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On-line version ISSN 1678-4456
Braz. J. Vet. Res. Anim. Sci. vol.35 n.2 São Paulo 1998
Leptospirosis on animal reproduction: IV. Serological findings in mares from six farms in Rio de Janeiro, Brazil (1993-1996)
Leptospirose em reprodução animal: IV. Achados sorológicos em éguas de seis propriedades no Rio de Janeiro, Brasil (1993-1996)
Departamento de Microbiologia e Parasitologia
Universidade Federal Fluminense
Rua Hernani Mello, 101
24210-130 Niterói RJ
From February 1993 until October 1996, 547 blood samples were collected from mares with reproductive problems in six farms of the state of Rio de Janeiro, Brazil, and tested by the microscopic agglutination test (MAT) with live antigens for antibodies to seven leptospira serovars. A total of 235 positive reactions (42.96%) at a minimum serum dilution of 1:100 were observed against one or more serovars, which in comparison to other studies developed in Brazil, may be considered as a high prevalence. On mares with anti-leptospiral antibodies, the predominant serovar was icterohaemorrhagiae, reactive on 102 (43.40%) samples, followed by 64 (27.23%) reactions against bratislava, and 34 (14.47%) against pomona. The high prevalence of antibodies to serovar icterohaemorrhagiae observed in this study could have been expected, because in Brazil about 36% of the population of the maintenance host of such strain, Rattus norvegicus, are leptospiral carriers, mainly to strains of the Icterohaemorrhagiae serogroup.
UNITERMS: Leptospirosis; Abortion; Reproduction; Horses.
Phisiopathology of reproductive problems and abortion determined by leptospirosis on cows and sows are well studied; however, the knowledge on equine leptospirosis, specially the reproductive pathogenesis of the disease, is still limited.
The association between leptospiral infection and equine disease has been controversial over a number of years, but it is now clear that it is an important cause of equine abortion3,6. The leptospiral infection in horses has been associated to uveitis16, keratitis17, fever and jaundice19 and, frequently, to abortions1,5.
Serological surveys showed that the disease occurs worldwide, but the predominant serovars of Leptospira interrogans in different countries may vary7. The prevalence of icterohaemorrhagiae in horse infection was refered in England10, Northern Ireland6 and Minnesota16. The serovar bratislava was found in Ontario11, Italy3 and Kentucky19. Ellis et al.7 suggested that bratislava is host-adapted to the horse, but the clinical significance of the infection with this serovar is still unclear11.
In Brazil, in spite of the enormous population of horses, there are few studies on equine leptospirosis. The first study was published on 19574, a serological survey on horses from São Paulo, and observed the predominance of serovar icterohaemorrhagiae. On 1960, Freitas et al.8 submitted 34 aborted fetuses to dark field and histological examination and confirmed the ocurrence of abortion associated with leptospirosis. On 1968, Santa Rosa et al.15 related the occurrence of antibodies against pomona and in 1974 Cordeiro et al.2 carried out a serological survey in 404 equine blood sera and registered the predominance of serovars pomona and tarassovi.
The isolation of Leptospira interrogans serovar icterohaemorrhagiae from the kidney of an aborted foetus was referred in 19819 and represents the first isolation of a leptospirae strain from a horse in Brazil. On 1986, Yasuda et al.22 reported the isolation of Leptospira biflexa serovar ranarum from an aborted equine foetus and discussed its importance in the aetiology of the abortion. The prevalence of icterohaemorragiae on mares with reproductive problems12 and in horses without clinical signs in São Paulo14 was presented in 1993.
The objectives of this study were to determine (1) the seroprevalence of Leptospira in mares with reproductive problems in Rio de Janeiro, Brazil and (2) the predominant serovars in those animals.
MATERIAL AND METHOD
From February 1993 until October 1996, 547 blood samples were collected from mares originated from different farms and with reproductive problems, such as estrous repetition, low conception rates, premature foaling and abortion, about 34 days after the occurrence of the abortion or the estrus repetition. In premature foaling cases, the blood samples were collected 3-5 days post-partum, and a second sample, when possible, 30 days after foaling. Sera were obtained by centrifugation and stored at -34ºC until being tested as a batch.
Sera were originated from six farms representing all geographic regions of the Rio de Janeiro State, and tested using the microscopic agglutination test (MAT) with live antigens7 for antibodies to seven leptospira serovars: andamana (CH11), pomona (Pomona), canicola (Hond Utrecht IV), icterohaemorrhagiae (RGA), hardjo (Hardjoprajitno), bratislava (Jez bratislava) and ballum (Mus 127). Titres were reported as the reciprocal of the highest dilution in which 50% of the leptospires were agglutinated. Any sample having a titre >100 to any of the seven serovars was considered positive. All samples reactive to more than one antigen at the dilution of 1:100 were subsequently titrated against reacting antigens using serial 2-fold dilutions of serum until the highest titre was obtained in order to determinate the infective serovar.
Of the 547 mares tested, 235 were positive at a minimum serum diluition of 1:100 against one or more Leptospira serovars, representing 42.96%. Titres to more than one serogroup were a common occurence.
