Anti-Neospora caninum antibodies in beef cattle from the northern region of Paraná state , Brazil

The presence of anti-Neospora caninum antibodies in beef cattle slaughtered in the northern region of the state of Paraná, Brazil, was evaluated. A total of 401 blood samples were collected; 281 samples from the municipality of Rolândia and 120 from the municipality of Borrazópolis, between April 2015 and November 2016. Of the total samples, 289 were from females and 112 from males, aged one and a half to eight years. Indirect fluorescent antibody test (IFAT) was performed, using a cut-off of 1:100. Variables were tabulated for statistical analyses (Fisher’s exact test and chi-square tests, p≤0.05). The analysis showed that of the 401 samples, 37 were positive for N. caninum, indicating a prevalence of 9.2 %, and observed titers were 1:100 (16), 1:200 (14), and 1:400 (7). The variables sex, age, and location did not differ statistically (p>0.05). Our results showed a sero-occurrence of N. caninum in cattle slaughtered in the northern region of the state of Paraná.


INTRODUCTION
Neosporosis, caused by Neospora caninum, is one of the major reasons for reproductive failure in cattle and is responsible for fetal mummification, still births, and miscarriages (MARQUES et al., 2011;DUBEY & SCHARES, 2011;IBRAHIM et al., 2012).It has been estimated that the disease is responsible for worldwide economic losses of around 1 billion US dollars per year, and a loss of more than 100 million US dollars in the Brazilian beef industry (REICHEL et al., 2013).
Neospora caninum is prevalent in all continents (DUBEY & SCHARES, 2011), including Brazil, where different studies have been conducted to detect antibodies in beef cattle, with the prevalence ranging from 9.1 % to 97.2 % (HASEGAWA et al., 2004;VIANNA et al., 2008;ANDREOTTI et al., 2010;AMARAL et al., 2012;SILVA et al., 2017).In the state of Paraná, different regions have been studied; the north central and southwest have a prevalence of antibodies against the parasite in beef cattle of up to 30.3 % and 13.2 %, respectively (MARQUES et al., 2011;MOURA et al., 2012;NASCIMENTO et al., 2014); however, different diagnostic techniques and cut-offs were used in these studies, which could explain the difference in prevalence rates.Moreover, seroprevalence of neosporosis may vary according to the type of cattle (beef versus dairy), mainly due to the differences between the production systems (DUBEY & SCHARES, 2011).

ABSTRACT:
The presence of anti-Neospora caninum antibodies in beef cattle slaughtered in the northern region of the state of Paraná, Brazil, was evaluated.A total of 401 blood samples were collected; 281 samples from the municipality of Rolândia and 120 from the municipality of Borrazópolis, between April 2015 and November 2016.Of the total samples, 289 were from females and 112 from males, aged one and a half to eight years.Indirect fluorescent antibody test (IFAT) was performed, using a cut-off of 1:100.Variables were tabulated for statistical analyses (Fisher's exact test and chi-square tests, p≤0.05).The analysis showed that of the 401 samples, 37 were positive for N. caninum, indicating a prevalence of 9.2 %, and observed titers were 1:100 ( 16), 1:200 ( 14), and 1:400 ( 7).The variables sex, age, and location did not differ statistically (p>0.05).Our results showed a sero-occurrence of N. caninum in cattle slaughtered in the northern region of the state of Paraná.Key words: Neosporosis, seroprevalence, IFAT, cattle, Paraná state.

PARASITOLOGY
As there are few studies on neosporosis in beef cattle from the northern region of Paraná state, the aim of the present study was to evaluate the occurrence of anti-N.caninum antibodies in beef cattle from slaughterhouses in the northern region of Paraná state.

Location and sampling
The sampling size was determined as 384, using the software EpiInfo, version 6, using a prevalence of 50 % and a 5 % confidence interval.Between April 2015 and November 2016, blood samples from 401 animals were collected, 221 samples from an abattoir in Rolândia and 180 samples from a slaughterhouse in Borrazópolis, both located at northern region of the state of Paraná, Brazil.The samples from beef cattle used in this study were from different municipalities in the northern region of Paraná State, including Borrazópolis, Cambé, Conselheiro Mairink, Floraí, Florestópolis, Guaraci, Ibiporã, Jaguapitã, Kaloré, Lobato, Londrina, Nova Londrina, Rolândia, Sabáudia, Sapopema, and Tamarana.Blood samples were collected in the slaughter line and serum samples stored at -20 °C until serological analysis.

