Molecular detection of Toxoplasma gondii and Neospora caninum in seabirds collected along the coast of Santa Catarina, Brazil.

Toxoplasma gondii and Neospora caninum are two closely related protozoans that infect a wide range of animals, including birds. However, the occurrence of N. caninum and T. gondii in seabirds is unknown. Therefore, this study aimed to determine the presence of T. gondii and N. caninum DNA in tissue samples of seabirds. Tissue samples of the pectoral muscles, heart, and brain were collected from 47 birds along the coastline of Santa Catarina State, SC, Brazil. The DNA was extracted from the tissues and screened using nested-PCR (nPCR) targeting internal transcribed spacer 1 (ITS1). T. gondii DNA was detected in tissues from seven seabirds (7/47, 14.8%), kelp gull (Larus dominicanus) (5/21), and Manx shearwater (Puffinus puffinus) (2/8). N. caninum DNA was detected in tissues of nine seabirds (9/47, 19.1%), the kelp gull (L. dominicanus) (4/21), Manx shearwater (P. puffinus) (2/8), neotropic cormorant (Phalacrocorax brasilianus) (1/4), brown booby (Sula leucogaster) (1/5), and white-chinned petrel (Procellaria aequinoctialis) (1/1); however, no co-infection was observed. In conclusion, this study showed the circulation of N. caninum and T. gondii in seabirds along the coastline of Santa Catarina State. Further studies are required to clarify the role of these birds in the epidemiology of neosporosis and toxoplasmosis.

Giardiasis in children and dogs, and the first report of assemblage E in dogs from northeastern Brazil.

Diagnosis is crucial for controlling giardiasis. We determined the prevalence and genetically characterize isolates of Giardia duodenalis of children and dogs from rural communities in northeastern Brazil. G. duodenalis cysts were concentrated by centrifugal flotation/sedimentation. Molecular characterization was carried out using the loci ssu-rRNA, bg, tpi, and gdh. By parasitological techniques, Giardia spp. infection was detected in 72/192 children (37.5%; 95% CI: 30.6%-44.7%) and 24/139 dogs (17.3%; 95% CI: 11.4%-24.6%). By molecular analysis, infection was detected in 60/141 children (42.5%; 95% CI: 34.3%-51.2%) and 26/92 dogs (28.3%; 95% CI: 19.4%-38.6%). The total prevalence of giardiasis was 54.9% in children (106/193; 95% CI: 47.1%-61.6%) and 32.9% in dogs (47/143; 95% CI: 25.2%-41.2%). Zoonotic assemblages A and B of G. duodenalis were detected in children, and assemblage E of G. duodenalis was detected in one child and two dogs. Parallel use of parasitological and molecular techniques proved to be a more effective strategy for detecting giardiasis in children and dogs from endemic areas.

Prevalence and risk factors of Eimeria spp. natural infection in sheep from northern Paraná, Brazil.

The present study aimed to perform an epidemiological and morphological identification of Eimeria infection in sheep in Brazil. Fecal samples from sheep were collected from 20 farms in northern Paraná, Brazil. An epidemiological questionnaire was used to evaluate the risk factors. Fecal samples containing oocysts per gram of feces (OoPG) ≥1000 were subjected to the modified Willis-Mollay method to perform oocyst identification. Sporulated oocysts were observed microscopically for morphological identification. A total of 807 fecal samples were collected. Based on the morphological characteristics of the sporulated oocysts, 10 species of Eimeria were identified, with main species observed: Eimeira ovinoidalis (98.1%), Eimeria crandallis (87.6%), Eimeria parva (79.1%), and Eimeria bakuensis (60.8%). Only 2.6% (7/268) of the sheep were infected with a single species, 4.8% (13/268) contained two different species, and 92.5% (248/268) were infected with three or more species. The analysis of risk factors showed that an intensive rearing, no rotation of pasture, dirt, and slatted floors, and age up to 12 months were associated with infection. This study showed a high prevalence of Eimeria natural infection in sheep from northern Paraná, Brazil. Furthermore, based on the risk factors, good management and hygiene practices must be employed to avoid infection.

Seroprevalence of Neospora caninum and risk factors associated with infection in water buffaloes (Bubalus bubalis) from Maranhão State, Brazil.

