Differential Expression of Immune Genes in the Rhipicephalus microplus Gut in Response to Theileria equi Infection.

Rhipicephalus microplus is the only tick species known to serve as a biological vector of Theileria equi for horses and other equids in Brazil. The protozoan T. equi is one of the causal agents of equine piroplasmosis, a major threat in horse breeding systems. Vector competence is closely linked to the pathogens' ability to evade tick defense mechanisms. However, knowledge of tick immune response against infections by hemoparasites of the Theileria genus is scarce. In the present study, the expression of genes involved in immune signaling pathways of R. microplus adults' guts when challenged with a high or low parasitic load of T. equi was evaluated. This research demonstrates divergences in the immune gene expression pattern linked to T. equi infection in R. microplus since the Toll, IMD, and JNK signaling pathways were transcriptionally repressed in the guts of adult ticks infected with T. equi. Moreover, the results showed that different infectious doses of T. equi induce differential gene expression of key components of immune signaling cascades in R. microplus gut, suggesting a link between the intensity of infection and the activation of tick immunity response. The present study adds knowledge to elucidate the gut immune signaling response of R. microplus to T. equi infection. In addition, the generated data can serve as a basis for further investigations to develop strategies for controlling and preventing equine piroplasmosis.

Dynamics of Theileria equi Infection in Rhipicephalus (Boophilus) microplus during the Parasitic Phase in a Chronically Infected Horse.

Experimental studies have demonstrated that Rhipicephalus (Boophilus) microplus transmits Theileria equi to horses. However, the degree and dynamics of this protozoan infection in the vector's organism have not been fully elucidated. Therefore, this study aimed to evaluate the infection rate and parasitic load of T. equi in R. (B.) microplus, the infection dynamics in this arthropod during experimental infestation in a horse chronically infected with T. equi, and to evaluate the trans-stadial and intrastadial transmission competence of T. equi by R. (B.) microplus. The experimental infestation period of R. (B.) microplus on the horse was 33 days, but males were found on the animal up to 60 days post-infestation. After the fifth day post-infestation, ticks and equine blood were collected every two days. Whole ticks from the same developmental stage collected in the same day were pooled. Adult ticks were dissected to extract salivary glands and gut. DNA extraction was performed for all the samples, and they were then submitted to qPCRs for T. equi diagnosis. Freshly molted nymphs collected as larvae in the horse and freshly molted males and females collected as nymphs in the horse showed equal to or greater than 75% positivity for T. equi, indicating a strong possibility of trans-stadial transmission. The longest permanence of the male ticks on the horse associated with the high positivity rate of this type of sample for T. equi indicate that the male may play a role in the intrastadial transmission of T. equi to infection-free horses. The salivary glands displayed 77.78% positivity for T. equi and presented a higher infection rate at the end of the experimental period (100% from 29 to 33 days post-infection). This study shows that R. (B.) microplus has high T. equi infection rates and that the infection rate and parasitic load increased over the experimental period. These findings confirm the importance of chronically infected horses with T. equi as a source of infection for R. (B.) microplus.

Molecular investigation of Babesia caballi in horses from the state of Rio de Janeiro, Brazil: Epidemiological aspects associated with the infection.

The epidemiological aspects of Babesia caballi infection were evaluated in 516 horse samples from Rio de Janeiro, Brazil. The presence and infestation level of ticks on horses, breed conditions, and animal management were evaluated on each farm through an epidemiological questionnaire. The gene that codes for rhoptry-associated protein-1 (RAP-1) of B. caballi was amplified by nested PCR (nPCR). Among the horses sampled, 17.2% (n = 89/516) presented B. caballi DNA. The characterized samples showed 99-100% similarity with other isolates of B. caballi based on the RAP-1 gene, available in GenBank. In the final logistic regression model, the variables associated with B. caballi infection in horses were as follows: age below two years (OR = 3.33; IC = 1.7-6.5), farms located in low altitudes (OR = 3.52; IC = 1.7-7.3) and Dermacentor nitens infestation (OR = 1.91; IC = 1.1-3.4). Furthermore, a high level of D. nitens infestation in horses was also a factor associated with positivity for B. caballi (OR = 2.11; IC = 1.25-3.54). In summary, young horses bred in low altitude regions characterized with high temperatures, and infested by D. nitens, mainly with a higher level of infestation, are more likely to be infected by B. caballi. This epidemiological study provides statical evidence that the D. nitens tick play a role as the biological vector of B. caballi in the studied region.

