Occurrence of enteric viruses causing clinical diarrhea in small ruminants in northern Indian plains: a reverse transcription PCR based molecular study.

Fowlpox virus (FPV) infects chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth, etc. The birds, affected with FPV, also show anemia and a ruffled appearance which are clinical symptoms of reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of reticuloendotheliosis virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the chorio-allantoic membrane (CAM) of 10-day-old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. However, the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

Repeatable and objective method for evaluating angiogenesis using real-time RT-PCR of endoglin expression in canine tumours.

Anti-angiogenic therapy is a cancer treatment strategy targeting new blood vessel formation. Microvessel density (MVD) is a histopathological method for evaluating angiogenesis and endoglin is used as an activated endothelial marker in human medicine. The assessment of the treatment effect using MVD is difficult because it is a non-repeatable method. To develop a repeatable method for evaluating angiogenesis, we investigated correlations among MVD, mRNA transcription levels of endothelial markers and angiogenesis factors, and confirmed the agreement of mRNA transcription levels between tissue samples and small samples obtained by fine needle aspiration (FNA). The various types of spontaneous tumours were collected from 51 dogs. MVD was assessed by immunostaining for von Willebrand factor (vWF). mRNA transcription levels of vWF, endoglin, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) were analysed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR). There were significant correlations between MVD and mRNA transcription levels of vWF, endoglin and VEGFR2. VEGFR2 was more strongly correlated with endoglin (P <.01, Rs = 0.649) than vWF (P <.01, Rs = 0.512), indicating that angiogenesis can be evaluated more accurately by the measurement of mRNA transcription levels of endoglin. The mRNA transcription levels in tissue and FNA samples were strongly correlated, suggesting that evaluating angiogenesis using FNA samples is possible. In conclusion, we developed a repeatable and objective method for angiogenesis evaluation using mRNA transcription levels of endothelial markers by FNA sampling.

Establishment of multiplex RT-PCR for differentiation between rabies virus with and that without mutation at position 333 of glycoprotein.

Rabid raccoon dogs (Nyctereutes procyonoides koreensis) have been responsible for animal rabies in South Korea since the 1990s. A recombinant rabies vaccine strain, designated as ERAGS, was constructed for use as a bait vaccine. Therefore, new means of differentiating ERAGS from other rabies virus (RABV) strains will be required in biological manufacturing and diagnostic service centers. In this study, we designed two specific primer sets for differentiation between ERAGS and other RABVs based on mutation in the RABV glycoprotein gene. Polymerase chain reaction analysis of the glycoprotein gene revealed two DNA bands of 383 bp and 583 bp in the ERAGS strain but a single DNA band of 383 bp in the field strains. The detection limits of multiplex reverse transcription polymerase chain reaction (RT-PCR) were 80 and 8 FAID50/reaction for the ERAGS and Evelyn-Rokitnicki-Abelseth strains, respectively. No cross-reactions were detected in the non-RABV reference viruses, including canine distemper virus, parvovirus, canine adenovirus type 1 and 2, and parainfluenza virus. The results of multiplex RT-PCR were 100% consistent with those of the fluorescent antibody test. Therefore, one-step multiplex RT-PCR is likely useful for differentiation between RABVs with and those without mutation at position 333 of the RABV glycoprotein gene.

Evaluation of polymorphisms in inflammatory mediator and cellular adhesion genes as risk factors for feline infectious peritonitis.

