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.     

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.     

Hypomagnesaemia in cattle

Leptospirosis is an important animal and human health problem in New Zealand, and the epidemiological and ecological aspects of infection in domestic species and free-living species are described. The characteristics of infection with a particular serovar are often considerably different in different species of animal and primary epidemiological investigations involve differentiating between maintenance-host populations and accidental-host populations for the serovars present in a particular ecosystem.

Cattle and pigs are maintenance hosts for hardjo and pomona respectively. Balcanica is maintained by possums (Trichosurus vulpecula) and ballum is maintained by the house mouse (Mus musculus), ship rat (Rattus rattus) and hedgehog (Erinaceus europaeus). In some regions, foci of copenhageni infection are maintained by Norway rats (Rattus norvegicus). Each serovar shows a high degree of parasitic adaption to its maintenance-host species and this is reflected in the nidality of leptospirosis in intensive farming environments. Accidental infections in domestic animals and man are the consequence of ecological associations with maintenance-host populations for particular serovars.

Control of leptospirosis in domestic animals depends on decreasing the prevalence of infection with serovars maintained within populations of domestic animals, and decreasing the degree of ecological association with free-living maintenance-host populations. Vaccination of cattle against hardjo. will considerably reduce the prevalence of infection in vaccinated herds and therefore decrease the prevalence of accidental hardjo infections in man.     

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.     

A serological survey of leptospirosis in cats

A serological survey of 225 cats from different parts of the NorthIsland was conducted using 11 leptospiral serovar antigens. Twenty (8.8%) of the sera gave titres to the microagglutination test.The prevalence of titres to the serovars were as follows: five (2.22%) to copenhageni, five (2.22%) to hardjo, four (1.77%) to ballum, four (1.77%) to pomona, one (0.44%) to balanica and one (0.44%) to canicola; no titres to tarassovi, bataviae, pyrogenes and australis were recorded. It is hypothesised that prey-predator transmission between cats and rodents is responsible for infections with serovars copenhageni and ballum. Infections with the other serovars are presumed to result from contact with infected animals or a contaminated environment.     

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.     

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.     

Experimental infection of the possum (Trichosurus vulpecula) with Leptospira interrogans serovar balcanica II. A comparison of laboratory techniques for the detection of leptospiraemia and leptospiruria

Leptospiruria in possums (Trichosurus vulpeculu) experimentally infected with balcanica was monitored for 13 months by dark-field microscopy, inoculation of hamsters and culture. Leptospiraemia was monitored by inoculation of hamsters and culture for the first 28 days post-inoculation.

Inoculation of hamsters for the detection of leptospiraemia had a sensitivity of 63.2%, whereas culture had a sensitivity of 36.8%. The use of hamsters enabled leptospires to be demonstrated in blood up to 15 days after inoculation by which time serum agglutination titres had reached peak levels.

Dark-field microscopy was the least sensitive method for the detection of leptospiruria (37.7%). Inoculation of hamsters and direct inoculation of media containing 400𝛍g 5-fluorouracil (5FU) had sensitivities higher than 70%. The incorporation of a high level of 5FU in media significantly improved isolation rates.

Differences were seen in the sensitivity of different methods at three different stages of leptospiruria. Inoculation of hamsters was the most sensitive method during the intial stage of leptospiruria, while direct inoculation of media containing 400𝛍g 5FU/ml was the most sensitive method during the chronic stage.     

Cross-Protection Between hardjo and balcanica

Abstract

Extract

Sir, — We were interested to see the results of the trial conducted by Flint and Liardet ⁽²⁾ Flint, S. H. and Liardet, D. M. 1981. Leptospira interrogans serovar hardjo vaccination against Leptospira interogans serovar balcanica infection. N.Z. vet. J., 29: 38–38. [Taylor &; Francis Online], [Web of Science ®] , [Google Scholar] which showed that the antibodies produced by hamsters in response to a hardjo bacterin protects them against balcanica infection. Their results complement those from a passive hamster protection test conducted by ourselves. In this test 10 hamsters were each injected intraperitoneally with 0.5 ml of bovine anti-Pithardjo serum. This serum had been collected from a heifer which had received a 2 ml dose of a commercial hardjo/pomona bacterin* * Leptavoid, Wellcome (N.Z.) Ltd. eight weeks previously and had a titre of 1:96 to hardjo. Twenty-four hours later these 10 hamsters were challenged: 5 received an intraperitoneal inoculation of approximately 2 x 10⁷ bakanica organisms and 5 received a similar inoculum of hardjo organisms. At the same time, 2 control groups of 5 hamsters were challenged with balcanica and hardjo by the same route. Leptospires could not be isolated from any of the challenged hamsters which received anti-hardjo serum when their kidneys were cultured 18 days post-inoculation. However, leptospires were isolated from the kidneys of 5 out of 5 control hamsters inoculated with hardjo and 4 out of 5 control hamsters inoculated with balcanica. An analogous trial using bovine anti-balcanica serum, collected from cattle experimentally infected with balcanica, gave almost identical results to the above trial.     

