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 Relationship of Tumor Microbiome and Oral Bacteria and Intestinal Dysbiosis in Canine Mammary Tumor.

Canine mammary tumor (CMT) is the most common tumor in dogs, with 50% of malignant cases, and lacks an effective therapeutic schedule, hence its early diagnosis is of great importance to achieve a good prognosis. Microbiota is believed to play important roles in systemic diseases, including cancers. In this study, 91 tumors, 21 oral and fecal samples in total were collected from dogs with CMTs, and 31 oral and 21 fecal samples from healthy dogs were collected as control. The intratumoral, oral and gut bacterial community of dogs with CMTs and healthy dogs was profiled by 16S rRNA high-throughput sequencing and bioinformatic methods. The predominant intratumoral microbes were Ralstonia, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Pseudomonas, unidentified_Chloroplast and Bacteroides at the genus level. In addition, our findings demonstrated striking changes in the composition of the oral and gut bacterium community in the dogs suffered from CMTs compared to the healthy dogs, with a significant increase of Bacteroides which also was the significant microbial biomarker in the oral and gut bacterium community. It showed that the Bacteroides was shared in the intratumoral, oral and intestinal bacterial microbiomes, confirming that microbiota might travel from the mouth to the intestine and finally to the distant mammary tumor tissue. This study provides a new microbiological idea for the treatment of canine mammary tumors, and also provides a theoretical basis for the study of human breast cancer.

Pseudorabies Virus: From Pathogenesis to Prevention Strategies.

Pseudorabies (PR), also called Aujeszky's disease (AD), is a highly infectious viral disease which is caused by pseudorabies virus (PRV). It has been nearly 200 years since the first PR case occurred. Currently, the virus can infect human beings and various mammals, including pigs, sheep, dogs, rabbits, rodents, cattle and cats, and among them, pigs are the only natural host of PRV infection. PRV is characterized by reproductive failure in pregnant sows, nervous disorders in newborn piglets, and respiratory distress in growing pigs, resulting in serious economic losses to the pig industry worldwide. Due to the extensive application of the attenuated vaccine containing the Bartha-K61 strain, PR was well controlled. With the variation of PRV strain, PR re-emerged and rapidly spread in some countries, especially China. Although researchers have been committed to the design of diagnostic methods and the development of vaccines in recent years, PR is still an important infectious disease and is widely prevalent in the global pig industry. In this review, we introduce the structural composition and life cycle of PRV virions and then discuss the latest findings on PRV pathogenesis, following the molecular characteristic of PRV and the summary of existing diagnosis methods. Subsequently, we also focus on the latest clinical progress in the prevention and control of PRV infection via the development of vaccines, traditional herbal medicines and novel small RNAs. Lastly, we provide an outlook on PRV eradication.

Epidemiological Investigation of Canine Mammary Tumors in Mainland China Between 2017 and 2021.

Epidemiological studies enable us to analyze disease behavior, define risk factors, and establish fundamental prognostic criteria. This study aimed to determine the epidemiological and clinical characteristics of canine tumors diagnosed during the years 2017-2021. The results showed that canine mammary tumors were the most common tumors, and their relative incidence for 5-years-total was 46.71% (504/1,079), with 48.41% (244/504) of benign, and 51.59% (260/504) of malignant. Pure breeds accounted for 84.13% (424/504) of submissions, and adult female dogs (9-12 years old) were most frequently involved, followed by 5-8-year-old females. Remarkably, 2.58% (13/504) occurred in the male dogs. In addition, a high prevalence of mammary tumors (77.38%, 390/504) was diagnosed in unneutered dogs, and different incidence rates were observed in different regions (Northeast, Southeast, Northwest and Southwest China). For clinical factors, the tumor size ranged from 0.5 to 28 cm, with the 0-5 cm being the most common tumor size (47.82%, 241/504), and malignant tumors (4.33 ± 2.88 cm, mean ± SD) were bigger than benign ones (3.06 ± 1.67 cm, mean ± SD) (p < 0.001). The incidence of single tumor (55.36%, 279/504) was higher than that of multiple tumors in dogs, while the latter had a higher incidence of malignant tumors (74.67%, 168/225). According to this study, we also found that canine mammary tumors were more common in the last two pairs of mammary glands. In addition, multiple linear regression analysis showed that there was linear significant relationship between three independent variables (age, tumor size, and tumor number) and histological properties of canine mammary tumor [(p>|t|) < 0.05]. This is the first retrospective statistical analysis of such a large dataset in China to reveal the link between epidemiological clinical risks and histological diagnosis. It aids in the improvement of the host's knowledge of canine tumor disorders and the early prevention of canine mammary tumors.

Isolation and Phylogenetic Analysis of Reemerging Pseudorabies Virus Within Pig Populations in Central China During 2012 to 2019.

