Piroplasmid infection is not associated with clinicopathological and laboratory abnormalities in cats from Midwestern Brazil.

Feline piroplasmids include the genera Babesia spp., Cytauxzoon spp., and Theileria spp. In Brazil, there are few reports regarding these hemoprotozoans; however, clinicopathological and molecular data are scarce. This study aimed to characterize the clinical relevance of these parasites through hematological, biochemical, and molecular approaches. For this purpose, 166 cats from Brasilia, Federal District, Midwestern Brazil, were screened using a quantitative polymerase chain reaction (qPCR) for piroplasmids based on the LSU4 mitochondrial gene, which resulted in an overall prevalence of 36/166 (21.7%). Twelve of 166 samples (7.2%) were positive for C. felis, while 19/166 (11.4%) were positive for Babesia vogeli. No samples tested positive for Theileria spp. Babesia vogeli and Cytauxzoon spp. LSU4 sequences showed identities of 97-100% and 99.3%, respectively, to US isolates. The hematological and biochemical findings did not differ significantly between the cats that tested positive and negative for piroplasmids. Although the lack of abnormalities in clinical and laboratory parameters does not eliminate the possibility that these cats were sick and recovered, it may suggest that the Brazilian strain of Cytauxzoon spp. is not as pathogenic as that from the USA, despite the high molecular identity with North American isolates.

Detection of Borrelia burgdorferi s.l., Anaplasma phagocytophilum and Babesia spp. in Dermacentor reticulatus ticks found within the city of Bialystok, Poland-first data.

Pathogens carried by ticks pose a threat to both human and animal health across the world. Typically associated with rural landscapes, ticks appear to adapt well to life in urban recreational areas. Although Dermacentor reticulatus is commonly found across Europe, data on the prevalence of pathogens in this tick species, in an urban environment, are very limited. PCR was used to examine 368 D. reticulatus individuals collected in the Zwierzyniecki Forest Nature Reserve in Bialystok, Poland. In total, 10.3% of ticks were infected, with Babesia spp. (9.2%), Anaplasma phagocytophilum (0.8%) and Borrelia burgdorferi sensu lato (0.3%). Rickettsia spp., Bartonella spp., and Coxiella burnetii were not detected. Sequence analysis for Babesia-positive samples identified 79.4% of them as Babesia canis, 8.8% as Babesia microti, 5.9% as Babesia spp., 2.9% as Babesia venatorum, and 2.9% as Babesia vogeli. Results obtained in this study indicate that D. reticulatus ticks found within the urban premises of the study area are infected with at least three pathogens and therefore are an important factor in public health risk for tick-borne diseases.

Molecular evidence of Babesia in pet cats in mainland China.

BACKGROUND: Babesia spp. are important emerging tick-borne protozoan hemoparasites, and pose a great impact on companion animals. Canine babesiosis has been well described worldwide, while felis babesiosis has primarily been reported from South Africa. To the best of our knowledge, Babesia spp. infections in dogs have been well elucidated in pet dog population in China, no report about Babesia spp. infection in cat population in mainland China. RESULTS: In this study, a total of 203 blood samples were collected from pet cats in Shenzhen city, and detected the presence of Babesia spp. with nested-PCR. Sequence comparison based on the 18S rRNA gene and ITS region revealed that three cats (1.48%) were infected with Babesia. vogeli. Notably, the sequences of ITS region obtained in this study shared the highest nucleotide identity with the sequence of B. vogeli strain isolated in cat from Taiwan. CONCLUSIONS: This study is the first report about babesiosis in domestic cats, and also provides molecular evidence of Babesia spp. infection in cat in mainland China. The data present in this study suggest B. vogeli may be circulating in cat population in mainland China. Further study to investigate the epidemiology of Babesia infection in cat nationwide is warranted.

Detection and molecular identification of Hepatozoon canis and Babesia vogeli from domestic dogs in Palestine.