OBJECTIVES: Feline infectious peritonitis (FIP) is a high mortality infectious disease. Single nucleotide polymorphisms (SNPs) in the genes encoding interferon gamma (IFNG), tumour necrosis factor alpha (TNFA) and dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN; CD209) have been associated with increased and decreased risk of developing FIP. This study was designed to determine whether these associations were present in a UK population of pedigree cats using samples from cats euthanased with a confirmed diagnosis (FIP, n = 22; non-FIP, n = 10) or clinically healthy cats over 11 years of age (n = 3). METHODS: DNA was extracted from tissue (n = 32) or blood (n = 3) and PCR performed for regions of IFNG, TNFA and CD209. PCR amplicons were sequenced, each SNP genotype was determined, and genotype/allele frequency for each SNP and FIP status were compared. RESULTS: No significant association was found between the genotype and FIP status for any SNP analysed. There was a trend for the heterozygous CT genotype at both IFNG g.401 and IFNG g.408 to be associated with FIP (P = 0.13), but this genotype was also found in a substantial proportion of non-FIP cats. There was also a trend for the heterozygous CT genotype at IFNG g.428 to be associated with FIP (P = 0.06), although most cats with FIP had the CC genotype at this locus. No associations were found between any allele at TNFA g.-421, CD209 g.1900, CD209 g.2276, CD209 g.2392 and CD209 g.2713 and FIP. CONCLUSIONS AND RELEVANCE: The use of the IFNG, TNFA and CD209 SNPs described to predict the risk of FIP cannot currently be recommended.

Development of a multiplex RT-PCR for the detection of major diarrhoeal viruses in pig herds in China.

The major enteric RNA viruses in pigs include porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (PRV-A), porcine kobuvirus (PKV), porcine sapovirus (PSaV) and porcine deltacoronavirus (PDCoV). For differential diagnosis, a multiplex RT-PCR method was established on the basis of the N genes of TGEV, PEDV and PDCoV, the VP7 gene of PRV-A, and the polyprotein genes of PKV and PSaV. This multiplex RT-PCR could specifically detect TGEV, PEDV, PDCoV, PRV-A, PKV and PSaV without cross-reaction to any other major viruses circulating in Chinese pig farms. The limit of detection of this method was as low as 100 -101  ng cDNA of each virus. A total of 398 swine faecal samples collected from nine provinces of China between October 2015 and April 2017 were analysed by this established multiplex RT-PCR. The results demonstrated that PDCoV (144/398), PSaV (114/398), PEDV (78/398) and PRV-A (70/398) were the main pathogens, but TGEV was not found in the pig herds in China. In addition, dual infections, for example, PDCoV + PSaV, PDCoV + PRV-A, PRA-V + PSaV and PEDV + PDCoV, and triple infections, for example, PDCoV + PRV-A + PSaV and PEDV + PDCoV + PKV, were found among the collected samples. The multiplex RT-PCR provided a valuable tool for the differential diagnosis of swine enteric viruses circulating in Chinese pig farms and will facilitate the prevention and control of swine diarrhoea in China.

Feline coronavirus with and without spike gene mutations detected by real-time RT-PCRs in cats with feline infectious peritonitis.

OBJECTIVES: Feline infectious peritonitis (FIP) emerges when feline coronaviruses (FCoVs) mutate within their host to a highly virulent biotype and the immune response is not able to control the infection. FCoV spike (S) gene mutations are considered to contribute to the change in virulence by enabling FCoV infection of and replication in macrophages. This study investigated the presence of FCoV with and without S gene mutations in cats with FIP using two different real-time RT-PCRs on different samples obtained under clinical conditions. METHODS: Fine-needle aspirates (FNAs) and incisional biopsies (IBs) of popliteal and mesenteric lymph nodes, liver, spleen, omentum and kidneys (each n = 20), EDTA blood (n = 13), buffy coat smears (n = 13), serum (n = 11), effusion (n = 14), cerebrospinal fluid (n = 16), aqueous humour (n = 20) and peritoneal lavage (n = 6) were obtained from 20 cats with FIP diagnosed by immunohistochemistry. Samples were examined by RT-PCR targeting the FCoV 7b gene, detecting all FCoV, and S gene mutation RT-PCR targeting mutations in nucleotides 23531 and 23537. The prevalence of FCoV detected in each sample type was calculated. RESULTS: In 20/20 cats, FCoV with S gene mutations was present in at least one sample, but there was variation in which sample was positive. FCoV with mutations in the S gene was most frequently found in effusion (64%, 95% confidence interval [CI] 39-89), followed by spleen, omentum and kidney IBs (50%, 95% CI 28-72), mesenteric lymph node IBs and FNAs (45%, 95% CI 23-67), and FNAs of spleen and liver and liver IBs (40%, 95% CI 19-62). CONCLUSIONS AND RELEVANCE: In these 20 cats with FIP, FCoVs with S gene mutations were found in every cat in at least one tissue or fluid sample. This highlights the association between mutated S gene and systemic FCoV spread. Examining a combination of different samples increased the probability of finding FCoV with the mutated S gene.