To understand the biological characteristics of the reemerging pseudorabies virus (PRV) strains, a total of 392 tissue samples were collected from diseased pigs during reemerging PR outbreaks between 2012 and 2019 on farms in central China where swine had been immunized with Bartha-K61 and 51 (13. 01%) were positive for the gE gene by PCR. Sixteen PRV strains were isolated and caused clinical symptoms and death in mice. Subsequently, gE, gC, gB, and gD complete genes were amplified from the 16 PRV isolates and sequenced. Phylogenetic analysis based on these four gene sequences shows that the 16 PRV isolates were more closely related to the Chinese PRV variants (after 2012) but genetically differed from early Chinese PRV isolates (before 2012). Sequence analysis reveals that PRV isolates exhibited amino acid insertions, substitutions, or deletions compared with early Chinese PRV isolates and European-American PRV strains. In addition, this is the first report that eight isolates (8/16) in this study harbor a unique amino acid substitution at position 280 (F to L) of the gC protein, and six isolates have an amino acid substitution at position 338 (A to V) of the gD protein compared with the Chinese PRV variants. The emulsion containing inactivated PRV NY isolate could provide complete protection against the NY isolate. This study might enrich our understanding of the evolution of reemerging PRV strains as well as pave the way for finding a model virus to develop a novel vaccine based on reemerging PRV strains.

Seroprevalence investigation and genetic analysis of pseudorabies virus within pig populations in Henan province of China during 2018-2019.

In late 2011, the outbreak of pseudorabies (PR) occurred in Bartha-K61-vaccinated pig farms and spread rapidly to many provinces of China, causing substantial economic losses to the swine industry. A total of 4708 pig serum samples from Henan province during 2018-2019 were collected to screen for the presence of pseudorabies virus (PRV) gE-specific antibodies, and phylogenetic analysis based on the gE gene of PRV was performed. Of the 4708 serum samples tested, 30.14% (1419/4708) were seropositive for PRV antibodies, based on PRV gE-coated enzyme-linked immunosorbent assay (ELISA), with slaughterhouses having the highest seroprevalence. The seropositive rates of PRV also varied with the region and the season. Phylogenetic analysis showed that three PRV isolates from this study were clustered in an independent branch together with the Chinese variant PRV strains (after 2012), and had a closer genetic relationship with the Chinese variant PRV strains, but differed genetically from the 4 early Chinese PRV strains and 4 European-American strains. This study suggests that three PRV isolates may belong to PRV variants, and the development of a novel vaccine against PRV variants is particularly urgent.

Molecular detection and phylogenetic analysis of Porcine circovirus 4 in Henan and Shanxi Provinces of China.

Porcine circovirus 4 (PCV4), a new circovirus with a distinct relationship to other circoviruses, was identified in 2019 in several pigs with severe clinical disease in Hunan Province, China. To investigate the epidemic profile and genetic diversity of the virus, 63 clinical samples were collected from 24 different pig farms in 14 cities in Henan and Shanxi Provinces, China, between February 2018 and December 2019, and the partial Cap gene of PCV4 was amplified by PCR. Among the 63 samples, 16 (25.40%) were positive for PCV4, and 50% (12/24) of the pig farms were positive for PCV4. PCV4 was detected in samples from pigs with different clinical presentations. One PCV4 strain (Henan-LY1-2019) was sequenced in this study, and shared 98.4% genomic nucleotide identity with PCV4 strain HNU-AHG1-2019 (accession no. MK986820) detected on a pig farm in Hunan Province in 2019. A phylogenetic analysis based on the genomes of Henan-LY1-2019 and 31 reference strains showed that the Henan-LY1-2019 strain together with PCV4 strain HNU-AHG1-2019 was grouped in a relatively independent sub-branch, and separated from other viruses in the genus Circovirus. The results of this study extend our understanding of the molecular epidemiology of PCV4.

Characteristics of the spike and ORF3 genes of porcine epidemic diarrhea virus in Henan and Shanxi provinces of China.

To investigate the epidemic characteristics of porcine epidemic diarrhea virus (PEDV), 135 clinical samples (including intestinal tissues and feces) were collected from diseased piglets during outbreaks of diarrhea from 2015 to 2019 on farms in Henan and Shanxi provinces of China where swine had been immunized with attenuated PEDV (CV777). A total of 86 clinical samples (86/135, 63.7%) were positive for PEDV by RT-PCR, and subsequently, the complete spike (S) and ORF3 genes of 32 PEDV samples were sequenced. Phylogenetic analysis showed that the 32 PEDV strains obtained in this study belonged to group 2 (pandemic variant strains) and had a close relationship to 17 Chinese strains after 2010, two South Korean strains (KNU-1305 and KNU-1807), three American strains (PC22A-P140.BI, USA/Colorado/2013, and USA/OK10240-6/2017) and a Mexican strain (PEDV/MEX/QRO/02/2017), but differed genetically from a South Korean strain (SM98), a European strain (Br1/87), a Chinese strain (LZC), and a vaccine strain (CV777). G2-a subgroup strains were the dominant pandemic variant strains circulating in Henan and Shanxi provinces of China. Furthermore, a cross-recombination event was identified in the S region of the SX/TY2/2017 strain, and the putative parental strains were the epidemic strains CH/GDGZ/2012 and CH/YZ1/2015, identified in China in 2012 and 2015, respectively. These results provide further information about PEDV evolution, which could improve our understanding of the circulation of PEDV in Henan and Shanxi provinces. This information will also be helpful for developing new strategies for prevention and control of variant strains.

