Molecular diversity and polyparasitism of avian trypanosomes in the Brazilian Atlantic Rainforest.

The current study proposes to investigate the diversity and phylogeny of trypanosomes parasitizing wild birds from the Brazilian Atlantic Forest. Cytological examination was carried out by light microscopy of blood smears and positive birds were selected for amplification of the 18S rDNA sequence through PCR. The resulting amplicons were subjected to purification, cloning, and sequencing analysis. Phylogenetic reconstruction was conducted, including all avian trypanosomes representative's lineages. A total of ten bird samples from species of Turdus flavipes (N=1/12), T. albicollis (N=1/8), Tachyphonus coronatus (N=6/121), Thamnophilus caerulescens (N=1/22) and Synallaxis spixi (N=1/8) were positive for Trypanosoma spp. In the six specimens of T. coronatus, five distinct lineages of Trypanosoma spp. 18S-rRNA were observed in ninety sequences obtained, and using the strategy of cloning independent PCR, it was possible to observe that two of them were related to T. avium (JB01/JB02), and three were closed related to T. bennetti (JB03/ JB04/JB05). Addionaly, all fifteen sequences obtained from T. caerulescens/ S. spixi/T. flavipes/T. albicollis were identical. The present research is the first study to access molecular diversity and polyparasitism by avian trypanosomes in Brazil. The current research exhibits the wide genetic variability in avian trypanosomes and its non-specific relationship with its avian hosts.

First record of Trypanosoma (Ornithotrypanum) infecting Neotropical birds.

Parasites play a pivotal role in ecosystem health, influencing human and zoonotic diseases, as well as biodiversity preservation. The genus Trypanosoma comprises approximately 500 species mostly found in wildlife animals. This study focuses on identifying trypanosomes found in the white-necked thrush (Turdus albicollis) and the yellow-legged thrush (Turdus flavipes) in the Neotropics. First, we demonstrate the utility of an 18S rDNA sequence-structure phylogeny as an alternative method for trypanosome classification, especially when gGAPDH sequences are unavailable. Subsequently, the sequence-structure phylogeny is employed to classify new trypanosome sequences discovered in wild birds, placing them within the Ornithotrypanum subgenus. This marks the first identification of Ornithotrypanum in Neotropical birds, contributing to the understanding of the distribution and ecological adaptation of avian trypanosomes. Beyond taxonomy, this study broadens our comprehension of the ecological implications of avian trypanosomes in the Neotropics, emphasizing the need for continued research in this field. These findings underscore the importance of alternative classification methods, which are essential to unravel the complex interactions between parasites, wildlife hosts, and their ecosystems.

Metarhizium pingshaense photolyase expression and virulence to Rhipicephalus microplus after UV-B exposure.

The current study examined the impact of ultraviolet (UV)-B radiation in Metarhizium pingshaense blastospores' photolyase expression and their virulence against Rhipicephalus microplus. Blastospores were exposed to UV under laboratory and field conditions. Ticks were treated topically with fungal suspension and exposed to UV-B in the laboratory for three consecutive days. The expression of cyclobutane pyrimidine dimmers (CPDs)-photolyase gene maphr1-2 in blastospores after UV exposure followed by white light exposure was accessed after 0, 8, 12, 24, 36, and 48 h. Average relative germination of blastospores 24 h after in vitro UV exposure was 8.4% lower than 48 h. Despite this, the relative germination of blastospores exposed to UV in the field 18 h (95.7 ± 0.3%) and 28 h (97.3 ± 0.8%) after exposure were not different (p > 0.05). Ticks treated with fungus and not exposed to UV exhibited 0% survival 10 days after the treatment, while fungus-treated ticks exposed to UV exhibited 50 ± 11.2% survival. Expression levels of maphr1-2 8, 12, and 24 h after UV-B exposure were not different from time zero. Maphr1-2 expression peak in M. pingshaense blastospores occurred 36 h after UV-B exposure, in the proposed conditions and times analyzed, suggesting repair mechanisms other than CPD-mediated-photoreactivation might be leading blastospores' germination from 0 to 24 h.

Molecular detection and phylogenetic analysis of Trypanosoma spp. in Neotropical primates from Rio de Janeiro State, Brazil / Detecção molecular e análise filogenética de Trypanosoma spp. em primatas neotropicais do estado do Rio de Janeiro, Brasil

Trypanosoma spp. infection is a problem in many tropical countries, infecting several animal species, including humans. The aim of the present study was to identify the Trypanosoma species in Neotropical primates from Rio de Janeiro state and compare the results with other reports both phylogenetically and geographically. Molecular detection was based on the 18 SSU gene. The sequences obtained in the PCR were sequenced and compared with others previously deposited in GenBank. These sequences were used to perform phylogenetic analysis and make a distribution map of primate species infected by Trypanosoma species in Brazil. Among 34 monkeys, five capuchin monkeys (Sapajus spp.) and one marmoset (Callithrix spp.) showed Trypanosoma spp. sequences in the same clade of Trypanosoma minasense and three capuchin monkeys' sequences were in the same clade of Trypanosoma cruzi. The Atlantic Forest and the Brazilian Amazon are the regions with the highest frequency of studies about Trypanosoma spp. and variety of Neotropical primate hosts. These are areas that deserve attention regarding the conservation of biodiversity, but it also makes evident the lack of studies with Neotropical primates in other regions of the country, as well as multidisciplinary studies to better understand the host pathogen relationships.

