Comparative transcriptome analysis of spleen of Newcastle Disease Virus (NDV) infected chicken and Japanese quail: a potential role of NF-κß pathway activation in NDV resistance.

Newcastle disease (ND) affects a few hundred avian species including chicken and several species of domestic and wild birds. The clinical outcome of Newcastle disease virus (NDV) infection ranges from mild to severe fatal disease depending on the NDV pathotype and the host species involved. Japanese quails serve as natural reservoirs of NDV and play important role in NDV epidemiology. While infection of chicken with velogenic NDV results in severe often fatal illness, the same infection in Japanese quails results in inapparent infection. The molecular basis of this contrasting clinical outcomes of NDV infection is not yet clearly known. We compared global gene expression in spleen of chicken and Japanese quails infected with lentogenic and velogenic NDVs. We found contrasting regulation of key genes associated with NF-κB pathway and T-cell activation between chicken and Japanese quails. Our data suggests association of NDV resistance in Japanese quails to activation of NF-κB pathway and T cell proliferation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00833-y.

Evidence linking the role of periodontal viruses in coronary artery disease with and without periodontitis.

BACKGROUND: Viruses are considered to be a newer family associated with inflammatory diseases. Yet the role of periodontal viruses in coronary artery diseases (CAD) remains unclear. Thus, the current study aims to evaluate the prevalence of periodontal viruses and compare the same in cardiac samples of CAD patients with and without periodontitis. METHODS: A total of 60 patients with CAD indicated for coronary artery bypass graft surgery (CABG) were included. These were grouped into 36 patients with healthy periodontium (CAD only) and 24 patients with periodontitis (CAD + P). The demographic variables, cardiac parameters and periodontal parameters were recorded. Cardiac tissue samples were collected during the CABG surgery and were analyzed by reverse transcriptase polymerase chain reaction for periodontal viruses such as Epstein-Barr virus (EBV), cytomegalovirus (CMV) and Herpes simplex virus. All the parameters were statistically analyzed. RESULTS: Among the demographic variables, age was statistically significant between the groups. Plaque index, bleeding index, probing depth, and clinical attachment level (CAL) were significantly higher in CAD+P group (P ˂0.05). Periodontal viruses such as EBV and CMV were significantly higher (62.5% and 75% respectively, P ˂0.05) in the cardiac samples of the CAD+P than CAD only (25% and 47.2%, respectively). A significant association between EBV and CAL was revealed by multiple logistic regression analysis. (B = 0.374, P = 0.046) CONCLUSIONS: The results revealed a higher prevalence of periodontal viruses such as EBV and CMV in CAD patients with periodontitis suggesting it as one of the risk factors for CAD. This is supported by the fact that severity of periodontal disease (CAL) is associated with the presence of EBV in coronary artery plaque samples in the current study.

A Defined Antigen Skin Test That Enables Implementation of BCG Vaccination for Control of Bovine Tuberculosis: Proof of Concept.

In most low- and middle-income countries (LMICs), bovine tuberculosis (bTB) remains endemic due to the absence of control programs. This is because successful bTB control and eradication programs have relied on test-and-slaughter strategies that are socioeconomically unfeasible in LMICs. While Bacillus Calmette-Guérin (BCG) vaccine-induced protection for cattle has long been documented in experimental and field trials, its use in control programs has been precluded by the inability to differentiate BCG-vaccinated from naturally infected animals using the OIE-prescribed purified protein derivative (PPD)-based tuberculin skin tests. In the current study, the diagnostic specificity and capability for differentiating infected from vaccinated animals (DIVA) of a novel defined antigen skin test (DST) in BCG-vaccinated (Bos taurus ssp. taurus x B. t. ssp. indicus) calves were compared with the performance of traditional PPD-tuberculin in both the skin test and in vitro interferon-gamma release assay (IGRA). The IFN-γ production from whole blood cells stimulated with both PPDs increased significantly from the 0 week baseline levels, while DST induced no measurable IFN-γ production in BCG-vaccinated calves. None of the 15 BCG-vaccinated calves were reactive with the DST skin test (100% specificity; one-tailed lower 95% CI: 82). In contrast, 10 of 15 BCG-vaccinated calves were classified as reactors with the PPD-based single intradermal test (SIT) (specificity in vaccinated animals = 33%; 95% CI: 12, 62). Taken together, the results provide strong evidence that the DST is highly specific and enables DIVA capability in both skin and IGRA assay format, thereby enabling the implementation of BCG vaccine-based bTB control, particularly in settings where test and slaughter remain unfeasible.

