Days Postinoculation Research Articles

Using a concurrent challenge with porcine circovirus 2 and porcine reproductive and respiratory syndrome virus to compare swine vaccination programs

The objectives of the present study were to evaluate the immune response of six commercial vaccines against PRRSV-2 and PCV2, administered as monovalent or combined products via intramuscular (IM) or intradermal (ID) routes. Seventy-two, 3-week-old pigs were randomly allocated into 8 treatments with 9 pigs each: IMPP0/PCVMH7, IDPP0/PCVMH7, IMING0/PCVMH7, IMPP0/PCVMH0, IDPP0/PCVMH0, IMTRF0, NV/CH, and NV/NC. IMPP0/PCVMH0 and IMPP0/PCVMH7 groups were IM vaccinated once with Prime Pac PRRS (MSD Animal Health, The Netherlands) at 0 days post-vaccination (DPV), followed by single IM vaccination with Porcilis PCV M Hyo (MSD Animal Health, The Netherlands) either at 0 or 7 DPV, respectively. IDPP0/PCVMH0 and IDPP0/PCVMH7 groups were ID vaccinated once with Prime Pac PRRS (MSD Animal Health, The Netherlands) at 0 DPV, followed by a single concurrent ID injection of Porcilis PCV ID (MSD Animal Health, The Netherlands) and Porcilis M Hyo ID ONCE (MSD Animal Health, The Netherlands) either at 0 or 7 DPV, respectively. The IMING0/PCVMH7 group was IM vaccinated once with Ingelvac PRRS MLV (Boehringer Ingelheim, Germany) at 0 DPV, and subsequently IM vaccinated with Ingelvac CircoFLEX (Boehringer Ingelheim, Germany) and Ingelvac MycoFLEX (Boehringer Ingelheim, Germany) at 7 DPV. The IMTRF0 group was IM vaccinated once with combined products of Ingelvac PRRS MLV (Boehringer Ingelheim, Germany), Ingelvac CircoFLEX (Boehringer Ingelheim, Germany), and Ingelvac MycoFLEX (Boehringer Ingelheim, Germany) at 0 DPV. The NV/CH and NV/NC groups were left unvaccinated. At 28 DPV (0 days post-challenge, DPC), pigs were intranasally inoculated with a 4 ml of mixed cell culture inoculum containing HP-PRRSV-2 (105.6 TCID50/ml) and PCV2d (105.0 TCID50/ml). Antibody response, IFN-γ-secreting cells (SC), and IL-10 secretion in supernatants of stimulated PBMC were monitored. Sera were collected and quantified for the PRRSV RNA and PCV2 DNA using qPCR. Three pigs from each group were necropsied at 7 DPC, lung lesions were evaluated. Tissues were collected and performed immunohistochemistry (IHC). Our study demonstrated that concurrent vaccination via the ID or the IM route did not introduce additional reactogenicity. We found no interference with the induction of immune response between vaccination timing. In terms of an immune response, ID vaccination resulted in significantly lower IL-10 levels and higher IFN-γ-SC values compared to the IM-vaccinated groups. In terms of clinical outcomes, only one IM-vaccinated group showed significantly better efficacy when antigens were injected separately compared with concurrently. While the vaccines were ID delivered, these effects disappeared. Our findings confirm that concurrent vaccination of PRRSV-2 MLV and PCV2 via either the IM or the ID routes could be a viable immunization strategy to assist with the control of PRDC. In situations where maximal efficacy is required, over all other factors, concurrent vaccination is possible with the ID route but might not be an ideal strategy if using the IM route.

FLU-v, a Broad-Spectrum Influenza Vaccine, Induces Cross-Reactive Cellular Immune Responses in Humans Measured by Dual IFN-γ and Granzyme B ELISpot Assay.

Previous reports demonstrated that FLU-v, a peptide-based broad-spectrum influenza vaccine candidate, induced antibody and cellular immune responses in humans. Here, we evaluate cellular effector functions and cross-reactivity. PBMC sampled pre- (day 0) and post-vaccination (days 42 and 180) from vaccine (n = 58) and placebo (n = 27) recipients were tested in vitro for responses to FLU-v and inactivated influenza strains (A/H3N2, A/H1N1, A/H5N1, A/H7N9, B/Yamagata) using IFN-γ and granzyme B ELISpot. FLU-v induced a significant increase in the number of IFN-γ- and granzyme-B-secreting cells responding to the vaccine antigens from pre-vaccination (medians: 5 SFU/106 cells for both markers) to day 42 (125 and 40 SFU/106 cells, p < 0.0001 for both) and day 180 (75 and 20 SFU/106 cells, p < 0.0001 and p = 0.0047). The fold increase from pre-vaccination to day 42 for IFN-γ-, granzyme-B-, and double-positive-secreting cells responding to FLU-v was significantly elevated compared to placebo (medians: 16.3-fold vs. 1.0-fold, p < 0.0001; 3.5-fold vs. 1.0-fold, p < 0.0001; 3.0-fold vs. 1.0-fold, p = 0.0012, respectively). Stimulation of PBMC with inactivated influenza strains showed significantly higher fold increases from pre-vaccination to day 42 in the vaccine group compared to placebo for IFN-γ-secreting cells reacting to H1N1 (medians: 2.3-fold vs. 0.8-fold, p = 0.0083), H3N2 (1.7-fold vs. 0.8-fold, p = 0.0178), and H5N1 (1.7-fold vs. 1.0-fold, p = 0.0441); for granzyme B secreting cells reacting to H1N1 (3.5-fold vs. 1.0-fold, p = 0.0075); and for double positive cells reacting to H1N1 (2.9-fold vs. 1.0-fold, p = 0.0219), H3N2 (1.7-fold vs. 0.9-fold, p = 0.0136), and the B strain (2.0-fold vs. 0.8-fold, p = 0.0227). The correlation observed between number of cells secreting IFN-γ or granzyme B in response to FLU-v and to the influenza strains supported vaccine-induced cross-reactivity. In conclusion, adjuvanted FLU-v vaccination induced cross-reactive cellular responses with cytotoxic capacity, further supporting the development of FLU-v as a broad-spectrum influenza vaccine.

