranaki in 1979-80 indicated that 62% (551/891) of the animals had evidence of Leptospira interrogans serovar hardjo infection as disclosed by the microscopic agglutination test. Titres to Leptospira interrogans serovar pomona were demonstrated in only 4% (23/591) of the animals examined. The high prevalence of hardjo infection is suggestive of an endemic infection whilst the low level to pomona is indicative of sporadic infection. In a detailed examination of 10 herds, 9 revealed high (55%-91%) prevalence of serological reactions to hardjo and the herd profiles of titres, indicated that the animals had become infected at one to two years of age. A field strain of hardjo from cattle as well as the usual laboratory strain (hardjoprajitno) was incorporated in the test but there were no significant differences between the results given by the two antigens.     

Seroprevalence and association with abortion of leptospirosis in cattle in Ontario.

Sera were collected using a systematic random sampling from 348 cattle herds in Ontario, in proportion to the cattle population in different areas. One cow in five from 296 dairy herds and one in three from 52 beef herds were sampled. The sera were analyzed for prevalence of antibodies to Leptospira interrogans serovar grippotyphosa, hardjo, icterohaemorhagiae and pomona using the microscopic agglutination test. Herd seroprevalence (one or more animals with titer greater than or equal to 80) in beef and dairy herds combined was grippotyphosa 2%, hardjo 13.8%, icterohaemorrhagiae 10.1% and pomona 25.8%; 39% of all herds showed evidence of leptospiral infection with one or more serovars; 44.2% of 52 beef herds had serological evidence of infection with serovar hardjo compared to 8.4% of 296 dairy herds (P less than 0.0001). Seroprevalence of other serovars was not significantly different between beef and dairy herds. The proportion of beef animals seropositive for hardjo and for pomona increased with age, particularly for hardjo; 26.5% of beef animals aged nine years or over were seropositive for hardjo. Dairy animals showed a significant rise of hardjo but not pomona titers with age. The seroprevalence of pomona infection was significantly higher in dairy cattle in eastern Ontario than in other regions. Thirty-four (6.1%) of 553 aborted bovine fetuses had leptospires detected by immunofluorescence techniques. Sixty-five percent of these fetuses were from submissions made between November and January. Leptospires were identified as serovar hardjo by specific immunofluorescence. There appeared, however, to be a paradoxical serological response in that eight aborting cows had antibody titers to pomona rather than hardjo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age and the ability of calves to respond to a leptospiral vaccine

The serological responses of calves at two different ages to a commercial hardjo/pomona vaccine were examined. Microscopic agglutination test (MAT) titres of a group of ten three-month-old calves and three groups of 11,12 and 12 six-month-old calves were monitored over a period of 28 weeks. The calves vaccinated at three months of age had a poorer serological response rate to vaccination (30% responded to pomona and 40% responded to hardjo) compared with those vaccinated at six months of age (91-100% responded to pomona and 83-91% responded to hardjo). Those three-month-old calves which did respond also produced a lower level of-antibody than six-month-old calves. It was concluded that in order to achieve adequate protection using commercially available leptospiral bacterins at the recommended dose, vaccination should not be undertaken routinely with animals less than six months of age. Where circumstances arise in which earlier vaccination is necessary, repeat vaccination for continued protection at six months or older is suggested.     

The influence of maternal antibody and age of calves on effective vaccination against Leptospira interrogans serovar hardjo

Twelve seronegative cows were vaccinated with an experimental bivalent Leptospira interrogans serovars hardjo and pomona vaccine late in their first pregnancy. Calves born of these dams were divided into 4 equal groups that received this vaccine at 4, 6, 10 and 18 weeks of age, respectively. Before vaccination the group geometric mean titres of maternally-derived circulating antibodies ranged from 2 to 25 for the microscopic agglutination (MA) test and 3 to 35 for enzyme-linked immunosorbent assay (ELISA) using a serovar hardjo outer envelope antigen. Post-vaccination peak titres were 645 to 1612 for MA and 562 to 1037 for ELISA, respectively. Calves vaccinated at the youngest age, had the highest pre-vaccination circulating maternal antibody titres, but showed the smallest rise in post-vaccination antibody titres. Circulating maternal antibody was detected in calves up to 13 weeks of age. All immunised calves were protected against a virulent challenge with serovar hardjo type Hardjobovis, regardless of their age or maternally-derived antibody titres. These findings indicate that calves as young as 4 weeks old, vaccinated in the presence of maternally-derived antibody, can be fully protected against homologous virulent challenge.     

