High serological and molecular prevalence of Ehrlichia canis and other vector-borne pathogens in dogs from Boa Vista Island, Cape Verde.

Despite the high global impacts of canine vector-borne diseases (CVBD) due to their wide distribution and zoonotic potential, the current epidemiological situation of CVBD in many tropical and subtropical regions remains unknown. This study examines the seroprevalence and molecular prevalence of Ehrlichia canis and other pathogens causing CVBDs (Leishmania infantum, Dirofilaria immitis, Babesia spp., Anaplasma spp. and Hepatozoon canis) in dogs living on the island of Boa Vista (Cape Verde Republic). Blood samples and infesting ticks were taken from 150 dogs across the island (stray, shelter, and pet dogs). Serum samples were tested using a rapid immunochromatographic test (Uranotest® Quattro) that detects antibodies against E. canis, L. infantum, Anaplasma spp. and D. immitis antigen. Levels of serum antibodies against E. canis were measured using the immunofluorescence antibody test (IFAT). In addition, tick-borne pathogens in blood samples (Anaplasma spp., Babesia spp., Hepatozoon spp., and Ehrlichia canis) were detected by microscopy observation and/or PCR plus sequencing. The seroprevalence of E. canis was extremely high at 82% (123/150), as revealed by both immunochromatography and IFAT. Most dogs returning a seropositive test result (82.92%; 102/123) had antibody titres > 1:1280 but showed no clinical signs or notable laboratory abnormalities. Of the 123 animals testing seropositive for E. canis, 67 (54.47%) also presented antibodies against Anaplasma spp., and 13 (10.56%) showed the presence of Hepatozoon spp. gamonts in the blood smear. Ehrlichia canis infection was detected in 17.1% (25/146) of dogs tested by direct sequencing of polymerase chain reaction (PCR) products. Co-infections were detected in seven of these dogs: four dogs tested PCR-positive for both E. canis and A. platys, two dogs tested positive for E. canis and Hepatozoon spp., and one dog tested positive for E. canis, A. platys and Hepatozoon spp. Rhipicephalus sanguineus sensu lato was the only tick species found infesting the canine study population. The high prevalence of tick-borne pathogens detected in dogs from Boa Vista Island highlights a need for improved control measures designed to prevent the transmission of these pathogens.

Rhipicephalus sanguineus from Hungarian dogs: Tick identification and detection of tick-borne pathogens.

The brown dog tick, Rhipicephalus sanguineus is a complex of tick species with an unsettled species concept. In Europe, R. sanguineus is considered mainly a Mediterranean tick with sporadic findings in central and northern Europe. R. sanguineus is known as a vector of a range of pathogens of medical and veterinary importance, most of which not yet reported as autochthonous in Hungary. A total of 1839 ticks collected by veterinarians from dogs and cats were obtained in Hungary. The study aims at precise determination of ticks identified as R. sanguineus and detection of pathogens in collected ticks. All ticks were morphologically determined and 169 individuals were identified as R. sanguineus. A subset of 15 ticks was selected for molecular analysis (16S rDNA, 12S rDNA, COI). Phylogenetic analyses invariably placed sequences of all three markers into a single haplotype identified as R. sanguineus sensu stricto. All 169 brown dog ticks were tested for the presence of A. platys, E. canis, R. conorii, B. vogeli and H. canis. None of the investigated ticks was positive for the screened pathogens, though A. phagocytophilum sequence was detected in a single tick.

Seasonal dynamics and genetic characterization of bovine arthropod-borne parasites in Nan Province, Thailand with molecular identification of Anaplasma platys and Trypanosoma theileri.

