Development of approaches for investigating the distribution of toxoplasma gondii infection in natural populations of animals and humans
Toxoplasma gondii is a globally distributed protozoan parasite that infects humans and a wide variety of warm-blooded animals. Although there are many surveys for T. gondii infection in mammals, little is known about the detailed distribution in localised natural populations. In this study we investigated host genotype and spatial location in relation to T. gondii infection and genotypes. We collected wood mice (Apodemus sylvaticus) from 4 sampling sites within a localised peri-aquatic woodland ecosystem which is relatively free of cats. Mice were genotyped using standard A. sylvaticus microsatellite markers and T. gondii and its genotypes were detected using 5 specific PCR based markers: SAG1, SAG2, SAG3, B1 and GRA6 directly from infected tissue. Of 126 wood mice collected, 44 samples gave positive reactions with T. gondii specific markers giving an infection rate of 34.92% (95% CI: 27.14%-43.59%). A total of 24/76 (31.58%, 95% CI: 22.19%-42.74%) males and 20/50 (40%, 95% CI: 27.59%-53.84%) female mice were found to be positive for T. gondii with no significant difference (P = 0.353). Juvenile, young adults and adults were infected at similar prevalences, respectively, 7/17 (41.18%), 27/65 (41.54%) and 10/44 (22.72%) with no significant age-prevalence effect (P = 0.23). Detailed analysis of the RFLP patterns and the DNA sequences for the SAG2, SAG3 and GRA6 loci showed a range of genotypes but, surprisingly, suggested that 30/44 (68.2%) infected mice had multiple genotypes (mixed infections) present. Results of genetic analysis of the mice showed that the collection consists of four genetically distinct populations. There was a significant difference in T. gondii infection in the different mouse genotypically derived populations (P=0.035) but not between geographically defined populations based on sampling location (P=0.29). In a parallel study, DNA was successfully collected from 88 human lung tissue samples. All samples showed successful amplification of the human α-tubulin gene and were used for T. gondii DNA detection. We used commonly used PCR markers (B1, SAG1, SAG2, SAG3, GRA6, APICO, L358, PK1, SAG1-Su, BTUB, alt.SAG2, c22-8 and c29-2), histological and immunohistochemical staining to confirm the presence of the parasite. All 88 tested samples were confirmed to be positive for T. gondii with markers B1, SAG1, SAG2 3’, SAG2 5’ and SAG3, giving a prevalence of 100% (95% CI: 95.82%-100%). From all successfully genotyped samples, 34 had single infection on all loci and 42 were of mixed infection on one or more loci with all three genotypes present. Type II genotype was the most predominant, followed by Type I and Type III. We detected 11 unusual genotypes. Immunohistochemistry was performed on 76 of the 88 tissue sections using commercial polyclonal antibodies produced in rabbits. All 76 sections were confirmed to be positive for T. gondii. A surprisingly high number of patients (96.05%) showed evidence of an active form of infection, as defined by the presence of tachyzoites or infected alveolar macrophages (or other cell types). Only three subjects (3.95%) had the dormant cyst stage as the only stage present. All 76 tissue sections were successfully stained with haematoxylin and eosin and observed under the light microscope. The presence of structures consistent with infection by the parasite was confirmed in 67 samples. All these patients are at risk of reactivation of chronic infection, leading to toxoplasmic encephalitis or pulmonary toxoplasmosis; which can complicate and delay their treatment or lead to death.
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