Development of a pan-Babesia FRET-qPCR and a survey of livestock from five Caribbean islands.

BACKGROUND: Babesia spp. are tick-borne protozoan hemoparasites and the second most common blood-borne parasites of mammals, in particular domestic animals. We used the Clustal Multiple Alignment program and 18S rRNA gene sequences of 22 Babesia species from GenBank to develop a PCR that could detect a wide variety of Babesia spp. in a single reaction. The pan-Babesia FRET-qPCR we developed reliably detected B. gibsoni, B. canis, B. vogeli, B. microti, B. bovis, and B. divergens under controlled conditions but did not react with closely related species, mainly Hepatozoon americanum, Theileria equi, and Toxoplasma gondii. RESULTS: When we tested the pan-Babesia FRET-qPCR on DNA of whole blood from 752 cattle, sheep, goats, donkeys and horses from five Caribbean islands, we detected Babesia spp. expected to be present in the animals, mainly B. bovis and B. bigemina in cattle and B. caballi in horses and donkeys. Further, we found that animals were not uncommonly infected with species of Babesia usually associated with other hosts, mainly B. vogeli and B. gibsoni in cattle, sheep and goats, B. rossi in goats, and B. caballi in goats and sheep. Finally, the pan-Babesia FRET-qPCR enabled us to identify unknown species of Babesia in cattle, goats, sheep and donkeys. CONCLUSIONS: Overall, 70 % (525/752) of the animals we tested were positive confirming earlier limited studies that infections with Babesia spp. are common in livestock in the Caribbean.

Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands.

BACKGROUND: The Ehrlichia are obligate intracellular Gram-negative tick-borne bacteria that are important human and animal pathogens. There is a need for assays to rapidly and reliably detect and differentiate the five generally recognized species into groups in a single reaction: E. canis, E. chaffeensis, E. ewingii, E. muris and E. ruminantium. METHODS: We developed primers and probes against the 16S rRNA gene to enable us to reliably detect the five major Ehrlichia spp. in a single FRET-qPCR. We tested the Ehrlichia FRET-qPCR on reference strains and on DNA from the blood of domestic ruminants from five Caribbean islands. The Ehrlichia present were determined using melting point analysis and by sequencing the Ehrlichia FRET-qPCR products as well as those of a nested PCR against the citrate synthase gene (gltA). RESULTS: Our Ehrlichia FRET-qPCR was negative for the closely related Anaplasma marginale and A. phagocytophilum but gave positive reactions with reference strains of the most generally recognized species and with other less characterized Ehrlichia of domestic ruminants, mainly E. ovina, the Panola Mountain Ehrlichia, and Ehrlichia sp. BOV2010. Melting point analysis revealed 4 distinct groups: E. ruminantium (T m ~55.8 °C); E. chaffeensis and E. ewingii (T m ~57.7 °C); E. canis, E. muris, E. ovina and Ehrlichia sp. BOV 2010 (T m ~62.0 °C); and the Panola Mountain Ehrlichia (T m ~65.5 °C). The detection limit of the FRET-qPCR was ~ 5 gene copies in a reaction and the sequences of the FRET-qPCR products were as expected. With DNA from domestic ruminants from the Caribbean we found 12.2 % (134/1,101) positive: cattle (76/385; 19.7 %), sheep (45/340; 13.2 %) and goats (13/376; 3.5 %). Melting point analysis and sequencing of the FRET-qPCR and nested PCR gltA products showed the Ehrlichia we detected were E. canis or very closely related organisms. CONCLUSIONS: In a single reaction, our Ehrlichia FRET-qPCR can detect the Ehrlichia spp. we studied and differentiate them into four groups. Domestic ruminants in the Caribbean are not uncommonly exposed to Ehrlichia, possibly E. canis or very closely related organisms.

Salmonella enterica prevalence in leatherback sea turtles (Dermochelys coriacea) in St. Kitts, West Indies.