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.     

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.     

Isolation of Mycobacterium bovis from brushtail possums with non-visible lesions

AIM: To determine the prevalence of Mycobacterium bovis infection in brushtail possums (Trichosurus vulpecula) that did not have macroscopic lesions of bovine tuberculosis, and to evaluate culture of pooled tissues from multiple possums as a method for determining the M. bovis-infection status of wildlife populations in New Zealand.

METHODS: Pools of selected tissues were collected from possums from four different populations known to be infected with M. bovis. Tissue pools from individual animals, and combined pools from multiple animals, were cultured for M. bovis.

RESULTS: In the four populations investigated, the prevalence of possums with macroscopic lesions confirmed by culture to be infected with M. bovis ranged from 1 to 19 (mean 31/283; 10.9)%. The prevalence of possums with non-visible lesions that were culture positive for M. bovis in the same populations ranged from 4 to 10 (mean 24/283; 8.5)%. The mean of the log10 cfu of M. bovis of the macroscopic lesions and of the culture-positive samples that did not have visible lesions was 3.85 (SE 0.26) and 1.46 (SE 0.26) log₁₀ cfu, respectively (p<0.01). Mycobacterium bovis was cultured from pools of 30–50 animals in the four populations studied.

CONCLUSIONS: The finding of M. bovis infection in possums with non-visible lesions identified a potential deficiency of declaring possum populations free of M. bovis on the basis of absence of macroscopic lesions. The culturing of pools of selected tissues from multiple animals without visible lesions can be used to reduce laboratory costs of possum surveys without a major reduction in the ability to detect M. bovis infection.     

A review of laboratory techniques and their use in the diagnosis of Leptospira interrogans serovar hardjo infection in cattle

SUMMARY This paper reviews the laboratory diagnosis of Leptospira hardjo infection in cattle. Two genotypes of L hardjo, Hardjoprajitno and Hardjobovis, have been identified in cattle, but only Hardjobovis has been isolated in Australia. There are problems with diagnosis and control of bovine leptospirosis. Infection is usually subclinical and the serological titres vary greatly in peak and duration. Leptospires may be excreted in urine for up to 18 months. Low microscopic agglutination test titres may be significant in unvaccinated herds as indicators of endemic infection. Vaccines differ in their composition, and their efficacy is difficult to evaluate. The serological response after vaccination is difficult to differentiate from the response after infection. Pregnant cows that become infected may abort, but this is usually after the serological response has peaked. Therefore, paired serum samples are of little use in diagnosing abortion caused by L hardjo. Fluorescent antibody techniques are more sensitive than dark field microscopy for detection of leptospires in urine and tissue samples. Techniques for culture have improved but are still difficult to perform and take 3 months or longer for results to be known. DNA probes and polymerase chain reaction tests are very sensitive and specific, quick to perform, and can be used on fluid and tissue samples.     

Liver fluke,Fasciola hepatica in New Zealand

Free-living members of the Mustelidae and feral cats (Felis catus) inhabiting farmland in the southern half of the North Island of New Zealand were examined for evidence of leptospiral infection. Nine stoats (Mustela erminea), 9 ferrets (Putorious putorious) and 4 weasels (Mustela nivalis) showed no serological or bacteriological evidence of infection. No leptospires were isolated from the kidneys of 11 feral cats but 2 animals had low titres to pomona and ballum respectively. All animals examined were from ecosystems where ballum infection was endemic in rodents; thus predator-chain transmission does not appear to be an important natural route of transmission for this serovar.     

