Molecular survey of Piroplasmida, Hepatozoon spp. and Anaplasmataceae in anemic and thrombocytopenic dogs from Uruguay.

Canine tick-borne diseases, such as babesiosis, rangeliosis, hepatozoonosis, anaplasmosis and ehrlichiosis, are of veterinarian relevance, causing mild or severe clinical cases that can lead to the death of the dog. The aim of this study was detecting tick-borne protozoan and rickettsial infections in dogs with anemia and/or thrombocytopenia in Uruguay. A total of 803 domestic dogs were evaluated, and 10% were found positive (detected by PCR) at least for one hemoparasite. Sequence analysis confirmed the presence of four hemoprotozoan species: Rangelia vitalii, Babesia vogeli, Hepatozoon canis and Hepatozoon americanum, and the rickettsial Anaplasma platys. The most detected hemoparasite was R. vitalii, followed by H. canis and A. platys. This is the first report of B. vogeli in Uruguay and the second report of H. americanum in dogs from South America. The results highlight the importance for veterinarians to include hemoparasitic diseases in their differential diagnosis of agents causing anemia and thrombocytopenia.

Molecular of Anaplasma marginale Theiler (Rickettsiales: Anaplasmataceae) in horseflies (Diptera: Tabanidae) in Uruguay.

Anaplasma marginale is transmitted biologically by infected ticks or mechanically by biting flies and contaminated fomites. In tick-free areas, such as southern Uruguay, horseflies could be the principal vectors of this pathogen for bovines, causing anaplasmosis. The objective of this work was to detect the presence of A. marginale by MSP-5 PCR and Sanger sequencing in the most prevalent species of horseflies obtained using different collection methods in Colonia, Tacuarembó and Paysandú, Uruguay. Eight horsefly species were tested (Dasybasis missionum, Poeciloderas lindneri, Tabanus campestris, T. claripennis, T. fuscofasciatus, T. platensis, T. tacuaremboensis and T. triangulum); four species were found to be positive for A. marginale, with D. missionum and P. lindneri having the most frequent infections, while only one individual each of T. fuscofasciatus and T. tacuaremboensis was positive. Both D. missionum and P. lindneri were positive for A. marginale in tick-free areas, and the implications are discussed in this report.

Characterization of strains of Anaplasma marginale from clinical cases in bovine using major surface protein 1a in Uruguay.

The major surface protein 1a (MSP1a) gene has been used to characterize Anaplasma marginale genetic diversity. This pathogen causes significant productivity and economic losses to the cattle industry. The objective of the present study was to report the first characterization of A. marginale genetic diversity in Uruguay based on MSP1a genotypes and their putative relationship with Rhipicephalus microplus. This cross-sectional study was conducted between 2016 and 2020. The study included whole blood samples from clinical cases of bovine anaplasmosis obtained from 30 outbreaks located in six Uruguay territorial departments. Diagnosis was performed using Giemsa-stained smears and confirmed by nested Polymerase Chance Reaction (nPCR) targeting the A. marginale major surface protein 5 gene. The genetic diversity of A. marginale strains was characterized by analyzing the microsatellite and tandem repeats of MSP1a. Based on the microsatellite structure, four genotypes were identified. Genotype E was the most prevalent. Analysis of MSP1a tandem repeats showed 28 different strains from the combination of 31 repeats, with τ-10-15 and α-ß-ß-ß-Γ being the most common. Repeats Γ, ß, α, and γ were associated with the absence of R. microplus with statistical significance (p < 0.05). Molecular observations showed that 46.7% of the strains identified in our samples lacked the ability to bind to tick cells; therefore, they were probably transmitted by other vectors. Strain genetic diversity provides valuable information for understanding the epidemiological behavior of A. marginale and could contribute to the development of effective vaccines for the control of this disease.

Description of bovine babesiosis and anaplasmosis outbreaks in northern Uruguay between 2016 and 2018.

