Evaluation of serum infection rate of four common Leptospira serotypes in traditional farms of Tarom city

Introduction: Leptospirosis is a zoonotic disease, which is caused by gram negative and aerobic leptospira interrogans and its different serovars. All the pathogenic leptospirae were formerly classified as members of the species Leptospira interrogans; the genus has recently been reorganized and pathogenic leptospirae are now identified in several species of Leptospira. Leptospirosis is significant occupational hazard in the animal husbandry in certain areas. The majority of infections remain asymptomatic (Constable et al 2017). However, leptospirosis as a cause of acute respiratory distress is becoming more frequently recognized. A wide variety of serological tests, which show varying degrees of serogroups and serovar specificity, have been described. Laboratory procedures are used in the diagnosis of leptospirosis but two tests have a role in veterinary diagnosis: the microscopic agglutination test (MAT) and ELISA. Cross-reactions caused by exposure to leptospiros of the same serogroup can occur in MAT, for example, infection by L. balcanica and L. medanensis can produce false positive L. hardjo reactions (Quinn et al 2002). The MAT has the disadvantages that it is tedious and time consuming, and the use of live culture imposes a risk of human infection.  Another disadvantage is the failure of the MAT to differentiate between titres after vaccination and those after natural infection, since the titres may be of similar magnitude. Uveitis is the most frequently encountered clinical manifestation of leptospirosis in animal, which appears to be mediated by autoimmune mechanisms involving cross reactivity between ocular tissues and leptospiral membrane proteins; however, abortion and stillbirth are serious problems. Many serological studies in different countries were conducted on leptospirosis disease which has mostly showed the highest percentage of infection in animal (Hassanpour et al 2009). The aim of the study is to determine the serovars of leptospira in Tarom area.
Material and methods: During June to September 2015 for this study, 200 blood samples randomly collected from jugular vein of different animal (47 males and 153 females) of Tarom area (Abbar, Chavarzag County and Daram, Dastjerde Village). None of these animals had been vaccinated against Leptospira and there was no history of leptospirosis-related symptoms or signs of the disease at the time of sampling. we have collected 10 ml of blood from the jugular vein of each animal. After taking blood, the tubes were stored at room temperature for 1 - 2 h, so that the blood clots are completely formed, then they were stored at 4˚C in refrigerator. Next morning these tubes were removed from the refrigerator and their serum was extracted using sterile Pasteur pipettes, and serums were transferred to Micro tubes. If there were red blood cells in the extracted serum, the serum would be centrifuged at 3000 rpm for 10 min, and then the pure serums were transferred to Micro tubes. It should be noted that during the transfer, the related numbers were inserted on Micro tubes. The serum micro tubes were frozen at -20˚C, so that the least damage occurs until performing the test. A few hours before MAT test, the samples were removed from the freezer and gradually melted at room temperature and tested by microscopic agglutination test (MAT). The serum samples were tested for antibodies to 4 live serovars of leptospira Canicola, leptospira Grippothyphosa, leptospira Pomona, and leptospira Icterohaemorrhagiae using the microscopic agglutination test. On the basis of age, these animal were divided in 4 groups (Results and discussion: In 48 samples (24 percent), including 23 cattles, 7 goats, 15 sheep and 3 horses among the 200 collected samples, infection with leptospira interrogans was detected. Leptospira Grippothyphosa has the highest percentage 39.59 (19 animal) including 10 cases of cattles, 7 sheep, and 2 horses, followed in descending order by Icterohaemorrhagiae (25%), Pomona (18.75%), and canicola (16.66%). Species of animal was significantly related to the prevalence of leptosprial antibodies. In serological tests for leptospirosis such as MAT, the results often indicate infection with more than one serovar. This may be the result of mixed serovar infection, but the existence of cross-reactivity in the MAT between the serovars is well known and can be excluded from this interpretation. The predominant Leptospira serovars giving rise serological reaction vary somewhat between countries. Haji Hajikolahi et al. (2005) reported that serovar Grippothyphosa is present in 33.33% of positive horses in Ahavaz area in Iran. In Urmia district, serovar Pomona is identified as causing about 16.2% of leptospiral infection (Abdollahpour et al 2016). The rate of infection in different genders were studied, in which the most affected gender was female animals (16.5 percent) included 16 cattle, 4 goats, 12 sheep, and 1 horse. There was   a significant difference between male and female seroprevalence (p < 0.05), which is in agreement with the report by Abdollahpour et al. (2013) concerning goats in Urmia; and hassanpour et al. (2009) concerning horses in Tabriz. There was no significant relationship between aging and the incidence of leptospiral infection in goats, sheep, and horses (P>0.05), but there was a significant difference between aging and the incidence of leptospiral infection in cattle (p < 0.05). The most general titer was 1:100 and the rate of infection in different ages were studied, in which the most affected age was 4-3 years, 23 animals (11.5 percent) including 11 cattles, 3 goats, 7 sheep, and 2 horses; which is in agreement with the report by Abdollahpour et al. (2016) concerning Buffalos in Urmia. In serological tests for leptospirosis such as MAT, the results often indicate infection with more than one serovar, but in this study, all samples showed a positive reaction to only one type of serovar. This may be the result of mixed serovar infection, but the existence of cross reactivity in the MAT between the serovars is well known and can be excluded from this interpretation. Leptospiral antibodies appear within a few days of infection and persist for weeks or months and, in some cases, years. Unfortunately, antibody titres may fall to undetectable levels, while animals remain chronically infected. To overcome this problem, sensitive methods are needed to detect the organism in urine or the genital tract of chronic carriers. The results were analyzed by chi-square to determine the difference between sexes and different groups of age and species of animal was significantly related to the prevalence of leptosprial antibodies. Therefore, the demonstration of leptospirae in the genital tract and or urine only must be interpreted with full consideration of the serological results and culture or detection of leptospirae in blood or body fluids, as these findings may indicate that the animals are carriers.
Conclusion: These results confirmed that leptospiral infection may exist in the animal population in Tarom region and the presence of antibodies in the absence of infection indicates exposure to the organism and must be acknowledged. In addition, these results confirmed that the majority of leptospiral infections are asymptomatic. Accordingly, prevention of animal leptospirosis must rely on good hygiene practices, minimization of rodent contact, and vaccination of other species of production and companion animals. In addition, these results confirmed that the majority of leptospiral infections are asymptomatic.