Isolation and characterization of a novel S1-gene insertion porcine epidemic diarrhea virus with low pathogenicity in newborn piglets.

Porcine epidemic diarrhea virus (PEDV) causes diarrhea and vomiting in piglets, leading to a mortality rate of 100%. Due to the high frequency of mutation, it is important to monitor the evolution of PEDV and develop potential vaccine candidates. In this study, two PEDV strains (ZJ2022 and ZQ2022) were identified by PCR. These strains were subsequently isolated, and their genome sequences, growth characteristics, and pathogenicity were compared. Phylogenetic and recombination analyses revealed that both strains belonged to GIIa-subgroup, and ZQ2022 was identified as a recombinant strain derived from ZJ2022. Further sequence analysis showed that the ZJ2022 strain had a modified top region of the S1 protein due to a three amino acid insertion (T380_Y380insGGE) in the S1 gene. According to the virus growth curve, ZJ2022 exhibited better cellular adaptation than ZQ2022, with higher viral titers from 8 hpi to 24 hpi. Additionally, ZQ2022 exhibited a high level of pathogenicity, causing severe diarrhea in piglets at 36 hpi and a 100% mortality rate by 96 hpi. In contrast, ZJ2022 showed lower pathogenicity, inducing severe diarrhea in piglets at 60 hpi, with a mortality rate of 60% at 96 hpi and 100% at 120 hpi. In summary, our findings provided evidence of the undergoing mutations in Chinese PEDV strains. Furthermore, the S gene insertion strain ZJ2022 exhibited strong cellular adaptability and low pathogenicity, making it a potential candidate strain for vaccine development.

Porcine epidemic diarrhea virus: A review of detection, inhibition of host gene expression and evasion of host innate immune.

As one of the most important swine enteropathogenic coronavirus, porcine epidemic diarrhea virus (PEDV) is the causative agent of an acute and devastating enteric disease that causes lethal watery diarrhea in suckling piglets. Recent progress in studying PEDV has revealed many intriguing findings on its prevalence and genetic evolution, rapid diagnosis, suppression of host gene expression, and suppression of the host innate immune system. Due to the continuous mutation of the PEDV genome, viral evasions from innate immune defenses and mixed infection with other coronaviruses, the spread of the virus is becoming wider and faster, making it even more necessary to prevent the infections caused by wild-type PEDV variants. It has also been reported that PEDV nsp1 is an essential virulence determinant and is critical for inhibiting host gene expression by structural and biochemical analyses. The inhibition of host protein synthesis employed by PEDV nsp1 may contribute to the regulation of host cell proliferation and immune evasion-related biological functions. In this review, we critically evaluate the recent studies on these aspects of PEDV and assess prospects in understanding the function of PEDV proteins in regulating host innate immune response and viral virulence.

Diagnosis of Canine Tumours and the Value of Combined Detection of VEGF, P53, SF and NLRP3 for the Early Diagnosis of Canine Mammary Carcinoma.

The average life of a dog is generally maintained at ten to fifteen years, and tumours are the predominant reason that leads to the death of dogs, especially canine mammary carcinoma. Therefore, early diagnosis of tumours is very important. In this study, tumor size, morphology, and texture could be seen through general clinical examination, tumor metastasis could be seen through imaging examination, inflammatory reactions could be seen through hematological examination, and abnormal cell morphology could be seen through cytological and histopathological examination. In the 269 malignant cases and 179 benign cases, we randomly selected 30 cases each, and an additional 30 healthy dogs were selected for the experiment (healthy dogs: dogs in good physical condition without any tumor or other diseases). We used RT-qPCR and ELISA to determine the relative expression of vascular endothelial growth factor (VEGF), tumor protein P53 (P53), serum ferritin (SF), and NOD-like receptor protein 3 (NLRP3) in 30 healthy dogs, 30 dogs with benign mammary tumours, and 30 dogs with malignant mammary tumours. In the results, the same expression trend was obtained both in serum and tissues, and the expression of the four markers was the highest in malignant mammary tumours, with highly significant differences compared with the benign and healthy/paracancerous groups. By plotting the ROC curves, it was found that the results of combined tests were better than a single test and the combination of the four markers was the best for the early diagnosis. In conclusion, this can assist the clinical early diagnosis to a certain extent, and also provides some references and assistance for the development of tumor detection kits in clinical practice.

