Toxoplasma gondii and Neospora caninum in invasive wild boars (Sus scrofa) and hunting dogs from Brazil.

The wild boar, an impactful invasive species in Brazil, is subject to population control activities, which often include the use of hunting dogs. Hunters commonly consume wild boar meat, which is also used to feed their dogs, posing a risk of Toxoplasma gondii infection for humans and both T. gondii and Neospora caninum for dogs. The study aimed to investigate the prevalence of infection in wild boars (n = 127) and hunting dogs (n = 73) from São Paulo, Rio Grande do Sul, and Paraná states. We employed histopathological, serological (indirect fluorescent antibody test), and molecular techniques (endpoint polymerase chain reaction). Histopathology slides of wild boar tissue (central nervous system, heart, skeletal muscle, liver, spleen, kidney, gastrointestinal tract, pancreas, lymph nodes, and thyroid) sections revealed no T. gondii or N. caninum cysts (0/47). Antibodies anti-T. gondii were detected in 35/108 (32.4%) and anti-N. caninum in 45/108 (41.7%) wild boars. Only 2/18 (11.1%) wild boar tissue homogenate samples tested positive for T. gondii on endpoint PCR. Hunting dogs showed antibodies against T. gondii in 62/73 (85%) and against N. caninum in 31/73 (42%). The presence of antibodies against T. gondii and N. caninum in wild boars and hunting dogs, along with T. gondii DNA detection in wild boars, indicates the circulation of these parasites. Educating hunters on preventing these foodborne diseases, including zoonotic risks, is crucial.

Correction: Mongruel et al. Expanding the Universe of Hemoplasmas: Multi-Locus Sequencing Reveals Putative Novel Hemoplasmas in Lowland Tapirs (Tapirus terrestris), the Largest Land Mammals in Brazil. Microorganisms 2022, 10, 614.

In the original publication [...].

Diversity of Ehrlichia spp., Anaplasma spp. and Neorickettsia spp. in vampire bats.

Although bats (Mammalia: Chiroptera) act as natural reservoirs for many zoonotic pathogens around the world, few studies have investigated the occurrence of Anaplasmataceae agents in bats, especially vampire bats. The family Anaplasmataceae (order Rickettsiales) encompasses obligate intracellular bacteria of the genera Anaplasma, Ehrlichia, Neorickettsia, Neoehrlichia, Wolbachia, and Allocryptoplasma. The present study aimed to investigate, using molecular techniques, the presence of species of Anaplasma, Ehrlichia, and Neorickettsia in vampire bats sampled in northern Brazil. Between 2017 and 2019, spleen samples were collected from vampire bats belonging to two species, Desmodus rotundus (n = 228) from the states of Pará (n = 207), Amazonas (n = 1), Roraima (n = 18) and Amapá (n = 3), and Diaemus youngii (n = 1) from Pará. Positivity rates of 5.2% (12/229), 3% (7/229), and 10.9% (25/229) were found in PCR assays for Anaplasma spp. (16S rRNA gene), Ehrlichia spp. (dsb gene) and Neorickettsia spp. (16S rRNA gene), respectively. The present study revealed, for the first time, the occurrence of Anaplasma spp. and different genotypes of Ehrlichia spp. in vampire bats from Brazil. While phylogenetic analyses based on the dsb and ftsZ genes of Ehrlichia and 16S rRNA of Anaplasma spp. revealed phylogenetic proximity of the genotypes detected in vampire bats with Anaplasmataceae agents associated with domestic ruminants, phylogenetic inferences based on the gltA and groEL genes evidenced the occurrence of genotypes apparently exclusive to bats. Neorickettsia sp. phylogenetically associated with N. risticii was also detected in vampire bats sampled in northern Brazil.

Novel Tick-Borne Anaplasmataceae Genotypes in Tropical Birds from the Brazilian Pantanal Wetland.

Despite numerous reports of Anaplasmataceae agents in mammals worldwide, few studies have investigated their occurrence in birds. The present study aimed to investigate the occurrence and molecular identity of Anaplasmataceae agents in birds from the Pantanal wetland, Brazil. Blood samples were collected from 93 different species. After DNA extraction, samples positive for the avian ß-actin gene were subjected to both a multiplex quantitative real-time (q)PCR for Anaplasma and Ehrlichia targeting the groEL gene and to a conventional PCR for Anaplasmataceae agents targeting the 16S rRNA gene. As a result, 37 (7.4%) birds were positive for Anaplasma spp. and 4 (0.8%) for Ehrlichia spp. in the qPCR assay; additionally, 13 (2.6%) were positive for Anaplasmataceae agents in the PCR targeting the 16S rRNA gene. The Ehrlichia 16S rRNA sequences detected in Arundinicola leucocephala, Ramphocelus carbo, and Elaenia albiceps were positioned closely to Ehrlichia sp. Magellanica. Ehrlichia dsb sequences detected in Agelasticus cyanopus and Basileuterus flaveolus grouped with Ehrlichia minasensis. The 16S rRNA genotypes detected in Crax fasciolata, Pitangus sulphuratus and Furnarius leucopus grouped with Candidatus Allocryptoplasma. The 23S-5S genotypes detected in C. fasciolata, Basileuterus flaveolus, and Saltator coerulescens were related to Anaplasma phagocytophilum. In conclusion, novel genotypes of Anaplasma, Ehrlichia, and Candidatus Allocryptoplasma were detected in birds from the Pantanal wetland.