Bovine babesiosis and anaplasmosis cause important economic losses in livestock production. In Uruguay, the main aetiological agents of bovine babesiosis and anaplasmosis are Babesia bovis, B. bigemina and Anaplasma marginale. The aim of this work was to describe the outbreaks of bovine babesiosis and anaplasmosis in northern Uruguay between 2016 and 2018. Convenience sampling was carried out. We worked with blood and organ samples from bovines with clinical signs and autopsy findings compatible with babesiosis and anaplasmosis. A total of 140 presumptive outbreaks were studied. Epidemiological information such as place, date of occurrence, age, number of sick and dead animals, clinical signs, autopsy findings, the presence of ticks and health management that involved injectables were registered. The diagnoses were carried out by blood and organ smears stained with Giemsa and confirmed by multiplex PCR. There were 83 (59.2%) positive outbreaks, comprising 35 (42.2%) A. marginale, 19 (22.9%) B. bigemina, 18 (21.7%) B. bovis and 11 (13.2%) mixed infections (Babesia spp. + A. marginale). Cows were the most commonly affected category. The clinical signs and autopsy findings with a significant association (p ≤ 0.05) were anaemia, pale mucous membranes, fever, jaundice, ataxia and aggressiveness, splenomegaly, and orange discolouration of the liver. Babesiosis had a seasonal occurrence, mainly in autumn, while anaplasmosis cases were recorded throughout the year. The use of injectable agents was associated with A. marginale transmission. This work contributes updated information about epidemiological and clinical patterns of bovine babesiosis and anaplasmosis in northern Uruguay, which is important for implementing preventive measures and control.

Validation of a multiplex PCR assay to detect Babesia spp. and Anaplasma marginale in cattle in Uruguay in the absence of a gold standard test.

Detection of bovine Babesia spp. and Anaplasma marginale is based on the reading of Giemsa-stained blood or organ smears, which can have low sensitivity. Our aim was to improve the detection of bovine Babesia spp. and A. marginale by validating a multiplex PCR (mPCR). We used 466 samples of blood and/or organs of animals with signs and presumptive autopsy findings of babesiosis or anaplasmosis. The primers in our mPCR amplified the rap-1a gene region of Babesia bovis and B. bigemina, and the msp-5 region of A. marginale. We used a Bayesian model with a non-informative priori distribution for the prevalence estimate and informative priori distribution for estimation of sensitivity and specificity. The sensitivity and specificity for smear detection of Babesia spp. were 68.6% and 99.1%, and for A. marginale 85.6% and 98.8%, respectively. Sensitivity and specificity for mPCR detection for Babesia spp. were 94.2% and 97.1%, and for A. marginale 95.2% and 92.7%, respectively. Our mPCR had good accuracy in detecting Babesia spp. and A. marginale, and would be a reliable test for veterinarians to choose the correct treatment for each agent.

Detection of a putative novel genotype of Anaplasma in gray-brocket deer (Mazama gouazoubira) from Uruguay.

Anaplasmataceae includes the genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachia, comprising a group of obligate intracellular bacteria. The genus Anaplasma has pathogenic species transmitted by ticks of veterinary and human health importance. Wild ungulates such as deer represent important reservoirs and amplifiers of Anaplasmataceae. The interaction between deer and domestic ruminants represents a serious problem due to the transmission of these pathogens through their ectoparasites. In the present study, we investigated the presence of Anaplasmataceae organisms in blood, tissues and tick samples of a gray-brocket deer (Mazama gouazoubira). The specimen was found dead in a farm in northeast Uruguay. PCRs targeting partial regions of 16S rRNA and groESL genes were carried out for Anaplasmataceae DNA detection. Moreover, several ectoparasites were identified: the chewing louse Tricholipeurus albimarginatus, the Neotropical deer louse fly Lipoptena mazamae, and the ticks Haemaphysalis juxtakochi and Rhipicephalus microplus. A consensus sequence of 1274 bp of 16S rRNA was generated for Anaplasma sp. from the M. gouazoubira blood sample. All ticks analysed by PCR assays were negative. No band was detected in any of the samples after PCR targeting groESL gene. Phylogenetic analysis using 16S rRNA partial gene sequences, clustered the putative novel genotype sequence obtained in this study, named Anaplasma sp. genotype Mazama-Uruguay, along with Anaplasma sp. detected in Mazama sp., Mazama americana and Mazama bororo, all deer species from Brazil. Furthermore, this cluster showed to be closely related to Anaplasma bovis sequences obtained from various ruminants and other mammals from several parts of the world. The pathogenicity as well as its infecting potential to other cervids or domestic ruminants is currently unknown. Further studies should be performed in order to characterize this novel species, especially targeting other genes.