A infecção por Trypanosoma spp. é um problema em muitos países tropicais, infectando várias espécies animais, incluindo humanos. O objetivo do presente estudo foi identificar as espécies de Trypanosoma em primatas neotropicais no estado Rio de Janeiro e comparar os resultados com outros relatos, tanto filogeneticamente quando geograficamente. A detecção molecular foi baseada no gene SSU 18. As sequências obtidas na PCR foram sequenciadas e comparadas com outras previamente depositadas no GenBank. Essas sequências foram utilizadas para análises filogenéticas e confeccionar um mapa de distribuição de espécies de primatas infectadas por espécies de Trypanosoma no Brasil. Entre 34 macacos, cinco macacos-prego (Sapajus spp.) e um sagui (Callithrix spp.) apresentaram sequências de Trypanosoma spp. no mesmo clado de Trypanosoma minasense e três sequências de macacos-prego estavam no mesmo clado de Trypanosoma cruzi. A Mata Atlântica e a Amazônia brasileira são as regiões com maior frequência de estudos sobre Trypanosoma spp. e variedade de primatas neotropicais hospedeiros. São áreas que merecem atenção no que se refere à conservação da biodiversidade, mas também evidencia a carência de estudos com PNH em outras regiões do país e de estudos multidisciplinares para melhor compreender as relações do patógeno hospedeiro.

Spatial distribution and molecular epidemiology of hemotropic Mycoplasma spp. and Mycoplasma haemocanis infection in dogs from Rio de Janeiro, Brazil.

This cross-sectional study aims to investigate the epidemiology and spatial distribution of hemotropic Mycoplasma spp. and Mycoplasma haemocanis in dogs from Rio de Janeiro, Brazil. Blood samples were collected at random from 437 household dogs. An epidemiological questionnaire was completed concerning the host characteristics as well as the environments in which they lived. A positivity frequency of 17.84% (78/437) was found for Mycoplasma spp. and 2% (9/437) for M. haemocanis in Rio de Janeiro, Brazil, through molecular detection based on the 16S rRNA sequence. According to the present study, dogs that live in households with the presence of rodents (odds ratio [OR] = 9.93; p-value = 0.02; confidence interval [CI]: 1.34-73.66) and wild animals (OR = 1.91; p-value = 0.03; CI: 1.06-3.42) are more likely to be infected with Mycoplasma spp.. Also, dogs with tick infestation (OR = 6.47; p-value = 0.007; CI: 1.63-25.60) have more chances to become infected with M. haemocanis. The spatial analysis disclosed a positive correlation between the Mycoplasma presence and tick infestation (global Moran index = 0.82; pseudo-p-value =0.001). The epidemiological findings support the hypothesis of Rhipicephalus sanguineus s.l. as the vector of M. haemocanis in the studied region and provide insightful information to prevent the Mycoplasma spp. infection in dogs from Rio de Janeiro.

Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis / Expressão dos genes icaA e icaD na formação de biofilme em isolados de Staphylococcus aureus de mastite subclínica bovina

Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.(AU)

Staphylococcus spp. desempenham um papel significativo na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais importante devido a alta prevalência e a dificuldade de tratamento in vivo que está relacionado à expressão dos fatores de virulência e formação de biofilme. Este estudo teve como objetivo detectar a expressão fenotípica da formação de biofilme em 20 cepas de S. aureus isoladas de mastite bovina e avaliar a expressão e regulação de genes envolvidos em sua produção. MALDI-TOF e ensaios de identificação fenogenotípica foram realizados para caracterizar os isolados. A produção fenotípica de biofilme e a presença dos genes icaA, icaD e bap foram avaliadas. O sistema Agr foi tipificado (agr I, agr II, agr III e agr IV) e seu regulador (agr RNAIII) foi detectado. Além disso, a PCR em tempo real (qPCR) foi realizada nos tempos determinados para quantificar a expressão dos genes icaA, icaD e hld em três isolados selecionados. Todas as 20 linhagens foram produtoras de biofilme e a maioria apresentava os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr do tipo II mostrou uma prevalência de 70%. A análise transcricional revelou aumento da expressão de genes ica às oito horas de crescimento. Estes resultados confirmam que a produção de polissacarídeos mediada pelos genes do operon icaADBC é um mecanismo essencial para a formação do biofilme e contribui para os estágios iniciais do crescimento bacteriano.(AU)

Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis

ABSTRACT: Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.