Phylogenetic inferences based on distinct molecular markers reveals a novel Babesia (Babesia pantanalensis nov. sp.) and a Hepatozoon americanum-related genotype in crab-eating foxes (Cerdocyon thous).

Piroplasmids and Hepatozoon spp. Are apicomplexan protozoa that may cause disease in several canid species. The present study aimed to expand the knowledge on the diversity of piroplasmids and Hepatozoon in crab-eating foxes (Cerdocyon thous; n = 12) sampled in the Pantanal of Mato Grosso do Sul State, central-western Brazil. PCR assays based on the 18S rRNA were used as screening. Three (25%) and 11 (91.7%) were positive for piroplasmids and Hepatozoon spp., respectively. Co-infection was found in three C. thous. Phylogenetic analyses based on the near-complete 18S rRNA, cox-1 and hsp70 genes evidenced the occurrence of a novel of Babesia spp. (namely Babesia pantanalensis nov. sp.) closely related to Rangelia vitalii and Babesia sp. 'Coco'. This finding was supported by the genetic divergence analysis which showed (i) high divergence, ranging from 4.17 to 5.62% for 18 S rRNA, 6.16% for hps70 and 4.91-9.25% for cox-1 and (ii) the genotype network (which displayed sequences separated from the previously described Piroplasmida species by median vectors and several mutational events). Also, phylogenetic analysis based on the 18S rRNA gene of Hepatozoon spp. positioned the sequences obtained herein in a clade phylogenetically related to Hepatozoon sp. 'Curupira 2', Hepatozoon sp. detected in domestic and wild canids from Uruguay and Hepatozoon americanum. The present study described Babesia pantanalensis nov sp. and Hepatozoon closely related to H. americanum in crab-eating foxes from Brazil. Moreover, the coinfection by piroplasmids and Hepatozoon sp. for the first time in crab-eating foxes strongly suggesting that this wild canid species potentially acts as a bio-accumulate of hemoprotozoan in wild environment.

Trypanosoma vivax in and outside cattle blood: Parasitological, molecular, and serological detection, reservoir tissues, histopathological lesions, and vertical transmission evaluation.

This study reports assessment of the sensitivity of diagnostic techniques to detect T. vivax in experimentally infected cattle. Additionally, it describes T. vivax extravascular parasitism during the acute and chronic phases of trypanosomosis and congenital transmission. The T. vivax diagnosis was compared using blood samples collected from the jugular, coccygeal and ear tip veins. For this study, 13 males and two females were infected with ≈ 1 × 106 viable T. vivax trypomastigotes (D0). One animal was kept as a negative control during the entire study. The 13 infected males were euthanized between 14 and 749 days post-infection (DPI). After confirming the cyclicity of both females (9 months of age), they were naturally mated with a bull. One female was euthanized at 840 DPI, and the other at 924 DPI. The two calves, one from each female, were euthanized at six months of age (924 DPI), and the negative control at 924 DPI. During this period, T. vivax in blood was assessed using direct methods (Woo test, cPCR, microscopic examination of fresh wet blood films and parasite quantification - Brener method), and serological methods (IFAT, ELISA, and IA). Tissue samples were collected from the liver, spleen, brain, cerebellum, heart, testicles, epididymis, kidneys, eyeballs, pre-scapular lymph nodes, ear tips, mammary glands, uterus, and ovaries. The protozoan DNA was examined using LAMP. There was no difference in the detection of T. vivax using the Woo test and Brener method among the jugular, coccygeal, and ear tip veins. The sensitivity of the detection methods varied depending on the disease phase. Direct methods (Woo test, Brener method, and cPCR) demonstrated higher sensitivity during the acute phase, while serological methods (IFAT, ELISA, and IA) were more sensitive during the chronic phase. Anti-T. vivax antibodies were detected up to 924 DPI. Tissue evaluation using LAMP demonstrated the presence of T. vivax DNA and associated histopathological changes up to 840 or 924 DPI. Only in mammary glands and ovaries was no DNA detected. The most frequently observed histopathological alteration was lymphohistioplasmocytic inflammatory infiltrate. No transplacental transmission of T. vivax was observed.

First molecular detection of Borrelia sp. in tapirs (Tapirus terrestris).

Borrelia theileri is a tick-borne spirochete causative agent of fever, apathy and reduced food consumption in cattle. Molecular diagnosis has expanded the understanding of Borrelia theileri with new hosts and geographical locations being described. The present study aimed to describe the first molecular detection of B. theileri in wild tapirs (Tapirus terrestris) from South America. Blood DNA samples obtained from 99 tapirs sampled in Pantanal (n = 61) and Cerrado (n = 38) biomes were screened using a qPCR assay based on the 16 S rRNA gene of Borrelia sp. Positive samples in the qPCR assay were subjected to PCR assays to allow characterization of fragments from 16 S rRNA and flaB genes. Two (2/99; 2.0%) animals from Pantanal biome were positive in the qPCR and one sample presented bands of expected size for the flaB protocol. Amplicons from this sample were successfully cloned and sequenced. In the phylogenetic analysis, Borrelia sp. from T. terrestris grouped together with B. theileri sequences previously detected in Rhipicephalus microplus ticks and cattle from Minas Gerais State in Brazil, Rhipicephalus geigyi from Mali, and R. microplus and Haemaphysalis sulcata from Pakistan. This finding contributes to our knowledge regarding susceptible hosts species for B. theileri. More studies are necessary to understand the potential effects of B. theileri on tapir's health.

