Evaluation of recombinant Babesia gibsoni thrombospondin-related adhesive protein (BgTRAP) for the sero-diagnosis of canine babesiosis.

Canine babesiosis, caused by Babesia gibsoni is one of the most significant tick-borne illnesses across the world. Light microscopy as well as polymerase chain reaction may fail in the diagnosis of disease when the level of parasitaemia is very low during subclinical and chronic cases. The serological techniques using a recombinant protein will be useful for the accurate and sensitive surveillance of the disease, especially in chronic cases. The present study describes the evaluation of recombinant N-terminal B. gibsoni Thrombospondin-related adhesive protein (BgTRAP) based indirect ELISA for the sero-diagnosis of B. gibsoni infection in dogs. A partial N-terminal BgTRAP gene (870 bp) of B. gibsoni, was expressed in Escherichia coli using a pET32a (+) vector. The recombinant BgTRAP based indirect ELISA was compared with the PCR targeting the same gene. A sensitivity and a specificity of 84% and 73.33% were observed in the indirect ELISA. The accuracy, positive predictive value and negative predictive value were 78.18%, 72.30%, 84.60% respectively. The rBgTRAP antigen did not show any cross-reactivity with sera from dogs infected with common helminth parasites viz. Ancylostoma caninum, Dirofilaria immitis, D. repens, Spirometra spp., Toxocara canis and haemoparasites like Trypanosoma evansi, Babesia vogeli, Hepatozoon canis and Ehrlichia canis.

Molecular detection of pathogens in the ova and unfed larvae of Rhipicephalus annulatus and Haemaphysalis bispinosa ticks infesting domestic cattle of south India.

This study aimed to investigate the presence of pathogens in the engorged ticks infesting domestic cattle, their ova, and unfed larvae. The engorged female ticks infesting domestic cattle of Wayanad district of Kerala, south India were collected and kept for oviposition. The dead females after the complete oviposition, their egg masses, and unfed larvae were screened for the presence of various pathogens by specific PCRs. The presence of Babesia bigemina, Anaplasma marginale, A. phagocytophilum, and Rickettsia spp. similar to R. raoultii was confirmed in Rhipicephalus annulatus ticks, their egg masses, and unfed larvae. Theileria orientalis was detected in Rh. annulatus females, but not in their egg masses or progenies. The presence of A. phagocytophilum and Rickettsia spp. similar to R. raoultii was confirmed in Haemaphysalis bispinosa ticks, their egg masses, and unfed larvae too. The presence of coinfections of B. bigemina with A. phagocytophilum and A. marginale were detected in Rh. annulatus ticks and their progenies.

Ticks and accompanying pathogens of domestic and wild animals of Kerala, South India.

The objective of the present study was to detect the chosen nucleotide DNA or RNA sequences of the pathogens in ticks of domestic and wild animals of Kerala, South India based on molecular techniques. Among 602 ticks collected, 413 were from bovines (cattle and buffalo), 26 from goats, 101 from dogs and 62 from wild animals. Amblyomma integrum, Am. gervaisi, Dermacentor auratus, Haemaphysalis bispinosa, Ha. intermedia, Ha. shimoga, Ha. spinigera, Rhipicephalus annulatus, Rh. microplus, Rh. haemaphysaloides and Rh. sanguineus s.l. were identified from various domestic and wild animals of Kerala. The cDNA synthesized from the RNA isolated from fully or partially engorged adult female/nymphal ticks was used as template for the specific polymerase chain reactions (PCR). Out of 602 ticks examined, nucleotide sequences of pathogens were detected in 28 ticks (4.65%). The nucleotide sequences of tick-borne pathogens like Theileria orientalis, Babesia vogeli, Hepatozoon canis, Anaplasma marginale, An. bovis, Rickettsia sp. closely related to Ri. raoultii, Ri. massiliae, Ri. africae and Ri. slovaca were detected. The identification of the previously unreported nucleotide sequences of rickettsial pathogens from India is of particular interest due to their zoonotic significance. The phylogenetic analysis of the major piroplasm surface protein (MPSP) gene of T. orientalis amplified from Rh. annulatus ticks revealed that they were genetically close to type 7, which belong to the highly pathogenic Ikeda group.

Molecular characterization of South Indian field isolates of bovine Babesia spp. and Anaplasma spp.

Ticks and tick-borne diseases (TTBDs) are considered major causes of economic loss in the livestock sector which incur an annual control cost estimated at US$ 498.7 million in India. Among these diseases, babesiosis, theileriosis and anaplasmosis are listed among the top ten livestock diseases in India and cause significant mortality and morbidity among cattle. However, molecular characterization of bovine Babesia and Anaplasma species are scant; thus, the aim of this study is to perform molecular characterization of field isolates of Babesia spp. and Anaplasma spp. infecting bovines in Kerala, South India. Blood smears and whole blood samples were collected from a total of 199 apparently healthy adult female cattle in Kerala. Based on microscopy, Babesia spp., Theileria orientalis and Anaplasma spp. organisms were detected in 9 (4.5%), 40 (20%) and 6 (3%) samples, respectively. Genus-specific polymerase chain reactions for amplification of 18S rRNA of Babesia spp. and 16S rRNA of Anaplasma spp. revealed positive results with 18 (9%) and 14 (7%) samples. The phylogenetic analysis of 18S rRNA gene sequences of Babesia spp. confirmed the existence of two different populations of Babesia spp. circulating in the blood of infected cattle viz., Babesia bigemina and a Babesia sp. genetically related to Babesia ovata. Further phylogenetic analysis using rap-1a sequences of isolates of B. bigemina revealed higher levels of genetic heterogeneity. However, the field isolates of B. bigemina displayed only slight heterogeneity when the rap-1c gene was examined. Polymerase chain reaction followed by sequencing and phylogenetic analysis of 16S rRNA gene of Anaplasma spp. revealed the existence of Anaplasma marginale, Anaplasma bovis and Anaplasma platys in bovines in South India. Based on msp4 gene sequences, all the field isolates of A. marginale from Kerala were clustered in a single clade with others isolated from around the world. To our knowledge, this study forms the first report on occurrence of B. ovata-like parasites and A. platys in cattle from India.