RESUMO: Staphylococcus spp. desempenham um papel significativo na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais importante devido a alta prevalência e a dificuldade de tratamento in vivo que está relacionado à expressão dos fatores de virulência e formação de biofilme. Este estudo teve como objetivo detectar a expressão fenotípica da formação de biofilme em 20 cepas de S. aureus isoladas de mastite bovina e avaliar a expressão e regulação de genes envolvidos em sua produção. MALDI-TOF e ensaios de identificação fenogenotípica foram realizados para caracterizar os isolados. A produção fenotípica de biofilme e a presença dos genes icaA, icaD e bap foram avaliadas. O sistema Agr foi tipificado (agr I, agr II, agr III e agr IV) e seu regulador (agr RNAIII) foi detectado. Além disso, a PCR em tempo real (qPCR) foi realizada nos tempos determinados para quantificar a expressão dos genes icaA, icaD e hld em três isolados selecionados. Todas as 20 linhagens foram produtoras de biofilme e a maioria apresentava os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr do tipo II mostrou uma prevalência de 70%. A análise transcricional revelou aumento da expressão de genes ica às oito horas de crescimento. Estes resultados confirmam que a produção de polissacarídeos mediada pelos genes do operon icaADBC é um mecanismo essencial para a formação do biofilme e contribui para os estágios iniciais do crescimento bacteriano.

Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis / Expressão dos genes icaA e icaD na formação de biofilme em isolados de Staphylococcus aureus de mastite subclínica bovina

Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.(AU)

Staphylococcus spp. desempenham um papel significativo na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais importante devido a alta prevalência e a dificuldade de tratamento in vivo que está relacionado à expressão dos fatores de virulência e formação de biofilme. Este estudo teve como objetivo detectar a expressão fenotípica da formação de biofilme em 20 cepas de S. aureus isoladas de mastite bovina e avaliar a expressão e regulação de genes envolvidos em sua produção. MALDI-TOF e ensaios de identificação fenogenotípica foram realizados para caracterizar os isolados. A produção fenotípica de biofilme e a presença dos genes icaA, icaD e bap foram avaliadas. O sistema Agr foi tipificado (agr I, agr II, agr III e agr IV) e seu regulador (agr RNAIII) foi detectado. Além disso, a PCR em tempo real (qPCR) foi realizada nos tempos determinados para quantificar a expressão dos genes icaA, icaD e hld em três isolados selecionados. Todas as 20 linhagens foram produtoras de biofilme e a maioria apresentava os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr do tipo II mostrou uma prevalência de 70%. A análise transcricional revelou aumento da expressão de genes ica às oito horas de crescimento. Estes resultados confirmam que a produção de polissacarídeos mediada pelos genes do operon icaADBC é um mecanismo essencial para a formação do biofilme e contribui para os estágios iniciais do crescimento bacteriano.(AU)

Parasitological and clinical-pathological findings in twelve outbreaks of acute trypanosomiasis in dairy cattle in Rio de Janeiro state, Brazil.

In Brazil, infection in cattle was first reported in the state of Pará, in 1944, and the presence of the parasite has already been recorded in several states. The purpose of this study was to report the clinical-pathological aspects of a natural infection by T. vivax in dairy cattle in the state of Rio de Janeiro. Twelve outbreaks of the infection were diagnosed in 11 municipalities from April 2016 to October 2018. All properties had acquired cattle from states where the disease had already been recorded and it was found that needles for oxytocin administration had been shared. These outbreaks were studied by visiting the properties to perform anamnesis, clinical exams and collection of material for laboratory diagnosis. Laboratory diagnosis was performed through parasitological, molecular and histopathological techniques. Animals with confirmed diagnosis for T. vivax showed anemia, lack of appetite, decreased milk production, weight loss, weakness, abortion, diarrhea and neurological signs. The main histological lesions found were meningoencephalitis and lymphohistiocytic myocarditis. In the central nervous system, the lesions were more severe in the brain compared to the spinal cord, being progressively more severe in the rostro-dorsal direction. Also, they were more accentuated in the white matter compared to the gray matter. Due to nonspecific clinical signs, laboratory tests were key for diagnosis. Trypanosomiasis in cattle herds in the state of Rio de Janeiro, Brazil, is of great concern because of its potential to cause economic losses.

Molecular diversity and coalescent species delimitation of avian haemosporidian parasites in an endemic bird species of South America.