Sero-monitoring of calf hood vaccination under brucellosis control program in selected states of India during 2016-18

The study aimed to evaluate the post-vaccination antibody response in sera of 4-8 months old female calves vaccinated with Brucella S19 vaccine under Brucellosis- Control Program (B-CP) initiated by Department of Animal Husbandry and Dairying (DAH&amp;D), Government of India during 2016-18. The antibody response was extremely good in three states [Telangana (82.53%), Himachal Pradesh (80.97%) and Maharashtra (74.02%)]. Higher antibody response was observed during 21-45 days post-vaccination (DPV) which indicated this period as appropriate for sampling to assess the antibody response. The knowledge acquired with respect to the post-vaccination sero-monitoring in this study will help the ongoing brucellosis control program under the flagship program of Government of India launched in 2019 as National Animal Disease Control Program (NADCP).

First report of Athelia rolfsii (=Sclerotium rolfsii) associated with southern blight disease of Macrotyloma uniflorum in India.

Horse gram (Macrotyloma uniflorum (Lam.) Verdc., Fabaceae) is an important legume crop cultivated widely in the arid and semiarid regions. During a survey carried out in the Mysore district (Karnataka, India, October 2021), horse gram plants showed the symptoms of southern blight disease. Disease incidence ranged from 20-27% in the approximate 52 hectares of horse gram fields evaluated. The symptoms initiated as tan lesions and the developing mycelial threads colonized the infected root-stem interface, causing girdling; lesions on leaves enlarged and developed into distinct spots. Infected parts (leaves & stem) (n=30) were collected in poly bags and incubated in a moist chamber overnight, followed by surface sterilization of small segments of stem, leaf with 2% NaOCl, rinsed with sterile water (SW), and placed onto the potato dextrose agar (PDA, HiMedia Lab, Mumbai) supplemented with chloramphenicol (40 mg/L). The plates were incubated at room temperature (28 ± 2°C) for 5-7 days. The fungal colonies developed from the diseased segments were sub-cultured through hyphal tipping to fresh PDA plates and pure cultures were obtained. Fungal colonies with dense, aerial whitish-cottony mycelia with uniformly globoid sclerotia (0.52.9 mm) were observed after 1012 days of incubation. Sclerotia were white in the beginning and turned to brown later. The average number of sclerotia produced per plate ranged from 112 to 320 (n = 20). To determine the identity of the isolated fungal pathogen, ITS-rDNA was amplified and sequenced using ITS1/ITS4 (White et al. 1990) primers. The amplified PCR product was purified and sequenced directly. The ITS sequences (OM037658 & OM037659) shared 100% (630/643bp) sequence similarity to Athelia rolfsii (KY640622.1, AB075298). The phylogenetic tree (Neighbour-Joining method) constructed based on ITS-rDNA region confirmed that the sequences shared a common clade with reference sequence of A. rolfsii. Thus the identity was confirmed based on micromorphology and phylogenetic analysis. Pathogenicity tests were conducted on a total of 20 plants (5-6 weeks old) in greenhouse conditions (at 28 ± 2°C and 70% relative humidity) by inoculating with sclerotia from 15 days old cultures on stem and leaves and 14 plants were found infected after 5 days of post-inoculation, while uninoculated control plants remained healthy. Similarly, detached leaf assay (Mahadevakumar et al., 2018) was performed under in vitro conditions at 28 ± 2°C in a moist chamber and 28 out of 30 leaves showed the leaf spot symptoms after 3-5 days of inoculation. Uninoculated control leaves remained healthy. The identity of the fungus was confirmed by morphology and molecular analysis after re-isolation. The occurrence as a pathogen on horse gram has not been previously reported elsewhere. This is the first report of southern blight disease caused by A. rolfsii on horse gram from India. Early diagnosis of this leaf spot disease will help the farmers to adopt suitable management practices to avoid loss in production.

The N-terminal Subunit of the Porcine Deltacoronavirus Spike Recombinant Protein (S1) Does Not Serologically Cross-react with Other Porcine Coronaviruses.

