Neospora caninum in free-range chickens ( Gallus gallus domesticus ) from southern Brazil

daniel_vetuel@hotmail.com Abstract Neospora caninum is an obligate intracellular parasite that can infect many domestic and wild animals, including birds. These animals are important sources for monitoring of environmental contamination, as they could become infected through sporulated oocysts; however, the real role of birds in the biological cycle of N. caninum remains uncertain. This study aimed to determine the prevalence of anti- N. caninum antibodies, evaluate associated factors, detect the parasite by molecular testing of free-range chickens from Brazil, and evaluate different techniques for its serological diagnosis. Blood samples of 366 chickens from 25 farms were collected for serological assays. The indirect fluorescent antibody test (IFAT) and the indirect enzyme-linked immunosorbent assay (ELISA) were used to detect anti- N. caninum antibodies. Chickens that tested seropositive by IFAT had their brain tissues and a pool of organs (heart, lung, and liver) submitted to PCR for molecular detection of the parasite. Out of 366 chickens, 65 (17.8%) and 163 (44.6%) were seropositive by IFAT and ELISA, respectively. Brain tissues (n=60) and the pools of organs (n=65) were negative in the PCR. Our results showed a high prevalence of antibodies in free-range chickens and that IFAT is the more sensitive technique for the detection of anti- N. caninum


Introduction
Neospora caninum is an Apicomplexa protozoan, of worldwide distribution, capable of infecting many domestic and wild animals, including birds (Dubey & Schares, 2011;Romero et al., 2016). The disease causes abortion in cattle and has considerable importance in cattle production, resulting in high economic losses worldwide (Reichel et al., 2013). In dogs, the disease is usually asymptomatic, although clinical manifestations that include muscular and neurological disorders have been described in young dogs (Silva & Machado, 2016).
In birds, serological evidence of N. caninum has been described in different species, including domestic and wild birds (Costa et al., 2008;Mineo et al., 2009;Gondim et al., 2010;Darwich et al., 2012;Salant et al., 2015;De Barros et al., 2017;Konell et al., 2019). A recent review showed that the seroprevalence in chickens (Gallus gallus domesticus) ranged from 7.2% to 83.6% according to the region (De Barros et al., 2018). The eating habits of birds increases their exposure to these pathogens, and since birds may be predated by dogs, chickens could contribute to the epidemiology of the parasite. Moreover, previous studies have reported a positive correlation between the presence of birds on the farm and increased reproductive problems in cattle that could be related to N. caninum (Bartels et al., 1999;Otranto et al., 2003).
Since the role of birds in the epidemiology of neosporosis is not fully understood, the present study aimed to detect N. caninum in free-range chickens from Brazil, and evaluate different techniques for its serological diagnosis.

Sampling and animals
Tissue (heart, brain, lung, and liver) and blood samples of 366 free-range chickens were collected between 2012 and 2014 from 25 farms of Paraná state, south Brazil ( Figure 1). For sample size calculation, we assumed a prevalence of 50%, a confidence level of 95%, and efficacy 5% by the Epi Info version 3.5.4. An epidemiological questionnaire was applied for each farmer to determine possible factors associated with exposure to N. caninum in chickens. All procedures involving animals were approved by the Ethics Committee of the State University of Londrina (protocol number 64/10).

Indirect fluorescent antibody test (IFAT)
Serum samples from chickens were tested by IFAT according to the methodology described previously (Conrad et al., 1993). Tachyzoites of NC-1 strain of N. caninum were used as antigen, while anti-chicken IgY conjugate labeled with fluorescein isothiocyanate (FITC) (F8888 -Sigma-Aldrich, USA) was used as a secondary antibody. Serum samples from chickens experimentally inoculated with N. caninum (NC-1 strain) and non-infected chickens were used as the positive and negative control, respectively. Samples that showed fluorescence over the total contour of the tachyzoite were considered positive. Since there are is not an established cut-off for chickens, we used the cut-off ≥16 to improve the sensitivity of the test.