The aim of this work was to determine the seroprevalence of Neospora caninum in buffaloes (Bubalus bubalis) from Maranhão State, Brazil, and identify risk factors associated with infection. In total, 338 buffaloes of different ages and both sexes from four farms were randomly selected. Information about the study region, animals and management was collected using an epidemiological questionnaire. Sera were subjected to an indirect fluorescent antibody test (IFAT) to detect anti-N caninum antibodies, while N. caninum DNA was detected using the polymerase chain reaction (PCR) assays. The overall seroprevalence of N. caninum in buffaloes was 27.5% (93/338), while DNA was detected in 3.0% (3/101) samples. The proportion of positive animals detected by IFAT and PCR simultaneously was 2.6% (2/77). The risk factors for N. caninum infection were contact with fomites (p = 0.022), management conditions (p = 0.005), calving interval of 20 months (p = 0.010) and deworming management (application 3 times a year in adults and calves, p = 0.020; change of anthelmintic group, p = 0.040). By multivariate analysis, management conditions was a risk factor for N. caninum infection with odds ratio of 2.2 (95% CI 1.0-4.6). This is the first report of the prevalence and risk factors for neosporosis in B. bubalis of Maranhão State, Brazil. Thus, N. caninum is widely distributed in buffalo herds in Maranhão, with management conditions being a risk factor for infection. Further studies should be conducted to elucidate the importance of buffaloes in the epidemiology of neosporosis in Maranhão State.

Prevalence and risk factors of Eimeria spp. natural infection in sheep from northern Paraná, Brazil / Prevalência e fatores de risco da infecção natural por Eimeira spp. em ovinos na região norte do Paraná, Brasil

Abstract The present study aimed to perform an epidemiological and morphological identification of Eimeria infection in sheep in Brazil. Fecal samples from sheep were collected from 20 farms in northern Paraná, Brazil. An epidemiological questionnaire was used to evaluate the risk factors. Fecal samples containing oocysts per gram of feces (OoPG) ≥1000 were subjected to the modified Willis-Mollay method to perform oocyst identification. Sporulated oocysts were observed microscopically for morphological identification. A total of 807 fecal samples were collected. Based on the morphological characteristics of the sporulated oocysts, 10 species of Eimeria were identified, with main species observed: Eimeira ovinoidalis (98.1%), Eimeria crandallis (87.6%), Eimeria parva (79.1%), and Eimeria bakuensis (60.8%). Only 2.6% (7/268) of the sheep were infected with a single species, 4.8% (13/268) contained two different species, and 92.5% (248/268) were infected with three or more species. The analysis of risk factors showed that an intensive rearing, no rotation of pasture, dirt, and slatted floors, and age up to 12 months were associated with infection. This study showed a high prevalence of Eimeria natural infection in sheep from northern Paraná, Brazil. Furthermore, based on the risk factors, good management and hygiene practices must be employed to avoid infection.

Resumo O presente estudo teve como objetivo realizar uma avaliação epidemiológica e morfométrica da infecção por Eimeria em ovinos no Brasil. Amostras fecais de ovinos foram coletadas em 20 fazendas no sul do Brasil. Um questionário epidemiológico foi utilizado para avaliar os fatores de risco. Amostras fecais, contendo oocistos por grama de fezes (OoPG) ≥1000, foram submetidas ao método de Willis-Mollay modificado para realizar a identificação de oocistos. Oocistos esporulados foram observados microscopicamente para identificação morfológica. Foram coletadas 807 amostras fecais. Com base nas características morfológicas e morfométricas dos oocistos esporulados, foram identificadas 10 espécies de Eimeria, com as principais espécies observadas: Eimeria ovinoidalis (98,1%), Eimeria crandallis (87,6%), Eimeria parva (79,1%) e Eimeria bakuensis (60,8%). Apenas 2,6% (7/268) dos ovinos estavam infectados com uma única espécie, 4,8% (13/268) continham duas espécies diferentes e 92,5% (248/268) estavam infectados com três ou mais espécies. A análise dos fatores de risco mostrou que uma criação intensiva, sem rotação de pasto, terra, piso de ripa e idade até 12 meses foram associadas à infecção. Este estudo mostrou uma alta prevalência de infecção natural por Eimeria em ovinos do norte do Paraná, Brasil. Além disso, com base nos fatores de risco, boas práticas de manejo e higiene devem ser empregadas para evitar infecções.

Evaluation of quantitative polymerase chain reaction for the detection of Toxoplasma gondii oocysts shed by cats.

