Epidemiological survey of leptospirosis in collared peccaries (Pecari tajacu Linnaeus, 1758) in legal breeding sites in some states in the Northeast region, Brazil

The collared peccary (Pecari tajacu Linnaeus, 1758) is considered an animal of economic viability for trading and potentially productive for meat, being important the knowledge about the health of this species. Thus, the objective of the research was to carry out a cross-sectional study of leptospirosis in captive collared peccary (Pecari tajacu) from the states of Paraíba, Rio Grande do Norte and Piauí, northeast region of Brazil, using serological and molecular techniques. Serum samples from 48 animals were tested using the microscopic agglutination test (MAT) technique. In the samples of vaginal and preputial fluid, the Polymerase Chain Reaction (PCR) was performed. Four animals (8.3%) were seroreactive for Leptospira sp. with reaction to serogroup Icterohaemorraghiae and negative in PCR. There was association between the occurrence of leptospirosis and the intensive breeding system (odds ratio=63.00; 95%CI=4.3910.6; P=0.002). The seroreactivity for leptospirosis suggests that, at some point, these animals were infected by sources of infection within the farm itself. It was also possible to observe the importance of knowing the serogroups prevalent in this species in the studied region, which allows the establishment of adequate strategies for its control, thus prioritizing the balance in the human-animal-environment relationship.


Introduction
The collared peccary (Pecari tajacu Linnaeus, 1758) is a medium-sized mammal belonging to the order Artiodactyla, suborder Suiformes and family Tayassuidae (Furtado, 2014). It is found from the southern United States, throughout Central America and in much of South America, especially in Brazil, where it is widespread (Margarido & Mangini, 2001;Sonner et al., 2004). It occupies several types of environments, such as tropical or temperate forests, deserts, and swamps, which highlights the euritopic character of this species, what makes these animals have no need for major environmental changes in order to implement a breeding site (Bodmer & Sowls, 1993).
Pecari tajacu stands out, in Brazil, as an animal of economic viability for trade and with great productive potential for meat, being the sixth most consumed exotic meat in the country (Ribeiro, Carvalho, Peruquetti, Medeiros & Freitas, 2016), with its commercial creation being legal according to the Normative Instruction (Annex 169/2008) from the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis [IBAMA] (2008).
Collared peccaries may be susceptible to the same infections that affect domestic swine and may act as reservoirs for some diseases such as leptospirosis, which may have a significant impact on the production and reproduction of the species (Furtado, 2014). Furthermore, the observation and study of animal pathologies enable the understanding of possible interactions with humans, which is of great importance in public health (Albuquerque et al., 2016).
According to Coleman (2000), Sales et al. (2012) and Centers for Disease Control and Prevention [CDC] (2019), leptospirosis is an acute to chronic zoonosis that affects several species of domestic animals, humans, and wild animals, such as artiodactyls. It is caused by an aerobic, mobile, spirited Leptospira, demanding in terms of microbiological culture (Genovez, 2016). However, data on infection by Leptospira spp. in the collared peccary are limited, mainly in the Northeast region of Brazil (Minervino et al., 2018).
Thus, the study of the epidemiology of leptospirosis in the species Pecari tajacu is important for understanding its role as a natural reservoir, characterizing the circulation of this infectious agent between wild and domestic species, supporting the development of actions that will minimize the negative impact of this for public health. Therefore, the objective of this work was to carry out a cross-sectional study of leptospirosis in collared peccaries (Pecari tajacu) in captivity in the States of Paraíba, Rio Grande do Norte and Piauí, Northeastern region of Brazil, using serological and molecular techniques.