Molecular evidence of Bartonella spp. in tropical wild birds from the Brazilian Pantanal, the largest wetland in South America.

Despite the worldwide occurrence of bartonellae in a broad range of mammal species, in which they usually cause a long-lasting erythrocytic bacteremia, few studies reported Bartonella spp. in avian hosts. The present work aimed to investigate the occurrence and molecular identity of Bartonella spp. infecting birds in the Pantanal wetland, central-western Brazil using a multigene approach. For this purpose, blood samples were collected from 517 individuals from 13 avian orders in the states of Mato Grosso and Mato Groso do Sul. DNA was extracted from avian blood and 500/517 (96.7%) samples were positive in a conventional PCR targeting the avian ß-actin gene. Nineteen (3.8%) out of 500 avian blood samples were positive in a qPCR assay for Bartonella spp. based on the nuoG gene. Among 19 avian blood DNA samples positive in the qPCR for Bartonella spp., 12 were also positive in the qPCR for Bartonella based on the 16S-23S RNA Intergenic region (ITS). In the PCR assays performed for molecular characterization, one 16S rRNA, three ribC, and one nuoG sequences were obtained. Based on BLASTn results, while 1 nuoG, 2 ribC, and 2 ITS sequences showed high identity to Bartonella henselae, one 16S rRNA and 2 ITS showed high similarity to Bartonella machadoae in the sampled birds. Bartonella spp. related to B. henselae and B. machadoae were detected, for the first time, in wild birds from the Brazilian Pantanal.

Molecular survey of hemoplasmas and Coxiella burnetii in vampire bats from northern Brazil.

In addition to zoonotic viral pathogens, bats can also harbor bacterial pathogens, including hemoplasmas (hemotropic mycoplasmas) and Coxiella burnetii. The present study aimed to investigate, using molecular techniques, the presence of hemoplasmas and C. burnetii in spleen samples from vampire bats in northern Brazil. For this purpose, between 2017 and 2019, spleen samples were collected from Desmodus rotundus (n = 228) and Diaemus youngii (n = 1) captured in the states of Pará (n = 207), Amazonas (n = 1), Roraima (n = 18) and Amapá (n = 3). DNA samples extracted from the bat spleen and positive in PCR for the endogenous gapdh gene were subjected to conventional PCR assays for the 16S rRNA, 23S rRNA and RNAse P genes from hemoplasmas and to qPCR based on the IS1111 gene element for C. burnetii. All spleen samples from vampire bats were negative in the qPCR for C. burnetii. Hemoplasmas were detected in 10 % (23/229) of spleen samples using a PCR based on the 16S rRNA gene. Of these, 21.73 % (5/23) were positive for the 23S rRNA gene and none for the RNAseP gene. The seven hemoplasma 16S rRNA sequences obtained were closely related to sequences previously identified in vampire bats from Belize, Peru and Brazil. The 23S rRNA sequence obtained revealed genetic proximity to hemoplasmas from non-hematophagous bats from Brazil and Belize. The analysis revealed different circulating genotypes among Brazilian vampire bats, in addition to a trend towards genera-specific hemoplasma genotypes. The present study contributes to the knowledge of the wide diversity of hemoplasmas in vampire bats.

Evaluation of mechanical transmission of Trypanosoma vivax by Stomoxys calcitrans in a region without a cyclic vector.

This work investigated the mechanical transmission of Trypanosoma vivax by Stomoxys calcitrans to cattle in a region without a cyclic vector. The study involved two experiments, one with calves experimentally infected with T. vivax, in the acute phase of trypanosomosis (Experiment 1) and the other in the chronic phase (Experiment 2). In both experiments, two transmission methods were used with flies that had not fed for 24 h or had never fed: (i) Method 1: flies released freely in cattle pens (≈3,300 flies/pen for 10 days); and (ii) Method 2: flies placed in a feeding chamber (12 flies/animal). To develop Method 1 in the two experiments (acute and chronic phases), T. vivax-positive animals were kept with T. vivax-negative animals. Periodically, the Brener method, Woo method, blood smears, cPCR, ELISA, IFAT, and Imunoteste® were performed to detect T. vivax in the animals. We also recorded the animals' head tossing and hoof stomping and the number of flies near the pens' inner walls. Subsequently, biological testing was performed using lambs. For Method 2 in both experiments, flies inside the feeding chamber first fed on T. vivax-positive animals and later on negative animals. In both experiments and methods, we examined the flies for the presence of T. vivax through blood smears and cPCR of the proboscis and abdomen. In Experiment 2 (chronic phase), a test was conducted to determine how long trypomastigotes forms could survive on the blood of animals with different levels of parasitemia. None of the animals (calves and lambs) became infected with T. vivax or showed antibodies against it. During the evaluation period, the animals in the presence of the flies exhibited more hoof stomping and head tossing compared to those without flies (control). Additionally, there was an increase in the number of flies in the pens during the experiment. Only in Experiment 1 (acute phase) were T. vivax trypomastigotes and DNA found in the abdomen of the flies but not in the proboscis. In Experiment 2 (chronic phase), higher concentrations of trypomastigotes per milliliter of blood were associated with a shorter the lifespan of this stage of the parasite. In conclusion, under the variable conditions of the experiments (hosts, number of flies, and level of parasitemia), S. calcitrans was unable to mechanically transmit T. vivax to cattle.