Dogs serve as hosts for a great number of parasites, which may affect their health and wellbeing. This study aimed to observe tick borne pathogens in dogs from Palestine including Hepatozoon canis and Babesia species. The prevalence of both H. canis and Babesia species infections in apparently healthy dogs, from ten districts of the West Bank was surveyed. DNA was extracted from blood samples obtained from dogs (n = 362) and ticks (n = 213) collected from dogs (n = 77). A primer set that amplifies a partial sequence of the Babesia and Hepatozoon 18S rRNA gene was used for PCR and the DNA sequences of the PCR products of all samples were determined. Twenty-nine (8·0%) of the dogs were found infected including 20 with H. canis (5·5%), seven with Babesia vogeli (1·9%) and two with undefined Babesia spp. (0·6%). Twelve Rhipicephalus sanguineus s.l ticks were pathogen-positive, including ten with H. canis (4·7%), one with B. vogeli (0·5%), and one with Hepatozoon felis (0·5%). The results indicated that a wide range of tick borne pathogens is circulating in the canine population in the surveyed region. This study is the first report on the prevalence of H. canis, B. vogeli and Babesia spp. in dogs in Palestine and its results will assist in the management of diseases associated with these blood parasites.

Molecular prevalence and genetic characterization of piroplasms in dogs from Tunisia.

In this study, the prevalence of piroplasms in dogs was assessed using polymerase chain reaction (PCR) to identify Babesia and Theileria species in 200 dogs from Northern and Central Tunisia between spring and autumn 2014. The overall molecular prevalence for piroplasms was 14·5% ± 0·05 (29/200); PCR detected 2 species, namely Babesia vogeli and Theileria annulata with an overall prevalence of 12·5 ± 0·04 and 2% ± 0·02, respectively. No differences in the molecular prevalences of B. vogeli were revealed for age and sex (P > 0·05). The molecular prevalence of B. vogeli was significantly higher in central Tunisia (26·5% ± 0·01) compared with the North (9·6% ± 0·04) (P 0·05). Comparison of the partial sequences of 18S rRNA and Tams 1 genes confirmed the presence of 2 novel B. vogeli and T. annulata genotypes. This is the first molecular detection of T. annulata and genetic characterization of dogs' piroplasms in Tunisia. Further studies are needed to better assess the epidemiological feature of piroplasms infection in North Africa.

A survey of tick-borne pathogens in dogs and their ticks in the Pantanal biome, Brazil.

Tick and blood samples collected from domestic dogs in the Brazilian Pantanal were tested by molecular methods for the presence of tick-borne protozoa and bacteria. Among 320 sampled dogs, 3.13% were infected by Babesia vogeli (Piroplasmida: Babesiidae), 8.75% by Hepatozoon canis (Eucoccidiorida: Hepatozoidae), 7.19% by Anaplasma platys (Rickettsiales: Anaplasmataceae), and 0.94% by an unclassified Anaplasma sp. In three tick species collected from dogs, the following tick-borne agents were detected: (a) B. vogeli, An. platys and Ehrlichia canis (Rickettsiales: Anaplasmataceae), infecting Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks; (b) H. canis, an unclassified Anaplasma sp. and Rickettsia amblyommii (Rickettsiales: Rickettsiaceae), infecting Amblyomma cajennense sensu lato (Ixodida: Ixodidae) ticks, and (c) Rickettsia sp. strain Atlantic rainforest, an emerging human pathogen, infecting Amblyomma ovale ticks. Molecular analysis, based on a mitochondrial gene, revealed that the Am. cajennense s.l. ticks of the present study corresponded to Amblyomma sculptum, a member of the Am. cajennense species complex, and that Rh. sanguineus s.l. belonged to the tropical lineage. Whereas dogs are exposed to a number of tick-borne bacterial and protozoan agents in the Pantanal biome, humans are potentially exposed to infection by spotted fever group rickettsiae (e.g. R. amblyommii and Rickettsia sp. strain Atlantic rainforest) because both Am. sculptum and Am. ovale are among the most important human-biting ticks in Brazil.

First detection and molecular identification of Babesia vogeli from Rhipicephalus sanguineus (Acari: Ixodidae) in Taiwan.

A total of 578 Rhipicephalus sanguineus ticks collected from dogs in Taiwan were examined for Babesia by species-specific polymerase chain reaction assay based on the 18S small subunit ribosomal RNA (ssrRNA) gene. Babesia DNA was detected in 1.04 % (6/578) of Rh. sanguineus ticks. Phylogenetic analysis revealed that these Babesia spp. were genetically linked to the same clade within the genospecies of Babesia vogeli and could be discriminated from other genospecies of Babesia. Intra-species analysis based on the genetic distance values indicated a lower level (0.079) compared with other genospecies of Babesia (GD > 0.094) and out-group protozoa (GD > 0.236). This study provides the first molecular evidence of B. vogeli detected and identified in various stages of Rh. sanguineus ticks removed from dogs in Taiwan. Detection of Rh. sanguineus in flat male ticks may imply the possible mechanism of transstadial transmission in Rh. sanguineus ticks. The vector competence and the diversity of Babesia species harbored by Rh. sanguineus ticks need to be further investigated.

