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.

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.

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.