Virulent species or strains of hematophagous borne pathogens such as Anaplasma spp., Babesia spp., Theileria spp., and Trypanosoma spp., are lethal to susceptible animals or reduce their productivity on a global scale. Nonetheless, efforts to diagnose the causative agents and assess the genotypic profiles as well as quantify the parasite burden of aforementioned parasites across seasons remain limited. Therefore, the present investigation sought to elucidate the genotypic composition of Anaplasma spp., Babesia spp., Theileria spp., and Trypanosoma spp. The findings revealed heightened infection rates during the summer, manifesting a correlation between Trypanosoma spp. infection and seasonal fluctuations. Among the identified pathogens, Anaplasma marginale emerged as the most dominant species, while the occurrence of Anaplasma platys in Thai cattle was confirmed via the sequencing of the groEL gene. Moreover, the study successfully identified two lineages of Trypanosoma theileri. The findings of this investigation offer valuable insights that can inform the development of preventive strategies for vector-borne diseases, such as considering the appropriate use of insect repellent, mosquito or insect nets, or eliminating breeding places for insects in each season.

Molecular survey of canine tick-borne pathogens in ticks and stray dogs in Dhaka city, Bangladesh.

Molecular surveillance of canine tick-borne pathogens (TBPs) in Bangladesh has constantly been undervalued. Therefore, the emergence of new pathogens often remains undetected. This study aimed to screen tick-borne pathogens in stray dogs and ticks in the Dhaka metropolitan area (DMA). Eighty-five dog blood and 53 ticks were collected in six city districts of DMA from September 2022 to January 2023. The ticks were identified by morphology. Screening of TBPs was performed by polymerase chain reaction (PCR), followed by sequencing. The PCR assays were conducted to analyze the 18S rRNA (Babesia gibsoni, B. vogeli, and Hepatozoon canis), 16S rRNA (Anaplasma phagocytophilum, A. platys, and A. bovis), gltA (Ehrlichia canis and Rickettsia spp.), flagellin B (Borrelia spp.) and 16-23S rRNA (Bartonella spp.). Three tick species, Rhipicephalus sanguineus (50/53), R. microplus (1/53), and Haemaphysalis bispinosa (2/53), were identified. Babesia gibsoni (38 out of 85) and A. platys (7 out of 85) were detected in dog blood. In contrast, four pathogens, B. gibsoni (1 out of 53), B. vogeli (1 out of 53), H. canis (22 out of 53), and A. platys (1 out of 53), were detected in the ticks. However, the detection rates of TBPs in dog blood and ticks were not correlated in this study. The phylogenetic analyses suggested that a single genotype for each of the four pathogens is circulating in DMA. This study reports the existence of B. vogeli, H. canis, and A. platys in Bangladesh for the first time.

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.

Molecular detection and associated risk factors of Anaplasma marginale, A. ovis and A. platys in sheep from Algeria with evidence of the absence of A. phagocytophilum.

Anaplasma species are obligate intracellular rickettsial pathogens that cause significant diseases in animals and humans. Despite their importance, limited information on Anaplasma infections in Algeria has been published thus far. This study aimed to assess the infection rate, characterize Anaplasma species, and identify associated risk factors in selected sheep farms across Oum El Bouaghi region in Algeria. In 2018, we collected 417 blood samples from sheep (Ovis aries) and performed molecular characterization of Anaplasma species infecting these animals. This characterization involved the use of 16S rRNA, msp2, rpoB, and msp5 genes, which were analyzed through nested PCR, qPCR, cPCR, DNA sequencing, and subsequent phylogenetic analysis. Our findings revealed infection rates of 12.7 % for Anaplasma species detected, with Anaplasma ovis at 10.8 %, Anaplasma marginale at 1.7 %, and Anaplasma platys at 0.2 %. Interestingly, all tested animals were found negative for Anaplasma phagocytophilum. Statistical analyses, including the Chi-square test and Fisher exact test, failed to establish any significant relationships (p > 0.05) between A. ovis and A. platys infections and variables such as age, sex, sampling season, and tick infestation level. However, A. marginale infection exhibited a significant association with age (p < 0.05), with a higher incidence observed in lambs (5.2 %) compared to other age groups. Remarkably, this study represents the first molecular detection of A. platys and A. marginale in Algerian sheep. These findings suggest that Algerian sheep may serve as potential reservoirs for these pathogens. This research contributes valuable insights into the prevalence and characteristics of Anaplasma infections in Algerian sheep populations, emphasizing the need for further investigation and enhanced surveillance to better understand and manage these diseases.

