Molecular detection of Ehrlichia spp., Anaplasma spp., and Bartonella spp. in dogs treated at a veterinary teaching clinic in Peru.

In recent years, vector-borne diseases have become widespread throughout the world and affect the health of humans and domestic animals. These diseases spread to areas where their primary vectors, fleas and ticks, thrive, particularly in tropical and subtropical climate regions, providing ideal conditions for their proliferation. The growing closeness between people and their pets increases the likelihood of bites from these ectoparasites, which represents a latent zoonotic risk. Therefore, the objective of the study was to determine the presence of Anaplasma spp., Ehrlichia spp., and Bartonella spp. in dogs treated at the Small Animal Clinic of the Faculty of Veterinary Medicine of the Universidad Nacional Mayor de San Marcos, located in Lima, Peru. Blood samples from 214 dogs were molecularly analyzed for hemopathogen detection. The results revealed prevalences of 2.6 % (6/214) for Anaplasma platys, 5.14 % (11/214) for Ehrlichia canis, and 0.46 % (1/214) for Bartonella rochalimae. No statistically significant relationship was found between the animal infection and the age, sex, breed, presence of fleas (Ctenocephalides felis) and ticks (Rhipicephalus sanguineus), and locality. This study reported molecularly for the first time the presence of A. platys, E. canis, and B. rochalimae in dogs from Lima city, and demonstrates the occurrence of zoonotic pathogens in pets treated at the veterinary clinic.

Development and evaluation of a TaqMan® real-time PCR assay for species-specific detection of Ehrlichia canis.

The aim of this work was to develop a real-time PCR assay with a TaqMan® probe that detects a species-specific part of the 16S rDNA gene of Ehrlichia canis. Canine blood samples (n = 207), collected and tested by a conventional PCR assay within a study conducted by De Salvo et al., were simultaneously analyzed with the novel designed real-time PCR, and the results of both assays were compared. The agreement between the two methods was 97.6 % with a kappa value of 0.92186. Hereby, the standard error was 0.034416 and the 95 % confidence interval from 0.8544 to 0.98931. While the conventional PCR assay showed false negative results (2.42 %; 5/207), the real-time PCR assays showed a specificity of 100 %. The results of the current study showed that the developed assay presents sensitivity and specificity for the detection of E. canis in blood samples, adding a new tool for the diagnosis of this pathogen.

Evaluating the circulation of Ehrlichia canis and Rickettsia spp. in domestic dogs from a semiarid region in Brazil.

Tick-borne diseases are important for animal and human health, because they can cause death if not diagnosed and treated early. Canine monocytic ehrlichiosis (CME) can cause high morbidity in dog populations. Rocky Mountain Spotted Fever (RMSF) is among the most virulent infectious in humans; dogs are also susceptible to infection. The aims of this study were to evaluate the presence of Ehrlichia canis and Rickettsia spp. infections in domestic dogs, and to identify tick species parasitizing dogs among urban areas of two municipalities (Sobral and Alcântaras) in the Ceará State, Northeastern Brazil. A total of 208 domiciled dogs was sampled. After clinical evaluation, blood samples and ticks were collected and submitted to Real-Time Polymerase Chain Reaction (RT-PCR) targeting E. canis DNA. Serum samples were screened by Indirect Immunofluorescence Assays (IFA) for antibodies against different strains of Rickettsia spp. previously recognized in Brazil. The results of this study indicate the molecular detection of E. canis in the state of Ceará, Brazil, where the proportion of canine infection in Sobral (9.9%) was higher than in Alcântaras (5.6%). Rhipicephalus sanguineus sensu lato was the prevalent tick species infesting the dogs in both municipalities (43.5 and 53.3%, respectively). Our serological results indicate that dogs of the study area were at low risk of exposure to these tick-borne Rickettsia spp. of the spotted fever group. Our study offers epidemiological data of these diseases to better understanding Rickettsiales epidemic and enzootic cycles in the Brazilian semiarid region, improving prevention and control measures.

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.

