In order to effectively prevent and treat sheep GIN infection, we need to deeply understand its epidemiological pattern. Infection rate is an essential indicator to determine the risk factors of population health, and a same infection intensity of different species may result different pathogenic potential. Therefore, accurate identification of different species and understanding of the parasitic epidemiology are the basis for developing sustainable parasite control measures. The conventional method of sheep GIN diagnosis is mostly by fecal examination. The nematode species are identified based on the biological characteristics of eggs and infectious larva found in fecal. For example, Trichuris and Nematodirus are easily identified according to their shapes and sizes. However, most species of Strongyloides are similar in size and shape, and usually cannot be accurately identified at the genus level, such as Haemonchus, Trichostrongylus, Teladorsagia, Cooperia, Bunostomum, etc. Therefore, fecal culture and morphometric analysis of the third stage larva (L3) must be conducted to identify the species, which requires considerable time and effort to identify the morphology of larva at various stages.
DNA molecular diagnostic technology has excellent specificity and sensitivity, and it is often used for the specific identification of GINs in livestock (Santos et al. 2020; Gasser 1999, 2006; Zarlenga et al. 2001). The most popular molecular markers are cytochrome c oxidase subunit I (cox1) of mitochondrial DNA (mtDNA), NADH dehydrogenase subunit 4 (Nad4) and the first and second internal transcribed spacers (ITS1 and ITS2, respectively) of ribosomal DNA (rDNA). Most studies consistently demonstrated that the ITS regions of rDNA could serve as reliable genetic markers for the specific identification of Strongyloides in domestic animals (Gasser 1999, 2006; Zarlenga et al. 2001; Chilton et al. 2004; Wimmer et al. 2004). The results of other studies have shown that the intraspecies sequence variation of ITS-1 and ITS-2 (usually < 1.5%) was much smaller than interspecies variation (Huby-Chilton et al. 2006). In this study, the sequence homological analysis of the three nematode species revealed intraspecific differences between 0.88% and 1.24%. By amplification and sequence analysis of the ITS-2 regions of parasite eggs or larva DNA, Santos et al. (2020) identified 12 species of seven genera, including Chabertia, Cooperia, Haemonchus, Oesophagostomum, Ostertagia, Teladorsagia, and Trichostrongylus. Elmahalawy (2018) used Droplet Digital™ PCR with designed ITS-2 primer/probe combinations of Haemonchus, Trichostrongylus, and Teladorsagiato and achieved effective results, distinguishing the most important sheep GINs in Sweden. In the present study, three pairs of ITS-2 primers targeting H.contortus, Trichostrongylus spp, and T. circumcincta were selected to amplify the fecal DNA and single larva DNA of naturally infected Kazakh sheep. As a result, single clear bands were obtained. In addition, no PCR amplification was detected using single larva DNA templates that belonged to different nematode species from the primers. It also provided evidence for the specificity of PCR. Through phylogenetic tree analysis, sequences of the same genus were clustered in one branch, while sequences of different genera were present in different branches, showing clear interspecific grouping. The ITS-2 sequences from different samples that located in the same branch of the same species cannot be effectively distinguished, therefore such intraspecies conserved genes with large interspecies differences could be used as ideal molecular markers for the taxonomic identification and evolutionary genetic research of various GIN species. In this study, Bunostomum trigoncephalum, Oesophagostomum, Nematodirus, and Marshallagia were all attempted to amplify respectively, but not all species could be detected. Presumably, it is because of the difference in infection levels of nematode species from different samples.
In this study, the same 93 fecal samples were used to carry out morphological identification of eggs and DNA molecular identification of hatched larva, respectively, and given different results. The infection rate tested by the saturated saline solution flotation method was 96.77%, whereas that by molecular identification was 100.00%, providing evidence for the high sensitivity of PCR. In addition, 37 samples were infected with all three dominant species as shown by saline solution flotation method, inconsistent with the results from molecular identification, which only showed three 3 triple infected fecal samples. Because of the similar size and shape of the eggs of Trichoencephalae, different species of nematodes may not be accurately distinguished when examining the eggs following the saturated saline solution flotation method. Different genera of nematodes may be mixed up too. For example, the egg size of H. contortus is (70–81) µm × (39–55) µm, and the egg size of Oesophagostomum. spp is (70–90) µm × (34–45) µm, and both of them are oval-shaped (Kong 2016; Zajac et al., 2012). Hence, the saturated saline solution flotation method may mistake the eggs of other species of nematodes as one of the three dominant species, thereby increasing the triple infection rate. In contrast, the primers for molecular identification were species-specific. The sequences of the obtained PCR products only identified nematodes of these three species and will not be mixed up with other species of nematodes. Therefore, molecular identification has a higher sensitivity that made the triple infection rate lower than the result of the saline solution flotation method. Other scholars have also conducted related research. For instance, to overcome the limitations of conventional methods such as fecal egg count (FEC) and/or larva culture (LC) in terms of sensitivity and specificity, Roeber et al. (2012)developed and evaluated a semi-automated, high-throughput multiplexed-tandem PCR (MT-PCR) platform that can be used for species- or genus-specific diagnosis of GIN infection within 24 hours, while the LC method requires 7–10 days. Hence the primary advantage of molecular identification is that it could run at least 96 samples in one day and eliminate any potential risk of “cross contamination”. Though the conventional saline solution flotation methods can diagnose an overt infection of Strongyloides within 1–2 days, only after the larva culture and microscopic identification by an experienced specialist, genus or species-specific diagnosis can be achieved. However, this culture method requires at least one week, and the fecal composition and culture conditions may cause significant changes in larva development, leading to bias in identification results (Roeber et al. 2011).