Does Perioperative Administration of Rabies Vaccine in Dogs Undergoing Surgical Sterilization Induce an Adequate Antibody Response?

High-volume spay/neuter events may facilitate access to free-roaming dogs to administer rabies vaccination, but important questions remain regarding the effect of surgery and anesthesia on the immune response to a vaccine administered in the perioperative period. This study evaluated the immunogenicity of primary rabies vaccination in dogs when administered during the immediate perioperative period at the time of surgical sterilization (ovariohysterectomy/orchidectomy). Healthy dogs of both sexes presenting for surgical sterilization who had never been vaccinated against rabies virus were eligible for enrollment in the study. Fifty dogs ranging in age from 5 to 96 months were enrolled and were vaccinated against rabies virus during the recovery period following anesthesia and surgery. Rabies virus neutralizing antibody (RVNA) titers were measured preoperatively and 28 days postoperatively. This cohort was compared to a historical control cohort of 57 dogs who received primary rabies vaccination for travel purposes and had RVNA titers measured at the same laboratory as the study group 28-35 days post-vaccination. After controlling for age and sex, there was no statistically significant difference in immunogenicity of a rabies vaccine administered to dogs during the perioperative period in comparison to dogs that received the rabies vaccine for travel alone in the absence of surgery. Perioperative administration of a rabies vaccine in dogs undergoing surgical sterilization induces an adequate antibody response. We recommend that rabies vaccine be administered perioperatively during spay/neuter campaigns in canine rabies endemic areas if other opportunities to access veterinary care and rabies vaccination are limited.

No evidence for sylvatic cycles of chikungunya, dengue and Zika viruses in African green monkeys (Chlorocebus aethiops sabaeus) on St. Kitts, West Indies.

BACKGROUND: Dengue, chikungunya and Zika viruses (DENV, CHIKV and ZIKV) are transmitted in sylvatic transmission cycles between non-human primates and forest (sylvan) mosquitoes in Africa and Asia. It remains unclear if sylvatic cycles exist or could establish themselves elsewhere and contribute to the epidemiology of these diseases. The Caribbean island of St. Kitts has a large African green monkey (AGM) (Chlorocebus aethiops sabaeus) population and is therefore ideally suited to investigate sylvatic cycles. METHODS: We tested 858 AGM sera by ELISA and PRNT for virus-specific antibodies and collected and identified 9704 potential arbovirus vector mosquitoes. Mosquitoes were homogenized in 513 pools for testing by viral isolation in cell culture and by multiplex RT-qPCR after RNA extraction to detect the presence of DENV, CHIKV and ZIKVs. DNA was extracted from 122 visibly blood-fed individual mosquitoes and a polymorphic region of the hydroxymethylbilane synthase gene (HMBS) was amplified by PCR to determine if mosquitoes had fed on AGMs or humans. RESULTS: All of the AGMs were negative for DENV, CHIKV or ZIKV antibodies. However, one AGM did have evidence of an undifferentiated Flavivirus infection. Similarly, DENV, CHIKV and ZIKV were not detected in any of the mosquito pools by PCR or culture. AGMs were not the source of any of the mosquito blood meals. CONCLUSION: Sylvatic cycles involving AGMs and DENV, CHIKV and ZIKV do not currently exist on St. Kitts.

Serosurvey for Brucella spp. and Coxiella burnetii in animals on Caribbean islands.

To determine the prevalence of antibodies to Brucella melitensis, Brucella abortus and Coxiella burnetii in animals on Caribbean islands we obtained sera from convenience samples of cattle (C), sheep (S), goats (G) and cats (F) from Dominica (C, S, G), Grenada (C, S, G), Montserrat (C, S, G), Puerto Rico (C), Nevis (C, S, G), St Kitts (C, S, G, F) and St Lucia (C, G). The sera were tested for antibodies against the Brucella spp. using commercial ELISA kits. Some sera were also tested at 1/80 for antibodies to C. burnetii using an indirect fluorescent antibody test. Positive sera were also tested at 1/640. None of 599 cattle, 462 sheep or 434 goats were positive in the Brucella ELISAs. None of 230 cattle had antibodies against C. burnetii, but one of 299 sheep was positive at 1/80 (Dominica - 1/54, 2%, 95% CI (0%-5.6%)), as were two of 314 goats, at 1/80 (Grenada - 1/53, 2%, 95% CI (0%-7.5%)) and 1/640 (St Kitts - 1/18, 5.6%, 95% CI (0%-16.7%)), and one of 34 cats, at 1/80 (St Kitts - 1/34; 3%, 95% CI (0%-8.8%)). Our data suggests that there is a very low prevalence or absence of B. melitensis and B. abortus on Caribbean islands. Coxiella burnetii, however, is present but it appears to be present on only some islands and then only at low levels. Overall, there appears to be a low threat to human and animal health from these organisms in the Caribbean.

Survey of vector-borne agents in feral cats and first report of Babesia gibsoni in cats on St Kitts, West Indies.