Microfluidic PCR and network analysis reveals complex tick-borne pathogen interactions in the tropics.

BACKGROUND: Ixodid ticks, particularly Rhipicephalus sanguineus s.l., are important vectors of various disease-causing agents in dogs and humans in Cuba. However, our understading of interactions among tick-borne pathogens (TBPs) in infected dogs or the vector R. sanguineus s.l. remains limited. This study integrates microfluidic-based high-throughput real-time PCR data, Yule's Q statistic, and network analysis to elucidate pathogen-pathogen interactions in dogs and ticks in tropical western Cuba. METHODS: A cross-sectional study involving 46 client-owned dogs was conducted. Blood samples were collected from these dogs, and ticks infesting the same dogs were morphologically and molecularly identified. Nucleic acids were extracted from both canine blood and tick samples. Microfluidic-based high-throughput real-time PCR was employed to detect 25 bacterial species, 10 parasite species, 6 bacterial genera, and 4 parasite taxa, as well as to confirm the identity of the collected ticks. Validation was performed through end-point PCR assays and DNA sequencing analysis. Yule's Q statistic and network analysis were used to analyse the associations between different TBP species based on binary presence-absence data. RESULTS: The study revealed a high prevalence of TBPs in both dogs and R. sanguineus s.l., the only tick species found on the dogs. Hepatozoon canis and Ehrlichia canis were among the most common pathogens detected. Co-infections were observed, notably between E. canis and H. canis. Significant correlations were found between the presence of Anaplasma platys and H. canis in both dogs and ticks. A complex co-occurrence network among haemoparasite species was identified, highlighting potential facilitative and inhibitory roles. Notably, H. canis was found as a highly interconnected node, exhibiting significant positive associations with various taxa, including A. platys, and E. canis, suggesting facilitative interactions among these pathogens. Phylogenetic analysis showed genetic diversity in the detected TBPs. CONCLUSIONS: Overall, this research enhances our understanding of TBPs in Cuba, providing insights into their prevalence, associations, and genetic diversity, with implications for disease surveillance and management.

Molecular Evidence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in Ticks (Ixodida: Ixodidae) Associated with Dogs (Carnivora: Canidae) from Nuevo Leon, Mexico.

Background: Ehrlichia canis is transmitted by ticks causing Canine monocytic ehrlichiosis, which is considered one of the most critical tickborne pathogens. Materials and Methods: This study aimed to identify by PCR technique E. canis in ticks associated with dogs from urban and rural homes in Nuevo Leon, Mexico. The study was conducted at 13 localities in eight municipalities from 2012 to 2021. Results: A total of 1873 ticks of three species were captured: Amblyomma tenellum, Dermacentor variabilis, and Rhipicephalus sanguineus s.l. The overall infection rate of E. canis in ticks was 59.12% (149/252). Of the 15 sequences, three haplotypes were identified. Conclusion: The urban transmission cycle of canine ehrlichiosis is demonstrated, where the potential vector is the tick R. sanguineus s.l.

Clinical and pathological factors associated with Ehrlichia canis in companion dogs.

INTRODUCTION: Canine monocytic ehrlichiosis (CME) is a disease caused by the Gram-negative bacteria Ehrlichia canis, a bacterium that affects domestic dogs but can also infect humans. The diagnosis implies a challenge due to its diversity in clinical manifestations. METHODOLOGY: The frequency of E. canis infection, risk factors, and clinical-pathological parameters associated with seropositivity were calculated with the PROC FREQ TABLES and PROC LOGISTIC procedures of the SAS statistical software. RESULTS: The study showed a seroprevalence of 26.62% (156/586). Association between seropositivity and risk factors was found. The age and the presence of ticks including clinical signs such as anorexia, seizures, cough, petechiae, epistaxis, and hematochezia, as well as multiple blood and biochemical alterations were analyzed. The logistic regression analysis showed a high predictive power (c = 0.98) for CME for thrombocytopenia, leukopenia, and anemia. CONCLUSIONS: The high prevalence of E. canis in endemic areas makes its diagnosis difficult. Thus, clinical signs must be considered, along with blood and biochemical alterations, as a possible predictor of the disease.

