Molecular characterization of Cryptosporidium spp. in Bactrian camels (Camelus bactrianus) from Yili Kazak Autonomous Prefecture of Xinjiang, China.

Introduction: Cryptosporidium spp. is a significant zoonotic parasite. The prevalence and infection characteristics of Cryptosporidium spp. in Bactrian camels in Yili Kazak Autonomous Prefecture have yet to be fully understood. Thus, the molecular epidemiology of cryptosporidiosis in camels was investigated in this region. Methods: A total of 1,455 fecal samples were collected from 6 counties in three regions (Altay, Tacheng, and Yili) in Yili Prefecture. Nested PCR targeting the small subunit ribosomal RNA (ssu rRNA) gene was used to identify the species or genotypes of Cryptosporidium infection in camels. For C. parvum positive samples, the subtypes were identified using the 60-kDa glycoprotein (gp60) gene. Results and discussion: The overall infection rate was 8.7% (126/1,455), ranging from 5.6% to 11.7% in different regions, and 4.2% to 15.8% in different counties. A significant difference was observed amongst the counties (p < 0.001). Three species were detected, namely C. andersoni (65.1%, 82/126), C. parvum (34.1%, 43/126), and C. occultus (0.8%, 1/126). Three C. parvum subtypes, If-like-A15G2 (n = 29), IIdA15G1 (n = 4), and IIdA19G1(n = 1) were detected, with If-like-A15G2 being the most prevalent subtype. Camels aged 3-12 months exhibited the highest infection rate (11.4%, 44/387), with no significant difference among age groups (p > 0.05). C. parvum was predominant in camels under 3 months, while C. andersoni prevailed in camels over 3 months. There was an extremely significant difference observed among seasons (p < 0.001), summer had the highest infection rates (16.9%, 61/360). This study collected nearly 1,500 samples and, for the first time, investigated Cryptosporidium spp. infection in camels based on different age groups and seasons. All three Cryptosporidiumspecies identified were zoonotic, posing a potential threat to human health and requiring close attention.

Fungi, including Pithomyces chartarum cause facial eczema and inflammation in grazing sheep in Western China.

Facial eczema is often found in flocks of grazing sheep in China. To investigate fungi species those cause disease and pathological roles. Forage and soil samples were collected during the pathogenic season and cultured. Samples were collected from regions with and without facial eczema affected sheep. Fungal isolation and identification, statistical analysis of fungal species and distribution were performed. Pathological changes, biochemical parameters of serum liver function and protection of inflammatory factors that tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-12 (IL-12) were observed. Fungal cultivation and identification showed that separation rate of Alternaria, Pithomyces chartarum, Fusarium and Aspergillus were higher, particularly, Pithomyces chartarum was significantly identical. Pathological anatomy and histology indicated that the disease likely attacked merino ewes with the age of 6 months old. The clinical manifestations were characterized by inflammational edema in face (ears and eyelids) and mandibular area. Postmortem examination of dead lambs showed enlargement of liver with yellow white patchs of necrotic lesion and tuberous sclerosis and fibrosis on section. Histologic examination of liver showed extravasated blood, severe lesion of liver cells and bile duct, and fatty degeneration. In sheep, fungal toxin induced the secretion of TNF-α, IL-6 and IL-12. These results revealed that Pithomyces chartarum maybe caused facial eczema and inflammation in sheep. The facial eczema was allergic eczema caused by hepatic dysfunction and hepatonecrosis.

Prokaryotic expression of the V protein of the peste des petits ruminants virus and development of an indirect ELISA.

In this study, we recombinantly expressed the V protein of the peste des petits ruminants virus (PPRV) and evaluated its diagnostic value for PPRV infection using an indirect ELISA (i-ELISA). The optimal concentration of the coated antigen of V protein was 15 ng/well at a serum dilution of 1:400, and the optimal positive threshold value was 0.233. A cross-reactivity assay showed that the V protein-based i-ELISA was specific to PPRV with consistent reproducibility and showed a specificity of 82.6% and a sensitivity of 100% with a virus neutralization test. Using the recombinant V protein as an antigen in ELISA is useful for seroepidemiological studies of PPRV infections.