Detection of Bartonella vinsonii, Anaplasma platys and Bartonella sp. in didelphis marsupialis, Pecari tajacu and Chelonoidis denticulate: Peru.

INTRODUCTION: Evidence suggest that wildlife Infectious diseases related to wildlife are of most importance because of the agents' capacity to spill over into humans from the wild reservoir. Among them, the bacteria Bartonella spp. and Anaplasma spp. are related to this zoonotic dynamic. OBJECTIVE: The primary goal of the present study was to determine the presence of pathogenic bacteria in kidney and liver tissues of Didelphis marsupialis; spleen, liver, and skin of Pecari tajacu; spleen, liver, and skin of Chelonoidis denticulata. METHODOLOGY: A PCR using universal and specific primers for 16 S rRNA, of Bartonella spp. with subsequent genetic sequencing were used. RESULTS: The results in this study indicate that Bartonella vinsonni was detected in the liver tissue of Didelphis marsupialis using both universal primers and those specific for Bartonella sp. Anaplasma platys was detected at the liver and spleen level using universal primers. Additionally, Bartonella spp. was found at the liver, spleen, and skin level in Pecari tajacu using the specific primers. Finally, using the universal and specific primers at the skin level, Bartonella spp. was evident in Chelonoidis denticulata. CONCLUSIONS: The presence of the DNA of the Bartonella vinsonii was detected at the liver tissue in Didelphis marsupialis. DNA of the Anaplasma platys and Bartonella spp. were identified at the spleen and liver level. This study also identified that DNA Bartonella spp. was detected in Pecari tajacu skin. Finally DNA of Bartonella spp. was evident in Chelonoidis denticulate skin. The findings of this study suggest that these bacteria are present in these animals and may be responsible for outbreaks.

Molecular identification of Anaplasma platys in cattle by nested PCR.

Background and Objectives: Anaplasmosis is a zoonotic disease caused by Gram-negative bacterium from Anaplasmataceae family. Anaplasma causes high economic losses worldwide. 16S rRNA analysis was used to diagnose Anaplasma platys in Cattle. Phylogenetic tree and estimation of evolutionary divergence between A. platys isolates were performed. Materials and Methods: A total of 60 blood samples were collected from a cattle farm in AL-Diwaniyah province. 16S rRNA gene was identified using nested PCR. Overall, 40% of cattle that were chosen to collect the blood were identified to be infected with A. platys. Results: The results have shown presence of targeting partial region of 16S rRNA gene in 24 samples out of 60. Sequencing results of 10 samples have revealed that the phylogenetic tree was divided in to two separate clades. Five isolates of A. platys-Iraq (accession no. OP646782, OP646783, OP646784, OP646790, and OP646791) were located in one clade with the A. platys-China (accession no. MN193068.1). While, five isolates (accession no. OP646785, OP646786, OP646787, OP646788, OP646789) were in different clade with two isolates of A. platys-Africa and A. platys-Zambia in distinct branches, close to the Rickettsiales. Conclusion: The phylogenetic study of A. platys sequences indicated that the isolates were collected from a cattle farm in Al-Dewaniyah were similar and close related to A. platys-China, A. platys-Zambia and A. platys-Africa). This study suggests that cattle can be considered a reservoir of A. platys.

Haematological Alterations Associated with Selected Vector-Borne Infections and Exposure in Dogs from Pereira, Risaralda, Colombia.