Different expression of Defensin-B gene in the endometrium of mares of different age during the breeding season.

BACKGROUND: Despite being one of the major causes of infertility in mares, the mechanisms responsible for equine endometrosis are still unclear and controversial. In the last few years, many investigations focused on local immune response modulation. Since it is generally accepted that endometrial fibrosis increases with age, we hypothesize that older mares could show altered local immune modulation, initiating a pro-inflammatory and tissue remodeling cascade of events that could lead to endometrosis. The aim of this study, indeed, is to evaluate and describe the local gene expression of genes involved in acute inflammatory response and fibrosis (COL1A1, COL3A1, TNFA, MMP9, IL6, TGFB1 and TGFBR1), together with others associated to immune modulation (DEFB4B, IDO1 and FOXP3), in uterine specimens from mares of different age. RESULTS: Twenty-five Standardbred mares were involved in the study with age ranging from 7 to 19 years (mean 10.40 ± 4.42). They were divided by age into two groups: G1 (n = 15, less than 10 years old) and G2 (N = 10, greater than 11 years old). Specimens from the uterus' right horn-body junction were collected and processed for histology evaluation and RT-qPCR assay.Gene expression of DEFB4B, MMP9 and TNFA was higher in younger mares, suggesting a balance in immune modulation and tissue remodeling. Interleukin-6 and COL3A1 gene expressions were greater in older animals, probably indicating inflammatory pathways activation and fibrosis increase. Although no differences in fibrosis and inflammation distribution could be found with histological examination among G1 and G2, our results suggest a possible involvement of DEF4BB in regulating the local immune response in younger mare's uterus (G1); age may contribute to the dis-regulation of DEFB4B transcription and, indirectly, influence the extracellular matrix homeostasis. Transcription of IDO1 and FOXP3 genes, instead, does not seem to be age related, or to be involved in local immune-response and tissue remodeling functions. CONCLUSIONS: Further investigations are needed in order to clarify the interactions between the expression of DEFB4B, IL6, TNFA, COL3A1 and MMP9 and other local signals of immune-modulation and tissue remodeling, in mares in a prospective study design.

Comparative measurement of FeLV load in hemolymphatic tissues of cats with hematologic cytopenias.

BACKGROUND: Feline leukemia virus (FeLV) is a serious viral infection in cats. FeLV is found in some tissues, such as spleen, lymph nodes and epithelial tissues. However, there is controversy about the organ in which the virus can be reliably detected in infected cats. The purpose of this study was to determine the level of viral infection in hemolymphatic tissues, including blood, bone marrow and spleen by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). RESULTS: A total of 31 cats with clinical signs of FeLV infection associated with at least a single lineage hematologic cytopenia were included in this study. Peripheral blood, bone marrow and spleen samples were obtained from each cat. Complete blood counts, biochemical tests, and a rapid test to detect FeLV p27 antigen in blood samples of cats were performed. Of 31 cats, 9 had anemia alone, 4 had thrombocytopenia alone, 2 had neutropenia alone, 9 had bicytopenia of anemia and thrombocytopenia, 3 had bicytopenia of anemia and neutropenia, and 4 had pancytopenia. FeLV RNA was then detected by RT-qPCR in the whole blood, bone marrow and spleen. Viral RNA copy numbers were detected in all cats by RT-qPCR whereas 24 out of 31 cats were positive for the serum FeLV antigen. We detected a significantly greater number of viral RNA in the spleen compared with the whole blood and bone marrow. CONCLUSION: Spleen is a site where FeLV is most frequently detected in cats with hematologic cytopenias.

