Leptospira interrogans serotype pomona infection in pigs: prevention of leptospiruria by immunisation before exposure to a natural infection

Fourteen of 24 pigs were immunised with repeated injections of killed cultures of Leptospira serotype pomona administered over a three-week period. The remaining 10 pigs served as controls.

Five days after immunisation all pigs were exposed to the same natural infection by being housed together for 12 weeks in a pen which received effluent from other pens containing infected pigs.

Neither leptospiruria nor kidney lesions were detected in any of the immunised pigs. In contrast, leptospiruria and kidney lesions occurred in all the controls.

The development of serum agglutinins after immunisation and before exposure suggested that this could be used as an index of adequate antigenic stimulation.     

Some Observations on the Diagnosis and Epidemiology of Leptospirosis in Swine

The results of combined epidemiological, clinical, serological, bacteriological and histopathological studies following an outbreak of disease caused by L. pomona on a farm stocked with cattle, sheep, pigs, goats and horses maintained for experimental purposes, are reported.

The incidence of infection was high in horses, cattle and pigs. A few low titres were seen in sheep. The goats were not infected. Apart from a single bovine abortion all the clinical symptoms observed occurred in pregnant sows. Seven of these aborted or gave birth to stillborn pigs within a six week period.

Fifteen species of wildlife were trapped or shot on the farm during the year following the outbreak. L. pomona was isolated from four skunks and a porcupine. Epidemiological studies indicated that wildlife reservoir hosts were the primary source of infection for the domestic livestock.

Leptospiruria and the serological response were studied in a group of eight infected sows. Microscopic agglutination titres of 10² or less could not be associated with leptospiruria and the duration of leptospiruria was found to range from a few weeks to over two years in individual sows. Direct dark-field examination of urine proved superior to guinea-pig inoculation as a method of detecting leptospiruria and it is suggested that the former technique could be adopted with advantage as a routine aid to diagnosis.

     

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.     

The efficacy of a leptospirosis vaccine in preventing leptospiruria in pigs

A commercially manufactured leptospirosis vaccine containing serovars pomona and hardjo and licensed for use in cattle and sheep was investigated to determine if it would prevent leptospiruria in pigs exposed to serovar pomona. Twenty piglets were each vaccinated twice at an interval of three weeks. Twenty other piglets were unvaccinated and served as controls. Three weeks after the second dose of vaccine all animals were exposed for 64 to 89 days to a natural infection with pomona. During the investigation blood samples were examined serologically and urine samples were examined by dark ground microscopy and cultured for the presence of leptospirae. Attempts were made to culture leptospirae from kidneys at slaughter. Kidneys were also examined histologically for evidence of leptospira infection. One vaccinated animal developed a respiratory disease. It was treated with antibiotics and removed from the trial. Leptosphuria was demonstrated in six of the remaining 19 vaccinated pigs and leptospirae were found in nine of 578 (1.5%) urine samples examined from these animals during the period of exposure. In contrast leptospiruria occurred in 19 of 20 unvaccinated pigs and leptospirae were found in 253 of 642 (39.4%) urine samples examined from these animals. Histopathological lesions consistent with leptospirosis were found in kidneys examined from two of 16 vaccinates and 17 of 18 non-vaccinates. Antibodies to serovar pomona were detected in 12 of 19 vaccinated pigs examined three weeks after the second dose of vaccine and before exposure to infection, and in all of 18 unvaccinated pigs examined after exposure to infection. It was concluded that use of this vaccine in pigs resulted in a significant degree of protection against leptospiruria.     

Experimental myrotheciotoxicosis in sheep and calves

Hamster inoculation, medium inoculation and microscopical methods were used for the detection of Leptospira interrogans serovar pomona organisms in bovine urine. Urine samples were collected from both naturally and experimentally infected animals during the period when leptospiruria could be expected.