Infections due to Ehrlichia, Anaplasma, Dirofilaria, Mycoplasma, Babesia and Hepatozoon continue to be highly prevalent in dogs, especially in tropical and subtropical areas, where vectors of many of them are present. However, many clinical aspects of dogs have not been characterized in detail, including assessing the haematological alterations associated with them, particularly in Colombia and Latin America. A group of 100 dogs with Ehrlichia, Anaplasma, Dirofilaria, Mycoplasma, Babesia and Hepatozoon infections/exposure were assessed by blood smear serology (SNAP4DX) and PCR in Pereira, Colombia. We performed blood counts to evaluate anaemia, leukopenia/leukocytosis, neutropenia, neutrophilia, lymphopenia/lymphocytosis, monocytosis, eosinophilia, and thrombocytopenia, among other alterations. Bivariate analyses were performed on Stata®14, with significant p < 0.05. From the total, 85% presented ≥1 infection (past or present), 66% with coinfections (≥2 pathogens) (Ehrlichia 75%), and 89% presented clinical alterations. A total of 100% showed anaemia, 70% thrombocytopenia, 61% monocytosis, and 47% neutropenia, among other alterations. Additionally, 11% presented pancytopenia and 59% bicytopenia. The median platelet count was lower in infected dogs (126,000 cells/µL) versus non-infected (221,000 cells/µL) (p = 0.003). Thrombocytopenia was higher among infected dogs (75%) versus non-infected (40%) (p = 0.006), with a 91% positive predictive value for infection. Median neutrophil count was lower in infected dogs (6591 cells/µL) versus non-infected (8804 cells/µL) (p = 0.013). Lymphocytosis occurred only among those infected (27%) (p = 0.022). Leukopenia was only observed among infected dogs (13%). Pancytopenia was only observed among infected dogs. Ehrlichiosis and other hematic infections have led to a significant burden of haematological alterations on infected dogs, including pancytopenia in a tenth of them, most with thrombocytopenia and all anemic.

MALDI-TOF MS Identification of Dromedary Camel Ticks and Detection of Associated Microorganisms, Southern Algeria.

This study used MALDI-TOF MS and molecular tools to identify tick species infesting camels from Tamanrasset in southern Algeria and to investigate their associated microorganisms. Ninety-one adult ticks were collected from nine camels and were morphologically identified as Hyalomma spp., Hyalomma dromedarii, Hyalomma excavatum, Hyalomma impeltatum and Hyalomma anatolicum. Next, the legs of all ticks were subjected to MALDI-TOF MS, and 88/91 specimens provided good-quality MS spectra. Our homemade MALDI-TOF MS arthropod spectra database was then updated with the new MS spectra of 14 specimens of molecularly confirmed species in this study. The spectra of the remaining tick specimens not included in the MS database were queried against the upgraded database. All 74 specimens were correctly identified by MALDI-TOF MS, with logarithmic score values ranging from 1.701 to 2.507, with median and mean values of 2.199 and 2.172 ± 0.169, respectively. One H. impeltatum and one H. dromedarii (2/91; 2.20%) tested positive by qPCR for Coxiella burnetii, the agent of Q fever. We also report the first detection of an Anaplasma sp. close to A. platys in H. dromedarii in Algeria and a potentially new Ehrlichia sp. in H. impeltatum.

Molecular detection of tick-borne pathogens in Rhipicephalus sanguineus sensu stricto collected from dogs in the steppe and high plateau regions of Algeria.

Epidemiology and distributions of canine tick-borne diseases as well as their veterinary and zoonotic significance are poorly understood in Algeria. The present study describes a molecular investigation of important tick-borne pathogens in Rhipicephalus sanguineus sensu stricto collected from domestic dogs in steppe and high plateau areas of central and eastern Algeria. In total, 1,043 ticks were collected from 147 dogs, including 756 ticks from 124 dogs in the steppe region of Djelfa and 287 ticks from 23 dogs in the high plateau area of Bordj Bou Arreridj. Ticks were divided into 384 pools (309 pools from Djelfa and 75 pools from Bordj Bou Arreridj) and tested for genomic materials of Crimean-Congo hemorrhagic fever virus (CCHFV) as well as DNA for Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, Rickettsia spp., Babesia spp., and Hepatozoon spp. using PCR, sequencing and phylogenetic analysis. Hepatozoon spp. was most prevalent, with 160 positive pools (41.7%), and 12 of these were sequenced and identified as Hepatozoon canis. Babesia spp. was detected in 50 samples (13.0%), of which 11 were sequenced and identified as Babesia vogeli. A. platys and E. canis were detected in 92 (24.0%) and 15 (3.9%) of tested samples, respectively. Rickettsia spp. were detected in 24 (6.3%) samples, including 11 samples identified as R. massiliae, 6 samples identified as R. conorii conorii, and 7 samples could not be identified to species level. All 384 pools tested negative for CCHFV and A. phagocytophilum. In addition to detection of R. conorii conorii, R. massiliae, and E. canis, the present study provides the first molecular data for occurrence of A. platys, B. vogeli, and H. canis in Rh. sanguineus s.s. infesting dogs in Algeria. Further large scale studies should be conducted to better understand the epidemiology, distributions, and importance of canine tick-borne pathogens in Algeria.

