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.     

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.     

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.     

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.     

A farming systems study of abortion in dairy cattle on the Atherton Tableland: 2. The pattern of infectious diseases

The role of infectious agents on dairy farms on the Atherton Tableland in tropical north Queensland was studied as part of a comprehensive investigation into the causes of bovine abortion. The prevalence of antibody in serums collected from 7 herds whose annual abortion rates ranged from 3% to 21% were as follows: Leptospira hardjo 49.9% (426/853), L. pomona 0.4% (3/851), bovine virus diarrhoea (BVD) 33.7% (35/104). Infectious bovine rhinotracheitis virus (IBR) 11.5% (12/105), Akabane virus 92.2% (95/103), Aino virus 62.1% (64/103), Chlamydia psittaci 3.1% (37/1004), Brucella abortus 0% (0/851), and Toxoplasma gondii 0% (0/105). Testing of serums against a wide range of leptospiral serotypes indicated that reactions occurring in the Hebdomadis and Sejroe serogroups were probably cross reactions with L. hardjo. Infection with L. hardjo and Akabane virus occurred prior to first mating and contact with Aino virus occurred during first pregnancy. Infection with BVD and IBR viruses was sporadic. The pathology and microbiology of 32 aborted foetuses from 24 Tableland herds (10 from the group of 19 farms under more intense study) were performed. Lesions associated with a Sarcocystis-like agent were present in 6, leptospires in 1, suspected toxic hepatosis in 2 and purulent bronchopneumonia (Staphylococcus aureus) in 1 foetus. No diagnoses were made in the remaining 22 foetuses (69%). Evidence for a common infectious cause of abortion in the population was inconclusive.     

The significance of leptospiral titres associated with bovine abortion

To investigate relationships between serological titres to 2 serovars, pomona (L. pomona) and hardjo (L. hardjo), of Leptospira interrogans and abortions, log linear and logit models were fitted to herd and individual cow data from cattle serologically negative for brucellosis. Serological titres to both serovars were significantly related to abortions in individual cows, with L. pomona having a stronger relationship than L. hardjo. L. hardjo was not significant when herd data were analysed. Differences between dairy and beef cattle in the serological titres found to both L. pomona and L. hardjo were detected when data sets of all cattle or cattle with no history of abortion were analysed. The beef/dairy differences may be due to different management practices and/or to different geographical distributions of both serovars and populations of beef and dairy cattle. If there are no cattle in a herd with a reciprocal titre of 3000 or greater for L. pomona, it is unlikely that L. pomona is associated with the abortion problem. There was no specific L. hardjo titre which separated high and low probabilities that the serum came from a cow or herd with an abortion history.     

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.     

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.     

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.     

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)     

Use of enzyme immunoassay in a serological survey of leptospirosis in sheep

A total of 731 serums, all from Merino rams from 20 farms, were tested for antibodies against Leptospira interrogans serovars hardjo, pomona and tarassovi using the microscopic agglutination test (MAT). The enzyme immunoassay (EIA) technique was used to test all serums for IgM and IgG antibodies to serovar hardjo. In the MAT, reactions to serovar hardjo were most common with 236 rams (32.3%) reacting at 1/100 or greater. Only 1.9% of serums reacted against serovar tarassovi and 1.1% against serovar pomona. The percentage of sheep with positive MAT reactions to serovar hardjo ranged from 0 0 to 94.9 between farms. When using EIA, 46 (6.2%) of the serums were positive for IgM antibody and 246 (33.6%) were positive for IgG antibody. Correlation of the EIA for detection of IgG antibody with the MAT was good. The EIA detection of IgG antibody was considered to be a good alternative test to the MAT for epidemiological studies in sheep.     

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.     

Seroprevalence of bovine leptospirosis in Garanhuns Municipal District,Pernambuco State,Brazil

The prevalence of Leptospira interrogans serovars in dairy cattle was determined by analyzing 464 serum samples from cows on 15 properties in Garanhuns municipal district, Pernambuco State, Brazil. A microscopic seroagglutination test including 12 serovars of Leptospira interrogans as antigens was used. Samples with titres 100 were considered positive. Two hundred and twenty-one (47.63%) of the samples were positive to one or more serovars. The prevalence of the serovars was hardjo (21.98%), bratislava (15.73%), castellonis (11.64%), tarassovi (10.56%), pyrogenes (1.72%), icterohaemorrhagiae (1.08%), pomona (0.86%), wolffi (0.86%), grippotyphosa (0.86%), djasiman (0.43%), canicola (0.21 %), and copenhageni (0.21%).     

