Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine.

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Exploring the therapeutic potential of allogeneic amniotic membrane for quality wound healing in rabbit model.

BACKGROUND: The amniotic membrane (AM) has shown immense potential in repairing wounds due to its great regenerative qualities. Although the role of AM as a biological scaffold in repairing wounds has been studied well, the tissue regenerative potential of AM-derived mesenchymal stem cells (MSCs) and conditioned media (CM) derived from it remains to be discovered as of now. Here, we examined the wound healing abilities of fresh and frozen thawed rabbit AM (rAM) along with the MSCs and their lyophilised CM in rabbits challenged with skin wounds. METHODS: To elucidate the role of rAM-MSCs and its CM in repairing the wound, we isolated it from the freshly derived placenta and characterised their differentiation potential by performing an in vitro tri-lineage differentiation assay besides other standard confirmations. We compared the wound repair capacities of rAM-MSCs and lyophilised CM with the fresh and cryopreserved AM at different timelines by applying them to excision wounds created in rabbits. RESULTS: By monitoring wound contractions and tissue histology of wounded skin at different time points after the application, we observed that rAM-MSCs and rAM-MSC-derived CM significantly promoted wound closure compared to the control group. We also observed that the wound closure capacity of rAM-MSCs and rAM-MSC-derived CM is as efficient as fresh and cryopreserved rAM. CONCLUSION: Our findings suggest that rAM-MSCs and rAM-MSC derived CM can be effectively used to treat skin wounds in animals and correctly delivered to the damaged tissue using AM as a bioscaffold, either fresh or frozen.

Pathomolecular epidemiology, antimicrobial resistance, and virulence genes of Streptococcus dysgalactiae subsp. equisimilis isolates from slaughtered pigs in India.

AIMS: We aimed to investigate the prevalence, pathology, and characterization of Streptococcus dysgalactiae subsp. equisimilis (SDSE) in slaughtered pigs of India. METHODS AND RESULTS: We collected 1254 morbid tissues (lungs-627 and spleen-627) and 627 heart-blood from 627 slaughtered pigs. The bacterial isolation, antibiogram, virulence gene profiling, and mouse pathogenicity testing were performed for the detection and characterization of SDSE. A total of 177 isolates (heart-blood-160 and tissues-17) were recovered from 627 slaughtered pigs with higher isolation rate in heart-blood (25.51%). The prevalence of SDSE was 11% in morbid tissues by polymerase chain reaction. Majority of isolates showed higher detection of streptolysin O, followed by streptokinase and extracellular phospholipase A virulence genes with higher degree of resistance to azithromycin, clindamycin, erythromycin, and penicillin antibiotics. Mouse pathogenicity testing confirmed virulence based on histopathological lesions and re-isolation of SDSE. CONCLUSIONS: Our findings highlight the high prevalence of SDSE in slaughtered pigs. The presence of virulence genes and mouse pathogenicity testing confirm their pathogenic potential.

Tularemia - a re-emerging disease with growing concern.

Tularemia caused by Gram-negative, coccobacillus bacterium, Francisella tularensis, is a highly infectious zoonotic disease. Human cases have been reported mainly from the United States, Nordic countries like Sweden and Finland, and some European and Asian countries. Naturally, the disease occurs in several vertebrates, particularly lagomorphs. Type A (subspecies tularensis) is more virulent and causes disease mainly in North America; type B (subspecies holarctica) is widespread, while subspecies mediasiatica is present in central Asia. F. tularensis is a possible bioweapon due to its lethality, low infectious dosage, and aerosol transmission. Small mammals like rabbits, hares, and muskrats are primary sources of human infections, but true reservoir of F. tularensis is unknown. Vector-borne tularemia primarily involves ticks and mosquitoes. The bacterial subspecies involved and mode of transmission determine the clinical picture. Early signs are flu-like illnesses that may evolve into different clinical forms of tularemia that may or may not include lymphadenopathy. Ulcero-glandular and glandular forms are acquired by arthropod bite or handling of infected animals, oculo-glandular form as a result of conjunctival infection, and oro-pharyngeal form by intake of contaminated food or water. Pulmonary form appears after inhalation of bacteria. Typhoidal form may occur after infection via different routes. Human-to-human transmission has not been known. Diagnosis can be achieved by serology, bacterial culture, and molecular methods. Treatment for tularemia typically entails use of quinolones, tetracyclines, or aminoglycosides. Preventive measures are necessary to avoid infection although difficult to implement. Research is underway for the development of effective live attenuated and subunit vaccines.