Felines are definitive hosts of Toxoplasma gondii and can shed oocysts in their feces, contaminating the environment. Sporulated oocysts are highly resistant to the environment and have higher infectivity, which are attributed to many toxoplasmosis outbreaks. The aim of the present study was to evaluate a quantitative polymerase chain reaction (qPCR) technique for the detection of T. gondii oocysts shed by cats. Twelve cats from a previous vaccine experiment were challenged orally with 600 cysts of the TgDoveBr8 strain on day 72. Fecal samples were collected daily using the centrifugal flotation technique, with microscopic examination (Sheather technique) and qPCR for 20 days after the challenge. Cats from all groups shed oocysts in their feces. Five negative cats in the Sheather were positive according to qPCR on the 3rd day post-inoculation (dpi). Oocysts were detected on the 4th dpi using the Sheather; however, there was no statistical difference between the two methods (p=0.1116). In addition, there was no statistically significant difference in oocyst shedding between the groups according to the Sheather technique (p=0.6534) and qPCR (p=0.9670). In conclusion, these results demonstrate that qPCR can be used as an alternative to the Sheather to detect and quantify T. gondii oocysts.

Epidemiology of canine visceral leishmaniasis in a vulnerable region in Brazil.

Visceral leishmaniasis (VL) is a neglected and endemic zoonosis that occurs throughout Brazil; nevertheless, few studies have focused on the early detection of the disease. The municipality of Ourinhos is a non-receptive, silent and vulnerable area for VL, where the seroprevalence of this disease has so far not been investigated. The present study aimed to determine the seroprevalence of canine VL in Ourinhos-SP, and to identify the presence of risk factors. Blood samples were obtained from 604 dogs during a rabies vaccination campaign together with application of a socioeconomic questionnaire, environmental and animal characteristics and tutor's knowledge about the disease. The samples were subjected to indirect ELISA and new samples were collected from reactive and suspect animals, including whole blood and lymph node aspiration evaluated by parasitological method, complete blood count and PCR. No animal was diagnosed as positive based on the combination of direct and indirect tests and the tutors' answers indicated little knowledge about leishmaniasis, being often confused with other diseases transmitted by arthropods; hence, according to the proposed methods, the presence of canine leishmaniasis in the city of Ourinhos was not confirmed and health education campaigns about the disease should be carried out.

Epidemiology of canine visceral leishmaniasis in a vulnerable region in Brazil / Soroepidemiologia da leishmaniose visceral canina em uma região vulnerável do Brasil

Abstract Visceral leishmaniasis (VL) is a neglected and endemic zoonosis that occurs throughout Brazil; nevertheless, few studies have focused on the early detection of the disease. The municipality of Ourinhos is a non-receptive, silent and vulnerable area for VL, where the seroprevalence of this disease has so far not been investigated. The present study aimed to determine the seroprevalence of canine VL in Ourinhos-SP, and to identify the presence of risk factors. Blood samples were obtained from 604 dogs during a rabies vaccination campaign together with application of a socioeconomic questionnaire, environmental and animal characteristics and tutor's knowledge about the disease. The samples were subjected to indirect ELISA and new samples were collected from reactive and suspect animals, including whole blood and lymph node aspiration evaluated by parasitological method, complete blood count and PCR. No animal was diagnosed as positive based on the combination of direct and indirect tests and the tutors' answers indicated little knowledge about leishmaniasis, being often confused with other diseases transmitted by arthropods; hence, according to the proposed methods, the presence of canine leishmaniasis in the city of Ourinhos was not confirmed and health education campaigns about the disease should be carried out.

Resumo A leishmaniose visceral (LV) é uma zoonose negligenciada e endêmica presente em todas as regiões do Brasil, mas mesmo assim poucos estudos têm objetivado a detecção inicial da doença. O município de Ourinhos - SP é uma área não receptiva, silenciosa e vulnerável à LV, não havendo até o momento estudos que tenham investigado a soroprevalência no município. Nesse sentido, o presente estudo objetivou determinar a soroprevalência da LV canina em Ourinhos-SP, bem como associar a presença de fatores de risco. Amostras sanguíneas de 604 cães foram obtidas juntamente com a aplicação de questionário socioeconômico, características ambientais e dos animais e conhecimento sobre a doença. As amostras foram submetidas à sorologia por ELISA e novas amostras coletadas de cães reagentes ou suspeitos foram analisadas por método parasitológico direto, hemograma e PCR. Nenhum animal foi considerado positivo na combinação de testes direto e indireto, e as respostas dos tutores indicaram pouco conhecimento sobre a leishmaniose, sendo muitas vezes confundida com outras doenças transmitidas por artrópodes. Dessa forma, de acordo com os métodos propostos, a presença de leishmaniose canina, na cidade de Ourinhos, não foi confirmada. Por isso campanhas de educação em saúde sobre a doença deveriam ser realizadas.