First study on molecular detection of three major canine tick-borne pathogens in subclinically infected dogs in Chiang Mai, Thailand.

Background and Aim: Canine tick-borne pathogens (CTBPs) are an important cause of morbidity in dogs in Thailand. This study aimed to evaluate the occurrence of three CTBPs in clinically normal, owned dogs to understand the risk for the general canine population. We also examined sex, age, tick infestation, and packed cell volume (PCV) of the animals in association with active infection of the CTBPs. Materials and Methods: A total of 139 dogs were included in the study. Blood samples were collected for thin blood smear, PCV and nested polymerase chain reaction (PCR) assay. Statistical analyses were performed to examine the association between individual factors and CTBP infection status determined by PCR. In addition, sensitivity, specificity, and Cohen's kappa were calculated to assess the utility of routine blood smear. Results: The PCR results showed that 31 dogs (22.3%) were infected with at least one of the three pathogens. The occurrence rate for Ehrlichia canis, Anaplasma platys, and Hepatozoon canis was 2.2% (3/139), 18.7% (24/139), and 2.8% (4/139), respectively. There were two cases of coinfection with A. platys and E. canis. The univariate analyses did not yield any associations between recorded variables and the active infection. Microscopic examination showed good sensitivity and agreement only for H. canis (Sn: 75%, 95% confidence interval: 24.9-98.7, k=0.85). Conclusion: Our findings confirmed the endemicity of the CTBPs in owned canine population in the study site. In-depth epidemiological investigation would be warranted to elucidate environmental risk factors for CTBP infection.

Seroprevalence canine survey for selected vector-borne pathogens and its relationship with poverty in metropolitan Pereira, Colombia, 2020.

Background: Tick-borne diseases (TBD) and dirofilariosis are currently not under surveillance in most Latin American countries. In addition, there is a significant lack of studies describing the current situation in most endemic areas, including Colombia. Therefore, seroprevalence studies are crucial for understanding the epidemiology of these vector-borne diseases. Methods: A serosurvey for TBD and dirofilariosis among 100 dogs was carried out in the municipality of Pereira, located in the Coffee-Triangle region, Colombia. Samples were tested using a rapid assay test system (SNAP® 4Dx®); based on an enzyme immunoassay technique' screening for antibodies to Anaplasma phagocytophilum/platys (sensitivity 99.1%)' Borrelia burgdorferi s.l. (98.8%), and Ehrlichia canis/ewingii (96.2%) by using specific antigens and checking for Dirofilaria immitis antigen based on specific antibodies (99.2%). Bivariate analyses were performed on Stata®14, significant p < 0.05. Findings: Global seroprevalence to the selected vector-borne pathogens was 74% (95%CI 65-83%). The highest seroprevalence was found for E. canis/ewingii (74%), followed by A. phagocytophilum/platys (16%). Seropositivity for Borrelia spp. and Dirofilaria spp. was 0%. All Anaplasma spp. seropositive dogs showed co-detection of Ehrlichia spp. (16%). Seroprevalence was significantly higher among dogs from families of lower socioeconomic status/level (I, 86%), followed by level II (74%), and III (36%) (p = 0.001). All dogs exhibiting anorexia (12%) were invariably seropositive (100%) (p = 0.029). Seroprevalence was higher among those showing mucocutaneous paleness (95%) compared to those without paleness (68%) (p = 0.013) (OR = 9.3; 95%CI 1.18-72.9). There was high variability in seroprevalence through the studied areas, ranging from 0% (La Libertad Park) up to Combia, Cesar Nader, Las Brisas and Saturno localities (100%) (p = 0.033). Interpretation: Given the high seroprevalence obtained in an area with documented ticks, there is a potential risk of zoonotic transmission to humans. Further seroprevalence studies in humans are needed to assess the prevalence of infections. Poverty is highly associated with these tick-borne pathogens in Pereira, as shown in the present study.

