Seroprevalence and associated risk factors for bovine leptospirosis in Egypt

Leptospirosis is caused by pathogenic bacteria of the genus Leptospira and is one of causative agents of reproductive problems leading to negative economic impact on bovine worldwide. The goal of this study was to investigate the seroprevalence of Leptospira spp. in cattle in some governorates of Egypt's Nile Delta and assess the risk factors for infection. A total of 410 serum samples were collected from cattle and examined using microscopic agglutination test. The overall seroprevalence was 10.2% and the most prevalent serovars were Icterohaemorrhagiae, Pomona and Canicola. In addition, the potential risk factors were associated Leptospira spp. infection were age, herd size, history of abortion, presence of dogs and rodent control. Thus, leptospirosis is common in dairy cattle in the Nile Delta and the presence of rodents in feed and dog-accessible pastures increases the risk of Leptospira spp. infection among animals.


Ethical statement
Benha University's ethics committee for animal research approved the study's methodology and techniques.All cattle owners provided informed consent to participate in the study.The Faculty of Veterinary Medicine's ethics committee guaranteed that all operations followed all applicable rules.The ARRIVE criteria were followed throughout the study process.

Study site
This study was performed during the period of March 2021 to February 2022 and cover three governorates (Kafr ElSheikh, Menofia and Qalyubia) situated at Nile Delta of Egypt, Fig. 1.The selected governorates are located at latitudes 31° 06′ 42″ N, 30.52°N, and 30.867°N, respectively, and at longitudes 30° 56′ 45″ E, 30.99°E, and 31.028°E.
A hot desert climate dominates the Nile Delta in general, but in its northernmost part, which is also the wettest region in Egypt, it has relatively moderate temperatures with a high of 31 °C in the summer, as is the case with all of the northern coast of Egypt.

Sample design and sampling
The sample size were determined using the following formula according 26 using the procedure for simple random sampling: where n is the sample size, P is the predicted prevalence 50%, Z = 1.96 with 95% confidence level, and d is the absolute error 5%.The calculated number of samples was 384 and increased to 410 to increase the precision.
In order to obtain serum, cattle blood samples were collected using vacuum tubes without anticoagulant through punctured the jugular vein and centrifuged at 3000 xg for ten minutes.the serum was stored at − 20 °C in 1.5-mL Eppendorf tubes till serological examination was completed.

Data collection
Cattle owners provided the database with their individual information to identify potential risk factors for leptospirosis seropositivity.At the time of blood sampling, each participant filled out a questionnaire.A number of variables were selected: (1) location (Kafr ElSheikh, Menofia and Qalyubia), (2) age (2, 2-3, and > 3 years), ( 3) sex (male and female), (4) herd size (50, 50-75, and > 75), (5) gestation status (pregnant and non-pregnant), ( 6) history of abortion (yes or no), (7) presence of dogs (yes or no), and 8) rodent control (yes or no).The samples were collected randomly from individual farmer, two medium herds and one large herd.

Serological diagnosis
In accordance with the recommendations of the World Organization for Animal Health (OIE), the serological diagnosis was carried out using a microscope equipped with a dark field condenser to conduct the microscopic agglutination test (MAT) as described by 27 .The panel of antigens utilized in this investigation contained seven common strains, taking into account the most common serovars of Leptospira interrogans in the country: Canicola, Hardjo, Pomona, Icterohaemorrhagiae, Grippotyphosa, Bratislava, and Copenhageni.A dilution of 1:50 was used for the initial testing of sera samples, and those with an agglutination level equal to or greater than 50% were further diluted.The final titration was calculated as the dilution at which 50% agglutination was detected.A titration of 1:50 indicated that the animals had been exposed to the causative agent.Titrations of 1:100 were regarded as positive for Leptospira infection.

Statistical analysis
The data from the questionnaires were analysed to identify potential risk factors for leptospirosis seropositivity.The analysis was done in two stages: univariate and multivariate.In the univariate analysis, each independent variable was crossed with the dependent variable (seropositivity), and those with a chi-square test P-value < 0.20 were subjected to multivariate logistic regression analysis [28][29][30][31][32][33] .A correlation analysis was used to confirm collinearity between independent variables; for those variables with substantial collinearity (correlation coefficient > 0.9).The statistical analysis was performed by SPSS software ver.24 (IBM < USA).
The univariate analysis for the variables associated to seropositivity for any Leptospira spp.serovar in cattle were presented in Table 2.The seroprevalence revealed non-significant (P > 0.05) association between locality, sex and gestation status and Leptospira seropositivity.
The seroprevalence rose with age and was substantially (P < 0.05) higher in cattle over 5 years old (15.8%), particularly in those raised in large herd sizes (37.1%).Furthermore, Leptospira seroprevalence in cattle increased significantly (P < 0.05) in animals with a history of miscarriage (16.4%), in animals living with dogs (18.7%), and in homes without rodent management (14.2%),Table 2.
The variables with P < 0.2 in univariate analysis were included in multivariate logistic regression model.The variables were identified as risk factors in multivariate model for Leptospira seropositivity were age more than five years (OR 7.24, P = 0.027), large herd size more than 75 (OR 30.53,P < 0.0001), animal with history of abortion (OR 1.49, P = 0.036), presence of dogs (OR 6.32, P < 0.0001) and absence of rodents control (OR 2.03, P = 0.010), Table 3.