Molecular Detection of Tick-Borne Pathogens in Dogs from Indigenous Communities, Amazon, Brazil.

Background: There are few reports of tick-borne pathogens infecting dogs living in indigenous communities of Brazil. Herein, we aimed to molecularly detect vector-borne pathogens in dogs from two indigenous communities in the Brazilian Amazon. Materials and Methods: We surveyed 327 dogs raised in Amazon region at 2 distinct indigenous ethnicities for the molecular detection of tick-borne pathogens (114 from Tapirapé and 213 from Karajá indigenous ethnicity). Whole blood samples were subjected to PCR and sequencing for Ehrlichia, Babesia, and Hepatozoon. Univariate and multivariate analyses were used to investigate the factors affecting the pathogen infection patterns in dogs. Results: Among the 327 blood samples, 40 were positive for Ehrlichia canis (12.2%), 2 for Anaplasma platys (0.61%), and 204 were positive for Hepatozoon canis (66.5%). Binary Logistic Regression showed association between E. canis infection and ethnicity (p = 0.010) and tick attachment (p = 0.041). Karajá dogs were 3.4 times (95% CI 1.3-8.5) more likely to be positive for E. canis than Tapirapé dogs. Dogs with ticks were 2.5 times more likely (95% CI 1.0-7.6) to be positive for E. canis than dogs without ticks. Conclusions: Our survey expands the knowledge regarding the presence of vector-borne pathogens in dogs from indigenous communities in the Amazon region.

Morphological and molecular identification of the brown dog tick in Mexico.

Ticks of the Rhipicephalus sanguineus complex are known as the brown dog ticks. This complex groups at least 12 species of ticks that are distributed worldwide. On the American continents, R. sanguineus sensu stricto (s.s.), is distributed in temperate areas, while Rhipicephalus sanguineus sensu lato (s.l.), also called "tropical lineage" is distributed in tropical regions. Previous analyses of brown dog ticks from Mexico have identified the so-called tropical lineage and the country generally has a climate more favorable for these ticks (> 20o C in average). In addition, some pathogens thought to be transmitted by this lineage (such as Ehrlichia canis, and Rickettsia rickettsii) are prevalent in Mexico. Herein we aim to contribute to the study of brown dog ticks by providing morphological identification and molecular analysis of mt 12S rDNA and 16S rDNA sequences from ticks collected from 12 states in Mexico. Our results indicate that the tropical lineage of R. sanguineus s.l., recently redescribed as R. linnaei is widely distributed in Mexico.

Correlation between canine biochemical analytes and TRP36 ELISA seropositivity for Ehrlichia canis in Brazil.

BACKGROUND: Synthetic peptides of tandem repeat proteins (TRPs) have been employed in the serologic analysis of canine monocytic ehrlichiosis (CME) and used in epidemiological studies in Brazil. Based on molecular studies of TRPs, different genotypes of Ehrlichia canis have been described, but data on their pathogenicity remain unknown. OBJECTIVES: To correlate hepatic, renal, and muscular alterations in relation to different genotypes of E. canis in naturally exposed dogs using enzyme-linked immunosorbent assay (ELISA) with TRP19 and TRP36 synthetic protein antigens. METHODS: Two hundred serum samples were subjected to ELISA with the antigens of TRP19 and three genotypes (US, Br, and CR) of TRP36 of E. canis circulating in Brazil. Positive sera were evaluated through eight biochemical parameters, and the results were evaluated by principal component analysis and canonical correlation. RESULTS: ELISA revealed that 47 (23.5%) serum samples reacted to the BrTRP36 peptide, 36 (18%) reacted to the TRP19 peptide, and 8 (4%) reacted to the USTRP36 and CRTRP36 peptides separately. The most frequent biochemical alterations observed were for CK (59.4%), ALB (31.8%), GLO (28.9%), TP (28.9%), ALP (26%), urea (24.6%), creatinine (14.4%), and ALT (14.4%). The most prominent diagnostic method in canonical correlation analysis was BrTRP36, followed by TRP19, which correlated with hyperglobulinemia and hypoalbuminemia. CONCLUSIONS: Antibodies that reacted against the Brazilian genotype of E. canis correlated positively with hyperglobulinemia and increases in serum urea and creatinine. According to our results, the Brazilian genotype of E. canis is related to the chronic phase of CME.

