EVIDENCE OF ANTIBODIES AGAINST DIFFERENT Leptospira spp. SEROGROUPS AND FACTORS ASSOCIATED WITH ITS PRESENCE IN SHEEP FROM VERACRUZ, MEXICO † DE ANTICUERPOS CONTRA DIFERENTES SEROGRUPOS SU EN OVEJAS DE VERACRUZ, MÉXICO]

spp., associated serogroups, and possible risk factors in the state of Veracruz, Mexico. Methodology . Blood samples were collected from 405 sheep in 55 farms located in 13 municipalities distributed along three ovine-producing regions in the state. Serological diagnosis was made by Microscopic Agglutination Technique (MAT). The prevalence and its confidence intervals (CI) were calculated using Vassarstats software, the Odds Ratio (OR) values were estimated with WinEpiscope. Results. An overall seroprevalence of 53.83% ( 95% CI:48.84-58.75), per municipality of 100% ( 95% CI :71.66-100) and per flock of 92.73% ( 95% CI: 81.58-97.65) were noted. The highest prevalence by region was 58.82% ( 95% CI :% :50.57-66.62) for Los Tuxtlas (p= 0.218) and per municipality 86.36% ( 95% CI:71.95-94.33) R. has an overall seroprevalence of 53.83% in the state of Veracruz, Mexico and several serogroups present. fueron región y municipio. Región de Los Tuxtlas (OR=1.92; IC 95% :1.08-3.43) y los municipios de Ángel R. Cabada (OR=15.5; IC 95% :5.1-47.2), Ayahualulco (OR=10.1; IC 95% :3.6- 28.7) y Coatzintla (OR=6.1; IC 95% : 30.3 – 95.0). Implicaciones: Este estudio explora la presencia de anticuerpos contra Leptospira spp. factores estado de Veracruz, México. Este es el primer reporte región. Se como hospedero de Leptospira spp. endémicas. Conclusiones. Leptospira spp. en ovinos tiene una seroprevalencia global de 53.83% en el estado de Veracruz, México y varios serogrupos presentes.


INTRODUCTION
Leptospirosis is an emerging disease that spreads at an alarming rate (Azhari et al., 2018). Leptospirosis is a zoonotic disease that affects humans, as well as wild and domestic animals. It is caused by a spirochete called Leptospira with more than 300 serotypes (Adler, 2015). In ovine, the disease generates productive and reproductive problems such as anorexia, depression, irritability, fever, polypnea, diarrhea, constipation, hemolytic disease, hemoglobinuria, anemia, jaundice, lameness or sudden drop in milk production, and epidemic abortions in acute states. Meanwhile, in subacute and chronic disease it can be observed infertility, increased number of services per conception, prolonged calving intervals, abortion, stillbirths, birth of weak animals and perinatal deaths (Consalter et al., 2019). All these effects translates into economic losses (Malone et al., 2010), being a serious problem, especially in the tropics, where seroprevalences of 54.9% have been found in Brazil (Eckstein et al., 2017), 44% in India (Balamurugan et al., 2021), 28.8 in Colombia (Parra et al., 2016), and 54.5% in Mexico (Arteaga-Troncoso et al., 2015).
Veracruz is the third state with the highest ovine production in Mexico. Only in 2019, 5,425 t of mutton were produced (SIAP, 2020). However, the condition in which ovine production develops is lacking adequate biosecurity measures, which in turn can determine the susceptibility of these animals to different serogroups of Leptospira spp. (Luna et al., 2018). Sheep can acquire Leptospira spp through the urine of infected rodents, by contact to infected animals or drinking water contaminated with the bacteria (Martins and Lilenbaum, 2017).
In particular, the serogroups Hardjo is the most frequent worldwide, although other serogroups such as Pomona, Ballum, Bratislava and Grippotyphosa have been found (Bautista et al., 2014). In Mexico, there are no official immunization protocols against Leptospira infection in sheep. Some of the factors that may determine a greater probability of presenting infection with Leptospira spp. in ovine are living with other animal species (Topazio et al., 2015), whether domestic or wild fauna, contact with aborted fetuses or placentas from infected animals, and even climatic conditions such as high temperature and humidity that favor the presence of the bacteria in the environment, among others (Arteaga-Troncoso et al., 2015). In Veracruz, the situation of ovine leptospirosis is unknown. Therefore, the objective of this study was to identify the seroprevalence of Leptospira spp., associated serogroups, and possible risk factors in the state of Veracruz, Mexico.