Piroplasmid Infections Among Domestic Dogs in the Mountain City of Rio de Janeiro, Brazil.

PURPOSE: The aim of the present study was to analyze the frequency of the piroplasmids in blood from dogs and ticks recovered from these animals in Teresópolis city, located in the mountain region of Rio de Janeiro state, Brazil. In addition to the clinical and hematological profile. METHODS: A total of 400 dogs attended in a veterinary clinic in this city between 2020 and 2021 were included. The blood was collected from the dogs, along with ticks and information on these dogs was obtained through a questionnaire applied to the owners. Thin-smear analyses and complete blood counts were performed. All forms characteristic of piroplasmids were measured and classified morphologically. The blood was also subjected to PCR assays based on the genes 18S rRNA and hsp70. In addition, the ixodid ticks were classified morphologically and subjected to PCR for piroplasmids research. The amplified products were sent for gene sequencing. RESULTS: Piroplasmids were detected in 2.3% of the dogs. The variables statistically associated with infections in these animals were hemorrhage/bleeding, jaundice, anisocytosis, activated monocytes and macroplatelets (p ≤ 0.05). Piriform, ring-shaped, oval and aberrant structures were viewed in erythrocytes, neutrophils and monocytes, with lengths greater than and less than 2.5 µm. The nine positive samples from these dogs were characterized as due to Rangelia vitalii. However, one sequence from B. vogeli was detected in a single adult specimen of R. sanguineus. CONCLUSION: Although circulation of two species of piroplasmids potentially infective for domestic dogs has been observed in the mountain city of Rio de Janeiro, infection due to R. vitalii was mostly seen in the dogs of the present study.

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.

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.

Molecular Survey of Tick-Borne Haemoparasites of Dogs by Multiplex Polymerase Chain Reaction from Punjab, India.

PURPOSE: Tick-transmitted parasites as Babesia gibsoni, Babesia vogeli, Ehrlichia canis, and Hepatozoon canis are major health concern for dogs. Owing to prevalence and infection severity, there is need of sensitive, specific, and affordable test for their simultaneous detection. METHODS: Prevalence of B. gibsoni, B. vogeli, E. canis, and H. canis infections was assessed on 719 blood samples by microscopy and multiplex PCR assay targeting 18S rRNA (B. gibsoni & H. canis), ITS1 & 5.8S rRNA (B. vogeli) and VirB9 gene (E. canis). An internal control (canine-actin) was also included to increase the accuracy of assay and effect of associated risk factors with disease prevalence was also studied. RESULTS: Microscopic prevalence of B. gibsoni, B. vogeli, E. canis and H. canis was 5.0%, 0.1%, 1.4% and 1.0%, respectively, whereas with multiplex PCR assay, the corresponding values were 8.9%, 1.1%, 2.6% and 5.1% besides concurrent infections of B. gibsoni & H. canis (0.4%), B. gibsoni & E. canis (0.4%), E. canis & H. canis (0.3%) and B. gibsoni & B. vogeli (0.1%). Analytical sensitivity of developed assay was 0.1pg (B. gibsoni & H. canis), 0.01pg (B. vogeli), and 1.0pg (E. canis). A ″fair″ (B. vogeli & H. canis) to ″substantial″ (B. gibsoni & E. canis) agreement between two tests was observed with data as statistically significant. Breed, sex and location were significantly associated with B. gibsoni infection. CONCLUSION: The developed multiplex PCR assay offers a potential solution to detect these pathogens simultaneously, aiding in timely diagnosis and effective disease management in suspected dogs.

A novel time-saving multiplex PCR assay for detecting and discriminating the most common canine Babesia species in Europe.