Pleural effusion-related Nocardia otitidiscaviarum, Anaplasma platys and Ehrlichia canis coinfection in a dog.

The coinfections by some microorganisms have been related to severe diseases in humans and animals, where immunosuppressive agents favor opportunistic behavior of other pathogens. A 4-month-old, female mixed-breed dog with a two-week history of inappetence, prostration, emaciation, and respiratory distress was admitted at a veterinary hospital in Brazil. Tachycardia, pale mucous membranes, severe respiratory distress, and a large number of ticks (Rhipicephalus sanguineus s.l.) in different body regions were observed at clinical examination. Hematological examination of dog showed leukocytosis, neutrophilia, mild anemia, and thrombocytopenia, whereas unremarkable values in biochemical tests. Thoracic radiography revealed a pleural effusion image. Blood and the pleural fluid (purulent aspect) samples were subjected to qPCR (16S rRNA and dsb genes) and sequencing, which identified Ehrlichia canis and Anaplasma platys coinfection. An aggregate of coccoid-to-branching or long filamentous microorganisms, surrounded by pyogranulomatous inflammatory reaction was seen at the cytology of the pleural fluid. Bacteriological culture of pleural effusion showed colonies compatible with the genus Nocardia, which revealed gram-positive filamentous organisms with a tendency of fragmentation and were identified as Nocardia otitidiscaviarum in mass spectrometry (MALDI-TOF MS). Therapy of N. otitidiscaviarum isolate using levofloxacin (supported by a previous in vitro susceptibility testing) and doxycycline for E. canis and A. platys resulted in complete resolution of the clinical picture. Here, we report for the first time a triple coinfection by Nocardia otitidiscaviarum, A. platys, and E. canis in a dog with pleural effusion, where debilitating or immunosuppressive conditions induced by A. platys and E. canis coinfection probably contributed to the opportunistic behavior of N. otitidiscaviarum.

Canine Parvovirus 2 in Free-Living Wild Mammals from Southern Brazil.

Pathogens from domestic canines represent a significant and constant threat to wildlife. This study looked for four common canine pathogens, Babesia vogeli, Ehrlichia canis, Leishmania infantum, and canine parvovirus 2 (CPV-2) in mammals from the Pampa Biome, southern Brazil. Animals killed by vehicular trauma on a road traversing this biome were evaluated over a 1-yr period. Tissues collected from 31 wild mammals and six dogs were further analyzed by specific real-time PCR assays for each pathogen. Babesia vogeli and L. infantum were not detected in any investigated animal. Ehrlichia canis was detected in one dog and CPV-2 in nine animals: four dogs, three white-eared opossums (Didelphis albiventris), one pampas fox (Lycalopex gymnocercus), and one brown rat (Rattus norvegicus). These results demonstrate the occurrence of important carnivore pathogens (E. canis and CPV-2) in domestic dogs and wild mammals from the Pampa Biome in southern Brazil.

Molecular detection and genetic characterization of Ehrlichia canis and Ehrlichia sp. in neotropical primates from Brazil.

