Molecular survey of Rickettsial organisms in ectoparasites from a dog shelter in Northern Mexico.

The objective of this study was to screen and identify rickettsial organisms in ectoparasites collected from dogs in a shelter in Gomez Palacio, Durango, Mexico. One hundred dogs were inspected for ectoparasites. All the dogs were parasitized with Rhipicephalus sanguineus ticks, three with Heterodoxus spiniger lice and one with Ctenocephalides felis fleas. DNA was extracted from the ectoparasites found on each dog, and PCR with the primers for the Anaplasmataceae 16S rRNA and citrate synthase gltA genes were performed. Eight DNA samples obtained from ticks, three from lice and one from fleas were positive to 16S rRNA. Only one sample from C. felis and one from H. spiniger were positive to gltA. Sequence analysis of amplified products from C. felis showed identity to Rickettsia felis, Wolbachia pipientis, and Wolbachia spp., while a sequence from H. spiniger showed identity to Wolbachia spp. Herein we report the molecular detection of R. felis, W. pipientis, and Wolbachia spp. in C. felis and H. spiniger in northern Mexico. These results contribute to the knowledge of the microorganisms present in ectoparasites from dogs in Mexico.

Molecular identification and characterization of Anaplasma platys and Ehrlichia canis in dogs in Mexico.

The tick-borne pathogens Ehrlichia canis and Anaplasma platys are the causative agents of canine monocytic ehrlichiosis (CME) and canine cyclic thrombocytopenia (CCT). Although molecular evidence of E. canis has been shown, phylogenetic analysis of this pathogen has not been performed and A. platys has not been identified in Mexico, where the tick vector Rhipicephalus sanguineus sensu lato (s.l.) is common. The aim of this research was to screen, identify and characterize E. canis and A. platys by PCR and phylogenetic analysis in dogs from La Comarca Lagunera, a region formed by three municipalities, Torreon, Gomez-Palacio and Lerdo, in the Northern states of Coahuila and Durango, Mexico. Blood samples and five engorged R. sanguineus s.l. ticks per animal were collected from 43 females and 57 male dogs presented to veterinary clinics or lived in the dog shelter from La Comarca Lagunera. All the sampled dogs were apparently healthy and PCR for Anaplasma 16S rRNA, Ehrlichia 16S rRNA, and E. canis trp36 were performed. PCR products were sequenced and used for phylogenetic analysis. PCR products were successfully amplified in 31% of the samples using primers for Anaplasma 16S rRNA, while 10% and 4% amplified products using primers for Ehrlichia 16S rRNA and E. canis trp36 respectively. Subsequent sequencing and phylogenetic analyses of these products showed that three samples corresponded to A. platys and four to E. canis. Based on the analysis of trp36 we confirmed that the E. canis strains isolated from Mexico belong to a conservative clade of E. canis and are closely related to strains from USA. In conclusion, this is the first molecular identification of A. platys and the first molecular characterization and phylogenetic study of both A. platys and E. canis in dogs in Mexico.

Infection and exposure to vector-borne pathogens in rural dogs and their ticks, Uganda.

BACKGROUND: In rural parts of Africa, dogs live in close association with humans and livestock, roam freely, and usually do not receive prophylactic measures. Thus, they are a source of infectious disease for humans and for wildlife such as protected carnivores. In 2011, an epidemiological study was carried out around three conservation areas in Uganda to detect the presence and determine the prevalence of vector-borne pathogens in rural dogs and associated ticks to evaluate the risk that these pathogens pose to humans and wildlife. METHODS: Serum samples (n = 105), blood smears (n = 43) and blood preserved on FTA cards (n = 38) and ticks (58 monospecific pools of Haemaphysalis leachi and Rhipicephalus praetextatus including 312 ticks from 52 dogs) were collected from dogs. Dog sera were tested by indirect immunofluorescence to detect the presence of antibodies against Rickettsia conorii and Ehrlichia canis. Antibodies against R. conorii were also examined by indirect enzyme immunoassay. Real time PCR for the detection of Rickettsia spp., Anaplasmataceae, Bartonella spp. and Babesia spp. was performed in DNA extracted from FTA cards and ticks. RESULTS: 99% of the dogs were seropositive to Rickettsia spp. and 29.5% to Ehrlichia spp. Molecular analyses revealed that 7.8% of the blood samples were infected with Babesia rossi, and all were negative for Rickettsia spp. and Ehrlichia spp. Ticks were infected with Rickettsia sp. (18.9%), including R. conorii and R. massiliae; Ehrlichia sp. (18.9%), including E. chaffeensis and Anaplasma platys; and B. rossi (1.7%). Bartonella spp. was not detected in any of the blood or tick samples. CONCLUSIONS: This study confirms the presence of previously undetected vector-borne pathogens of humans and animals in East Africa. We recommend that dog owners in rural Uganda be advised to protect their animals against ectoparasites to prevent the transmission of pathogens to humans and wildlife.

Molecular and immunological characterization of three strains of Anaplasma marginale grown in cultured tick cells.

Anaplasma marginale is an economically important tick-borne pathogen of cattle that causes bovine anaplasmosis. A wide range of geographic strains of A. marginale have been isolated from cattle, several of which have been characterized using genomics and proteomics. While many of these strains have been propagated in tick lines, comparative analyses after propagation in tick cells have not been reported. The overall purpose of this research therefore was to compare the degree of conservation of selected genes after propagation in tick cell culture among A. marginale strains from the U.S. (the Virginia strain) and Brazil (UFMG1 and UFMG2 strains). The genes studied herein included those which encode the proteins HSP70 and SODB involved in heat shock and stress responses, respectively, and two genes that encode major surface proteins MSP4 and MSP5. Strain identities were first confirmed by sequencing the tandem repeats of the msp1a gene which encodes for the adhesin, MSP1a. The results of these studies demonstrated that the genes encoding for both stress response and heat shock proteins were highly conserved among the three A. marginale strains. Antibodies specific for MSP4, MSP5, SODB and HSP70 proteins were used to further characterize the A. marginale strains, and they reacted with all of these strains propagated in tick cell culture, providing further evidence for antigenic conservation. Although antigenic differences were not found among the three A. marginale strains, multi-locus sequence analysis (MLSA) performed with nucleotide sequences of these genes demonstrated that the A. marginale Brazilian and U.S. strains fall in different clades. These results showed that phylogenetically distant strains of A. marginale are antigenically conserved, even after several in vitro passages, supporting the use of some of the above conserved proteins as candidates for universal vaccines.