Molecular and serological detection of Anaplasma spp. in small ruminants in an area of Cerrado Biome in northeastern Brazil.

Anaplasmosis, caused by bacteria of the genus Anaplasma, is an important tick-borne disease that causes economic losses to livestock farms in many countries. Even though Anaplasma spp. have been detected in goats and sheep worldwide, few studies investigate the occurrence and genetic identity of these agents in small ruminants from Brazil. Thus, this work aimed to detect and determine the genetic identity of Anaplasma spp. in small ruminants from the Baixo Parnaíba region, state of Maranhão, northeastern Brazil. For this purpose, blood samples were collected from 161 animals (91 goats; 70 sheep) from 4 municipalities in the Baixo Parnaíba region. Sheep and goat serum samples were subjected to recombinant membrane surface protein (MSP5)-based iELISA. Whole blood samples were subject to DNA extraction and molecular diagnosis using PCR assays for Anaplasma spp. targeting msp1ß, msp1α, 16S rRNA and msp4 genes. Positive samples were sequenced and then subjected to Anaplasma marginale msp1α genetic diversity analysis and phylogenetic inferences based on the 16S rRNA and msp4 genes. The serological survey detected the presence of anti-A. marginale IgG antibodies in 18 animals (11.1%): 2.9% (2/70) sheep and 17.4% (16/91) goats. Anaplasma marginale DNA was detected in 2 goats (1.2%) using qPCR based on the msp1ß gene. Two distinct A. marginale msp1α strains, namely α ß and α ß ΓγΓγΓγΓγ were found in the infected goats, each one found in a different animal, both belonging to the H genotype. Phylogenetic analysis based on the 16S rRNA gene showed the sequences positioned in three different clades and grouped with sequences from 'Candidatus Anaplasma boleense', A. platys and A. marginale. Phylogenetic inferences based on the msp4 gene positioned the sequence variants in the A. marginale clade. The present work represents the first molecular detection of sequence variants phylogenetic associated to 'Candidatus Anaplasma boleense' and A. platys and α ß and α ß ΓγΓγΓγΓγ in goats from Brazil.

Artificial feeding of Ornithodoros fonsecai and O. brasiliensis (Acari: Argasidae) and investigation of the Transstadial perpetuation of Anaplasma marginale

Anaplasma marginale is a Gram-negative, obligate intraerythrocytic bacterium that causes bovine anaplasmosis. While hard ticks of the genera Dermacentor and Rhipicephalus can be biological vectors, transmitting this pathogen via saliva during blood meals, blood-sucking insects, and fomites play a role as mechanical vectors. Little is known about the interaction between Anaplasma marginale and Argasidae ticks. Among soft ticks, Ornithodoros fonsecai (Labruna and Venzal) and Ornithodoros brasiliensis Aragão inhabit environments surrounding localities where many cases of bovine anaplasmosis have been reported. Ticks of the species O. fonsecai parasitize bats, while O. brasiliensis can parasitize different vertebrate species. Therefore, the present study aimed to feed third-instar nymphs artificially (N3) of O. fonsecai and O. brasiliensis using blood samples obtained from a calf naturally infected with A. marginale and rabbit blood added to A. marginale-containing bovine erythrocytes, to investigate the ability of these nymphs to acquire, infect and transstadially perpetuate this agent. For the artificial feeding system, adapted chambers and parafilm membranes were used. Nymphs of both tick species were submitted to different replications weighed before and after each feeding. Blood samples and molted ticks were submitted to DNA extraction, quantitative real-time PCR for the msp1β gene to detect A. marginale DNA, while a semi-nested polymerase chain reaction for the msp1α gene was performed for genotyping. Using calf blood naturally infected with A. marginale, among the three artificial feeding replications performed with O. fonsecai and O. brasiliensis nymphs, the DNA of A. marginale was detected in both nymphs after 30–50 days of molting. For artificial feeding with rabbit blood added to bovine erythrocytes containing A. marginale, the DNA of this pathogen was also detected in both nymph species. As for the assay for the msp1α gene, strains were found Is9; 78 24-2; 25; 23; α; and β. It was concluded that nymphs (N3) of O. fonsecai and O. brasiliensis could feed artificially through a parafilm membrane using blood from calves and rabbits infected by A. marginale. The DNA of A. marginale was detected in nymphs fed artificially of both tick species studied after molt. However, further studies are needed to confirm transstadial perpetuation in other instars and their host transmission capacity.