A European perspective of phylogenomics, sublineages, geographical distribution, epidemiology, and mutational landscape of mpox virus: Emergence pattern may help to fight the next public health emergency in Europe.

BACKGROUND: The 2022 outbreak of the mpox virus (previously monkeypox virus, MPXV) in non-epidemic regions has created a global issue. The emergence of MPXV was first reported in Europe, which was described as the MPXV epicenter, however, no reports are available to illustrate its outbreak patterns in Europe. METHODS: The study used numerous in silico and statistical methods to analyze hMPXV1 in European countries. Here, we used different bioinformatics servers and software to evaluate the spread of hMPXV1 in European countries. For analysis, we use various advanced servers like Nextstrain, Taxonium, MpoxSpectrum, etc. Similarly, for the statistical model, we used PAST software. RESULTS: The phylogenetic tree was depicted to illustrate the origin and evolution of hMPXV1 using vas number of genome sequences (n = 675). We found several sublineages in Europe, indicating microevolution. The scatter plot reveals the clustering patterns of the newly developed lineages in Europe. We developed statistical models for the monthly total relative frequency counts of these sublineages. The epidemiology of MPX in Europe was examined in an attempt to capture the epidemiological pattern, total cases, and deaths. Our Study noted the highest number of cases was in Spain (7500 cases) and the second-highest in France (4114 cases). The third highest number of cases was in the UK (3730 cases), which was very similar to Germany (3677 cases). Finally, we noted the mutational landscape throughout European genomes. Significant mutations were observed at the nucleotide and protein levels. We identified several unique homoplastic mutations in Europe. CONCLUSION: This study reveals several essential aspects of the European outbreak. It might help to eradicate the virus in Europe, assist in strategy formation to fight against the virus, and support working against the next public health emergency in Europe.

Revealing the structural and molecular interaction landscape of the favipiravir-RTP and SARS-CoV-2 RdRp complex through integrative bioinformatics: Insights for developing potent drugs targeting SARS-CoV-2 and other viruses.

BACKGROUND: The global research community has made considerable progress in therapeutic and vaccine research during the COVID-19 pandemic. Several therapeutics have been repurposed for the treatment of COVID-19. One such compound is, favipiravir, which was approved for the treatment of influenza viruses, including drug-resistant influenza. Despite the limited information on its molecular activity, clinical trials have attempted to determine the effectiveness of favipiravir in patients with mild to moderate COVID-19. Here, we report the structural and molecular interaction landscape of the macromolecular complex of favipiravir-RTP and SARS-CoV-2 RdRp with the RNA chain. METHODS: Integrative bioinformatics was used to reveal the structural and molecular interaction landscapes of two macromolecular complexes retrieved from RCSB PDB. RESULTS: We analyzed the interactive residues, H-bonds, and interaction interfaces to evaluate the structural and molecular interaction landscapes of the two macromolecular complexes. We found seven and six H-bonds in the first and second interaction landscapes, respectively. The maximum bond length is 3.79 Å. In the hydrophobic interactions, five residues (Asp618, Asp760, Thr687, Asp623, and Val557) were associated with the first complex and two residues (Lys73 and Tyr217) were associated with the second complex. The mobilities, collective motion, and B-factor of the two macromolecular complexes were analyzed. Finally, we developed different models, including trees, clusters, and heat maps of antiviral molecules, to evaluate the therapeutic status of favipiravir as an antiviral drug. CONCLUSIONS: The results revealed the structural and molecular interaction landscape of the binding mode of favipiravir with the nsp7-nsp8-nsp12-RNA SARS-CoV-2 RdRp complex. Our findings can help future researchers in understanding the mechanism underlying viral action and guide the design of nucleotide analogs that mimic favipiravir and exhibit greater potency as antiviral drugs against SARS-CoV-2 and other infectious viruses. Thus, our work can help in preparing for future epidemics and pandemics.

Pathological and molecular identification of Mycobacterium avium infection in a loft of domestic pigeons (Columba livia var. domestica) from India.