Evaluation of quantitative polymerase chain reaction for the detection of Toxoplasma gondii oocysts shed by cats / Avaliação da reação em cadeia da polimerase quantitativa para a detecção de oocistos de Toxoplasma gondii eliminados por gatos

Abstract Felines are definitive hosts of Toxoplasma gondii and can shed oocysts in their feces, contaminating the environment. Sporulated oocysts are highly resistant to the environment and have higher infectivity, which are attributed to many toxoplasmosis outbreaks. The aim of the present study was to evaluate a quantitative polymerase chain reaction (qPCR) technique for the detection of T. gondii oocysts shed by cats. Twelve cats from a previous vaccine experiment were challenged orally with 600 cysts of the TgDoveBr8 strain on day 72. Fecal samples were collected daily using the centrifugal flotation technique, with microscopic examination (Sheather technique) and qPCR for 20 days after the challenge. Cats from all groups shed oocysts in their feces. Five negative cats in the Sheather were positive according to qPCR on the 3rd day post-inoculation (dpi). Oocysts were detected on the 4th dpi using the Sheather; however, there was no statistical difference between the two methods (p=0.1116). In addition, there was no statistically significant difference in oocyst shedding between the groups according to the Sheather technique (p=0.6534) and qPCR (p=0.9670). In conclusion, these results demonstrate that qPCR can be used as an alternative to the Sheather to detect and quantify T. gondii oocysts.

Resumo Felinos são hospedeiros definitivos do Toxoplasma gondii e podem eliminar oocistos nas fezes, contaminando o meio ambiente. Oocistos esporulados são altamente resistentes ao meio ambiente com elevada infectividade, sendo atribuído a muitos surtos de toxoplasmose. O objetivo do estudo foi avaliar a reação em cadeia da polimerase quantitativa (qPCR) para a detecção de oocistos de T. gondii eliminados por gatos. Doze gatos de um experimento prévio de vacina foram desafiados por via oral com 600 cistos da cepa TgDoveBr8 no dia 72. Amostras fecais foram coletadas diariamente pela técnica de centrifugo-flutuação seguida de exame microscópico (técnica de Sheather) e qPCR por 20 dias após desafio. Gatos de todos os grupos eliminam oocistos nas fezes. Cinco gatos negativos na técnica Sheather foram positivos de acordo com a qPCR no 3º dia pós-inoculação (dpi). Oocistos foram detectados no 4º dpi no Sheather; entretanto, não houve diferença estatística entre os dois métodos (p=0,1116). Não houve diferença estatisticamente significativa na eliminação de oocistos entre os grupos de acordo com a técnica de Sheather (p = 0,6534) e qPCR (p = 0,9670). Em conclusão, esses resultados demonstram que qPCR pode ser usada como uma alternativa ao Sheather para detectar e quantificar oocistos de T. gondii.

Seroprevalence of Toxoplasma gondii, Neospora caninum, and Leishmania spp. in hunting dogs from Mato Grosso do Sul, Brazil / Soroprevalência de Toxoplasma gondii, Neospora caninum e Leishmania spp. em cães de caça do Mato Grosso do Sul, Brasil

ABSTRACT: Toxoplasmosis, neosporosis, and leishmaniasis are important diseases of worldwide distribution and can affect both pets and humans. Hunting dogs have been trained to hunt domestic and wild animals, which makes them more exposed to parasitic infections. The present study aimed to evaluate the seroprevalence of Toxoplasma gondii, Neospora caninum, and Leishmania spp. in hunting dogs from a rural area in Mato Grosso do Sul, Brazil. Serum samples were collected from 39 American Foxhound dogs, and the sex and age variables were recorded. Serum samples were subjected to an indirect fluorescent antibody test (IFAT) to detect antibodies. Seroprevalence was 35.9%, 15.4%, and 2.6% for Toxoplasma gondii, Neospora caninum e Leishmania spp., respectively. There was no statistical difference between genders for these diseases (P>0.05). Results demonstrated a circulation of these protozoa in hunting dogs in a rural area of ​​the state of Mato Grosso do Sul, which can contribute to the epidemiology of these diseases.