Haemoproteus spp. and Plasmodium spp. are blood parasites that occur in birds worldwide. Identifying the species within this group is complex, especially in wild birds that present low parasitemia when captured, making morphological identification very difficult. Thus, the use of alternative tools to identify species may be useful in the elucidation of the distribution of parasites that circulate in bird populations. The objectives of this study were to determine the prevalence and parasitemia of the genera Plasmodium and Haemoproteus in Tachyphonus coronatus in the Atlantic Forest, Brazil, and to evaluate the molecular diversity, geographic distribution, and specificity of these parasites based on coalescent species delimitation methods. Microscopic analysis, PCR, cyt b gene sequencing, phylogenetic analysis and coalescent species delimitation using single-locus algorithms were performed (Poisson tree process (PTP) and multi-rate Poisson tree process (MPTP) methods). The analyses were performed in 117 avian host individuals. The prevalence was 55.5% for Plasmodium and 1.7% for Haemoproteus, with a mean parasitemia of 0.06%. Twenty-five Plasmodium and two Haemoproteus lineages were recovered. The MPTP method recovered seven different evolutionarily significant units (ESUs) of Plasmodium and one of Haemoproteus, whereas PTP presented fourteen ESUs of Plasmodium and one of Haemoproteus. The MPTP was more consistent with current taxonomy, while PTP overestimated the number of lineages. These ESUs are widely distributed and have already been found in 22 orders of birds that, all together, inhabit every continent, except Antarctica. The computational methods of species delimitation proved to be effective in cases where the classification of Haemosporida based just on morphology is insufficient.

Genetic Diversity of Theileria equi From Horses In Different Regions of Brazil Based On the 18S rRNA Gene.

Equine piroplasmosis stands out among the diseases that affect Equidae in Brazil and the world. It is caused by the protozoa Theileria equi and Babesia caballi. The objective of the present study was to carry out the molecular characterization of T. equi using equine blood samples collected in the 5 geographic regions of Brazil. Samples from all over the country were tested for the presence of T. equi by real-time PCR. The 18S rRNA sequences (∼1,600 bp) obtained from 23 samples taken from naturally infected horses were characterized by sequencing and analyzed to identify the genotypes and the possible sites of genetic variability. Thirteen different T. equi 18S rRNA sequences were identified, and 2 different genotypes were demonstrated to be in circulation in Brazil. Alignment entropy analysis demonstrated the existence of three hypervariable regions (V2, V4, and V8) within the 18S rRNA sequence of T. equi. The V2 region is located between nucleotides 63 and 75, V4 is located between nucleotides 524 and 586, and V8 is located between nucleotides 1,208 and 1,226. The hypervariable region V4 demonstrated the greatest variation within the 18S rRNA sequence of T. equi. Phylogenetic analysis based on the 18S rRNA sequences revealed the formation of 3 distinct clades (A, B, and C). The Brazilian samples belonged to 2 clades (A and C). The present study describes the characterization and heterogeneity of the circulating T. equi 18S rRNA sequences in Brazil. The results confirm that the country is an endemic area for the disease, and they indicate that at least 2 distinct T. equi genotypes are naturally infecting equines in Brazil.

Comparison of heat shock protein 70 kDa and 18S rDNA genes for molecular detection and phylogenetic analysis of Babesia vogeli from whole blood of naturally infected dogs.

A total of 300 blood samples of domiciliated dogs in rural and urban areas of southeast Rio de Janeiro State, Brazil, were used to compare the 18S ribosomal DNA region (18S rDNA) and the heat shock protein 70 kDa (hsp70) gene for molecular detection of Babesia vogeli and to perform a phylogenetic study comparing the two genes for B. vogeli classification. Using conventional polymerase chain reaction (cPCR) of 18S rDNA and hsp70 sequences, we were able to detect B. vogeli with the same sensitivity (96.15%) and specificity (99.63%). However, sequencing revealed one false positive (Rangelia sp.) for 18S rDNA that was not detected by hsp70. This is the first report of an organism closely related to the Rangelia vitalii parasite of dogs in Brazil. In the hsp70-cPCR and hsp70-qPCR comparison, 15.66% of samples were considered positive by quantitative (q)PCR, significantly more than was detected by cPCR (8.66%). In addition to the high conservation of the 18S rDNA, phylogenetic analysis showed that the hsp70 gene can be used to describe phylogenetic relationships between canine piroplasmids with more accuracy than 18S rDNA. According to these findings, the qPCR method has greater sensitivity than cPCR for detection of B. vogeli in naturally infected dogs. The hsp70-qPCR detection limit was 10 copies, with an efficiency of 100.30% and a determination coefficient (R2) of 0.998. The development of this qPCR method provides a highly sensitive approach for B. vogeli molecular detection and a tool that is capable of quantifying parasitemia levels in whole blood samples from dogs. The primers and probes were designed to be specific for B. vogeli, though analytical specificity of the assay has not been tested in vitro with DNA of certain Babesia species that infect dogs. The hsp70 gene is a precise molecular marker for Babesia phylogeny, especially species that infect dogs.

Molecular epidemiology of Babesia vogeli in dogs from the southeastern region of Rio de Janeiro, Brazil.