Molecular cloning, characterisation, and expression analysis of adipocyte fatty acid binding protein gene in Xupu goose (Anser cygnoides domesticus).

1. Adipocyte fatty acid binding protein (A-FABP) plays a key role in fatty acid uptake and intracellular transport. The objective of the present study was to identify and characterise the A-FABP gene in Xupu goose.2. The full-length cDNA of goose A-FABP gene was cloned from the liver tissue using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The distribution of the goose A-FABP in different tissues was determined by quantitative real-time PCR (qRT-PCR).3. The results showed that the full-length cDNA sequence of goose A-FABP was 657 bp, containing a 5'-UTR of 52 bp, a 3'-UTR of 206 bp and an open reading frame (ORF) of 399 bp, which encoded a polypeptide of 132 amino acids (AA).4. The AA sequence of goose A-FABP showed 76.52%, 75.00%, 93.18% and 99.24% identities with previously described homologues from humans (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), and duck (Anas platyrhynchos), respectively, and phylogenetic analysis revealed a close relationship among them. The transcript of Xupu goose A-FABP was ubiquitously expressed in all tested tissues, and showed a high-level expression in abdominal fat, sebum and liver.5. A significant positive correlation was identified between A-FABP mRNA abundance in the three adipose tissues and liver weight, ratio of liver to body weight, TG content, and VLDL concentration in the plasma of Xupu goose. A significant negative correlation was observed between the mRNA level of A-FABP and HDL concentration in the plasma of Xupu goose.6. These findings provide a foundation for further research on the function and mechanism of A-FABP in the fat deposition process.

Selection of reference genes for miRNA quantitative PCR and its application in miR-34a/Sirtuin-1 mediated energy metabolism in Megalobrama amblycephala.

MiRNAs are small, non-coding RNAs that downregulate gene expression at post-transcriptional levels. They have emerged as important regulators involved in metabolism, immunity, and cancer. Real-time quantitative PCR (RT-qPCR) is an effective and main method for quantifying target miRNA. For robust RT-qPCR method, suitable reference genes play crucial roles in data normalization. Blunt snout bream (Megalobrama amblycephala) is an economically important aquaculture species; however, no reference genes dedicated for qPCR method has been identified for this species so far. The objective of this study was to screen stable reference genes for miRNA RT-qPCR and demonstrated its application in energy metabolism in blunt snout bream. The stabilities of ten potential reference genes (miR-21-1-5p, miR-107a-3p, miR-222a-3p, miR-146a-5p, miR-101a-3p, miR-22a-3p, miR-103-3p, miR-456-3p, miR-221-3p, and U6 (RNU6A)) were evaluated in nine tissues (brain, muscle, liver, skin, spleen, heart, gill, intestine, and eye) under normal condition and in three tissues (liver, intestine, and spleen) under four stresses (heat stress, ammonia stress, bacterial challenge, and glycolipid stress). Using GeNorm, NormFinder, and RefFinder softwares, we discovered that different tissues and stresses are both important variability factors for the expression stability of miRNAs. After verifying miR-34a/Sirtuin-1 expressions in high-carbohydrate diet-induced blunt snout bream, we eventually identified that the most stable reference gene in this species was miR-221-3p, and the best combination of reference genes were miR-221-3p and miR-103-3p.

Diagnosis of non-effusive feline infectious peritonitis by reverse transcriptase quantitative PCR from mesenteric lymph node fine-needle aspirates.

