First report of four rare strongylid species infecting endangered Przewalski's horses (Equus ferus przewalskii) in Xinjiang, China.

BACKGROUND: The Przewalski's horse (Equus ferus przewalskii) is the only surviving wild horse species in the world. A significant population of Przewalski's horses resides in Xinjiang, China. Parasitosis poses a considerable threat to the conservation of this endangered species. Yet, there is limited information on the nematode parasites that infect these species. To deepen our understanding of parasitic fauna affecting wild horses, we identified the intestinal nematodes of Przewalski's horses in Xinjiang and added new barcode sequences to a public database. METHODS: Between 2018 and 2021, nematodes were collected from 104 dewormed Przewalski's horses in Xinjiang. Each nematode was morphologically identified to the species level, and selected species underwent DNA extraction. The extracted DNA was used for molecular identification through the internal transcribed spacer 2 (ITS2) genetic marker. RESULTS: A total of 3758 strongylids were identified. To the best of our knowledge, this is the first study to identify four specific parasitic nematodes (Oesophagodontus robustus, Bidentostomum ivashkini, Skrjabinodentus caragandicus, Petrovinema skrjabini) and to obtain the ITS2 genetic marker for P. skrjabini. CONCLUSIONS: The ITS2 genetic marker for P. skrjabini enriches our understanding of the genetic characteristics of this species and expands the body of knowledge on parasitic nematodes. Our findings extend the known host range of four strongylid species, thereby improving our understanding of the relationship between Przewalski's horses and strongylids. This, in turn, aids in the enhanced conservation of this endangered species. This study introduces new instances of parasitic infections in wild animals and offers the DNA sequence of P. skrjabini as a valuable resource for molecular techniques in nematode diagnosis among wildlife.

Assessment of Adaptation Status of Reintroduced Equus Przewalskii Based on Comparative Analysis of Fecal Bacteria with Those of Captive E. Przewalskii, Domestic Horse and Mongolian Wild Ass.

Intestinal microbiota play an important role in the survival of the host. However, no study to date has elucidated the adjustment of intestinal microbiota of the host during rewilding. Thus, this study aims to describe the intestinal bacterial community of reintroduced Przewalski's horse (RPH) after being released into their original habitat for approximately 20 years in comparison with that of captive Przewalski's horse (CPH), sympatric domestic horse (DH) and Mongolian wild ass (MWA) by sequencing the 16S rRNA gene. The results showed that the prevalent bacterial communities were different among CPHs, RPHs, DHs and MWAs at the family level. NMDS and ANOSIM analysis showed that the pattern of bacterial community composition in captive equines was distinct from that in the wild groups. It is shown that some bacteria had significant differences among different taxa (p < 0.001), such as Firmicutes, Bacteroidetes, Armatimonadetes, Clostrida, Bacteroidia, Clostridiales, Bacteroidales, Rikenellaceae and Bacteroidales_UCG-001. These bacteria were associated with the transition from in captive to in the wild (CPH and RPH), which reflected the change of environmental conditions. Meanwhile, Proteobacteria, Clostridia, Bacilli, Negativicutes, Gammaproteobacteria, Clostridiales, Bacillales, Selenomonadales, Pseudomonadales and Planococcaceae were the changed groups among RPHs, MWAs and DHs, which are related to feeding habits and diseases. Our results clearly showed the differences between intestinal microbiota in reintroduced animals and wild animals and led us to understand the survival state of reintroduced animals in the wild.

The Role of Intestinal Microbial Metabolites in the Immunity of Equine Animals Infected With Horse Botflies.

The microbiota and its metabolites play an important role in regulating the host metabolism and immunity. However, the underlying mechanism is still not well studied. Thus, we conducted the LC-MS/MS analysis and RNA-seq analysis on Equus przewalskii with and without horse botfly infestation to determine the metabolites produced by intestinal microbiota in feces and differentially expressed genes (DEGs) related to the immune response in blood and attempted to link them together. The results showed that parasite infection could change the composition of microbial metabolites. These identified metabolites could be divided into six categories, including compounds with biological roles, bioactive peptides, endocrine-disrupting compounds, pesticides, phytochemical compounds, and lipids. The three pathways involving most metabolites were lipid metabolism, amino acid metabolism, and biosynthesis of other secondary metabolites. The significant differences between the host with and without parasites were shown in 31 metabolites with known functions, which were related to physiological activities of the host. For the gene analysis, we found that parasite infection could alarm the host immune response. The gene of "cathepsin W" involved in innate and adaptive immune responses was upregulated. The two genes of the following functions were downregulated: "protein S100-A8" and "protein S100-A9-like isoform X2" involved in chemokine and cytokine production, the toll-like receptor signaling pathway, and immune and inflammatory responses. GO and KEGG analyses showed that immune-related functions of defense response and Th17 cell differentiation had significant differences between the host with and without parasites, respectively. Last, the relationship between metabolites and genes was determined in this study. The purine metabolism and pyrimidine metabolism contained the most altered metabolites and DEGs, which mainly influenced the conversion of ATP, ADP, AMP, GTP, GMP, GDP, UTP, UDP, UMP, dTTP, dTDP, dTMP, and RNA. Thus, it could be concluded that parasitic infection can change the intestinal microbial metabolic activity and enhance immune response of the host through the pathway of purine and pyrimidine metabolism. This results will be a valuable contribution to understanding the bidirectional association of the parasite, intestinal microbiota, and host.