SEROLOGICAL TITRES OF DAIRY COWS OVER A 63-WEEK PERIOD FOLLOWING NATURAL INFECTION WITH LEPTOSPIRA INTERROGANS SEROVAR HARDJO

SUMMARY Leptospirosis due to Leptospira interrogans serovar hardjo, occurred in a dairy herd of 104 cows and produced signs of mastitis, characterised by a sudden decrease in milk production and uniformly flaccid udders, in 11 cows. The microscopic agglutination (MA) test was used to determine antibody titres to serovar hardjo in all cows in the herd at 6, 33 and 63 weeks after the initial outbreak. The prevalence of MA antibodies to hardjo at week 6 was highest in cows in the youngest age groups and lowest in cows in the oldest age group. Over the 63-week period MA antibody titres to hardjo declined in 54 out of 62 seropositive cows, remained unchanged in 6 cows, and increased slightly in 2 cows. Fourteen of 59 cows (23.7%) with MA titres greater than 100 at week 6 had titres greater than 100 by week 63. Included in this group of 14 were 5 of 7 cows that had been affected with atypical mastitis. The distribution of serological titres to hardjo showed bimodal configurations at weeks 6 and 33 with the second peaks occurring at titres greater than 100, while the configuration was linear at week 63. MA titres to hardjo were also determined for 22 heifers 1, 16 and 36 weeks after they were added to the herd. No clinical signs of leptospirosis were observed in the heifers; however, the distribution of MA titres showed bimodal configurations at weeks 16 and 33 with second peaks occurring at titres greater than 100. Leptospiras were isolated from the urine of 2 seropositive heifers 16 weeks after their introduction to the herd, and cross-agglutinin absorption tests performed on one of the isolates indicated that it was identical to serovar hardjo.     

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.     

The prevalence of antibodies to serovars of Leptospira interrogans in horses

SUMMARY Serum samples from 272 horses, some III, were tested by the microscopic agglutination test for the presence of antibody to 12 serovars of Leptospira interrogans. Serums from 41.5% of horses reacted to one or more of the serovars tested; the most common reactions were to L. interrogans serovar ballum (15.1%), L. interrogans serovar autumnalis (11.8%), L. interrogans serovar icterohaemorrhagiae (9.9%), L. interrogans serovar pomona (8.1%) and L. interrogans serovar hardjo (7.7%).     

Mycobacterium avium subsp. paratuberculosis infection in wildlife on three deer farms with a history of Johne's disease

Abstract

AIM: To determine the prevalence of Mycobacterium avium subsp. paratuberculosis (Map) infection in wildlife, in pastoral landscapes with a recent history of clinical Johne's disease in livestock.

METHODS: A total of 449 wild mammals and birds from three farms in the South Island of New Zealand with recent histories of clinical Johne's disease in their deer herds were trapped and examined for gross pathological changes in the gastrointestinal tract. Additionally, individual mesenteric lymph nodes from 380 mammals, and segments of gastrointestinal tract from 32 birds were excised, homogenised and cultured for viable Map bacilli. The prevalence of Map infection was then calculated for the various species. Faecal samples from those mammals which had culture-positive tissues were further cultured for the presence of Map.

RESULTS: Gross pathological changes were identified in the gastrointestinal tract of four brushtail possums, one cat, six ferrets, 12 hares, six hedgehogs, three rabbits, one stoat, and one paradise shelduck. Infection with Map in the gastrointestinal tract was confirmed in only three of these cases, one each of brushtail possums, hares and hedgehogs. In contrast, Map infection in the absence of gross pathological changes was frequently recorded in enteric tract tissues of mammals and birds. Among mammals, Map infection was recorded in 18/73 (25%) brushtail possums, 4/23 (17%) cats, 15/42 (36%) hedgehogs and 29/113 (26%) rabbits. Among birds, intestinal tract tissue Map infection was recorded in 3/17 (18%) paradise shelducks. Among 64 of the 74 mammals which had Map culture-positive tissues, 38% (n=5) of hedgehogs and 11% (n=3) of rabbits also had culture-positive faecal samples.

CONCLUSIONS: This study is the first to identify that Map infection can be prevalent in wildlife in New Zealand. There was a high prevalence of Map infection among both scavenging and grazing wild animals. Both mammals and birds are capable of harbouring viable Map organisms in their gastrointestinal tract; further, viable Map was excreted into the environment via faeces by hedgehogs and rabbits.

CLINICAL RELEVANCE: Previous studies overseas have postulated a role of wildlife as reservoirs of Map infection and possible vectors of Johne's disease to livestock. Here, brushtail possums, hedgehogs and rabbits and in particular were identified as potential wildlife hosts for Map infection in NewZealand. This suggests that several wildlife species could contribute to the persistence of Map infection within a wildlife/livestock complex, and potentially, perhaps more importantly, to the spread of infection between farms.     

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).