Porcine deltacoronavirus (PDCoV), belonging to family Coronaviridae and genus Deltacoronavirus, is a major enteric pathogen in swine. Accurate PDCoV diagnosis relying on laboratory testing and antibody detection is an important approach. This study evaluated the potential of the receptor-binding subunit of the PDCoV spike protein (S1), generated using a mammalian expression system, for specific antibody detection via indirect enzyme-linked immunosorbent assay (ELISA). Serum samples were collected at day post-inoculation (DPI) −7 to 42, from pigs (n = 83) experimentally inoculated with different porcine coronaviruses (PorCoV). The diagnostic sensitivity of the PDCoV S1-based ELISA was evaluated using serum samples (n = 72) from PDCoV-inoculated animals. The diagnostic specificity and potential cross-reactivity of the assay was evaluated on PorCoV-negative samples (n = 345) and samples collected from pigs experimentally inoculated with other PorCoVs (n = 472). The overall diagnostic performance, time of detection, and detection rate over time varied across different S/P cut-offs, estimated by Receiver Operating Characteristic (ROC) curve analysis. The higher detection rate in the PDCoV group was observed after DPI 21. An S/P cut-off of 0.25 provided 100% specificity with no serological cross-reactivity against other PorCoV. These results support the use of S1 protein-based ELISA for accurate detection of PDCoV infections, transference of maternal antibodies, or active surveillance.

Plantago lagopus extract as a green fungicide induces systemic resistance against Rhizoctonia root rot disease in tomato plants.

Extensive use of chemical control agents and fungicides typically leads to numerous risks to human health and the environment. Using plant extracts as natural substances represents a dual key for the environment and sustainable food production, as it reduces the input of synthetic pesticides into the environment and/or controls plant pathogens. For the first time, a Plantago lagopus ethanolic extract has been characterized and evaluated for its protective and curative effects against Rhizoctonia solani in tomato plants. The results showed that P. lagopus extract (10 μg/ml) completely inhibited R. solani mycelial growth in vitro. At 20 days of post fungal inoculation, the results demonstrated that using P. lagopus extract (100 μg/ml) in vivo enhanced tomato plant growth by significantly increasing shoot and root parameters in protective and curative treatments. Furthermore, the protective and curative treatments significantly reduced the disease index by 18.66 and 38.66%, respectively. Induction of systemic resistance with upregulation of PR-1 and PR-2 and a significant increase in the transcriptional levels of PR-3 and CHS in all P. lagopus extract-treated tomato plants were reported compared to untreated plants. HPLC analysis showed that the most common polyphenolic components detected in P. lagopus extract were rutin (74206.3 mg/kg), naringenin (2388.74 mg/kg), quercetin (1249.13 mg/kg), and p-hydroxybenzoic acid (1035.87 mg/kg). In addition, the ellagic acid (798.47 mg/kg), vanillic acid (752.55 mg/kg), catechol (648.89 mg/kg), cinnamic acid (332.51 mg/kg), ferulic acid (296.32 mg/kg), benzoic acid (295.95 mg/kg), and chlorogenic acid (116.63 mg/kg) were also reported. Our study is the first to show that P. lagopus extract can help plants fight off R. solani fungal infection. Furthermore, the findings imply that using the P. lagopus extract as a natural biocontrol agent could be a sustainable strategy to manage plant fungal diseases.

The production and influence of anti-Vibrio parahaemolytics IgY against experimental infection of V. parahaemolyticus in cultured Fenneropenaeus indicus

The increasing number of antibiotic-resistant bacteria emphasizes the need to find alternatives to complement antibiotics. Immunotherapy may also be used as a complementary treatment against pathogens that are difficult to treat with traditional antibiotics. Eggs are normal dietary components and there is practically no risk of toxic side effects of IgY given orally. In the present study, pathogenic Vibrio parahaemolyticus was isolated from infected shrimp and studied their virulence factors including LD50 (by challenging with Fenneropenaeus indicus), proteolytic and hemolytic activities. The edible antibody IgY was raised by injecting the antigen of Extra Cellular Products (ECP) of V. parahaemolyticus to Gallus gallus domesticus during layoff period with and without the herbal immunoadjuvants, Asparagus racemosus and Glycine max (V.p wo: V. parahaemolyticus ECP without adjuvant; V.p A: V. parahaemolyticus ECP with A. racemosus and V.p G: V. parahaemolyticus ECP with G. max). Eggs were collected after five weeks of immunization and anti- V. parahaemolyticus IgY was extracted and purified. Physicochemical properties of the immunized Chickens' serum and anti- V. parahaemolyticus IgY's cross reactivity, growth inhibition assay, single radial immunodiffusion assay and bacterial agglutination were studied. The results revealed that, the serum protein parameters were significantly (P ≤ 0.001) increased in experimental groups from control group. The antibody raised with immunoadjuvants had significantly (P ≤ 0.001) higher cross reactivity, growth inhibition, single radial immunoassay and bacterial agglutination when compared with and without immunoadjuvant and control groups. Further the control and experimental anti-V. parahaemolytics IgY coated artificial diets were fed to F. indicus for 60 days. After 30 and 60 dpv (days of post vaccination), shrimps from each groups were challenged with virulent V. parahaemolyticus and studied the survival, haematological and immunological parameters. The IgY coated diets (V. p A and V.p G) fed shrimps had decreased cumulative mortality, significantly (P ≤ 0.001) improved coagulase activity, total haemocyte count and oxyhaemocyanin. The immunological parameters such as prophenoloxidase, intracellular anion production, lysozyme production and phagocytosis also improved significantly (P ≤ 0.001) in IgY treated shrimps.