Potential of ovine Wharton jelly derived mesenchymal stem cells to transdifferentiate into neuronal phenotype for application in neuroregenerative therapy.

Introduction: The transdifferentiation potential of mesenchymal stem cells (MSCs) is not limited to mesodermal derivatives but also to other cell types such as neuronal cells under appropriate cell culture conditions.Materials and methods: The present study characterizes the differentiation of Wharton's jelly (WJ) derived MSCs using neuronal conditioned medium (NCM) collected from cultured foetal brain cells.Results: After induction with NCM to neuronal stem cells (NSC), the WJ MSCs showed profound morphological changes showing multiple neurites extending from the cell body containing reminiscent of Nissl substance and single long axon-like processes. In RT PCR and immunocytochemistry, the induced neuronal cells showed a strong positive expression of neuronal markers Nestin, ß III tubulin and GFAP indicated that, the cells were reactive to NCM for differentiation. A significant (p < 0.01) increase in the level of secretome BDNF was observed in NCM suggests that the BDNF could play a key role in the transdifferentiation of WJMSCs to NSCs.Conclusion: These results support the potential of ovine MSCs isolated from umbilical cord WJ of abattoir derived foetuses to differentiate into neuronal stem cells and also provide a valuable experimental data for NSC transplant research in veterinary medicine.

An immuno-chromatographic lateral flow assay (LFA) for rapid on-the-farm detection of classical swine fever virus (CSFV).

Classical swine fever (CSF) is a highly contagious and potentially fatal disease of domestic pigs. Classical swine fever is routinely diagnosed by clinical signs, serology, detection of CSF virus (CSFV) nucleic acid by PCR and virus isolation. Most of the current CSF diagnostic methods are expensive and have an extended turnaround time. In the majority of the CSF endemic countries, lack of easy access to diagnostic facilities is a major problem for swine producers trying to obtain early diagnosis and often results in the entire herd being infected. The acute form of CSF can show non-specific signs of illness, leaving CSF often undiagnosed. Hence there is an urgent need for a rapid and reliable pen side diagnostic assay for the better detection and control of this economically important disease of swine. We developed an immuno-chromatographic lateral flow assay (LFA) for on the farm detection of CSFV. A CSFV isolate [CSFV/AP/TRP2/2009 (TS2)] of genotype 1.1 was used for the production of monoclonal antibodies (mAbs) for the LFA's development. The virus detection level of the LFA device was 36.8 TCID50/ml of CSFV. The sensitivity and specificity of LFA in comparison with PCR were 80.36% and 87.10%, respectively. The positive and negative predictive values of the LFA device were 91.84% and 87.10%, respectively. In conclusion, the CSFV-LFA is a reliable and convenient resource for preliminary on the farm detection of classic swine fever.

Co-expression of sialic acid receptors compatible with avian and human influenza virus binding in emus (Dromaius novaehollandiae).

Influenza A viruses (IAVs) continue to threaten animal and human health with constant emergence of novel variants. While aquatic birds are a major reservoir of most IAVs, the role of other terrestrial birds in the evolution of IAVs is becoming increasingly evident. Since 2006, several reports of IAV isolations from emus have surfaced and avian influenza infection of emus can lead to the selection of mammalian like PB2-E627K and PB2-D701N mutants. However, the potential of emus to be co-infected with avian and mammalian IAVs is not yet understood. As a first step, we investigated sialic acid (SA) receptor distribution across major organs and body systems of emu and found a widespread co-expression of both SAα2,3Gal and SAα2,6Gal receptors in various tissues that are compatible with avian and human IAV binding. Our results suggest that emus could allow genetic recombination and hence play an important role in the evolution of IAVs.

Differential detection of avian oncogenic viruses in poultry layer farms and Turkeys by use of multiplex PCR.