Construction and immunogenicity of a gE/gI/TK-deleted PRV based on porcine pseudorabies virus variant.

Pseudorabies (PR) caused by re-emerging pseudorabies virus (PRV) variant has outbroken among PRV vaccine-immunized swine herds on many Chinese pig farms, with severe socioeconomic consequences since late 2011. Here, a gE/gI/TK-deleted recombinant virus (rPRV NY-gE-/gI-/TK-) was constructed based on PRV NY strain from 2012 through homologous DNA recombination and gene-editing technology termed clustered regularly interspaced palindromic repeats (CRISPR)/associated (Cas9) system. The rPRV NY-gE-/gI-/TK- strain showed similar growth kinetics to the parental PRV NY strain in vitro, and was safe for mice. Sixty mice were injected subcutaneously (s.c.) twice with 106.0 TCID50 of rPRV NY-gE-/gI-/TK- and DMEM, respectively, with two-week interval. The levels of PRV gB antibodies and neutralizing antibodies against PRV NY in mice immunized with rPRV NY-gE-/gI-/TK- were higher than those in the DMEM control group. The number of T lymphocyte subclasses CD3+, CD4+ and CD8+ in rPRV NY-gE-/gI-/TK--immunized mice was higher than that in DMEM-injected mice. After challenge with 106.0 TCID50 PRV NY at 42 dpi, all rPRV NY-gE-/gI-/TK--immunized mice survived without exhibiting any pathological lesions in different tissues and intranuclear eosinophilic inclusions of the brain, and the viral genomic copy numbers in various organs of mice were obviously lower than DMEM group. These results showed the rPRV NY-gE-/gI-/TK- could be a promising next-generation vaccine to control now epidemic PR in China.

Characterization of a recombinant pseudorabies virus expressing porcine parvovirus VP2 protein and porcine IL-6.

BACKGROUND: Porcine parvovirus (PPV) and pseudorabies virus (PRV) are the important etiological agents of swine infectious diseases, resulting in huge economic losses to the Chinese swine industry. Interleukin-6 (IL-6) has the roles to support host immune response to infections as a pleiotropic cytokine. It is essential to construct a live attenuated vaccine-based recombinant PRV that expresses PPV VP2 protein and porcine IL-6 for prevention and control of PRV and PPV. METHODS: The recombinant plasmid, pGVP2-IL6, was constructed by porcine IL-6 gene substituting for EGFP gene of the PRV transfer plasmid pGVP2-EGFP containing VP2 gene of PPV. Plasmid pGVP2-IL6 was transfected into swine testicle cells pre-infected with the virus rPRV-VP2-EGFP strain through homologous recombination and plaque purification to generate a recombinant virus rPRV-VP2-IL6. The recombinant PRV was further identified by PCR and DNA sequencing, and the expression of the VP2 protein and porcine IL-6 was analyzed by reverse transcription-PCR (RT-PCR) and Western blot. The virus titer was calculated according to Reed and Muench method. The immunogenicity of the recombinant virus was preliminarily evaluated in mice by intramuscular administration twice with the rPRV-VP2-IL6 at 4-week intervals. RESULTS: A recombinant virus rPRV-VP2-IL6 was successfully constructed and confirmed in this study. The properties of rPRV-VP2-IL6 were similar to the parental virus HB98 in terms of growth curve, morphogenesis and virus plaque sizes, and rPRV-VP2-IL6 was proliferated in different cell types. It induced specific antibodies against PPV as well as a strong increase of PPV-specific lymphocyte proliferation responses in mice immunized with rPRV-VP2-IL6, and provided partial protection against the virulent PPV challenge. rPRV-VP2-IL6 also induced a high level of neutralizing antibodies against PRV, and significantly reduced the mortality rate of (1 of 10) following virulent PRV challenge compared with the control (10 of 10). CONCLUSIONS: The recombinant rPRV-VP2-IL6 might be a potential candidate vaccine against PRV and PPV infections in pigs.

Simultaneous detection of classical swine fever virus and porcine circovirus 3 by SYBR green I-based duplex real-time fluorescence quantitative PCR.

