Intramammary immunization of pregnant mice with staphylococcal protein a reduces the post-challenge mammary gland bacterial load but not pathology

Protein A, encoded by the spa gene, is one of the major immune evading MSCRAMM of S. aureus, demonstrated to be prevalent in a significant percentage of clinical bovine mastitis isolates in Australia. Given its' reported significance in biofilm formation and the superior performance of S. aureus biofilm versus planktonic vaccine in the mouse mastitis model, it was of interest to determine the immunogenicity and protective potential of Protein A as a potential vaccine candidate against bovine mastitis using the mouse mastitis model. Pregnant Balb/c mice were immunised with Protein A emulsified in an alum-based adjuvant by subcutaneous (s/c) or intramammary (i/mam) routes. While humoral immune response of mice post-immunization were determined using indirect ELISA, cell-mediated immune response was assessed by estimation of interferon-gamma (IFN-γ) produced by protein A-stimulated splenocyte supernatants. Protective potential of Protein A against experimental mastitis was determined by challenge of immunized versus sham-vaccinated mice by i/mam route, based upon manifestation of clinical symptoms, total bacterial load and histopathological damage to mammary glands. Significantly (p<0.05) higher levels of IgG1 isotype were produced in mice immunized by the s/c route. In contrast, significantly higher levels of the antibody isotype IgG2a were produced in mice immunized by the i/mam route (p<0.05). There was significant reduction (p<0.05) in bacterial loads of the mammary glands of mice immunized by Protein A regardless of the route of immunization, with medium level of clinical symptoms observed up to day 3 post-challenge. However, Protein A vaccine failed to protect immunized mice post-challenge with biofilm producing encapsulated S. aureus via i/mam route, regardless of the route of immunization, as measured by the level of mammary tissue damage. It was concluded that, Protein A in its' native state was apparently not a suitable candidate for inclusion in a cell-free vaccine formulation against mastitis

A comparative study of enumeration techniques for free-roaming dogs in rural Baramati, District Pune, India

The presence of unvaccinated free-roaming dogs (FRD) amidst human settlements is a major contributor to the high incidence of rabies in countries such as India, where the disease is endemic. Estimating FRD population size is crucial to the planning and evaluation of interventions, such as mass immunisation against rabies. Enumeration techniques for FRD are resource intensive and can vary from simple direct counts to statistically complex capture-recapture techniques primarily developed for ecological studies. In this study we compared eight capture-recapture enumeration methods (Lincoln–Petersen’s index, Chapman’s correction estimate, Beck’s method, Schumacher-Eschmeyer method, Regression method, Mark-resight logit normal method, Huggin’s closed capture models and Application SuperDuplicates on-line tool) using direct count data collected from Shirsuphal village of Baramati town in Western India, to recommend a method which yields a reasonably accurate count to use for effective vaccination coverage against rabies with minimal resource inputs. A total of 263 unique dogs were sighted at least once over 6 observation occasions with no new dogs sighted on the 7th occasion. Besides this direct count, the methods that do not account for individual heterogeneity yielded population estimates in the range of 248–270, which likely underestimate the real FRD population size. Higher estimates were obtained using the Huggin’s Mh-Jackknife (437 ± 33), Huggin’s Mth-Chao (391 ± 26), Huggin’s Mh-Chao (385 ± 30), models and Application “SuperDuplicates” tool (392 ± 20) and were considered more robust. When the sampling effort was reduced to only two surveys, the Application SuperDuplicates online tool gave the closest estimate of 349 ± 36, which is 74% of the estimated highest population of free-roaming dogs in Shirsuphal village. This method may thus be considered the most reliable method for estimating the FRD population with minimal inputs (two surveys conducted on consecutive days)

Performance Evaluation of Cabbage Cultivars under Open Field Cultivation in High Altitude of Tawang Arunachal Pradesh

The study has been undertaken to evaluate the performance of locally available commercial cultivars of cabbage in open field during summer season of 2019 at Defence Research Laboratory Research and Development Centre Tawang (Arunachal Pradesh). The commercial cultivars of these vegetables were evaluated based on the growth parameters and their yield performance under open field with paired row system of planting. It was found that the cultivar Green Express performance was outstanding among test edcultivars and it superseded cv. Royal Ball BC-51 and cv. Blue Jayes in terms of growth parameters as well as marketable yield (2.62 kg/m2) and biological yield (3.47 kg/m2). The cv. Royal Ball BC-86 was also found next best performing cultivar after cv. Green Express as former was at par with growth and yield parameter and harvest maturity of later. The head of cv. Green express attended harvest maturity (149.25 days) approximately one week earlier as compared to cv. Blue Jayes (155.0 days). However, the productivity of cultivars Green Express and Royal Ball BC-86 was found within the range of national average, therefore, these two cultivars could be recommended for the commercial cultivation with paired row system of planting under open field cultivation during summer season in high altitude areas of Tawang (Arunachal Pradesh)