Avian oncogenic viruses include Marek's disease virus (MDV), a highly contagious herpesvirus, as well as retroviruses such as avian leukosis virus (ALV) subgroups A to J and reticuloendotheliosis virus (REV). In this study, we examined the incidence of these viruses in suspected samples collected from poultry layer farms of South India, mainly in the Namakkal district of Tamil Nadu, a highly dense poultry-growing area in India. The histopathology-positive tissue sections were identified and further confirmed by immunohistochemistry using virus-specific antibodies. The viruses belonging to all 3 groups (MDV, ALV, and REV) were isolated in a cell culture system and confirmed by immunofluorescence using virus-specific antibodies. PCR appeared to be the method of choice for rapid and accurate diagnosis of these viruses. The multiplex PCR primers specific to MDV, ALV, REV, and chicken DNA were designed for rapid differential diagnosis. The specificity of the primers was checked by amplification of DNA from virus-infected cell culture in comparison with uninfected samples, and sensitivity was evaluated by calculating the minimum copy number at which amplification occurs in the cloned PCR products. The sequences of the amplicons were compared by BLAST analysis. PCR tests demonstrated the presence of single, dual, or triple viruses in some of the samples. Of 169 samples screened by multiplex PCR, 9 samples were positive for MDV, 17 samples were positive for ALV, 12 samples were positive for REV, and 17 samples were positive for both ALV and REV. Three samples were positive for all three viruses. ALV-positive samples were further subjected to subgroup-specific PCR, which gave positive results for subgroups B and D but not for subgroup J. Multiplex PCR appeared to be a useful technique for rapid differential diagnosis of avian oncogenic viruses and detection of multiple infections of avian oncogenic viruses under field conditions.

Rapid detection of the Marek's disease viral genome in chicken feathers by loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) method for the rapid detection of serotype 1 Marek's disease virus (MDV) was developed. The method used a set of three pairs of primers to amplify the MEQ gene for detecting serotype 1 MDV. The MDV LAMP method did not cross-react with serotype 2 and serotype 3, nor did the LAMP primers have binding sites for the common avian DNA viruses (reticuloendotheliosis virus, chicken anemia virus, subgroup J of the avian leukosis virus). Additionally, the assay could detect up to 10 copies of the MEQ gene in the MD viral genome, and it had 10 times higher sensitivity than the traditional PCR methods. The LAMP master mix was stable for 90 days at -20°C. Furthermore, the efficiency of LAMP for detection of serotype 1 MDV in clinical samples was comparable to those of PCR and viral isolation. The LAMP procedure is simple and does not rely on any special equipment. The detection of serotype 1 MDV by LAMP will be useful for detecting and controlling oncogenic Marek's disease.

Evaluation of immune responses and protective efficacy in a goat model following immunization with a coctail of recombinant antigens and a polyprotein of Mycobacterium avium subsp. paratuberculosis.

The protective efficacy of four recombinant antigens (85A, 85B, superoxide dismutase [SOD], and a fusion polypeptide [Map74F]) of Mycobacterium avium subsp. paratuberculosis (MAP) along with the adjuvant dimethydioctadecyl ammonium bromide (DDA) was assessed in a goat challenge model. Animals were immunized with the four antigens with adjuvant DDA (Group I, eight goat kids) or without the adjuvant (Group II, eight goat kids) or adjuvant only (Group III, nine goat kids). Animals were boostered 3 weeks after the primary vaccination and challenged 3 weeks after the booster. Significant antigen-specific lymphoproliferation was observed in the immunized animals 3 weeks after the booster immunization. This response increased further at 4 weeks after the booster. Similarly, antigen-specific IFN-gamma responses increased in the immunized animals 3 weeks after the booster. The response was significantly higher for 85A and Map74F at 10 weeks after primary vaccination (APV) in Group I animals compared to the other two groups. CD4+ T-cell populations were higher in the vaccinated animals from 6 to 10 weeks APV than those of the control animals. A significant increase in recombinant antigen-specific IFN-gamma gene expression was detected in the vaccinated animals. At necropsy (38 weeks APV), our multicomponent subunit vaccine imparted a significant protection in terms of reduction of MAP burden in target organs as compared to sham-immunized goats. This study indicates that our multicomponent subunit vaccine induced a good Th1 response and conferred protection against MAP infection in a goat challenge model.

Immunization with a DNA vaccine cocktail induces a Th1 response and protects mice against Mycobacterium avium subsp. paratuberculosis challenge.