Comparative genomic analysis of 127 Escherichia coli strains isolated from domestic animals with diarrhea in China.

BACKGROUND: Escherichia coli is an important pathogen that causes diarrhea in both humans and animals. To determine the relationships between putative virulence factors and pathotypes or host taxa, many molecular studies on diarrhea-associated E. coli have been reported. However, little is known regarding genome-wide variation of E. coli from animal hosts. In this study, we performed whole genome sequencing of 127 E. coli isolates from sheep and swine with diarrhea in China. We compared isolates to explore the phylogenomic relatedness based on host origin. We explored the relationships of putative virulence factors across host taxa and pathotypes. Antimicrobial resistance was also tested. RESULTS: The E. coli genomes in this study were diverse with clear differences in the SNP, MLST, and O serotypes. Seven putative virulence factors (VFs) were prevalent (> 95%) across the isolates, including Hcp, csgC, dsdA, feoB, fepA, guaA, and malX. Sixteen putative VFs showed significantly different distributions (P < 0.05) in strains from sheep and swine and were primarily adhesion- and toxin-related genes. Some putative VFs were co-occurrent in some specific pathotypes and O serotypes. The distribution of 4525 accessory genes of the 127 strains significantly differed (P < 0.05) between isolates obtained from the two animal species. The 127 animal isolates sequenced in this study were each classified into one of five pathotypes: EAEC, ETEC, STEC, DAEC, and EPEC, with 66.9% of isolates belonging to EAEC. Analysis of stx subtypes and a minimum spanning tree based on MLST revealed that STEC isolates from sheep and EAEC isolates from sheep and swine have low potential to infect humans. Antibiotic resistance analysis showed that the E. coli isolates were highly resistant to ampicillin and doxycycline. Isolates from southeast China were more resistant to antibiotics than isolates from northwest China. Additionally, the plasmid-mediated colist in resistance gene mcr-1 was detected in 15 isolates, including 4 from sheep in Qinghai and 11 from swine in Jiangsu. CONCLUSIONS: Our study provides insight into the genomes of E. coli isolated from animal sources. Distinguishable differences between swine and sheep isolates at the genomic level provides a baseline for future investigations of animal E. coli pathogens.

[Characterization of ibeB gene of meningitic Escherichia coli strains in calves from Xinjiang].

Objective: To understand the molecular biology information of ibeB gene of meningitic Escherichia coli isolates in calves. Methods: The strain used was isolated from the brain and liver tissue of calves died from Meningitis. It was identified to be an O161-K99-STa pathogenic Escherichia coli strain and named as bovine-EN and bovine-EG. Based on the sequence of ibeB gene of meningitic Escherichia coli K1 RS218 strain in GenBank, a pair of primers was designed and the ibeB gene was cloned from isolates by PCR. Part molecular biology information of ibeB among different strains was compared. Results: The sequence length of isolates ibeB gene was 1500 bp, containing a 1371 bp open reading frame (ORF) encoding 457 amino acids. Bioinformatics analysis showed that the nucleotide and amino acid homology of ibeB gene of bovine-EN strain shared 90.5% and 96.9% identity with Escherichia coli K1 RS218 ibeB gene, respectively, while bovine-EG strain shared 99.4% and 100.0% identity with Escherichia coli K12 respectively. The ibeB gene of bovine-E strains encoded water-soluble protein whose molecular weight was 50.26 kDa and isoelectric point was 6.05. This protein contained a signal peptide A but no transmembrane domain. Subcellular localization of ibeB belonged to the secreted protein, which secretory signal path site (SP) proportion was 0.939. Conclusion: The ibeB gene was cloned from meningitic E. coli isolates and had higher homology and similar biological characteristics with meningitis E. coli K1 RS218ibeB, which belongs to extraintestinal pathogenic Escherichia coli.

