GENETIC AND MORPHOLOGIC CHARACTERIZATION OF DIASCHISTORCHIS PANDUS (DIGENEA: PRONOCEPHALIDAE) TREMATODES EXTRACTED FROM HAWKSBILL TURTLES, ERETMOCHELYS IMBRICATA (TESTUDINES: CHELONIIDAE), IN GRENADA, WEST INDIES.

The hawksbill turtle Eretmochelys imbricata is a critically endangered species with a worldwide distribution. Limited information is available about the naturally occurring intestinal parasites of this species and what impact these parasites may have on the health of the hawksbill turtle. Diaschistorchis pandus was identified postmortem in 5 hawksbill turtles from Grenada, West Indies, using morphologic characterization. Sanger sequencing was performed for conserved ribosomal regions (5.8S, ITS2, 28S) and the mitochondrial cytochrome c oxidase subunit 1 gene (COI). Phylogenetic analysis of the 28S rRNA gene sequence data shows D. pandus clustering with other trematodes in the family Pronocephalidae, corroborating morphological classification. No genetic sequences have been previously reported for this trematode species, which has limited the collection of objective epidemiological data about this parasite of marine turtles.

An analysis of time trends for canine heartworm disease in Grenada and its associated risk factors based on veterinary clinical pathology laboratory data base records between 2003 and 2015.

Canine heartworm (CHW) disease is a common parasitic infection in dogs in the Caribbean islands. However, studies on temporal trends and risk factors are limited for this region. This study represents the time trends in laboratory prevalence and risk factors of canine heartworm infections between 2003 and 2015. In this case series, 662 cases of laboratory-diagnosed heartworm were compared to 662 dogs without a laboratory diagnosis of heartworm (controls). One hundred and seventy two frozen serum positive samples were later analyzed for heartworm antigens using Heska solo® Step CH, and all 172 cases of microfilariae were confirmed as Dirofilaria immitis. Annual prevalence, linear trends and odds ratios (OR) for CHW were estimated using EPIINFO version 7 at a significance level of α = 0.05. Logistic regression was used to assess the association of CHW with variables showing a statistically significant univariate relationship. Laboratory prevalence of CHW decreased from 72 cases per 1000 dogs per year in 2003 to 15 cases per 1000 dogs per year in 2015 [X2 for linear trend = 151.8, p < 0.0001], with the occurrence of an epidemic of CHW between 2008 and 2010. The odds of CHW were higher among adult dogs [(OR) = 3.9 (95% CI, 2.9-7.0)] and geriatric dogs [OR = 2.1 (95% CI, 1.1-4.3)] compared to puppies. The odds of CHW were higher [OR = 1.3 (95% CI, 1.1-1.6)] among male dogs than female dogs, but the odds for CHW were lower among neutered dogs [OR = 0.4 (95% CI, 0.2 - 0.6)] compared to intact dogs. The odds of a dog being diagnosed with CHW were elevated [OR = 4.1 (95% CI, 3.2-5.2)] during the dry season compared to the rainy season. Our findings show that canine heartworm is extensive in laboratory submissions in Grenada. However, the laboratory prevalence of CHW decreased between 2003 and 2015, with an epidemic occurring between 2008 and 2010. Age of the dog, sex, neutered status, and seasonality of diagnosis were significantly associated CHW in Grenada.

Ectoparasites of brown rats (Rattus norvegicus) in Grenada, West Indies.

BACKGROUND AND AIM: Arthropod ectoparasites (mites, lice, ticks, and fleas) on common house rats or brown rats (Rattus norvegicus) are known to transmit zoonotic pathogens and diminish rat health. To the best of our knowledge, there is no published information regarding the prevalence of ectoparasites on R. norvegicus in Grenada. This study aimed to determine the prevalence and types of ectoparasites present on brown rats from Grenada. MATERIALS AND METHODS: One hundred sixty-eight rats were collected live from the parishes of St. George and St. David, Grenada, from May to July 2017. Following euthanasia, external parasites were collected using fine combs, thumb forceps, and paper tape. Tape samples and free specimens were placed in containers with 70% ethanol. External parasites were evaluated using dissection microscopy. RESULTS: Over 2000 ectoparasites were collected from 149 of the 168 trapped rats (88.7%). Ectoparasites identified included mesostigmatid mites (found on 84.6% of infested rats), fur mites in the families Atopomelidae and Listrophoridae (67.1%), Polyplax spp. lice (6.7%), fleas (3.4%), an unidentified larval tick (0.7%), and a mite in the family Myobiidae (0.7%). Infestation rates were higher in St. David Parish (86/89; 96.6%) than in St. George Parish (63/79; 79.7%) (p=0.001). When comparing sex and age, males had a marginal increase in infestation rate (83/89; 93.3%) compared to females (66/79; 83.5%) (p=0.054), and adults had an infestation rate of 90.7% (97/107) compared to juvenile rats who had a 66.7% (14/21) infestation rate. CONCLUSION: Brown rats in Grenada are heavily infested with ectoparasites, including known vectors of pathogens. Future studies will examine the prevalence of zoonotic pathogens in these arthropods.

Identification of Helminth Parasites from Selar crumenophthalmus in Grenada, West Indies.

HIGHLIGHTS: Helminths in Selar crumenophthalmus fish were identified by PCR. Two helminth taxa were identified: Anisakis typica and an unknown acanthocephalan. Neither taxon of helminth identified is zoonotic. To our knowledge, this is the first report of either type of helminth in fish in 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.

Canine generalized demodicosis treated with varying doses of a 2.5% moxidectin+10% imidacloprid spot-on and oral ivermectin: parasiticidal effects and long-term treatment outcomes.