RESUMO: Toxoplasmose, neosporose e leishmaniose são importantes doenças de distribuição mundial e podem afetar tanto os animais de companhia quanto os humanos. Os cães de caça têm sido treinados para caçar animais domésticos e selvagens, o que torna esses animais mais expostos a infecções parasitárias. O presente estudo teve como objetivo avaliar a soroprevalência de Toxoplasma gondii, Neospora caninum e Leishmania spp. em cães de caça de área rural do Mato Grosso do Sul, Brasil. Foram coletadas amostras de soro de 39 cães da raça Foxhound-americano e as variáveis sexo e idade foram registradas. As amostras de soro foram submetidas a reação de imunofluorescência indireta (RIFI) para detecção dos anticorpos. A soroprevalência foi de 35,9%, 15,4% e 2,6% para Toxoplasma gondii, Neospora caninum e Leishmania spp., respectivamente. Não foi observado diferença estatística entre os sexos para todas as doenças (P>0.05). Os resultados demonstram circulação desses protozoários em cães de caça de uma área rural do estado do Mato Grosso do Sul, o que pode contribuir para a epidemiologia dessas doenças.

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

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 antibodies.

Protection against Toxoplasma gondii cysts in pigs immunized with rROP2 plus Iscomatrix.

This study aimed to evaluate the humoral immune response in pigs immunized intranasally and intramuscularly with recombinant Toxoplasma gondii rROP2 protein in combination with the adjuvant Iscomatrix. Twelve mixed breed pigs divided into three groups (n=4) were used, G1 received recombinant ROP2 proteins (200 µg/dose) plus Iscomatrix, G2 received PBS plus Iscomatrix, and G3 as the control group. The intranasal (IN) and intramuscular (IM) routes were used. Animals were challenged orally with VEG strain oocysts and treated on day three after challenge. Fever, anorexia, and prostration were the clinical signs observed in all animals. All the G1 animals produced antibodies above the cut-off on the day of the challenge, while the G2 and G3 remained below the cut-off. Better partial protection against parasitemia and cyst tissue formation was observed in G1 than G3. The protection factors against tissue cyst formation were 40.0% and 6.1% for G1 and G2, respectively, compared to G3. In conclusion, there were not systemic antibody responses in pigs with IN immunization with rROP2+Iscomatrix; however, after IM immunization, those animals produced higher titers than animal controls. We associated these results with partial protection obtained against parasitemia and tissue cysts formation.

First molecular detection of Haemoproteus spp. and Plasmodium spp. in eared doves (Zenaida auriculata) in Brazil.

The aim of this study was to verify the presence and identify the species of haemosporidian parasites in eared doves (Zenaida auriculata) in Brazil. Two hundred and eleven male and female eared doves were trap-captured in four different regions of Londrina city, in southern Brazil. Whole blood was collected in EDTA tubes through heart puncture after euthanasia in a CO2 chamber. A nested PCR targeting the mitochondrial cytochrome b gene (cyt b) of Haemoproteus spp./Plasmodium spp. was performed, followed by an enzymatic digestion to identify the genus. Phylogenetic trees were constructed to determine the closely related species. Out of 211 eared doves, 209 (99.05%) were positive for Haemoproteus spp. and/or Plasmodium spp. RFLP analysis showed that 72.72% (152/209) of eared doves were positive only for Haemoproteus spp., 6.22% (13/209) were positive only for Plasmodium spp., and 21.05% (44/209) of eared doves had mixed infections. Genetic analysis found four samples that were homologous with Haemoproteus multipigmentatus and one that was homologous with Plasmodium sp. This is the first molecular study of hemoparasites from eared doves in Brazil, and it is also the first description of H. multipigmentatus and Plasmodium spp. infection in eared doves in Brazil.

First study of Cryptosporidium spp. occurrence in eared doves (Zenaida auriculata).

