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.     

Serological studies on leptospirosis in cattle in east central Alabama.

Serological surveys of leptospiral antibodies in cattle were carried out in Macon and the surrounding counties of East Central Alabama. A total of 286 bovine serum samples were screened for the presence of antibodies against live antigens from twelve pathogenic leptospiral serotypes using a microscopic agglutination test. The most frequently encountered serotypes were Leptospira hardjo (47%), Leptospira wolffi (34%), Leptospira canicola (12%), Leptospira pomona (10%) and Leptospira ballum (10%). Leptospira autumnalis, Leptospira grippotyphosa, Leptospira icterohemorrhagiae, Leptospira pyrogenes and Leptospira tarassovi were observed in less than 5% of the samples.     

THE USE OF A FORMOLISED ANTIGEN AS A SCREENING TEST FOR LEPTOSPIRAL ANTIBODIES IN HORSES

Six hundred and thirty-two equine serums were examined for the presence of leptospiral antibodies. A positive reaction to one or more antigenic pools of a formolised leptospiral antigen (used in the rapid macroscopic slide agglutination test) was recorded in 41% of cases.
One hundred samples were tested with 5 formolised antigen pools and 19 live antigens (by the microscopic agglutination test). Of 20 samples in which the live antigen test suggested leptospiral infection with serotypes known to occur in the region, 17 (85%) were confirmed with the formolised antigens.
When the results of both tests were compared, there was agreement in 42 samples (30 positive and 12 negative). Forty-one samples produced equivocal results and 17 gave doubtful reactions to the formolised antigens, 15 of which were negative to the live antigens.
Dilution of the serums 1:1 with normal saline or heat inactivation had no effect in increasing the specificity between the formolised and live antigens. Agreement between operators in the use of the formolised antigen was poor. It is concluded that the formolised antigen has too wide a divergence to be of use for screening horse serums.     

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.     

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

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

Serologic evidence of equine leptospirosis in the northeast United States.

Serologic testing for leptospiral antibody was conducted with the macroscopic agglutination test on 1,346 equine serum samples. These were collected from clinically normal horses in 123 purebred herds in the Northeast. Sixty-eight samples (5%) from the population tested reacted at significant levels (1:40 or higher) to one or more of the 5 serotype antigens used. These reactors were from 38 (31%) of the herds tested. Reactions to serotype pomona predominated in 25 (72%) of these 38 herds. Smaller numbers of herds had reactors to canicola, icterohemorrhagiae and grippotyphosa. No significant reactions to serotype hardjo were detected.     

Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs.

Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources, expertise, or facilities to perform the microscopic agglutination test (MAT). Thus, there is a need for a rapid and simple serological test that could be used in any diagnostic laboratory. In this study, a genus-specific, heat-stable antigenic preparation from Leptospira interrogans serovar pomona was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of leptospiral antibodies in dog sera. This antigenic preparation reacted with rabbit antisera against L. interrogans serovars bratislava, autumnalis, icterohaemorrhagiae and pomona and with rabbit antiserum against L. kirschneri serovar grippotyphosa. The ELISA showed a relative specificity of 95.6% with 158 dog sera which were negative at a dilution of 1:100 in the MAT for serovars pomona, bratislava, icterohaemorrhagiae, autumnalis, hardjo, and grippotyphosa. The relative sensitivity of this assay with 21 dog sera that revealed serovars MAT titres of > or =100 to different serovars was 100%. This assay is easily standardized, technically more advantageous than MAT, and uses an antigenic preparation that can be routinely prepared in large amounts. It was concluded that this ELISA is sufficiently sensitive test to be used as an initial screening test for the detection of leptospiral antibodies in canine sera, with subsequent confirmation of positive test results with the MAT.     

