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.
Infection due to the zoonotic protozoan parasite Toxoplasma gondii is worldwide in distribution. All warm-blooded animals including mammals and birds are suceptible to T. gondii (Robert-Gangneux and Dardé, 2012). Cats are important in the life cycle of T. gondii because they are the only known definitive hosts capable of shedding environmentally resistant oocysts in nature (Dubey, 2010). Sporulated oocyts serve as a source of infection for a wide range of intermediate hosts.
Free-range chickens are important in the epidemiology of T. gondii because they feed from the ground, thus getting exposed to different genotypes of T. gondii oocyts. Serological surveys revealed high T. gondii prevalence backyard chickens worldwide (Dubey, 2010).
Initially, T. gondii was considered clonal with low genetic diversity and grouped into 3 major lineages: types I, II, and III (Howe and Sibley, 1995). However, research has revealed that the genetic diversity of T. gondii is far greater than previously appreciated; the population structure of T. gondii is strongly subdivided by geographic region and by the existence of non-clonal lineages in some regions such as South America (Shwab et al., 2014). Importantly, some of these South American lineages are associated with severe ocular disease, suggesting that differences in clinical severity may be influenced by the parasite genotype (Khan et al., 2006).
There is limited information about genotypes and genetic diversity of T. gondii in free-range chickens in the Caribbean region. The objectives of this study were to isolate and speciate T. gondii in free-range chickens from Grenada by using the 10 PCR-RFLP markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico (Su et al., 2010).
MATERIALS AND METHODS
Location of the study
Grenada is a Caribbean island located 12 degrees north of the equator. This island consists of a warm, humid climate that averages annual temperatures ranging from 24 to 31 C. The 2 bodies of water that surround Grenada are the Caribbean Sea to the west and the Atlantic Ocean to the east.
Naturally infected backyard chickens
One hundred and forty-five backyard chickens from 5 parishes of Grenada were purchased, captured, and transported to the veterinary pathology diagnostic laboratory for necropsy, following humane euthanasia. Heart and blood were collected from each chicken. The blood was centrifuged at 2,500 g to separate serum. Sera and hearts were stored at 2 C for 2–3 days before they were shipped to the Animal Parasitic Diseases Laboratory (APDL), United States Department of Agriculture, Maryland, in order to evaluate T. gondii infection.
Sera from free-range chickens were tested for IgG antibodies to T. gondii by the modified agglutination test (MAT) as described by Dubey (2010). Sera were diluted 2-fold serially from 1:25 to 1:200. Titers equal or greater than 1:25 are considered positive. The MAT was found to be highly accurate for the detection of T. gondii infection in chickens (Dubey et al., 2016a).
Bioassay in mice
Heart samples from the 39 seropositive free-range chickens were bioassayed in outbred albino Swiss mice (National Cancer Institute, Bethesda, Maryland) following a previously published method (Dubey, 2010). For this, whole myocardial tissue (∼10–15 g) from each backyard chicken was homogenized in normal saline, digested in pepsin, centrifuged at 1,200 g, suspended in saline (0.85% aqueous NaCl), and neutralized with sodium bicarbonate and centrifuged at 1,200 g. The sediment was resuspended in normal saline containing 1,000 units of penicillin G and 100 μg of streptomycin per ml and the homogenate inoculated into 2 outbreed Swiss Webster mice. The mice that survived were bled 2 mo later and their sera examined for IgG antibodies to T. gondii using MAT at a serum dilution of 1:25. Brains of all surviving mice were examined for the presence of tissue cysts by brain squash smear preparations. Mice with no demonstrable antibodies to T. gondii and tissue cysts were considered not infected.
In vitro cultivation of T. gondii and RFLP genotyping
Mouse brain tissues infected with T. gondii isolates were seeded on to African green monkey kidney fibroblast cells (CV-1 cell line) culture flasks, and tachyzoites were harvested from the medium. DNA was extracted from cell-cultured tachyzoites, and genotyping was performed using 10 PCR-RFLP genetic markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, as described previously (Su et al., 2010). Appropriate positive and negative controls were included in each electrophoresis gel run. Designation of the T. gondii genotypes was done by referring to a T. gondii data base ( http://toxodb.org/), as described by Kissinger et al. (2003).
Out of the 145 free-range chickens, 39 (26.9%) were seropositive for T. gondii (Table I). Bioassay in mice of the 32 seropositive chicken hearts yielded 20 isolates designated as TgCkGr37 to TgCkGr57 (Table II). These isolates were further propagated in cell culture (CV-1 cells fibroblasts). Genotyping of T. gondii revealed 4 genotypes including ToxoDB PCR-RFLP no. 2 (clonal Type III, 15 isolates), no. 7 (atypical, 1 isolate), no. 13 (atypical, 3 isolates), and no. 259 (atypical and new, 1 isolate, Table III). Type III was significantly more prevalent than the rest of the genotypes. Phenotypically, all the isolates were avirulent for SW mice.