Prevalence of Mycoplasma bovis Infection in calves and dairy cows in Western Australia

Mycoplasma bovis (M. bovis) can cause a multitude of diseases in cattle, with detrimental effects on the farm economy and the welfare of both adult and young cattle. The objective of this study was to determine the prevalence of M. bovis in adult cows and calves in the south-west region of Western Australia. A cross-sectional study was conducted on 29 dairy farms with 699 apparently healthy adult lactating cows and 495 young calves during 2019–2020. Nasal swabs and blood samples collected from the animals and bulk tank milk (BTM) samples were assessed for M. bovis-specific proteins and antibodies by using polymerase chain reaction (PCR) and Mycoplasma immunogenic lipase A- Enzyme-Linked Immune Sorbent Assay (MilA ELISA). A seroprevalence of 42.5% (95% CI: 38.9–46.2) and 61% (95% CI: 56.6–65.2) was found in adult lactating cows and calves, respectively. The herd-level seroprevalence of M. bovis ranged from 4% (95% CI: 07–19.5) to 92% (95% CI: 75.0–97.8) in adult lactating cows and 25% (95% CI: 10.2–49.5) to 87% (95% CI: 67.9–95.5) for calves in these farms. None of the BTM and nasal swab samples were positive for M. bovis, indicating an absence of any current active infections on the farms. The female calves and pure Holstein–Friesian animals are twice as likely to be seropositive for M. bovis compared to male calves (OR 2.4; 95% CI: 1.7–3.5) and Holstein–Friesian crossbred calves (OR 2.4; 95% CI: 1.7–3.5). The high seroprevalence in both adult and young cattle in the southwest dairy farms of Western Australia warrants more effective farm biosecurity measures and further evaluation of the current prevention and management measures practiced on the farms

TWEAK/Fn14 signalling promotes cholangiocarcinoma niche formation and progression.

Background & Aims: Cholangiocarcinoma (CCA) is a cancer of the hepatic bile ducts that is rarely resectable and is associated with poor prognosis. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is known to signal via its receptor fibroblast growth factor-inducible 14 (Fn14) and induce cholangiocyte and myofibroblast proliferation in liver injury. We aimed to characterise its role in CCA. Methods: The expression of the TWEAK ligand and Fn14 receptor was assessed immunohistochemically and by bulk RNA and single cell transcriptomics of human liver tissue. Spatiotemporal dynamics of pathway regulation were comprehensively analysed in rat and mouse models of thioacetamide (TAA)-mediated CCA. Flow cytometry, qPCR and proteomic analyses of CCA cell lines and conditioned medium experiments with primary macrophages were performed to evaluate the downstream functions of TWEAK/Fn14. In vivo pathway manipulation was assessed via TWEAK overexpression in NICD/AKT-induced CCA or genetic Fn14 knockout during TAA-mediated carcinogenesis. Results: Our data reveal TWEAK and Fn14 overexpression in multiple human CCA cohorts, and Fn14 upregulation in early TAA-induced carcinogenesis. TWEAK regulated the secretion of factors from CC-SW-1 and SNU-1079 CCA cells, inducing polarisation of proinflammatory CD206+ macrophages. Pharmacological blocking of the TWEAK downstream target chemokine monocyte chemoattractant protein 1 (MCP-1 or CCL2) significantly reduced CCA xenograft growth, while TWEAK overexpression drove cancer-associated fibroblast proliferation and collagen deposition in the tumour niche. Genetic Fn14 ablation significantly reduced inflammatory, fibrogenic and ductular responses during carcinogenic TAA-mediated injury. Conclusion: These novel data provide evidence for the action of TWEAK/Fn14 on macrophage recruitment and phenotype, and cancer-associated fibroblast proliferation in CCA. Targeting TWEAK/Fn14 and its downstream signals may provide a means to inhibit CCA niche development and tumour growth. Lay summary: Cholangiocarcinoma is an aggressive, chemotherapy-resistant liver cancer. Interactions between tumour cells and cells that form a supportive environment for the tumour to grow are a source of this aggressiveness and resistance to chemotherapy. Herein, we describe interactions between tumour cells and their supportive environment via a chemical messenger, TWEAK and its receptor Fn14. TWEAK/Fn14 alters the recruitment and type of immune cells in tumours, increases the growth of cancer-associated fibroblasts in the tumour environment, and is a potential target to reduce tumour formation

Phenotypic PIA-dependent biofilm production by clinical non-typeable staphylococcus aureus is not associated with the intensity of inflammation in mammary gland: A pilot study using mouse mastitis model