The Anaplasmataceae family includes obligate, arthropod-transmitted intracellular bacteria that can be zoonotic and potentially fatal. Studies focusing on the interaction between neotropical primates and the agents of this family are scarce. The present study aimed to identify agents of the Anaplasmataceae family in the whole blood of free-living and captive neotropical primates in the State of Mato Grosso, Central-West Brazil. Thirty-eight samples of six nonhuman primate (NHP) species were collected in seven municipalities and analysed through polymerase chain reaction (PCR), nucleotide sequencing, and phylogenetic analysis of the dsb, groEL, 16S rRNA, and gltA genes. DNA fragments similar to those of Ehrlichia canis were detected in Sapajus apella and Ehrlichia chaffeensis from Mico melanurus. The sequences generated in this study and homologous sequences retrieved from GenBank® were used for phylogenetic analyses to characterize the Ehrlichial agents detected in NHPs. The agents were then grouped into clades corresponding to different isolates from the NHP species. In addition, an Anaplasma sp. closely related to Anaplasma marginale was identified in two S. apella individuals. These findings shed light on the susceptibility of neotropical NHPs to Anaplasmataceae agents. These bacteria are known to be transmitted by ticks, which can also serve as possible sources of infection for other animals, including humans.

Detecção molecular de Anaplasma platys, Ehrlichia canise Babesia spp. em cães atendidos em clínica da região metropolitana do estado do Rio de Janeiro / Molecular detection of Anaplasma platys, Ehrlichia canis and Babesia spp. in dogs served at a clinic in the metropolitan region of the state of Rio de Janeiro

O presente estudo teve como objetivo detectar por meio da Reação em cadeia da Polimerase (PCR) a frequência de Ehrlichiacanis, Babesia spp. e Anaplasma platys em cães, relacionando a prevalência dos achados hematológicos aos resultados positivos pela PCR. Foram avaliadas 209 amostras de sangue de cães atendidos em clínica veterinária particular do município de Queimados, RJ, Brasil, no período de julho a outubro de 2014. Foram realizados hemograma completo e extração de DNA para técnica de PCR. Do total de 209 animais, 19,1% (40/209) animais apresentaram resultado positivo para hemoparasitos pela técnica de PCR. Destes, 52,5% (21/40) foram positivos para E. canis, 27,5% (11/40) positivos para Babesia spp. e 10% (4/40) positivos para A. platys. Quatro animais (1,91%), dos 209 testados, foram positivos para pelo menos dois agentes, caracterizando assim coinfecção. Dos 40 cães positivos para algum dos agentes testados, 25 (62,5%) estavam trombocitopênicos. Ou seja, 15 cães (37,5%) reagiram positivos para hemoparasitos, mas não apresentavam trombocitopenia. A anemia foi um achado comum, sobretudo nas infecções por Babesia spp., 100% (11/11) e E.canis, 90,5% (19/21). A técnica de PCR foi um importante método diferencial na detecção das principais hemoparasitoses caninas, juntamente com os achados clínicos e hematológicos para o diagnóstico preciso da infecção em questão.

The present study aimed to detect, by means of Polimerase chain reaction (PCR), the frequency of Ehrlichia canis, Babesia spp. and Anaplasma platys in dogs, relating the prevalence of hematological findings to positive PCR results. A total of 209 blood samples from dogs treated at a private veterinary clinic in the city of Queimados, RJ, Brazil, from July to October 2014 were evaluated. Complete blood count and DNA extraction were performed for the PCR technique. Of the total of 209 animals, 19.1% (40/209) animals were positive for hemoparasites by the PCR technique. Of these, 52.5% (21/40) were positive for E. canis, 27.5% (11/40) were positive for Babesia spp. and 10% (4/40) positive for A. platys. Four animals (1.91%) of the 209 tested were positive for at least two agents, thus characterizing coinfection. Of the 40 dogs positive for any of the agents tested, 25 (62.5%) were thrombocytopenic. That is, 15 dogs (37.5%) were positive for hemoparasites, but did not have thrombocytopenia. Anemia was a common finding, especially in infections by Babesia spp., 100% (11/11) and E. canis, 90.5% (19/21). The PCR technique was an important differential method in the detection of the main canine hemoparasitoses, together with the clinical and hematological findings for the accurate diagnosis of the infection in question.

Molecular characterization of Ehrlichia canis and Babesia vogeli reveals multiple genogroups associated with clinical traits in dogs from urban areas of Colombia.

