Single nucleotide polymorphisms (SNPs) and indels identified from whole-genome re-sequencing of four Chinese donkey breeds.

This paper represents the fundamental report of the survey of genome-wide changes of four Chinese indigenous donkey breeds, Dezhou (DZ), Guangling (GL), North China (NC), and Shandong Little donkey (SDL), and the findings will prove usefully for identification of biomarkers that perhaps predict or characterize the growth and coat color patterns. Three genomic regions in CYP3A12, TUBGCP5, and GSTA1 genes, were identified as putative selective sweeps in all researched donkey populations. The loci of candidate genes that may have contributed to the phenotypes in body size (ACSL4, MSI2, ADRA1B, and CDKL5) and coat color patterns (KITLG and TBX3) in donkey populations would be found in underlying strong selection signatures when compared between large and small donkey types, and between different coat colors. The results of the phylogenetic analysis, FST, and principal component analysis (PCA) supported that each population cannot clearly deviate from each other, showing no obvious population structure. We can conclude from the population history that the formation processes between DZS and NC, GL, and SDL are completely different. The genetic variants discovered here provide a rich resource to help identify potential genomic markers and their associated molecular mechanisms that impact economically important traits for Chinese donkey breeding programs.

Recent advances on the regulation of bacterial biofilm formation by herbal medicines.

Biofilm formation is a fundamental part of life cycles of bacteria which affects various aspects of bacterial-host interactions including the development of drug resistance and chronic infections. In clinical settings, biofilm-related infections are becoming increasingly difficult to treat due to tolerance to antibiotics. Bacterial biofilm formation is regulated by different external and internal factors, among which quorum sensing (QS) signals and nucleotide-based second messengers play important roles. In recent years, different kinds of anti-biofilm agents have been discovered, among which are the Chinese herbal medicines (CHMs). CHMs or traditional Chinese medicines have long been utilized to combat various diseases around the world and many of them have the ability to inhibit, impair or decrease bacterial biofilm formation either through regulation of bacterial QS system or nucleotide-based second messengers. In this review, we describe the research progresses of different chemical classes of CHMs on the regulation of bacterial biofilm formation. Though the molecular mechanisms on the regulation of bacterial biofilm formation by CHMs have not been fully understood and there are still a lot of work that need to be performed, these studies contribute to the development of effective biofilm inhibitors and will provide a novel treatment strategy to control biofilm-related infections.

Comparison of gut microflora of donkeys in high and low altitude areas.

Donkeys' gut microbe is critical for their health and adaptation to the environment. Little research has been conducted on the donkey gut microbiome compared with other domestic animals. The Tibetan Plateau is an extreme environment. In this study, 6 Qinghai donkeys (QH) from the Tibetan Plateau and 6 Dezhou donkeys (DZ) were investigated, and the contents of 4 parts-stomach, small intestine, cecum, and rectum-were collected. 16S rRNA sequencing and metagenomic sequencing were used to analyze the composition and diversity of gut microbial communities in donkeys. The results showed that the flora diversity and richness of the hindgut were significantly higher than those of the foregut (p < 0.01), with no sex differences, and the community structure and composition of the same or adjacent regions (stomach, small intestine, cecum, and rectum) were similar. Besides, the flora diversity and richness of QH on the Tibetan Plateau were significantly higher than those of DZ (p < 0.05). The major pathways associated with QH were signal transduction mechanisms and carbohydrate transport and metabolism, and Bacteroidales were the major contributors to these functions. Our study provides novel insights into the contribution of microbiomes to the adaptive evolution of donkeys.

A reassortant G3P[12] rotavirus A strain associated with severe enteritis in donkeys (Equus asinus).