Non-typeable (NT) Staphylococcus aureus strains are associated with chronic bovine mastitis. This study investigates the impact of biofilm formation by clinical NT S. aureus on cytokine production and mammary tissue damage by using a mouse mastitis model. Mice infected with two different NT S. aureus strains with strong and weak biofilm forming potential demonstrated identical clinical symptoms (moderate), minimal inflammatory infiltrates, and tissue damage (level 1 histopathological changes) in the mammary glands. However, the S. aureus load in the mammary glands of mice and the level of pro-inflammatory cytokines (IL-1β, IL-6, IL-12, IL-17 and IFN-γ) in serum were significantly higher (p ≤ 0.05) in those infected with the strong biofilm forming NT S. aureus strain. The level of IL-6 in sera samples of these mice was extremely high (15,479.9 ± 532 Pg/mL). Furthermore, these mice died in 24h of post infection compared to 30 h in the weak biofilm forming NT S. aureus infected group. The study demonstrates no association between the strength of PIA (polysaccharide intercellular adhesion)-dependent biofilm production by clinical NT S. aureus and mammary gland pathology in a mouse mastitis model. However, the role of biofilm in the virulence of S. aureus advancing the time of mortality in mice warrants further investigation

Validation of application SuperDuplicates (AS) enumeration tool for free-roaming dogs (FRD) in urban settings of Panchkula Municipal Corporation in North India

A cost-effective estimation of the number of free-roaming dogs is an essential prerequisite for the control of rabies in countries where the disease is endemic, as vaccination of at least 70% of the population is recommended to effectively control the disease. Although estimating the population size through sight-resight based maximum likelihood methodology generates an estimate closest to the actual size, it requires at least five survey efforts to achieve this. In a rural setting in India, a reliable estimate of at least 70% of the likely true population of free-roaming dogs was obtained with the Application SuperDuplicates shinyapp online tool using a photographic sight-resight technique through just two surveys. We tested the wider applicability of this method by validating its use in urban settings in India. Sight-resight surveys of free-roaming dogs were conducted in 15 sectors of the Panchkula Municipal Corporation in north India during September- October 2016. A total of 1,408 unique dogs were identified through 3,465 sightings on 14 survey tracks. The estimates obtained by the Application SuperDuplicates shinyapp online tool after two surveys were compared with the maximum likelihood estimates and it was found that the former, after two surveys, provided an estimate that was at least 70% of that obtained by the latter after 5–6 surveys. Thus, the Application SuperDuplicates shinyapp online tool provides an efficient means for estimating the minimum number of free-roaming dogs to vaccinate with a considerably lower effort than the traditional mark-resight based methods. We recommend use of this tool for estimating the vaccination target of free-roaming dogs prior to undertaking mass vaccination efforts against rabies

Prevalence of failure of passive transfer of immunity in dairy calves in a Mediterranean pasture-based production system of the south-west region of Western Australia

The objective of this study was to estimate the prevalence of failure of passive transfer of immunity (FPTI) in dairy calves in the south-west region of Western Australia herds. A cross-sectional study was conducted in 26/140 dairy farms and serum samples were collected from 495 healthy 2–7 day-old calves. A radial immunodiffusion (RID) test was used to determine the concentration of serum IgG and calves were classified as having FPTI if the IgG concentration was less than 10 mg/mL. Estimation of FPTI was also assessed using two indirect methods using serum total protein (STP) and a brix refractometer. The estimated prevalence of FPTI was found to be 8.7% (43 calves out of 495) by RID with the concentration of IgG ranging between 0 and 6.2 mg/ml. The STP was found to vary from 46 to 96 mg/mL and using a cut-off point of 55 mg/mL the calf level prevalence was estimated as 7.1% (33 calves). Using the brix refractometer, the prevalence was found to be 13.1% (65 calves) with the refractometer reading ranging 6–14% of IgG. In the present study there was no association between calf-level factors (age, sex and breed) and FPTI. There was a higher correlation of the RID test results and the STP results compared to the RID and brix refractometer results. It is concluded that the prevalence of FPTI in dairy calves in the south-west region of Western Australia is low (8.7%) and the brix refractometer is not a reliable indirect method for determining passive transfer of immunity to calves

The role of liver progenitor cells during liver regeneration, fibrogenesis and carcinogenesis

The growing worldwide challenge of cirrhosis and hepatocellular carcinoma due to increasing prevalence of excessive alcohol consumption, viral hepatitis, obesity, and the metabolic syndrome has sparked interest in stem cell-like liver progenitor cells (LPCs) as potential candidates for cell therapy and tissue engineering, as an alternative approach to whole organ transplantation. However, LPCs always proliferate in chronic liver diseases with a predisposition to cancer; they have been suggested to play major roles in driving fibrosis, disease progression, and may even represent tumor-initiating cells. Hence, a greater understanding of the factors that govern their activation, communication with other hepatic cell types, and bipotential differentiation as opposed to their potential transformation is needed before their therapeutic potential can be harnessed

THU-077-Fibro-protective molecular and cellular mechanisms emerge during resolution from thioacetamide-induced fibrosis

Poster presentatio