Characterization of the Rhipicephalus (Boophilus) microplus Sialotranscriptome Profile in Response to Theileria equi Infection.

This study intends to characterize the sialotranscriptome profile of Rhipicephalus (Boophilus) microplus in response to Theileria equi and identify genes of interest with differential genomic expression, indicating relevant targets in the tick-protozoan interactions. The experimental design consisted of RNA sequencing from uninfected and T. equi-infected R. microplus salivary glands (SGs) to obtain transcriptomic profiles for characterization and comparison. A total of 288,952 transcripts were obtained from both tick profiles, 3456 transcripts (p < 0.05) differentially expressed in response to T. equi infection. The uninfected SGs' registered 231,179 transcripts, of which 155,359 were annotated. The most transcribed sequences were female-specific histamine binding protein and lipocalins. Regarding the T. equi-infected SGs, from the 238,964 assembled transcripts, 163,564 were annotated. The most transcribed sequences were histone demethylase JARID1 and Y-box-binding protein. Five transcripts (cystatin, arginase, nuclear factor κB kinase inhibitor subunit ß (IκB), IκB delta, lysosomal-trafficking regulator, and reeler protein) presented the gene ontology (GO) category "response to protozoan" and were exclusively displayed in the T. equi-infected profile. The transcriptome of T. equi was also analyzed, registering 4728 hits. The study's genetic and molecular information would be of great value for future studies and biotechnological applications envisaging disease control.

Immature ticks on wild birds and the molecular detection of a novel Rickettsia strain in the Ibitipoca State Park, southeastern Brazil.

Birds are recognized hosts of ticks, especially for the immature stages which may harbor various species and strains of Rickettsia. To explore landscapes inhabited by birds and their ticks would expand the knowledge on host-parasite relationships and the rickettsiae. The aim of this paper was to record the diversity of ticks collected on wild birds and assess the phylogenetic position of a novel Rickettsia strain detected in immature ticks. Birds were captured in the Ibitipoca State Park, located in the Minas Gerais state, southeastern Brazil, as part of a long-term research project on the ecology of ticks, birds and Rickettsia. We found three tick species parasitizing birds: Amblyomma aureolatum (63 larvae, 10 nymphs), Haemaphysalis leporispalustris (28 larvae, seven nymphs) and Amblyomma romarioi (27 larvae). Among these, A. aureolatum was the most abundant species including 54% (73/135) of the collected ticks. New tick-host records were: A. romarioi on Turdus amaurochalinus and H. leporispalustris on Thamnophilus caerulescens, Saltator similis and Zonotrichia capensis. Of the 82 ticks tested for Rickettsia spp. by PCR, two larvae (2.5%) of A. romarioi were infected with 'Candidatus Rickettsia paranaensis', a novel putative Rickettsia species closely related to Rickettsia africae, Rickettsia sibirica and Rickettsia parkeri, as corroborated by our phylogenetic analysis. Finally, we present a list of all records of immature stages of H. leporispalustris on passerine birds in Brazil.

Author Correction: Detection of Neorickettsia risticii, the agent of Potomac horse fever, in horses from Rio de Janeiro, Brazil.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Detection of Neorickettsia risticii, the agent of Potomac horse fever, in horses from Rio de Janeiro, Brazil.

