Keshaw Tiwari

Seroprevalence of Toxoplasma gondii in Pigs, Sheep, Goats, and Cattle from Grenada and Carriacou, West Indies

abstract:  Prevalence of Toxoplasma gondii infection in pregnant women in Grenada is considered high. Little is known of the epidemiology of T. gondii infection in Caribbean Islands. Serum samples of 750 food animals in Grenada and Carriacou were tested for antibodies to T. gondii by the modified agglutination test (MAT). Antibodies to T. gondii (MAT, 1∶25 or higher) were found in 23.1% of 247 pigs, 44.1% of 204 sheep, 42.8% of 180 goats, and 8.4% of 119 cattle. Seroprevalence increased with age, indicating postnatal acquisition of T. gondii. Antibody titers of 1∶200 or higher were present in 65 of 90 seropositive sheep, 61 of 77 seropositive goats, and 23 of 57 seropositive pigs. However, none of the cattle had a MAT titer of 1∶200, suggesting that bovines are a poor host for T. gondii. Results indicate that pigs, sheep, and goats could be important sources of T. gondii infection if their meat is consumed undercooked.

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.

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 domestic dogs (Canis familiaris) from Grenada, West Indies revealed high genetic variability.

Stray dogs are considered as sentinels in the epidemiology of Toxoplasma gondii because they are carnivores and eat a variety of foods, including garbage. In the present study, tissues and sera of 249 stray dogs (Canis familiaris) from Grenada, West Indies were examined for T. gondii infection. Sera were examined for antibodies to T. gondii by the modified agglutination test (MAT); 89 (35.7%) of 249 were seropositive with titers of 25 in seven dogs, 50 in 22 dogs, 100 in 22 dogs, 200 or higher in 38 dogs. Hearts of 76 seropositive dogs were bioassayed in mice. Viable T. gondii was isolated from 12 dogs; these isolates were designated TgDogGr1 to TgDogGr12. These isolates were further propagated in cell culture and DNA isolated from cell culture derived tachyzoites of 12 isolates was genotyped using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The results revealed six genotypes, including ToxoDB PCR-RFLP #1, #2, #3, #7, #13 and #224, with 1, 6, 1, 2, 1 and 1 isolates, respectively. The result supports previous findings that T. gondii population genetics is highly diverse in Grenada.

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.

Spirocercosis in owned and stray dogs in Grenada

The aim of this retrospective study was to estimate the prevalence of Spirocerca lupi and its associated lesions in owned and stray dogs in Grenada. During 2001-2011 necropsies were carried out on 1022 owned and 450 stray dogs at the pathology diagnostic laboratory, School of Veterinary Medicine, St. Georges University, Grenada. Lesions due to S. lupi characterized by focal to multifocal granulomatous esophagitis with aneurysms, mineralized plaques and nodules in the adjacent thoracic aorta were found in 90 (8.8%; 95% confidence interval, 7.1-10.5%) of owned dogs and 64 (14.2%; 95% CI, 11.2-17.6%) of stray dogs. Stray dogs were significantly more affected by spirocercosis than owned dogs (p=0.0022). Of the 90 owned dogs with spirocercosis, 3 dogs had aberrant migration to the thoracic vertebral column with resultant spondylitis; 1 dog each had aberrant migration involving the stomach and the lung. Two dogs had ruptured aorta with hemothorax. Among the 64 stray dogs with spirocercosis, one dog had an esophageal granuloma that transformed into a fibroblastic osteosarcoma; spondylitis due to aberrant migration of S. lupi and hypertrophic osteopathy. We report spirocercosis for the first time in the dogs from a tropical island of Grenada.

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.

Antimicrobial susceptibility profiles of Escherichia coli recovered from feces of young healthy domestic pigs in Grenada, West Indies.

Aim: To determine the antimicrobial susceptibility profiles of E. coli isolates of porcine origin in Grenada, West Indies. Study Design: During the period of May to July, 2014, rectal swab samples were collected from pigs of six weeks to 12 week of age, from randomly selected small scale pig farms from the six parishes of Grenada and analyzed in the bacteriology lab in the Pathobiology Department, School of Veterinary Medicine, St. George’s University, Grenada. Methodology: A total of 180 rectal swab samples were examined for the presence of E. coli by culture. All the isolates were tested against 12 antibiotics using the standard Kirby-Bauer disc diffusion method. Results: All the 180 tested pigs were culture positive for E. coli . Antimicrobial susceptibility tests against 12 drugs showed susceptibility of all the E. coli isolates to amoxicillin-clavulanic acid, cefotaxime, ceftazidime, ciprofloxacin, imipenem, and gentamicin. Rate of resistance to tetracycline

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.

Abstract 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.

Retrospective study of canine heartworm disease with caval syndrome in Grenada, West Indies

Canine heartworm disease caused by Dirofilaria immitis is an important disease of dogs. The aim of this retrospective study was to estimate the prevalence of canine heartworm disease and evaluate cases of caval syndrome in dogs submitted for necropsy in Grenada. Out of 1617 dogs necropsied over a period of 13 years (2001-2013), 249 were found to be infected with D. immitis; giving an overall prevalence of 15.4% (95% confidence interval, 13.6% to 17.1%). There was no significant difference between male and female dogs with respect to canine heartworm infection (p = 0.3). During this period, the annual prevalence of canine heartworm disease was 22% in 2001 before slightly declining to an average of 18% in 2002-2003 and peaking at 26.8% in 2004-2005. From 2006 onwards, annual prevalence rates have steadily been decreasing; reaching the lowest (9%) in 2013. Among the 249 positive cases, 33 (13.2%) of the dogs had caval syndrome. Caval syndrome cases presented with concurrent clinical signs and were associated with cardio-pulmonary and hepatic gross lesions at necropsy. Aberrant migration of D. immitis was also noted in 2 dogs with caval syndrome. This is the first report which presents the findings of canine heartworm disease with caval syndrome in Grenada.