Ehrlichia canis and Babesia vogeli are vector-borne pathogens that infect blood cells and produce the diseases Canine Monocytic Ehrlichiosis (CME) and Babesiosis in dogs. Considering the lack of studies on these pathogens in Colombia, this study aims to determine the molecular prevalence and genetic characterization of E. canis and Babesia spp., in dogs from the Metropolitan Area of Bucaramanga (MAB), Santander, a region with one of the greatest pet densities in Colombia. One hundred eighty-five dogs were surveyed and analyzed through molecular, clinical, and hematological approaches. The molecular detection of E. canis and Babesia spp., was performed by conventional PCR targeting the dsb and 18S rRNA genes, respectively. To identify genogroups, E. canis positive samples underwent a hemi-nested PCR of the trp36 gene, and the PCR products were subsequently sequenced. Molecular analyses showed a prevalence of 13% (24/185; CI 95%, 8.1 - 18.0%) and 1.09% (2/185; CI 95,% -0.43 - 2.6%) for E. canis and B. vogeli respectively, as well as the presence of the genogroups US (USA), BR (Brazil), and CR (Costa Rica), in 62.5, 16.6, and 16.6% of E. canis positive samples, respectively. Values of hematocrit, hemoglobin, platelets, erythrocytes, white blood cell (WBC) count, lymphocytes, and eosinophils showed significant differences between animals infected with the different genogroups of E. canis (p< 0.05). In contrast, hematocrit values, hemoglobin, platelets, red blood cells, and creatine kinase MB isoenzyme (CK-MB) were lower in B. vogeli positive animals. Statistical analysis indicated that E. canis infection was associated with specific socioeconomic sectors as well as with some household features (p< 0.05). In conclusion, our results present evidence of the circulation of multiple genogroups of E. canis in the MAB, which is associated with different geographical origins and clinical traits. Epidemiological analyses suggest a need to increase molecular surveillance and prevention campaigns especially in lower socioeconomic sectors.

Dogs naturally infected by Rangelia vitalii, Babesia canis vogeli, and Ehrlichia canis in São Paulo, Brazil / Cães naturalmente infectados por Rangelia vitalii, Babesia canis vogeli, e Ehrlichia canis em São Paulo, Brasil

Several agents can cause hemoparasitic diseases in dogs, and blood-sucking arthropods transmit these diseases. These agents can cause several clinical manifestations and, in some cases, can kill the host. Because these agents are essential in animal health, this study aims to detect the frequency of Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, and Rangelia vitalii by real-time PCR and Babesia vogeli in dogs in the southern region of the city of São Paulo, São Paulo. Of the 98 dog samples, 18 (18.4%) tested positive with real-time polymerase chain reaction for at least one studied agent. Of these 18 samples, 17 tested positive for a single agent (11.2% for B. canis vogeli, 1.02% for R. vitalii, and 5.1% for E. canis), and one showed co-infection with B. canis vogeli and R. vitalii. The results demonstrate the presence of hemoparasites in the studied animals, which can influence the quality and life expectancy of these animals. The Rangeliadetection warns small animal clinicians to include it as a differential diagnosis for hemoparasitosis.(AU)

As hemoparasitoses em cães podem ser causadas por diversos agentes, sendo essas doenças transmitidas por artrópodes hematófagos. Esses agentes podem causar diversas manifestações clínicas e, em alguns casos, podem matar o hospedeiro. Este estudo teve como objetivo detectar por PCR em tempo real a frequência de Ehrlichia canis, Rickettsia rickettsii, Anaplasma platys, Rangelia vitalii e Babesia canis vogeli em amostras de cães da zona sul da cidade de São Paulo, Brasil. Das 98 amostras de cães, 18 (18,4%) testaram positivo com reação em cadeia da polimerase em tempo real para pelo menos um agente estudado. Destas 18 amostras, 17 testaram positivo para um único agente (11,2% para B. canis vogeli, 1,02% para R. vitalii e 5,1% para E. canis), e uma apresentou coinfecção com B. canis vogeli e R. vitalii. Os resultados demonstram a presença de hemoparasitas nos animais estudados, o que pode influenciar a qualidade e a expectativa de vida desses animais. Além disso, é o primeiro relato da detecção de R. vitalli na zona sul de São Paulo e serve de alerta para os clínicos de pequenos animais incluírem esse agente como diagnóstico diferencial para as hemoparasitoses.(AU)