Cryptosporidium is a protozoan parasite with a wide range of hosts, including humans. However, only a few Cryptosporidium species have been described in birds (C. meleagridis, C. baileyi, C. galli and C. avium). The aim of this study was to investigate the occurrence of Cryptosporidium spp. in feces of eared doves (Zenaida auriculata), followed by molecular characterization of the parasite. A total of 196 animals of both sexes were trap-captured; the animals were culled and the intestinal contents were collected for DNA extraction. After extraction, a nested-PCR (nPCR), which amplifies a fragment of the 18S rRNA gene of Cryptosporidium spp., was performed. The amplicons obtained were purified and sequenced. PCR analysis revealed that 30 animals (15.3%) were positive for Cryptosporidium spp. There was no significant sex-dependent enrichment of Cryptosporidium occurrence (p > 0.05). Only 15 out of the 30 positive samples were successfully sequenced and their species determined, of which, 13 (86.7%) and 2 (13.3%) were C. meleagridis and C. galli, respectively. Herein, we present for the first time a molecular characterization of Cryptosporidium from feces of eared doves (Z. auriculata) and propose that these birds are a potential source of C. meleagridis infection in humans.

First study of Cryptosporidium spp. occurrence in eared doves (Zenaida auriculata) / Primeiro estudo de ocorrência de Cryptosporidium spp. em pomba-de-bando (Zenaida auriculata)

Abstract Cryptosporidium is a protozoan parasite with a wide range of hosts, including humans. However, only a few Cryptosporidium species have been described in birds (C. meleagridis, C. baileyi, C. galli and C. avium). The aim of this study was to investigate the occurrence of Cryptosporidium spp. in feces of eared doves (Zenaida auriculata), followed by molecular characterization of the parasite. A total of 196 animals of both sexes were trap-captured; the animals were culled and the intestinal contents were collected for DNA extraction. After extraction, a nested-PCR (nPCR), which amplifies a fragment of the 18S rRNA gene of Cryptosporidium spp., was performed. The amplicons obtained were purified and sequenced. PCR analysis revealed that 30 animals (15.3%) were positive for Cryptosporidium spp. There was no significant sex-dependent enrichment of Cryptosporidium occurrence (p > 0.05). Only 15 out of the 30 positive samples were successfully sequenced and their species determined, of which, 13 (86.7%) and 2 (13.3%) were C. meleagridis and C. galli, respectively. Herein, we present for the first time a molecular characterization of Cryptosporidium from feces of eared doves (Z. auriculata) and propose that these birds are a potential source of C. meleagridis infection in humans.

Resumo Cryptosporidium é um protozoário com uma grande variedade de hospedeiros, incluindo os seres humanos. No entanto, poucas espécies têm sido descritas em aves (Cryptosporidium meleagridis, C. baileyi, C. galli e C. avium). O objetivo do presente estudo foi investigar a ocorrência de Cryptosporidium spp. em fezes de pombas-de-bando (Zenaida auriculata), e realizar a caracterização molecular dos isolados. Um total de 196 animais de ambos os sexos foram capturados, eutanasiados e o conteúdo intestinal recolhido para extração de DNA. Após a extração, realizou-se uma nested-PCR (nPCR), que amplifica um fragmento do gene 18S rRNA do Cryptosporidium spp.. Os fragmentos obtidos foram purificados e encaminhados para sequenciamento. Os resultados da n-PCR revelaram 30 animais (15.3%) positivos para Cryptosporidium spp.. Quanto ao sexo dos animais não foram observadas diferenças estatísticas significativas (p > 0.05). Somente 15 de 30 amostras positivas foram sequenciadas com sucesso e as espécies determinadas, das quais, 13 (86.7%) e 2 (13.3%) foram C. meleagridis e C. galli, respectivamente. Esse é o primeiro estudo com caracterização molecular de Cryptosporidium de fezes de pombas-de-bando (Z. auriculata), e propõe serem esses animais potenciais fonte de infecção de C. meleagridis para humanos.

Anti-Neospora caninum antibodies in beef cattle from the northern region of Paraná state, Brazil / Anticorpos anti-Neospora caninum em bovinos de corte da região norte do estado do Paraná, Brasil

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.

