Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from the Caribbean.

Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming.

Development of a Multiplex PCR and Magnetic DNA Capture Assay for Detecting Six Species Pathogens of the Genera Anaplasma and Ehrlichia in Canine, Bovine, Caprine and Ovine Blood Samples from Grenada, West Indies.

Infections with tick-borne pathogens belonging to Anaplasma/Ehrlichia in various vertebrate hosts are a persistent problem resulting in nonspecific clinical signs during early infection. Diagnosis of single and multi-infections with these pathogens, causing diseases in companion/agricultural animals and people, remains a challenge. Traditional methods of diagnosis, such as microscopy and serology, have low sensitivity and specificity. Polymerase chain reaction (PCR) assays are widely used to detect early-phase infections, since these have high sensitivity and specificity. We report the development and validation of an assay involving PCR followed by magnetic capture method using species-specific oligonucleotides to detect six Anaplasma/Ehrlichia species pathogens in canine, bovine, caprine, and ovine blood samples. Overall, the assay application to 455 samples detected 30.1% (137/455) positives for one or more out of six screened pathogens. Single-pathogen infections were observed in 94.9% (130/137) of the positive samples, while co-infections were detected in 5.1% (7/137). Anaplasma marginale infection in cattle had the highest detection rate (34.4%), followed by canines positive for Anaplasma platys (16.4%) and Ehrlichia canis (13.9%). The assay aided in documenting the first molecular evidence for A. marginale in cattle and small ruminants and Ehrlichia chaffeensis and Ehrlichia ewingii in dogs in the Caribbean island of Grenada.

Antimicrobial resistance patterns of commensal Escherichia coli isolated from feces of non-diarrheic dogs in Grenada, West Indies.

BACKGROUND AND AIM: There is currently no published information on the prevalence and antimicrobial susceptibility patterns of commensal Escherichia coli in dogs of Grenada origin. Monitoring antimicrobial resistance helps in the empirical selection of antibiotics. This study determined the occurrence of E. coli including the O157:H7 serotype in feces of non-diarrheic dogs of Grenada origin and the antibiotic resistance pattern of the E. coli isolates. MATERIALS AND METHODS: Fecal samples from 142 of the 144 (98.6%) dogs were culture positive for E. coli. Selection of up to three colonies from each of the 142 E. coli-positive samples yielded a total of 402 E. coli isolates, which were analyzed for the presence of non-sorbitol fermenting colonies, and O157-agglutination. RESULTS: Of the 402 E. coli isolates, 30 (7.5%) were non-sorbitol fermenters. However, none of the 402 isolates gave a positive reaction (O157:H7) to the E. coli O157:H7 latex kit. Antimicrobial susceptibility tests against 12 antibiotics revealed low resistance rates to all the tested antibiotics except for tetracycline (Te) (23.4%), cephalothin (CF) (13.2%), and ampicillin (AM) (7.7%). Thirty-nine out of the 402 (9.7%), E. coli isolates were resistant to two or more antibiotics of different classes. CONCLUSION: This is the first report of isolation and antimicrobial susceptibilities of commensal E. coli from non-diarrheic dogs in Grenada. Some of the isolates (39/402 isolates, 9.7%) were resistant to multiple antibiotics. This study showed that presently, dogs in Grenada should not be considered a reservoir for the E. coli O157:H7 serotype and for multiple antibiotic-resistant E. coli strains. Among the 402 E. coli isolates, the resistance rate to drugs other than Te, CF, and AM was very low.

Serovars and antimicrobial resistance of non-typhoidal Salmonella isolated from non-diarrhoeic dogs in Grenada, West Indies.

Non-typhoidal salmonellosis remains an important public health problem worldwide. Dogs may harbour Salmonella in their intestines and can easily shed Salmonella in the environment with the possibility of transmission to humans. Thus, monitoring is essential to understand the role of dogs in zoonotic transmission. The objectives of this study were to determine the shedding of Salmonella by owned, apparently healthy dogs in Grenada, West Indies, to identify the serovars, and to examine their antimicrobial susceptibility profiles. Faecal samples collected during August to October, 2016 from 144 non-diarrhoeic owned dogs were examined by enrichment and selective culture for the presence of Salmonella spp. Eight (5.6%) of the tested animals were culture positive, yielding 35 Salmonella isolates that belonged to six serovars of Salmonella enterica subspecies enterica. These were serovars Arechavaleta from two dogs, Arechavaleta and Montevideo from one dog, and Javiana, Rubislaw, Braenderup and Kiambu from one dog each. All these serovars have been reported as causes of human salmonellosis globally. Antimicrobial susceptibility tests on 35 isolates showed absence of resistance to the currently used drugs for cases of human salmonellosis, including ciprofloxacin and cefotaxime. One isolate (2.9%) was resistant to neomycin, two isolates (5.7%) showed intermediate susceptibility to neomycin, and another (2.9%) had intermediate susceptibility to tetracycline. This is the first report of isolation and antimicrobial susceptibilities of non-typhoidal Salmonella serovars from dogs in Grenada. This study shows that dogs in Grenada may be involved in the epidemiology of salmonellosis.