Diagnóstico hematológico de coinfecção por Ehrlichia spp. e Hepatozoon canis em cães no semiárido da Paraíba / Hematogical diagnosis of coinfection by Ehrlichia spp. and Hepatozoon canis in dogs in the semi-arid of Paraiba

Dentre as hemoparasitoses com maior frequência de transmissão por carrapatos pode-se destacar a Erliquiose, a Hepatozoonose, a Anaplasmose e a Babesiose. A coinfecção por esses agentes são frequentes na rotina clínica; no entanto, poucas são as descrições sobre a ocorrência simultânea desses agentes em hemogramas. Sendo assim, o objetivo deste trabalho foi relatar os casos de quatro cães diagnosticados com coinfecção por Ehrlichia spp. e Hepatozoon canis, os quais foram atendidos no Hospital Veterinário Adílio Santos de Azevedo (HV-ASA) do Instituto Federal de Educação, Ciência e Tecnologia da Paraíba (IFPB), campus Sousa. Durante a avaliação clínica, relatou-se que os pacientes apresentavam, de maneira em comum, oligodipsia e hiporexia, além de histórico de presença de carrapatos. Em seguida, foram realizados hemogramas, que revelaram que todos os animais apresentavam anemia leve a moderada. Além disso, notou-se que o animal 1 estava com trombocitopenia e os animais 2 e 3, neutrofilia. Na pesquisa de hemoparasitas em esfregaço sanguíneo, observou-se coinfecção por H. canis e E. spp. nos quatro animais. Desta forma, este relato demonstrou a importância do uso do hemograma e da pesquisa dos agentes causadores das hemoparasitoses, através do esfregaço sanguíneo. Ressalte-se que o esfregaço sanguíneo, embora seja um método inespecífico e de baixa sensibilidade, pode e deve ser adotado como rotina, por ser uma técnica simples, rápida, barata, pouco invasiva e de diagnóstico definitivo.

Among the hemoparasitoses with the highest frequency of transmission by ticks, ehrlichiosis, hepatozoonosis, anaplasmosis, and babesiosis may be highlighted. The coinfection with these agents is frequent in clinical routine; however, there are few descriptions of the simultaneous occurrence of these agents in blood counts. Therefore, this work aimed to report the cases of four dogs diagnosed with Ehrlichia spp. and Hepatozoon canis, which were treated at the Adílio Santos de Azevedo Veterinary Hospital (HV-ASA), of the Federal Institute of Education, Science, and Technology of Paraíba (IFPB), Sousa campus. During the clinical evaluation, it was reported that the patients had oligodipsia and hyporexia in common, in addition to a history of tick presence. Then, blood counts were performed, which revealed that all animals had mild to moderate anemia. In addition, it was noted that animal 1 had thrombocytopenia and animals 2 and 3 had neutrophilia. In the investigation of hemoparasites in blood smears, co-infection with H. canis and E. spp. was observed in the four animals. Thus, this report demonstrated the importance of using the blood count and researching the causative agents of hemoparasitosis, through the blood smear. It should be noted that the blood smear, although it is a non-specific and low-sensitivity method, can and should be adopted as a routine, as it is a simple, fast, inexpensive, minimally invasive, and definitive diagnostic technique.

Molecular detection of vector-borne pathogens in semen from dogs in southeastern Brazil.