Discussion
Leptospirosis is a global zoonotic threat and information on the disease's epidemiology and the variables that contribute to its incidence is very important to improve the control level of leptospirosis 34 .In particular, few studies to our knowledge have been considered the epidemiological situation of leptospirosis in cattle in Dakhalia governorates but no data about its prevalence in other governorates of Nile Delta.Therefore, one of the major aim of this study is determination the seroprevalence of Leptospira spp. in cattle in three Egyptian governorates and assess its associated potential risk variables.In this study, the seroprevalence of Leptospira spp. in cattle raising the three studied governorates in Nile Delta (Kafr ElSheikh, Menofia and Qalyubia) was 10.2% (95% CI 7.66-13.55).In another Nile Delta governorate, cattle seroprevalence was estimated to be 39.33% 25 .As a result, the findings emphasise the significance of this disease in the country and the necessity to develop effective control measures to lower its incidence.
Alternatively, lower prevalences have been reported in some countries, it was 3% in North Eastern India 40 , 3.2% in Poland 41 , 13% in Tanzania 42 , 20.3% in Sri Lanka 43 , 31.3% in Brazil 44 , and 24.48% in southwestern Ethiopia 45 .www.nature.com/scientificreports/Several factors may contribute to this variation, including geography, husbandry practices, management, sampling and diagnostic method, natural immunity, and disease resistance 9,14,30,32,33,[45][46][47] .In addition, high densities of infected cows with Leptospira spp.might lead to environmental contamination and disease spreading since they could serve as reservoirs and spread infection to other animals residing in the same habitat 48 .
Interestingly, the most prevalent serovars among examined cattle in the present study were Icterohaemorrhagiae (2.9%), Pomona (2.2%) and Canicola (1.9%).These findings are in accordance with previous findings reported by 49 and 50 , they found the most common serovars in cattle Pomona and Icterohaemorrhagiae.Moreover, Icterohaemorrhagiae and Pomona serogroups are associated to animal interaction with various animal species that serve as reservoirs for the diseases 51 .
In the present study, the seroprevalence of Leptospira spp.did not varied between studied governorates because all of them situated in the Nile Delta and have the same climatic features and topographic characters 52 .Moreover, Marzok, et al. 52 found that the most prevalent serovars in Egypt was Icterohaemorrhagiae, Canicola and Pomona.
Similar to previous findings of dos Santos, et al. 44 , but in contrast with findings of Parvez, et al. 53 , the seroprevalence of Leptospira spp.increased significantly with age.In addition, in an Indian investigation, Sudharma and Veena 54 observed that the seroprevalence was not correlated with animal age.This might be attributable to the fact that exposure to Leptospira becomes more common as old cattle, and that seropositivity can remain for a very long period 1, 25 .
The present findings revealed that the females were more seropositivity for Leptospira spp.than males, this consistent with previous findings of El-Deeb, et al. 25 and Ijaz, et al. 55 .However, many previous studies have shown that males are more likely to contract leptospirosis than females without a significant variation 56,57 .There is no clear explanation for these findings and reported differences in relation to sex 57 .The result of present study might be contributed to most of the samples examined were collected from female cows which give its potential influence.
Leptospira spp.seroprevalence significantly increased in large herd size in accordance with prior findings of Benseghir, et al. 58 .This finding may be explained by inadequate sanitation facilities, difficulty in monitoring hygienic practices on large herds compared to small herds and Leptospiral infection spread rapidly in overcrowded farms which have poor management and sanitation application 4,35,44,55 .
In the current study, the prevalence of Leptospira spp. was higher in cattle suffered from history of abortion or second semester of pregnancy.The findings confirm previous reports that Leptospira spp.present chronically in bovines and can lead to sexual dysfunction, low fertility, and abortion 59,60 .
The presence of dogs increased the prevalence of Leptospira spp. in cattle, which come in agreement with previous findings of Fávero, et al. 49 .Moreover, Leptospira spp.were more prevalent in cattle raising farm which have poor management and rodent control.Similar findings were concluded by Motto, et al. 42 .Rodents are mostly recognized epidemiologically for spreading various pathogenic Leptospira and contaminating pasture 61 , and as a result, animals may contract leptospirosis during grazing 62 .

Conclusion
The results of present study confirmed that Leptospira spp.present among cattle in Nile Delta of Egypt, contributed as cause of abortion in pregnant animals.The multivariate logistic regression model identified age, herd size, history of abortion and control of rodents as potential risk factors for Leptospira spp.infection.The identification of species and biovars, the understanding of transmission cycles, and the implementation of preventative and control measures are critical, particularly for dairy cows, as well as identifying alternatives to management practices that could spread disease to people or animals.

Figure 1 .
Figure 1.MAP illustrated the governorates under the study (MAP generated by QGIS software).

Table 1 .
The prevalence of Leptospira in relation to different serovars.

Table 2 .
Prevalence of Leptospira spp. in cattle in relation to different variables.*The result considered significant if P < 0.05.χ2: Chi-square, df: degree of freedom , P: P value.

Table 3 .
Multivariate analysis for risk factors associated with Leptospira spp.infection.B Logistic regression coefficient, SE Standard error, OR Odds ratio, CI Confidence interval.