Problems associated with the serological diagnosis of Leptospira interrogans serovar hardjo infection in bovine populations

Data combining sequential bacteriology and serology from a longitudinal study of a dairy herd were used to demonstrate the limitations of serology as a diagnostic method in cross-sectional sampling of bovine populations. Whole-herd point serological prevalences showed considerable variation over a two-year sampling period (38.8 to 76.2 per cent), and this was mainly due to varying age-specific prevalence. Owing to the rapid decline in titres and the varying persistence of infection, point serological prevalences failed to approximate to cumulative infection rates (either past or present) at different times of the year. A higher estimate of the number of susceptible animals in the herd than is the case results in inaccurate information on true incidence rates and can confuse assessments of the susceptibility of different age groups, especially if only small numbers are sampled. A sampling exercise demonstrated that a 10-cow sample usually provided little useful information other than establishing the presence or absence of hardjo in the herd. Increasing the sample size markedly improved epidemiological information, investigations of clinical disease, assessments of vaccination needs and public health tracebacks. Preferably 10 sera from each of the yearling, first calver, second calver and older age groups should be tested. Serology was an inadequate indicator of infection in individual animals. Group geometric mean titres taken from a mean serological response curve were shown to have limited application in the interpretation of field data, unless infection had occurred in the previous two months.     

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.     

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.     

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.     

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<0.0l) 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.     

Development of a control strategy for Leptospira hardjo infection in a closed beef herd.

Serological evidence of infection with a leptospire belonging to the Sejroe serogroup was identified in a closed population of Luing cattle in the west of Scotland, and the geographical isolation of the population presented an opportunity to control and possibly eradicate the infection in a large beef herd farmed under extensive conditions. Serological and bacteriological studies revealed that infection was present at a high level throughout the herd, and that the infecting serovar was hardjo. Unlike endemic hardjo infection in dairy herds, new infections were still occurring in older age-groups. Investigations of other domestic and free-living species sharing the habitat demonstrated that the maintenance of an endemic focus of hardjo was restricted to the cattle. Changes in management to prevent the transmission of infection to successive cohorts of young animals were impractical and risky, and antibiotic treatment followed by removal to clean pasture failed to prevent new cases. Thus vaccination offered the only means of control and possible eradication, and the epidemiological characteristics of the infection dictated that the programme be applied to the whole herd.     

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.     

Antibody responses to natural Dictyocaulus viviparus exposure in first year grazing cattle using vaccination or different kinds of anthelmintic treatment.

In two field trials vaccination or different anthelmintic treatments against Dictyocaulus viviparus infections were used in 6 groups of first year grazing cattle. The antibody response to lungworm infections was determined using an ELISA. Cattle treated once or repeatedly at long intervals with levamisole developed clinical signs of dictyocaulosis. The detection of anti-D. viviparus antibodies at the end of the grazing season confirmed that these anthelmintic treatments were not able to prevent lungworm infections. Cattle that received strategically administered treatments with ivermectin remained clinically healthy. These cattle were seronegative until the end of the trial which proved the efficacy of the strategic control. Cattle that received an intraruminal slow release bolus did not develop clinical disease. However one animal shed lungworm larvae and the herd became seropositive at the end of the grazing season indicating a history of infection. The serological examination of a cattle herd at the end of the grazing season is able to demonstrate a history of lungworm infections and to determine the efficacy of anthelmintic control measures.     

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.     

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 .     

LEPTOSPIRA INTERROGANS SEROVAR HARDJO INFECTION OF CATTLE

A survey of normal cattle in the Southern Victorian statistical divisions revealed that microscopic agglutination titres to L. hardjo occur at high frequency and are distributed throughout the cattle population. These titres are difficult to interpret as they may represent recent or old exposure, with or without disease. L. hardjo infection of dairy cattle was studied in 4 herds using the microscopic agglutination and complement-fixation tests. Statistical comparisons of individual titres obtained indicated that the sensitivity of the complement-fixation test was satisfactory for diagnostic purposes, but the test was unable to differentiate between current or past infections.     