Hemoparasitic diseases are prominent in domestic animals, particularly in Brazil, a tropical country with a wide range of vectors. This study investigated the epidemiology of Babesia vogeli in the whole blood of dogs from the southeastern region of Rio de Janeiro, Brazil. Whole blood samples from 390 dogs were screened for the presence of B. vogeli DNA by qPCR using the heat shock protein 70 kDa (hsp70) gene of B. vogeli. Characteristics related to the host and its environment were collected using a questionnaire. Bivariate analysis was used to evaluate each factor individually. A phi correlation test was used to verify collinearity. The variables with p < .1 and a low or moderate correlation with the other variables were selected for the multivariate analysis. Multiple models were created, and the best logistic regression model was chosen using the Akaike Information Criterion (AIC). The final model was used to determine which variables were closely related to B. vogeli infections in dogs. Of the 390 dog blood samples, 15.66% were positive for B. vogeli. The variables cat contact, age, shelter, street or woods access, tick infestation and fur lengthwere included in the final model. Per the logistic regression analysis, three variables explained B. vogeli detection in dogs: age (odds ratio [OR] = 2.12; p-value <.05; confidence interval [CI]: 1.13-3.96), tick infestation (OR = 2.08; p-value <.05; CI: 1.10-3.93) and shelter (OR = 2.22; p-value <.05; CI: 1.16-4.26). These variables were determined to be associated with B. vogeli detection in domiciled dogs in the southeastern region of Rio de Janeiro, Brazil. These data indicate that the age of the animal, the presence of ticks and the lack of shelter directly affect the epidemiology of B. vogeli.

Epidemiological survey of Neorickettsia risticii in equids from the State of Rio de Janeiro, Brazil / Levantamento epidemiológico de Neorickettsia risticii em equídeos do Estado do Rio de Janeiro

Equine neorickettsiosis (EN), also known as Potomac Horse Fever, is a non-contagious disease caused by the bacterium Neorickettsia risticii of the Anaplasmataceae family. The objectives of this study were to detect the presence of anti-N. risticii antibodies by the indirect immunofluorescence assay (IFA) and of its DNA by qPCR in equids at high and low altitude regions in the State of Rio de Janeiro, Brazil, and to identify factors associated with seropositive equids by multiple logistic regression analysis. The frequency of anti-N. risticii antibodies was 16.05% (n=113/704). The animal age and breeding region were the factors that influenced the seropositivity rate for N. risticii in the equids (p<0.05). Equids from the lowland region had higher seropositivity (p<0.05; OR=5.87) compared to those of the mountain region. The presence of snails on the farm was a factor associated with this result (p<0.05; OR=2.88). In the lowland region, age of the animal and site of breeding were protective factors for the detection of antibodies anti-N. risticii in equids, with lower frequency of seropositivity in younger animals (p<0.05; OR=0.06) and in animals raised in dry areas (p<0.05; OR=0.22). The presence of the target DNA of N. risticii by qPCR was not observed in any of the samples tested. The existence of seropositive equids for N. risticii demonstrates a possible circulation of this agent in the studied area, and that the age related characteristics and equids breeding region are important factors regarding seropositivity in the State of Rio de Janeiro.(AU)

A Neorickettisiose equina (NE), também conhecida como Febre do Cavalo de Potomac, é uma doença não contagiosa causada pela bactéria Neorickettsia risticii da família Anaplasmataceae. Os objetivos deste estudo foram detectar a presença de anticorpos anti-N. risticii através da reação de Imunofluorescência Indireta (RIFI) e do DNA dessa bactéria através da qPCR em equídeos de regiões de alta e baixa altitude no Estado do Rio de Janeiro, Brasil; e identificar os fatores associados com a soropositividade dos equídeos através da análise de regressão logística múltipla. A frequência de anticorpos anti-N. risticii foi de 16,05% (n=113/704). Observou-se que a idade e a região de criação foram os fatores que influenciaram a taxa de soropositividade para N. risticii nos equídeos (p<0,05). Equídeos da região de baixada apresentaram maior soropositividade (p<0,05; OR=5,87) quando comparado aos criados em região de montanha. A presença de caramujos na propriedade foi um fator associado a este resultado (p<0,05; OR=2,88). Na região de baixada, animais mais jovens (p<0,05; OR=0,06), criados em áreas secas (p<0,05; OR=0,22) demonstraram serem fatores de proteção na detecção de anticorpos anti-N. risticii. Não foi observada a presença do DNA-alvo de N. risticii através da qPCR em nenhuma das amostras testadas. A existência de equídeos soropositivos para N. risticii demonstra a possível circulação desse agente na área estudada, e as características inerentes a idade e a região de criação dos equídeos são fatores importantes relacionados à soropositividade no estado do Rio de Janeiro.(AU)

A new quantitative PCR method for the detection of Anaplasma platys in dogs based on the citrate synthase gene.