Assessment of Different Infectious Bovine Rhinotracheitis Marker Vaccines in Calves.

Three commercially available infectious bovine rhinotracheitis (IBR) live marker vaccines were evaluated for their ability to provide clinical protection to vaccinated calves against wild-type (wt) Bovine alphaherpesvirus-1 (BoHV-1) challenge and their possible effect on wt BoHV-1 latency reactivation following the challenge. On 35 post-vaccination days (PVDs), all animals were challenged with wt BoHV-1. Only the calves in the control group developed severe forms of IBR. The reactivation of latent BoHV-1 was induced by dexamethasone (DMS) treatment on 28 post-challenge days (PCDs). All animals showed IBR clinical signs on three post-DMS treatment days (PDTDs). On PVD 14, all vaccinated animals developed neutralizing antibodies (NAs), whereas in control animals, the NAs appeared post-challenge. The positivity for glycoprotein-B (gB) was detected using real-time polymerase chain reactions in all animals from PCDs 1 to 7. In contrast, the gB-positivity was observed in the immunized calves from PDTDs 3 to 10. Positive expression of gD and gE was observed in nasal swabs of all calves on PDTD 7. These findings suggested that the IBR marker vaccines evaluated in this study protected against wt BoHV-1-induced disease but not against wt BoHV-1-induced latency reactivation, indicating the necessity of developing new products to protect animals from wt BoHV-1-induced latency.

Dairy Cows Experimentally Infected With Bovine Leukemia Virus Showed an Increased Milk Production in Lactation Numbers 3-4: A 4-Year Longitudinal Study.

Bovine leukemia virus (BLV) is widespread in global cattle populations, but the effects of its infection on milk quantity and quality have not been clearly elucidated in animal models. In this study, 30 healthy first-lactation cows were selected from ≈2,988 cows in a BLV-free farm with the same criteria of parity, age, lactation number, as well as milk yield, SCS, and composition (fat, protein, and lactose). Subsequently, these cows were randomly assigned to the intervention (n = 15) or control (n = 15) group, and reared in different cowsheds. Cows in the intervention group were inoculated with 1 × phosphate-buffered solution (PBS) resuspended in peripheral blood mononuclear cells (PBMC) from a BLV-positive cow, while the controls were inoculated with the inactivated PBMC from the same individual. From June 2016 to July 2021, milk weight (kg) was automatically recorded by milk sensors, and milk SCS and composition were originated from monthly performed dairy herd improvement (DHI) testing. Fluorescence resonance energy transfer (FRET)–qPCR and ELISA showed that cows in the intervention group were successfully infected with BLV, while cows in the control group were free of BLV for the entire period. At 45 days post-inoculation (DPI), the numbers of whole blood cells (WBCs) (P = 0.010), lymphocytes (LYMs) (P = 0.002), and monocytes (MNCs) (P = 0.001) and the expression levels of IFN-γ (P = 0.013), IL-10 (P = 0.031), and IL-12p70 (P = 0.008) increased significantly in the BLV infected cows compared to the non-infected. In lactation numbers 2–4, the intervention group had significantly higher overall milk yield (P < 0.001), fat (P = 0.031), and protein (P = 0.050) than the control group, while milk SCS (P = 0.038) and lactose (P = 0.036) decreased significantly. Further analysis indicated that BLV infection was associated with increased milk yield at each lactation stage in lactation numbers 3–4 (P = 0.021 or P < 0.001), but not with SCS and milk composition. Together, this 4-year longitudinal study revealed that artificial inoculation of BLV increased the milk yield in cows in this BLV challenge model.

Oral SARS-CoV-2 Inoculation Causes Nasal Viral Infection Leading to Olfactory Bulb Infection: An Experimental Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections can cause long-lasting anosmia, but the impact of SARS-CoV-2 infection, which can spread to the nasal cavity via the oral route, on the olfactory receptor neuron (ORN) lineage and olfactory bulb (OB) remains undetermined. Using Syrian hamsters, we explored whether oral SARS-CoV-2 inoculation can lead to nasal viral infection, examined how SARS-CoV-2 affects the ORN lineage by site, and investigated whether SARS-CoV-2 infection can spread to the OB and induce inflammation. On post-inoculation day 7, SARS-CoV-2 presence was confirmed in the lateral area (OCAM-positive) but not the nasal septum of NQO1-positive and OCAM-positive areas. The virus was observed partially infiltrating the olfactory epithelium, and ORN progenitor cells, immature ORNs, and mature ORNs were fewer than in controls. The virus was found in the olfactory nerve bundles to the OB, suggesting the nasal cavity as a route for SARS-CoV-2 brain infection. We demonstrated that transoral SARS-CoV-2 infection can spread from the nasal cavity to the central nervous system and the possibility of central olfactory dysfunction due to SARS-CoV-2 infection. The virus was localized at the infection site and could damage all ORN-lineage cells.