Salmonella spp. are gram-negative bacteria capable of causing diseases in a wide range of aquatic and terrestrial animals, including humans. Sea and terrestrial turtles have been recognized as carriers of this zoonotic pathogen. In this project, conventional and molecular diagnostic methods were combined to investigate the prevalence of Salmonella enterica in leatherback sea turtles (Dermochelys coriacea) that used the island of St. Kitts, West Indies as a nesting ground during 2011 (n = 21). Isolates obtained from selective media were screened and colonies suspected of being Salmonella spp. were confirmed by fluorescence resonance energy transfer polymerase chain reaction. The prevalence of S. enterica within this sample population during this period was found to be 14.2%. Moreover, due to the increasing risk of antibiotic resistance in enteric bacteria, antimicrobial susceptibility was investigated in all recovered Salmonella spp. isolates utilizing the broth microdilution method. All isolates were susceptible to the lowest concentration of kanamycin, gentamicin, ciprofloxacin, enrofloxacin, nalidixic acid, and trimethoprim/sulfamethoxazole tested. Further research should be pursued to understand the interaction of this bacterial pathogen with the environment, host, and other microbial communities, and to further develop faster, more sensitive, and more specific diagnostic methods.

Ehrlichiosis, babesiosis, anaplasmosis and hepatozoonosis in dogs from St. Kitts, West Indies.

BACKGROUND: Although tick-borne diseases are important causes of morbidity and mortality in dogs in tropical areas, there is little information on the agents causing these infections in the Caribbean. METHODOLOGY: We used PCRs to test blood from a cross-section of dogs on St Kitts for Ehrlichia (E.) canis, Babesia (B.) spp., Anaplasma (A.) spp. and Hepatozoon (H.) spp. Antibodies against E. canis and A. phagocytophilum/platys were detected using commercial immunochromatography tests. Records of the dogs were examined retrospectively to obtain clinical and laboratory data. PRINCIPAL FINDINGS: There was serological and/or PCR evidence of infections of dogs with E. canis (27%; 46/170), Babesia spp. (24%; 90/372) including B. canis vogeli (12%; 43/372) and B. gibsoni (10%; 36/372), A. platys (11%; 17/157) and H. canis (6%; 15/266). We could not identify the Babesia sp. detected in nine dogs. There was evidence of multiple infections with dual infections with E. canis and B. canis vogeli (8%; 14/179) or B. gibsoni (7%; 11/170) being the most common. There was agreement between immunochromatography and PCR test results for E. canis for 87% of dogs. Only 13% of exposed dogs had signs of a tick-borne disease and 38% had laboratory abnormalities. All 10 dogs presenting for a recheck after treatment of E. canis with doxycycline were apparently healthy although all remained seropositive and six still had laboratory abnormalities despite an average of two treatments with the most recent being around 12 months previously. Infections with Babesia spp. were also mainly subclinical with only 6% (4/67) showing clinical signs and 13% (9/67) having laboratory abnormalities. Similarly, animals with evidence of infections with A. platys and H. canis were largely apparently healthy with only occasional laboratory abnormalities. CONCLUSIONS: Dogs are commonly infected with tick-borne pathogens in the Caribbean with most having no clinical signs or laboratory abnormalities.

Identification of feline immunodeficiency virus subtype-B on St. Kitts, West Indies by quantitative PCR.

INTRODUCTION: Although antibodies to the feline immunodeficiency virus (FIV) have been detected by SNAP assay in cats from St. Kitts, there have been no molecular studies to further confirm the infection and determine the FIV subtypes present. METHODOLOGY: Total nucleic acids were extracted from EDTA whole blood specimens from 35 cats, followed by quantitative fluorescence resonance energy transfer (FRET) PCR under a six-channel LightCycler 2.0 Instrument with Software version 4.1. RESULTS: Four of 11 stray cats (36 %) but none of 24 owned cats were FIV positive by real-time PCR.  High-resolution melting curve analysis indicated that all four positive cats were infected with FIV subtype-B. CONCLUSIONS: This is the first molecular characterization of FIV subtypes on St. Kitts and the results confirm the high prevalence of FIV infection in stray cats on the island.