Serologic prevalence of leptospiral infection in the horses in Kurdistan Province in Iran

taken randomly from jugular veins of all mares and stallions using a 10 cc special syringe. Of 7-10 cc blood taken per horse, 1-3 cc was used to prepare serum in the laboratory. After collecting of blood samples, determining numbers were written on the tube labels to facilitate access to the sample data in the next stages. The determined age groups were < 2 years (n = 34, 20.4%), 2-4 years (n = 66, 39.5%), 4-6 years (n = 43, 25.7%), and > 6 years (n = 24, 14.4%). Samples were collected from cities Sanandaj (42 mares, 27 stallions), Bijar (24 mares, 18 stallions), Ghorveh (13 mares, 13 stallions), Kamyaran (5 mares, 11 stallions), and Marivan (7 mares, 7 stallions), with total numbers of 69, 42, 26, 16, and 14 for both sexes in each city, respectively. Three Kurdish, Arab, and hybrid races were also investigated each comprising 118 (70.65%), 14 (8.38%), and 35 (20.95%) horses, respectively.

At the same time with sampling horses, such data as gender, age, race, and history of related diseases were recorded for individual horses. Accuracy of data was ensured through follow up of recorded health information by a veterinarian and inquiries from responsible horse fancier.

After serum isolation, samples were kept frozen and sent to the leptospirosis laboratory of the Veterinary Hospital, Faculty of Veterinary Medicine, University of Tehran, for examinations. The infection with different leptospira serovars was examined by MAT to determine average contamination. 

Statistical analysis

Results of infection level were expressed descriptively and were compared between groups using SPSS 24 software. Infection level was compared between male and female horses by t-test and between age groups, races, and cities with analysis of variance (ANOVA). Racial differences, disease history, and the presence of various serovars were assessed through the Kruskal-Wallis test.

significantly different among different cities of the province (P > 0.05) (Table 5).

Table 1. Frequency and comparison of serologic infection with different leptospirosis strains in the experimental horses based on sex.

Table 2. Comparison of serologic infection with different leptospirosis strains in the experimental horses.

                            *Significant at P < 0.05

Table 3. Frequency of horses infected with different leptospirosis strains based on age groups and infection levels among them.

Discussion

Although the literature available at Iranian scientific centers suggest the spread of infection in most regions of the country, no research has so far been conducted on the serologic prevalence of leptospirosis in horses of Kurdistan Province. To determine the disease status in the equine population of the region, samples were collected from horse-riding clubs in this region to examine the presence of leptospira antibodies serologically against six important leptospira serotypes.

A total of 167 blood samples was prepared from horse farms in the above regions and undergone the serologic tests for serologic prevalence of leptospirosis based on MAT test as a commonly used method for serologic diagnosis of leptospirosis. According to our observations, the leptospiral infection may be present in the equine population of Kurdistan Province. The microorganism itself has to be identified to detect the presence of either infection or asymptomatic antibody alone.

The presence of 16 (9.6%) and 151 (90.4%) positive and negative samples, respectively, out of 167 samples can be attributed to the presence of moisture and rats in some local stables and contact of some horses with such animals as goats, sheep, and cows as reservoirs of leptospira (Pilgrim and Threlfall, 1999).

In serologic-based examinations at different regions, leptospiral infections of 20.6-33.6% and 13.5% were reported in the USA and India, respectively (Roth and Gleckman, 1985; Park et al., 1992; Sheoran et al., 2000). A study in Ahvaz reported leptospiral infections of

27.88% in horses and of 40% in donkeys (Haji Hajikolaeiet al., 2005). Serologic leptospiral infection level was found to be 41.05% in horses of Tabriz, and 42.68% of stallions and 30.77% of mares were positive serologically with significant differences between the two sexes, but the ages and races of horses were not significantly correlated with leptospiral infection levels (Hassanpour et al., 2009). The study also reported that the highest infection (46.15%) was caused by Pomona serovar, followed by Grippotyphosa (41.03%), Icterohaemorrhagiae (17.95%), Canicola (12.82%), and Hardjo (2.56%). In research in Ardabil, a leptospiral infection prevalence of 7.77% was noticed in horses of the region (Khoushehet al., 2012).

