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.

Detection of serum antibodies against Leptospira spp. in brown rats (Rattus norvegicus) from Grenada, West Indies.

BACKGROUND AND AIM: Leptospirosis is an emerging disease of animals and humans. Among rodents brown rats (Rattus norvegicus) are an important reservoir of bacteria Leptospira. There is a paucity of information on reservoirs of Leptospira in Grenada. This study was conducted to estimate the prevalence of antibodies against Leptospira spp. in brown rats in a densely human populated area of Grenada. MATERIALS AND METHODS: Blood samples from 169 brown rats were collected and sera screened for antibodies against Leptospira spp. using enzyme-linked immunosorbent assay. RESULTS: Among a total of 169 brown rats trapped in two parishes in Grenada, 77/169 (45.5%) were positive for Leptospira spp. antibodies. A significant difference in seropositive population of brown rats between two collection sites was observed. No differences were found between sex and age of seropositive rats. CONCLUSION: Due to the close contact of brown rats with humans in Grenada, rats should be considered a high-risk factor in transmission of Leptospira to humans. Appropriate preventive measures should be instituted to prevent the transmission of Leptospira infection to humans.

Prevalence of antibodies against visceralizing Leishmania spp. in brown rats from Grenada, West Indies.

BACKGROUND AND AIM: Leishmania spp. are known to cause disease in man and animals. Rats are considered important reservoir hosts and transmission takes place through the bite of female sand fly, Phlebotomus spp. To the best of our knowledge, there is no published information on Leishmania infection in rats in Grenada. This study was conducted to estimate the antibodies for visceralizing Leishmania spp. (VL) in rats (Rattus norvegicus) from Grenada. MATERIALS AND METHODS: A total of 146 brown rats (R. norvegicus) were trapped live from two parishes (St. George and St. David) in Grenada. Following anesthesia, blood was collected from the heart through thoracic puncture. The serum was collected after the centrifugation of blood. Serum was tested for antibodies to VL. with a commercially available immunochromatographic dipstick test which is licensed for use in animals and humans. RESULTS: The seroprevalence of antibodies against Leishmania spp. was found in 34 of 146 rats (23.3%; CI 95% from 16.70 to 30.99). No significant differences were found between sexes and young or adults. The prevalence between parishes (St. George and St. David) was also not significant. CONCLUSION: The results show that rats (R. norvegicus) in Grenada are exposed to Leishmania spp. The rats could play an important role in the transmission of leishmaniasis to humans and other animals in Grenada.

Role of Rats ( Rattus norvegicus) in the Epidemiology of Toxoplasma gondii Infection in Grenada, West Indies.

Rodents are known to be reservoir hosts of Toxoplasma gondii infection for other animals, such as cats and pigs. From February to July 2017, 167 rats ( Rattus norvegicus) were trapped in Grenada, and serum, heart, skeletal muscle, and brain were examined for T. gondii infection by serological examination (modified agglutination test, 1:25) for T. gondii antibodies and for viable parasites by bioassay in mice. Samples of heart, skeletal muscle, and brain of all rats were bioassayed in Swiss Webster (SW) outbred albino mice and interferon gamma gene knockout (KO) mice. Toxoplasma gondii was isolated from heart and brain from 1 rat; this was the only seropositive rat. The T. gondii strain was avirulent for SW mice but killed KO mice. Tissue cysts were detected in the brains of SW mice, and tachyzoites were detected in the lungs of KO mice that died of acute toxoplasmosis. The strain was propagated in cell culture, and DNA derived from cell-cultured tachyzoites was genotyped using the 10 PCR restriction fragment length polymorphisms (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The strain was a clonal Type III (ToxoDB genotype no. 2) strain. Although the prevalence of T. gondii in humans and animals in Grenada is high, rats seem to have little importance in the transmission of T. gondii on this island.

Giardiasis: Serum antibodies and coproantigens in brown rats (Rattus norvegicus) from Grenada, West Indies.