Anaplasma platys is an obligate intracellular bacterium that primarily affects dogs, but it can also infect humans. Our study aimed to standardize a quantitative real-time (q)PCR method using the citrate synthase gene (gltA) as a specific target for A. platys detection in naturally infected dogs. Primers (gltA84F and gltA84R) and probe (PLATYSp) were designed to amplify an 84-bp fragment based on the gltA gene sequences of A. platys available in GenBank. A total of 186 dog blood samples originating from the Brazilian state of Rio de Janeiro were tested by qPCR. Additionally, the same samples were tested by cytology and a nested (n)PCR that targeted the 16S ribosomal DNA to determine the performance of our qPCR method compared to these existing techniques. Among the samples tested with qPCR, 17.2% were considered positive, significantly more than detected by nPCR (14.0%). Under optical microscopy, inclusions were observed in platelets of 25.3% of the samples, and among these samples, only 33.9% were identified as positive for A. platys using qPCR. The qPCR technique proved to be more specific than cytology and to have superior sensitivity to nPCR for detecting A. platys in dogs. The development of this new qPCR method contributes to the advancement of research involving A. platys Furthermore, it can be used to quantify the presence of this bacterium to evaluate the treatment of infected animals, or even as a more sensitive and specific tool for situations indicating possible clinical disease but with negative cytology.

Epidemiological survey of Neorickettsia risticii in equids from the State of Rio de Janeiro, Brazil / Levantamento epidemiológico de Neorickettsia risticii em equídeos do Estado do Rio de Janeiro

Equine neorickettsiosis (EN), also known as Potomac Horse Fever, is a non-contagious disease caused by the bacterium Neorickettsia risticii of the Anaplasmataceae family. The objectives of this study were to detect the presence of anti-N. risticii antibodies by the indirect immunofluorescence assay (IFA) and of its DNA by qPCR in equids at high and low altitude regions in the State of Rio de Janeiro, Brazil, and to identify factors associated with seropositive equids by multiple logistic regression analysis. The frequency of anti-N. risticii antibodies was 16.05% (n=113/704). The animal age and breeding region were the factors that influenced the seropositivity rate for N. risticii in the equids (p < 0.05). Equids from the lowland region had higher seropositivity (p < 0.05; OR=5.87) compared to those of the mountain region. The presence of snails on the farm was a factor associated with this result (p < 0.05; OR=2.88). In the lowland region, age of the animal and site of breeding were protective factors for the detection of antibodies anti-N. risticii in equids, with lower frequency of seropositivity in younger animals (p < 0.05; OR=0.06) and in animals raised in dry areas (p < 0.05; OR=0.22). The presence of the target DNA of N. risticii by qPCR was not observed in any of the samples tested. The existence of seropositive equids for N. risticii demonstrates a possible circulation of this agent in the studied area, and that the age related characteristics and equids breeding region are important factors regarding seropositivity in the State of Rio de Janeiro.(AU)

A Neorickettisiose equina (NE), também conhecida como Febre do Cavalo de Potomac, é uma doença não contagiosa causada pela bactéria Neorickettsia risticii da família Anaplasmataceae. Os objetivos deste estudo foram detectar a presença de anticorpos anti-N. risticii através da reação de Imunofluorescência Indireta (RIFI) e do DNA dessa bactéria através da qPCR em equídeos de regiões de alta e baixa altitude no Estado do Rio de Janeiro, Brasil; e identificar os fatores associados com a soropositividade dos equídeos através da análise de regressão logística múltipla. A frequência de anticorpos anti-N. risticii foi de 16,05% (n=113/704). Observou-se que a idade e a região de criação foram os fatores que influenciaram a taxa de soropositividade para N. risticii nos equídeos (p < 0,05). Equídeos da região de baixada apresentaram maior soropositividade (p < 0,05; OR=5,87) quando comparado aos criados em região de montanha. A presença de caramujos na propriedade foi um fator associado a este resultado (p < 0,05; OR=2,88). Na região de baixada, animais mais jovens (p < 0,05; OR=0,06), criados em áreas secas (p < 0,05; OR=0,22) demonstraram serem fatores de proteção na detecção de anticorpos anti-N. risticii. Não foi observada a presença do DNA-alvo de N. risticii através da qPCR em nenhuma das amostras testadas. A existência de equídeos soropositivos para N. risticii demonstra a possível circulação desse agente na área estudada, e as características inerentes a idade e a região de criação dos equídeos são fatores importantes relacionados à soropositividade no estado do Rio de Janeiro.

Reproductive efficiency of asymptomatic Theileria equi carriers mares submitted to an embryo transfer program / Eficiência reprodutiva de éguas portadoras assintomáticas de Theileira equi submetidas a um programa de transferência de embriões

This study aimed to assess and evaluate the effects of Theileria equi infection on embryonic recovery, gestation and early embryonic loss. Thirteen Mangalarga Marchador Theileria equi positive donors (diagnosed through nested-PCR) and 40 embryos receptors were used. Donors were submitted to two embryo collections in two consecutive estrous cycles (GId); after, the same mares were treated with imidocarb dipropionate (1.2mg/kg IM.) in order to collect more embryos in two more estrous cycles (GIId). Receptors were divided into two groups (control and with treated) with 20 animals each, where one group was the control (GIr) and the other one (GIIr) treated with 1.2mg/kg IM of imidocarb dipropionate assessing the gestation rate at 15, 30, 45 and 60 days. After 52 embryo collections, the embryonic recovery rates were 53.84% (14/26) and 65.38% (17/26) (p> 0.05) for GId and GIId, respectively. The gestation rate was 70% (14/20) (p>0.05) at 15, 30, 45 and 60 days in group GIr and for GIIr was 85% (17/20) (p>0.05) at 15 days, 80% (16/20) (p>0.05) at 30, 45 and 60 days. The treatment with imidocarb dipropionate did not cause significant improvement in the reproductive efficiency at an ET program.(AU)