BACKGROUND: In contrast to horses, the only evidence suggesting gastrointestinal disease in neonatal donkeys is associated with Group A rotaviruses (RVAs) is the detection of viral antigens by ELISA in just 1 of 82 symptomatic donkey foals. No additional, more comprehensive investigations have been conducted, and RVAs if circulating in donkey populations have not been molecularly characterised. OBJECTIVES: To investigate if RVAs are associated with an outbreak of severe enteritis in neonatal donkeys and if associated determine the genotype(s) along with the phylogenetic relationship to RVA strains circulating in horses. STUDY DESIGN: Cross-sectional. METHODS: RT-PCR-based techniques were used for RVA diagnosis and gene amplification. Statistical significance was determined by Chi-square and Fisher's exact two-sided tests. Genotyping was performed by RotaC and phylogenetic analysis by neighbour joining. RESULTS: In 2019, acute enteritis occurred in 119 of 206 donkey foals (≤4 months) at two intensive donkey farms in the Shandong province of China. The highest morbidity (68.1%), mortality (29.5%) and fatality levels (45.5%) occurred in foals in the 30-89 day, 30-59 day and 0-29 day age groups respectively. RVA gene sequences were detected in 107 (89.9%) of the symptomatic individuals while further analysis demonstrated the outbreak was associated with the same G3P[12] RVA strain designated RVA/Donkey-wt/CHN/Don01/2019/G3P[12]. Although the VP4 gene of Don01 exhibited close phylogenetic relationships with equivalent RVA sequences commonly circulating in horses, encoding VP7 was more closely associated with sequences isolated from bats suggesting this new donkey strain arose via an intergenogroup reassortment event. MAIN LIMITATIONS: Actual prevalence not determined because <7% of asymptomatic donkey foals were included in this study. The complete genomic sequence of RVA/Donkey-wt/CHN/Don01/2019/G3P[12] remains to be determined. CONCLUSIONS: Valuable new information about the molecular epidemiology of rotaviruses in different equid species is provided by isolation and molecular characterisation of a novel RVA strain from neonatal donkeys.

Therapeutic Efficacy of Phage PIZ SAE-01E2 against Abortion Caused by Salmonella enterica Serovar Abortusequi in Mice.

Salmonella enterica subsp. enterica serovar Abortusequi is a frequently reported pathogen causing abortion in mares. In this study, the preventive and therapeutic effects of phage PIZ SAE-01E2 against S Abortusequi in a mouse model of abortion were investigated. Phage PIZ SAE-01E2 was stable at different temperatures (4 to 70°C) and pH values (pH 4 to 10) and could lyse the majority of the Salmonella serogroup O:4 and O:9 strains tested (25/28). There was no lysogeny-related, toxin, or antibiotic resistance-related gene in the genome of PIZ SAE-01E2. All of these characteristics indicate that PIZ SAE-01E2 has the potential for use in phage therapy. In in vivo experiments, 2 × 103 CFU/mouse of S Abortusequi ATCC 9842 was sufficient to lead to murine abortion (gestational day 14.5) within 48 h. A single intraperitoneal inoculation of PIZ SAE-01E2 (108 PFU/mouse, multiplicity of infection = 105) 1 h before or after S Abortusequi challenge provided effective protection to all pregnant mice (10/10). After 24 h of treatment with phage PIZ SAE-01E2, the bacterial loads in both the placenta and the uterus of the infected mice were significantly decreased (<102 CFU/g) compared to those in the placenta and the uterus of the mice in the control group (>106 CFU/g). In addition, the levels of inflammatory cytokines in the placenta and blood of the mice in the phage administration groups were significantly reduced (P < 0.05) compared to those in the placenta and blood of the mice in the control group. Altogether, these findings indicate that PIZ SAE-01E2 shows the potential to block abortions induced by S Abortusequi in vivoIMPORTANCES Abortusequi is an important pathogen that can induce abortions in mares. Although S Abortusequi has been well controlled in Europe and the United States due to strict breeding and health policies, it is still widespread in African and Asian countries and has proven difficult to control. In China, abortions caused by S Abortusequi have also been reported in donkeys. So far, there is no commercial vaccine. Thus, exploiting alternative efficient and safe strategies to control S Abortusequi infection is essential. In this study, a new lytic phage, PIZ SAE-01E2, infecting S Abortusequi was isolated, and the characteristics of PIZ SAE-01E2 indicated that it has the potential for use in phage therapy. A single intraperitoneal inoculation of PIZ SAE-01E2 before or after S Abortusequi challenge provided effective protection to all pregnant mice. Thus, PIZ SAE-01E2 showed the potential to block abortions induced by S Abortusequi in vivo.