The dominant leptospira serovars inducing serologic responses are different in various countries. For example, Pomona with 30.5% and 12.47% in Queensland and California, respectively, Australis with 16.2, 16.6, 53.3, and 22.3% in Ohio, England, Northern Ireland, and the USA, respectively, Bratislava, Copenhagen, and Pyogenes with 21.3% in the Republic of Ireland, and Pomona with 48.7% in India were reported as the most prevalent serovars among horses (Vermaet al., 1977; Egan and Yearsley, 1986; Parket al., 1992; Pilgrim and Threlfall, 1999). Grippotyphosa serovar reportedly accounted for 33.33% and 49.51% of serum positive cases among horses and donkeys studied in Ahvaz region (Haji Hajikolaei et al., 2005). Bratislava-related abortion was found to account for about 25% of abortions caused by Leptospira (Egan and Yearsley, 1986).

Studies have been conducted in various countries concerning Leptospira serovars through such methods as MAT and PCR. In Brazil, for instance, a study on the prevalence of Leptospira serovars in horses indicated an antibody presence of 32.7% in general infection, with Serjoe being the most abundant serovar (15.9%). Research has shown relationships between serum response with age, sex, activity (exercise, rest, or both), nutrition, complement use, waste depot, and the presence of felines in the environment (Peixoto Ribeiroet al., 2018). In England, 30 horses with equine recurrent uveitis (ERU) and 43 controls were analyzed to determine recurrent EUR inducing serovars. In the ERU group, only two cases were diagnosed with a c-value > 4 (a Leptospira induced uveitis event, 6.7%), along with the detection of serovars Hardjo and Javanica. There was no serologic difference between uveitis prevalence (65.5%) and control group (41.9%; P = 0.11). No significant difference was observed between Leptospira ҆s positive group (100%) and Leptospira-free uveitis (P = 0.52); (Malalanaet al., 2017).

In a post-flood study, 276 thoroughbred horses (aged 2-5 years) were examined in Rio de Janeiro, Brazil. The animals abided in flood for 72 hours, after which blood samples were collected for serologic tests. Assuming the flood incidence as day zero, the horses were sampled on days 20 and 35. On Day 20, 132 (47%) horses were seroactive with 200 titers, and 23 (31%) animals had increased antibody titers on Day 35. In addition, 34 urine samples for culture and PCR analysis were collected from serum positive horses on Day 35. The Copenhagen serovar had the highest incidence (88.8%). Even so, none of the urine samples were positive in the culture, and 12 (35.2%) samples were positive in PCR analysis. This seems to be the first report of leptospirosis incidence in urban horses (Hamond et al., 2012).

Some reports suggest the presence of leptospirosis-induced hemolytic anemia in horses. In a blood profile analysis, serum biochemistry and mineral factors were analyzed on days zero, 7, and 21. Significant reductions were observed in RBC, MCHC, Hb, PCV, platelets, RDW, and TLC (total leucocyte count, including neutrophils, eosinophils, basophils, lymphocytes, and monocytes). MCV also increased significantly indicating the incidence of hemolytic anemia in the samples (Sohail, 2017). Biochemical assays revealed significant rises of ALT, AST, ALP, BUN, and creatinine anemia in the samples, as evidence of liver and kidney damages. Significant decreases were recorded in serum concentrations of sodium, potassium, calcium, magnesium, phosphorus, copper, and zinc, indicating renal tubular injury (Sohail, 2017).

A general 5-week survey confirmed the presence of five leptoserovars in horses of a northern island in New Zealand. Three different groups of mares (non-pregnant, pregnant, or foal-bearing) were investigated in each farm(Bengtsson, 2018). 

For the first time in Belgium, the presence of equine Leptospira was confirmed and compared using MAT and real-time PCR methods in 2018. Of 66 eyes, Leptospira strains were prevalent in 20 (30.33%) samples in the ERU group. DNA was positive in both 11 aqueous humors and 17 vitreous bodies of 8 Leptospira positive horses. Vitreous bodies and aqueous humors were separately positive in 9 and 3 horses, respectively. A correlation of 0.47 was obtained between aqueous humors and vitreous bodies in the PCR diagnosis of Leptospira, which was interpreted as a low association. All the eyes were negative in the control group (Sauvage et al., 2019).