Effect of testing protocol and within-pen prevalence on the detection of Mycoplasma hyopneumoniae DNA in oral fluid samples

Combinations of 2 nucleic acid extractions and 3 Mycoplasma hyopneumoniae (MHP) PCRs (namely Protocol 1, 2, 3, and 4) were compared in terms of the probability of detecting DNA in pen-based oral fluid samples as a function of within-pen MHP prevalence. Oral fluid samples were created by randomly assigning 39 7-week old pigs to one of 5 pens, i.e., negative control pen (3 pigs) and 4 pens of 9 pigs each that differed in the proportion of MHP-inoculated pigs (1, 3, 6, or 9). Deep tracheal swabs were collected twice weekly to establish individual pig MHP infection status and derive within-pen prevalence estimation. On DPI 3, tracheal swabs from 15 of 19 inoculated pigs were MHP DNA positive. Oral fluids (n = 320) were collected daily from − 4 to 59 days post inoculation (DPI). Using a piecewise exponential model to account for within-pen transmission dynamics followed by a mixed-effect logistic regression, the probability of detecting MHP DNA in oral fluids was positively associated with within-pen prevalence (P < 0.0001) and differed among test protocols. MHP DNA was detected in 173 oral fluid samples with Protocol 3 versus 148, 134, and 101 with Protocols 4, 2, and 1, respectively. At 100% within-pen prevalence, the probability of detecting MHP DNA in oral fluids was highest using Protocol 3 (95.7%), followed by Protocols 4 (70.1%), 2 (60.1%), and 1 (34.0%). The fact that PCR protocols performed differently suggests that further improvements in extraction methods and MHP PCRs are possible. In the field, the dynamics of MHP infections should be taken into account if using oral fluid samples in surveillance.

Sympathetic Modulation of Oral Cancer Pain via Adrenergic Signaling in Trigeminal Primary Afferent Neurons

Head and neck squamous cell carcinoma (HNSCC) often codevelops with severe functional pain that significantly impacts patients' quality of life. Previous research demonstrated dense innervation of sympathetic and sensory nerves within the oral tumor microenvironment. We hypothesize that nociceptive afferents innervating oral cancer develop adrenergic sensitivity resulting in sympathetically maintained pain. Patient-reported pain in tandem with patient blood and tumor samples were used to explore the relationship between tumor-innervating sympathetic nerves and cancer pain. A syngeneic tongue transplant mouse oral cancer (MOC2) model and an orofacial behavioral assay using gnaw-time as an index of pain were used to quantify cancer-induced nociceptive behavior. qPCR was performed on trigeminal ganglia (TGs) from MOC2-tumor-bearing mice to investigate the effect of oral cancer on adrenergic receptor gene expression. Sympathetic nerve fibers, identified using tyrosine hydroxylase (TH) immunoreactivity, comprised 4.73± 1.3% of total nerve area across 13 HNSCC tumor samples. We found a positive correlation (r2=0.309) between TH nerve presence and patient-reported pain using the FACT-H&N questionnaire. Furthermore, we quantified a 2-fold increase in platelet NE concentration isolated from HSNCC patient plasma compared to healthy controls and there was a positive correlation (r2=0.687) between NE concentration and patient reported pain. MOC2 tumor progression evoked orofacial nociceptive behavior in female mice; there was a 198.8±20.5% and 478.5±146.8% increase in gnaw-time at post inoculation day (PID) 7 and 14 respectively compared to sham mice. PID14 was also associated with significant changes in adrenergic gene expression in TG from tumor-bearing female compared to control mice; there was a 2-fold increase in genes for the following adrenergic receptor subtypes: Adra1b, Adra2b, and Adrb2. Future investigation using adrenergic blockade will further support the role of sympathetic modulation of oral cancer pain and allow for better and more focused treatment options for these patients. Grant support from NIH NIDCR R01DE030892.

Sensory Neurotransmitter CGRP Modulates Oral Cancer Growth and Cancer-associated Immune Response

Head and neck squamous cell carcinoma (HNSCC) induces severe pain due in part to activation of primary afferent neurons by cancer-secreted mediators. Local neurotransmitter release (e.g., calcitonin gene-related peptide (CGRP)) from trigeminal neurons innervating the cancer has been linked to tumorigenesis. We hypothesize that CGRP exerts a dual effect on both cancer-associated pain and tumor progression, suggesting that CGRP may be a promising therapeutic target in HNSCC treatment. We used human tumor tissue and patient-reported outcomes to explore the relationship between CGRP+ sensory nerve innervation and cancer pain in patients. To determine CGRP receptor expression on tumor cells, immunohistochemistry and PCR were performed on human and mouse oral cancer cell lines. We used a syngeneic tongue tumor transplant mouse model of oral cancer and a global Calca knockout mouse (i.e. CGRP-KO) to investigate the impact of CGRP signaling on tumor growth and the associate immune response in vivo. We found prominent CGRP-immunoreactive sensory nerve presence innervating human HNSCC tumor tissue, which positively correlated to patient-reported pain (r2=0.357). Furthermore, human HNSCC cell lines expressed 3-fold more CGRP receptor, RAMP1, compared to a non-tumorigenic keratinocyte cell line. In tumor-bearing CGRP-KO mice, we found a significant reduction in tumor size at post-inoculation days 7 and 14 compared to wildtype. We also found a 4-fold increase in tumor infiltrating RAMP1-expressing CD4+ T cells, as well as a 5-fold increase cytotoxic CD8+ T cells and NK1.1+ NK cells in tumor tissue CGRP-KO mice compared to wildtype. This preliminary data suggests that CGRP signaling from sensory neurons may increase cancer associated pain and tumor progression. Further knowledge regarding the relationship between sensory neurons and cancer could allow for the repurposing clinically available nervous system drugs (e.g., anti-CGRP antibodies) for the treatment of cancer and cancer pain. Grant support from the Rita Allen Foundation.