Identification of bovine papillomavirus type 1 and 2 from bovine anogenital fibropapillomas.

Papillomavirus (PV) is a well-known pathogen associated with epithelial and mucosal neoplastic diseases. In contrast to human PVs, characterization of animal PVs from the aspect of anogenital neoplasm is still on a learning curve. In the present study, two vulval and one anal warts, histologically diagnosed as fibropapillomas, excised from dairy cattle were analyzed. PCR and sequencing revealed that bovine papillomavirus type 1 (BPV-1) and BPV-2 were detected from anal and vulval fibropapillomas, respectively. Immunohistochemistry detected PV antigen in a few differentiated keratinocytes of one vulval case. Reverse-transcriptase PCR detected the early region, but not the late region of BPV mRNA in all three cases. The present study will provide new insight into the relationship between BPV and anogenital papilloma in cattle.

Teat papillomatosis in dairy herds: First detection of bovine papillomavirus type 10 in China.

Teat papillomatosis is one important infectious disease affecting cattle health and results in significant economic losses especially in the dairy industry. Although there is a large number of commercial cattle herds in China, limited information is available for molecular epidemiological investigation of bovine papillomaviruses (BPVs). In October 2017, an outbreak of teat papillomatosis occurred in the Shandong Province of China. Samples were collected and diagnosed with PCR, and 3 full-length viral genomes were amplified from tissue samples collected from 3 outbreak farms. Analysis results revealed that the outbreak was associated with BPV type 10. This is the first report of BPV-10 infection in China and will contribute to the molecular epidemiological study of the disease.

Coinfection of a lingual lesion with bovine papular stomatitis virus and bovine papillomavirus.

To date, there have been no reports of coinfection with bovine papular stomatitis virus (BPSV) and bovine papillomavirus (BPV) in the same lesion. In the present study, one lingual papilloma-like sample was collected at an abattoir from the tongue of a 31-month-old Japanese black cow. Coinfection with BPSV and BPV was confirmed by histopathology, immunohistochemistry, PCR and RT-PCR. The evidence for coinfection with BPSV and BPV in the same lesion and an association of BPV with lingual papillomatosis will contribute to future epidemiological studies of these two viruses.

A novel NADC30-like porcine reproductive and respiratory syndrome virus (PRRSV) plays a limited role in the pathogenicity of porcine circoviruses (PCV2 and PCV3) and PRRSV co-infection.

Co-infection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCVs) is commonly observed under field conditions and elicits more severe diseases than any singular infection. In this study, the co-infection of PRRSV, PCV2 and PCV3 was analyzed in tissue samples collected from 150 pigs from April 2016 to April 2018. PRRSV, PCV2 and PCV3 was detected in 55 (36.67%), 43 (28.67%) and 3 (2%) of 150 pigs respectively. Remarkably, one lung sample (SD17-36) collected from a diseased pig was co-infected with PRRSV, PCV2 and PCV3. The complete genomes of SD17-36 viruses of PRRSV, PCV2 and PCV3 were determined, which belong to the subgroups of NADC30-like PRRSV, PCV2d and PCV3a respectively. Sequence comparison showed that PRRSV SD17-36 isolate contains a N33 deletion in GP5. Animal challenge study showed that the novel NADC30-like PRRSV SD17-36 isolate is low pathogenic. Our results indicate that the co-infection of PRRSV and PCVs might cause diseases even when PRRSV plays a limited role in the pathogenicity of the co-infection.

Evaluation of DNA vaccine encoding BCSP31 surface protein of Brucella abortus for protective immunity.