Vector-borne pathogens (VBPs) are primarily transmitted by arthropod vectors, but secondary ways of transmission have been described, including via venereal route. Nonetheless, there is still limited research on possible sexual transmission of VBPs in dogs. We molecularly investigated the presence of vector-borne pathogens in semen from dogs living in an area where these agents are endemic. Upon PCR testing, seven out of 22 (31.8%) semen samples tested positive for at least one VBP, whereas simultaneous positivity to two or more pathogens was detected in three (13.6%) dogs. Among pathogens detected in semen, Trypanosoma cruzi (n = 1) and Leishmania infantum (n = 3) were identified to species level by restriction fragment length polymorphism analysis. Attempts to sequence PCR products from other pathogens were unsuccessful, but coupled epidemiological and molecular data suggest the presence of Anaplasma platys (n = 5), Babesia vogeli (n = 1) and Ehrlichia canis (n = 1) in semen from dogs. Further experimental studies would be needed to confirm the sexual transmission hypothesis for these VBPs and also the possible implications of these findings for canine reproduction.

Use of a recombinant positive control in the diagnostic of canine Ehrlichiosis from 16sRNA gen of Ehrlichia canis in Mexico City.

Ehrlichia canis has gained importance over the years as a zoonotic bacterium, nevertheless in Mexico is unknown the extent of the problem in animals and public health. The country had a few studies carried out locally using serology and molecular tests as diagnostic methods. Ehrlichiosis is not considered endemic in the central valley of Mexico, because the climatic conditions in the region have not allowed the vector (Rhipicephalus sanguineus) to establish itself adequately, therefore, diagnosis is not used in clinical practice in this area. A nested PCR (nPCR) offers rapid results with high sensitivity and specificity regardless of cost. The use of a recombinant positive control provides the advantage of timely diagnosis, follow-up treatment and allows the clinician to decide. In this work, the nPCR reported by Wen et al. (J Clin Microbiol 35(7):1852-2185, 1997) was used for the diagnosis of E. canis by modifying the reaction conditions to improve the detection of the test. We constructed a recombinant positive control to nPCR as diagnostic technique for E. canis, also we modified the reaction conditions to improve detection of the test which allowed the diagnosis of E. canis in dogs in the Mexican Republic using 53 samples from dogs with positive serological diagnosis of Ehrlichiosis, some of them from the valley of Mexico. Currently, this nPCR is offered to public at the Faculty of Veterinary Medicine and Zootechnics of the National Autonomous University of Mexico at an accessible cost and allows to begin to generate epidemiological information to know distribution of the bacterium.

Novel Ehrlichia canis genogroup in dogs with canine ehrlichiosis in Cuba.

BACKGROUND: Canine monocytic ehrlichiosis (CME) is caused by the tick-borne pathogen Ehrlichia canis, an obligate intracellular Gram-negative bacterium of the family Anaplasmataceae with tropism for canine monocytes and macrophages. The trp36 gene, which encodes for the major immunoreactive protein TRP36 in E. canis, has been successfully used to characterize the genetic diversity of this pathogen in different regions of the world. Based on trp36 sequence analysis, four E. canis genogroups, United States (US), Taiwan (TWN), Brazil (BR) and Costa Rica (CR), have been identified. The aim of this study was to characterize the genetic diversity of E. canis in Cuba based on the trp36 gene. METHODS: Whole blood samples (n = 8) were collected from dogs found to be infested with the tick vector Rhipicephalus sanguineus sensu lato (s.l.) and/or presenting clinical signs and symptoms of CME. Total DNA was extracted from the blood samples and trp36 fragments were amplified by PCR. Nucleotide and protein sequences were compared using alignments and phylogenetic analysis. RESULTS: Four of the trp36 sequences obtained (n = 8) fall within the phylogenetic cluster grouping the US genogroup E. canis strains. The other E. canis trp36 sequences formed a separate and well-supported clade (94% bootstrap value) that is phylogenetically distant from the other major groups and thus represents a new genogroup, herein designated as the 'Cuba (CUB) genogroup'. Notably, dogs infected with the CUB genogroup presented frequent hemorrhagic lesions. CONCLUSIONS: The results of this study suggest that genetic diversification of E. canis in Cuba is associated with the emergence of E. canis strains with increased virulence.