Rickettsia felis (Rickettsiales: Rickettsiaceae) is an emergent human pathogen that causes febrile illnesses in various parts of the world. This study describes the identification and growth characteristics of a R. felis-like organism (designated as Rickettsia sp. TH2014) cultured from Ctenocephalides orientis fleas in rural Malaysia. In this study, culturing of rickettsiae from filtered triturated flea lysates was performed in Aedes albopictus C6/36 cells. Cytopathic effects were observed from one of the samples 4 d post-inoculation. Electron microscopy revealed actively replicating intracytosolic coccobacillary organisms in the rickettsia-infected cells. Sequence analysis of amplified citrate synthase (gltA) gene fragment shows complete match of the rickettsia with Rickettsia sp. Rf31 in Southeast Asia, and 'Candidatus Rickettsia senegalensis' strain PU01-02 in Africa. The whole-genome sequence of Rickettsia sp. TH2014 was determined and assembled. The estimated genome size and guanine + cytosine content of the rickettsia are 1.37 Mb and 32.9%, respectively. The high values of average nucleotide identity and tetra-nucleotide signature correlation index obtained from pairwise genome comparison study suggest the identification of the rickettsia as R. felis. The whole-genome single-nucleotide polymorphism analysis demonstrates close genetic relatedness of the rickettsia with R. felis and Rickettsia asemboensis. However, based on sequence analyses of rickettsial genes (16S rDNA, gltA, ompB, and sca4), Rickettsia sp. TH2014 is found to be distinct from R. felis and R. asemboensis. The sequence analyses reveal that Rickettsia sp. TH2014 is highly similar to 'Ca. Rickettsia senegalensis' detected in fleas from Africa, Asia, and North America. Further investigation to provide insights on pathogenic potential and transmission dynamics of the rickettsia is warranted.
Flea-borne pathogens were screened from 100 individual cat fleas using a PCR approach, of which 38 % were infected with at least one bacterium. Overall, 28 % of the flea samples were positive for Bartonella as inferred from ITS DNA region. Of these, 25 % (7/28) were identified as Bartonella clarridgeiae, 42.9 % (12/28) as Bartonella henselae consisted of two different strains, and 32.1 % (9/28) as Bartonella koehlerae, which was detected for the first time in Malaysia. Sequencing of gltA amplicons detected Rickettsia DNA in 14 % of cat flea samples, all of them identified as Rickettsia asembonensis (100 %). None of the flea samples were positive for Mycoplasma DNA in 16S rRNA gene detection. Four fleas were co-infected with Bartonella and Rickettsia DNAs. Statistical analyses reveal no significant association between bacterial infection and mtDNA diversity of the cat flea. Nevertheless, in all types of pathogen infections, infected populations demonstrated lower nucleotide and haplotype diversities compared to uninfected populations. Moreover, lower haplotype numbers were observed in infected populations.
Rural communities in Malaysia have been shown to be exposed to Coxiella, Borrelia and rickettsial infections in previous seroprevalence studies. Further research is necessary to identify the actual causative agents and the potential vectors of these infections. The arthropods parasitizing peri-domestic animals in these communities may serve as the vector in transmitting arthropod-borne and zoonotic agents to the humans. Molecular screening of bacterial and zoonotic pathogens from ticks and fleas collected from dogs, cats and chickens from six rural communities in Malaysia was undertaken. These communities were made up of mainly the indigenous people of Malaysia, known as the Orang Asli, as well as settlers in oil palm plantations. The presence of Coxiella burnetii, Borrelia, and rickettsial agents, including Rickettsia and Anaplasma, was investigated by performing polymerase chain reaction (PCR) and DNA sequencing. Candidatus Rickettsia senegalensis was detected in one out of eight pools of Ctenocephalides felis fleas. A relapsing fever group Borrelia sp. was identified from one of seven Haemaphysalis hystricis ticks tested. The results from the PCR screening for Anaplasma unexpectedly revealed the presence of Candidatus Midichloria sp., a potential tick endosymbiont, in two out of fourteen Haemaphysalis wellingtoni ticks tested. C. burnetii was not detected in any of the samples tested. The findings here provide evidence for the presence of potentially novel strains of rickettsial and borrelial agents in which their impact on public health risks among the rural communities in Malaysia merit further investigation. The detection of a potential endosymbiont of ticks also suggest that the presence of tick endosymbionts in the region is not fully explored.
Fleas of the genus Ctenocephalides serve as vectors for a number of rickettsial zoonoses, including Rickettsia felis. There are currently no published reports of the presence and distribution of R. felis in India, however, the ubiquitous distribution of its vector Ctenocephalides felis, makes it possible that the pathogen is endemic to the region. This study investigates the occurrence of Rickettsia spp. infection in various subspecies of C. felis infesting dogs from urban areas of Mumbai, Delhi and Rajasthan in India.
Rickettsioses are emerging zoonotic diseases reported worldwide. In spite of the serological evidence of spotted fever group rickettsioses in febrile patients in Malaysia, limited studies have been conducted to identify the animal reservoirs and vectors of rickettsioses. This study investigated the presence of rickettsiae in the tissue homogenates of 95 wild rats and 589 animal ectoparasites. Using PCR assays targeting the citrate synthase gene (gltA), rickettsial DNA was detected in the tissue homogenates of 13 (13.7%) wild rats. Sequence analysis of the gltA amplicons showed 98.6-100% similarity with those of Rickettsia honei/R. conorii/R. raoultii (Rickettsiales: Rickettsiaceae). Sequence analysis of outer membrane protein A gene (ompA) identified Rickettsia sp. TCM1 strain from two rats. No rickettsia was detected from Laelaps mites, Rhipicephalus sanguineus and Haemaphysalis bispinosa ticks, and Felicola subrostratus lice in this study. R. felis was identified from 32.2% of 177 Ctenocephalides felis fleas. Sequence analysis of the gltA amplicons revealed two genotypes of R. felis (Rf31 and RF2125) in the fleas. As wild rats and cat fleas play an important role in the enzoonotic maintenance of rickettsiae, control of rodent and flea populations may be able to reduce transmission of rickettsioses in the local setting.