Anaplasmosis outbreaks in taurine and zebu cattle in Brazil: causes, treatments, and Anaplasma marginale genetic diversity.

This study aimed to investigate seven outbreaks of A. marginale infection in two regions of Brazil, affecting taurine, zebu, and crossbred cattle. We assessed the possible causes, treatment measures, and genetic diversity of A. marginale. These outbreaks occurred in two states (Goiás: outbreaks 1-7; Mato Grosso do Sul: outbreak 3), breeds (Holstein, Nellore, and crossbreed), age groups (beef cattle: 18-25 days old and 7-8 months; dairy cattle: 18-25 days old, 13-14 months, and cow after the first birth) and rearing systems (feedlot, pasture, pen in a wood shaving bedding system and compost bedded-pack barns). Metaphylactic or prophylactic treatments varied according to outbreak (imidocarb dipropionate: outbreaks 1-4 and 6; enrofloxacin: outbreaks 5 and 7; diminazene diaceturate: outbreak 5). In outbreaks 6 and 7, the packed cell volume was monitored. In all outbreaks, the practice of needle/syringe sharing was discontinued. For outbreaks 1-3, clinical signs and mortality (range, 4.8-13.3%) occurred 36-45 days after entry into the feedlot. In outbreak 4, A. marginale was diagnosed in 66.2% of the calves (bacteremia, 0-4.5%), with a mortality of 8.6%. Among nursing calves aged 60 days during outbreak 5, 53.8% were infected with A. marginale, with average bacteremia of 2.7% (range, 0-21.3%), and a mortality of 13.8%. In dairy heifers aged 14 months, raised in paddocks lacking vegetation cover and infested with R. microplus, then transitioned to a rotational grazing system also infested with R. microplus, the A. marginale bacteremia ranged from 3.2 to 6.7%, with a mortality of 20%. Before monitoring during outbreak 7, the mortality was 17.9%, but no further deaths were observed after monitoring initiation. In conclusion, possible causes triggering the outbreaks included primary tick infestation, needle/syringe sharing, and stress factors which may have affected the immunological statues of animals in the feedlots. Control measures performed in all outbreaks were effective. The partial msp4 gene sequences of A. marginale generated herein belonged to two haplotypes, but further research would be needed to investigate if this finding has any clinical significance.

One Health Approach on Ehrlichia canis: Serosurvey of Owners and Dogs, Molecular Detection in Ticks, and Associated Risk Factors in Tick-Infested Households of Southern Brazil.

Background: Ehrlichia canis has been the main hemopathogen affecting domestic dogs in Brazil. Even though tick-infested dogs may lead to household infestation and predispose human exposure and public health concern, no comprehensive study has surveyed humans, dogs, and environmental ticks altogether. Materials and Methods: Accordingly, the present study aimed to assess tick-infested households, identify tick species, perform serological (immunofluorescence assay) and molecular (PCR and q-PCR) detection of Ehrlichia in ticks, in the eighth biggest metropolitan area of Brazil. Results: Between 2007 and 2020, 233/5973 (3.9%) out of all complaints were from tick-infested households of 200 different addresses. Overall, 370/552 (67.0%) ticks were collected and identified as adult and 182/552 (33.0%) as immature forms of Rhipicephalus sanguineus s.l. complex; a single tick from one owner, a female tick of Amblyomma sculptum; and 395 ticks from dogs, 319/395 (80.8%) adult and 72/395 (18.2%) immature forms of Rhipicephalus spp., and 4/395 (1.01%) female Amblyomma aureolatum. Overall, 2/135 (1.5%) owners and 13/136 (9.6%) dogs were seropositive for E. canis. The DNA of Anaplasmataceae family was molecularly detected in 16/50 (32.0%) R. sanguineus s.l. As expected, the number of monthly tick infestation complaints were directly associated, and mean (p = 0.01), maximum (p = 0.011), and minimum (p = 0.008) temperature were statistically significant and had a low positive correlation (0.24, 0.23, and 0.24, respectively). In addition, complaints were highly associated to all socioeconomic variables (p < 0.001), with the exception of the presence of vacant lots. Conclusions: Despite low samplings and human negative results, areas with low-income with adequate temperature and urban agglomerations have been shown to be associated risks for tick infestations, predisposing tick-borne diseases. In conclusion, monitoring should always be conducted in such areas, including One Health approach with serosurvey of owners and dogs, along with identification and molecular screening of ticks.

Susceptibility of Amblyomma sculptum, Vector of Rickettsia rickettsii, Ticks from a National Park and an Experimental Farm to Different Synthetic Acaricides.