Complete genomic characterization of a porcine reproductive and respiratory syndrome virus isolate in Xinjiang province of China.

Porcine reproductive and respiratory syndrome (PRRS) is a swine disease of major economic importance that causes reproductive and respiratory problems in pigs. In the present study, one strain of porcine reproductive and respiratory syndrome virus (PRRSV) was isolated in Xinjiang province, Northwest China, designated XJu-1. The full-length genome of XJu-1 was found to be 14,987 nucleotides in length, including the poly(A) tail. Comparative analysis with the genomic sequences of type 2 isolates revealed that XJu-1 shared 87.2-99.2 % identity with these isolates, but only 60.4 % with the type 1 virus-Lelystad Virus, indicating that this new Chinese isolate was closely related to the North American PRRSV genotype. XJu-1 was a novel strain with unique deletions in NSP2 region, namely that 150-amino acid deletion in NSP2. The genomic variations of XJu-1 strain provided the basis for further studies of virulence determinants and evolution for PRRSVs.

Molecular characterization of a signal-regulated kinase homolog from Echinococcus granulosus.

BACKGROUND: Cystic echinococcosis due to Echinococcus granulosus (E. granulosus) is one of the most important chronic helminthic diseases, especially in sheep/cattle-raising regions. The larval stage of the parasite forms a cyst that grows in the liver, lung, or other organs of the host. To ensure a long life in the host tissues, the parasite establishes complex inter-cellular communication systems between its host to allow its differentiation toward each larval stage. Recent studies have reported that this communication is associated with the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade in helminth parasites, and in particular that these protein kinases might serve as effective targets for a novel chemotherapy for cystic echinococcosis. The aim of the present study investigated the biological function of a novel ERK ortholog from E. granulosus, EgERK. METHODS: DNA encoding EgERK was isolated from protoscolices of E. granulosus and analyzed using the LA Taq polymerase chain reaction (PCR) approach and bioinformatics. Reverse transcription PCR (RT-PCR) was used to determine the transcription level of the gene at two different larval tissues. Western blotting was used to detect levels of EgERK protein. The expression profile of EgERK in protoscolices was examined by immunofluorescence. RESULTS: We cloned the entire Egerk genomic locus from E. granulosus. In addition, two alternatively spliced transcripts of Egerk, Egerk-A, and Egerk-B were identified. Egerk-A was found to constitutively expressed at the transcriptional and protein levels in two different larval tissues (cyst membranes and protoscolices). Egerk-A was expressed in the tegumental structures, hooklets, and suckers and in the tissue surrounding the rostellum of E. granulosus protoscolices. CONCLUSIONS: We have cloned the genomic DNA of a novel ERK ortholog from E. granulosus, EgERK (GenBank ID HQ585923), and found that it is constitutively expressed in cyst membrane and protoscolex. These findings will be useful in further study of the biological functions of the gene in the growth and development of Echinococcus and will contribute to research on novel anti-echinococcosis drug targets.

Analysis of genetic diversity and phylogenetic relationship of red deer subspecies in XinJiang, China.