Inoculation into hamsters of 0.5 ml of urine from each of 14 samples resulted in 5 positives. The inoculation of 0.025 ml of the same sample into semi-solid EMJH medium gave 10 positives. From a cumulative total of 46 urine samples, 28 were positive using dark-ground microscopy of centrifuged urine deposits and 35 were positive using media inoculation. A cumulative total of 126 urine samples were examined, after centrifugation, under dark-ground microscopy and leptospirae were detected in 60.

Fluorouracil (100 mg/ml) proved to be beneficial for successful isolations in media, whereas neomycin (5 mg/ml) did not.     

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.     

the use of a hardjo-pomona vaccine to prevent leptospiruria in cattle exposed to natural challenge with leptospira interrogans serovar hardjo

The efficacy of the hardjo component of a hardjo-pomona vaccine was evaluated in yearling heifers under conditions of natural challenge in a commercial dairy herd when endemic hardjo infection was present. Eight heifers received 2 doses of vaccine 4 weeks apart and were run with 10 unvaccinated heifers for a period of 56 weeks. Results from the culture of urinesamples showed that the vaccine either prevented leptospiruria to a significant degree (P<0.05) or, if it developed, greatly shortened its duration (P<0.01). Leptospires were cultured on an average of 5 occasions (range 3 to 8) from each of the infected controls and on only one occasion each from 2 of the vaccinates. Fifty one to 56 weeks after commencement of the trial, 9 of the unvaccinated animals were excreting leptospires while none of the vaccinates were leptospiruric at that time.

It is concluded that an appropriate vaccination programme could prevent the maintenance of hardjo infection in the herd.     

Leptospira interrogans serovar pomona vaccines with different adjuvants in cattle

Three groups of four heifers were vaccinated twice, 11 weeks apart. One group received a commercial pomona vaccine with aluminium hydroxide adjuvant, the second a similar experimental vaccine, and the third a Freund's compete adjuvant (FCA) vaccine. After 47 weeks, the heifers were challenged with at least 65 × 10⁸ virulent serovar pomona organisms.

All vaccinated animals resisted the challenge, and leptospirae were only found in urine from unvaccinated controls.

The outcome of the challenge was not predictable from microscopic agglutination, cold and warm complement fixation, and growth inhibition titres.

The FCA vaccine gave rise to considerably higher antibody responses than the two aluminium hydroxide vaccines, which gave similar responses.     

EVALUATION OF A HARDJO-POMONA VACCINE TO PREVENT LEPTOSPIRURIA IN CATTLE EXPOSED TO A NATURAL CHALLENGE WITH LEPTOSPIRA INTERROGANS SEROVAR HARDJO

SUMMARY The efficacy of a vaccine against Leptospira interrogans serovar pomona and Leptospira interrogans serovar hardjo was evaluated in a group of dairy heifers that were serologically negative at the time of vaccination and later subjected to natural challenge with L. interogans serovar hardjo. Thirty-nine heifers were vaccinated twice, at a one-month interval, with a commercially prepared bivalent vaccine, while 43 unvaccinated heifers were used as controls. After vaccination, microscopic agglutination (MA) titres of serums to L. interrogans serovar hardjo ranged from 32 to 512, and those to L. interrogans serovar pomona ranged from 32 to 2048. Titres resulting from vaccination were short-lived and after the first vaccination the serums of 95% of vaccinated heifers did not react in the MA test by 24 weeks. The first indication of infection in the heifers was noted at week 6, and by week 16, elevated MA titres (≥128) to L. interrogans serovar hardjo had occurred in 62% of unvaccinated heifers and had increased to 85% by week 24. At week 18, 18% of the vaccinated heifers and 56% of the unvaccinated heifers had leptospiruria (p<0.01); after 22 weeks, 13% of the vaccinated heifers and 58% of the unvaccinated heifers showed evidence of leptospiruria (p<0.01).     

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.     

Clinical and biochemical changes in sheep inoculated with leptospira interrogans serotype pomona

Clinical and biochemical changes occurring in whole blood, urine, plasma and serum of sheep experimentally infected with Leptospira interrogans serotype pomona were studied.

