First report of Eimeria and Entamoeba infection in alpacas (Vicugna pacos) in Shanxi Province, northern China.

Intestinal protozoa Eimeria and Entamoeba can infect many animal species including alpacas. However, data on the prevalence and pathogenicity of species of the two genera Eimeria and Entamoeba in alpacas in China is scarce. The current study was carried out to investigate the prevalence of Eimeria and Entamoeba in alpacas in two cities (Taiyuan and Xinzhou) in Shanxi Province, northern China, using PCR-based approaches. Eimeria spp. were only found in Taiyuan city, and the overall prevalence was 1.64%. All samples collected from male alpacas were PCR-negative for Eimeria. Four Eimeria-positive samples were tested positive as Eimeria lamae. The molecular prevalence of Entamoeba in alpacas was 18.03% (66/366), including 16.39% (50/305) in alpacas from Taiyuan city and 26.23% (16/61) from Xinzhou city, respectively. The Entamoeba prevalence in male alpacas (25.00%) was significantly higher than that in female alpacas (15.69%). Entamoeba bovis was the predominant species, and no Entamoeba histolytica infection was detected. Nine unique SSU rRNA gene sequences of Entamoeba were obtained which formed a new cluster. The results showed that sex and location might be the risk factors associated with prevalence of Eimeria spp., and sex might be the risk factor associated with prevalence of Entamoeba spp.. This is the first report of Entamoeba in alpacas worldwide. These findings expand our understanding of the prevalence and genetic diversity of Eimeria and Entamoeba in alpacas.

Serological evidence of Toxoplasma gondii and Chlamydia infection in alpacas (Vicugna pacos) in Shanxi Province, northern China.

Toxoplasma gondii, Neospora caninum, Chlamydia and bluetongue virus (BTV) are four important pathogens which can cause reproductive loss. The present study was conducted to estimate the seroprevalence of T. gondii, N. caninum, Chlamydia and BTV in alpacas in Shanxi Province, northern China. A total of 251 serum samples were collected from alpacas, and antibodies against T. gondii and Chlamydia were examined by the modified agglutination test (MAT) and indirect hemagglutination assay (IHA), respectively. Antibodies to N. caninum and BTV were determined by using the commercially available competitive enzyme-linked immunosorbent assay (cELISA) kits, respectively. The overall seroprevalence of T. gondii was 9.16% (95% CI 5.59-12.73) in the three sampled counties, of which, no T. gondii-seropositive samples were detected in alpacas in Fanshi County. Gender differences in the T. gondii seroprevalence were observed. The overall Chlamydia seroprevalence was 13.94% (95% CI: 9.66-18.22), and there was a statistically significant difference in Chlamydia seroprevalence in alpacas between the two counties, Jiexiu and Fanshi. All serum samples tested negative for N. caninum and BTV antibodies, respectively. To our knowledge, this is the first report of T. gondii and Chlamydia seroprevalence in alpacas in China, which provides baseline information for controlling T. gondii and Chlamydia infection in alpacas in China.

Prevalence and genotype distribution of Enterocytozoon bieneusi in farmed raccoon dogs (Nyctereutes procyonoides) in Shandong Province, eastern China.

Enterocytozoon bieneusi is a common microsporidian species, which can infect humans and various species of animals. However, little is known about E. bieneusi prevalence and genotypes in farmed raccoon dogs (Nyctereutes procyonoides) in Shandong Province, China. In this study, a total of 356 fecal samples were collected from farmed raccoon dogs in Weihai, Weifang, and Yantai cities in Shandong Province, China. A total of 23 (6.5%) samples were E. bieneusi-positive by nested PCR amplification of the internal transcribed spacer (ITS) region of ribosomal DNA. Statistical analysis showed that E. bieneusi prevalence in male raccoon dogs was higher than that in female raccoon dogs, and the highest E. bieneusi prevalence was detected in adult raccoon dogs. Sequence analysis revealed four known E. bieneusi genotypes (D, type IV, CHG1, and Peru8), and type IV (11/23) was the predominant genotype. The genotypes type IV, Peru8, and CHG1 were reported in raccoon dogs for the first time in China. Phylogenetic analysis showed that three human-pathogenic genotypes (D, type IV, and Peru8) were clustered into group 1, and the CHG1 belonged to group 2. These findings expand the current understanding of E. bieneusi prevalence and genotype distribution in raccoon dogs in China. Our study also shows that raccoon dogs are hosts for E. bieneusi belonging to several genotypes, including zoonotic ones, highlighting the possibility of transmission of this pathogen between raccoon dogs and humans.

Molecular characterization of Eimeria spp. and Blastocystis in rabbits in Shandong Province, China.

Eimeria spp. and Blastocystis are the common parasites that parasitize the intestinal tract of rabbits, which can seriously threaten the health of rabbits and lead to economic losses to the rabbit industry. However, information about the prevalence and transmission of these two parasites in rabbits is limited in China. The objective of this study was to survey the prevalence of Eimeria spp. and Blastocystis in rabbits in Shandong Province. A total of 616 rabbit fecal samples were collected from two cities (Rizhao and Weihai) in Shandong Province, eastern China, and Eimeria spp. and Blastocystis were identified by polymerase chain reaction based on species-specific markers. The prevalence of Eimeria spp. was 20% (123/616) and the Blastocystis prevalence was 0.97% (6/616). Five different Eimeria species (Eimeria intestinalis, E. perforans, E. magna, E. media, and E. irresidua) and the ST4 subtype of Blastocystis were identified in rabbits by sequence analysis. This is the first report of Blastocystis prevalence and subtype ST4 in rabbits in Shandong Province. The findings provide baseline data for the prevention and control of Eimeria spp. and Blastocystis in rabbits in Shandong Province, China.