Enzyme-linked immunosorbent assay (ELISA)
The ELISA was performed according to the methodology described previously (Paré et al., 1995) with few modifications. Tachyzoites of NC-1 strain grown in Vero cells were used to produce the total antigen. The tachyzoites obtained were passed through 27G needles, centrifuged at 10,000 g for 10 min, and the pellet was mixed with ceramic beads via vortexing three times for 10 min each. The resultant solution was centrifuged under the same conditions as described before, and the pellet was discarded. Finally, the protein concentration was quantified by a commercial kit (Pierce™ BCA, Thermo Fisher Scientific, USA), and the antigen was stored at -20°C until further analysis.
Flat-bottom 96-well polystyrene microtitration plates (Nunc-Immuno Plate Maxisorp, Denmark) were coated with 100 µl of crude antigen (2.5 µg/ml) diluted in 0.1 M carbonate buffer (pH 9.6) and incubated at 4ºC overnight. After incubation, the plates were washed three times with PBS-Tween 20 (50 mM Tris, 100 mM NaCl, and 0.05% Tween 20), blocked with carbonate buffer plus 8% nonfat dry milk and incubated at 37°C for one hour. Serum samples were diluted 1:100 in PBS-Tween 20% plus 5% nonfat dry milk, and 100 µl of this mixture were added in duplicate into the microplates and incubated for one hour at 37°C. After another washing cycle, 100 µl peroxidaselabeled anti-chicken IgY antibody (A9046; Sigma-Aldrich, USA) diluted in PBS-Tween 20% plus 5% nonfat dry milk was added in each well, followed by incubation for one hour at 37°C.
The plates were washed three times, and the peroxidase activity was revealed by adding 100 µl of tetramethylbenzidine solution (TMB Single Solution; Thermo Fisher Scientific, USA). Then, 50 µl of hydrochloric acid (1N) was used to stop the reaction, and the optical density (OD) was read at 450 nm through a microplate reader (iMark Microplate Absorbance Reader, Bio-Rad, USA). Serum samples from chickens experimentally inoculated with N. caninum (NC-1 strain) (positive control) and non-infected chickens (negative control) were added to all plates. The OD was corrected to control plate-to-plate variation, and the cut-off was calculated by the OD mean of the negative controls plus two times the standard deviation (Garcia et al., 2006b). The corrected OD were grouped into nine levels, where each subsequent level was determined by an increment of 35% (Machado et al., 1997).

Enzymatic digestion
Pooled organs (10 g of each tissue, including heart, lung, and liver) were digested as previously described (Dubey, 1998). Briefly, the organs were crushed in saline solution and added to artificial digestive fluid (0.78 g pepsin, 0.5 g NaCl, 0.7 ml HCl and 150 ml of distilled water) for one hour at 37ºC. The product of digestion was filtered and centrifuged at 1,200 g for 10 min. The supernatant was discarded, and the pellet neutralized with a sodium bicarbonate solution 1.2%. The solution was resuspended in physiological solution and centrifuged again. Then the supernatant was discarded, the solution was resuspended in 1 ml, with 1000 UI of penicillin and 100 µg of streptomycin per ml added and stored at -20ºC until molecular analysis.

DNA extraction
Animals considered positive by IFAT had their tissue samples submitted to polymerase chain reaction (PCR). Samples of 60 brains (25 mg) and 65 pools of organs (heart, lung, and liver) (300 µl of enzymatic digestion) were used for DNA extraction. The samples were homogenized and transferred to microtubes with an equal volume of extraction buffer (200 mM NaCl, 20 mM Tris, 50 mM EDTA, proteinase K 1 mg/ml and 2% SDS), followed by incubation at 56ºC for one hour. Then, with buffered phenol (300 µl) added, it was centrifuged at 13,000 g for 5 min. The supernatant was transferred for another microtube, and with phenol:chloroform:isoamyl alcohol added, it was centrifuged at 13,000 g for 5 min (Garcia et al., 2006a). Then, DNA was precipitated by sodium acetate and ethanol (Sambrook et al., 1989).

PCR
For the molecular detection of N. caninum, a conventional PCR, targeting the Nc5 gene, was performed using the primers Np6 plus (5'-CTCGCCAGTCAACCTACGTCTTCT-3') and Np21 plus (5'-GGGTGTGCGTCCAATCCTGTAAC-3'), which amplify a fragment of 337 bp (Hughes et al., 2006). The reaction was performed in 25 µl using 100 ng/µl of DNA, 25 mM MgCl2, 200 µM dNTP (Thermo Fisher Scientific, USA), 1 µM of each primer, 1x PCR buffer (20 mM Tris-HCl, 50 mM KCl, pH 8.4) 1.25 U of Taq-DNA polymerase (Thermo Fisher Scientific, USA) and ultrapure sterile H 2 O. The thermocycler program was run in appliance TC-512 (Techne, USA) according to the program: 95ºC for 5 min for initial denaturation, 35 cycles of 95ºC for 30 sec for denaturation, 63ºC for 30 sec for annealing, and 72ºC for 1 min for the extension. A final extension at 72ºC for 5 min was included. Positive and negative controls were added in all reactions. PCR products were submitted to electrophoresis in agarose gel 1.5% stained with Sybr® Safe DNA Gel Stain (Thermo Fisher Scientific, USA), visualized and photo-documented under UV (L -PIX EX, Loccus Biotechnology, Brazil).

Statistical analysis
The kappa coefficient was used to evaluate the agreement between the serological techniques, and the interpretation was performed as previously described (Landis & Koch, 1977). Sensitivity and specificity were calculated using IFAT as the gold standard (Casartelli-Alves et al., 2014). All the variables were analyzed by chisquare test or Fisher's exact test using a computer software (GraphPad Prism, v.6.0), and p-values ≤ 0.05 were considered significant.