Amblyomma sculptum is a relevant tick species from a One Health perspective, playing an important role as a vector of Rickettsia rickettsii, the main agent of spotted fever rickettsiosis in Brazil. In this study, we evaluated the susceptibility of two A. sculptum populations from Goiás state (midwestern Brazil) to different acaricides. The first tick population (GYN strain) originated from an experimental farm, where the ticks are annually exposed to acaricides. The second (PNE strain) was collected in a national park (Emas National Park), where the ticks had not been exposed to acaricides. Immersion tests were conducted with 21-day-old laboratory-reared larvae and nymphs originating from adult ticks collected in the areas mentioned above. The chosen acaricides were two synthetic pyrethroids (cypermethrin and deltamethrin), one organophosphate (chlorfenvinphos), one formamidine (amitraz), and two combinations of pyrethroids and organophosphates (cypermethrin, chlorpyrifos and citronellal; cypermethrin, fenthion and chlorpyrifos). Mortality data were used to determine the lethal concentration (LC) values at which 50%, 90%, and 99% of the ticks died (LC50, LC90, and LC99, respectively), and resistance ratios (RR) were calculated based on the LC values. The RR revealed differences between the acaricide-exposed (GYN) and unexposed (PNE) tick strains. The PNE strain larvae and nymphs were susceptible to all the tested acaricides. The GYN strain larvae were tolerant to cypermethrin, whereas the nymphs were tolerant to deltamethrin, chlorfenvinphos, and the combination of cypermethrin, chlorpyrifos, and citronellal (2 < RR ≤ 10). The GYN strain nymphs were resistant to amitraz (RR > 10). This is the first report of A. sculptum nymphs with resistance to amitraz and tolerance to deltamethrin, chlorfenvinphos, and the combination of cypermethrin, chlorpyrifos, and citronellal.

Phylogenetic Inferences Based on Distinct Molecular Markers Confirm a Novel Babesia Species (Babesia goianiaensis nov. sp.) in Capybaras (Hydrochoerus hydrochaeris) and Associated Ticks.

Piroplasmids (order Piroplasmida) are a diverse group of tick-borne protozoa that may cause disease in animals and occasionally in humans. Novel Piroplasmida clades and species have been found in wild animals from Brazil based on the phylogenetic assessment of near-complete 18S rRNA, mitochondrial and heat-shock protein genes. For instance, a putative novel Babesia species has been detected in capybaras and Amblyomma ticks in three Brazilian states. The present work aimed to describe, using phylogenetic assessments based on distinct molecular markers, this novel Babesia species in capybaras and associated ticks (Amblyomma sculptum and Amblyomma dubitatum) sampled in Goiânia city, Goiás state, midwestern Brazil. While the phylogenetic analysis based on both near-complete 18S rRNA and hsp-70 genes positioned the sequences obtained from capybara blood samples into a new clade sister to the Babesia sensu stricto clade, the phylogenetic inference based on the COX-3 amino acid positioned the obtained sequences from capybara blood samples and A. sculptum ticks also into a clade sister to the Theileria sensu stricto clade, highlighting the inappropriateness of this marker inferring evolutionary relationships among piroplasmids. Pairwise distance analysis demonstrated that the divergence rates between the 18S rRNA sequences detected in capybaras and other Piroplasmida already described were very high and ranged from 9.4 to 12.9%. Genotype analysis based on the near-full 18S rRNA sequences of the Piroplasmida detected in capybaras and associated ticks demonstrated the occurrence of high genotype diversity at an intra-species level. In conclusion, phylogenetic analyses based on distinct molecular markers supported the description of Babesia goianiaensis nov. sp. in capybaras and associated Amblyomma ticks. Additionally, a novel phylogenetic clade, apart from the previously described ones, was described in the present study and contributed to untangling the complex evolutionary history of the Piroplasmida.

Detection of Rickettsia spp. in Animals and Ticks in Midwestern Brazil, Where Human Cases of Rickettsiosis Were Reported.