Molecular detection and genotype distribution of Enterocytozoon bieneusi in farmed silver foxes (Vulpes vulpes) and arctic foxes (Vulpes lagopus) in Shandong Province, eastern China.

Enterocytozoon bieneusi is an opportunistic enteric pathogen which can infect a wide range of animal species and humans. It is the most diagnosed species of Microsporidia in humans and has an impact on public health. Many infected animals including foxes may be a potential source for transmitting E. bieneusi to humans. However, limited information is available on the E. bieneusi prevalence and genotypes in farmed foxes in China. Therefore, in the present study, 344 fresh fecal samples were collected from farmed foxes (Vulpes vulpes and Vulpes lagopus) in Shandong Province, and the prevalence and genotypes of E. bieneusi were examined based on sequence analysis of the ribosomal internal transcribed spacer (ITS) region. The overall E. bieneusi prevalence was 9% (31/344); of them, 6.5% (9/138) in farmed silver foxes (V. vulpes) and 10.7% (22/206) in farmed arctic foxes (V. lagopus). Moreover, four known (Hum-q1, NCF2, HND-1, and Type IV) and two novel E. bieneusi genotypes (SDF1 and SDF2) were identified in farmed foxes in the present study. All of the E. bieneusi genotypes belonged to the zoonotic group based on phylogenetic analysis. In addition, 2, 4, 0, and 11 samples were successfully amplified at MS1, MS3, MS4, and MS7 loci, respectively. The present study reveals E. bieneusi prevalence and genotype distribution in farmed foxes in Shandong Province and enlarged the host and geographic information of E. bieneusi in China.

Prevalence and Subtypes of Blastocystis in Alpacas, Vicugna pacos in Shanxi Province, China.

Blastocystis, an enteric protist, has been reported to be an important cause of protozoal gastrointestinal manifestations in humans and animals worldwide. Animals harboring certain Blastocystis subtypes (STs) may serve as a potential source of human infection. However, information about the prevalence and genetic diversity of Blastocystis in alpacas is limited. In the present study, a total of 366 fecal samples from alpacas in Shanxi Province, northern China, were examined for Blastocystis by PCR amplification of the small subunit rRNA gene, followed by sequencing and phylogenetic analysis. The prevalence of Blastocystis in alpacas was 23.8%, and gender difference in the prevalence of Blastocystiswas observed. The most predominant Blastocystis ST was ST10, followed by ST14 and ST5. The detection of ST5, a potentially zoonotic genotype, indicates that alpacas harboring ST5 could be a potential source of human infection with Blastocystis. These data provide new insight into the prevalence and genetic diversity of Blastocystis in alpacas.

Prevalence and multilocus genotypes of Enterocytozoon bieneusi in alpacas (Vicugna pacos) in Shanxi Province, northern China.

Enterocytozoon bieneusi is a single-celled obligate pathogen that seriously threatens animal and public health. However, information on the prevalence and genotypes of E. bieneusi in alpacas in China is limited. In the present study, 366 fresh fecal samples from alpacas in Shanxi Province, northern China, were collected to detect E. bieneusi by nested PCR amplification of the internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA). The overall prevalence of E. bieneusi in alpacas was 4.4% (16/366), including 3.9% (12/305) in Yangqu County and 6.6% (4/61) in Dai county, respectively. Four known genotypes were identified, namely ALP1, ALP3, P, and SH11, all of which belong to the zoonotic group 1 by phylogenetic analysis. Moreover, ITS-positive samples were further characterized by PCR amplification of other four targets, including three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4). Multilocus sequence typing (MLST) showed that 5, 2, 3, and 3 types were identified at MS1, MS3, MS7, and MS4 loci, respectively, representing eight multilocus genotypes (MLGs). These findings contribute to the improved understanding of the prevalence and genotypes of E. bieneusi in alpacas in China and have important implications for controlling E. bieneusi infections in animals and humans.

Expression and immunoreactivity of a recombinant multi-epitope antigen designed based on four major structural proteins of avian infectious bronchitis virus.

The development of rapid, simple, and sensitive diagnostic methods for identification of avian infectious bronchitis virus (IBV) is crucial for the effective control of avian infectious bronchitis. In the present study, a tandemly arranged multiepitope peptide (named SEMN) was designed with four antigenic regions derived from four major structural proteins of IBV. Then, we performed codon optimization of SEMN gene by changing the codon-adaptation index from 0.45 to 0.94 and expressed the optimized gene in codon bias-adjusted Escherichia coli Rosetta (DE3), followed by determination of the immunoreactivity of the purified protein. Bioinformatics analysis of SEMN showed a high antigenicity, surface probability and hydrophilicity. The recombinant protein rSEMN was expressed both in soluble forms and as inclusion bodies, and the molecular weight of rSEMN was about 39 kDa. The preliminary diagnostic performance of rSEMN was confirmed by Western blotting analysis using chicken anti-IBV polyclonal antibodies. Further studies are needed to evaluate the immunogenicity in animal models and to give a final assessment of the diagnostic utility of this recombinant multi-epitope antigen.