The epidemiological interpretation of serological responses to leptospiral serovars in sheep

Serum samples were examined for evidence of leptospiral agglutinins from 928 sheep from 45 lines and kidneys from 12 of these lines for evidence of leptospiral infection. All sheep had been submitted for slaughter at meat works in the Manawatu. Serological results were analysed using the results at a minimum serum dilution in the microscopic agglutination test (MAT) of 1:24 and at a minimum dilution of 1:48. It was shown that a minimum dilution of 1:24 resulted in many non-specific or cross-reactions. A minimum dilution of 1:48 was more accurate for detecting the serological prevalence of specific agglutinins to leptospires in ovine sera. Twenty percent of the sheep had titres of 1:48 or greater to hardjo, 3.8% to pomona, 2.6% to tarassovi, 2.3% to copenhageni and 2.7% to ballum. No titres of 1:48 or greater to australis were detected. Serovar hardjo was isolated from the kidneys of three animals in one line. Eighteen months later 291 serum samples and 95 urine samples were collected from live animals on the property from which the three hardjo infected animals originated. No titres to hardjo were detected in the sera of lambs, but a serological prevalence of 44% and 84% to this serovar was demonstrated in the hoggets and ewes respectively. No leptospires were demonstrated in any of the urine samples. These results show that sporadic infection of sheep with hardjo can occur but they also indicate that infection with this serovar is not endemic and that sheep are unlikely to act as maintenance hosts for hardjo in New Zealand.     

Development and initial evaluation of an indirect enzyme-linked immunosorbent assay for the detection of Leptospira interrogans serovar hardjo antibodies in bovine sera.

Outer sheath antigen from Leptospira interrogans serovar hardjo type hardjoprajitno and acetic acid extracted antigens from serovar hardjo types hardjoprajitno and hardjobovis were evaluated in an immunoassay for ability to detect hyperimmune rabbit serum to serovar hardjo. The degree of cross-reactivity with hyperimmune rabbit sera to L. interrogans serovars pomona, copenhageni, grippotyphosa, canicola and sejroe, and Leptospira biflexa serovar patoc was also measured for each antigen. All of the antigens reacted with the antiserum to L. interrogans serovar hardjo. The outer sheath antigen however, also showed wide cross-reactivity with the antisera to all of the serovars of L. interrogans tested and with the antiserum to L. biflexa serovar patoc. The acetic acid extracted antigen from either type hardjoprajitno, or type hardjobovis, showed a high degree of specificity for serovar hardjo antiserum. The hardjobovis acetic acid extracted antigen was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting, and was incorporated into an indirect ELISA for detection of anti-serovar hardjo antibodies in bovine serum. This ELISA showed a relative specificity of 100% with 156 bovine sera which were negative at a dilution of 1:100 in the microscopic agglutination test (MAT) for L. interrogans serovars hardjo, pomona, sejroe, icterohaemorrhagiae, copenhageni, canicola, and grippotyphosa. The relative sensitivity of this assay with 192 bovine sera which had serovar hardjo MAT titres of > or = 100 was 95.3% (95% confidence limit = 2.99%). The degree of cross-reactivity with 289 bovine sera which had serovar pomona MAT titres of > or = 100 (with no detectable serovar hardjo MAT titres) was approximately 1.0%. This assay was: easily standardized, scored objectively, repeatable, semi-automated and used a non-hazardous antigen that can be routinely prepared in gram amounts.     

Leptospirosis: I. Clinical investigation of the infection in dairy cattle in the Waikato district of New Zealand

An investigation was made into the prevalence of leptospiral infection in cattle. An area 50 km radius was selected in a region where leptospirosis was reputedly common. Farmers volunteered 250 herds with 39 500 cows for testing and 7 500 animals were selected and sampled. Twenty-nine cows (0.4%) on 14 (5.6%) of the farms had leptospiruria at the first examination. Leptospirae were cultured from the urines of nine of these animals and all were Leptospira interrogans serovar hardjo. Serologically 12.5% of cows had titres of 1:200 or greater to hardjo and 3.5% titres of 1:200 or greater to pomona. In the Spring of 1977, there was evidence of clinical leptospirosis in calves associated with only one of the herds and no clinical leptospirosis in the 250 lactating herds, although leptospiral titres were found in 88% of them. This indicated that clinical disease was much less common than infection. We concluded that leptospirosis was of minor economic importance in dairy cattle, although it could be significant in individual herds, and a health hazard to farm workers.     

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.     

Leptospirosis: II. Investigation of clinical disease in dairy cattle in the Waikato district of New Zealand

Investigations were carried out in 1975, 1976 and 1977 in 16 dairy herds where leptospiral abortions were suspected and in five other herds where clinical disease was not present. Both Leptospira interrogans serovars pomona and hardjo were isolated from cattle in herds with leptospirosis, but only pomona was recovered from those that had aborted. There was no evidence that hardjo caused clinical disease in dairy cattle in the Waikato district. It was found that 73% of the cows that aborted and 19% of other animals in the same herds had microscopic agglutination test titres to pomona of 1:2,000 or greater. By contrast, only 2% of cattle in herds without clinical evidence of leptospirosis had such titres. One cow retained a titre of 1:2,000 or greater to pomona for 7 months; titres of this order had a shorter duration in other cows. Leptospiruria occurred in 50% of cows that had aborted and in 9% of in-contact cows in the same herds. Only 0.7% of cows had leptospiruria in the herds with no clinical disease. Ten of 35 cows shedding pomona still had leptospiruria one month later. It was concluded that clinical leptospirosis should be diagnosed by testing a sample of the herd, rather than just individual cows, because of the variability and persistence of leptospiruria and serological titres in cows with and without clinical signs. Although hardjo is common in cattle in the Waikato district, it was not found to cause abortion in cattle.     