Brazilian spotted fever (BSF) is the most important tick-borne diseases affecting humans in Brazil. Cases of BSF have recently been reported in the Goiás state, midwestern Brazil. All cases have been confirmed by reference laboratories by seroconversion to Rickettsia rickettsii antigens. Because serological cross-reactions among different rickettsial species that belong to the spotted fever group (SFG) are common, the agent responsible for BSF cases in Goiás remains unknown. From March 2020 to April 2022, ticks and plasma were collected from dogs, horses and capybaras (Hydrochoerus hydrochaeris), and from the vegetation in an area where BSF cases have been reported and two areas under epidemiological surveillance in Goiás. Horses were infested by Amblyomma sculptum, Dermacentor nitens and Rhipicephalus microplus; dogs by Rhipicephalus sanguineus sensu lato (s.l.), Amblyomma ovale and A. sculptum, and capybaras by A. sculptum and Amblyomma dubitatum. Adults of A. sculptum, A. dubitatum, Amblyomma rotundatum and immature stages of A. sculptum and A. dubitatum, and Amblyomma spp. were collected from the vegetation. DNA of Rickettsia that did not belong to the SFG was detected in A. dubitatum, which was identified by DNA sequencing as Rickettsia bellii. Seroreactivity to SFG and Rickettsia bellii antigens was detected in 25.4% (42/165) of dogs, 22.7% (10/44) of horses and 41.2% (7/17) of capybaras, with higher titers for R. bellii in dogs and capybaras. The seropositivity of animals to SFG Rickettsia spp. antigens demonstrates the circulation of SFG rickettsiae in the region. Further research is needed to fully determine the agent responsible for rickettsiosis cases in this area.

First molecular detection of Borrelia theileri subclinical infection in a cow from Brazil.

Borrelia theileri is a relapsing fever group Borrelia that is transmitted to cattle by ticks of the genus Rhipicephalus. In this study, we describe the first molecular detection of B. theileri subclinical infection in a cow in Brazil. During the examination of stained blood smears of 10 cows from a farm with a recent history of fatal Trypanosoma vivax trypanosomiasis, spirochete-like structures were incidentally detected in one of the cows. The animal presented good body score, normal hematocrit and normal-colored ocular mucosa. Temperature, heart rate and respiratory rate were all normal. The animal was infested by ticks, which were morphologically identified as Rhipicephalus microplus. The diagnosis was confirmed by testing DNA extracted from a blood sample using a PCR targeting a ≈ 650 bp fragment of the flagellin B (flaB) gene of Borrelia spp. The partial flaB sequence obtained showed 99.83% similarity with B. theileri. Phylogenetically, the flaB partial sequence generated herein clustered with other B. theileri sequences, being separated from B. lonestari. This is the first molecular detection of B. theileri subclinical infection in a cow in Brazil. The possible implications of this finding are discussed.

Rickettsial Infection in Ticks from a National Park in the Cerrado Biome, Midwestern Brazil.

This study was carried out from February 2020 to September 2021 in Parque Nacional das Emas (PNE), a national park located in the Cerrado biome, midwestern Brazil, as well as in surrounding rural properties. Serum and tick samples were collected from dogs, terrestrial small mammals, and humans. Ticks were also collected from the environment. Dogs were infested with Rhipicephalus linnaei adults, whereas small mammals were infested by immature stages of Amblyomma spp., Amblyomma triste, Amblyomma dubitatum, and Amblyomma coelebs. Ticks collected from vegetation belonged to several species of the genus Amblyomma, including A. coelebs, A. dubitatum, Amblyomma naponense, Amblyomma sculptum, and A. triste. Two Rickettsia species were molecularly detected in ticks: Rickettsia parkeri in A. triste from the vegetation and a Rickettsia sp. (designated Rickettsia sp. strain PNE) in A. sculptum and A. triste collected from lowland tapirs (Tapirus terrestris). Based on short gltA gene fragments, this rickettsial organism showed 99.7-100% to Rickettsia tillamookensis. Seroreactivity to Rickettsia antigens was detected in 21.9% of dogs, 15.4% of small mammals, and 23.5% of humans. The present study reveals the richness of ticks and demonstrates the circulation of rickettsial agents in one of the largest conservation units in the Cerrado biome in Brazil. To our knowledge, this is the first report of a rickettsial phylogenetically related to R. tillamookensis in Brazil.

Epidemiology of Rickettsia spp. in Atlantic rainforest areas of island and seashore mainland, southern Brazil.