In the present study, the SYBR green I-based duplex quantitative polymerase chain reaction (qPCR) was developed for simultaneous detection of classical swine fever virus (CSFV) and porcine circovirus 3 (PCV3). The assay was used to detect both CSFV and PCV3 in one sample by their distinct melting temperatures (melting peaks at 87°C for CSFV and 81.5 °C for PCV3), and no specific fluorescence signals were detected for other non-targeted porcine pathogens. The assay had a high degree of linearity (R2 > 0.998) with the detection limits of 23 copies/µL for CSFV and 36 copies/µL for PCV3, and exhibited high repeatability and reproducibility with a low coefficient of variation below 2.0% in both intra- and inter-assay. In this study, 130 clinical samples collected from sick pigs in the field were tested by this assay with the positive rates of 9.23% (12/130) for CSFV and 21.54% (28/130) for PCV3 respectively, and the positive rate of CSFV and PCV3 co-infection was 6.92% (9/130). Our results showed that the developed method was a reliable diagnostic tool to monitor and survey CSFV, PCV3 and CSFV/PCV3 co-infection in the field.

Analysis of genetic variation of porcine circovirus type 2 within pig populations in central China.

In order to investigate the genetic diversity of porcine circovirus type 2 (PCV2), 284 clinical tissue samples were collected from different pig farms in central China from 2015 to 2017. A total of 162 tissue samples (162/284, 57.04%) were positive for PCV2 by PCR, and subsequently, the complete genome of 36 of these PCV2 samples was cloned and sequenced. The sequencing results showed that 37 complete PCV2 sequences were obtained from 36 PCV2-positive clinical samples. These PCV2 strains were relatively conserved and extremely homologous to the representative classical PCV2 strains. Of these, 20 PCV2 strains belonged to genotype PCV2d, 14 belonged to PCV2b, and three others belonged to PCV2a. Coinfection with PCV2b and PCV2d was identified in one sample (DF-2). These results show that PCV2d may be gradually replacing PCV2b as the predominant PCV2 genotype in central China, and that other genotypes also exist in individual regions. The results of this study will aid in our understanding of the molecular epidemiology of PCV2.

Development of a SYBR green I-based duplex real-time fluorescence quantitative PCR assay for the simultaneous detection of porcine epidemic diarrhea virus and porcine circovirus 3.

The development of a rapid, specific, and sensitive SYBR Green I-based duplex real-time quantitative PCR assay is described for the simultaneous detection of porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 3 (PCV3). The assay specifically detected PEDV and PCV3, with no fluorescence detected for other non-targeted pig pathogens. The assay showed a good linear relationship, and the limits of detection for this assay were 34.6 copies/µL and 61.2 copies/µL for PEDV and PCV3, respectively. The assay exhibited high repeatability and reproducibility, with intra-assay and inter-assay variation coefficients less than 2.0%. A clinical evaluation using intestinal tissue and fecal samples from piglets suffering from diarrhea at different pig farms in China revealed that the singular infection rates of PEDV and PCV3 were 43.94% (29/66) and 16.67% (11/66), respectively, while the co-infection rate of PCV3 with PEDV was 27.27% (18/66). The results indicate this assay is a rapid and reliable diagnostic tool for PEDV and PCV3 monitoring and surveillance in the field, and provides technical support for the quantitative detection of clinical samples infected or co-infected with PEDV and PCV3.

Detection and phylogenetic analysis of porcine circovirus type 3 in central China.

Porcine circovirus type 3 (PCV3) is the pathogen responsible for a new infectious disease that was first reported in 2016 in the United States. To further investigate the epidemic profile and genetic diversity of the virus, one hundred and seventy clinical samples (110 tissue samples and 60 serum samples) were collected from 41 different pig farms in 14 cities in central China, and a SYBR Green I-based quantitative real-time PCR method was developed to detect PCV3. The partial cap genes of four field strains from four different farms were sequenced and analysed. The results showed the detection limit was 2.19 × 101 genome copies/µl. Fifty-three of 170 samples were detected as positive for PCV3, giving a PCV3-positive rate of 31.18%, with 48.78% (20/41) of pig farms harbouring PCV3, which varied from 20% to 42.86% between 2013 and 2017. PCV3 could be detected in samples from pigs with different clinical presentations, and the PCV3-positive rates varied for these different clinical presentations. The partial capsid genes of four PCV3 strains (designated YZ, LY-03, NY and SP) shared 96.3%-99.4% nucleotide identity with those available in GenBank. Phylogenetic analysis based on the capsid gene of 32 PCV3 strains showed that the four PCV3 strains in this study were clustered with the China/GD2016 and South Korea Ku-1606 strains. The results of this study will aid our understanding of the molecular epidemiology of PCV3.