OBJECTIVES: The aim of this study was to evaluate a feline coronavirus (FCoV) reverse transcriptase quantitative PCR (RT-qPCR) on fine-needle aspirates (FNAs) from mesenteric lymph nodes (MLNs) collected in sterile saline for the purpose of diagnosing non-effusive feline infectious peritonitis (FIP) in cats. METHODS: First, the ability of the assay to detect viral RNA in MLN FNA preparations compared with MLN biopsy preparations was assessed in matched samples from eight cats. Second, a panel of MLN FNA samples was collected from a series of cats representing non-effusive FIP cases (n = 20), FCoV-seropositive individuals (n = 8) and FCoV-seronegative individuals (n = 18). Disease status of the animals was determined using a combination of gross pathology, histopathology and/or 'FIP profile', consisting of serology, clinical pathology and clinical signs. RESULTS: Viral RNA was detected in 18/20 non-effusive FIP cases; it was not detected in two cases that presented with neurological FIP. Samples from 18 seronegative non-FIP control cats and 7/8 samples from seropositive non-FIP control cats contained no detectable viral RNA. Thus, as a method for diagnosing non-effusive FIP, MLN FNA RT-qPCR had an overall sensitivity of 90.0% and specificity of 96.1%. CONCLUSIONS AND RELEVANCE: In cases with a high index of suspicion of disease, RT-qPCR targeting FCoV in MLN FNA can provide important information to support the ante-mortem diagnosis of non-effusive FIP. Importantly, viral RNA can be reliably detected in MLN FNA samples in saline submitted via the national mail service. When applied in combination with biochemistry, haematology and serological tests in cases with a high index of suspicion of disease, the results of this assay may be used to support a diagnosis of non-effusive FIP.

Expression of the Short Form of RON/STK in Feline Mammary Carcinoma.

RON is a tyrosine kinase receptor activated by the macrophage-stimulating protein (MSP) ligand that is overexpressed in human breast cancer. In humans, RON protein can be present in different isoforms, and the most studied isoform is represented by the short form of RON ( sf-RON), which is generated by an alternative promoter located in intron 10 of the RON complementary DNA (cDNA). It plays an important role in breast cancer progression. Considering the many similarities between feline mammary carcinoma (FMC) and human breast cancer, the aim of this study was to investigate the expression of both RON and MSP in FMCs and to identify the presence of the sf-RON transcript. Tissue samples of spontaneous mammary tumors were collected from 60 queens (10 benign lesions, 50 carcinomas). All of the samples were tested for RON and MSP expression by immunohistochemistry; moreover, RNA was extracted from paraffin-embedded tissue samples, and the cDNA was tested by reverse transcription-polymerase chain reaction (RT-PCR) to identify the presence of sf-RON. Immunohistochemistry detected the expression of RON and MSP in 34 of 50 (68%) and 29 of 50 (58%) FMCs, respectively. RT-PCR revealed the presence of the short-form in 18 of 47 (38%) FMCs. This form originates, as in humans, from an alternative promoter (P2), and it codes for the proper feline short form ( sf-RON). sf-RON expression was associated with poorly differentiated tumors and with a shorter disease-free ( P < .05; hazard ratio [HR], 2.2) period and a shorter survival ( P < .05; HR, 2.2). These results support FMC as a suitable model in comparative oncology and identify sf-RON expression as potential predictor of outcomes for this disease.

Benzo(a)pyrene suppresses tracheal antimicrobial peptide gene expression in bovine tracheal epithelial cells.

Respiratory disease is an important cause of morbidity and mortality in cetaceans, which are also threatened by environmental degradation caused by crude oil spills. Following oil spills, cetaceans at the water surface may inhale droplets of oil containing toxic polycyclic aromatic hydrocarbons (PAHs), which could potentially alter respiratory immunity via activation of the aryl hydrocarbon receptor (AHR) and its subsequent interaction with nuclear factor kappa B (NF-κB). ß-defensins are antimicrobial peptides secreted by airway epithelial cells and their expression is known to be dependent on NF-κB. We hypothesized that PAHs may suppress the expression of ß-defensins, and thereby contribute to the pathogenesis of pneumonia. This hypothesis was modeled by measuring the in vitro effects of benzo(a)pyrene (BAP), phenanthrene, and naphthalene on tracheal antimicrobial peptide (TAP) gene expression in bovine tracheal epithelial cells. Stimulation with lipopolysaccharide (LPS) induced 20 ± 17-fold (mean ± SD) increased TAP gene expression. Exposure of tracheal epithelial cells to 5 µM BAP for 4 or 8 h, followed by incubation with a combination of LPS and 5 µM BAP for another 16 h, significantly (P = 0.002) suppressed LPS-induced TAP gene expression by 40.6 ± 21.8% (mean ± SD) in tracheal epithelial cells from 9 calves tested. BAP-induced suppression of TAP gene expression coincided with induction of cytochrome P450 1A1 gene expression. In contrast, phenanthrene and naphthalene had no consistent effect, and exposure to PAHs did not significantly affect constitutive TAP gene expression (i.e. without LPS). These findings characterize the suppressive effects of BAP-a toxic pollutant found in crude oil-on this respiratory innate immune response.