Sampling and field activities
The research was carried out with a qualitative and quantitative described by Pereira et al. (2018). The methodological protocols for this study were approved by the Sistema de Autorização e Informação em Biodiversidade (SISBio under n° 36263-5) and the Comitê de Ética em Pesquisa no Uso de Animais (CEUA) of the Centro de Saúde e Tecnologia Rural Research, Society and Development, v. 10, n. 3, e47810313421, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i3.13421 After physical containment of the animal with the handgrip and/or chemical with midazolam 0.3 to 0.5 mg / kg, IM (Furtado, 2014), blood samples were collected by puncture of the lateral saphenous vein and transported in isothermal boxes to the Laboratório de Doenças Transmissíveis at the Centro de Saúde e Tecnologia Rural of the Universidade Federal de Campina Grande (LDT / CSTR / UFCG), in Patos, tate of Paraíba, where they were drained and stored at -20° C until serological tests were performed. Collections of vaginal and preputial fluid were also made with the aid of sterile swabs, which were stored in microtubes containing 1.5 ml of 0.9 % sodium chloride solution and sent to the Laboratório de Biologia Molecular do Semiárido (BIOMOL/CSTR/UFCG), in Patos, Paraíba, for DNA extraction and subsequent Polymerase Chain Reaction (PCR). An epidemiological questionnaire was applied to those responsible for the breeding of collared peccaries, which was designed to offer variables that sought to verify the absence or presence of some practices and conditions that may act as a parameter indicating the chances of animals being seroreactive for the disease.

Serological diagnosis of Leptospira spp.
The serological diagnosis of leptospirosis was performed using the microscopic agglutination test (MAT) (Cordeiro, Oliveira & Vieira, 2017) and those with 50 % or more agglutination were titrated by examining a series of geometric dilutions of ratio two. The serum titer was the reciprocal of the highest dilution that showed a positive result. Antigens were examined under a dark field microscope, prior to testing, to verify mobility and the presence of autoagglutination or possible contaminants.

Molecular diagnosis of Leptospira spp.
The DNA contained in the vaginal and foreskin swabs was extracted using the Dneasy Blood and Tissue Kit (Qiagen, Hilden, Germany), following the recommendations from the manufacturer. The polymerase chain reaction (PCR) was performed with the primers LipL32-45F (5′-AAG CAT TAC CGC TGG TG-3 ′) and LipL32-286R (5′-GAA CTC CCA TTT CAG CGA TT-3 ′), to amplify the LipL32 gene, which is specific for pathogenic Leptospira spp.. As a positive control, Pomona serovar Kennewicki sample was used and ultrapure water was used as a negative control (Stoddard, Gee, Wilkins, McCaustland & Hoffmaster, 2009).

Statistical analysis
The analysis of factors associated with seroreactivity for Leptospira spp. was performed with data collected from epidemiological questionnaires applied to breeders and was carried out in two stages: univariate and multivariate analysis.
In the univariable analysis, two groups of animals were formed -reactive and non-reactive -which were compared against the analyzed variables: sex (male, female); age (3-12 months, 13-24 months, 25-48 months, > 48 months); general management: Type of destination (preservation, commercial), breeding system (intensive, semi-intensive, extensive), access to water (natural source, drinking fountains), water source (water company, tubular well) contact with other animals (no, yes); sanitary/reproductive management: deworming (no, yes), quarantine (no, yes), separate picket for females in the delivery and/or postpartum phase (no, yes), there is control of rats (no, yes), animal has contact with dams/flooded areas (no, yes), tests for leptospirosis diagnosis (no, yes), natural mating (no, yes); and infrastructure: management center (no, yes), type of enclosure (beaten earth floor, slatted, cemented, other).
Those variables that presented P ≤ 0.2 by the chi-square test or Fisher's exact test (Zar, 2010) were selected for multivariate analysis, using multiple logistic regression (Hosmer & Lemeshow, 2000). The level of statistical significance was set at 0.05 and calculations were performed using the Statistical Package for the Social Sciences (SPSS) software ® version 20.0.

Results
From 48 samples submitted to the MAT, four (8.33 %) were positive, being two females and one male from site A and one female from site D for anti-Leptospira spp. Agglutinins, showing seroreactivity for the serogroup Icterohaemorraghiae with titers ranging from 1:50 to 1:200 (Table 1). The results of the analysis of risk factors are presented in Table 2, where the selected variables (P≤ 0.2) for the multiple analysis were: sex, breeding system, contact with other animals, quarantine, separate paddock for females in the delivery phase and/or postpartum, natural mating and type of enclosure. In the multiple analysis, it was verified that intensively bred animals had a higher chance of seroreactivity (odds ratio=63.00; 95% IC=4.3-910.6; P=0.002) (Table 3).  In the molecular analysis for the LipL32 gene of Leptospira spp., through samples of vaginal and preputial fluid, all animals were negative in PCR. In the differential diagnosis for Brucella abortus, there were no positive reactions, excluding the need for a confirmatory test of 2-mercaptoethanol (2-ME).