MATERIALS AND METHODS
The study was carried out in the state of Veracruz, which is located on the coastal strip of the Gulf of Mexico between 17° 09´ and 22° 28´ of north latitude and 93° 36' and 98° 39' of west longitude. The 13 municipalities with the highest sheep inventory in the state were included in the study. All of them belonged to the Rural Development Districts (RDD) 002 Tuxpan, RDD 003 Martínez de la Torre, RDD 004 Coatepec and RDD 003 San Andrés Tuxtla ( Figure  1). The present work was a multistage and stratified cross-sectional epidemiological study. The universe was that of the 2018 state ovine inventory corresponding to 698,520 heads (SIAP, 2021). Sample size was calculated according to the formula provided by Thrusfield (2005), for an estimated prevalence of 50% and 95% confidence: = 2 2 where: n = sample size, z = 1.96 for 95% confidence, 2.56 for 99%, p = expected prevalence, q = 1-p, b = accuracy or admitted error.
In total, 405 blood samples were collected from sheep in 55 farms from 13 municipalities, located in three sheep-producing regions. The inclusion criteria were: females older than three months old, age and studs destined for service and absence of previous vaccination against leptospirosis as declared by owner. Meanwhile, the exclusion criteria were: lambs younger than three months old and males not destined for breeding rams.
A general questionnaire was applied for each farm, including variables such as: productive system (classified according to degree of confinement and/or grazing used as intensive, semi-intensive, and extensive), productive status (lambs, ewes, pregnant ewes, rams, lactating ewes), water sources (domestic service network, river, deep well) and contact with flood areas (yes / no). The concurrence on farm presence of domestic, wildlife, and noxious fauna was also considered. Additionally, an individual questionnaire collected data on each sampled sheep, on subjects such as breed, age, sex, productive status, body condition, health status, and presence of clinical signs suggestive of leptospirosis. This individual data was used to test for possible risk factor at the individual level.
A blood sample of 7 mL was taken by puncture of the jugular vein on each selected animal. The samples were identified and transported to the Microbiology Laboratory of the Faculty of Veterinary Medicine, University of Veracruz in Mexico where they were centrifuged at 1,000 X g for 15 minutes to obtain serum and aliquots of them were made and then stored at -20° C until processed by serological test.
The serological diagnosis was made in the Leptospira and Leptospirosis Laboratory of the Universidad Autónoma Metropolitana, Unidad Xochimilco, in Mexico City using the Microscopic Agglutination Technique (MAT), which has a sensitivity of 98.2% and a specificity of 96.4% (WHO, 2003). Serial double dilutions of each serum were made starting at 1:50, the antibody titer was determined at the highest serum dilution where 50% or more agglutination was observed. Titers of 1:100 were considered as positives. Prevalences and their 95% confidence interval (95%CI) were obtained with the Vassarstats online program. Chi-square was used to test differences among several variables such as region, municipality, productive system, productive status, water source, and exposure to flood areas. Risk factors were estimated by Odds Ratio (OR) with WinEpi software.