Results
Out of 366 serum samples, 65 (17.8%) were positive by IFAT (Table 1), with the following titers: 16 (86.2%), 64 (12.3%) and 256 (1.5%). The occurrence of anti-N. caninum antibodies on each farm ranged from 7% to 62%, while 72% (18/25) of the visited farms had at least one positive animal. According to the ELISA test, 163 (44.6%) samples were positive, with the most frequent OD level among the positive samples at level four (  comparison between the serological techniques showed that ELISA had lower sensitivity and specificity than IFAT, presenting 49% and 56%, respectively. The kappa coefficient of 0.036 demonstrates a slight agreement between the techniques. Factors associated with exposure to N. caninum in chickens were slaughter above 90 days (p=0.02), contact with animal wastes (p=0.05), and chickens raised free all-day (p=0.005) ( Table 3). Through PCR, no samples were considered positive.

Discussion
We observed that 17.8% of free-range chickens from Paraná State from Brazil were seropositive for N. caninum, demonstrating that the parasite is circulating in this region, and chickens are exposed to parasitic stages. Previous studies on free-range chickens in Brazil have also described a similar prevalence by IFAT, in Bahia with 17 -23.5% of seropositivity (Costa et al., 2008;Gonçalves et al., 2012), 36.5% in Rio Grande do Sul (Camillo et al., 2015), and 20% in São Paulo (Martins et al., 2011). In other countries, the seroprevalence ranged from 23.1% to 41.5% (Martins et al., 2011;Feng et al., 2016;Rocchigiani et al., 2017). A similar prevalence was detected in waterfowl birds (17.5%, 32/182) from Italy, indicating that, as these bird species feed directly on the ground, they have a higher seroprevalence (Nardoni et al., 2019). However, in an urban park from Brazil, only one goose (Anser sp.) (out of 149) was found to be seropositive to N. caninum, suggesting that this seroprevalence may be due to a low seroconversion rate or a different type of feed in this species (Konell et al., 2019).
Based on the variable analysis, slaughter above 90 days was a variable associated with exposure to N. caninum in chickens. This result indicates that chickens more than 90 days of age have a higher chance of infection due to longer lifespan; however, a previous study showed no significant association between the N. caninum infection and age of chickens (Feng et al., 2016). Chickens that have contact with animal wastes and raised free all-day were also associated with infection. We suggest that these variables contribute to a higher exposition to the parasitic stages present in the environment since outdoor chickens have higher seropositivity (Costa et al., 2008). The presence of dogs on the farm showed no significant association with seropositivity in chickens; however, the presence of wild canids could contribute to the epidemiology of neosporosis (Melendez et al., 2020).
In the present study, we evaluated the ELISA technique using the total antigen; however, it showed a low sensitivity and specificity. Additionally, the Kappa coefficient showed a slight agreement with IFAT and ELISA, which makes this technique not appropriated for the screening test in epidemiological studies in chickens since cross-reaction with apicomplexan parasites may be present . The protein disulfide isomerase (PDI), heat-shock protein (HSP70), and ribosomal proteins (RP1) have been identified as cross-reactive antigens and could be related to the low specificity of the serological test (Liao et al., 2005;Zhang et al., 2011). An ELISA using the surface antigen 1 of N. caninum (NcSAG1t) was tested in serum from chickens, and was a good option for serological diagnosis of neosporosis in animals since the use of species-specific recombinant proteins avoids cross-reaction (Ibrahim, 2013;Gondim et al., 2017). An agglutination test (N-MAT) showed high sensitivity and specificity for naturally and experimentally infected chickens and could be an alternative tool (Sayari et al., 2016).
According to molecular analyses, no samples were positive. In chickens from China, no positive samples have been observed by molecular analyses in myocardium digestion liquids (n=25) (Feng et al., 2016). However, the presence of N. caninum DNA in chickens was first reported in the brain of six animals, confirming that chickens are natural intermediate hosts of the parasite (Costa et al., 2008). In wild birds that tested seropositive with a low titer of the antibody, a high rate of positive PCR samples was detected, indicating that these birds could also be involved in the epidemiology of N. caninum (Nardoni et al., 2019). DNA of N. caninum was also detected in brain and heart tissues from wild waterfowl from Italy, showing that these animals can become infected and could contribute to the spread of the parasite. (Rocchigiani et al., 2017).
Animals experimentally infected with tachyzoites and oocysts of two different strains of N. caninum yielded negative results at PCR (Oliveira et al., 2018); however, this resistance to chronic infection could be related to the strain of parasite as the parasite had a low capacity for multiplication at temperatures above 39ºC to 41.5ºC (Rezende- Gondim et al., 2017). Since we could not detect positive samples by PCR, we suggested that chickens were infected by a low dose of parasites, or they are resistant to chronic infection, being the tissue cysts eliminated due to high corporal temperature of free-range chickens. Moreover, previous studies have demonstrated that chickens present a transient N. caninum infection, being seropositive without the establishment of infection (Furuta et al., 2007;Munhoz et al., 2014).

Conclusions
Our results showed that N. caninum is circulating among free-range chickens on the studied farms; however, the importance of these animals on the epidemiology of neosporosis should be elucidated. The ELISA demonstrated a slight agreement with IFAT, being the IFAT a more appropriate technique for epidemiological studies.