Discussion
The diagnosis of leptospirosis has already been described in some wild animals, including collared peccaries (Pecari tajacu), from various regions of Brazil and the USA (Luna-Alvarez, Moles-Cervantes, Torres- Barranca & Gual-Sill, 1996;Ito et al., 1998;Jori, Galvez, Mendoza, Cespedes & Mayor, 2009), but this is the first study in the Northeast region of Brazil for this species. The four positive animals reacted to the serogroup Icterohaemorraghiae, which has been identified as the main responsible for the clinical cases of human leptospirosis in Brazil and the rodent Rattus norvegicus is considered the most relevant host (Bharti et al., 2003;Souza et al., 2006;Martins & Spink, 2020). There is an important role for rodents in the transmission of serogroup Icterohaemorraghiae to wild animals that live in captivity, since they are present in agglomeration sites, making contact more favorable, according to the Corrêa et al. (2004) and Ministério da Saúde [MS] (2019).
Regarding the seroreactivity of the collared peccary for leptospirosis, a research was carried out in Pecari tajacu from Pará, through MAT, in which four (9.8 %) animals were positive for the serogroup Autumnalis (Mayor et al., 2006). In Mexico, two collared peccaries were positive for the Icterohaemorrhagie and Panama serogroups ((Luna-Alvarez, Moles-Cervantes, Torres-Barranca & Gual-Sill, 1996). In Colombia it was reported that 39 (78 %) free-living collared peccaries showed a high prevalence for serogroups Australis, Icterohaemorrhagiae, Grippotyphosa, Canicola, and Pomona (Montenegro et al., 2018). Thus, it is possible to indicate the participation of these animals as links in the epidemiological chain of the disease and, probably, the lower prevalence in this study may be associated with the better sanitary control offered to captive animals (8.33 %), differently from free-living ones.
The highest frequency of positive Pecari tajacu was identified at the zoo (site A), in which the breeding system is intensive. However, the high value of the odds ratio does not reflect the real chance that an intensively bred animal will be seroreactive and this was probably due to the small number of samples included in this study. However, as the animals are confined, humidity may favor the maintenance of the bacteria in the breeding paddocks, as well as the accumulation of water near the enclosures. In addition to the contact of rodents with the collared peccaries due to the breeding sites, which are usually located in the urban perimeter with areas of extensive vegetation, facilitating the survival of these synanthropic animals.
In the molecular analysis for Leptospira spp. this research differs from another carried out using three captive collared peccaries in the municipality of Ilha Solteira in the state of São Paulo, where one (33.3 %) Pecari tajacu was positive for PCR, indicating infection with the pathogen, without developing the disease (Paixão et al., 2011). It should be noted that the excretion of Leptospira spp. in the urine is intermittent; thus, negative results in PCR do not exclude the possibility that the animal is a host. Therefore, periodic tests may be necessary for the proper detection of all carriers in a breeding site (Lilenbaum et al., 2009).

Conclusion
The seroreactivity for leptospirosis suggests that at some point these animals were infected through sources within the intensive environment itself, such as contact with synanthropic hosts or pools of water. In these cases, it is recommended to detect these sources in the environment, combined with corrective measures, in addition to a new serological investigation to verify the ineffectiveness or not of the intervention in the environment.
In addition, it was possible to observe the importance of knowing the serogroups prevalent in this species in three states investigated in the Northeast region, which allows the establishment of appropriate strategies for effective control, thus prioritizing the balance in the human-animal-environment interaction.
For future research on leptospirosis in collared peccaries, if possible, it is suggested to schedule collections in the rainy periods as it is more conducive to the transmission of the disease at this time of the year. In addition, in the event of the death of an animal, it should investigate the identification of bacteria in the tissues through immunohistochemistry or other anatomopathological analyzes.