Contribution to the knowledge of Neotrichodectes (Nasuicola) pallidus (Piaget, 1880) (Phthiraptera: Trichodectidae)

The species in the genus Neotrichodectes (Phthiraptera: Ischnocera) infest carnivores. Neotrichodectes (Nasuicola) pallidus (Piaget, 1880), which has been primarily found parasitizing Procyonidae mammals, has been recorded in ring-tailed coatis (Nasua nasua) in the Brazilian states of Minas Gerais, Pernambuco, Santa Catarina, Rio Grande do Sul and Pernambuco. We report a new record of N. pallidus in coatis in the state of Mato Grosso do Sul, central-western Brazil, using morphological (Light and Scanning Electronic Microscopy) and molecular approaches (PCR, sequencing and phylogenetic analysis). Coatis were sampled in two peri-urban areas of Campo Grande city, Mato Grosso do Sul state, Brazil, between March 2018 and March 2019, as well as in November 2021. Lice were collected and examined under light and Scanning Electron Microscopy. DNA was also extracted from nymphs and adults and submitted to PCR assays based on the 18S rRNA and cox-1 genes for molecular characterization. One hundred and one coatis were sampled from 2018 to 2019 and 20 coatis in 2021 [when the intensity of infestation (II) was not accessed]. Twenty-six coatis (26/101–25.7%) were infested with at least one louse, with a total of 59 lice collected in 2018–2019. The II ranged from one to seven lice (mean 2.2 ± SD 1.7). The louse species was confirmed based on the following morphological characteristics: female gonapophyses rounded with the setae along anterior region but not in the medial margin; the male genitalia with a parameral arch not extending beyond the endometrial plate. The same ornamentation was observed on the abdomen of the females, males, and nymphs. The nymphs and the eggs were described in detail for the first time. The obtained 18S rRNA and cox1 sequences from N. pallidus clustered in a clade with other sequences of Ischnocera species. In the present study, a new record of the louse N. pallidus in central-western Brazil was provided, along with new insights into the morphological features of this species, with the first morphology contribution of nymphal and eggs stages.

Evaluation of techniques for diagnosis of Trypanosoma vivax infections in naturally infected cattle in the Zona da Mata Mineira / Avaliação de técnicas para diagnóstico de infecções por Trypanosoma vivax em bovinos naturalmente infectados na Zona da Mata Mineira

Abstract This study aimed to evaluate diagnostic techniques for trypanosomiasis, caused by Trypanosoma vivax, in naturally infected cattle in Minas Gerais, Zona da Mata. The deaths of six lactating cows with similar clinical conditions—characterized by hyporexia, hypogalactia, and recumbency—had been reported from one property. Initially, two animals were examined and diagnosed with trypanosomiasis through identification of the protozoan in a blood smear. After the initial diagnosis, all lactating cows (n=37) on the property were examined, and blood samples were collected for tests including whole blood smear, buffy coat smear, Woo's technique, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR). Woo's test, buffy coat smears, and whole blood smears indicated that 4/37 (10.81%) animals were positive for trypanosomiasis, whereas ELISA and PCR indicated that 33/37 (89.19%) and 27/37 (72.97%) animals, respectively, were positive. The agreement obtained between parasitological techniques was classified as high, while between ELISA and PCR, no agreement. In conclusion, parasitological techniques have a low capacity to identify infected animals in the chronic stage of T. vivax infection. Therefore, techniques such as PCR and/or ELISA should be used to minimize the occurrence of false negatives.

Resumo Este estudo objetiva avaliar as técnicas de diagnóstico da tripanossomíase, causada pelo Trypanosoma vivax, em bovinos naturalmente infectados, em Minas Gerais, Zona da Mata. A morte de seis vacas em lactação com condições clínicas semelhantes - caracterizadas por hiporexia, hipogalaxia e decúbito - foi relatada em uma propriedade. Inicialmente, dois animais foram examinados e diagnosticados com tripanossomíase através da identificação do protozoário em esfregaço sanguíneo. Após o diagnóstico inicial, todas as vacas em lactação (n = 37) na propriedade foram examinadas, e amostras de sangue foram coletadas para testes, incluindo esfregaço de sangue total, esfregaço de capa leucocitária, técnica de Woo, ensaio imunoenzimático (ELISA) e reação em cadeia da polimerase (PCR). O teste de Woo, os esfregaços de capa leucocitária e de sangue total indicaram que 4/37 (10,81%) animais foram positivos para tripanossomíase, enquanto ELISA e PCR indicaram que 33/37 (89,19%) e 27/37 (72,97%) animais, respectivamente, foram positivos. A concordância entre técnicas parasitológicas foi classificada como alta, enquanto entre ELISA e PCR, sem concordância. As técnicas parasitológicas apresentam baixa capacidade para identificar animais infectados na fase crônica da infecção por T. vivax. Dessa forma, técnicas como PCR e/ou ELISA devem ser utilizadas para minimizar a ocorrência de falsos negativos.

Follow-up of dairy cattle naturally infected by Trypanosoma vivax after treatment with isometamidium chloride / Acompanhamento de bovinos leiteiros naturalmente infectados com Trypanosoma vivax após tratamento com cloreto de isometamidium

Abstract Trypanosoma vivax infections cause nonspecific clinical signs in cattle associated with aparasitemic intervals, making disease diagnosis a challenge. In Brazil, diminazene aceturate and isometamidium chloride (ISM) are available to treat bovine trypanosomosis. The objective of this study was to follow-up, by molecular and serological techniques, dairy cattle naturally infected by T. vivax after ISM treatment. Thirty cattle naturally infected with T. vivax received two applications of ISM, at a dosage of 1.0 mg/kg intramuscularly, on days 0 and 150. For T. vivax diagnosis, EDTA-blood and serum samples were evaluated on 0, 7, 15, 30, 60, 90, 120, 150, 180, 210, and 240 days after treatment PCR, Loop-mediated isothermal amplification (LAMP) and ELISA. Animals with persistent detection of T. vivax DNA by both PCR and LAMP were found and continuous detection of anti-T. vivax IgG antibodies by ELISA, suggesting the presence of T. vivax resistance to ISM. The combination of LAMP and ELISA tests can prevent misdiagnosis of the parasite clearance in treated cattle, contributing to better disease control. This is the first experiment that demonstrates the persistence infection of T. vivax under ISM treatment in a natural infected herd and evidence of ISM chemotherapy-resistant T. vivax in Brazil.