Mycobacterium avium is a zoonotic pathogen associated with a wide range of pulmonary and extrapulmonary manifestations in a range of host species like humans, animals, and birds. The disease is more common in the avian population, and opportunistic infections have been reported in immune-compromised or debilitated animals and humans. This study reports the pathological and molecular identification of Mycobacterium avium causing avian mycobacteriosis in a loft of domestic pigeons (Columba livia var. domestica). Out of 30 pigeons aged 2-3 years, ten adult racing pigeons revealed a severe chronic and debilitating disease followed by death. The clinical signs included chronic emaciation, dullness, ruffled feathers, lameness, and greenish, watery diarrhea. Post-mortem examination of birds revealed multifocal gray- to yellow-colored raised nodules in the liver parenchyma, spleen, lungs, intestines, bone marrow, and joints. Avian mycobacteriosis was suspected based on the tissue impression smears stained by Ziehl-Neelsen staining. Histopathological examination also revealed multifocal granulomatous lesions in affected organs, which is characteristic of avian mycobacteriosis. The PCR analysis based on 16S rRNA, IS1245, and IS901 regions suggested the presence of Mycobacterium avium infection belonging to either subspecies avium or sylvaticum. This is the first detailed report of avian mycobacteriosis in pigeons from India, warranting a strict surveillance program to identify the carrier status of these microorganisms in the pigeons, which may prove a fatal zoonotic infection in humans.

A retrospective study showing a high rate of seropositivity against SARS-CoV-2 in wild felines in India.

We report a high rate of seropositivity against SARS-CoV-2 in wild felines in India. Seropositivity was determined by microneutralization and plaque reduction neutralization assays in captive Asiatic lions, leopards, and Bengal tigers. The rate of seropositivity was positively correlated with that of the incidence in humans, suggesting the occurrence of large spillover events.

Pathological and molecular studies on elephant endotheliotropic herpesvirus haemorrhagic disease among captive and free-range Asian elephants in India.

In the present research pathology and molecular diagnosis of elephant endotheliotropic herpes virus-haemorrhagic disease (EEHV-HD) among Asian elephants was studied. Out of 76 cases, 20 were positive for EEHV infection in PANPOL and POL1 based semi-nested PCR. Out of 20 samples, 10 samples were fatal cases of EEHV-HD while 10 were of either subclinical or latent infection. Acute onset haemorrhagic disease with EEHV-HD had anorexia, facial and neck swelling, cyanotic buccal mucosa and tongue, nasal and ocular discharge, and colic. The hallmark of gross finding in all cases were severe haemorrhagic lesions in the internal organs viz. cyanosis of tongue with multifocal petechial haemorrhages, diffuse epicardial and endocardial haemorrhages, swollen liver (rounded edges) with parenchymal haemorrhages, serosal and mucosal haemorrhages in gastrointestinal tract, congested kidneys with corticomedullary haemorrhages, highly congested meninges, and brain capillaries with haemorrhages. Microscopic findings in all the cases had severe vascular changes in the visceral organs. Microthrombi was present in the vasculature of tongue, heart, lung, liver, kidney, and brain. The endothelial lining of most of the blood vessels were swollen with apoptotic changes. Amphophilic to basophilic intranuclear inclusion bodies were observed in the endothelial cells. Immunostaining using anti-EEHV DNAPOL hyperimmune sera revealed intense positive signals in the endothelium of blood vessels and their walls. Quantification of viral load in necropsy tissue samples revealed highest in the heart (7.4 × 106/µg of sample) and least in the brain (9 × 103/µg of sample). The PCR amplicons from EEHV1 specific genes (POL1(U38) and TER were subjected to partial genome sequencing which had 99.9% similarity with the EEHV1A subtype. It was concluded that Asian elephants in India are latently infected for EEHV1 and in all the fatal EEHV-HD cases, EEHV1A subtype was the causative agent with characteristic pathomorphological changes in visceral organs.

The first record of Heterakis gallinarum as a cause of fatal nodular typhlitis in golden pheasants (Chrysolophus pictus) in India.