Serology
Serum samples were analyzed using an indirect fluorescent antibody test to detect anti-N.caninum antibodies according to the methodology described by CONRAD et al. (1993).Tachyzoites of the Nc-1 strain previously cultivated in Vero cells in our lab were used as the antigen.Serum samples obtained from experimentally infected cattle with N. caninum and from healthy cattle were used as positive and negative controls, respectively, and included in all slides.The conjugate used was anti-bovine IgG FITC (whole molecule, Sigma-Aldrich ® , St. Louis, Missouri, USA) and serum samples that showed fluorescence through all parasite contours and titers ≥ 100 were considered positive.Positive samples were titrated until the maximum dilution titer was reached.

Statistical analysis
Association between the variables was analyzed using a chi-square test (x²) with a significance level of 5 % using the Epi Info software, version 6.04b (DEAN et al., 1994).P-values ≤0.05 were considered significant.
Previous studies of N. caninum seroprevalence performed in Brazil have shown a prevalence ranging from 9.1% to 97.2% (CERQUEIRA-CÉZAR et al., 2017).Although, RAGOZO et al. (2003) in Minas Gerais State and AGUIAR et al. (2006) in Rondônia State observed a similar of prevalence using IFAT of 9.5% and 11.1%, respectively, these studies used a cut-off of 25, which is different to our study.Other studies, also using IFAT and beef cattle, have described higher prevalences, ranging from 29.6% to 62.5%; however, care should be taken when comparing the studies, since the animals were from different regions, and might have had different management, and the cut-off used in each study might have varied (MELO et al., 2006;ANDREOTTI et al., 2010;SILVA et al., 2017).Epidemiological studies have indicated considerable differences in the prevalence of neosporosis between different countries, regions, beef and dairy cattle, and types of production.However, it is difficult to compare these results, mainly due to the differences in serological techniques and cut-offs used (DUBEY & SCHARES, 2011).
The age of animals used in this study ranged from one and a half to eight years old, with most of them less than three years old (Table 1).The age of animals did not statistically influence the occurrence of N. caninum antibodies (p>0.05);although, the occurrence differed numerically (Table 1).These results do not allow us to identify whether horizontal or vertical transmission is the main route of infection and maintenance of the parasite in beef cattle herds in the region.
In a previous study performed in Pará State, it was not possible to observe a relationship between age or seropositivity and neosporosis (SILVA et al., 2015).In another study performed in Peru, the authors did not find a correlation between age or the origin of the animals and seropositivity (p>0.05).Risk factors associated with N. caninum infection in cattle were the presence of more than three dogs in the herd, the disposal of animal waste in the environment, and the herd being composed of more than one hundred animals (PORTOCARRERO et al., 2015).MARQUES et al. (2011) analyzed 159 samples obtained from the northern region of Paraná State and observed that the prevalence of N. caninum in dairy cattle did not proportionally increase with the age of the infected animals, corroborating the results of the present study.Some of the municipalities had a prevalence below the average (9.2 %), including the cities Florestópolis, Ibiporã, Londrina, Sapopema, and Tamarana, where no sero-occurrence of the parasite was observed (Table 1).However, no statistical significance was observed.This could be due to the lower number of collected samples.In contrast, in the municipality Sabáudia and Conselheiro Mairink, a high percentage of positive animals was observed.Geographical differences in the distribution of animals infected by N. caninum has already been reported in other studies and may be related to the relative abundance of the definitive host (canids) and the type of production (NASIR et al., 2012).

CONCLUSION
Our results showed a sero-occurrence of N. caninum in beef cattle slaughtered in the northern region of the state of Paraná, showing that epidemiological studies performed in slaughterhouses could be used as a model for regional serum occurrence of N. caninum in beef cattle.

Table 1 -
Results of indirect immunofluorescence antibody test (IFAT) for Neospora caninum in beef cattle from the north region of the state of Paraná, Brazil.