Resumo Em bovinos, infecções por Trypanosoma vivax geram sinais clínicos inespecíficos que, associados a intervalos aparasitêmicos, faz com que o diagnóstico da enfermidade seja desafiador. No Brasil, somente aceturato de diaminazeno e cloridrato de isometamidum (ISM) estão disponíveis para o tratamento da tripanossomose bovina. Este trabalho teve como objetivo acompanhar bovinos leiteiros naturalmente infectados por T. vivax, após o tratamento com ISM por meio de técnicas moleculares e sorológica. Foram utilizados 30 bovinos naturalmente infectados com T. vivax, sendo estes tratados com duas aplicações de ISM, na dosagem de 1,0 mg/kg por via intramuscular profunda, nos dias 0 e 150. Foram avaliadas, para diagnóstico de T. vivax, amostras de sangue acrescido de EDTA e soro, colhidas nos 0, 7, 15, 30, 60, 90, 120, 150, 180, 210 e 240 dias após os tratamentos pela reação em cadeia da polimerase (PCR), amplificação circular isotérmica do DNA (LAMP) e ensaio de imunoabsorção enzimático (ELISA). Verificou-se a presença de animais com persistência na detecção de DNA de T. vivax pela PCR e LAMP, bem como detecção contínua de anticorpos IgG anti-T. vivax pelo método de ELISA, sugerindo a presença de resistência de T. vivax ao ISM. A combinação dos testes LAMP e ELISA pode evitar falsos diagnósticos da eliminação do parasita nos bovinos tratados, contribuindo para um melhor controle da doença. Este é o primeiro experimento que demonstra infecção persistente do T. vivax em rebanho naturalmente infectado, tratado com ISM, e evidencia possível resistência ao quimioterápico no Brasil.

Genomic analysis on Brazilian strains of Anaplasma marginale / Análise genômica de cepas Brasileiras de Anaplasma marginale

Abstract Anaplasma marginale is a vector-borne pathogen that causes a disease known as anaplasmosis. No sequenced genomes of Brazilian strains are yet available. The aim of this work was to compare whole genomes of Brazilian strains of A. marginale (Palmeira and Jaboticabal) with genomes of strains from other regions (USA and Australia strains). Genome sequencing of Brazilian strains was performed by means of next-generation sequencing. Reads were mapped using the genome of the Florida strain of A. marginale as a reference sequence. Single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) were identified. The data showed that two Brazilian strains grouped together in one particular clade, which grouped in a larger American group together with North American strains. Moreover, some important differences in surface proteins between the two Brazilian isolates can be discerned. These results shed light on the evolutionary history of A. marginale and provide the first genome information on South American isolates. Assessing the genome sequences of strains from different regions is essential for increasing knowledge of the pan-genome of this bacteria.

Resumo Anaplasma marginale é um patógeno transmitido por vetores que causam uma doença conhecida como anaplasmose. Até a presente data, não há genomas sequenciados de cepas brasileiras. O objetivo deste estudo foi comparar o genoma completo das cepas brasileiras de A. marginale (Palmeira e Jaboticabal) com os genomas de cepas de outras regiões (cepas dos EUA e Austrália). As sequências dos genomas das cepas brasileiras foram obtidas mediante sequenciamento de nova geração. As "reads" foram mapeadas usando-se como referência o genoma de A. marginale da cepa Florida. Foram identificados polimorfismos de nucleotídeo único (SNPs) e analisadas inserções/deleções (INDELs). As duas linhagens brasileiras se agruparam em um clado particular que, por sua vez, agrupou-se em um grupo maior junto com as linhagens norte-americanas. Além disso, foram identificadas diferenças significativas nas proteínas de superfície entre os dois isolados brasileiros. Esses resultados lançam luz sobre a história evolutiva de A. marginale e fornecem as primeiras informações de genomas de isolados sul-americanos. Avaliar as sequências de genomas de cepas de diferentes regiões é essencial para aumentar o conhecimento do pan-genoma dessa bactéria.

High level of infection by Toxoplasma gondii in pigs slaughtered in the city of São Luís, Maranhão / Alto nível de infecção por Toxoplasma gondii em suínos abatidos na cidade de São Luís, Maranhão

Abstract A serological, molecular and histopathological study was carried out in order to investigate occurrences of Toxoplasma gondii in pigs slaughtered with and without inspection service. Serum samples were collected from 60 pigs to detect anti-T. gondii antibody by indirect fluorescent antibody (IFAT). Tongue, masseter and diaphragm fragments were also collected for parasite DNA detection by means of the polymerase chain reaction (PCR) and histopathological analysis. The serological results showed that 77% (44/60) of the pigs were positive. Regarding PCR, 66.67% (40/60) were positive for T. gondii. Among the tissues evaluated, the diaphragm was the one with the highest frequency of positivity (40%; 24/60), followed by the masseter (38.33%; 23/60) and tongue (33.3%; 20/60). Histopathological changes were only observed in the diaphragm, which presented inflammatory infiltrates of lymphohistiocytic and neutrophilic types. These results not only show the potential threat of T. gondii to human health, but also demonstrate the dynamic epidemiological situation of toxoplasmosis in pigs in the city of São Luís, providing support for food security regarding pigs and for T. gondii control programs in Brazil.