Advocate(®) (2.5% moxidectin+10% imidacloprid) (Bayer HealthCare, Leverkusen, Germany) is a multiparasiticidal spot-on authorized for treating canine demodicosis in many countries. This blinded, randomized three-phase clinical trial compared its efficacy employing different dosing regimens with that of ivermectin. In the blinded first phase, 58 dogs suffering from generalized demodicosis were randomly assigned to one of four groups and treated with monthly, biweekly or weekly applications of Advocate(®), or with oral ivermectin (IVR) at 500 µg/kg daily. Dogs were evaluated clinically and multiple skin scrapings undertaken every 4 weeks until parasitological cure was achieved (defined as two consecutive series of deep skin scrapings at monthly intervals negative for all life forms). Forty dogs completed the 16-week initial blinded phase, with 5 cases achieving parasitological cure. Five dogs were deemed treatment failures and subsequently treated with ivermectin. The treatment protocol was then changed for the remaining 35 dogs and this cross-over phase (Phase 2) was maintained for a further 8 weeks with an additional 9 dogs achieving parasitological cure. Thereafter, all remaining animals were treated with IVR until cured (Phase 3). Overall, 26 dogs achieved parasitological cure during the clinical investigation. Of these, 23 remained disease-free for at least 12 months while two were lost to follow up and one died of unrelated causes. A total of 32 (55.2%) dogs were withdrawn at various stages of the investigation including the 5 dogs that were judged treatment failures. Other reasons for withdrawal included: non-compliance, lost to follow-up, ivermectin toxicity or reasons unrelated to the investigation. No adverse effects were attributable to the use of Advocate(®). Parasiticidal efficacy was assessed by changes in mite counts (live adult, juvenile and egg) and skin lesion extent & severity scores. Statistical significance was assessed using ANCOVA with initial mite counts or skin scores used as the covariate to account for variations in disease severity. Planned pairwise comparisons were used to identify differences between treatment groups. The efficacy of Advocate(®) increased with its rate of application across all measures of efficacy. Although ivermectin was shown to be more effective than Advocate(®) applied once weekly, both treatment protocols produced clinically satisfactory results. It was concluded that weekly application of Advocate(®) can be recommended as effective for the treatment of canine generalized demodicosis without the potential for toxicity associated with ivermectin.

Survey of helminths, ectoparasites, and chytrid fungus of an introduced population of cane toads, Rhinella marina (Anura: Bufonidae), from Grenada, West Indies.

One hundred specimens of Rhinella marina , (Anura: Bufonidae) collected in St. George's parish, Grenada, from September 2010 to August 2011, were examined for the presence of ectoparasites and helminths. Ninety-five (95%) were parasitized by 1 or more parasite species. Nine species of parasites were found: 1 digenean, 2 acanthocephalans, 4 nematodes, 1 arthropod and 1 pentastome. The endoparasites represented 98.9% of the total number of parasite specimens collected. Grenada represents a new locality record for Mesocoelium monas, Raillietiella frenatus, Pseudoacanthacephalus sp., Aplectana sp., Physocephalus sp., Acanthacephala cystacanth, and Physalopteridae larvae. The digenean M. monas occurred with the highest prevalence of 82%, contrasting many studies of R. marina where nematodes dominate the parasite infracommunity. Female toads were found to have a significantly higher prevalence of Amblyomma dissimile than male toads. Only 2 parasites exhibited a significant difference between wet and dry season with Parapharyngodon grenadensis prevalence highest in the wet season and A. dissimile prevalence highest during the dry season. Additionally, A. dissimile was significantly more abundant during the dry season.

New species of Parapharyngodon (Nematoda: Pharyngodonidae) in Rhinella marina (Anura: Bufonidae) from Grenada, West Indies.

Parapharyngodon grenadaensis n. sp. (Nematoda: Pharyngodonidae) from the large intestine of the cane toad, Rhinella marina, is described and illustrated. Parapharyngodon grenadaensis n. sp. is the 48th species assigned to the genus and the 16th species from the Neotropical region. It differs from other species in the genus by possessing 4 pairs of caudal papillae, an echinate anterior cloacal lip, and a blunt spicule of 67-104 µm. This is only the second report of R. marina harboring a species of Parapharyngodon.

Treatment of canine-generalized demodicosis: a blind, randomized clinical trial comparing the efficacy of Advocate(Bayer Animal Health) with ivermectin.

Advocate (moxidectin 2.5% + imidacloprid 10%) is a multiparasiticidal agent authorized for treating canine demodicosis in many countries. This blind, randomized clinical trial assessed the efficacy of Advocate at varying treatment intervals and compared it with that of oral ivermectin. Fifty dogs with generalized demodicosis were randomly assigned to one of four treatment groups: oral ivermectin (500 microg/kg once daily), Advocate applied at the authorized dose monthly (ADV1), every 2 weeks (ADV2) or weekly (ADV4). Each dog was evaluated every 4 weeks for 4 months or until negative scrapings at all sites resulted on two successive evaluations (parasitological cure). Miticidal efficacy was determined through deep skin scrapings taken from the same three sites on each occasion. Total numbers of live and dead adult mites, juveniles and eggs were determined. Thirty-five dogs completed the 4-month trial. Parasiticidal efficacy was assessed using several parameters including reduction in live adult mite counts. ancova analysis for this parameter confirmed that there were differences in efficacy among the treatment groups (P < 0.002). Tukey-Kramer all pairwise multiple comparison tests revealed that ADV4 was more effective than ADV1 (P = 0.016). Ivermectin was more effective than ADV1 (P = 0.003). Both ivermectin and ADV4 showed clinically substantial reductions in adult mite counts (89% for ADV4 and 98% for ivermectin). In conclusion, the efficacy of Advocate increased with the rate of application and weekly application may represent a new approach to the treatment of caninegeneralized demodicosis.