Spatial distribution of Gasterophilus pecorum (Diptera) eggs in the desert steppe of the Kalamaili Nature Reserve (Xinjiang, China).

BACKGROUND: The dominant Gasterophilus species in the desert steppe (Xinjiang, China) Gasterophilus pecorum poses a serious threat to the reintroduced Przewalski's horses. We investigated the distribution pattern of G. pecorum eggs in June 2017. METHODS: Two sampling methods, transect and grid, were used, and the results were analyzed via geostatistics by semivariance. The nest quadrat was used to determine the optimal quadrat size. RESULTS: Eggs were found in 99 quadrats (63.1%) and 187 clusters (1.5%) of Stipa caucasica on the steppe. The mean oviposition count of a cluster was 3.8 ± 1.6. Three-eggs is the mode of which females oviposit on each ovigerous S. caucasica (22.0%). Semivariogram analysis revealed that the distance of spatial dependence for eggs was 921 m, 1233 m and 1097 m for transect 1, transect 2 and grid methods, respectively, while spatial continuity was 62%, 77% and 57.0% for transect 1, transect 2 and grid, respectively. The eggs showed a patchy, aggregated distribution pattern. This suggested the spherical model is most applicable. The proportion of ovigerous S. caucasica was significantly correlated with the distance from water resources (r = - 0.382, p = 0). CONCLUSION: Our findings indicated that diversification of G. pecorum oviposition was a new adaptative strategy for its survival in the desert steppe ecological niche. This made it more efficient at infecting hosts in the local environment. Areas surrounding water resources, especially around the drinking paths of equids (500 m radius surrounding the water), were concentrated epidemic areas. It is suggested that more attention to be paid to the ecological characteristics of G. pecorum in order to develop control measures that would reduce the infection risk for Przewalski's horses.

Analysis of volatiles from feces of released Przewalski's horse (Equus przewalskii) in Gasterophilus pecorum (Diptera: Gasterophilidae) spawning habitat.

The absolute dominant species that infests wild population of Przewalski's horse (Equus przewalskii) is Gasterophilus pecorum, and feces of released Przewalski's horse, a habitat odor, plays an important role in mating and ovipositing locations of G. pecorum. To screen out unique volatiles for attracting G. pecorum, volatiles from fresh feces of released horses at stages of pre-oviposition (PREO), oviposition (OVIP), and post-oviposition (POSO) of G. pecorum, and feces with three different freshness states (i.e., Fresh, Semi-fresh, and Dry) at OVIP were collected by dynamic headspace adsorption and determined by automatic thermal desorption GC-MS. Results show that there were significant differences in fecal volatiles within both test conditions. Of the five most abundant volatiles from the five individual samples, the most important volatile was ammonium acetate at OVIP/Fresh, followed by acetophenone (Semi-fresh), toluene (PREO, OVIP and POSO), butanoic acid (OVIP and Semi-fresh), acetic acid (PREO, POSO and Semi-fresh), 1,6-octadiene,3,7-dimethyl-,(S)- (PREO, OVIP and POSO), 1,5,9-undecatriene,2,6,10-trimethyl-,(Z)- (PREO and Semi-fresh) and caprolactam (all conditions), which seem to be critical substances in oviposition process of G. pecorum. The findings may be beneficial to development of G. pecorum attractants, facilitating prevention and control of infection by G. pecorum to released Przewalski's horse.

Effects of Gasterophilus pecorum infestation on the intestinal microbiota of the rewilded Przewalski's horses in China.

Horse botflies have been a threat to the Przewalski's horses in the Kalamaili Nature Reserve in Xinjiang of China since their reintroduction to the original range. As larvae of these parasites could infest the intestine of a horse for months, they could interact with and alter the structure and composition of its intestinal microbiota, affecting adversely its health. Nonetheless, there are no such studies on the rewilded Przewalski's horses yet. For the first time, this study characterizes the composition of the intestinal microbiota of 7 rewilded Przewalski's horses infected severely by Gasterophilus pecorum following and prior to their anthelmintic treatment. Bioinformatics analyses of the sequence data obtained by amplicon high throughput sequencing of bacterial 16S rRNA genes showed that G. pecorum infestation significantly increased the richness of the intestinal microbial community but not its diversity. Firmicutes and Bacteroidetes were found the dominant phyla as in other animals, and the parasitic infestation decreased the F/B ratio largely by over 50%. Large reduction in relative abundances of the two genera Streptococcus and Lactobacillus observed with G. pecorum infestation suggested possible changes in colic and digestion related conditions of the infected horses. Variations on the relative abundance of the genus groups known to be pathogenic or symbiotic showed that adverse impact of the G. pecorum infestation could be associated with reduction of the symbiotic genera Lactobacillus and Bifidobacterium that are probiotics and able to promote immunity against parasitic infection.