Resumo Realizou-se um estudo sorológico, molecular e histopatológico com o objetivo de verificar a ocorrência de Toxoplasma gondii em suínos abatidos com e sem serviço de inspeção. Foram coletados soros de 60 suínos para a pesquisa de anticorpos anti-T. gondii pela reação de imunofluorescência indireta (RIFI). Também foram coletados fragmentos de língua, masseter e diafragma para a detecção do DNA do parasito por meio da reação em cadeia da polimerase (PCR) e análise histopatológica. A análise sorológica demonstrou que 77% (44/60) dos suínos apresentaram anticorpos anti-T. gondii. Com relação ao PCR, 66,67% (40/60) foram positivos para T. gondii. Dentre os tecidos avaliados, o diafragma foi o que obteve maior frequência de positividade (40%; 24/60), seguidos de masseter (38,33%; 23/60) e língua (33,3%; 20/60). Alterações histopatológicas foram observadas apenas no diafragma, que apresentou infiltrado inflamatório do tipo linfohistiocitário e neutrofílico. Esses resultados não evidenciam apenas a ameaça potencial de T. gondii à saúde humana, mas também demonstram a dinâmica situação epidemiológica da toxoplasmose em suínos na região da cidade de São Luís, fornecendo suporte para a segurança alimentar de suínos e programas de controle de T. gondii no país.

Detection of Trypanosoma vivax in tissues of experimentally infected goats: what is the role of adipose tissue in the life cycle of this protozoon? / Detecção de Trypanosoma vivax em tecidos de caprinos experimentalmente infectados: qual é o papel do tecido adiposo no ciclo de vida desse protozoário?

Abstract Trypanosomiasis, caused by Trypanosoma vivax, is responsible for great economic losses among livestock in Africa and South America. During the life cycle of these parasites, they may present different morphological, metabolic and physiological characteristics depending on the interactions that are encountered at each point of their life cycle. Although T. vivax is frequently reported in the circulation of its mammalian hosts, it has the ability to migrate to the tissues of these individuals. However, this characteristic is poorly understood. In this context, we aimed to investigate the presence of T. vivax and the changes caused in different tissues of experimentally infected goats. Despite the animals were not perfused before tissues collection, using different approaches, we demonstrated its presence in different samples, including in the adipose tissue and skin of infected animals. In addition, a mononuclear inflammatory reaction, mostly characterized by an infiltrate of lymphocytes, plasma cells and macrophages were observed. The results highlight the possibility that, like other trypanosomatids, T. vivax may use these tissues during its life cycle. Future studies aiming to elucidate the length of time for which T. vivax remains active in these sites, and whether it uses these sites as a refuge from trypanocidal drugs, and whether it is capable of recolonizing the blood circulation, are much needed.

Resumo A tripanossomíase, causada por Trypanosoma vivax, é responsável por grandes perdas econômicas na bovinocultura da África e da América do Sul. Durante seu ciclo de vida, o parasita pode apresentar diferentes características morfológicas, metabólicas e fisiológicas em função das interações que ele encontra em cada ponto do seu ciclo. Embora o T. vivax seja reportado, frequentemente, na circulação dos seus hospedeiros mamíferos, o protozoário tem a capacidade de migrar para os tecidos desses indivíduos. Entretanto, essa característica é pobremente conhecida. Neste contexto, o objetivo foi verificar a presença, assim como as alterações causadas pelo T. vivax nos diferentes tecidos de caprinos experimentalmente infectados. Apesar dos animais não terem sido perfundidos antes da coleta dos tecidos, utilizando-se diferentes abordagens, foi evidenciada a presença do T. vivax em diferentes amostras teciduais, incluindo no tecido adiposo e pele dos animais infectados. Além disso, foi observada reação inflamatória mononuclear, caracterizada majoritariamente por infiltrado de linfócitos, plasmócitos e macrófagos. Os resultados evidenciam a possibilidade de que, assim como outros tripanossomatídeos, T. vivax pode usar esses tecidos durante o seu ciclo de vida. São necessários futuros estudos, objetivando elucidar o período em que o T. vivax permanece ativo nesses sítios, se ele utiliza esses locais como refúgio das drogas tripanocidas, e se ele é capaz de recolonizar a circulação sanguínea.