Of the 235 positive mares, 102 (43.40%) reacted against serovar icterohaemorrhagiae, followed by 64 (27.23%) against bratislava, 34 (14.47%) against pomona, 17 (7.24%) against ballum, eight (3.40%) against hardjo, six (2.56%) against canicola and four (1.70%) against andamana, a Leptospira biflexa serovar (Fig. 1). Reactions to serovar andamana, canicola and ballum, unusual in equine samples, were considered as coagglutinations, and are not included on Fig. 1. With 98 mares, a second serum specimen was obtained, and at least a four fold - increase of titers was observed to serovar icterohaemorrhagiae in 63 (64.28%) samples, represented by 29 with 400, 25 with 800 titres, five with 1600 and four samples that reached 3200. To serovar pomona nine (9.18%) samples presented titres of 400 (four samples), 800 (four samples) and 1600 (one sample). The titres found with the second sample can be observed on Fig. 2.
Prevalence of Leptospira serovars in mares with reproductive problems from six farms in
Distribution of titres on the second sample obtained from mares with reproductive problems in Rio de Janeiro, Brazil (1993-1996).
As the diagnosis of equine leptospirosis by culture or by demonstrating leptospira in equine tissue is limited19, the serological evidences in mares with reproductive problems, specially with a historical of abortions, although not confirmed through statistical methods, suggests the existence of a relationship between leptospirosis and reproduction problems in horses, as it was observed in Northern Ireland6 and in Kentucky as well1.
The percentage of reactors was 42.96%. A similar finding was observed in Kentucky, where Williams et al.19 reported 42.2% of positive horses. In England, Hathaway et al.10 reported 34.6% of reactivity and, in Australia, sera from 41.5% of the horses reacted to one or more of the tested serovars18. Therefore, it was lower than with those observed in Northern Ireland by Ellias et al.7, that found antibodies in 89.1% of the tested sera.
In comparison to other studies developed in Brazil, 42.96% of reactors may be considered a high prevalence, since they found positivities of 4.53%9, 10.11%21 and 26.92%2. This could be explained by the non-inclusion of serovar bratislava as an antigen in those surveys, since in the present study the seroreactivity to this serovar represented 11.70% of the total of samples.
Although equine leptospirosis is a widespread infection, the spectrum of leptospiral titres in horses is probably a reflection of exposure to serotypes maintained by other animals in the same geographical region10, consequently may vary significantly by region19.
The greatest number of positive horses in this study reacted with serovar icterohaemorrhagiae, which is maintained by rats. Ellis et al.7 found that 26% of mares had antibodies to a serovar of Icterohaemorrhagiae serogroup in Northern Ireland; Williams et al.19 related in central Kentucky, USA an average of 21.5% of seropositivity to that serovar, and, in Brazil, it was reported the predominance of icterohaemorrhagiae on serological surveys accomplished on horses9,14. The incidental infections of horses by strains maintained by other hosts are important, and there is a considerable opportunity of transmission between rats and housed horses6. So, the high prevalence of antibodies to serovar icterohaemorrhagiae in mares from Rio de Janeiro could have been expected, since about 36% of the population of Rattus norvegicus are leptospirae carriers in Brazil, with the predominance to strains of Icterohaemorrhagiae serogroup13.
The serovar bratislava was suggested to be host-adapted to the horse7, what could explain the high prevalence of antibodies usually observed with this serovar. However, isolations data are still insufficient to support this fact19. In this study, 27.23% of positive mares reacted to bratislava, making it the second most prevalent serovar to icterohaemorrhagiae in terms of frequency of occurrence. Since host-adapted strains causes little pathogenicity in this host20, a higher prevalence of non-adapted and, consequently, more agressive strains could be expected in those mares with reproductive problems and abortions.
The serovar pomona was found in many parts of the world, and is linked to periodic ophthalmia, jaundice and abortion in horses1,16,17,19. It is maintained in cattle and swine, and the horse is an incidental host. In Brazil, pomona has been recognized as an important agent in animal leptospirosis, including cattle, swine and horses8. In 1968, Santa Rosa et al.15 related pomona as the most frequent serovar on equine leptospirosis, with rates of 13.6% of reactivity. In 1974, Cordeiro et al.2 worked with horses of the state of Minas Gerais and also observed predominance of pomona, with 26.15% of positivity.
Knowledge of the role and importance of leptospirae infections in horses is necessary in order to allow the understanding of the epidemiology of the disease all over the world, since economic losses due to abortions and low reproductive efficiency on mares infected with leptospirae strains have still not been calculated.
De fevereiro de 1993 a outubro de 1996, 547 amostras sangüíneas foram colhidas de éguas com problemas reprodutivos de seis propriedades no Estado do Rio de Janeiro, Brasil, e testadas pela técnica da soroaglutinação microscópica (MAT) com antígenos vivos para anticorpos perante sete sorotipos de leptospiras. Um total de 235 reações positivas (42,96%) a um título mínimo de 100 foram observadas perante um ou mais sorotipos de leptospira, o que, em comparação com outros estudos já realizados no Brasil, pode ser considerado uma alta prevalência. Em éguas com anticorpos antileptospiras, o sorotipo predominante foi icterohaemorrhagiae, reativo em 102 (43,40%) amostras, seguido de 64 (27,23%) reações positivas para bratislava e 34 (14,47%) perante pomona.
UNITERMOS: Leptospirose; Aborto; Reprodução; Eqüinos.
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