In Europe, most cases of canine babesiosis are caused by Babesia canis, Babesia vogeli (large piroplasms) and Babesia vulpes (small piroplasm). Molecular diagnosis is recommended due to its high sensitivity. Species identification after sequencing allows applying a rapid and efficient treatment, leading to a better prognosis; however, it is expensive and time-consuming. Thus, the objective of the present study was to develop a time-saving multiplex polymerase chain reaction (PCR) for simultaneously detecting and discriminating between large and small forms without sequence analysis. A new multiplex PCR was designed and tested using blood samples from 79 dogs showing clinical signs compatible with babesiosis which were previously analysed using blood smears and molecular methods. Multiplex PCR successfully discriminated between both Babesia groups showing bands of 700 and 890 bp for B. canis/B. vogeli and B. vulpes, respectively. No significant differences in the results of both PCR were detected and a substantial agreement between protocols (κ = 0.64) was found. Our multiplex PCR represents a reliable tool for detecting infections by the major Babesia spp. in dogs from Europe. Since no sequence analysis is required for identifying the species involved, this PCR allows the rapid administration of an appropriate treatment, thus improving the survival rate of the infected animals. In addition, it will represent a helpful tool for unravelling the real prevalence and distribution of B. vulpes and its implication in clinical cases.

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.

Canine piroplasmids: Molecular detection and laboratory characterization in dogs from Brasilia, Brazil, with the first molecular evidence of dog exposure to a novel opossum-associated Babesia sp.

Canine piroplasmid infections can be caused by Babesia spp., Theileria spp. and Rangelia vitalii. In Brazil, canine babesiosis caused by Babesia vogeli is endemic and reported throughout the country. On the other hand, Rangeliosis caused by R. vitalii has only been described so far in the South and Southeast regions. Despite that, studies analyzing the laboratory and molecular characterization of these hemoprotozoa are still scarce. To investigate the occurrence, the laboratory features, the molecular characterization, and the diversity of piroplasmids from Midwestern Brazil, a survey was performed using blood samples obtained from 276 domestic dogs from Brasília, Federal District, Midwestern Brazil. A broad-range quantitative PCR (qPCR) targeting the mitochondrial large subunit ribosomal DNA (LSU4) was used to detect piroplasmid DNA. The overall molecular occurrence of piroplasmids was 11.2% (31/276), with 9.7% (27/276) of the sequences identified as Babesia vogeli (98-100% identity to B. vogeli isolate from the USA). Based on a partial 18S rRNA sequence pairwise alignment (-250 bp), 1.4% (4/276) of the sequences showed only 76.8% identity with B. vogeli but 100% identity with opossum-associated Babesia sp. (MW290046-53). These findings suggest the exposure of dogs from Brazil to a recently described Babesia sp. isolated from white-eared opossum. None of the analyzed dogs was positive for Theileria spp. or R. vitalii. Subsequently, all positive sequences were submitted to three additional PCR assays based on the 18S rRNA, cox-1, and cytb genes, aiming at performing a haplotype network analysis. Haplotype network using cox-1 sequences showed the presence of six different haplotypes of B. vogeli; one of them was shared with isolates from Brazil, the USA, and India. When including animals co-infected with other vector-borne diseases, piroplasmid-positive dogs had 2.3 times higher chance of having thrombocytopenia than the negative ones. The molecular results demonstrated that the compared Babesia vogeli sequences showed a low variability as well as evidence of exposure to a putative novel opossum-associated Babesia sp. in dogs from Midwestern Brazil.

Co-infections of major tick-borne pathogens of dogs in Andhra Pradesh, South India.

Implementing hemoprotozoan control strategies in dogs has become difficult because of the co-infections. A multiplex polymerase chain reaction (PCR) was carried out for simultaneous detection of the co-infections of Babesia gibsoni, B. vogeli, Hepatozoon canis and Ehrlichia canis from dogs (N = 442) in Andhra Pradesh, South India. The co-infection combinations were classified as (i) B. gibsoni + B. vogeli + E. canis + H. canis (BEH), (ii) B. gibsoni + B. vogeli + E. canis (BE), (iii) B. gibsoni + B. vogeli + H. canis (BH) and (iv) E. canis + H. canis (EH) groups. The parasite-specific multiplex PCR amplified 18S rRNA gene of B. gibsoni, B. vogeli and H. canis and VirB9 gene of E. canis. The age, gender, breed, medium, living condition and region of dogs were studied as risk factors for co-infections using logistic regression model. Among the co-infections, the incidence was 1.81%, 9.28%, 0.69% and 0.90% for BEH, BE, BH and EH infections, respectively. Young age (< one year), females, mongrels, rural dogs, kennel dogs and presence of ticks were the identified risk factors for overall prevalence of tick-borne pathogens. The incidence of infection was less in rainy season, especially in dogs with a previous acaricidal treatment. The study concludes that the multiplex PCR assay could simultaneously detect natural co-infections in dogs, emphasizing the need for the assay in epidemiological studies to reveal the real pattern of pathogens and select pathogen-specific treatment protocols.