High genetic diversity of Anaplasma marginale infecting dairy cattle in northeastern Brazil / Alta diversidade genética de Anaplasma marginale em bovinos de leite no nordeste do Brasil

Abstract Anaplasma marginale is an obligate intracellular Gram-negative bacterium found in ruminants' erythrocytes and is the etiological agent of bovine anaplasmosis. The bacterium's genetic diversity has been characterized based on sequences of major surface proteins (MSPs), such as MSP1α. The aim of the present study was to investigate the genetic diversity of A. marginale in cattle in the state of Maranhão, northeastern Brazil. To this end, 343 blood samples were harvested and subjected to iELISA assays using the recombinant surface protein MSP5. Out of 343 blood samples, 235 (68.5%) were randomly chosen and submitted to DNA extraction, qPCR and conventional PCR targeting the msp1α gene to determine amino acid sequences and classify the genotypes. The iELISA results showed 81.34% seropositivity (279/343), whereas qPCR revealed 224 positive samples (95.32%). Among these qPCR-positive samples, 67.4% (151/224) were also positive in the cPCR. Among the 50 obtained sequences, 21 strains had not been previously reported. Regarding the genotypes, H (26/50) and E (18/50) were identified most often, while genotypes F and C were only identified twice each and B and G once each. In conclusion, high prevalence and genetic diversity for A. marginale were observed in dairy cattle herds in the state of Maranhão.

Resumo Anaplasma marginale é uma bactéria Gram-negativa intracelular obrigatória de eritrócitos de ruminantes e responsável pela anaplasmose bovina. A diversidade genética de A. marginale tem sido caracterizada com base nas sequências das principais proteínas de superfície (MSPs), como a MSP1α. O objetivo deste estudo foi investigar a diversidade genética de A. marginale em bovinos no estado do Maranhão, Nordeste do Brasil. Dessa forma, 343 amostras de sangue foram submetidas ao ensaio iELISA, utilizando-se a proteína recombinante MSP5. Das 343 amostras de sangue, 235 (68,5%) foram escolhidas aleatoriamente e submetidas à extração de DNA, qPCR e PCR convencional para gene msp1α, para determinação das sequências de aminoácidos e classificação dos genótipos. Os resultados do iELISA mostraram 81,34% de soropositividade (279/343), enquanto qPCR revelou 224 amostras positivas (95,32%). Dentre estas na qPCR, 67,4% (151/224) mostraram-se positivas no PCR convencional. Das 50 sequências obtidas, 21 cepas não haviam sido relatadas anteriormente. Em relação aos genótipos, H (26/50) e E (18/50) foram os mais frequentes, enquanto os genótipos F e C foram identificados apenas duas vezes cada, e B e G uma vez cada. Em conclusão, alta prevalência e marcante diversidade genética de A. marginale foram observadas em rebanhos leiteiros no estado do Maranhão.

Genetic diversity of Hepatozoon spp. in rodents from Chile / Diversidade genética de Hepatozoon spp. em roedores do Chile

Abstract This study aimed to investigate the genetic diversity of Hepatozoon spp. in rodents from Valdivia, Chile. A total of 74 rodents (synanthropic n=38; wild n=36) were trapped in Valdivia. We performed conventional PCR assays for Apicomplexa organisms targeting two overlapping 18S rDNA gene fragments (600 bp and 900 bp) followed by sequencing of selected amplicons. Hepatozoon spp. occurrence was 82.43% (61/74). Twelve sequences obtained from the 600 bp and ten from the 900 bp 18S rDNA fragments were identified as Hepatozoon sp. Six sequences obtained from 18S rDNA-based overlapping PCR protocols were used for concatenated (1,400 bp) phylogenetic, haplotype and distance analyses. Hepatozoon spp. 18S rDNA concatenated sequences from the present study were detected in Oligoryzomys longicaudatus, Rattus norvegicus, Mus musculus, and Abrothrix longipilis grouped with Hepatozoon species earlier described in rodents and reptiles from Chile and Brazil. Nucleotide polymorphism of the six 18S rDNA sequences (1,400 bp) from this study, and other Chilean sequences from rodents and rodent's ticks, showed high diversity with a total of nine Chilean haplotypes. Three haplotypes from Valdivia were identified for the first time in this study, suggesting the circulation of novel haplotypes in rodents from southern Chile.

Resumo Este estudo teve como objetivo investigar a diversidade genética de Hepatozoon spp. em roedores de Valdivia, Chile. Um total de 74 roedores (sinantrópicos n=38; selvagens n=36) foram capturados. PCR convencional foi realizada para organismos Apicomplexa, visando dois fragmentos sobrepostos do gene 18S rDNA (600 bp e 900 bp), seguida pelo sequenciamento de amplicons selecionados. A ocorrência de Hepatozoon spp. foi de 82,43% (61/74). Doze sequências obtidas dos fragmentos de 18S rDNA de 600 pb e dez dos fragmentos de 18S rDNA de 900 pb foram identificadas como Hepatozoon sp. Seis sequências obtidas, a partir de protocolos de PCR sobrepostos, foram usadas para análises filogenéticas (1.400 bp), de haplótipos e de distância. Sequências concatenadas 18S rDNA do presente estudo foram detectadas em Oligoryzomys longicaudatus, Rattus norvegicus, Mus musculus e Abrothrix longipilis e agrupadas com Hepatozoon descrito em roedores e répteis do Chile e do Brasil. A análise de polimorfismos das seis sequências deste estudo, junto com outras sequências chilenas de roedores e carrapatos de roedores, mostrou alta diversidade com um total de nove haplótipos no Chile. Três haplótipos detectados em Valdivia foram identificados pela primeira vez neste estudo, sugerindo que novos haplótipos circulam em roedores do sul do Chile.