Background: Heterakidosis is one of the most prevalent parasitic diseases in birds, the caecae of a variety of wild and domestic birds are infected with these nematodes. In pheasants, nodular typhlitis is a lethal disease caused mainly by infection with Heterakis isolonche alone or in conjunction with Heterakis gallinarum. H. gallinarum has long been recognized to infect birds with low pathogenicity, with only a few fatal cases previously reported. Case description: This paper describes a case of fatal nodular typhlitis due to H. gallinarum in a male and female pair of adult golden pheasants (Chrysolophus pictus) from a zoological garden in Uttar Pradesh, India. Findings/treatment and outcome: The caecum had multiple serosal and mucosal nodules, the majority of which were found to contain various stages of parasites embedded in the center along with the free forms in the caecal contents. Histopathologically, these nodules were generally represented by granulomas centered on necrotic parasite debris, with the occasional reactive fibrous hyperplastic tissue reaction. Based on the morphology and nematode-specific internal transcribed spacer (ITS) ITS1-5.8 rRNA-ITS2 region-based PCR, the nematode was identified as H. gallinarum. The presence of H. gallinarum was further confirmed by sequencing the ITS region followed by phylogenetic analysis. According to the author's best knowledge, this is the first instance of H. gallinarum being linked to nodular typhlitis in pheasants in India. Conclusion: Our findings confirm that H. gallinarum, other than H. isolonche, can induce severe nodular typhlitis with a fatal outcome in pheasants.

Clinicopathological investigation of nutritional osteodystrophia fibrosa in a flock of young stall-fed goats.

Background: Osteodystrophia fibrosa (ODF) is a metabolic disorder affecting the skeletal system, causing progressive loss of calcified bone mass and its replacement with fibrous tissue, which may be a sequel to primary or secondary hyperparathyroidism. This report intends to document the clinicopathological findings of ODF in a flock of young goats fed primarily on a wheat bran-rich diet. Case description: In a flock of 50 stall-fed goats aged 1 to 2 years, seven were clinically presented with bilateral facial enlargement, leading to dyspnea and difficulty in prehension and mastication. Among the seven clinically affected goats, four died in 2 months. Findings/treatment and outcome: The clinical examination revealed bilateral mandibular enlargement and limb deformities. On radiography, the maxilla and mandible had decreased radiopacity. Fine needle aspiration cytology (FNAC) from the affected bones showed occasional fibroblasts and individual osteoclasts clusters. On necropsy, the enlarged mandible revealed a meaty consistency. Undecalcified histological sections of the mandible showed severe osteopenia, multiple osteoclasts, Howship's lacunae, and extensive fibroplasia. Dietary corrective measures led to the prevention of ODF in the rest of the flock. Conclusion: Excessive wheat bran feeding in stallfed goats might have led to calcium and phosphorus imbalance, resulting in nutritional secondary hyperparathyroidism and subsequent skeletal deformities. FNAC of the affected bones, gross and histological findings provide a clinicopathological diagnosis of ODF.

Detection of SARS-CoV-2 in a free ranging leopard (Panthera pardus fusca) in India.

We report an incidence of natural infection of SARS-CoV-2 in free-ranging Indian leopard (Panthera pardus fusca). The case was detected during routine screening. Post-mortem and laboratory examination suggested virus-induced interstitial pneumonia. Viral genome could be detected in various organs including brain, lung, spleen, and lymph nodes by real-time PCR. Whole-genome sequence analysis confirmed infection of Pango lineage B.1.617.2 of SARS-CoV-2. Till now, only Asiatic lions have been reported to be infected by SARS-CoV-2 in India. Infections in animals were detected during peak phase of pandemic and all the cases were captive with close contacts with humans, whereas the present case was observed when human cases were significantly low. No tangible evidence linked to widespread infection in the wild population and the incidence seems to be isolated case. High nucleotide sequence homology with prevailing viruses in humans suggested spillover infection to the animal. This report underlines the need for intensive screening of wild animals for keeping track of the virus evolution and development of carrier status of SARS-CoV-2 among wildlife species. Supplementary Information: The online version contains supplementary material available at 10.1007/s10344-022-01608-4.

Pathomorphology, immunohistochemical, and molecular detection of an atypical porcine dermatitis and nephropathy syndrome (PDNS) due to PCV-2d-2 in naturally affected grower pigs of India.