Polymorphisms for seven microsatellite loci in three red deer subspecies (9 populations) found in XinJiang were detected by polymerase chain reaction (PCR), 12% nondenaturation polyacrylamide gel electrophoresis and the Sanguinetti silver staining method. Numbers of alleles, average effective numbers of alleles (E) and the average rate of homozygosity, allelic frequencies of seven microsatellite loci, polymorphism information content (PIC), mean heterozygosity (H) and genetic distances among the populations were calculated for each population. Dendrograms were constructed based on genetic distances by the neighbor-joining method (NJ), utilizing molecular evolutionary genetics analysis software PHYLIP (3.6). The phylogenetic tree was constructed based on allelic frequencies using maximum likelihood (ML); the bootstrap value was estimated by bootstrap test in the tree. Lastly, phylogenesis was analyzed. The results showed that four of the seven microsatellite loci were highly polymorphic, but BMS2508 and Celjp0023 showed no polymorphism and BM5004 was a neutral polymorphism. It is our conclusion that the four microsatellite loci are effective DNA markers for the analysis of genetic diversity and phylogenetic relationships among the three red deer subspecies. The mean PIC, H and E-values across the microsatellite loci were 0.5393, 0.5736 and 2.64, which showed that these microsatellite loci are effective DNA markers for the genetic analysis of red deer. C.e. songaricus populations from Regiment 104, 151 and Hami are clustered together. C.e. yarkandensis populations from Regiment 35, Xaya and Alaer are clustered together. These two clusters also cluster together. Lastly, C.e. sibiricus populations from Burqin, Regiment 188 and the first two clusters were clustered together. The phylogenetic relationship among different red deer populations is consistent with the known origin, history of breeding and geographic distributions of populations.

Polymorphisms in mannose-binding lectin (MBL) gene and their association with MBL protein levels in serum in the Hu sheep.

Mannose-binding lectin (MBL) is the archetypical pathogen recognition protein of the innate immune defence. In humans, three frequently occurring single nucleotide polymorphisms (SNPs) in the coding region of MBL gene are associated with the abnormal polymerization, decreased serum concentration and strongly impaired function of MBL protein. To understand whether or not SNPs in MBL gene are associated with serum concentration of MBL in sheep, we investigated 105 individuals of the Hu sheep by PCR single-strand conformation polymorphism (SSCP) analysis, DNA sequencing, and enzyme-linked immunosorbent assay. SSCP analyses of PCR amplicons from a 194-bp section of the exon-I region of the MBL gene revealed four patterns: A, B, C and D. In comparison with the sequences of the full-length MBL gene of sheep (GenBank accession numbers FJ977629 and AM933378; reference sequence hereafter), pattern A has a 3-bp deletion, a 6-bp deletion and 42 SNPs. Pattern B has 3 SNPs, pattern C has 2 SNPs, whereas pattern D is identical to the reference sequence. Twenty-four of the 47 SNPs of the four patters are synonymous whereas the other 23 SNPs are non-synonymous. The two deletions in the pattern A result in deletions of amino acids but there are no frame shifts in the putative MBL protein. The concentration of MBL protein in serum ranges from 1571 to 3657 µg/L in the Hu sheep. Our statistic analyses showed that patterns A and B are associated with reduced MBL protein level in serum, whereas pattern C is associated with increased MBL protein level in serum (P<0.05) in the Hu sheep.

[Molecular typing and immunoblotting of 17 Mycoplasma ovipneumoniae isolates from China].

OBJECTIVE: To study the heterogeneity and immunogenic variability among Mycoplasma ovipneumoniae (M. ovipneumoniae) isolates from different regions of China. METHODS: The heterogeneity of 17 strains of M. ovipneumoniae isolated from 8 regions of China was studied by the amplified fragment length polymorphism (AFLP) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The software NTsys-2. 10e was used to analyze the profiles obtained from the AFLP and SDS-PAGE. The proteins reacted with the antiserum against M. ovipneumoniae type strain Y98 were then detected by Western-blot. RESULTS: Seventeen strains of M. ovipneumoniae were divided into 8 AFLP groups based on the source regions when the coefficient was 0.78. They were also divided into 8 SDS-PAGE groups based on the source regions when the coefficient was 0.85. A total of 6 immunogenic proteins were detected within 8 strains of M. ovipneumoniae, and their molecular weights were 105 kDa, 83 kDa, 65 kDa, 42 kDa, 40 kDa or 26 kDa, respectively. Interestingly, the 83 kDa and 40 kDa proteins were conserved in all the 8 isolates. CONCLUSION: M. ovipneumoniae isolates from some regions of China were genetically different, but the 83 kDa and 40 kDa antigenic proteins were conserved among the tested isolates. This study can provide some insights for the diagnosis and vaccine development of the disease caused by M. ovipneumoniae.