The Immunogenicity and Safety of Influenza Vaccines among Indonesian Older adult

BACKGROUND: Immunosenescence, frailty, sarcopenia, and Vitamin D deficiency were thought of having a negative influence on adequate immune response following vaccination. Epidemiological data related to influenza vaccination and its immunogenicity and safety in Indonesia are still scarce. AIM: This study aimed to assess the immunogenicity and safety of the influenza vaccine in older adults in Indonesia and common related conditions. METHODS: This study was a one-group, pre-test, post-test, quasi-experimental study. The vaccine used was a trivalent type. Immunogenicity was assessed according to rate of seropositivity and seroconversion based on hemagglutination inhibition antibody titer. Immunization safety was assessed according to adverse events following immunization (AEFI). RESULTS: A total of 227 subjects were involved in this study. Antibody titers were significantly increased after 28-day influenza vaccination for all strains (p &lt; 0.001). Seropositive rate in 28-day post-vaccination for A/H1N1, A/H3N2, and influenza B was 98.7%, 99.1%, and 97.4%, respectively. Meanwhile, the seroconversion rate for A/H1N1, A/H3N2, and influenza B strains was 54.2%, 66.1%, and 60.4%, respectively. The conditions of the frailty status, sarcopenia, and Vitamin D level were not associated with either seroconversion or seropositive status, both at baseline and endpoint. AEFI was occurred on seven subjects (3.08%) with pain at the injection site and one subject (0.44%) with arm tenderness. CONCLUSION: Influenza vaccination showed adequate immune response and safety regardless of the frailty status, sarcopenia, or Vitamin D level. This result strengthened the importance of influenza vaccine administration in Indonesia older adults.

Antibody response of sheep to simultaneous vaccination of foot-and-mouth disease, peste des petits ruminants, sheep pox and goat pox, and bluetongue vaccines.

There are many infectious animal diseases in T urkey and generally, vaccination is the primarly control strategy to combat them. However, it is difficult to apply all vaccines in a definite period in the field due to limitations of the labor and finance. Rapid vaccination and effective use of labor can be possible with the help of simultaneous vaccine administrations. The study aims to show the effects of simultaneous foot-and-mouth disease (FMD), peste des petits ruminants (PPR), sheep pox and goat pox (SGP), and bluetongue (BT) vaccine administration on the antibody response of sheep. For this aim, 30 sheep were divided into one experiment and 5 control groups. Blood samples were collected in each group at 0, 30 and 60 days post-vaccination (DPV). Immune response was measured with virus neutralization test (VNT) and, liquid phase blocking ELISA (LPBE) for FMDV; VNT for BTV and PPR. A live virus challenge study was performed to determine the immune response of SGP vaccine. As a result, antibody titers for each vaccine agent decreased on 60 DPV with the simultaneous vaccination except FMD. The difference between means of antibody titer decrease with single and simultaneous vaccinations is significant especially for BTV and PPR vaccines at 60DPV (p<0.05). Briefly, this decreasing immune response of three live vaccines can be explained with the development of the interference, administration of these vaccines from the same injection site, the effect of cytokines, especially IL-10 effect of SGP vaccine. It was concluded that four vaccines can not be used simultaneously in sheep.

Thermoresistant Newcastle disease vaccine effectively protects SPF, native, and commercial chickens in challenge with virulent virus

BackgroundDue to the more stability and a better homogenecity in immune response, the use of thermoresistant vaccines in different chicken types has been increased.ObjectiveThis study aimed to evaluate the efficacy of a newly developed Newcastle disease vaccine (ND.TR.IR) originating from I‐2 strain in specific pathogen‐free (SPF) and native and broiler chickens.MethodsFollowing determination of pathogenicity indices on the candidate seed, three efficacy examinations were conducted. In the first experiment, 120 1‐day‐old SPF chickens were randomly allocated to six groups and either vaccinated with ND.TR.IR via eye drop at 1, 7, and 21 days of age (V1, V7, and V21), or considered as non‐vaccinated control groups (C1, C7, and C21). At 20th post‐vaccination day, sera hemagglutination inhibition (HI) antibody titres against ND virus (NDV) were measured and then the chickens were challenged by virulent NDV (vNDV). In the second and third experiments, the efficacy of ND.TR.IR vaccine was compared to routine vaccination program (B1 and LaSota) in native and broiler chickens that were vaccinated at 10 and 20 days of age, respectively. The HI antibody titres were measured on 10, 20, 30, and 40 days of age, and also challenge efficacy test with vNDV was conducted on 30 days of age.ResultsThe studied virus, as a vaccinal seed, complied with the pathogenicity indices of avirulent NDV and molecular identity of I‐2 strain. In the efficacy evaluation trials, the vaccinated chickens had higher HI antibody titres against NDV compared with their corresponding control chickens (p < 0.05). Results of the challenge tests indicated 95% and 100% protection against vNDV in native, SPF, and broiler‐vaccinated chickens, respectively.ConclusionsThe present findings indicated that administration of ND.TR.IR induced appropriate HI antibody titres against NDV in SPF, native, and broiler chickens associated with good protection in efficacy test.