This study aims to report the presence of Neorickettsia risticii DNA in blood samples from naturally infected horses in Rio de Janeiro, provide clinicopathological findings related to the infection, and report the phylogenetic diversity of the 16S rDNA of N. risticii in order to evaluate its heterogeneity. Real-time quantitative polymerase chain reaction (qPCR) was performed to investigate the presence of N. risticii in samples collected from horses (n = 187). Five positive samples were found in the molecular screening. Hypoalbuminemia and high levels of creatine kinase and lactate dehydrogenase were the predominant findings in the biochemical analysis. The sequences were similar to those of N. risticii. Phylogenetic analysis revealed genotype segregation based on the geographical distribution in the N. risticii sequence clade. Dendrograms constructed with five hypervariable regions revealed that V4 distinguished Neorickettsia at the species level and produced a phylogeny that best represented the phylogeny obtained with the complete 16S rDNA sequence. This is the first report of N. risticii DNA in the blood of Brazilian horses based on sequences deposited in GenBank. Further studies are necessary to clarify the epidemiological chain of this vector-borne parasite in order to determine and establish appropriate preventive measures in the equine trading market.

Multi-locus phylogenetic analysis groups the New World bacterium Rickettsia sp. strain ApPR with the Old World species R. africae; proposal of "Candidatus Rickettsia paranaensis".

Rickettsia parkeri sensu stricto (s.s.) is an emerging human pathogen in the Americas. Comprehension of the etiology of R. parkeri infections in South America is complicated by the existence of genetic variants (Atlantic rainforest, NOD and Parvitarsum) of this species that are associated with specific groups of Amblyomma ticks. The rickettsial bacterium strain ApPR was first reported in Amblyomma parkeri ticks in Southern Brazil in 2012 and was considered, based on sequencing of fragments of the gltA, htrA, ompA and ompB genes, to represent yet another genetic variant of R. parkeri. In the current work, a multi-locus phylogenetic analysis employing additional genes and intragenic regions was performed using DNA extracted from (a) larvae of A. parkeri and Amblyomma species haplotype Nazaré ticks collected from wild birds, (b) a nymph of Amblyomma sp. haplotype Nazaré recovered from a monkey (Callicebus nigrifons), representing the first report of that tick parasitizing a non-human primate and (c) from a cultured isolate of ApPR, isolated from colony-reared adults of Amblyomma geayi. Phylogenetic inference performed using Maximum-likelihood (ML), Maximum Parsimony (MP) and Bayesian (B) methods, consistently placed strain ApPR outside the New World R. parkeri complex and instead grouped it in proximity to the Old World species Rickettsia africae and Rickettsia sibirica. Estimates of evolutionary divergence provided additional support for the inferred phylogenetic relationship. Given the clear evolutionary distance between strain ApPR and R. parkeri we propose the recognition of "Candidatus Rickettsia paranaensis".

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.

Molecular evidence of Anaplasma phagocytophilum and Theileria equi coinfection in horses from Rio de Janeiro, Brazil.

The present study aims to determine the frequencies of Theileria equi and Anaplasma phagocytophilum antibodies among horses from the state of Rio de Janeiro, Brazil, and to detect the presence of DNA of these pathogens through molecular methods. A total of 98 serum samples of horses from the municipality of Seropedica were tested by indirect immunofluorescence antibody (IFA) to detect anti-A. phagocytophilum and anti-T. equi IgG antibodies. In addition, quantitative real-time PCR (qPCR) was used to detect these pathogens in the DNA extracted from the whole blood and buffy coat of horses. Bivariate analysis and odds ratio were performed to verify the possible association between positivity and characteristics related to the horses. As evaluated by IFA and qPCR, the frequency of animals that tested positive for T. equi was 89.8% (n = 88/98) and 91.8% (n = 90/98), whereas A. phagocytophilum was 17.4% (n = 17/98) and 1.0% (n = 1/98), respectively. Serological evidence of exposure to A. phagocytophilum and T. equi was observed in 16.3% (n = 16/98) of the horses; however, exposure was confirmed by qPCR in only 1.0% (n = 1/98). No statistical association was found in the bivariate and odds ratio analysis. This is the first study reporting the molecular detection of A. phagocytophilum DNA in horses from the state of Rio de Janeiro, and also the coinfection of A. phagocytophilum and T. equi in a horse from Brazil confirmed by molecular methods. Equine granulocytic anaplasmosis is circulating in Brazilian horses, together with T. equi, and should be included in the differential diagnosis of tick-borne diseases.