Molecular Identification Of Entamoeba Spp. Among Macaques In Southwest China

Objective Various Entamoeba species are frequently found in the feces of captive and wild nonhuman primates (NHP). Although the majority of these Entamoeba species are considered to be harmless, care should be taken when E. histolytica, the causative agents of amebiasis, or E.nuttalli, a potentially virulent species, is involved. E.histolytica or E.nuttalli infection in these primates is not only a serious problem for animal health but also has zoonotic potential, which make it important to discriminate E.histolytica. and E.nuttalli from other non-pathogenic amoeba. Most of the previous studies were based on the detection of cysts or trophozoites in feces by using light microscopy. However, it is difficult to distinguish E.histolytica and E.nuttalli from other Entamoeba species based on morphological features. For this purpose, molecular methods, such as PCR approach, are more appropriate. In this study, the objective was to identify the Entamoeba spp. in a large population of captive Macaca fascicularis and wild Macaca mulatta from Southwest China.Methods A total of955stool samples were obtained, including152and88from captive Macaca fascicularis in two colonies located in Guilin and Nanning, respectively,120and595from wild Macaca mulatta in two colonies located in Guangxi and Guizhou, respectively. Entamoeba cysts were detected after concentration. Genomic DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen) or FastDNA SPIN kit for soil (MP BIO) according the instructions of their manufacturers. The genomic DNA was then subjected to PCR amplification using primer sets specific for E.histolytica, E.dispar, E.nuttalli, E.chattoni, and E.coli targeting the18S ribosomal RNA gene. The sizes of the PCR products were then confirmed by electrophoresis on2%agarose gels. PCR products of the18S ribosomal RNA gene were purified using a gel extraction kit (Qiagen), then subjected to sequencing after inserted into the pMD-19T vector. Sequence data were analyzed using ClustalX Verl.83.Results All, except39, of the samples were positive for at least one Entamoeba species by PCR. The number of infected samples comprised100%,98.0%,93.4%, and61.3%of the total for E.chattoni,70.3%,81.2%,94.1%, and73.9%of the total for E.coli, in Guangxi, Guizhou, Guilin, and Nanning, respectively. Positive rates of E.dispar infection are82.9%,51.1%, and13.3%in Guilin, Nanning, and Guangxi, respectively. E.nuttalli DNA was detected in a large proportion (62.5%in Guangxi and61.3%in Guizhou) of the samples from wild Macaca mulatta while negative in all of the samples of captive Macaca fascicularis. However, no specific fragments were amplified from any of the samples using primer sets specific for E.histolytica. Sequence analysis of the partial18S ribosomal RNA gene amplified using primer sets specific for E.nuttalli showed a0.2%(1/452) difference in sequence, compared with the reported E.nuttalli NASA6in the overlapping region. Nucleotide sequence of the amplicon for E.chattoni was identical to AF149912in all of the samples, while that for E.dispar was identical to AB282661in all of the samples. Sequence analyses revealed the presence of two E.coli sequences which were identical to FR686448(Subtype2) and FR686413(Subtype1) respectively and a novel E.coli sequence.Conclusion This study describes the molecular identification of Entamoeba species from a large population of captive Macaca fascicularis and wild Macaca mulatta in Southwest China based on an Entamoeba species specific PCR approach. This study shows virulent E.histolytica is absent in these four colonies while high rate of E.nuttalli infection in wild Macaca mulatta in Southwest China. High prevalence of E.nuttalli, E.chattoni, E.coli, and E.dispar infections suggest that extensive transmission of Entamoeba occurs in the colonies. A novel strain of E.coli which was common in China was isolated. The monitoring of captive Macaca fascicularis and wild Macaca mulatta, coming into frequent contact with humans, is important for animal health and for prevention of zoonosis.