Complete diagnosis of leptospirosis in tropical reproductive cattle

Leptospirosis is a worlwide zoonosis of great impact in both animal and public health. Bovine leptospirosis is commonly manifested by reproductive disorders, such as abortion, stillbirth and infertility; causing depletion of the economic balance of livestock farms, along with representing a health risk problem for farm workers. In view of these consequences, we aimed to evaluate the sanitary status of tropical cattle and their role as reproductive disseminators of leptospirosis. We analyzed blood and semen samples from 11 brazilian herds by three diagnostic methods -Culture, Microscopic Agglutination Test and Polymerase Chain Reaction. All animals were negative for bacteriological culture in Fletcher’s semisolid medium; 66% (264/400) animals were seropositive to at least one of 19 serovars (17 serogroups) of Leptospira spp. by MAT, given that 42.4% and 5.3% of animals presented titers against brazilians isolates Guaricura and Nupezo, respectively; furthermore, five animals were positive by PCR in blood and/or culture samples and three semen samples were positive by PCR (one of them also seropositive). These results highlight the coexistence of both disease's stages (acute and chronic) in the same environment, thus alert for venereal dissemination of leptospirosis, aggravating their sanitary condition and fomenting economic losses. We, authors, recommend the adoption of prophylactic measures, such as systemic vaccination, treatment of animals and improvement of hygienic-sanitary conditions.


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Among reproductive diseases that affect livestock, leptospirosis has a high degree of 44 importance, specially in tropical countries (1). This zoonosis is caused by members of 45 genus Leptospira spp., which currently includes 22 species and more than 300 46 serovars (2). Bovine leptospirosis represents an animal health problem, given its 47 common manifestation like chronic reproductive disorders, such as abortion, stillbirth 48 and infertility (3,4). Clinical signs of the acute presentation and mortality are most 49 frequent in calves (5). Furthermore, leptospirosis also represents a public health risk for 50 farm workers along with decrease in economic balance of livestock farms (6). For 51 diagnosis of leptospirosis, many current options are avaliable depending on sample 52 type and clinical phase of disease, each method has its own advantages and 53 disadvantages. Microscopic aglutination test -MAT, remains the reference test for 54 routine diagnosis (7,8). Concordance among positive MAT results and positive 55 outcomes by other techniques has proved that MAT is an efficient diagnostic test for 56 prediction of infecting serogroup (9); althought, another study proved that cattle may 57 not react against their own isolate by MAT, demanding caution when evaluating 58 negative MAT results for carrier status (10). Countries with strong livestock production, 59 like Brazil, have a particular concern on elucidate important features regarding 60 leptospirosis. Many research groups aim to explain the dissemination and risk factors 61 of this disease (11). Throughout the years, the interest on understanding leptospirosis 62 pathogenesis has led to studies envolving possible transmission routes. Venereal 63 transmission has attracted attention since the report of genital Hardjo infection in 64 naturally infected cattle (12), and presence of Leptospira spp. was demonstrated in 65 bovine semen by PCR (13), proving that the bacteria can also be established in 66 gonads, suggesting possible sexual transmission. These informations should be 67 considere when assessing disease's control programs, along with endemic serovars, 68 antibiotic and vaccination availability, besides epidemiological studies (14,15). In this 69 context, we aimed to evaluate the sanitary status of this zoonosis in clinically 70 asymptomatic cattle from a tropical region of Brazil. 71 72

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The area chosen for this study was the microregion of Bauru, which is a country town 74 of São Paulo, Brazil. Bauru is a tropical area known for its hot temperature, typically 75 from 59 F to 86 F; with extreme seasonal variation in rainfall, the least rain falls in 76 winter and the most rain falls around summer, encompassing up to 12 inches. 77 In order to estimate sample size for this study, we used the online program Epi Info TD 78 (http://www.openepi.com), with confidence interval of 95% and based on a preview 79 study that found prevalence of 58.7% in the city of Bauru, years before (16). Sample 80 size calculated for disease frequency was 373 samples. 81 We analyzed blood samples of 11 herds from five farms (representing 400 animals); as 82 to semen samples from two herds. Cattle were raised for reproductive purposes and 83 shared water fountain and pastures among most herds studied. All farms presented 84 historic of reproductive failures and no vaccination program installed for leptospirosis. 85

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Analysis by PCR of semen displayed some interesting results as well. Two bulls were 147 positive for confirmatory PCR of negative culture (dark-field microscopy); three bulls 148 were positive for semen PCR and for semen culture confirmation PCR; and other two 149 bulls were positive solely for culture confirmation PCR, but not for direct semen PCR. 150 Among these positive bulls, three were also positive by MAT (table 3). 151 152

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In order to evaluate sanitary state of the disease and not just merely exposure to the 157 bacteria, we used serovars from pathogenic species of Leptospira spp., one serovar 158 representative from each serogroup and also two native brazilian isolates, serovar 159 Guaricura (Leptospira santarosai serogroup Sejroe) and serovar Nupezo (Leptospira 8 canicola serogroup Canicola). Serovar Nupezo showed reactivity when compared to 161 serovar Canicola, supporting the inclusion of local strains for more sensitive MAT 162 results. Serogroup Sejroe had most positive outcomes, however serogroups 163 Autumnalis and Hebdomadis were also quite reactive, suggesting possible contact 164 among cattle with wild animals, given that only serogroup Sejroe is known to be 165 adaptive to cattle and the other two serogroups have been reported in wild animals 166 (20,21). Several studies around Brazilian territory showed the magnitude and 167 importance of leptospirosis in this tropical region, and despite variety of these studies' 168 outcomes, the seriousness can't be disguised (

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Considering that 66% (264/400) of cattle were seropositive, furthermore six animals 173 were positive for PCR of blood and/or culture of blood, as three bulls were also positive 174 by confirmatory PCR of semen cultures (one of them confirmed by semen PCR as 175 well), we can suggest there is an imminent present exposure of cattle to the disease in 9 those farms, the same ones that constitute a representation of the extensive livestock 177 production system in this tropical region. 178 PCR outcomes also showed trustworthiness when compared to culture, given that 179 results that were negative by culture were positive by PCR, which was no surprise 180 considering difficultness of visualization of spirochetes by dark-field microscopy (7). 181 Surprise came when comparing PCR results between negative cultures and samples, 182 animals were positive on blood culture but not on whole blood or vice versa, suggesting 183 that there could be an inhibitor in blood that was diluted when cultured. Similar results 184 occurred with two semen samples, thus same explanation can be granted (27).