Non-fatal cases of rickettsial infection with different clinical features than the classic BSF (Brazilian Spotted Fever) have been reported in seashore areas of Paraná state, southern Brazil. In addition, Amblyomma ovale tick infected by Rickettsia parkeri strain Atlantic rainforest has been also described in this area. Accordingly, the aim of this study was to investigate the occurrence of anti-Rickettsia spp. antibodies in human and dog populations, and Rickettsia spp. infection in ticks from oceanic islands and seashore mainland cities of southern Brazil. Serum samples were collected from 328 persons and their 282 dogs from three islands and two seashore mainland cities. A total of 211 ticks were collected from dogs, identified as A. ovale and R. sanguineus. In overall, 40 of 328 (12.2%) human samples were seropositive for Rickettsia spp., including 21 of 190 (11.1%) on islands and 19 of 138 (13.7%) on seashore mainland, and 62 of 282 (22.0%) dog samples, including 31 of 153 (20.3%) on islands and 31 of 129 (24.0%) in seashore mainland areas. In overall, nine of 82 (11.0%) ticks were positive to real-time PCR assay targeting a fragment of the rickettsial gltA gene, including two of 64 (3.1%) Rickettsia sanguineus and seven of 18 (38.9%) A. ovale, of which four were infected with the R. parkeri strain Atlantic rainforest. Despite no association between risk factors and Rickettsia spp. seropositivity was found in human beings, access to natural areas (p = .011) and tick infestation (p = .004) was significantly associated to dog seropositivity. The serological and molecular findings herein have confirmed previous tick and clinical case reports and enlarged the geographical occurrence of A. ovale infected by R. parkeri strain Atlantic rainforest in oceanic islands and seashore mainland cities of Paraná State, indicating a new likely transmission area of this new rickettsial infection in human beings and dogs of southern Brazil.

Detection of an Undescribed Babesia sp. in Capybaras and Amblyomma Ticks in Central-Western Brazil.

Capybaras (Hydrochoerus hydrochaeris) are the largest rodents on Earth. While capybaras are hosts for various tick species, there is limited information regarding the tick-borne pathogens they can carry. We investigated the presence of piroplasmids and Ehrlichia spp. in capybaras and their associated ticks in two peri-urban areas in Goiás state, central-western Brazil. Blood samples collected from 23 capybaras were used to investigate the presence of piroplasmids and Ehrlichia spp. in stained-blood smears and by PCR. Ticks collected from the capybaras were identified morphologically and also tested using PCR for the same pathogens. A total of 955 ticks were collected, including 822 (86.1%) Amblyomma sculptum, 132 (13.8%) Amblyomma dubitatum, and one (0.1%) unidentified larva of Amblyomma sp. Neither the capybaras nor ticks were positive for Ehrlichia spp. However, a stained-blood smear examination revealed the presence of ring-stage and pyriform-shaped merozoites in the erythrocytes of one (4.4%) capybara. In the same way, 47.8% (11/23) and 19.9% (36/181) of blood samples and ticks, respectively, were positive for piroplasmids in the PCR. We successfully sequenced a partial 18S rRNA gene fragment of four samples (two capybaras, one A. sculptum, and one A. dubitatum), and the phylogenetic reconstruction disclosed that the organism reported in the present study clusters within the genus Babesia. Further research is required for a formal delineation of this species (designated as Babesia sp. strain Capybara) and to investigate the hypothesis of A. dubitatum and A. sculptum ticks being vectors.

Natural infection and molecular detection of Cytauxzoon felis in a free-ranging Puma concolor in the state of Goiás, Brazil / Infecção natural e detecção molecular de Cytauxzoon felis em Puma concolor de vida livre no estado de Goiás, Brasil

The puma (Puma concolor Linnaeus, 1771), the most widely distributed felid species in the Americas, can be found in all Brazilian biomes. Nevertheless, few studies have focused on hemoparasites in this species. Cytauxzoon felis, a hemoparasite that can infect domestic cats, has also been described in wild felids in Brazil. To the best of our knowledge, this study is the first to diagnose the natural infection and molecular detection of C. felis in a P. concolor in the state of Goiás. This animal presented non-regenerative anemia and inclusion suggestive of piroplasmids within red blood cells. The amplified 551 bp fragment of partial Piroplasmida 18S rRNA gene sequence was 100% identical to corresponding sequences of C. felis available in GenBank. No specific treatment for cytauxzoonosis was administered, and after rehabilitation, the animal was reintroduced into the wild. This finding provides some evidence that P. concolor may act as a natural host of the parasite. The epidemiology, vector and pathogenicity of this hemoparasite in wild and domestic cats in Brazil deserves further investigation.

O puma (Puma concolor Linnaeus, 1771) tem a maior distribuição entre os felídeos das Américas e é encontrado em todos os biomas do Brasil. No entanto, poucos estudos têm se concentrado nos hemoparasitos nesta espécie. Cytauxzoon felis, um hemoparasito que pode infectar gatos domésticos, também foi descrito em felídeos selvagens no Brasil. A saber, este estudo é o primeiro diagnóstico de infecção natural e detecção molecular de C. felis em um P. concolor do estado de Goiás. Este animal apresentou anemia arregenerativa e inclusão de piroplasmídeos nos glóbulos vermelhos. A amplificação do fragmento de 551 pb da sequência parcial do gene Piroplasmorida 18S rRNA foi 100% idêntica a sequências correspondentes de C. felis disponíveis no GenBank. Nenhum tratamento específico para citauxzoonose foi administrado e, após a reabilitação, o animal foi reintroduzido na natureza. Essa descoberta fornece algumas evidências de que P. concolor pode atuar como um hospedeiro natural do parasito. A epidemiologia, vetor e patogenicidade deste hemoparasito em gatos selvagens e domésticos no Brasil merecem uma investigação mais aprofundada.