Natural exposure of bats in Grenada to rabies virus.

Introduction: Grenada is a rabies endemic country, where terrestrial rabies is maintained in the small Indian mongoose (Herpestes auropunctatus). The role of bats in the epidemiology of rabies in Grenada is unknown. A 1974 report described one rabies virus positive Jamaican fruit bat (Artibeus jamaicensis), and a high seroprevalence in this species. In the current study, the natural exposure to rabies virus in Grenadian bats was re-evaluated. It is postulated that bats serve as a natural rabies reservoir, probably circulating a bat-specific rabies virus variant. Material and methods: Bats were trapped in 2015 in all six parishes of Grenada using mist- and hand nets. For the detection of rabies virus in brain tissue, the direct fluorescent antibody test (dFAT) and the reverse transcription polymerase chain reaction (RT-PCR) were used. Serum neutralizing antibodies were determined using the fluorescent antibody virus neutralization test (FAVN). Results and discussion: Brain tissue and sera from 111 insectivorous and frugivorous bats belonging to four species were tested (52 Artibeus jamaicensis, two Artibeus lituratus, 33 Glossophaga longirostris, 24 Molossus molossus). Rabies virus antigen and genomic RNA were not detected in brain tissues. Rabies virus neutralizing antibodies were detected in the sera of eight A. jamaicensis in four of the six parishes. Bats in Grenada continue to show natural exposure to rabies virus. As rabies virus was not isolated in this study, serology alone is not sufficient to determine the strain of rabies virus circulating in A. jamaicensis bats in Grenada. Conclusion: Artibeus jamaicensis appears to play a role as a reservoir bat species, which is of public health concern in Grenada. Dispersion of bats to neighboring islands is possible and serological bat surveys should be initiated in these neighboring states, especially in those areas that are free of rabies in terrestrial mammals.

Campylobacter Species Isolated from Pigs in Grenada Exhibited Novel Clones: Genotypes and Antimicrobial Resistance Profiles of Sequence Types.

Infections caused by Campylobacter species pose a severe threat to public health worldwide. However, in Grenada, the occurrence and characteristics of Campylobacter in food animals, including pigs, remain mostly unknown. In this study, we identified the sequence types (STs) of Campylobacter from young healthy pigs in Grenada and compared the results with previous studies in Grenada and other countries. Antimicrobial resistance patterns and diversity of the Campylobacter clones were evaluated. Ninety-nine Campylobacter isolates (97 Campylobacter coli and 2 Campylobacter jejuni) were analyzed by multilocus sequence typing. Eighteen previously reported STs and 13 novel STs were identified. Of the 18 previously reported STs, eight STs (ST-854, -887, -1068, -1096, -1445, -1446, 1556, and -1579) have been associated with human gastroenteritis in different geographical regions. Among these 18 previously reported STs, ST-1428, -1096, -1450, and -1058 predominated and accounted for 18.2%, 14.1%, 11.1%, and 8.1% of all isolates, respectively. Of the 13 novel STs, ST-7675 predominated and accounted for 20% (4 of 20 isolates), followed by ST-7678, -7682, and -7691, each accounting for 10% (2 of 20 isolates). Antimicrobial resistance testing using Epsilometer test revealed a low resistance rate (1-3%) of all C. coli/jejuni STs to all antimicrobials except for tetracycline (1-10.1%). Some of the C. coli STs (13 STs, 24/99 isolates, 24.2%) were resistant to multiple antimicrobials. This is the first report on antimicrobial resistance and multidrug resistance patterns associated with Campylobacter STs recovered from swine in Grenada. This study showed that pigs in Grenada are not major reservoirs for STs of C. coli and C. jejuni that are associated with human gastroenteritis worldwide.