AIM: Giardia is a serious zoonotic parasite, which causes diarrheal disease in humans and animals including rodents. The purpose of this study was to estimate the prevalence of Giardia spp. in brown rats (Rattus norvegicus) in Grenada. MATERIALS AND METHODS: Intestinal contents from 99 and serum samples from 169 brown rats (R. norvegicus) from Grenada were collected. These samples were examined for the Giardia coproantigens using Cryptosporidium/Giardia Quik Chek assay (Tech lab® Inc., USA), and the serum was screened through an enzyme-linked immunosorbent assay (ELISA) test kit for Giardia antibody (anti-GD) ELISA kit (MyBioSource, San Diego, CA, USA). RESULT: Giardia coproantigens were positive in 17.17% (95% confidence interval [CI]; 10.33-26.06%) rats, whereas 55% (95% CI: 47.20-62.68) were positive with serum antibodies (anti-GD) to Giardia. CONCLUSION: The prevalence of Giardia spp. in brown rats in Grenada was moderate based on the presence of coproantigens in the intestinal contents and antibody in serum. The findings of Giardia infections and prevalence in brown rats will help veterinarians and physicians to better plan diagnostic and preventative strategies. This is the first report of prevalence of Giardia in brown rats in Grenada.

Sarcocystis cymruensis: discovery in Western Hemisphere in the Brown rat (Rattus norvegicus) from Grenada, West Indies: redescription, molecular characterization, and transmission to IFN-γ gene knockout mice via sporocysts from experimentally infected domestic cat (Felis catus).

Rodents are intermediate hosts for many species of Sarcocystis. Little is known of Sarcocystis cymruensis that uses the Brown rat (Rattus norvegicus) as intermediate hosts and the domestic cat (Felis catus) as experimental definitive host. Here, we identified and described Sarcocystis cymruensis in naturally infected R. norvegicus from Grenada, West Indies. Rats (n = 167) were trapped in various locations in two parishes (St. George and St. David). Microscopic, thin (< 1 µm) walled, slender sarcocysts were found in 11 of 156 (7.0%) rats skeletal muscles by squash examination. A laboratory-raised cat fed naturally infected rat tissues excreted sporocysts that were infectious for interferon gamma gene knockout (KO) mice, but not to Swiss Webster outbred albino mice. All inoculated mice remained asymptomatic, and microscopic S. cymruensis-like sarcocysts were found in the muscles of KO mice euthanized on day 70, 116, and 189 post inoculation (p.i.). Sarcocysts from infected KO mice were infective for cats at day 116 but not at 70 days p.i. By transmission electron microscopy, the sarcocyst wall was "type 1a." Detailed morphological description of the cyst wall, metrocytes, and bradyzoites is given for the first time. Additionally, molecular data on S. cymruensis are presented also for the first time. Molecular characterization of sarcocysts 18S rDNA and 28S rDNA, ITS-1, and cox1 loci showed the highest similarity with S. rodentifelis and S. muris. In conclusion, the present study described the natural infection of S. cymruensis in Brown rat for the first time in a Caribbean country and provided its molecular characteristics.

Gastrointestinal parasites in small ruminants from Grenada, West Indies: A coprological survey and a review of necropsy cases.

Gastrointestinal parasites are important in small ruminant farming because they can impact negatively on the productivity of animals. The objectives of the present study were to estimate the prevalence of gastrointestinal parasites and to assess mortality attributable to gastrointestinal parasite infection in sheep and goats. We collected fecal samples from 114 sheep and 292 goats from 34 farms for coprological examination. In addition, we evaluated necropsy records for sheep and goats that were submitted from 2002 to 2016 to the pathology diagnostic laboratory in the School of Veterinary Medicine at St. George's University, Grenada. Out of 406 small ruminant (292 goat and 114 sheep) fecal samples examined, 385 were positive for gastrointestinal parasites, giving an overall prevalence of 95% (95% confidence interval (CI) 92% to 97%). All the 34 farms visited were found to have positive animals to at least one type of gastrointestinal parasite; 100% herd prevalence (95% CI: 88% to 100%). Among the 292 goat fecal samples examined, 285 were positive for gastrointestinal parasites (98%; 95% CI: 95% to 99%) whereas the proportion of positive fecal samples in sheep was 88% (95% CI: 80% to 93%). There was a significant difference in prevalence of gastrointestinal parasites between sheep and goats (p = .0002). The proportion of infection with coccidia in goats and sheep was 76% and 75%, respectively. For helminthes, the proportions were as follows: Moniezia spp., 14% in goats and 4% in sheep; Strongyloides spp., 36% in goats and 21% in sheep; strongyle type eggs 89% in goats and 66% in sheep. Mixed infections in both sheep and goats were more common (92%) than single ones (8%). Out of 220 necropsy records evaluated, 29% of mortality was due to Haemonchus contortus infection. Moniezia spp and Oesophagostomum spp. were also commonly found.

Seroprevalence of antibodies to astrovirus in chickens in Grenada, West Indies.