RESUMO: A presença de anticorpos anti-Neospora caninum em bovinos de corte, abatidos na região norte do estado do Paraná, Brasil, foi avaliada. Foram coletadas 401 amostras de sangue, sendo 281 amostras no município de Rolândia e 120 no municipio de Borrazopolis, entre os meses de abril de 2015 e novembro de 2016. Do total de amostras, 289 eram de fêmeas e 112 amostras de machos, na faixa etária de um ano e meio até oito anos de idade. Foi realizada a reação da imunofluorescência indireta (RIFI) utilizando ponte de corte de 1:100. Em seguida, foram tabulados as variáveis para análise estatística (testes exato de Fisher e do qui-quadrado, p≤0,05). A análise mostrou que das 401 amostras, 37 foram positivas para N. caninum, indicando uma prevalência de 9,2 % e os títulos observados foram 1:100 (16), 1:200 (14) e 1:400 (7). As variáveis sexo, idade e local não diferiram estatisticamente (p>0,05). Nossos resultados demonstram uma soro-ocorrência de N. caninum em bovinos abatidos na região norte do Paraná.

Prevalence of Eimeria spp. in calves from dairy farms in northern Paraná state, Brazil.

Bovine coccidiosis is a disease of major importance in cattle herds across the world. The disorder mainly affects young calves, and E. bovis and E. zuernii are considered the most pathogenic species of the genus, however, E. alabamensis have been described in grazing calves. In this study, the prevalence of Eimeria spp. was evaluated in calves on dairy farms in the northern region of the state of Paraná, Brazil. Four hundred calves on 44 dairy farms were tested for the presence of coccidian oocysts. The positives were re-examined and the oocysts were morphometrically analyzed for species identification. All the farms were contaminated and 205 animals (51.25%) presented Eimeria spp. oocysts. Among these, 146 animals (71.22%) were co-infected by two or more species of coccidia. Ten species of Eimeria were identified: E. bovis (in 30.25% of the positive samples), E. alabamensis (26.75%), E. zuernii (22.00%), E. ellipsoidalis (18.50%), E. auburnensis (13.75%), E. canadensis (8.00%), E. cylindrica (7.25%), E. subspherica (5.00%), E. bukidnonensis (3.00%) and E. brasiliensis (0.75%). This study demonstrates the high prevalence of Eimeria spp. in the northern region of Paraná, Brazil, and detection for the first time in our region the pathogenic species E. alabamensis.

Prevalence of Eimeria spp. in calves from dairy farms in northern Paraná state, Brazil / Prevalência de Eimeria spp. em bezerros de propriedades leiteiras do norte do estado do Paraná, Brasil

Abstract Bovine coccidiosis is a disease of major importance in cattle herds across the world. The disorder mainly affects young calves, and E. bovis and E. zuernii are considered the most pathogenic species of the genus, however, E. alabamensis have been described in grazing calves. In this study, the prevalence of Eimeria spp. was evaluated in calves on dairy farms in the northern region of the state of Paraná, Brazil. Four hundred calves on 44 dairy farms were tested for the presence of coccidian oocysts. The positives were re-examined and the oocysts were morphometrically analyzed for species identification. All the farms were contaminated and 205 animals (51.25%) presented Eimeria spp. oocysts. Among these, 146 animals (71.22%) were co-infected by two or more species of coccidia. Ten species of Eimeria were identified: E. bovis (in 30.25% of the positive samples), E. alabamensis (26.75%), E. zuernii (22.00%), E. ellipsoidalis (18.50%), E. auburnensis (13.75%), E. canadensis (8.00%), E. cylindrica (7.25%), E. subspherica (5.00%), E. bukidnonensis (3.00%) and E. brasiliensis (0.75%). This study demonstrates the high prevalence of Eimeria spp. in the northern region of Paraná, Brazil, and detection for the first time in our region the pathogenic species E. alabamensis.

Resumo A coccidiose bovina é uma doença de grande importância em rebanhos ao redor do mundo. A desordem afeta principalmente bezerros jovens, e E. bovis e E. zuernii consideradas as espécies mais patogênicas deste gênero, causando grave enterite em animais infectados. No entanto, casos de E. alabamensis foram descritos em bezerros mantidos a pasto. No presente estudo, a prevalência de Eimeria spp. foi avaliada em bezerros de gado leiteiro da região norte do estado do Paraná, Brasil. Quatrocentos bezerros foram amostrados e testados para a presença de oocistos de coccídios. Os positivos foram re-examinados e os oocistos analisados morfologicamente para identificação da espécie. Todas as fazendas estavam contaminadas e 205 (51,25%) animais apresentaram oocistos de Eimeria spp. Destes, 146 (71,22%) animais estava co-infectados por duas ou mais espécies de coccídio. Dez espécies de Eimeria foram identificadas: E. bovis (30,25% de amostras positivas), E. alabamensis (26,75%), E. zuernii (22,00%), E. ellipsoidalis (18,50%), E. auburnensis (13,75%), E. canadensis (8,10%), E. cylindrica (8,00%), E. subspherica (5,00%), E. bukidnonensis (3,00%) e E. brasiliensis (0,75%). Este estudo demonstra a alta prevalência de Eimeria spp. na região norte do estado do Paraná, Brasil, e a detecção, pela primeira vez, de E. alabamensis.

