Factors associated with the seroprevalence of Neospora caninum (Apicomplexa: Toxoplasmatinae) in sheep from the State of Sergipe, Brazil

Copyright Mendonça et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted non-commercial use, distribution, and reproduction in any medium provided the original work is properly cited. Factors associated with the seroprevalence of Neospora caninum (Apicomplexa: Toxoplasmatinae) in sheep from the State of Sergipe, Brazil


Introduction
Neosporosis is significant in sheep production because it triggers disorders related to reproduction, and, consequently, economic loss related to abortion, neonatal mortality and the birth of weak lambs (Faria et al., 2010;Pinto et al., 2012). The prevalence of infection in Brazilian sheep flocks varies from 1.8% in Rio Grande do Norte (Soares et al., 2009) to 64.2% in Pernambuco (Tembue et al., 2011).
The risk factors for this species are not widely known, but some studies have identified a relationship between seropositivity and property size, ingestion of backwater, animal slaughter on the property, viscera exposed to dogs and the presence of reproductive problems (Faria et al., 2010;Machado et al., 2011;Munhóz et al., 2010). Considering the relevance of this disease and the scarcity of epidemiological information in Sergipe, the aim of this study was to identify the main factors related to the frequency of antibodies against N. caninum in a sheep flock from the State of Sergipe.
Producers were selected by means of non-probabilistic convenience sampling; due to the absence of a representative list of sheep producers in the state, random sampling would have been impractical. In order to determine the sample population, municipalities that held the largest sheep flocks of each region of the State were selected (Figure 1). In total, 19 were selected: six are located in the mesoregion of the Sergipe Backlands (83,728 animals), six in Agreste of Sergipe (89,688 animals) and seven in Eastern Sergipe (20,373),comprising 193,789 animals,or 78.23% of the sheep population of the State (247,703).
The minimum number of samples to be tested (383) was calculated using the statistical software EPI INFO version 3.5.1 (Centers for Disease Control and Prevention, 2017), considering an expected prevalence of 50%, sampling error of 5% and confidence level of 95%, for a population of 193,789 animals. The number of properties visited was estimated according to the minimum amount of samples collected in each municipality, which was determined in proportion to the flock, whereas an average of 15 samples was established for each property.
Sample collection was approved by the Ethics Committee on Animal Use, pursuant to file number (CEUA/UESC-39/2009). Blood samples were collected by means of external jugular vein puncture. Such samples were centrifuged at 1600 x g for 10 minutes, whereas sera were separated, inserted into microtubes and frozen at -20 °C. During the visit, a semi-structured interview was conducted to collect data related to the animal, producer, property, production system, and health and feeding management.
The indirect immunofluorescence technique was used to determine antibodies against N. caninum. Slides suitable for indirect immunofluorescence were used. They were impregnated with antigens of the N. caninum NcBA strain of positive control serum, obtained by means of blood collection from a six-month-old lamb, 30 days after the inoculation of N. Caninum tachizoites (1x10 6 ), and a negative control serum for N. caninum. The initial dilution consisted of 1:50 (Figliuolo et al., 2004), and positive samples underwent sequential dilutions with a base of two until the reaction result was negative. The analysis was performed with an epifluorescence microscope Binocular BX 51 (Olympus TM ). Samples in which the microscopic field presented more than 50% tachyzoites with total peripheral fluorescence were considered positive.
To identify risk factors associated with N. caninum infection, a bivariate analysis was performed with a Chi-squared test and Fisher's exact test, with a significance level of 5%, using the statistical software EPI INFO version 3.5.1 (Centers for Disease Control and Prevention, 2017). All variables with p≤0.2 in the bivariate analysis underwent a collinear analysis determined by a Spearman's test, using the software BioEstat 5.0 (Ayres et al., 2007). Subsequently, an unconditional logistic regression multivariate analysis was conducted using the software EPI INFO version 3.5.1.
The prevalence of antibodies against N. caninum in sheep in Brazil ranges from 1.8% in Rio Grande do Norte (Soares et al., 2009) to 64.2% in Pernambuco (Tembue et al., 2011). Among the studies conducted in Brazil with identical techniques and cut-off points, similar results were found by Faria et al. (2010) in Alagoas (9.6%) and Munhóz et al. (2010) in São Paulo (13.9%). However, the prevalence verified in this study was higher than the rates identified by Salaberry et al. (2010) in Minas Gerais (8.1%) and Figliuolo et al. (2004) in São Paulo (9.2%), and lower than the rates found by Andreotti et al. (2009) in farms in the city of Campo Grande, central-western Brazil (30.8%), by Rossi et al. (2011) in Minas Gerais (47.1%) and by Tembue et al. (2011) in Pernambuco (64.2%). Variations by location might be related to differences in the region, climate, animal age, sample size (Dubey et al., 2011), production systems (Melo et al., 2001), or even pathogenic synergism (Melo et al., 2004).
The variables "age", "presence of bovines on the property", "exchange/borrowing of breeders", "frequent deworming", "contact with dogs", "ingestion of water straight from the source", "compacted soil stall", "natural mating" and "presence of a sheep caretaker" presented p-values lower than 0.20 (Table 2). Based on collinearity, the "presence of a sheep caretaker" and "exchange/borrowing of breeders" presented a p-value higher than 0.8, such that "presence of a sheep caretaker" was excluded from the initial model of the unconditional logistic regression. The final logistic regression model demonstrated that "contact with dogs" acted as a protective factor (OR= 0.323, p<0.001), whereas "exchange/borrowing of breeders" acted as a risk factor for infection by N. caninum (OR= 22.287,p<0.001).
Factors associated with the seroprevalence of Neospora caninum (Apicomplexa: Toxoplasmatinae) in sheep from the State of Sergipe, Brazil  Recent studies on occurrence and risk factors have demonstrated that N. caninum infects sheep in several other regions of the world. In Italy, Gazzonis et al. (2016) found an occurrence of 19.3%, and demonstrated the well-known variables that affect the presence of N. caninum in sheep herds, such as the rearing system and farm size; small and family-run farms were at a higher risk, and outcomes from the survey mainly indicated that these farms should implement proper sanitary measures and monitor the spread of the infection among small ruminants. In China, Nie et al. (2018) found a seroprevalence of 8.4%, and observed that the seroprevalence of N. caninum in male Tibetan sheep (10.8%) was significantly higher than in females (7.4%; p<0.01); this is probably related to the different hormone levels between males and females. According to Azevedo Filho et al. (2017), vertical transmission (11%) was clearly detected in a sheep flock.
The presence of dogs on the property was a significant factor (p<0.001), whereby positive rates were lower with their presence, so dogs were considered a protective factor in this study. The function of the definitive host is known in the epidemiology of infection by N. caninum; however, the presence of dogs might limit the entrance of stray and/or wild dogs, reducing the contamination of the environment with oocysts (Barling et al., 2001). Moreover, the idea of the congenital transmission of this parasite is sustained, since only two of the assessed properties applied artificial reproduction methods and the majority reported reproductive problems, such as the birth of weak lambs, and, mainly, abortions (Pinto et al., 2012). The results also indicate a dissemination of N. caninum in the sheep flocks of Sergipe and restraining the exchange/borrowing of breeders between flocks is suggested based on the possibility of N. caninum dissemination.