Research progress of good markers for canine mammary carcinoma.

PURPOSE: Mammary gland tumors are the most common neoplastic diseases in elderly female dogs, about 50% of which are considered to be malignant. Canine mammary tumors are similar to human breast cancers in many respects, so canine mammary tumors are frequently studied alongside human breast cancer. This article mentioned KI-67, HER-2, COX-2, BRCA1, BRCA2, P53, CA15-3, MicroRNA, Top2α and so on. All these markers are expected to have an important role in the clinic. METHODS: Existing markers of canine mammary carcinoma are reviewed, and the expression of each marker and its diagnostic role for this tumor are described in detail. RESULTS: This article introduced several effective markers of canine mammary tumors, among them, antigen KI-67 (KI-67), human epidermal growth factor receptor 2 (HER-2), cyclooxygenase 2 (COX-2) are promising and can be detected in both serum and tissue samples. Breast cancer caused by mutations in the breast cancer 1 gene (BRCA1) and breast cancer 2 gene (BRCA2) is also a hot topic of research. In addition to the above symbols, tumor protein p53 (p53), cancer antigen15-3 (CA15-3), MicroRNA (miRNA), topoisomerase πα (Top2α), proliferating cell nuclear antigen (PCNA), epidermal growth factor receptor (EGFR) and E-cadherin will also be involved in this paper. We will also mention Mammaglobin, which has been rarely reported so far.

Isolation, Identification and Antimicrobial Resistance Analysis of Canine Oral and Intestinal Escherichia coli Resistant to Colistin.

In recent years, the antimicrobial resistance in Escherichia coli has gradually developed into a global problem. These resistant bacteria could be transmitted to humans through animal feces in the environment or direct contact with pets, leading to a problem in bacterial treatment for humans and animals. Now, the antibiotic resistance of oral and intestinal microbiota from dog origins remains unclear in China. Therefore, this study first analyzed the current colistin resistance of oral and intestinal microbiota from dog origins in mainland China. A total of 536 samples were collected from dogs in mainland China and, respectively, cultured on the SS and MacConkey agar plate containing colistin (4 µg/mL) to obtain bacteria, and the antibiotic-resistance phenotype of Escherichia coli was investigated for nine antibiotics. Results showed that a total of 2259 colistin-resistant bacteria were isolated from samples and identified, and among them, the isolated rate of Escherichia coli (34.01%, 769/2259) was relatively higher than that of other bacteria. Subsequently, it was found that the resistance of these Escherichia coli was very severe by exploring its resistance to different antibiotics, particularly to three common antibiotics in a clinic which were ceftriaxone, ampicillin and trimethoprim/sulfamethoxazole, with the resistance rates of 60.60% (466/769), 57.22% (440/769), and 53.06% (408/769), respectively. Moreover, the simultaneous resistance of Escherichia coli to one or more antibiotics was determined, and 69.96% (538/769) strains have defined the resistance to both two or more antibiotics, and even 13 of Escherichia coli strains that were resistant to all nine antibiotics, indicating that the Escherichia coli from dog origins has severe antibiotic resistance in the clinic. In conclusion, this study guided the use of antibiotics and could draw attention to antibiotic resistance in veterinary clinical treatment for animals in the future.

Characterization of a gE/gI/TK gene-deleted pseudorabies virus variant expressing the Cap protein of porcine circovirus type 2d.