AIM: Chicken astroviruses (CAstV) are known to cause mild gastroenteritis, growth depression, and even mortality in poultry, especially in chickens, turkeys, and ducks. To the best our knowledge, there is no published information on CAstV in Grenada. This study was conducted to determine the prevalence of astrovirus in chickens in Grenada. MATERIALS AND METHODS: Blood samples from 366 indigenous chickens and 92 commercial chicken layers were collected from all parishes of the island and tested for antibodies against CAstV using commercial enzyme-linked immunosorbent assay. RESULTS: The seroprevalence of antibodies against astrovirus was 57.6% (95%, Confidence interval [CI]: 47.4-67.2) in commercial layers and 61.5% (95%, CI: 56.4-66.3) in indigenous chickens. The results show the presence of infection throughout the island. CONCLUSION: The results show the infection with CAstV in approximately half of the chicken population in Grenada. This is the first report on the prevalence of CAstV in chickens in Grenada and the Caribbean region.

Cysticercus fasciolaris in Brown Rats (Rattus norvegicus) in Grenada, West Indies.

Cat is the definitive host of Taenia taeniaeformis (T. taeniaeformis). Cysticercus fasciolaris (C. fasciolaris), the larval stage of T. taeniaeformis, develops in small rodents which act as intermediate host. The aim of this study was to estimate the prevalence of C. fasciolaris in brown rats (Rattus norvegicus) in the densely human populated parishes, St. George's and St. David's of Grenada, West Indies. One hundred and seventy rats were trapped near the residential areas from May to July, 2017 and examined for C. fasciolaris in their liver. Of the 170 rats 115 (67.6%, CI 95% from 60.1 to 74.6) were positive for the larval stage of T. taeniaeformis. One to three cysts were observed in each liver, containing a single larva in each cyst. The prevalence was 77.9% in St. George and 59.1% in St. David which is a significant difference (p < 0.05) between the two parishes under study. Based on gender, prevalence in males was 60.9% and females 74.7%. Significant difference was observed between young and adult rats (p = 0.03). Prevalence in young rats was 45.0% compared to adults (70.7%). Further study of risk assessment in the cat population in areas of the present research is strongly suggested.

Isolation and RFLP Genotyping of Toxoplasma gondii in Free-Range Chickens (Gallus domesticus) in Grenada, West Indies, Revealed Widespread and Dominance of Clonal Type III Parasites.

The objectives of the present cross-sectional study were to isolate and genotype Toxoplasma gondii in free-range chickens from Grenada, West Indies. Using the modified agglutination test, antibodies to T. gondii were found in 39 (26.9%) of 145 free-range chickens with titers of 25 in 7 chickens, 50 in 6 chickens, 100 in 2 chickens, and 200 or higher in 24 chickens. The hearts of the 39 seropositive chickens were bioassayed in mice; viable T. gondii was isolated from 20 and further propagated in cell culture. Genotyping of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed 4 genotypes, including ToxoDB PCR-RFLP no. 2 (Type III), no. 7, no. 13, and no. 259 (new). These results indicated that T. gondii population genetics in free-range chickens seems to be moderately diverse with ToxoDB no. 2 (Type III) as the most frequent (15/20 = 75%) compared to other genotypes in Grenada.

The phylogeography of rabies in Grenada, West Indies, and implications for control.

In Grenada, West Indies, rabies is endemic, and is thought to be maintained in a wildlife host, the small Indian mongoose (Herpestes auropunctatus) with occasional spillover into other hosts. Therefore, the present study was undertaken to improve understanding of rabies epidemiology in Grenada and to inform rabies control policy. Mongooses were trapped island-wide between April 2011 and March 2013 and examined for the presence of Rabies virus (RABV) antigen using the direct fluorescent antibody test (dFAT) and PCR, and for serum neutralizing antibodies (SNA) using the fluorescent antibody virus neutralization test (FAVN). An additional cohort of brain samples from clinical rabies suspects submitted between April 2011 and March 2014 were also investigated for the presence of virus. Two of the 171 (1.7%) live-trapped mongooses were RABV positive by FAT and PCR, and 20 (11.7%) had SNAs. Rabies was diagnosed in 31 of the submitted animals with suspicious clinical signs: 16 mongooses, 12 dogs, 2 cats and 1 goat. Our investigation has revealed that rabies infection spread from the northeast to the southwest of Grenada within the study period. Phylogenetic analysis revealed that the viruses from Grenada formed a monophyletic clade within the cosmopolitan lineage with a common ancestor predicted to have occurred recently (6-23 years ago), and are distinct from those found in Cuba and Puerto Rico, where mongoose rabies is also endemic. These data suggest that it is likely that this specific strain of RABV was imported from European regions rather than the Americas. These data contribute essential information for any potential rabies control program in Grenada and demonstrate the importance of a sound evidence base for planning interventions.