[Isolation and characterization of Hantavirus carried by rodents in Huludao, Liaoning province].

OBJECTIVE: To investigate the Hantavirus infection and their genotype in rodents in Huludao. METHODS: Rodents were collected from the main epidemic areas to detect antigen of Hantavirus in rat lungs by indirect immunofluorescence assay. Antigen-positive samples were inoculated onto cultures of confluent Vero E6 cells for the isolation of virus. The genotypes of viruses in all antigen-positive samples were identified by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: 200 rats were collected in the main epidemic areas, and 11 Hantavirus-positive samples were tested. The positive rate of Hantavirus in rats was 5.5%. Three strains of Hantavirus were isolated in Vero E6 cell culture. Data from the phylogenetic trees constructed by partial S segment (620-999 nt) or partial G1 segment (180-580 nt) showed that the three isolates carried by rats from Huludao were all genetic subtype SEOV 3. Furthermore, the phylogenetic tree constructed by partial G2 segment (2003-2302 nt) divided SEOV strains into 7 genetic subtypes, and the three isolates were having a closer evolutionary relationship with isolates CP211, ch302 and dc501 from Beijing, and the isolates SD10 and SD227 form Shandong. CONCLUSION: Data indicated that the rate of carrying virus was high and the main genetic subtype of Hantavirus was S3 of Seoul virus in Huludao area.

[Polymorphism analysis of MHC-DRB3 gene in Dolang sheep with PCR-RFLP].

MHC is a chromosomal region consisting of a group of closely linked loci which are highly polymorphic, and plays a central role in the immune system. The restrictive polymorphism of MHC-DRB3 exon2 in Dolang sheep was Analyzed by PCR-RFLPs. The results revealed extensive polymorphisms 2, 2 and 6 RFLP types of PCR products were found with enzymes TaqI, PstI and HaeIII respectively. Considering all restrictive pattern, 24 alleles for DRB3 locus were found in Dolang sheep.

[Microsatellite analysis of genetic diversity and phylogenetic relationship of eight sheep breeds in Xinjiang].

To reveal the genetic diversity and syseighttemic relationship of main sheep breeds in north Xingjiang, the genetic polymorphisms of 10 microsatellites in 8 sheep breeds and one first filial generation (F1) in north Xinjiang were studied by means of PCR, polyacrylamide gel electrophoresis and silver staining. Number of alleles, average effective number of alleles (E) and average rates of homozygote of each breeds were counted. According to allele frequencies of ten microsatellites, polymorphism information content (PIC), mean heterozygosity (h) and genetic distances were calculated for each breeds. By using the Neighbor-joining method of Molecular Evolutionary Genetics Analysis software, a dendrogram was obtained based on genetic distances. Another dendrogram was obtained by Maximum Likelihood method in PHYLIP (3.6) software. The bootstrap values were evaluated for each crunode of the dendrogram by means of bootstrap test. The systemic relationship was analyzed as well. The results showed that 8 of 10 microsatellite loci were highly polymorphic, but BM1824 and MAF65 were low and medium polymorphic respectively, so the 8 microsatellite loci were effective markers for analysis of genetic relationship among sheep breeds. The average PIC (0.5631), h (0.5721) and E(2.9) of the whole population was all lower than those of other sheep breeds reported in the documents, which showed the gene polymorphisms and genetic diversity in these sheep breeds are relative rare. The genetic distances of native Aletai, Kazak and Bashibai sheeps in Xingjiang from foreign sheep breeds and cultivated breeds bearing foreign bloodline are relatively large. Consequently, they clustered in two groups. The phylogenetic relationship between different sheep breeds was in accordance with their resource, breeding history, differentiation and localities.