Porcine circovirus type 2 (PCV2) plays a key role in the etiology of PCV2-associated disease (PCVAD), and its predominant strain is PCV2d which is not completely controlled by most commercially available vaccines against PCV2a strains. Pseudorabies (PR) caused by pseudorabies virus (PRV) variants re-emerged in Bartha-K61 vaccine-immunized swine herds in late 2011, which brought considerable losses to the global pig husbandry. Therefore, it is significantly important to develop a safe and effective vaccine against both PCV2d and PRV infection. In the present study, the PCV2d ORF2 gene was amplified by PCR, and cloned into the BamHI site of PRV transfer plasmid pG vector to obtain the recombinant transfer plasmid pG-PCV2dCap-EGFP. Subsequently, it was transfected into ST cells infected with the three gene deleted PRV variant strain NY-gE-/gI-/TK- to generate a recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+/EGFP+, and then the EGFP gene was knocked out to harvest the rPRV NY-gE-/gI-/TK-/PCV2dCap+ using gene-editing technology termed CRISPR/Cas9 system. The recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ had similar genetic stability and proliferation characteristics to the parental PRV as indicated by PCR and one-step growth curve test, and the expression of Cap was validated by Western blot. In animal experiment, higher PCV2-specific ELISA antibodies and detectable PCV2-specific neutralizing antibodies could be elicited in mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ compared to commercial PCV2 inactivated vaccine. Moreover, the recombinant virus rPRV NY-gE-/gI-/TK-/PCV2dCap+ significantly reduced the viral loads in the hearts, livers, spleens, lungs, and kidneys in mice following a virulent PCV2d challenge. Mice immunized with rPRV NY-gE-/gI-/TK-/PCV2dCap+ developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. Together, the rPRV NY-gE-/gI-/TK-/PCV2dCap+ recombinant strain has strong immunogenicity.

Identification of canine mammary tumor-associated metabolites using untargeted metabolomics.

The canine mammary tumor is the most common tumor type in female dogs and seriously threatens their life. Currently, no effective treatments are available for this condition. Hence, it is essential to identify biomarkers that positively influence the early diagnosis and treatment and prognosis of this disease. To provide a basis for early diagnosis of canine breast tumors, in this study, 23 dogs with mammary tumors were identified via histopathological examination combined with ancillary diagnoses via blood examinations and diagnostic imaging. The canine mammary tumor and tumor-adjacent healthy tissues were collected, and their metabolites were identified utilizing a UHPLC-qTOF-MS-based untargeted metabolomics approach. The metabolic results revealed a total of 979 ion features in the positive polarity mode and 371 ion features in the negative polarity mode in the tissues of two groups; among them, 536 differential metabolites (385 in the positive and 151 in the negative polarity mode) were analyzed by PCA and PLS-DA. Subsequently, the enrichment pathways purine metabolism, alpha-linolenic acid metabolism, vitamin B6 metabolism, and fatty acid biosynthesis were analyzed using Metaboanalyst 4.0, which suggested that these pathways were valuable diagnostic and therapeutic targets. Moreover, the receiver operating characteristic curves further confirmed 13Z,16Z-docosadienoic acid, 23-nordeoxycholic acid, and (±)12(13)-DiHOME as expected candidate biomarkers of canine mammary tumors. In conclusion, the discovery of tumor biomarkers based on untargeted metabolomics is informative for pathological mechanism studies and facilitates the early diagnosis of canine mammary tumors.

A study on the correlation between intrauterine microbiota and uterine pyogenesis in dogs.

Pyometra is a common and high-incidence reproductive system disease in female dogs, and its development involves both hormonal and bacterial factors. Characterization of the endometrial microbiome in healthy dogs and diseased dogs with pyometra remains unclear at present, however. In this study, dogs with pyometra were identified based on the clinical examinations, hematology examinations, vaginal smears and uterine histopathology. The endometrial samples of healthy dogs (n = 30) and diseased dogs (n = 41) were then collected and sequenced by 16S rRNA high-throughput sequencing technology. Dogs with pyometra suffered from inflammation, and their endometrial microbial diversity (ACE and Chao 1 indices) was significantly lower than that of healthy dogs (P < 0.05). The endometrial samples of both groups were enriched in four phyla (Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria), with a greater abundance of Firmicutes in diseased dogs (P < 0.05). At the genus level, the most prevalent microbes in diseased dogs belonged to Pseudomonas, Escherichia-Shigella, Mycoplasma, Enterococcus, Haemophilus, Vibrio and Ralstonia, with lower levels of Mycoplasma, Enterococcus and Haemophilus in the healthy control. Principal co-ordinates analysis and non-metric multi-dimensional scaling showed that the endometrial microbiome of diseased dogs clustered separately from that of the healthy controls (P < 0.05). In the LDA effect size analysis, 18 members of the endometrial microbiome were screened. Of these, the bacterial species Pseudomonas_aeruginosa and microbes within the genera Mycoplasma, Enterococcus and Haemophilus were found to be enriched in the uteruses of diseased dogs. Furthermore, the Random Forests model further confirmed that Mycoplasma and Haemophilus could be considered as biomarkers of diseased endometrium. In conclusion, this study provided a theoretical basis for the development of probiotic preparation in the future.