Porcine circovirus type 2 (PCV-2) is of great economic significance to porcine industry worldwide. PCV-2 variants and genotypes, alternating world over, are the etiological agent of several clinical syndromes such as porcine dermatitis and nephropathy syndrome (PDNS), post-weaning multi-systemic wasting syndrome (PMWS) and others in pigs. This study is reporting an atypical manifestation of PDNS in twelve grower pigs, 3- to - 4.5 months age and either sex, died of the disease, with predominant lesions of nephropathy and no obvious clinical lesions in skin. Necropsy examination of pigs showed lesions of petechial -to- ecchymotic hemorrhages in the kidneys and in the right auricular musculature of the hearts. Microscopic lesions in H & E sections of the kidneys showed acute glomerulonephritis, interstitial nephritis, and vasculitis, but the skin morphology and architecture remained unaltered in contrast to the pathognomonic lesions of PDNS described in the literature. Other syndromic associations of PDNS in these cases included-perimyocarditis, interstitial pneumonia, depleted lymphoid tissues, tonsillitis, enteritis, and meningo-encephalitis. The lesional sites in duplicate paraffin tissue sections of kidneys, heart, lungs, spleen, lymph nodes, intestine, and brain demonstrated PCV-2 antigen in the cytoplasm of cells as highlighted by the intense immunolabeling on IHC staining. The PCV-2 positive organs reconfirmed by PCR, targeting ORF2 gene, which yielded 481bp size of products. The sequencing results of 481bp products on phylogenetic analysis showed 94% similarity with that of PCV-2 sequences in the database that grouped into PCV2d-2 genotype. The present report confirms, probably for the first time, the atypical PDNS cases due to PCV2d-2 genotype in naturally affected grower pigs of India.

Complete Genome Sequence of a Virulent Duck Enteritis Virus (DEV/India/IVRI-2016) Isolated from Southern India.

Molecular characterization of Indian isolates of duck enteritis virus (DEV) so far has been limited to a few selected genomic regions. Here, we report the complete genome sequence of an isolate, DEV/India/IVRI-2016, from southern India that is 158,091 bp in length.

Detection of porcine enteric picornaviruses from faecal samples of Indian pigs.

Porcine enteric picornaviruses often consequence diarrhoea and nervous complications in pig and pose enormous loss to pig farming. The present study expands the limited Indian data of porcine enteric picornaviruses which is needed for the early implementation of control measures and to check further outbreaks. A total of 398 porcine faecal samples from Uttar Pradesh, Madhya Pradesh, Chhattisgarh and Jharkhand state of India were screened for porcine teschovirus (PTV), porcine sapelovirus (PSV) and enterovirus G (EV-G) by reverse transcriptase-polymerase chain reaction (RT-PCR) using 5'UTR-specific primers. The prevalence of PTV, PSV and EV-G was found to be 12.81% (51/398), 5.77% (23/398) and 24.37% (97/398), respectively. EV-G was relatively higher in circulation in Indian pigs among all the included enteric picornaviruses. Conversely, the concurrent infection of more than one enteric picornavirus was also frequent. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-022-00756-0.

Strategies to tackle SARS-CoV-2 Mu, a newly classified variant of interest likely to resist currently available COVID-19 vaccines.

Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have recently been reported in many countries. These have exacerbated the coronavirus disease 2019 (COVID-19)-induced global health threats and hindered COVID-19 vaccine development and therapeutic progress. This commentary discusses the potential risk of the newly classified Mu variant of interest, seeming a highly vaccine-resistant variant, and the approaches that can be adopted to tackle this variant based on the available evidence. The SARS-CoV-2 B.1.621 (Mu variant) lineage has shown approximately ten times higher resistance to neutralizing sera obtained from COVID-19 survivors or BNT161b2-vaccinated people than the parenteral B.1 lineage. Several urgent and long-term strategic plans, including quick genomic surveillance for uncovering the genetic characteristics of the variants, equitable global mass vaccination, booster dose administration if required, and strict implementation of public health measures or non-pharmaceutical interventions, must be undertaken concertedly to restrict further infections, mutations, or recombination of the SARS-CoV-2 virus and its deadly strains.

Twin combination of Omicron and Delta variants triggering a tsunami wave of ever high surges in COVID-19 cases: A challenging global threat with a special focus on the Indian subcontinent.

The emergence of Omicron (B.1.1.529) variant of SARS-CoV-2 has resulted into a very massive surge in COVID-19 cases worldwide. Due to continuous emergence of multiple variants of SARS-CoV-2, the ongoing pandemic has caused severe morbidity and mortality in last two years. The rate of infectivity of Omicron variant is much higher than Delta variant and in a very quick time Omicron has displaced the Delta variant and now become a dominant variant across the globe. The twin combination of Omicron and Delta variant is triggering a Tsunami wave of ever high surges in COVID-19 cases worldwide. This article highlights the global threats and challenges posed by Omicron, and strategies to counter it with a particular focus on Indian sub-continent.