First Report of Coniella granati Associated with Dieback of Rose (Rosa sp.) in India.

Rose (Rosa sp.) is an important floricultural crop largely cultivated in the Karnataka state of southern India. A field survey conducted in Devanahalli, Bangalore rural during October, 2019 revealed rose plants (cv. Arka Pride) showing die-back symptoms with a disease incidence of 7% in an area of 30 hectare rose field. Die-back was characterized by the development of necrotic lesions that started at the tip of shoots and progressed downwards leading to the death of young shoots. Pycnidia were found on young shoots showing necrotic lesions. Die-back affected shoots (n=10) were surface sterilized with 2% NaOCl for 2 min, rinsed thrice in sterile distilled water, and plated on potato dextrose agar (PDA) medium amended with chloramphenicol (40mg/L). The plates were incubated at 28±2 °C and pure cultures were obtained by hyphal tipping. Fungal colonies with white aerial mycelia along with pycnidia were observed on eight samples. Colony growth rate was at 4.2 ± 0.4 mm/day with an average colony diameter of 47.2 ± 3.8 mm after 7 days (12/12 h light and dark period). Pycnidia were solitary and globose. Conidia were hyaline, single celled ellipsoid to fusiform, measured 12.4 - 16.78 × 3.2 - 5.1 μm (n=50). Based on cultural and morphological features, the fungus was identified as Coniella granati (Van Niekerk et al., 2004; Mahadevakumar et al., 2019) (Figure S1). Further, two representative isolates (NLD-2021-1 & NLD-2021-2) were used for molecular identification based on barcoding of ITS-rDNA, LSU and tef1 regions amplified using primer pairs described in Alvarez et al., 2016. PCR amplified products were sequenced and the consensus sequence were analyzed through BLASTn search which showed ITS (NLD-2021-1: 611/611bp - MH860368 & NLD-2021-2: 573/573bp - KU147239), LSU (NLD-2021-1: 783/906bp - MH872113 & NLD-2021-2: 844/1172bp - KX833400) and tef1 (NLD-2021-1 & NLD-2021-2: 548/548bp -KX833682) sequences shared 100% sequence similarity with C. granati respectively. The nucleotide sequences were deposited in GenBank with the accession numbers ITS: MW898436, MW898437; LSU: MZ895475, MZ895476 and tef1 MW916275, MW916276 respectively. The phylogenetic tree constructed based on combined ITS-LSU-tef1 loci confirmed that the sequences shared a common clade with C. granati (Figure S2). Further, pathogenicity tests were conducted on conducted twice on healthy rose plants (Arka Pride; 60 days old after grafting) maintained in a greenhouse at 28±2°C and 70% relative humidity. Whole plant inoculation was followed by spraying the conidial suspension (10ml / plant) of C. granati (NLD-2021-1) (3 × 106 conidia/ml, amended with Tween20 at 2%) on 15 plants. A total of 10 healthy plants inoculated with sterile distilled water amended with Tween20 at 2% served as control. Inoculated plants were covered with polythene bags for 48 h to maintain relative humidity. Die-back symptoms were observed on young shoots after 15-18 days of post inoculation (DPI), and control plants remained disease free after 15 DPI. The pathogen was re-isolated on PDA from 10 diseased plants and identity was confirmed through morphological methods. Coniella granati is an important phytopathogen known to cause die-back and twig blight of pomegranate globally and other economically important crops (Cintora-Martinez et al. 2017). No reports are available on the association of C. granati with dieback of rose (Farr & Rossman, 2021), which should help in developing appropriate disease management strategies.

First Report of Aspergillus versicolor Associated with Fruit Rot Disease of Tomato (Solanum lycopersicum) from India.