Molecular discrimination and genetic diversity of three common tick-borne pathogens in dogs in Thailand.

There was little information regarding the occurrence of canine vector-borne disease (CVBDs) in shelter dogs in Thailand. This work is the first report regarding a molecular method used to determine the occurrence and genetic diversity of three canine tick-borne pathogens (TBPs) (Hepatozoon canis, Anaplasma platys and Ehrlichia canis) in blood samples from 275 shelter dogs in the north and central areas of Thailand. The PCR results based on the 18S rRNA and 16S rRNA genes showed that 71 (25.82%) dogs were positive for at least a TBP. The overall occurrence rates of H. canis, A. platys and E. canis infections were 1.81, 16.36 and 7.64%, respectively. For the phylogenetic analysis, A. platys 16S rRNA gene was genetically diverse, while H. canis 18S rRNA and E. canis 16S rRNA genes were conserved. The haplotype diversity exhibited 12 and 2 haplotypes as well as 78 and 178 polymorphic sites of A. platys and E. canis 16S rRNA genes, respectively. Our findings could be used to improve the understanding of phylogeny and genetic diversity of TBP rRNA genes and used to ameliorate the diagnosis and control programmes for the diseases in Thailand.

Molecular detection and phylogeny of Ehrlichia canis and Anaplasma platys in naturally infected dogs in Central and Northeast Thailand.

Background and Aim: Ehrlichia canis and Anaplasma platys are tick-borne, Gram-negative bacteria that cause canine monocytic ehrlichiosis and canine cyclic thrombocytopenia, respectively. These diseases are of great importance and are distributed globally. This study aimed to create new primers for the identification of E. canis and A. platys in naturally infected dogs using polymerase chain reaction (PCR), DNA sequencing, and phylogenetic analysis using the 16S rDNA and gltA genes. Materials and Methods: In total, 120 blood samples were collected from dogs in three different locations (Saraburi, Buriram, and Nakhon Ratchasima provinces) in Central and Northeast Thailand. The molecular prevalence of E. canis and A. platys was assessed using PCR targeting the 16S rDNA and gltA genes. All positive PCR amplicons were sequenced, and phylogenetic trees were constructed based on the maximum likelihood method. Results: Ehrlichia canis had an overall molecular prevalence of 15.8% based on the 16S rDNA gene, compared to 8.3% based on the gltA gene. In addition, the overall molecular prevalence of A. platys using the 16S rDNA gene was 10.8%, while the prevalence rate was 5.8% using the gltA gene. Coinfection was 0.8% in Saraburi province. The partial sequences of the 16S rDNA and gltA genes of E. canis and A. platys in dogs in Central and Northeast Thailand showed 96.75%-100% identity to reference sequences in GenBank. Phylogenetic analysis of the 16S rDNA and gltA genes revealed that E. canis and A. platys sequences were clearly grouped into their own clades. Conclusion: This study demonstrated the molecular prevalence of E. canis and A. platys in Central and Northeast Thailand. The 16S rDNA and gltA genes were useful for the diagnosis of E. canis and A. platys. Based on the phylogenetic analysis, the partial sequences of the 16S rDNA and gltA genes in E. canis and A. platys were related to prior Thai strains and those from other countries.