Este estudo teve por objetivo avaliar a influência da infecção por Theileria equi nas taxas de recuperação embrionária, gestação e perda embrionária precoce. Foram utilizadas 13 doadoras e 40 receptoras de embrião da raça Mangalarga Marchador, positivas para Theileria equi através da técnica de nested-PCR. Nas doadoras foram realizados duas coletas de embriões em dois ciclos estrais consecutivos (GId), em sequência, esses mesmos animais foram tratados com dipropionato de imidocarb (1,2mg/kg IM.) para realização de mais duas coletas de embriões em dois ciclos estrais (GIId). As receptoras foram divididas em dois grupos de 20 animais cada, onde um grupo foi o controle (GIr) e, o outro grupo, foi tratado (GIIr) com 1,2mg/ Kg IM de dipropionato de imidocarb, com intuito de avaliar a taxa de gestação aos 15, 30, 45 e 60 dias. Após a realização de 52 coletas de embrião, as taxas de recuperação embrionária foram de 53,84% (14/26) e 65,38% (17/26) (p> 0,05) para GId e GIId, respectivamente. A taxa de gestação foi de 70% (14/20) (p>0,05) aos 15, 30, 45 e 60 dias no grupo GIr e para o GIIr foi 85% (17/20) (p>0,05) aos 15 dias, 80% (16/20) (p>0,05) aos 30, 45 e 60 dias. O tratamento com dipropionato de imidocarb não promoveu melhora significativa na eficiência reprodutiva em um programa de TE.(AU)

Reproductive efficiency of asymptomatic Theileria equi carriers mares submitted to an embryo transfer program / Eficiência reprodutiva de éguas portadoras assintomáticas de Theileira equi submetidas a um programa de transferência de embriões

This study aimed to assess and evaluate the effects of Theileria equi infection on embryonic recovery, gestation and early embryonic loss. Thirteen Mangalarga Marchador Theileria equi positive donors (diagnosed through nested-PCR) and 40 embryos receptors were used. Donors were submitted to two embryo collections in two consecutive estrous cycles (GId); after, the same mares were treated with imidocarb dipropionate (1.2mg/kg IM.) in order to collect more embryos in two more estrous cycles (GIId). Receptors were divided into two groups (control and with treated) with 20 animals each, where one group was the control (GIr) and the other one (GIIr) treated with 1.2mg/kg IM of imidocarb dipropionate assessing the gestation rate at 15, 30, 45 and 60 days. After 52 embryo collections, the embryonic recovery rates were 53.84% (14/26) and 65.38% (17/26) (p> 0.05) for GId and GIId, respectively. The gestation rate was 70% (14/20) (p>0.05) at 15, 30, 45 and 60 days in group GIr and for GIIr was 85% (17/20) (p>0.05) at 15 days, 80% (16/20) (p>0.05) at 30, 45 and 60 days. The treatment with imidocarb dipropionate did not cause significant improvement in the reproductive efficiency at an ET program.

Este estudo teve por objetivo avaliar a influência da infecção por Theileria equi nas taxas de recuperação embrionária, gestação e perda embrionária precoce. Foram utilizadas 13 doadoras e 40 receptoras de embrião da raça Mangalarga Marchador, positivas para Theileria equi através da técnica de nested-PCR. Nas doadoras foram realizados duas coletas de embriões em dois ciclos estrais consecutivos (GId), em sequência, esses mesmos animais foram tratados com dipropionato de imidocarb (1,2mg/kg IM.) para realização de mais duas coletas de embriões em dois ciclos estrais (GIId). As receptoras foram divididas em dois grupos de 20 animais cada, onde um grupo foi o controle (GIr) e, o outro grupo, foi tratado (GIIr) com 1,2mg/ Kg IM de dipropionato de imidocarb, com intuito de avaliar a taxa de gestação aos 15, 30, 45 e 60 dias. Após a realização de 52 coletas de embrião, as taxas de recuperação embrionária foram de 53,84% (14/26) e 65,38% (17/26) (p> 0,05) para GId e GIId, respectivamente. A taxa de gestação foi de 70% (14/20) (p>0,05) aos 15, 30, 45 e 60 dias no grupo GIr e para o GIIr foi 85% (17/20) (p>0,05) aos 15 dias, 80% (16/20) (p>0,05) aos 30, 45 e 60 dias. O tratamento com dipropionato de imidocarb não promoveu melhora significativa na eficiência reprodutiva em um programa de TE.