Brucellosis is a highly contagious and zoonotic disease and has a considerable impact on animal health and economy of a country, principally in Pakistan, where rural income largely depends upon livestock farming and dairy products. The disease burden is more in underdeveloped/developing countries due to the low economy and limited access to the diagnostic facilities. In Pakistan, the prevalence of Brucella abortus is very high, so it is the need of the hour to control this disease through more advanced methods. This study was designed with the aim to construct the DNA based vaccine of gene encoding antigenic surface protein (BCSP31). For this purpose, the BCSP31 gene was amplified, purified and ligated in pTZ57 R/T (cloning vector). Dubbed BCSP31-pTZ57 R/T vector was transformed into competent cells (DH5α). After plasmid extraction, the plasmid and pET-28a vector was restricted with EcoRI and BamHI. Again, ligation was done and dubbed pET-28a-BCSP31 transformed into E. coli (BL21). After expression, the protein was purified and used for evaluation of immunogenic response. The protective and immunogenic efficacy of the vaccine was evaluated in rabbits (n = 20). The rabbits were divided into four equal groups. Groups A-C were given purified protein diluted in normal saline @ 750, 1500 and 3000 µg/0.2 mL, respectively through intraconjunctival route. Group D was given 0.2 mL normal saline through intraconjunctival route. Specific immunoglobulin G (IgG) responses were measured through indirect ELISA on a weekly basis. The titer of IgG against the antigen was significantly (p < 0.05) higher in vaccinated groups A-C as compared to group D (control group) in a dose dependent manner. Moreover, log units of protection produced by DNA based vaccine in the rabbits (3.02) also indicated the protective efficacy of the DNA vaccine against B. abortus challenge. The response of this vaccine in rabbit suggested its potential effectiveness against Brucella abortus in large animals.

Full genome analysis of bovine papillomavirus type 1 derived from a calf with severe cutaneous multiple papillomatosis.

Severe papillomatosis occasionally causes astasia leading to euthanizing cattle. There are currently a limited number of reports on virologic approach in severe bovine papillomatosis. Here we report a full genome characterization of bovine papillomavirus type 1 (BPV-1) from the case of severe papillomatosis. A calf developed numerous papillomas on the skin and some nodules in the upper gastrointestinal tract at seven months old. The skin lesion was diagnosed as the epithelial papilloma with BPV antigen expression, while the gastrointestinal lesions were diagnosed as the fibropapilloma without BPV antigen. Full genome analysis revealed that BPV-1s detected in all the lesions were exactly the same. Compared with the reference BPV-1 sequence, there was a single nucleotide insertion in the upstream regulatory region.

Epidemiology of chlamydial infection and disease in a free-ranging koala (Phascolarctos cinereus) population.

Chlamydial disease continues to be one of the main factors threatening the long-term survival of the koala (Phascolarctos cinereus). Despite this, large epidemiological studies of chlamydial infection and disease in wild koala populations are lacking. A better understanding of the prevalence, transmission and pathogenesis is needed to improve control measures, such as the development of vaccines. We investigated the prevalence of Chlamydia pecorum infection and disease in 160 koalas in a peri-urban wild population in Queensland, Australia and found that 31% of koalas were Chlamydia PCR positive and 28% had clinically detectable chlamydial disease. Most infections were at the urogenital site (27%; both males and females) with only 14% at the ocular site. Interestingly, we found that 27% (4/15) of koalas considered to be sexually immature (9-13 months) were already infected with C. pecorum, suggesting that a significant percentage of animals are infected directly from their mother. Ocular infection levels were less prevalent with increasing age (8% in koalas older than 4 years), whereas the prevalence of urogenital tract infections remained high into older age (26% in koalas older than 4 years), suggesting that, after mother-to-young transmission, C. pecorum is predominantly a sexually transmitted infection. While 28% of koalas in this population had clinically detectable chlamydial disease (primarily urogenital tract disease), many PCR positive koalas had no detectable disease and importantly, not all diseased animals were PCR positive. We also observed higher chlamydial loads in koalas who were C. pecorum infected without clinical disease than in koalas who were C. pecorum infected with clinical disease. These results shed light on the potential mechanisms of transmission of C. pecorum in koalas and also guide future control measures, such as vaccination.

Isolation of equine papillomavirus type 1 from racing horse in Japan.

Although many studies have been conducted worldwide for Equus caballus papillomavirus (EcPV), limited information is available on the virus in Japan. We recently collected one classical viral papillomatosis sample (E150904) from a racing horse in Japan. Papillomavirus infection was confirmed by histopathology, immunohistochemistry and PCR assays, and the sample was diagnosed as epithelial papilloma. Full-length genome of the virus was cloned and sequenced. It was 7,613 bp in length and had the same genome organization with reported EcPV-1. Moreover, phylogenetic analysis based on L1 gene revealed that the infection was caused by a variant of EcPV-1. This is the first report of EcPV infection in Japan, and would further contribute to the molecular epidemiological and pathological studies for EcPV.