Diversity of Anaplasma species and importance of mixed infections in roe deer from Spain.

Although wildlife can act as reservoirs of some Anaplasma species, studies on the presence and distribution of Anaplasma spp. in wild cervids are mainly limited and focused on zoonotic species. In order to identify the Anaplasma species in roe deer from Spain and to detect co-infections, 224 spleen samples were tested for Anaplasma spp. using a commercial qPCR; positive samples were further characterized using generic 16S rRNA primers and species-specific primers targeting the msp2 and groEL genes. Anaplasma DNA was detected in the 50.9% of samples, and four Anaplasma species were identified. Anaplasma phagocytophilum (43.8%) was predominant, followed by Anaplasma bovis (13.8%), Anaplasma capra (5.8%) and Anaplasma ovis (2.2%). In addition, strains similar to Anaplasma platys were found in nine animals. Most positive roe deer (71.9%) were infected with a single Anaplasma species, whereas co-infections with two (19.3%) or three (8.8%) Anaplasma species were also found. This study confirms the widespread occurrence of Anaplasma spp. in roe deer from Spain, being the first report of A. platys-like strains and A. capra in this cervid; it is also the first report of A. capra in Spain. The detection of Anaplasma species pathogenic for humans and/or domestic animals in roe deer suggests that this cervid may play a role in the sylvatic cycle of these bacteria contributing to the appearance of clinical anaplasmosis cases. In addition, co-infections are common in roe deer revealing that Anaplasma species specific PCR assays are essential for a reliable identification as well as for determining their real prevalence.

First report on molecular prevalence and identification of Anaplasma platys in dogs in Khon Kaen, Thailand.

BACKGROUND AND AIM: Anaplasma platys is a blood parasite that infects platelets, causing thrombocytopenia. Rhipicephalus sanguineus ticks are believed to transmit A. platys. To identify A. platys, nested polymerase chain reaction (PCR) has proven to be an effective diagnostic tool. In this study, the molecular prevalence of A. platys infection in dogs was investigated for the 1st time in the Khon Kaen region of Thailand. The association between risk factors and A. platys infection was also evaluated. MATERIALS AND METHODS: A total of 130 blood samples were collected from dogs in Khon Kaen, Thailand. DNA from the samples was extracted and nested PCR was applied for molecular analysis. Platelet count and packed cell volume (PCV) levels were measured. Platelet counts were categorized into four grades: Non-thrombocytopenia (platelets >200,000 cells/µL), mild thrombocytopenia (platelets 150,000-200,000 cells/µL), moderate thrombocytopenia (platelets 100,000-150,000 cells/µL), and severe thrombocytopenia (platelets <100,000 cells/µL). Four categories for PCV levels of >37%, 30-37%, 20-29%, and <20% were defined as no anemia, mild anemia, moderate anemia, and severe anemia, respectively. DNA sequencing was analyzed using BTSeq™ (Barcode-Tagged Sequencing; CELEMICS, Seoul, South Korea) for similarity index. RESULTS: Among the 130 samples, 9 (6.9%) were positive for A. platys infection. There was an association between low platelet count and infection (p<0.05). PCV level was also associated with A. platys infection (p<0.05). DNA sequencing results of the nine positive samples showed similarity to known sequences of A. platys with 99.36-100% nucleotide identity. These results suggested low genetic diversity in A. platys infecting dogs in the Khon Kaen area. CONCLUSION: By amplifying 16S rRNA, A. platys infection was detected in the blood of Thai dogs. Further work should be performed to identify risk factors potentially associated with A. platys infection in dogs in Khon Kaen. Other related factors should also be considered, such as location and breeding, as well as the environmental characteristics of each locality. In addition, sampling a larger number of animals may reveal predictors for the positivity of A. platys in dogs in this region.

Epidemiology, Diagnosis, and Control of Canine Infectious Cyclic Thrombocytopenia and Granulocytic Anaplasmosis: Emerging Diseases of Veterinary and Public Health Significance.