Relationship between the pathology of bovine intestinal tissue and current diagnostic tests for Johne's disease.

Johne's disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon translocation from the lumen of the small intestine, mycobacteria have the ability to thwart innate defense mechanisms and persist within the macrophage in the lamina propria. In an effort to understand how the pathology of disease is reflected in current diagnostic tests, immunofluorescent (IFA) labeling was performed to quantitate macrophage and MAP numbers in the ileum of infected cattle and correlate results with common methods for diagnosis of MAP infection; including ELISA, IFN-γ assay, RT-PCR, culture of MAP, and histological classification of tissue sections. Predictive models for clinical and subclinical disease states, histopathology acid-fast (AF), MAP location, granulomatous inflammation and type classifications, as well as macrophage, MAP and macrophages with intracellular MAP IFA labeling were successfully developed. The combination of macrophage number and ELISA were the best predictors of clinical disease state, while macrophage number was the best and only significant predictor of subclinical disease state. Fecal culture and number of MAP were the best predictors of granulomatous inflammation, and of combined AF, MAP location and granuloma type, respectively. Additionally, fecal culture and tissue culture were the best predictors of numbers of macrophages and MAP, respectively, while both ELISA and tissue culture were the best predictors of number of macrophages with intracellular MAP.

The biological characteristics of sheep umbilical cord mesenchymal stem cells.

Although mesenchymal stem cells (MSCs) are now regarded as a promising cell resource for tissue repair and regeneration, the optimal source of MSCs has not yet been determined. The objective of this study was to provide a theoretical basis for the clinical application of umbilical cord mesenchymal stem cells (UCMSCs) in the future. Umbilical cord is an easily obtainable tissue resource, which is one reason that it has become a candidate resource for mesenchymal stem cells. In this study, we analyzed the biological characteristics of UCMSCs, such as their multiple differentiation and clone-forming ability, through morphological observation, reverse transcription polymerase chain reaction (RT-PCR), growth curve, positive rate test, and immunophenotype. Umbilical cord MSCs were successfully isolated and passaged to 29 generations. The results from RT-PCR showed that UCMSCs were positive for CD29, CD44, CD73, but negative for CD34. The expression of the stem cell marker nucleostemin and tenocyte-related markers showed similar positive results with CD44, CD73, and CD90. In addition, UCMSCs can be induced to differentiate into osteoblasts, adipocytes, or chondrocytes. Our study showed that UCMSCs not only have the ability to self-renew, but also have the potential to differentiate into multiple lineages. In general, we concluded that UCMSCs are a reliable source for use in cell therapy.