The impact of temperature on the life cycle of Gasterophilus pecorum in northwest China.

BACKGROUND: The departure of the mature larvae of the horse stomach bot fly from the host indicates the beginning of a new infection period. Gasterophilus pecorum is the dominant bot fly species in the desert steppe of the Kalamaili Nature Reserve (KNR) of northwest China as a result of its particular biological characteristics. The population dynamics of G. pecorum were studied to elucidate the population development of this species in the arid desert steppe. METHODS: Larvae in the freshly excreted feces of tracked Przewalski's horses (Equus przewalskii) were collected and recorded. The larval pupation experiments were carried out under natural conditions. RESULTS: There was a positive correlation between the survival rate and the number of larvae expelled (r = 0.630, p < 0.01); the correlation indicated that the species had characteristic peaks of occurrence. The main periods during which mature larvae were expelled in the feces were from early April to early May (peak I) and from mid-August to early September (peak II); the larval population curve showed a sudden increase and gradual decrease at both peaks. Under the higher temperatures of peak II, the adults developing from the larvae had a higher survival rate, higher pupation rate, higher emergence rate and shorter eclosion period than those developing from peak I larvae. Although G. pecorum has only one generation per year, its occurrence peaked twice annually, i.e. the studied population has a bimodal distribution, which doubles parasitic pressure on the local host. This phenomenon is very rarely recorded in studies on insect life history, and especially in those on parasite epidemiology. CONCLUSION: The period during which G. pecorum larvae are naturally expelled from the host exceeds 7 months in KNR, which indicates that there is potentially a long period during which hosts can become infected with this parasite. The phenomenon of two annual peaks of larvae expelled in feces is important as it provides one explanation for the high rate of equine myiasis in KNR.

First reports of nasal and traumatic myiasis infection in endangered Przewalski's horses (Equus ferus przewalskii).

Myiasis has great economic and medical importance. However, myiasis in wildlife that is caused by oestroid flies is relatively rarely recorded compared with that in humans and domestic animals. Recently, during our research on the conservation of Przewalski's horse (PH), we observed two new records of oestroid flies parasitizing wildlife in China. The first is the horse nasal bot fly, Rhinoestrus sp. (Diptera: Oestridae), found in a dead PH from Kalamaili Nature Reserve. One morphotype (R. purpureus-like) was identified. The second is the Wohlfahrt's wound myiasis fly, Wohlfahrtia magnifica (Schiner, 1862) (Diptera: Sarcophagidae), which was collected from an open wound of a PH in the Wild Horse Breeding Research Centre. These observations extend the records of known hosts of these two oestroid myiasis agents. To the knowledge of the authors, infestation by Rhinoestrus and Wohlfahrtia larvae causing myiasis in wildlife has not been reported in China previously.

Genetic diversity of common Gasterophilus spp. from distinct habitats in China.

BACKGROUND: Gasterophilus species are widely distributed around the world. The larvae of these flies parasitize the digestive tract of equids and cause damage, hindering horse breeding and protection of endangered species. However, study of the genetic structure of geographically distinct Gasterophilus populations is lacking. Here, we analyzed the genetic diversity of Gasterophilus pecorum, G. intestinalis, G. nasalis and G. nigricornis from three typical grasslands (meadow, desert and alpine steppes) in China as compared to published sequences from Italy, Poland and China (Daqing and Yili), based on the mitochondrial cytochrome c oxidase cox1 and cox2 gene sequences. RESULTS: Haplotype diversity and nucleotide diversity of mitochondrial genes was generally high in all Gasterophilus populations. Due to the unique natural climatic conditions of the alpine steppe, there were high levels of genetic differentiation among different geographical populations of G. pecorum and G. nasalis, indicating that environmental variations influenced population genetic structure. Frequent exchanges between meadow and desert steppe Gasterophilus species resulted in low genetic differentiation. The highest exchange rates were found among G. intestinalis populations. Genetic differentiation was only observed on a large geographical scale, which was confirmed by analyzing population genetic structure. Three species, G. pecorum, G. intestinalis and G. nasalis, from meadow steppe showed a high emigration rate, indicating that the direction of Gasterophilus dispersal in China was from east to west. CONCLUSIONS: Our results show that the four Gasterophilus species have a high level of genetic diversity and different degrees of genetic differentiation and gene flow among different populations of the same species, reflecting their potential to adapt to the environment and the environmental impact on genetic structure. Knowledge of the genetic structure, population history, and migration will help understand the occurrence and prevalence of gasterophilosis and provide a basis for controlling the local spread of Gasterophilus spp.