Arthropathy associated with Brucella abortus strain 19 vaccination in cattle. I. Examination of field cases

Thirty cows presenting with lameness and persistent serological reactions to Brucella abortus had chronic granulomatous arthropathy of the femorotibial and occasionally other joints. Attempts to culture Brucella or other pathogens gave negative results but organisms of Brucella morphology were seen in fluorescent antibody-stained cryostat sections of synovial tissue. The synovial fluids contained high titres of antibodies to B. abortus and Yersinia enterocolitica O:9 and had elevated total protein and immunoglobulin concentrations showing an oligoclonal electrophoretic profile. Immune complexes and rheumatoid factor were detected in some of the fluids.     

A STUDY OF THE ACCESSORY SEX GLANDS OF BULLS EN ABATTOIRS IN NORTHERN AUSTRALIA

The accessory sex glands of 521 slaughtered bulls were examined for lesions; normal anatomical observations were also made.
The average weights and measurements (length and width) of the seminal vesicles and bulbourethral glands of 110 bulls were 34.1 g and 9.1 × 2.5 cm, and 4.6 g and 2.9 × 1.9 cm, respectively. Weights of both glands were correlated to age, but bulbourethral gland weight was more closely correlated to bodyweight than age. The average length and diameter of the ampulla was 11.1 and 1.0 cm, respectively.
Seminal vesiculitis and ampullitis were found in 9.0% and 3.8% of bulls respectively. The incidence of both conditions was highest in old bulls. Mesonephric duct anomalies (observed in 1.7% of bulls) and other congenital and inflammatory lesions of the accessory sex glands were also recorded.
Macroscopically normal accessory sex glands of 86 bulls were studied histologically and a high percentage of all glands showed inflammatory changes.
Attempts to isolate viral agents, Mycoplasma, and Chlamydia from accessory sex glands were unsuccessful. Corynebacterium pyogenes was isolated from two cases of seminal vesiculitis. IBR-IPV virus, Campylobacter fetus subsp. venerealis and Tritrichomonas foetus were frequently isolated from the prepuce of bulls, many of which also had positive serological titres to Leptospira pomona and Leptospira hardjo.
No one factor capable of causing bull infertility was consistently identified, and the incidence figures for observed conditions of the accessory sex glands were generally comparable to those found in bull populations elsewhere.     

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.     

Antibody response to genus- and serovar-specific leptospiral antigens in Leptospira-infected cows

In cows inoculated with Leptospira interrogans serovar pomona or hardjo, the 2-mercaptoethanol-sensitive microscopic agglutination test (MAT) antibody to the serovar appeared 3 to 8 days after inoculation and peaked at 10 to 20 days, whereas the 2-mercaptoethanol-resistant MAT antibody was predominant at 35 to 80 days. A persistent antibody response, probably associated with serovar-specific leptospiral antigens, was detected in the cows inoculated with serovar pomona, using a sonicated or an alkaline-extracted antigen derived from serovar pomona in the enzyme-linked immunosorbent assay (ELISA). In contrast, a short-lived antibody response to the same antigens was demonstrated in cows inoculated with serovar hardjo, probably more typical of the response to genus-specific leptospiral antigens. Antigens derived from L biflexa serovar patoc only detected the latter type of antibody response in cows inoculated with serovar pomona or hardjo. Correlative studies revealed that the antigens derived from serovar patoc seem to be genus specific and serologically closely related, but not identical. The antigens derived from serovar pomona were genus specific on the basis of the early antibody response to leptospiral inoculation in the cows, but serovar specific based on the subsequent more persistent response to leptospiral inoculation. These antigens were also serologically closely related, but not identical. Examination of sera from cows that aborted and were MAT-positive for serovar pomona or hardjo revealed a more serovar-specific antibody response, indicating that there may have been a less recent leptospiral antigenic stimulus, thus emphasizing the caution with which results of the ELISA and other serologic assays for the detection of bovine leptospirosis must be interpreted.     

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.     

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 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.