The first study of molecular prevalence and species characterization of Cryptosporidium in free-range chicken (Gallus gallus domesticus) from Brazil / Primeiro estudo de prevalência molecular e caracterização de espécies de Cryptosporidium em galinhas (Gallus gallus domesticus) colonial/caipiras do Brasil

Abstract Rearing free-range chicken is based on grazing feeding patterns, and these animals could be potential environmental contaminants of Cryptosporidium oocysts for humans and other animals. Therefore, the present study aimed to evaluate the molecular prevalence of Cryptosporidium spp. in free-range chickens from Brazil. A total of 351 fecal samples from chickens were examined from 20 farms. For detection of Cryptosporidium spp., 18S rRNA gene fragments were amplified using a nested PCR reaction. Positive samples were sent for sequencing. The overall prevalence of Cryptosporidium was 25.6% (95% CI = 21.2% - 30.6%). Sequencing of the amplified fragments allowed for the identification of three species: C. meleagridis in 57 (62.6%), C. baileyi in 15 (16.4%), C. parvum in 3 (3.2%) samples, and a new Cryptosporidium genotype (C. genotype BrPR1) in 3 (3.2%) samples. Cryptosporidium genotype BrPR1 has not yet been classified as a species, and its host spectrum is not known. Cryptosporidium, including zoonotic species, exists at a high prevalence in free-range chickens within the region studied.

Resumo A criação de galinhas no estilo colonial/caipira é baseada em padrões de alimentação de pastagem, o que as torna potenciais contaminantes ambientais de oocistos de Cryptosporidium para humanos e outros animais. Portanto, o presente estudo teve como objetivo avaliar a prevalência molecular de Cryptosporidium spp. em galinhas criadas em sistema colonial/caipira. Um total de 351 amostras de fezes de frangos foram examinadas em 20 fazendas. Para a detecção de Cryptosporidium spp., os fragmentos do gene rRNA 18S foram amplificados utilizando-se a reação de nested-PCR. A prevalência global de Cryposporidium foi de 25,6% (IC 95% = 21,2% - 30,6%). O sequenciamento dos fragmentos amplificados permitiu a identificação de três espécies que infectam aves: C. meleagridis em 57 (62,6%), C. baileyi em 15 (16,4%), C. parvum em 3 (3,2%) amostras, bem como, um novo genótipo de Cryptosporidium (C. genótipo BrPR1) foi identificado em 3 (3,2%) amostras. Cryptosporidium genotipo BrPR1 não foi ainda classificado como uma espécie, e seu espectro de hospedeiros é desconhecido. O presente trabalho permitiu concluir que Cryptosporidium, incluindo espécies zoonóticas, existe com alta prevalência em galinhas criadas em sistema colonial/caipira na região estudada.

The first study of molecular prevalence and species characterization of Cryptosporidium in free-range chicken (Gallus gallus domesticus) from Brazil.

Rearing free-range chicken is based on grazing feeding patterns, and these animals could be potential environmental contaminants of Cryptosporidium oocysts for humans and other animals. Therefore, the present study aimed to evaluate the molecular prevalence of Cryptosporidium spp. in free-range chickens from Brazil. A total of 351 fecal samples from chickens were examined from 20 farms. For detection of Cryptosporidium spp., 18S rRNA gene fragments were amplified using a nested PCR reaction. Positive samples were sent for sequencing. The overall prevalence of Cryptosporidium was 25.6% (95% CI = 21.2% - 30.6%). Sequencing of the amplified fragments allowed for the identification of three species: C. meleagridis in 57 (62.6%), C. baileyi in 15 (16.4%), C. parvum in 3 (3.2%) samples, and a new Cryptosporidium genotype (C. genotype BrPR1) in 3 (3.2%) samples. Cryptosporidium genotype BrPR1 has not yet been classified as a species, and its host spectrum is not known. Cryptosporidium, including zoonotic species, exists at a high prevalence in free-range chickens within the region studied.