Seroprevalence and hematological abnormalities associated with Ehrlichia canis in dogs referred to a veterinary teaching hospital in central-western Brazil / Soroprevalência e alterações hematológicas associadas com Ehrlichia canis em cães encaminhados a um hospital veterinário-escola no centro-oeste do Brasil

Ticks are significant parasites of dogs in the tropics, where tick-borne pathogens are highly prevalent, especially in areas where tick control measures are frequently neglected. This study investigated the seroprevalence and hematological abnormalities associated with Ehrlichia canis in dogs referred to a veterinary teaching hospital in Central-western Brazil. Out of 264 dogs tested for anti-Ehrlichia canis antibodies by an indirect immunofluorescence assay (IFA), 59.1% (156/264) were positive. Seropositivity was significantly associated to anemia and thrombocytopenia, alone or in combination, and to leukopenia. Conversely, there were no differences in terms of seroprevalence according to sex, breed and age. This study demonstrated that dogs referred to a veterinary teaching hospital in Central-western Brazil are highly exposed to E. canis and that seropositive dogs are more likely to present hematological abnormalities, particularly anemia, thrombocytopenia and leukopenia. To our knowledge, this is the first study on detection of anti-E. canis antibodies by means of IFA among dogs in the state of Goiás. These findings highlighted the need for increasing awareness among dog owners regarding tick control measures in Central-western Brazil, ultimately to reduce the risk of exposure to E. canis and other tick-borne pathogens.

Carrapatos são importantes parasitos de cães nos trópicos, onde patógenos transmitidos por carrapatos são altamente prevalentes, especialmente em áreas onde as medidas de controle de carrapatos são frequentemente negligenciadas. O estudo investigou a soroprevalência e as anormalidades hematológicas associadas à Ehrlichia canis em cães encaminhados para um hospital veterinário-escola no Centro-oeste do Brasil. Dos 264 cães testados para anticorpos anti-Ehrlichia canis por meio da reação de imunofluorescência indireta (RIFI), 59.1% (156/264) foram positivos. A soropositividade foi associada significativamente à anemia e trombocitopenia, isoladamente ou em combinação, e à leucopenia. Por outro lado, não houve diferenças quanto à soroprevalência segundo sexo, raça e idade. Este estudo demonstrou que cães encaminhados a um hospital veterinário-escola na região Centro-oeste do Brasil são altamente expostos à E. canis, e que cães soropositivos têm maior probabilidade de apresentar alterações hematológicas, principalmente anemia, trombocitopenia e leucopenia. Para o nosso conhecimento, este é o primeiro estudo sobre a detecção de anticorpos anti-E. canis por meio da RIFI em cães do estado de Goiás. Essas descobertas destacam a necessidade de aumentar a conscientização entre os proprietários de cães em relação às medidas de controle do carrapato no Centro-oeste do Brasil, em última análise, para reduzir o risco de exposição ao E. canis e outros patógenos transmitidos por carrapatos.

Seasonal dynamics of Amblyomma sculptum in two areas of the Cerrado biome midwestern Brazil, where human cases of rickettsiosis have been reported.

Amblyomma sculptum is a common human-biting tick in Brazil, where it plays an important role as a vector of Rickettsia rickettsii, the agent of the Brazilian spotted fever. Herein, we studied the seasonal dynamics of A. sculptum in an urban area of the Cerrado biome in midwestern Brazil, where human rickettsiosis is endemic. Ticks were collected in two sites located within the campus of Federal University of Goiás. The collections were done by dragging, flagging and visual search. In total, 117,685 ticks were collected, including 100,627 Amblyomma spp. larvae, 10,055 nymphs and 6977 adults of A. sculptum, and one nymph and 25 adults of Amblyomma dubitatum. The highest peak of larvae occurred in June 2018 and in July 2019, whereas nymphs peaked in July 2018 and September 2019. Adults reached their highest numbers in March 2018 and November 2019. These data suggest that A. sculptum develops one generation per year in this urban area of the Cerrado biome in midwestern Brazil. Interestingly, the peak of nymphs occurred during the same period of all confirmed cases of rickettsiosis in Goiás, suggesting a possible relationship between the seasonal dynamics of this tick stage and rickettsiosis transmission in this state.