Detection and Molecular Characterization of Canine Babesiosis Causative Agent Babesia canis in Naturally Infected Dogs in the Dobrogea Area (Southeastern Romania).

Canine babesiosis is an emerging tick-borne disease of major veterinary concern in Europe. Its prevalence has increased in the last two decades and is spreading rapidly toward the north. The aim of this study was to investigate the genetic diversity of Babesia spp. strains isolated from naturally infected dogs in a tick-endemic area (Dobrogea) in southeastern Romania. For this purpose, a total of twenty-three samples from dogs diagnosed with various clinical forms of babesiosis, evaluated by means of clinical history, physical examination, and hematological tests, were subjected to a molecular investigation using PCR, sequencing analysis, and genetic characterization. A microscopic examination of thin Diff-quick-stained blood smears revealed large intra-erythrocytic Babesia piroplasms in all dogs. The PCR and sequencing analysis results indicated the presence of Babesia canis in 22 dogs (95.7%) and Babesia vogeli in 1 dog (4.3%). Among the B. canis isolates, two genotypes were distinguished based on two nucleotide substitutions (GA→AG) observed in the 18S rRNA gene sequences (at positions 609 and 610), with the AG genotype predominating (54.5% of samples), while the GA variant was identified in 9.1% of samples. In the remaining isolates (36.4%), both variants were identified. The B. vogeli-positive dog also tested positive for antibodies against Ehrlichia canis and displayed severe disease. This study reports, for the first time, the presence of genetically heterogenic B. canis strains in dogs with clinical babesiosis in Romania. These findings provide a basis for future studies on the relationship between the genetic structure of the causative agents of canine babesiosis in Romania and the course of the disease.

The Diverse Pathogenicity of Various Babesia Parasite Species That Infect Dogs.

Babesia species infect a very wide range of mammal hosts across the globe, and zoonotic infections are of growing concern. Several species of the Babesia genus infect dogs, and some of these cause significant morbidity and mortality. The Apicomplexan parasite resides within the red cell and infections result in direct damage to the host through intra- and extravascular hemolysis. An exuberant inflammatory response by the host to some species of Babesia parasites also results in significant collateral damage to the host. Canine infections have been the subject of many studies as the well-being of these companion animals is increasingly threatened by the spread of tick vectors and an increasingly mobile dog population. There are currently no widely available and effective vaccines, and effective treatment can be challenging. Understanding disease pathogenesis underlies the development of new treatments. The varying pathogenicity of the various Babesia parasite species that infect dogs offers an opportunity to explore the molecular basis for the wide range of diseases caused by infection with this parasite genus. In this review, we focus on what has been reported about the clinical presentation of Babesia-infected dogs in an attempt to compare the severity of disease caused by different Babesia species.

Prevalence and geographic distribution of Babesia conradae and detection of Babesia vogeli in free-ranging California coyotes (Canis latrans).

Babesia species are intraerythrocytic piroplasms that can result in disease characterized by hemolytic anemia and thrombocytopenia. Of the 5 species that are known to infect canids in the United States, Babesia conradae is most frequently diagnosed in California, and Babesia vogeli is prevalent in the US. Despite the recent re-emergence of B. conradae, the mechanism of transmission is not known. Coyotes (Canis latrans) have been a proposed reservoir of disease, and previous work has shown that dogs with known aggressive interactions with coyotes are at greater risk for infection. This study aimed to determine the prevalence of B. conradae in wild coyote populations in California to assess the viability of coyotes as a potential source of infection for domestic dogs. Four hundred and sixty-one splenic samples were obtained during post-mortem examination of coyote carcasses from Southern California, Fresno, and Hopland. Demographic data including age, sex, cause of death, and urbanity were collected for each coyote. DNA was extracted from samples and amplified using real-time PCR with primers specific for the B. conradae ITS-2 gene. The 18S gene was amplified and sequenced using conventional PCR primers specific to the Babesia genus from any coyotes positive for B. conradae. In total, 22 coyotes tested positive for B. conradae in Fresno (n = 15), Orange (n = 4), San Bernardino (n = 1), and Los Angeles counties (n = 1) with an overall prevalence of 4.8%. Coyotes from Fresno (P<.01) and rural coyotes (P<.01) were significantly more likely to be infected with B. conradae. Ten of 14 samples sequenced were 99-100% homologous to B. conradae, and 4 samples were 100% homologous with B. vogeli DNA indicating co-infection with both pathogens. This study demonstrates that coyotes can become infected and harbor B. conradae and B. vogeli and should be investigated as a possible source of infection in domestic dogs.