BACKGROUND: As there is little data on vector-borne diseases of cats in the Caribbean region and even around the world, we tested feral cats from St Kitts by PCR to detect infections with Babesia, Ehrlichia and spotted fever group Rickettsia (SFGR) and surveyed them for antibodies to Rickettsia rickettsii and Ehrlichia canis. RESULTS: Whole blood was collected from apparently healthy feral cats during spay/ neuter campaigns on St Kitts in 2011 (N = 68) and 2014 (N = 52). Sera from the 52 cats from 2014 were used to detect antibodies to Ehrlichia canis and Rickettsia rickettsii using indirect fluorescent antibody tests and DNA extracted from whole blood of a total of 119 cats (68 from 2011, and 51 from 2014) was used for PCRs for Babesia, Ehrlichia and Rickettsia. We could not amplify DNA of SFG Rickettsia in any of the samples but found DNA of E. canis in 5% (6/119), Babesia vogeli in 13% (15/119), Babesia gibsoni in 4% (5/119), mixed infections with B. gibsoni and B. vogeli in 3% (3/119), and a poorly characterized Babesia sp. in 1% (1/119). Overall, 10% of the 52 cats we tested by IFA for E. canis were positive while 42% we tested by indirect fluorescent antibody (IFA) for R. rickettsii antigens were positive. CONCLUSIONS: Our study provides the first evidence that cats can be infected with B. gibsoni and also indicates that cats in the Caribbean may be commonly exposed to other vector-borne agents including SFGR, E. canis and B. vogeli. Animal health workers should be alerted to the possibility of clinical infections in their patients while public health workers should be alerted to the possibility that zoonotic SFGR are likely circulating in the region.

First Molecular Characterization of Bovine Leukemia Virus Infections in the Caribbean.

Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leucosis. To investigate the presence and genetic variability of BLV in the Caribbean for the first time, we preformed fluorescence resonance energy transfer (FRET)-PCR for the pol of BLV on DNA from whole blood of cattle from Dominica, Montserrat, Nevis and St. Kitts. Standard PCRs with primers for the env were used for phylogenetic analysis of BLV in positive animals. We found FRET-PCR positive cattle (12.6%, 41/325) on Dominica (5.2%; 4/77) and St. Kitts (19.2%; 37/193) but not on Montserrat (0%, 0/12) or Nevis (0%, 0/43). Positive animals were cows on farms where animals were raised intensively. Phylogenetic analysis using the neighbor-joining (NJ) method on partial and full-length env sequences obtained for strains from Dominica (n = 2) and St. Kitts (n = 5) and those available in GenBank (n = 90) (genotypes 1-10) revealed the Caribbean strains belonged to genotype 1 (98-100% sequence homology). Ours is the first molecular characterization of BLV infections in the Caribbean and the first description of genotype 1 in the region.

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.

First report of Babesia gibsoni in Central America and survey for vector-borne infections in dogs from Nicaragua.

BACKGROUND: Although many vector-borne diseases are important causes of morbidity and mortality in dogs in tropical areas and potential zoonoses, there is little information on these conditions in Central America. METHODS: Seven qPCRs for vector-borne pathogens were performed on a Roche LightCycler PCR Instrument to investigate their prevalence in a convenience sample of whole blood samples from apparently healthy dogs in Nicaragua. Also, a qPCR targeting the canine hydroxymethylbilane synthase (HMBS) gene was used as an endogenous internal control and verified the quality and quantity of DNA in the samples was appropriate for the study. RESULTS: We found DNA of Rickettsia felis (5%), Babesia spp. (26%), Hepatozoon canis (51%), Anaplasma platys (13%) and Ehrlichia canis (56%) in the 39 dogs studied. The qPCRs for Coxiella burnetii and Dirofilaria immitis were negative. Of the 30 (80%) dogs that were positive by qPCR, 12 (31%) were positive for one agent, 11 (28%) for two, 3 (8%) for three, and 4 (10%) for four agents. CONCLUSIONS: This is the first report of B. gibsoni in dogs from Central America and the first recording of vector-borne agents in dogs from Nicaragua. Dogs in Nicaragua are commonly infected with a variety of vector-borne pathogens, some of which may also infect people.

Molecular detection of Dirofilaria immitis, Hepatozoon canis, Babesia spp., Anaplasma platys and Ehrlichia canis in dogs on Costa Rica.

Although vector-borne diseases are important causes of morbidity and mortality in dogs in tropical areas, there is little information on these conditions in Costa Rica. In PCRs of blood from dogs in Costa Rica, we did not detect DNAs of Rickettsia (R.) felis and Coxiella (C.) burnetii but we did find evidence of infection with Dirofilaria (D.) immitis (9/40, 22.5%), Hepatozoon (H.) canis (15/40, 37.5%), Babesia spp. (10/40, 25%; 2 with B. gibsoni and 8 with B. vogeli), Anaplasma (A.) platys (3/40, 7.5%) and Ehrlichia (E.) canis (20/40, 50%). Nine dogs (22.5%) were free of any vector-borne pathogens while 14 (35%) were infected with a single pathogen, 11 (27.5%) with two, 4 (10%) with three, 1 (2.5%) with four, and 1 (2.5%) with five pathogens. Dogs in Costa Rica are commonly infected with vector-borne agents.

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.

Tick-borne pathogens and disease in dogs on St. Kitts, West Indies.

Between 2009 and 2011, we conducted a case-control study of ticks and tick-associated pathogens affecting dogs on the island of St. Kitts, eastern Caribbean, including 55 cases of clinically suspected tick-borne disease (TBD) and 110 presumably healthy animals presented for elective surgeries. Rhipicephalus sanguineus caused year-round infestations of dogs, and 36% of the dogs in the study were infested at the time of examination. Overall, 62% of suspected TBD cases and 24% of presumably healthy dogs tested positive by PCR for infections with: Anaplasma platys (0% and 4%), Babesia canis vogeli (20% and 6%), Babesia gibsoni (18% and 5%), Ehrlichia canis (35% and 7%), and Hepatozoon canis (5% and 2%). Co-infections were documented in 15% of these PCR-positive dogs. Antibodies against A. platys or E. canis were noted in 36% of the dogs. Thrombocytopenia was the most common sign of infection, followed by anemia. This is the first detection of A. platys, B. canis vogeli, or H. canis on St. Kitts and the first detection of B. gibsoni in the Caribbean. We conclude that tick-borne pathogens of dogs are highly prevalent in this region and may present in dogs that appear healthy, in spite of hematologic abnormalities that may increase surgical risk.

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.