Several antigens of Mycobacterium avium subsp. paratuberculosis have been studied as vaccine components and their immunogenicity has been evaluated. Previously, we reported that 85 antigen complex (85A, 85B, and 85C), superoxide dismutase (SOD), and 35kDa protein could induce significant lymphocyte proliferation as well as the elaboration of Th1-associated cytokines including interferon gamma (IFN-gamma), interleukin-2 (IL-2), IL-12 and tumor necrosis factor alpha (TNF-alpha). Based on these results, we cloned and expressed 85A, 85B, 85C, SOD, and 35kDa-protein genes into the eukaryotic expression plasmid pVR1020. C57BL/6 mice were immunized three times intramuscularly with the recombinant DNA cocktail and pVR1020 DNA alone as control. A significant reduction in the bacterial burden in the spleen and liver of mice immunized with the DNA cocktail as compared to the vector control group was found. Also, the relative severity of the liver and spleen histopathology paralleled the MAP culture results, more granulomas and acid-fast bacilli in the vector control animals. Moreover, mice immunized with the DNA cocktail developed both CD4(+) and CD8(+) T cell responses to the recombinant antigens and showed significant lymphocyte proliferation. The Th1 response related cytokine (IFN-gamma) levels increased in splenocytes obtained from immunized animals. These results indicate that the use of a recombinant DNA vaccine can provide protective immunity against mycobacterial infection by inducing a Th1 response.

Immune responses in mice to Mycobacterium avium subsp. paratuberculosis following vaccination with a novel 74F recombinant polyprotein.

Johne's disease (JD) is a chronic infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Here, we report the cloning and expression of a 74kDa recombinant polyprotein (Map74F) and its protective efficacy against MAP infection in mice. Map74F was generated by the sequential linkage of the ORFs of the approximately 17.6-kDa C-terminal fragment of Map3527 to the full-length ORF of Map1519, followed at the C-terminus with approximately 14.6-kDa N-terminal portion of Map3527. Mice immunized with Map74F had a significant IgG1 response but not IgG2a. In immunized animals, the IgG1/IgG2a ratio increased until 4 weeks after MAP challenge. The ratio decreased from 8 weeks indicating a shift to a Th1 response. Antigen specific IFN-gamma response, CD3+ and CD4+ T cells increased significantly in immunized mice. Following challenge, MAP burden was significantly lower in liver, spleen and mesenteric lymph nodes of immunized animals compared to control animals indicating protection against MAP infection. This was further evident by the improved liver and spleen pathology of the immunized animals, which had fewer granulomas and lower numbers of acid-fast bacilli. Results of this study indicated that immunization of mice with Map74F protected mice against MAP infection.

Vaccination with recombinant Mycobacterium avium subsp. paratuberculosis proteins induces differential immune responses and protects calves against infection by oral challenge.

We previously reported the in vitro cellular immune responses to recombinant antigens (rAgs) of Mycobacterium avium subsp. paratuberculosis (MAP). Here we report the differential immune responses and protective efficacy of four rAgs of MAP (85A, 85B, 85C, and superoxide dismutase (SOD)) used with two adjuvants (monophosphoryl lipid A (MPLA) containing synthetic trehalose dicorynomycolate, cell wall skeleton (MPLA) and bovine IL-12), against MAP challenge in calves. Group I was administered the four rAgs with MPLA and IL-12. Group II was administered the four rAgs and MPLA. Group III received MPLA and IL-12, and Group IV MPLA. rAgs induced significant lymphoproliferative responses in vaccinated animals (Groups I and II). All the rAgs induced significant IFN-gamma production from 11 to 23 wk after primary vaccination (APV), except for SOD. Significant increases were noted in CD3(+), CD4(+), CD8(+), CD21(+), CD25(+), and gammadelta(+) cells against all four rAgs in vaccinated animals. rAg-specific expression of IL-2, IL-12p40, IFN-gamma and TNF-alpha was significantly higher in the two vaccinated groups. Culture results found 4/8 animals in Group I, 3/8 animals in Group II, and 3/4 animals in Groups III and IV were positive for MAP in one or more tissues. Among the seven positive animals in Groups I and II, all but one had had <10CFU. Isolation was confined to one tissue in these animals, except in one animal in which MAP was isolated from two tissues. In the control groups (III and IV), MAP was cultured from up to five different tissues with >250CFU. Preliminary data from this study indicates that all four rAgs induced a good Th1 response and conferred protection against MAP infection in calves.