This review highlights the diagnostic methods used, the control strategies adopted, and the global epidemiological status of canine cyclic thrombocytopenia and granulocytic anaplasmosis at the animal-human interface. Canine anaplasmosis is an important worldwide disease, mainly caused by Anaplasma platys and A. phagocytophilum with zoonotic implications. A. platys chiefly infects platelets in canids, while A. phagocytophilum is the most common zoonotic pathogen infecting neutrophils of various vertebrate hosts. Diagnosis is based on the identification of clinical signs, the recognition of intracellular inclusions observed by microscopic observation of stained blood smear, and/or methods detecting antibodies or nucleic acids, although DNA sequencing is usually required to confirm the pathogenic strain. Serological cross-reactivity is the main problem in serodiagnosis. Prevalence varies from area to area depending on tick exposure. Tetracyclines are significant drugs for human and animal anaplasmosis. No universal vaccine is yet available that protects against diverse geographic strains. The control of canine anaplasmosis therefore relies on the detection of vectors/reservoirs, control of tick vectors, and prevention of iatrogenic/mechanical transmission. The control strategies for human anaplasmosis include reducing high-risk tick contact activities (such as gardening and hiking), careful blood transfusion, by passing immunosuppression, recognizing, and control of reservoirs/vectors.

Molecular detection and risk factors for Anaplasma platys infection in dogs from Egypt.

BACKGROUND: Anaplasma platys is a tick-borne bacterium which infects blood platelets of dogs, causing canine cyclic thrombocytopenia. The disease is distributed worldwide, particularly in the tropics and subtropics, but information on the epidemiology of A. platys infection in dogs is fragmentary in many countries, including Egypt. In this study, we investigated the prevalence and risk factors associated with A. platys infection in dogs from Egypt. METHODS: A conventional PCR targeting a fragment of the 16S rRNA gene of A. platys was used to screen 500 dogs from five North Egyptian governorates. DNA sequencing and phylogenetic analysis were performed for one of the positive samples. RESULTS: The overall prevalence of A. platys in the studied dogs was 6.4%. Females of the German shepherd breed without veterinary care had higher odds for A. platys positivity. High tick infestation and lack of anti-tick treatment were also identified as risk factors for A. platys infection. Phylogenetic analysis revealed that the sequence obtained herein was closely related to sequences from Egypt, South Africa and Uruguay. CONCLUSIONS: This is the first large-scale epidemiological study of A. platys in Egypt, where female German shepherd dogs without veterinary care, as well as dogs with high tick infestation and without anti-tick treatment are at a higher risk of infection.

Novel High-Throughput Multiplex qPCRs for the Detection of Canine Vector-Borne Pathogens in the Asia-Pacific.

The Asia-Pacific hosts a large diversity of canine vector-borne pathogens (VBPs) with some of the most common and most pathogenic, generating significant mortality as well as a spectrum of health impacts on local dog populations. The VBPs Anaplasma platys, Babesia gibsoni, Babesia vogeli, Ehrlichia canis, Hepatozoon canis and haemotropic Mycoplasma spp. are all endemic throughout the region, with many exhibiting shifting geographical distributions that warrant urgent attention. Moreover, many of these species cause similar clinical signs when parasitising canine hosts, whilst knowledge of the exact pathogen is critical to ensure treatment is effective. This is complicated by frequent coinfection that can exacerbate pathology. Here, we describe the development, optimisation and validation of two novel quadruplex Taq-Man based real-time PCRs (qPCRs) for the specific and sensitive detection of the aforementioned VBPs. To ensure accurate evaluation of diagnostic performance, results of our qPCRs were evaluated on field samples from Thai dogs and compared with both conventional PCR (cPCR) results and next-generation sequencing (NGS) metabarcoding. Our qPCRs were found to be more sensitive at detecting canine VBP than cPCR and generated results similar to those achieved by NGS. These qPCRs will provide a valuable high-throughput diagnostic tool available to epidemiologists, researchers and clinicians for the diagnosis of key canine VBPs in the Asia-Pacific and further afield.