Papillomavirus in yaks: the isolates of bovine papillomavirus type 1 have a high possibility of belonging to a novel type.

Although papillomaviruses (PVs) have been widely reported in vertebrates, there have been only a few PV reports in yaks (Bos grunniens). In 2012, Bam et al. reported bovine papillomavirus type 1 (BPV-1) and BPV-2 associated with cutaneous papillomatosis in yaks, which provided genomic and pathology information for yak PVs. However, nucleotide identity and phylogenic analyses revealed that there are two isolates with a high possibility of belonging to a novel type that is not BPV-1. The argument was thought to be caused by type-specific primers. Our analysis showed that BPV-1 type-specific primers can detect not only BPV-1 but also other PVs. It suggests that identification results using type-specific primers should be confirmed with more robust methods in molecular epidemiological studies.

Comparative analysis of LTR and structural genes in an equine infectious anemia virus strain isolated from a feral horse in Japan.

Although equine infectious anemia virus (EIAV) poses a major threat to the equine industry worldwide, the molecular epidemiology of this virus is poorly understood. Recently, an EIAV strain (EIAVMiyazaki2011-A) representing a new monophyletic group was discovered in feral horses in southern Japan. In the present study, the EIAVMiyazaki2011-A proviral genome is compared with evolutionarily divergent EIAV isolates to investigate conservation of functional elements or motifs within the long terminal repeats (LTRs) and structural genes. This analysis represents a significant step forward in increasing understanding of the molecular conservation and variation between geographically distinct strains of this equine lentivirus.

Bovine papillomavirus type 10 with a deletion associated with a lingual papilloma in a cow.

Different types of papillomavirus usually cause papillomas in specific tissues. Previously, bovine papillomavirus (BPV) type 10 has been associated specifically with cutaneous papillomas in cattle. In this study, BPV-10 was detected in a papilloma on the tongue of a cow. Whole genome analysis demonstrated that the sequence of this BPV-10 strain had a 129 base pair deletion in the E1 open reading frame, which was confirmed by Southern blot analysis, PCR and reverse transcriptase-PCR.

Initial detection of a circular genome deletion in a naturally bovine papillomavirus-infected sample.

Bovine papillomavirus type 12 (BPV-12) was recently identified in epithelial papillomas on the cattle tongue in our previous study. Along with the full-length genome, one deleted circular genome, named BPV-12-del, was detected in the same papilloma lesion. BPV-12-del is 3363 base pairs in length, and a total of 3,384 base pairs, including E1, E2 and E4 genes and partial E7 and L2 ORFs, were deleted compared with the complete genome. Real-time PCR results showed that the percentage of BPV-12-del was 42% of the total genomes in the sample. Southern Blot analysis also confirmed the presence of the deleted genome. This is the first report describing a circular genome deletion detected in a naturally BPV-infected sample.

Bos grunniens papillomavirus type 1: a novel deltapapillomavirus associated with fibropapilloma in yak.

Papillomaviruses (PVs) have been widely identified among vertebrates, but have not yet been reported to infect yaks. We report, for the first time, a novel deltapapillomavirus that was associated with fibropapilloma in yak herds on the Qinghai-Tibetan Plateau. Six skin papilloma samples were collected and examined using histopathology, immunohistochemistry and PCR assays. The samples were identified as fibropapilloma and were found to contain PV antigens. Sequencing of diagnostic PCR products and the full-length genome revealed that all samples were infected with the same PV type. The whole virus genome was 7946 bp in length and possessed the common PV genomic organization. The virus was identified as a novel PV type and designated Bos grunniens papillomavirus type 1 (BgPV-1) based on the nucleotide sequence alignment of the L1 ORF. It is classified in the Delta-4 species of the genus Deltapapillomavirus based on phylogenetic analysis of the L1 ORF. Identification of this novel PV type provides further information about the pathology, development of diagnostic methods and evolutionary studies of the family Papillomaviridae.