Development and validation of multiplex SYBR Green real-time PCR assays for detection and molecular surveillance of four tick-borne canine haemoparasites.

Two multiplex SYBR Green based real-time PCR assays were standardized and evaluated to detect DNA from four canine haemoparasites (Babesia gibsoni, Babesia vogeli, Ehrlichia canis and Hepatozoon canis), along with internal controls from dogs from selected districts of Punjab state, India. Amplicons of 126 bp, 337 bp, 234 bp and 106 bp corresponding to B. gibsoni (18S rRNA gene), B. vogeli (18S rRNA gene), E. canis (virB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. Microscopic evaluation of 200 blood samples from dogs revealed the prevalence of B. gibsoni, E. canis and H. canis as 1.5%, 1.5% and 1.0%, respectively, while with the multiplex real-time PCR assays the values for B. gibsoni, B. vogeli, E. canis, and H. canis were 8.0%, 1.5%, 3.5% and 23.5%, respectively, with concurrent infections of B. gibsoni and H. canis (3.5%); E. canis and H. canis (2.0%) and B. gibsoni, B. vogeli, E. canis, and H. canis (0.5%). The diagnostic sensitivity of the multiplex real-time PCR assays with respect to microscopy in the detection of B. gibsoni, E. canis and H. canis was 100% while the specificity for B. gibsoni, B. vogeli, E. canis, and H. canis was 93%, 100%, 98% and 77%, respectively, revealing the respective strength of agreement as ″fair″, ″slight″, ″moderate″ and ″slight″ by kappa value statistics, and the data were statistically significant, for detection of B. gibsoni and E. canis infections, by Fisher's exact test. The analytical sensitivity of the multiplex PCR assays in detection of DNAs was 8.59 × 105 and 9.9 × 106 copies for B. vogeli and E. canis, respectively, and 1.15 × 106 and 3.41 × 105 copies for B. gibsoni and H. canis, respectively. Assessment of risk factors viz. age, sex, breed, season and locations showed no significant association with the prevalence of these haemoparasites except for B. vogeli, E. canis and H. canis where significant associations were found for location, age and breed, respectively by multiplex real-time PCR assays.

Microscopic Detection, Hematological Evaluation and Molecular Characterization of Piroplasms from Naturally Infected Dogs in Rio de Janeiro, Brazil.

PURPOSE: This study aimed to analyze the frequency of piroplasmids in the blood of dogs in Rio de Janeiro, compare the performance of microscopic techniques, assess the risk factors associated with infections and also molecularly and morphologically characterize the piroplasmids identified. METHODS: In all, 407 blood samples were collected from dogs between 2018 and 2019. These were subjected to microscopic parasitological techniques for thin and thick smears, stained with Giemsa and using a rapid staining kit. The slides were read under an optical microscope and the protozoa were characterized morphometrically. In addition, the blood samples were subjected to molecular characterization for diagnosing piroplasmid species using primers that amplified the gene 18S rRNA. RESULTS: Piroplasmids were detected in 38 (9.3%) samples. Of these, 33 samples presented nucleotide sequences compatible with Babesia vogeli. Most of the positive samples were young, male, defined breeds dogs that had been attended in clinics in São Gonçalo city. Thrombocytopenia and leukopenia were the hematological alterations more observed in positive samples, but positive samples without alterations were also detected. The sex was the only variable that showed statistical differences. Males dogs being more often infected than females (p < 0.05). The microscope slides mostly showed piriform and oval merozoites measuring greater than 2.5 µm in length, which were compatible with B. vogeli. However, smaller forms were also identified, thus demonstrating the polymorphic nature of this parasite. CONCLUSION: Babesia vogeli was detected in blood samples from dogs in the metropolitan cities of Rio de Janeiro by molecular techniques in different parasite morphotypes.