Half-time and fractional clearance rates of sulphobromophthalein, phenosulphophthalein and chromium (^5lCr) EDTA were also measured to assess functional damage to the liverand kidney.

The results suggested that the biochemical changes occurring following pomona infection were due mainly to haemolysis of erythrocytes and to mild impairment of tubular and glomerular kidney function. No alteration in liver function was observed: Analyses of isocitrate dehydrogenase, arginase, and total bilirubin in serum and measurements of kidney glomerular and tubular function appeared to be the most useful biochemical indicators of pomona infection in sheep.     

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.     

A leptospirosis outbreak in a piggery

A newly established piggery experienced an abortion storm that was diagnosed as being caused by Leptospira pomona. Serumantibody titres and urine culture indicated the rapid spread of infection within the piggery, and established the period since infection began. This showed the origin of infection to beone of the piggeries from which stock were purchased.

Histological and microbiological studies indicated the continuing presence of L. pomona as the cause of abortion. Initial treatment with dihydrostreptomycin and “Leptovax” had no effect on preventing infection, and the subsequent use of “Terramycin” caused little modification to the spread of infection or prevention of abortions. The spread of leptospirosis through the herd resulted in a marked increase in the number of piglets aborted or born dead and, subsequently, a poor survival rate of piglets reared through to slaughter. Efforts are being made to control leptospirosis within the piggery, but eradication is currently considered to be almost impossible.     

LEPTOSPIRA INTERROGANS SEROTYPE HARDJO OUTBREAK IN A VICTORIAN DAIRY HERD AND ASSOCIATED INFECTION IN MAN

SUMMARY Leptospirosis associated with probable L. hardjo infection was investigated in a dairy herd in a coastal district of Western Victoria. Thirty-six of 110 cows suffered leptospiruria and mastitis characterised by flaccid udders and abnormal milk. One of two media used enabled the isolation of the organism from infected guinea pigs inoculated with fresh urine. Microscopic agglutination titres to L. hardjo were elevated during the outbreak. There was an associated human infection.     

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.     

The epidemiology of Leptospira interrogans serovar pomona in grower pig herds

An epidemiological study on four farms showed that leptospiral infection became apparent in piglets from 12 weeks of age. The intensity of leptospiruria was greatest in the first 3-4 weeks of infection and from then on declined and became intermittent. Factors affecting the cultural and serological prevalence of infection of the piglets were found to be the standard of hygiene and variations in the titre of the dam. Dams with high titres gave better protection to their young than did those with low titres. The spread of infection within piggeries was encouraged by mixing infected with uninfected pigs, which resulted in epidemics within the pens. Transmission from infected to susceptible grower pigs continuously occurred in grower houses, with a constant proportion of pigs becoming infected each week.     

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 balcanica 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 litre to hardjo but not pomona.

A fourth calf died with severe nephritis but a diagnosis of leptospirosis was not confirmed in this case.     

Experimental infection of the possum (Trichosurus vulpecula) with Leptospira interrogans serovar balcanica I. Characteristics of infection

The serological, bacteriological and histopathological characteristics of experimental infection with serovar balcanica in possums (Trichosurus vulpecula) are described, and the possum is shown to be a potential maintenance host for this organism. Serum agglutination titres were maintained at almost constant levels for longer than a year, and leptospiruria was present in 50% of animals for a similar period. A paradoxical reaction to hardjo antigen was found in sera from all possums infected with balcanica.

Comparative studies were conducted using recently isolated field strains of serovars hardjo and ballum. Young possums seronegative to Hebdomadis group titres were insusceptible to challenge with hardjo, and the pathogenesis of ballum infection was characteristic of leptospiral infection in an accidental host.

The occurrence of antibodies in the urine of possums infected with balcanica is also described.     

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 be 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 he discounted.     

Prevention of Leptospira interogans 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.