Identification of Canine Pyometra-Associated Metabolites Using Untargeted Metabolomics.

Canine pyometra frequently occurs in middle-aged to older intact bitches, which seriously affects the life of dogs and brings an economic loss to their owners. Hence, finding a key metabolite is very important for the diagnosis and development of a new safe and effective therapy for the disease. In this study, dogs with pyometra were identified by blood examinations, laboratory analyses and diagnostic imaging, and fifteen endometrium tissues of sick dogs with pyometra and fifteen controls were collected and their metabolites were identified utilizing a UHPLC-qTOF-MS-based untargeted metabolomics approach. The results indicated that the elevated inflammatory cells were observed in dogs with pyometra, suggesting that sick dogs suffered systemic inflammation. In the untargeted metabolic profile, 705 ion features in the positive polarity mode and 414 ion features in the negative polarity mode were obtained in endometrium tissues of sick dogs with pyometra, with a total of 275 differential metabolites (173 in positive and 102 in negative polarity modes). Moreover, the multivariate statistical analyses such as PCA and PLS-DA also showed that the metabolites were significantly different between the two groups. Then, these differential metabolites were subjected to pathway analysis using Metaboanalyst 4.0, and Galactose metabolism, cAMP signaling pathway and Glycerophospholipid metabolism were enriched, proving some insights into the metabolic changes during pyometra. Moreover, the receiver operating characteristic curves further confirmed kynurenic acid was expected to be a candidate biomarker of canine pyometra. In conclusion, this study provided a new idea for exploring early diagnosis methods and a safe and effective therapy for canine pyometra.

The Analysis of E-Cadherin, N-Cadherin, Vimentin, HER-2, CEA, CA15-3 and SF Expression in the Diagnosis of Canine Mammary Tumors.

Canine mammary tumors (CMTs) are one of the most common tumors in female dogs, and they are associated with a poor prognosis owing to their high rate of recurrence and metastasis rates, which make their diagnosis especially important in clinical veterinary medicine. In this study, the characteristics of tumors were observed in dogs suffering from CMTs, and clinical diagnosis and histopathology were used to identify tumors. Furthermore, the expression levels of tumor markers for CMTs were analyzed by enzyme-linked immunosorbent assay (ELISA) and quantitative PCR (qPCR). Upon clinical examination, dogs with CMTs displayed a distinct and irregular mass in the mammary gland, and X-ray (Latero-lateral and ventro-dorsal views) and ultrasonography of the abdomen revealed a moderately echogenic mass at the mammary gland with slightly stronger density than the surrounding tissue. A total of 30 tumors were identified by histopathology, 11 benign and 19 malignant. Changes in some indicators in dogs suffering from CMTs and healthy dogs suggested that there were multiple direct or paraneoplastic changes associated with tumors that could be detected/suspected by hematological examination, and ELISA revealed the HER-2 serum concentrations were significantly different between healthy animals and those with malignant tumors. qPCR indicated that tumor markers N-cadherin, Vimentin, HER-2, CEA, CA15-3 and SF were higher in dogs with malignant tumors than healthy dogs, with a low level of E-cadherin in malignant tumors. This study verified that serological tests and molecular biological tests were essential to the early diagnosis, treatment and prognosis of dogs with tumors.