Bien que les cellules souches mésenchymateuses (CSMs) soient maintenant considérées comme une ressource prometteuse de cellules pour la réparation tissulaire et la régénération, la source optimale de CSMs n'a pas encore été déterminée. L'objectif de la présente étude était de fournir une base théorique pour l'application clinique de cellules souches mésenchymateuses de cordon ombilical (CSMCO) dans le futur. Le cordon ombilical est une ressource tissulaire pouvant être obtenue facilement, une des raisons pour laquelle il est devenu un candidat pour les CSMs. Dans cette étude nous avons analysé les caractéristiques biologiques des CSMCO, telles que leur différenciation multiple et la capacité à former des clones, par des observations morphologiques, par réaction d'amplification en chaîne par la polymérase avec la transcriptase réverse (ACP-TR), courbe de croissance, test de ratio positif, et immunophénotype. Les CSMCO ont été isolées avec succès et des passages obtenus jusqu'à la 29e génération. Les résultats d'ACP-TR ont montré que les CSMCO étaient positives pour CD29, CD44, CD73, mais négative pour CD34. L'expression de nucléostémine, un marqueur de cellule souche, et de marqueurs apparentés aux ténocytes ont montré des résultats positifs similaires à ceux de CD44, CD73, et CD90. De plus, les CSMCO peuvent être induites à se différencier en ostéoblastes, adipocytes, ou chondrocytes. Notre étude a démontré que les CSMCO ont non seulement la capacité de s'auto-renouveler, mais ont également le potentiel de se différencier en des lignées multiples. En général, nous avons conclu que les CSMCO sont une source fiable pour utilisation en thérapie cellulaire.(Traduit par Docteur Serge Messier).

Molecular characterization of a porcine teschovirus HuN-1 isolate proliferating in PK-15 cell.

BACKGROUND: Porcine teschoviruses (PTVs) are small non-enveloped viruses with single-stranded, positive sense genomic RNA, belonging to the family Picornaviridae. Natural infections of teschoviruses are limited to pigs. RESULTS: In this study, a PTV HuN-1 was found that it could be proliferated in PK-15 cell, and it came from the pig fecal samples from Hunan province, in central China. The complete genome of the HuN-1 was amplified by RT-PCR and sequenced. The complete genome of HuN-1 isolate is 7098 nt, which shares the highest sequence identity (85.9%) with the PTV 8 strain of Jilin/2003/2 and Fuyu/2009/2. The HuN-1 isolate contains only one ORF (from 320 to 7039 nt) coding a 2240 amino acid polyprotein. Aligned sequences show that more mutations occurred in the structural region than in the nonstructural region. Phylogenetic analysis showed that HuN-1 isolate did not clustered with the hitherto reported strains, according to P1 sequences, forming a subgroup in the PTV cluster. CONCLUSION: In this study, complete genome of PTV HuN-1 was cloned and sequenced. Detection and characterization of further PTV strains from different geographic areas are important to understand the worldwide distribution and heterogeneity (serotype) of PTVs and their association with symptomatic infections in pigs.

Identification and isolation of infective filamentous particles in Infectious Salmon Anemia Virus (ISAV).

The infectious salmon anemia virus (ISAV) is an aquatic pathogen that is a member of the Orthomyxoviridae family with lethal hemorrhagic potential. Although it affects other species of salmonid fish, ISAV only causes disease in Atlantic salmon (Salmo salar) specimens in sea water. In spite of the fact that the virus has been described as enveloped with icosahedral symmetry, viral like particles with anomalous morphology have been observed in field samples, this we have not been able to recover then in adequate quantities for full demonstration. We report a procedure to concentrate and recover these novel forms of the virus, comparing two cell lines from different origins, demonstrating that these forms were preferentially expressed in cells of epithelial origin.

Cross-sectional quantitative RT-PCR study of feline coronavirus viremia and replication in peripheral blood of healthy shelter cats in Southern California.