Serologic survey for leptospirosis in Arizona beef cattle in 1981

Sera from 1,215 beef cattle in Arizona were evaluated by leptospiral microscopic agglutination test in 1981. Over 25% had agglutinins to greater than or equal to 1 of 5 serovars of Leptospira interrogans used as antigens (canicola, grippotyphosa, hardjo, icterohaemorrhagiae, and pomona) at a titer of greater than or equal to 1:100, and 8.2% had titers of greater than or equal to 1:400 to greater than or equal to 1 serovars. The most common serovar to which reactions were detected was hardjo; agglutinins were detected at titers of greater than or equal to 1:100 in 14.3% and of greater than or equal to 1:400 in 5.5%. Cross reactions were rare at serum dilutions greater than or equal to 1:100 (2%) and extremely rare at greater than or equal to 1:400 (0.7%). Because vaccination with leptospiral bacterins is seldom practiced in Arizona beef cattle, a titer of greater than or equal to 1:100 may be useful in estimating incidence and prevalence of the disease and as an aid to diagnosis of leptospirosis.     

Sero-prevalence of agglutinins to Listeria monocytogenes in Nigerian domestic animals

A survey using tube agglutination test was conducted to determine the antibody prevalence to Listeria monocytogenes serotypes 1/2a, 1/2b, 1/2c, 3a and 4b in 1,190 serum samples of 8 animals species from various sources in Kano and Kaduna states of Nigeria. Following absorption with Staphylococcus aureus antigen to remove cross-reacting agglutinins, 52 (68.4 p. 100) of the horse samples were positive. Twenty-six (36.1 p. 100) pig, 52 (20.8 p. 100) cattle, 50 (20.0 p. 100) goat, 20 (20.0 p. 100) dog, serum samples were also positive. Free-ranging chickens had an antibody prevalence of 18 (32.1 p. 100) while those intensively managed had 3 (6.8 p. 100), a difference found to be statistically significant (P less than or equal to 0.01; X2). Sheep sera collected from Zaria abattoir had a prevalence of 30 (14.7 p. 100) while those from Ahmadu Bello University, Veterinary Teaching Hospital had 6 (13.0 p. 100) prevalence. The prevalence in camel was 4 (4.3 p. 100). Overall, of the 1,190 serum samples tested, 26 (21.9 p. 100) were sero-positive for L. monocytogenes agglutinins. Each species of animal tested for L. monocytogenes was positive for all five serotypes, except camel which was negative for serotype 3a. Fourty-four (53.0 p. 100) samples were positive at a titre of greater than or equal to 480 for serotypes 1/2a, 60 (58.3 p. 100) for 1/2b, 57 (52.3 p. 100) for 1/2c, 7 (13.7 p. 100) for 3a and 23 (39.0 p. 100) for 4b. It is concluded that L. monocytogenes infection is widespread in domestic animals in Nigeria.     

Bovine leptospirosis: the effects of vaccination on serological responses as determined by complement fixation and microscopic agglutination tests

Fifty-one calves were divided into six trial groups of seven to eleven animals and vaccinated with a commercial leptospiral vaccine containing serovars pomona and hardjo. Vaccinations were given at 6,7,14 or 21 months of age and animals in various groups were vaccinated on one to four occasions. Antibody responses were determined by the microscopic agglutination test (MAT) and the complement fixation test (CFT) at one to four weeks intervals until 66 weeks after the start of the trial. Fifty percent or greater agglutination in serum diluted 1:100 or more and 50% or greater fixation of complement in serum diluted 1:20 or more were considered positive titres in the MAT or CFI respectively. Positive titres were still present in some animals six weeks after vaccination at 21 months of age. In other cases MAT titres (range 1:100-1:3000) persisted for 7-23 weeks and CFI titres (range 1:20-1540) persisted for 1-14 weeks. Marked individual variation in serological findings occurred using either test. In general the number of animals producing a positive titre, and the magnitude and persistence of titres was related to the number of doses of vaccine given. It was concluded that for diagnostic purposes neither the CFT nor the MAT could reliably differentiate titres due to vaccination from those following natural infections.     

Serodiagnosis of leptospirosis in pigs using an axial filament enzyme-linked immunosorbent assay.