Occurrences of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from four districts of Tocantins state, Brazilian Legal Amazon Region / Ocorrência de anticorpos anti-Toxoplasma gondii e anti-Neospora caninum em ovelhas de quatro municípios do estado do Tocantins, Região da Amazônia Legal Brasileira

Toxoplasmosis and neosporosis have been recognized as economically important diseases with considerable impact on the livestock industry. Little is known concerning the occurrence of Toxoplasma gondii and Neospora caninum in sheep from Tocantins state, Brazil. Here, we investigated antibodies against these parasites and associated factors in 182 sheep from Araguaína, Santa Terezinha do Tocantins, Arguianópolis and Palmeiras do Tocantins districts, Tocantins. Sheep sera were assayed for T. gondii and N. caninum IgG antibodies by indirect fluorescence antibody test (IFAT), using cut-off point at a dilution of 1:40 and 1:25 respectively. The prevalence of seropositive animal for T. gondii was 13.74% and 13.74% for N. caninum. None of the characteristics studied including reproductive problems, presence of cats, presence of dogs and veterinary care (p>0.05) was associated with occurrence of T. gondii or N. caninum infection. Only breed was identified as associated factor for the occurrence of toxoplasmosis in sheep (p<0.05). The present study is the first report on serum occurrence of T. gondii and N. caninum in sheep from the state of Tocantins, Brazil.(AU)

Toxoplasmose e Neosporose são reconhecidas por doenças economicamente importantes com impacto considerável na indústria pecuária. Pouco se sabe sobre a ocorrência de Toxoplasma gondii e Neospora caninum em ovelhas do estado do Tocantins, Brasil. Foram investigados a ocorrência de anticorpos contra estes parasitos e fatores associados em 182 ovelhas das cidades de Araguaína, Santa Terezinha do Tocantins, Arguianópolis e Palmeiras do Tocantins, Tocantins. Os soro das ovelhas foram testados para anticorpos IgG anti-T. gondii e anti-N. caninum pela Reação de Imunofluorescência Indireta (RIFI), usando os pontos de corte na diluição de 1:40 e 1:25, respectivamente. A prevalência de animais soropositivos para T. gondii foi de 13.74% e para N. caninum, 13.74%. Nenhuma das características estudadas incluindo problemas reprodutivos, presença de gatos, presença de cães e cuidados veterinários (p>0.05) foram associadas com a ocorrência infecção por T. gondii ou N. caninum. Somente raça foi identificada como fator associado à ocorrência de toxoplasmose em ovelhas (p<0.05). O presente trabalho é o primeiro relato da ocorrência sérica de T. gondii e N. caninum em ovelhas do estado do Tocantins, Brasil.(AU)

Occurrences of anti-toxoplasma gondii and anti-neospora caninum antibodies in sheep from four districts of tocantins state, brazilian legal amazon region

Toxoplasmosis and neosporosis have been recognized as economically important diseases with considerable impact on the livestock industry. Little is known concerning the occurrence of Toxoplasma gondii and Neospora caninum in sheep from Tocantins state, Brazil. Here, we investigated antibodies against these parasites and associated factors in 182 sheep from Araguaína, Santa Terezinha do Tocantins, Arguianópolis and Palmeiras do Tocantins districts, Tocantins. Sheep sera were assayed for T. gondii and N. caninum IgG antibodies by indirect fluorescence antibody test (IFAT), using cut-off point at a dilution of 1:40 and 1:25 respectively. The prevalence of seropositive animal for T. gondii was 13.74% and 13.74% for N. caninum. None of the characteristics studied including reproductive problems, presence of cats, presence of dogs and veterinary care (p>0.05) was associated with occurrence of T. gondii or N. caninum infection. Only breed was identified as associated factor for the occurrence of toxoplasmosis in sheep (p<0.05). The present study is the first report on serum occurrence of T. gondii and N. caninum in sheep from the state of Tocantins, Brazil.

Toxoplasmose e Neosporose são reconhecidas por doenças economicamente importantes com impacto considerável na indústria pecuária. Pouco se sabe sobre a ocorrência de Toxoplasma gondii e Neospora caninum em ovelhas do estado do Tocantins, Brasil. Foram investigados a ocorrência de anticorpos contra estes parasitos e fatores associados em 182 ovelhas das cidades de Araguaína, Santa Terezinha do Tocantins, Arguianópolis e Palmeiras do Tocantins, Tocantins. Os soro das ovelhas foram testados para anticorpos IgG anti-T. gondii e anti-N. caninum pela Reação de Imunofluorescência Indireta (RIFI), usando os pontos de corte na diluição de 1:40 e 1:25, respectivamente. A prevalência de animais soropositivos para T. gondii foi de 13.74% e para N. caninum, 13.74%. Nenhuma das características estudadas incluindo problemas reprodutivos, presença de gatos, presença de cães e cuidados veterinários (p>0.05) foram associadas com a ocorrência infecção por T. gondii ou N. caninum. Somente raça foi identificada como fator associado à ocorrência de toxoplasmose em ovelhas (p<0.05). O presente trabalho é o primeiro relato da ocorrência sérica de T. gondii e N. caninum em ovelhas do estado do Tocantins, Brasil.