Prevention of Leptospira interrogans serovar pomona infection in cattle

A commercial hardjo-pomona vaccine which has previously been shown to be effective against hardjo infection was tested against pomona. Following challenge all 11 six-month-old non-vaccinated calves seroconverted and pomona was isolated from blood or urine on at least one occasion from nine of them. Pomona was isolated once only, on the third day after challenge, from the blood of one of 11 vaccinated calves.     

Dual infections of red deer (Cervus elaphus) by Leptospira interrogans serovars copenhageni and hardjo

On a property in the Nelson District, blood and urine samples were taken from red deer (Cervus elaphus) from which low (<1:100) antibody titres to serovar copenhageni and suspected leptospiral abortions had previously been reported. A total of 27 hinds were sampled. Microscopic agglutination test (MAT) titres in sera ranging from 1:32 to 1:128 were found in six animals. Thirteen leptospiral isolations were made from nine of the 27 urine samples. Four of these were typed as copenhageni and nine as hardjo. Two cultures were prepared from each urine sample and hardjo and copenhageni were both isolated from single urine samples from two animals. None of the 27 deer had serum MAT titres at 1:32 or above to copenhageni.     

Epidemiology of leptospirosis in dairy farm workers in the Manawatu. Part II. A case-control study of high and low risk farms

Subsequent to a cross-sectional serological survey of Manawatu dairy farm workers, a case-control study was carried out to investigate the correlation between titres to leptospiral serovars in workers and those in cattle in their herds. A total of 52 herds was investigated, 25 of which were high risk where milkers had titres of 1:96 or greater, and 27 were case-controls where milkers had no detectable agglutinin titres at a minimum serum dilution of 1:24. The serological prevalence of titres to hardjo in cattle on high risk farms (76.5%) was significantly higher (P<0.05) than on the case-control farms (60.0%). The geometric mean titres of seropositive cattle on high risk farms were also significantly higher (P<0.01) than in the cattle from the case-control farms, especially in the younger cohorts. These findings suggest that there was active endemic hardjo infection in the two- to three-year-old cattle on the high risk farms. Titres to pomona were demonstrated in only 5.2% of the cattle from both types of farm. Workers with titres to pomona tended to be from farms on which stock, especially calves, were bought-in and pigs were kept. Conventional measures for protecting milkers from contact with infected urine appeared to be ineffective and it is concluded that prevention of leptospirosis in dairy farm workers can only be achieved by elimination of infection in the herd by vaccination of cattle.     

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 .     

Experimental infection of lactating goats with Leptospira interrogans serovars pomona and hardjo

Pathogenesis of Leptospira interrogans serovars pomona and hardjo was evaluated in 14 lactating goats. Although mild clinical signs of leptospiral infection characterized by pyrexia and reduction in milk yield appeared in some animals, a consistent clinical pattern was not observed in the inoculated animals. The pomona serovar was isolated from the kidney of 1 of the 4 goats inoculated with serovar pomona. The hardjo serovar (strain UI 750) was isolated in the rabbit serum-supplemented bovine albumin polysorbate-80 liquid medium only from the mammary gland of 1 of 4 goats at 13 days after inoculation with serovar hardjo. The positive culture was detected after an 8-month incubation period.     

The long term efficacy of a Hardjo-pomona vaccine in preventing leptospiruria in cattle exposed to natural challenge with Leptospira interrogans serovar hardjo

The long term efficacy of a commercially prepared bivalent vaccine against Leptospira interrogans serovar hardjo and L. interrogans serovar pomona was evaluated in a group of 82 dairy heifers exposed to natural challenge with L. interrogans serovar hardjo for up to 55 weeks after calfhood vaccination. Nineteen heifers were vaccinated twice with a one-month interval, at calfhood (9 to 10 months). A further 18 heifers received a similar calfhood vaccination regimen plus a third injection at adulthood (22 to 23 months), while 19 heifers were vaccinated twice at adulthood only and finally 22 heifers were used as unvaccinated controls. At 55 weeks after calfhood vaccination and prior to adulthood vaccination, only 2.7% of the vaccinated heifers were found to have leptospiruria compared with 58.5% of the unvaccinated heifers (p less than 0.0001). Microscopic agglutination (MA) titres at the same time in the unvaccinated heifers ranged from 32 to 4096 while those vaccinated at calfhood ranged from 32 to 64. Adult vaccination of infected animals did not significantly reduce leptospiruria . Prior to adulthood vaccination, 9 of 19 heifers had leptospiruria , in comparison to 4 of 15 after adulthood vaccination. At the same sampling periods 15 of 22 controls had leptospiruria in comparison to 4 of 9 subsequently tested.