Of 91 mares and 76 stallions from a total of 167 samples herein, 12 (13.18%) mares and four (5.26%) stallions were Leptospira

positive, with no significant differences between serologic infections of mares and stallions (P > 0.05). Age groups of < 2, 2-4, and 4-6 years presented one (2.94%), seven (10.60%), and eight (18.6%) positive samples. No positive samples were found in horses aged over six years, with no significant differences among the age groups.

The Kurdish race was the most abundant among the sampled horses due to its high abundance in the region. The Arab and hybrid horses were also samples in limited numbers. Among Leptospira ҆s positive horses, there were all-mare, one stallion and four mares, and three stallions and one mare among leptospira-positive Kurdish, Arab, and hybrid races, respectively. Significant differences were recorded among the genera and races of horses infected with different leptoserovars (P < 0.05).

In this study, different sexes were not significantly different in terms of serologic infection with Leptospira. In a study in Tabriz, on the other hand, significant differences were observed in serologic infection between stallions (42.68%) and mares (30.77%) (Hassanpouret al. 2009).

In the current investigation, Icterohaemorrhagiae and Australis were the most and least abundant strains, respectively, and Canicola and Grippotyphosa were of intermediate abundance. Hardjo and Pomona were not observed among positive samples. The dominant leptospira serovars inducing serologic responses are different in various countries. For instance, Pomona with 30.5% and 12.47% in Queensland and California, respectively, Australis with 16.2, 16.6, 53.3, and 22.3% in Ohio, England, Northern Ireland, and the USA, respectively, Bratislava, Copenhagen, and Pyogenes with 21.3% in the Republic of Ireland, and Pomona with 48.7% in India were reported as the most prevalent serovars among horses (Ellis et al., 1983; Roth and Gleckman, 1985; Park et al., 1992; Sheoran et al., 2000). Grippotyphosa serovar reportedly accounted for 33.33% and 49.51% of positive serum cases among horses and donkeys studied in Ahvaz region(Haji Hajikolaei et al., 2005). Hassanpour et al. (2009) reported that Pomona serovar was present in 38.96% of positive serum cases among horses, donkeys, and mules in East Azerbaijan Province (Hassanpouret al., 2009). The same authors reported that 46.15% of positive serum cases were related to Pomona serovar among horses in the Tabriz region. In Ardabil, Hardjo serovar comprised 43% of serum positive cases among horses (Hassanpour et al., 2012). Bratislava-caused abortion was detected to be responsible for about 25% of abortions induced by Leptospira(Elliset al., 1983).

The highest and lowest Leptospira infection levels were recorded in Sanandaj and Bijar, respectively, with no significant differences among the sampled cities of Kurdistan Province (P > 0.05).

Among positive samples, 15 (93.75%) and one (6.25%) cases were infected with one and two serotypes, respectively. Hassanpour et al. (2009) noticed that eight cases of experimental samples (20.51% of positive serum cases) were positive for more than one serotype (Hassanpour et al., 2009). In serologic tests of leptospirosis such as MAT, the results usually determine infection about more than one serovar (Ellis et al., 1983; Roth and Gleckman, 1985; Park et al., 1992; Hartskeerl et al., 2004). This may result from simultaneous infection with several serovars, but the presence of cross- reactivity between serovars can deviate the report of the MAT test.

History of urogenital diseases, abortion, or ocular infections were observed in 12 out of 16 positive samples, which accounted for 75% of positive specimens, with significant differences (P < 0.05).

Conclusion

Our findings demonstrate that leptospiral infection may be present in the equine population of Kurdistan Province, and the presence of antibodies in the absence of infection necessitates the detection of this pathogen. The results also indicate that some leptospiral infections are hidden without clinical symptoms; hence preventive measures are necessary to control the disease in different regions of western Iran and Kurdistan Province.

Ethical approval

No ethical approval was obtained because this study did not involve a prospective evaluation, did not involve laboratory animals, and only involved non-invasive procedures (e.g. serum samples).

Acknowledgments

Special thanks to the Leptospirosis Lab of the University of Tehran (Leptospira Research Laboratory located in Veterinary Research and Teaching Hospital (VRTH) in Faculty of Veterinary Medicine, University of Tehran.) for collaborating on this project.

Conflicts of Interest

The authors declare no conflicts of interest statement.