Tomato (Solanum lycopersicum L.) is an important vegetable crop and cultivated throughout India. During 2019-20, Karnataka produced 2163 tonnes of tomato, accounting for 10.51 percent of total production (https://agricoop.gov.in/horticulture-reports). During a survey conducted in September 2019 in southern Karnataka region, severe fungi fruit rot symptoms were observed on at harvest in a 18 hectare production operation. The incidence of fruit rot disease was estimated around 12–14% in an area of 18 hectares (~62 fields). The necrotic lesions on infected fruit were covered with. The fruit rot was characterised by necrosis lesions filled with black fungal fruiting bodies, which later became soft and fully necrotic. A total of 20 samples were used for isolation and diagnostics. Infected fruit rot samples were surface sterilized with 2% NaOCl for 2 min, rinsed thrice in sterile distilled water, and plated on potato dextrose agar (PDA) medium amended with chloramphenicol (40mg/L). The plates were incubated at 28±2 °C and pure cultures were obtained by hyphal tip excision. On PDA medium, fungal colonies were initially white, but later turned to yellow green, often intermixed with pink sections. They produced sulcate and centrally raised sporulating conidiophores which were hyaline or slightly pigmented with smooth walls. Vesicles produced on conidiophores were sub-globose to ellipsoidal with phialides borne on metulae. Conidia were globose, brownish and conspicuously echinulate. On the basis morphological and cultural characteristics, the causative pathogen was identified to be Aspergillus versicolor (Jurjevic et al., 2012). Further molecular characterization was also done by amplifying internal transcribed spacer (ITS) region using universal ITS1-ITS4 primers with three representative isolates (White et al. 1990). Genomic DNA was isolated using CTAB using 15 days old culture followed by PCR amplification using ITS1-ITS4 primer pair and sequencing. The sequences analysis had 100% (561/561 base pairs) similarity with the reference sequences of Aspergillus versicolor (KU318417.1, MT798844.1) according to BLAST analysis in GenBank. The representative sequence of ITS-rDNA for three isolates viz., A. versicolor NCM_1, NCM_2 &amp; NCM_3 were deposited in GenBank (Accession number: MZ520551.1 – MZ520553.1). Pathogenicity tests were conducted on 10 healthy mature fruit of tomato F1 hybrid (INDAM 3003) using wound inoculation (conidial suspension 3 × 106 conidia mL−1). Several tomato fruit (n=5) were only inoculated with sterile water to serve as controls. The experiments were conducted in triplicates with 10 fruits each and repeated three times (Mahadevakumar et al. 2019). Within 7 days of post-inoculation, fruit rot symptoms similar to those in the original out break in Karnataka were observed on all inoculated fruit. Control fruit remained asymptomatic even after 10 days. The pathogen was reisolated from diseased fruit and the identity was confirmed based on morphology and ITS sequencing. A. versicolor has been reported as a post-harvest fungal pathogen on other various vegetable crops (Aidoo, 1993). Tomato fruit rots are known to be a major problem (Nizamani et al., 2021) in the region. However, there no reports of tomato fruit rot caused by A. versicolor in India (Farr and Rossman, 2021), which warrants additional attention as a potential threat to regional production.

Comparative Assessment of Seroconversion in Poultry Vaccinated with Two Avian Influenza Vaccines

FromAugust to December 2020, a new outbreak of H5 avian influenza caused anotherserious animal health emergency in Kazakhstan, leading to the deaths or cullingof more than 500 000 chickens. This outbreak renewed interest in developingprevention strategies for this re-emerging infection. In this study, weevaluated poultry seroconversion levels after two H5 vaccines. Regardless of age, productivity, and schemeof vaccination Super Nick layers (a total of 368 heads) received a single doseof an inactivated whole H5 vaccine or a baculovirus-derived H5 vaccine,respectively. We assessed theseroconversion by Hemagglutination Inhibition (HI) and Enzyme-LinkedImmunosorbent (ELISA) assays. Cloacal and tracheal swabs were tested forInfluenza A Virus (AIV) by real-time PCR to monitor the field AIV circulation.The analysis of 368 serum samples showed that the inactivated vaccine provideda significantly higher humoral immune response when compared to thebaculovirus-derived vaccine as evaluated by both ELISA and HI in 30, 60, and120 Days Post-Vaccination (DPV). Thus, our study demonstrates that under farmconditions classical Inactivated Avian Influenza (AIV) vaccine induces a higherseroconversion level against the H5N1 virus predicting better protectionagainst field infection, than a baculovirus-derived H5 vaccine.

Immune gene expression and protective effects in goldfish (Carassius auratus L.) immunized with formalin-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine

The goldfish hematopoietic necrosis viral disease (GHNVD) has led to worldwide economic losses in goldfish aquaculture. The present study has focused on the development of an inactivated vaccine for the cyprinid herpesvirus (CyHV-2) and to check the immunogenicity of the vaccine in the host. The fantail goldfish fin (FtGF) cell line was used in the propagation of the CyHV-2 and the viral titer obtained were of 107.8 TCID50/ml. Followed by the virus was inactivated using 0.1% formalin for 2 days. Various concentrations of formalin-inactivated CyHV-2 (1%, 0.7%, 0.5%, 0.3% and 0.1%) were studied in the FtGF cell line. Morphological changes were observed in the FtGF cell line in all other concentrations of formalin except 0.1% formalin-inactivated CyHV-2 vaccine. The goldfishes were intraperitoneally injected with 300 μl of vaccine and various immune gene responses were studied for a period of 30 days. The gene expression of the adaptive markers CD8, CD4, IFN-ϒ, the cytokines (IL-10, IL-12) was studied in kidney and spleen tissues. Formalin-inactivated CyHV-2 vaccine showed a significant up-regulation of the genes CD8 and IFN-ϒ by the 6th hr post-vaccination onwards. The experimental fish were challenged intraperitoneally with CyHV-2 virus of concentration 107.8 TCID50/ml after 30 days of post-vaccination. A significant difference in cumulative mortality rate was observed for the vaccinated fishes from the unvaccinated fishes. The relative percent survival for formalin immunized fish was 74.03%. Our results have proven that the formalin-inactivated vaccines were efficient and it resulted in triggering the immune gene expression in goldfish. The development and further enhanced studies for this vaccine will lead to a promising low-cost commercial vaccine for CyHV-2 viral infection.