Toxoplasma gondii seroprevalence and mouse bioassay of chicken samples from Grenada.
Total bioassay (total isolated).
Toxoplasma gondii isolation from myocardium of chickens from Grenada.
No. of mice T. gondii positive/no. of mice inoculated.
PCR-RFLP genotyping of viable T. gondii isolates from chicken from Grenada.
Free-range chickens are important in the epidemiology of T. gondii because they feed from the ground and their infection with T. gondii is considered as an indicator of the level of environmental contamination (Dubey et al., 2005).
In the previous study of T. gondii in free-range chickens, molecular characterization of the 35 isolates using restriction fragment length polymorphism (RFLP) at 1 locus, SAG 2, revealed 29 isolates as type III, 1 isolate as type I, and 4 isolates as type II (Dubey et al., 2005). Subsequently, only 9 out of the 35 cryopreserved isolates from the aforementioned study were revived in cell culture and evaluated further using RFLP on 11 loci (SAG 1, SAG 2, alt.SAG 2, SAG 3, BTUB, GRA 6, L 358, PK 1, C22-8, C 29-2, and Apico); revealing 4 non-clonal (atypical) and 5 type III genotypes (Rajendran et al., 2012). The present study based on a large sample size sheds more light on the genotypes and molecular diversity of T. gondii in free-range chickens in Grenada.
In the present study, type III appears to be the most predominant strain, but diversity of T. gondii can be considered to be moderate. A related study on T. gondii genotyping in stray dogs in Grenada reported type III as the most common and with high diversity (Dubey et al., 2013). In rats from Grenada, the only isolate was also a type III (Dubey et al., 2006). A study on molecular characterization of T. gondii in mongoose (Herpestes auropunctatus) in Grenada revealed 3 genotypes, of which one was a type III (ToxoDB no. 2), 2 ToxoDB no. 7, and 1 ToxoDB no. 216 (Choudhary et al., 2013). In addition, the study in stray dogs in Grenada revealed 1 new genotype designated ToxoDB no. 224 (Dubey et al., 2013). In the present study, we also report a new genotype (ToxoDB no. 259) in chickens. Taken together, finding of new genotypes in the current study and the previous one in stray dogs has shed more light on the population diversity of T. gondii in Grenadian animals in particular and worldwide in general.
A study of toxoplasmosis in cats in St. Kitts and Nevis, West Indies, revealed types III and II, and 2 unique genotypes from the 7 isolates (Dubey et al., 2009). Similarly, a recent study based on limited molecular characterization of T. gondii DNA directly from tissues of small ruminants and pigs in St. Kitts and Nevis, West Indies, revealed Type III as the most predominant genotype (Hamilton et al., 2015). A recent study in dogs on the same island revealed 6 isolates of which 4 were type III and 2 were atypical (Dubey et al., 2016b).
Overall, the findings in our present study agree with the general trend that genetic diversity of T. gondii in Central and South America is high (Schwab et al., 2014), and interestingly the clonal type III is widespread and dominant in this area.
Regarding the virulence of T. gondii, type I genotype is highly virulent, whereas types II and III are not virulent in mice (Robert-Gangneux and Dardé, 2012). Recent studies investigating the genetic nature of virulent strains of T. gondii have shown that ROP18 and ROP5 gene allele types in T. gondii are highly predictive of virulence in mice (Dubey et al., 2014; Shwab et al., 2016). In the present study, none of the mice inoculated with the T. gondii chicken isolates from Grenada died, suggesting that all the isolates including the non-clonal ones were not virulent in mice. This is in contrast to studies in South America where the atypical strains exhibited virulence in mice and in humans (Dubey et al., 2005; Khan et al., 2006). A further molecular study on virulence of T. gondii strains found in Grenada, targeting 4 gene loci (ROP18, ROP5, ROP16, and ROP17), is recommended.
Genotyping analysis of 88 T. gondii isolates in immunocompromised patients in France revealed all 3 clonal genotypes and non-clonal ones as causal agents of illness (Ajzenberg et al., 2009). Given the emerging problem of HIV infection in Grenada in particular and the Caribbean in general, T. gondii will continue to pose a major threat as an opportunistic infectious agent in this group of people. In addition, free-range chickens contribute significantly to household food security as a source of meat and eggs in Grenada. In light of the findings in the present study, people should be advised to adequately cook free-range chicken meat before its consumption.
We would like to heartily thank SGU, a small grant initiative for funding the study. The technical assistance of Ray Samuel, Julia Coughlin, and Zachary Pearl is also highly appreciated.
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