Comparison of serological and molecular techniques to detect Toxoplasma gondii in free-range chickens (Gallus gallus domesticus).

The present study aimed to compare different indirect and direct diagnostic techniques to diagnose Toxoplasma gondii in free-range chickens. Samples of 386 chickens obtained from 24 Paraná properties were used for serological analysis by indirect fluorescent antibody test (IFAT), modified agglutination test (MAT), and enzyme-linked immunosorbent assay (ELISA). Animals positive by IFAT and/or MAT had their tissues submitted to the mouse bioassay, and those who were positive in this technique had their blood, tissues, and acidic pepsin tissue digestion submitted to PCR (conventional, nested, and quantitative-PCR (qPCR)). One hundred and nineteen chickens (30.8 %) were positive in at least one of the serological tests, being 102 (26.4 %) in the IFAT, 64 (16.6 %) in the MAT, and 62 (16.0 %) in the ELISA. The IFAT was used as a gold standard, and the MAT showed higher sensitivity (46.0 %) and specificity (94.0) compared to ELISA (43.5 % and 93.6 %, respectively). Ninety samples of eighteen chickens positive in the mouse bioassay were subjected to PCR, and according to molecular tests, the conventional PCR detected the T. gondii DNA in 30 % (27/90) of the samples, in 38.8 % (35/90) with nested-PCR and 40.0 % (36/90) with real-time. According to molecular analyzes, the sensitivity was higher in ITS1 nested-PCR (69.4 %) and specificity in conventional PCR-529bp (90.7 %), using the qPCR as the gold standard. MAT and ELISA had similarities in concordance analyzes. The IFAT was the serological technique with the highest agreement with the mouse bioassay, and serological tests in parallel showed to be a good screening option for the isolation of T. gondii in chick tissues. The PCR markers effectively detected the parasite DNA, and the heart was the tissue with the highest number of positives samples. The conventional PCR had sensitivity similar to nested-PCR and qPCR and could be a cheaper alternative to diagnose T. gondii infection in chicken tissues.

Toxoplasma gondii: prevalence and characterization of new genotypes in free-range chickens from south Brazil.

Toxoplasma gondii is an intracellular parasite that can infect all warm-blooded animals including humans. Recent studies showed that T. gondii strains from South America are genetically diverse. The present work aimed to determine T. gondii prevalence in free-ranging chicken in northwest Parana state in Brazil by two serological tests, to isolate the parasites from seropositive chickens and to genotype the isolates. Antibodies to T. gondii in 386 serum samples from 24 farms were investigated by immunofluorescence antibody assay (IFA) and modified agglutination test (MAT). Samples having titers ≥ 16 were considered positive for both tests. Among the 386 serum samples, 102 (26.4%) were positive for IFA, 64 (16.6%) were positive for MAT, 47 (12.2%) were positive in both tests, and 119 (30.8%) were positive in at least one of the two tests. Brain and pool of heart, lung, and liver from the 119 seropositive chickens were used for mouse bioassay to isolate the parasites. Thirty eight (31.9%) of these seropositive chickens were considered positives in mouse bioassay and 18 isolates were obtained. The isolates were characterized by 10 PCR-RFLP genetic markers including SAG1, SAG2 (5'-3'SAG2, alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. Results of genotyping were compared with the genotypes in ToxoDB database. It revealed ten genotypes, including ToxoDB PCR-RFLP genotypes #6 (n = 2), #19 (n = 1), #21 (n = 2), #111 (n = 2), #152 (n = 1), and #175 (n = 1) and four new types not described before. Our results confirmed a high genetic diversity of this parasite in southern Brazil and also showed that the use of two serological tests in combination can improve the chance of T. gondii isolation. More studies should be taken to determine the zoonotic potential of chickens in the transmission of T. gondii.

Survey of Neospora caninum in eared doves (Zenaida auriculata) in Southern Brazil.

Neosporosis is an infectious disease caused by Neospora caninum, a protozoan parasite that has worldwide distribution and is responsible for enormous economic losses in cattle. Birds are considered a good bioindicator of environmental contamination, since they feed on the ground, being exposed to N. caninum oocysts. The aim of this study was to determine the occurrence of antibodies against N. caninum and to verify the presence of parasite DNA in brain from free-ranging eared doves (Zenaida auriculata) from Southern Brazil. For this purpose, blood and brain samples were collected from 249 doves for ELISA and PCR analysis respectively. The prevalence of N. caninum antibodies in doves was 31.72% (79/249) and detection of parasite DNA was not observed in none of birds. This is the first report of antibodies against N. caninum in doves Z. auriculata, what show us that these birds had previously contact with the parasite but since no N. caninum DNA was detected, more studies should be performed to elucidate the real importance of doves in the epidemiologic cycle of the N. caninum.