The axial filament (AF) from Leptospira interrogans serovar canicola was isolated by cesium chloride density gradient centrifugation of 2% sarcosyl treated whole cells. Isolation of AF was confirmed by electron microscopic examination, by protein-A immunogold labelling, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting. Analysis by SDS-PAGE of the purified preparation showed relatively weak bands of molecular size 41 kDa and 21 kDa, and strong bands of 35 kDa and 34.5 kDa. Immunoblot analysis using antiserum to the AF against sonicated leptospires of a variety of serovars showed prominent reaction against the 41, 35, and 34.5 kDa protein bands, as well as against minor bands of molecular weight 43, 39, and 37 kDa. Antisera prepared against leptospiral serovars also identified minor bands at 33 and 32 kDa. Immunoblots with antiserum to whole cells of serovar bratislava detected the 35 and 34.5 kDa AF bands of Borrelia burgdorferi moderately and of Treponema hyodysenteriae only slightly in comparison to leptospiral AF. Antibody to B. burgdorferi did not detect the leptospiral AF antigen. Immunoblots with antiserum to T. hyodysenteriae showed a marked reaction with a 41 kDa band of B. burgdorferi but only a very minor reaction with leptospiral AF. The AF was tested in an AF-ELISA against sera from 260 pigs, many of which reacted in the microscopic agglutination test (MAT) against one or more leptospiral serovars. A sensitivity of 97.1% and a specificity of 93.1% was determined in comparison to the MAT. Only moderate correlation was observed between titres detected in the AF-ELISA and the MAT (r = 0.4). When sonicated whole cells (WC) of serovar canicola were used in an ELISA (WC-ELISA), high correlation was observed between AF-ELISA and WC-ELISA (r = 0.97). These findings show that the AF-ELISA can be used effectively as a species-specific antigen for the serological diagnosis of leptospirosis in swine and that sonicated whole cells can substitute excellently for purified AF as the antigen source. These findings may be extrapolated to the use of AF in immunodiagnosis of leptospirosis in other species.     

Evaluation of a recombinant LigB protein of Leptospira interrogans serovar Canicola in an enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis

A recombinant leptospiral lipoprotein, LigB, was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLigB IgG ELISA). The standard reference test (Microscopic agglutination test, MAT) of 200 serum samples from cattle suspected of leptospirosis showed that 95 (47.5%) samples had positive agglutination titres, which ranged from 100 to 1600. In rLigB IgG ELISA, 49% of the samples were positive. Sensitivity of IgG ELISA for 95 bovine sera, which had MAT titres of greater than or equal to 100, were 100%. ELISA showed a specificity of 97.1% with 105 bovine sera, which were negative at a 1:50 dilution in MAT for Leptospira interrogans serovars. The results of ELISA and MAT correspond very good. When analytical specificity of IgG ELISA was evaluated using bovine serum samples from animals showing the serum antibodies to other pathogens, no cross-reaction was observed. Thus the recombinant LigB IgG ELISA can be used instead of the MAT as an aid to the diagnosis of bovine leptospirosis.     

我国鸭疫里氏杆菌血清型的鉴定

１９９７年１月－１９９８年３月,从北京市２０个商品鸭场自然病死的北京白鸭和河南省与上海市部分鸭场的樱桃谷鸭分离到２７６株鸭疫里氏杆菌,采用凝集试验和琼脂扩散沉淀试验,对其进行了血清型的研究。其中７０株细菌为１型,６４株为２型,其余１４２株怀１,２,型参考菌株的抗血清发生反应。     

Some leptospira agglutinins detected in domestic animals in British Columbia.

During a period of six years 7,555 bovine sera, 421 canine sera, 251 porcine sera and 135 equine sera were tested for agglutinins to Leptospira interrogans serotypes canicola, grippotyphosa, hardjo, icterohemorrhagiae, pomona and sejroe. The bovine sera reacted predominantly with hardjo and/or sejroe at a rate of 15% compared to 3.5% with pomona. Breeding or abortion problems were associated with pomona but not with sejroe/hardjo agglutinins. The canine sera reacted to canicola (9.9%y and icterohemorrhagiae (5.4%), tcted predominantly with canicola (8.9%) and icterohemorrhagiae (8.1%).     