Phylogenetic analysis of Hepatozoon spp. (Apicomplexa: Hepatozoidae) infecting Philodryas patagoniensis (Serpentes: Dipsadidae) in Uruguay.

The purpose of this study was to perform a phylogenetic analysis of Hepatozoon spp. infecting Philodryas patagoniensis in Uruguay. Twenty-five road-killed specimens of P. patagoniensis from ten departments were obtained. Samples of blood and/or heart tissue were taken. Polymerase chain reaction (PCR) assay was carried out amplifying a specific target region of the 18S rRNA gene of Hepatozoon spp. Eighteen out of twenty-five samples were positive to Hepatozoon spp., which gave an overall prevalence of 72%. Phylogenetic analyses with the obtained sequences were carried out to determine the relationship with closely related species found in the region. The results revealed that samples were split into two clades with a high bootstrap support. Clade I was formed with Hepatozoon spp. sequences obtained in this study from P. patagoniensis, Hepatozoon cuestensis from Crotalus durissus terrificus and Hepatozoon musa from Philodryas nattereri, and Hepatozoon spp. retrieved from Cerdocyon thous, Hemidactylus mabouia, and Phyllopezus pollicaris from Brazil, respectively. Clade II was grouped with Hepatozoon cevapii and Hepatozoon massardii, both species described for C. d. terrificus from Brazil. This is the first report of Hepatozoon spp. in snakes from Uruguay.

Probability of Rhipicephalus microplus introduction into farms by cattle movement using a Bayesian Belief Network.

Attempts to eliminate Rhipicephalus microplus from Uruguay have been unsuccessful, and, currently, the country is divided into two areas: a tick-free area and a tick-infested area. In the tick-infested area, different farms face different situations. Some farms are in regions where, due to environmental conditions or a lack of infrastructure, it is difficult to eliminate R. microplus, and the only option is to control it. In contrast, other farms can attempt complete removal. Before deciding whether a farmer should attempt to eliminate R. microplus, the probability of reintroduction must be evaluated. The objective of this study was to develop a probabilistic model based on a Bayesian Belief Network (BBN) to assess the likelihood of a farm becoming infested with R. microplus via the introduction of tick-infested cattle. Only the tick-infested area was considered in the development of this model. Nine variables related to environmental conditions and biosecurity measures, with a focus on cattle movement, were considered. Three different sources of data were used to populate the BBN model: data from the literature; a representative national survey from 2016; and a survey developed to identify biosecurity practices on farms. Model sensitivity and specificity were assessed, and an overall accuracy of 92% was obtained. The model was applied to 33 farms located in the tick-infested area. For one farm, the probability of introduction of R. microplus was 1%; for three farms, the probability was between 21% and 34%; for seven farms, it was between 66% and 76%; and for 22 farms, the probability was greater than 83%. This model was useful for estimating the probability of the introduction of R. microplus into farms, making it possible to assess the impact that the evaluated biosecurity measures have on the probability of introduction and, thus, guiding more objective decision making about the control or elimination of R. microplus from farms.