Effect of nematode infections and management practices on growth performance of calves on commercial dairy farms

Liveweight of calves on 86 dairy farms was measured at the end of the grazing season and related per herd to the level of exposure to nematode infection estimated in October and December. There were significant between-herd variations in all the serological infection parameters measured. On average 20.5 larvae per gram faeces (geometric mean) were found in October. Faecal samples of 20.5% of the herds contained lungworm larvae. Liveweight of calves deviated per herd from -59.8 kg to +52.2 kg from an age-adjusted population mean after their first grazing season. Growth performance was significantly related negatively to several serological and parasitological parameters. Data were fitted by means of both linear and segmented curvilinear regression. By combining infection parameters 19% of the variation in growth performance among herds could be explained. Infection parameters involved were antibody titre against Cooperia spp., egg output and lungworm larval count. It was found that antibody titres were significantly correlated positively to herd age, while pepsinogen values and egg output were negatively correlated to age. Combining supplementary feeding and anthelmintic treatment during the grazing season with the infection parameters into one model explained approximately 30% of the observed variation in growth performance among herds. It was shown that these findings were consistent with those of a similar study conducted on the same farms a year earlier, although there were clear differences between the years. Finally, significant positive relations were found between the levels of exposure to nematode parasites within farms between two consecutive years.     

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.     

The Successful Management of Leptospirosa hardjo Infection in a Beef Herd in Northern Ontario

Abortion, premature calving, hemolytic anemia and fatal hematuria were associated with high levels (titer > 10(-4)) of antibody to Leptospira interrogans serovar hardjo and with isolation of hardjo in a herd of 265 beef cattle in the Great Clay Belt of northern Ontario. This herd was bred by artificial insemination, after heat detection by vasectomized bulls. The antibody prevalence rate in the herd was 54 to 60% over a five year period. The rate tended to reach 100% by age three years and to be below 5% in yearlings, which were raised in isolation from older cattle. Hardjo was isolated from the urine of a cow that aborted in the eighth month of pregnancy, and from kidneys of yearling steers which had been exposed to an older cow. Maternal antibody levels in calves paralleled those in their dams, protecting calves while they were being naturally exposed to infection, thus contributing to the achievement of balance between host and parasite. A controlled vaccination trial was conducted in 50 initially seronegative yearling steers and heifers. Serological response to vaccine was limited to a maximum agglutinin titer of 10(-2) in 8% of vaccinated cattle. Vaccination reduced the infection rate from 86% in the controls to 46% in the treated group, indirectly reducing the number of calves for which colostral antibody against hardjo would be available. A vaccination program was not implemented in the herd. Hardjo infection appeared to die out over a period of six years following the initial five year study period, with antibody prevalence falling from 60% to 0.7% and reactors persisting only in two eight year old cows. Decline in infection was coincident with changes in management which protected heifers from exposure to infection until their third pregnancy, and which probably lowered the reservoir of infection by increased culling from older age classes.     

LEPTOSPIRA SEROGROUP HEBDOMADIS INFECTION AS AN AUSTRALIAN ZOONOSIS

Two cases of leptospirosis due to infection with a member of the Hebdomadis serogroup are described in farm workers on a Victorian dairy farm. The source of infection appeared to be the milking herd which had elevated serum antibody titres against a member of the Hebdomadis serogroup. Agglutinin-absorption testing of one patient's serum indicated that the infecting serovar was hardjo .
A survey of 1,144 cattle entering abattoirs throughout the State indicated that 44.3% of these animals showed serological evidence of past or present infection with leptospira of the Hebdomadis serogroup and the potential risk for people with occupational contact with cattle is emphasised. The importance of considering leptospirosis in the differential diagnosis of all patients with influenza-like symptoms or pyrexia of unknown origin and having occupational contact with animals, especially cattle, is discussed.     

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.     

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)     

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.     

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.     

Leptospirosis associated with serovars hardjo and pomona in red deer calves (Cervus elaphus)

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.