Serological studies on leptospirosis in domestic animals in Quebec.

During a period of 30 months, from January 1977 to June 1979, Leptospira agglutinins were detected in 355 (6%) of 5841 bovine sera, 52 (10.1%) of 511 porcine sera, one (5%) of 20 equine sera and one (12.5%) of eight canine sera. Bovine, porcine and equine sera reacted predominantly with L. pomona. Reactors to L. hardjo/sejroe, L. icterohaemorrhagiae and L. grippotyphosa were also detected in cattle. One porcine serum reacted with L. grippotyphosa and one canine serum with L. icterohaemorrhagiae. Al the sera originated from suspected cases of leptospirosis.     

Non-specific seroreactions against Brucella abortus in ruminants in New Zealand and the presence of Yersinia enterocolitica 0:9

The level of non-specific reactions found in the brucellosis serology of ruminants in New Zealand was very low until July 1992. This changed when, in an export consignment of 1071 deer, 35% reacted in the Brucella abortus tube agglutination test with titres varying from 50 to 200 IU. The reactors were also positive in the Rose-Bengal agglutination test and most of them reacted in the complement fixation test with titres varying from 10 to 80 IU. Yersinia enterocolitica 0:9 was later isolated from one deer of this consignment. It was the first isolate of this serotype recovered in New Zealand from an animal. Shortly after, false reactors occurred more frequently than before in sera from Brucella abortus accredited free cattle herds. As the involvement of Yersinia enterocolitica 0:9 was suspected in these cases, faecal samples from reactors and in-contact animals were cultured for this organism. Yersinia enterocolitica 0:9 was isolated from nine of 19 herds showing one or more false Brucella abortus seroreactions. Prior to 1990, Yersinia enterocolitica serotype 0:9 had not been isolated in New Zealand, despite the recovery of a number of other bio- or serotypes of the organism from humans and animals. From 1990 onward, serotype 0:9 began to be isolated from human faecal samples with increasing frequency. Since the first isolations from deer and cattle in 1992, it has now also been recovered from a cat and an alpaca and from cattle without any association with false positive Brucella abortus reactions. All serotype 0:9 isolates were of biotype 2.     

Toxoplasma gondii and Chlamydophila abortus in caprine abortions in Tobago: a sero-epidemiological study

A sero-epidemiological study was conducted on a goat farm that experienced an abortion epidemic in the 2005 breeding season in Tobago. Serum samples of goats (aborting and non-aborting) and cats were collected, in addition to the use of stored sera from the farm sampled in 2003 and 2004. Farm records on the reproductive and mortality rates for year 2003, 2004 and 2005 were also reviewed. The sera were screened for Toxoplasma gondii antibodies using the latex agglutination test (LAT), Chlamydophila abortus with an enzyme-linked immunosorbent assay (ELISA) and Brucella abortus using the buffered plate agglutination test (BPAT). Farm records revealed that for the period 2003-2005, the average kid per doe rate decreased from 2.1 to 1.5, the mortality rate increased from 6.3% in 2002 to 19.4% in 2004 and the fertility rate decreased from 98-99% (2002-2004) to 89% (2005). There was a dramatic increase in the abortion rate from <1% (2002, 2003 and 2004) to 29.2% (2005). Of a total of 161 sera tested comprising 12 from 2003, 89 from 2004 and 70 from 2005, 0 (0.0%), 21 (23.6%) and 45 (64.3%) were positive for T. gondii agglutinins (i.e. titres > or =1 : 64) and the differences were statistically significant (P < 0.05; chi(2)). Of all serum samples tested, only 1 (1.1%) of 89 from 2004 was positive for C. abortus while all the sera tested were negative for B. abortus. Amongst the 24 does which aborted in 2005 and were available for testing in mid-2005, 15 (62.5%) had reciprocal titres of > or =1 : 2048, three (12.5%) each had titres of 1 : 1024, 1 : 256 and < or =1 : 16 i.e. negative. The seroprevalence and titres of does that aborted, 20 (87.0%) of 23, all with titres > or =1 : 256 suggesting current infection, were statistically significantly (P < 0.05; chi(2)) higher than was detected amongst does that delivered normal kids, 25 (53.25) of 47 with 22 (48.8%) having titres of > or =1 : 256. One (50.0%) of two cats caught and tested was seropositive with a reciprocal titre of 128. This is considered the first documentation of T. gondii agglutinins in caprine abortion as well the detection of C. abortus antibodies from livestock in Trinidad. It is concluded that of the three zoonotic abortifacient pathogens tested for, T. gondii appeared to have played some aetiological role in the abortion epidemic investigated.     