RESULTS AND DISCUSSION
The general seroprevalence obtained from MAT was 53.83% (95%CI: 48.84-58.75). Out of the 55 farms analyzed, 92.73% (95%CI: 81.58-97.65) had at least one seropositive animal. Although sheep are generally considered to be resistant to Leptospira spp. infection and low prevalence rates have been found worldwide (Ellis, 2015), in this study, a seroprevalence higher than expected was recorded. Although, animals did not exhibit any clinical signs suggestive of leptospirosis, sheep could play a role as a maintenance host for this pathogen (Arteaga-Troncoso et al., 2015). It has been recognized that the circulation of individuals without clinical signs in endemic areas is important in the epidemiology of Leptospira spp. because they are not identified and act as transmitters for other animals (Almeida et al., 2019). In fact, in Santa Catarina, Brazil, Topazio et al. (2015) recognized the presence of ovine as a risk factor for the spread of Leptospira spp. to caprine.
Los Tuxtlas Region had the highest seroprevalence (58.8%, OR=1.9). In this region, Ángel R. Cabada municipality had the highest prevalence among all the municipalities (86.4%, OR=1.9), but there were others with a prevalence over the average (Catemaco, 57%; San Andrés Tuxtla; 56.3%) ( Table 1). Sheep in the municipalities of Ángel R. Cabada, Ayahualulco, and Coatzintla had over six times more chances of resulting seropositive against to Leptospira spp. than sheep from other tested municipalities in the state of Veracruz. The climate in this region is warm-humidregular with an average temperature of 25°C. Agricultural activities occupy more than 84% of its surface and only 5.4% is jungle, while water bodies represent 2.8% of the total area in this region. Papaloapan, one of the 10 main rivers in the state of Veracruz, crosses the region, which also exhibits several lakes and lagoons. The tropism of Leptospira spp. in tropical and subtropical areas for tributaries of natural or artificial water (Ansdell, 2017), could explain the high prevalence found in sheep of this region and municipalities. Martins and Lilenbaum (2014) noted that tropical conditions are an ideal scenario for the maintenance and spreading of leptospires, as a result of the concurrence of abundant rainfall and the diversity of wild and domestic reservoirs that may contribute to disseminate the agent. For other domestic ruminants residing in tropical areas, the presence of rodents triplicates the chances of the occurrence of seroreactive animals (Higino et al., 2013). The environmental conditions that Leptospira spp. requires to persist relies on multiple variables such as soil and water pH, temperature, and even environmental microbial communities (Barragan et al., 2017).
Serovar Hardjo had the highest prevalence with 45.9% (95%CI: 41.02-50.92), while serovar Grippotyphosa had the lowest one with 0.2% (95%CI: 0.01-1.59) ( Table 2). Machado et al. (2016) argued that sheep show evidence of being positive to a small number of serogroups. Twelve serogroups were used for this study but, except for serovar Pomona, seropositive animals were found to all the used serogroups. Indeed, the prevalence for the serovar Harjo is high, but for the other ten serovares was mostly below 4%. No previous study on the presence of leptospirosis in sheep has been done in the state of Veracruz, but one research that was conducted in goats of 14 municipalities of central Veracruz showed an overall seroprevalence of 23.40% and seroreaction against seven serovars were found (Peña-Ramírez et al., 2011).
Infection in domestic species, such as ovine, with more than one serovar may be due to grazing and intra-species transmission of the agent within the flock (Garba, 2017). Larger herd size was the common risk factor for Leptospira spp. seroprevalence when several livestock species were grazing together (beef cattle, sheep, and deer) in New Zealand (Subharat et al., 2012;Dreyfus et al., 2018). In northeastern Brazil, Campos et al. (2017) suggested the possibility of interspecies Leptospira transmission among cattle, sheep, and goats when consorted rearing occurred, so they concluded that the presence of dogs, rodents, and wild animals (reservoirs), and the raising of different species are considered important factors in the epidemiology of Leptospira spp. Wild animals that inhabit large areas have contact with flooded sites and feed on rodents that may be infected with Leptospira spp. (Viera et al., 2017). Moreover, although the water sources supplying ovine farms and sheep access to flooded areas were not relevant in this study, taking biosecurity measures should not be neglected because these conditions are ideal for Leptospira spp. transmission.

Table 1. Seroprevalence against Leptospira spp. by region and municipality in sheep from Veracruz, Mexico.
Region: Chi-square= 4.957, p=0.175; Municipality: Chi-square=53.163, p=0.000, C.I. =Confidence interval.   (Martins & Lilenbaum, 2017). In this study, a higher frequency of antibodies was demonstrated for sheep in a semi-intensive system; that is to say, those animals that combine pen feeding and grazing. In this regard, it has been pointed out that the probability of ovine becoming infected is higher in semi-intensive systems, which they determined as a risk factor (Machado et al., 2016). Whereas in intensive flocks, clinical infections are more often produced by serovar Hardjo (Ellis, 2015). Therefore, the transmission of Leptospira spp. depends on the coexistence with other species such as cattle, as well as on external environmental factors, such as rain and the accumulation of surface water (Correia et al., 2017). In general, the state of Veracruz complies with the environmental and geographical conditions necessary for spreading Leptospira spp.

CONCLUSIONS
Leptospira spp. has a high seroprevalence in sheep and is widely distributed on farms in the state of Veracruz, Mexico. The most frequent serovar is Hardjo, but other serovars could be also found. The high Leptospira spp. seroprevalence in sheep found prevented the identification of associated risk factors.
Funding. The study was financed with resources of FUNPROVER (Fundación Produce Veracruz) under grant 30-2009-0986 for the project "Comprehensive study on the main etiological agents that affect the production of small ruminants". Also, CONACYT provided a scholarship for doctoral studies of the first author under contract 781426.

Conflict of interests.
The authors hereby declare that they have no conflict of interest.