Genotypes, Antibiotic Resistance, and ST-8 Genetic Clone in Campylobacter Isolates from Sheep and Goats in Grenada.

Rectal swabs from 155 sheep and 252 goats from Grenada were evaluated to determine the prevalence of Campylobacter spp., antibiotic resistance, and multilocus sequence types. Fifteen Campylobacter isolates were obtained (14 C. jejuni and 1 C. coli). The prevalence (3.7%) did not differ significantly between sheep (4.5%) and goats (3.2%). Among the seven antimicrobials tested, resistance was only detected for tetracycline (30.8%) and metronidazole (38.5%). Campylobacter isolates showed no significant difference between sheep and goats for type of antimicrobial resistance or percent of resistant isolates. Twelve of the isolates were successfully genotyped consisting of four recognized clonal complexes and three novel sequence types. Importantly, one isolate from one goat was identified as the C. jejuni sequence type-8, a zoonotic and tetracycline-resistant clone reported to be a highly virulent clone associated with ovine abortion in the USA. Although most samples were from comingled sheep and goat production units, there were no shared sequence types between these two host species. None of the sequence types identified in this study have previously been reported in poultry in Grenada, suggesting sheep- and goat-specific Campylobacter clones in Grenada. This is the first report of genotyping of Campylobacter isolates from sheep and goats in the Eastern Caribbean.

Mycobacterium avium subspecies paratuberculosis confirmed following serological surveillance of small ruminants in Grenada, West Indies.

Surveillance for Mycobacterium avium subsp. paratuberculosis (Map) infection in small ruminants of Grenada was undertaken using a commercial enzyme-linked immunosorbent assay (ELISA). Among the 479 sheep tested, 11 (2.3%) were ELISA positive while only 1 out of 260 goats (0.3%) was ELISA positive. Five of the 12 ELISA-positive animals were also positive in a commercial agar gel immunodiffusion (AGID) assay, and 4 of these showed acid-fast rods consistent with Map in fecal smears. Two sheep that were test-positive by ELISA, AGID, and fecal smears were euthanized and necropsied. Both had gross and histological lesions of paratuberculosis affecting the ileocecal area of small intestines and adjacent lymph nodes. These tissues were successfully cultured in 2 of 3 variants of Middlebrook 7H10 medium. The identity of acid-fast organisms isolated from the tissues was confirmed as Map by multiplex conventional polymerase chain reaction. Using IS1311 amplification and Hinf I restriction digest analysis, isolates were identified as cattle (C) strains of Map. The current study describes Map infection in Grenada and confirms the presence of C type in sheep on the island of Carriacou. The low seroprevalence in clinically normal animals on the islands of Grenada and Carriacou suggests that control measures implemented in the near future may have a good chance of preventing spread of the infection.

Isolation and RFLP genotyping of Toxoplasma gondii from the mongoose (Herpestes auropunctatus) in Grenada, West Indies.

Little is known of the genetic diversity and epidemiology of Toxoplasma gondii infection in wildlife in Caribbean Islands. The prevalence and genetic diversity of T. gondii in mongooses (Herpestes auropunctatus) was investigated. During 2011 and 2012, 91 mongooses were trapped in different parts of Grenada, bled, euthanized, and examined at necropsy. Antibodies to T. gondii were found in 27 mongooses tested by the modified agglutination test (cut-off titer 25). Muscles (heart, tongue, neck) of 25 of the seropositive mongooses were bioassayed for T. gondii infection in mice. Viable T. gondii was isolated by bioassay in mice from four mongooses with MAT titers of 1:50 in two, 1:200 for one, and 1:400 for one mongoose. The four T. gondii isolates were further propagated in cell culture. Strain typing of T. gondii DNA extracted from cell-cultured tachyzoites using the 10 PCR-restriction fragment length polymorphism (RFLP) markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed one isolate belongs to the Type III (ToxoDB #2) lineage, two to ToxoDB#7 lineage, and one to the ToxoDB #216 lineage. This is the first report of T. gondii isolation and genotyping in H. auropunctatus worldwide.