Objectives The objectives of this study were to determine the prevalence of feline coronavirus (FCoV) viremia, and its replication in peripheral blood using quantitative RT-PCR (qRT-PCR) methodology in a population of 205 healthy shelter cats in Southern California, as well as to assess any possible connection to longitudinal development of feline infectious peritonitis (FIP). Methods The study was performed on buffy-coat samples from EDTA-anticoagulated whole blood samples of 205 healthy shelter cats. From 50 of these cats, fecal samples were also examined. FCoV genomic and subgenomic RNA in the buffy coats was amplified by a total FCoV RNA qRT-PCR. Evidence for FCoV replication in peripheral blood and feces was obtained by M gene mRNA qRT-PCR. Results Nine of 205 cats (4.4%) were viremic by the total FCoV RNA qRT-PCR, and one of these cats had evidence of peripheral FCoV blood replication by an FCoV mRNA qRT-PCR. The single cat with peripheral blood replication had a unique partial M gene sequence distinct from positive controls and previously published FCoV sequences. Neither seven of the nine viremic cats with follow-up nor the single cat with replicating FCoV with positive qRT-PCR results developed signs compatible with FIP within 6 months of sample collection. Conclusions and relevance FCoV viremia and peripheral blood replication in healthy shelter cats have a low prevalence and do not correlate with later development of FIP in this study population, but larger case-control studies evaluating the prognostic accuracy of the qRT-PCR assays are needed.

Steroidogenic factor-1 inverse agonists as a treatment option for canine hypercortisolism: in vitro study.

Hypercortisolism is one of the most commonly diagnosed endocrinopathies in dogs, and new targeted medical treatment options are desirable. Steroidogenic factor-1 (SF-1), an orphan nuclear hormone receptor, is a key regulator of adrenal steroidogenesis, development, and growth. In pituitary-dependent hypercortisolism (PDH), high plasma ACTH concentrations increase the transcriptional activity of SF-1. In adrenal-dependent hypercortisolism, SF-1 expression is significantly greater in dogs with recurrence after adrenalectomy than in those without recurrence. Inhibition of SF-1 could therefore be an interesting treatment option in canine spontaneous hypercortisolism. We determined the effects of 3 SF-1 inverse agonists, compounds IsoQ A, #31, and #32, on cortisol production, on the messenger RNA (mRNA) expression of steroidogenic enzymes and SFs, and on cell viability, in primary adrenocortical cell cultures of 8 normal adrenal glands and of 3 cortisol-secreting adrenocortical tumors (ATs). To mimic PDH, the normal adrenocortical cell cultures were stimulated with ACTH. The results show that only compound #31 inhibited cortisol production and SF-1 target gene expression in non-ACTH-stimulated and ACTH-stimulated normal adrenocortical cells but did not affect cell viability. In the AT cell cultures, the effects of #31 on cortisol production and target gene expression were variable, possibly caused by a difference in the SF-1 mRNA expressions of the primary tumors. In conclusion, inhibition of SF-1 activity shows much promise as a future treatment for canine hypercortisolism.

Immunohistochemical and quantitative RT-PCR methods to assess RANK expression in normal and neoplastic canine mammary gland.

The receptor activator of nuclear factor-kB ( RANK) gene is found in both human and murine mammary epithelial cells and in human cancer cell lines. We analyzed RANK expression in normal and proliferative canine mammary tissue samples ( n = 47) and cell lines ( n = 10), and identified its expression in epithelial cell populations. The correlation of RANK protein with clinicopathologic parameters was also studied. A double immunohistochemical method using RANK and p63 antibodies was applied to 33 tissue samples to analyze RANK protein expression and its possible co-expression with p63 protein, the latter used to identify myoepithelial (ME) cells (p63-positive) or luminal epithelial (LE) cells (p63-negative). RANK protein expression was found in ~75% of the tissue samples analyzed, at a similar level in all of the histologic types studied: dysplasias (4 of 4, 100%), malignant tumors (13 of 17, 76%), normal glands (12 of 17, 70%), and benign tumors (6 of 9, 67%). ME and LE cells expressed RANK protein at a similar level. A higher level of RANK protein expression was found in older animals (≥10 y, p = 0.027). Quantitative RT-PCR was applied to 6 ME (1 normal and 5 neoplastic) and 4 LE (1 normal and 3 neoplastic) primary cell lines. The RANK gene was found at similar expression levels in all canine mammary ME and LE cell lines studied. We found RANK expression in normal, dysplastic, and neoplastic canine mammary tissues and cell lines, in both ME and LE cell populations.