A STUDY OF NEPHRITIS OF BEEF CATTLE IN NORTH QUEENSLAND

The incidence of subclinical nephritis in beef cattle slaughtered at a Townsville abattoir during the period 1970-73 was 3.8 percent. A rising incidence coincided with the wet summer of 1973-74 reaching a peak of 8.2 percent thereafter in June 1974. All but 1 of 100 cases macroscopically identified as nephritis and studied histologically proved to be of interstitial type in which lymphocytic infiltration predominated. Follicular lymphoid hyperplasia was also a common feature. The exceptional case showed proliferative glomerulonephritis. Leptospires were isolated from 16 percent of cases cultured, and observed in 8 percent of specimens examined histologically. All of the 8 isolates subjected to serogrouping were L. pomona. Of the animals tested serologically against 2 serotypes 66.2 percent were positive to L. pomona (33.8 percent), L. hardjo (18.9 percent) or both (13.5 percent). The total incidence is significantly higher than in other random surveys carried out in the region. These findings suggest that Leptospira sp. are a major cause of bovine nephritis. Since other pathogens endemic in the area, for example, Babesia and Theileria sp., may be capable of causing nephritis in cattle an accurate assessment of the aetiology of that form of renal disease in north Queensland cannot yet be made.     

The vaginal mucus agglutination test in the diagnosis of bovine brucellosis

Of 1140 vaginal mucus agglutination tests (VMAT) on specimens obtained in 1971-72 from 663 dairy cows in seven herds infected with brucellosis, 97 were positive. When the VMAT was positive one or more serological tests were also positive. Of the 97 corresponding serum agglutination tests 80 sera had titres of more than 533 international units. Only 69.8 per cent of VMAT from serologically positive cows were positive. No evidence was found of non-specific agglutinins in vaginal mucus and positive VMAT reactions appeared to be specific for field infection. Three cows showed evidence of local agglutinins in the vagina. Hence herd testing by VMAT has no advantage over tests of blood serum but the test could be an aid in establishing whether individual cattle are infected.     

An old disease with a new face: canine leptospirosis does not lose its relevance

The clinical features of the disease are presented based on retrospective analysis of the records of eleven dogs diagnosed with leptospirosis using clinical signs and results of the microagglutination test (MAT) between 1991 and 1996. Additionally, Leptospira titres were determined in 30 healthy dogs and 20 hospitalised dogs without clinical or laboratory evidence of leptospirosis. A positive titre for L. grippotyphosa, L. pomona, L. bratislava, L. australis, L. icterohaemorrhagiae and/or L. canicola was found in 16 normal dogs and only one hospitalised patient. Eight of these dogs had titres of > or = 1:800. Only one of them had been vaccinated shortly before sampling. These results suggest that many dogs from the surroundings of Bern, Switzerland have contact with various Leptospira interrogans serovars. In ten healthy dogs, the Leptospira titre was determined before and four weeks after vaccination with leptospiral antigen. Only two of the dogs showed a serologically measurable response to the antigen contained in the vaccine. In dogs MAT titers presumably do not reliably reflect the immune status against leptospiral infections.