Isolation and propagation of classical swine fever virus in porcine Wharton's Jelly mesenchymal stem cells.

Classical Swine Fever (CSF) is an extremely infectious and deadly disease of pigs and wild boars caused by the CSF virus (CSFV) which is a member of the Pestivirus genus and the family Flaviviridae. This study was designed to detect the permissibility and replication of CSFV in mesenchymal stem cells (MSCs) monolayer derived from Porcine Wharton's jelly. Porcine Wharton's jelly MSCs (pWJ-MSCs) were ex vivo expanded and propagated for more than 81 generations and third passage pWJ-MSCs were characterized as per standard criteria i.e., growth characteristics, trilineage differentiation potential and molecular characterization for pluripotency and stem cell surface markers. Porcine WJ tissue samples found negative for CSFV by RT-PCR test were processed further for the isolation of pWJ-MSCs and CSFV was propagated over the characterized pWJ-MSCs monolayer. No cytopathic effect was observed, which was consistent with non-cytopathic nature of CSFV. The replication of CSFV in pWJ-MSCs was affirmed by RT-PCR and demonstration of viral antigen in the cytoplasm of virus infected cells by immuno-staining technique. In total, three different CSFV isolates were propagated in pWJ-MSCs. Primary pWJ-MSCs permitted CSFV replication to good titer. To the best of our information, this is the first ever report of isolation of CSFV in pWJ-MSCs.

Comparative analysis of the immunomodulatory potential of caprine fetal adnexa derived mesenchymal stem cells.

The caprine mesenchymal stem cells (MSCs) derived from fetal adnexa are highly proliferative. These cells possess tri-lineage differentiation potential and express MSC surface antigens and pluripotency markers with a wound-healing potential. This present study was conducted to compare the immunomodulatory potential of caprine MSCs derived from the fetal adnexa. Mid-gestation caprine uteri (2-3 months) were collected from the abattoir to isolate MSCs from amniotic fluid (cAF), amniotic sac (cAS), Wharton's jelly (cWJ) and cord blood (cCB), which were expanded and characterized at the 3rd passage. These MSCs were then stimulated with inflammatory cytokines (IFN-γ and TNF-α) to assess the percentage of inhibition produced on peripheral blood mononuclear cells (PBMCs) proliferation. The percentage of inhibition on activated PBMCs proliferation produced by cWJ MSCs and cAS MSCs was significantly higher than cCB and cAF MSCs. The relative mRNA expression profile and immunofluorescent localization of different immunomodulatory cytokines and growth factors were conducted upon stimulation. The mRNA expression profile of a set of different cytokines and growth factors in each caprine fetal adnexa MSCs were modulated. Indoleamine 2, 3 dioxygenase appeared to be the major immunomodulator in cWJ, cAF, and cCB MSCs whereas inducible nitric oxide synthase in cAS MSCs. This study suggests that caprine MSCs derived from fetal adnexa display variable immunomodulatory potential, which appears to be modulated by different molecules among sources.

Development of a Taqman-based real-time PCR assay for detection of porcine sapelovirus infection in pigs.

The objective of the present study was to develop a rapid, simple, specific and sensitive Taqman-based real-time PCR assay for porcine sapelovirus (PSV) detection. Specific primers and probe were designed from the five untranslated regions (UTRs) of the viral genome. The detection limit of the real-time PCR was 102 copies. The specificity of the Taqman real-time PCR assay was evaluated using other animal viruses and nuclease free water as a negative control. Strong fluorescent signals were obtained only in the detection of PSV real-time PCR and conventional RT-PCR were preformed simultaneously on 90 faecal samples. Based on conventional RT-PCR study 17.7% (16/90) of the faecal samples were positive for PSV. Whereas 21 of 90 samples (23.3%) were positive by real-time RT-PCR. The results showed that real-time PCR was more sensitive than the conventional RT-PCR assay. In conclusion, the Taqman real-time PCR assay for detection of PSV developed, herein, is sensitive, specific, and reliable. This assay will be useful for clinical diagnosis, epidemiological, and pathogenesis studies.