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.

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-1ß, 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.

Using Dairy Value Chains to Identify Production Constraints and Biosecurity Risks.

The dairy industry plays an important role in the economy and food security of India. A study of the dairy value chains was conducted in Punjab, India, to identify production constraints and biosecurity risks. Focus group discussions and key informant interviews were conducted during 2018-2019 with a total of 119 participants comprising veterinarians (41), paraveterinarians (15), veterinary academics (12), dairy farmers (46) and key informants (5). Input and output value chains were created, and potential risk nodes were identified that could facilitate the transmission of pathogens between animals, farms and villages. The majority of the participants were male (93%), middle-aged (68%) or worked in rural areas (75%). Most of the farmers self-cultivated their green fodder (82%), used the wheat straw from their own fields (60%) but purchased commercial feed (63%). Artificial insemination was used by 85% of farmers for cattle, but only 68% for buffaloes. Most of the farmers (76%) reported getting their animals vaccinated against foot-and-mouth disease and hemorrhagic septicemia. Animals were sold and purchased without any health certification and testing in most cases. Adoption of biosecurity measures by farmers and the use of personal protective equipment by veterinary personnel were very low. We recommend conducting epidemiological studies to further characterize the identified risk nodes, training of veterinary practitioners and farmers to ensure adequate biosecurity practices and the appropriate use of personal protective equipment.

Author Correction: Demographic characteristics of free-roaming dogs (FRD) in rural and urban India following a photographic sight-resight survey.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Demographic characteristics of free-roaming dogs (FRD) in rural and urban India following a photographic sight-resight survey.

An understanding of the core demographic characteristics of the sub-populations of FRD is essential to effectively implement both rabies control interventions through mass vaccination of FRD, and dog population control programmes. This study compares the data obtained following photographic sight-resight surveys in rural (Shirsuphal village in west India) and urban (Municipal Corporation Panchkula in north India) locations . A total of 263 and 1408  FRD were seen at least once through 617 and 3465 sightings in the rural and urban sites, respectively. The rural location had a lower proportion of females (OR 0.5, 95% CI 0.4-0.7) and a higher proportion of poor and fair conditioned dogs (OR 1.8, 95% CI 1.3-2.3) compared to the urban setting. The rural site also had fewer active FRD (OR 0.6, 95% CI 0.5-0.7) and FRD were less likely to be sighted within 20 m of garbage points (OR 0.3, 95% CI 0.2-0.3) compared to the urban site. The demographic composition of the FRD population was found to vary within the urban location, with the odds of sighting a de-sexed dog being significantly higher in residential areas compared to other areas. The study underlines the importance of knowing the demographic composition of FRD for implementation of effective interventions against rabies. Fewer female dogs in the rural location indicate that spaying could be an effective tool for dog population management in this setting, while presence of dogs within 20 m of garbage points in urban settings highlights that an improved garbage management may reduce the carrying capacity of the urban locality resulting in smaller FRD population. It is concluded that quick and low cost surveys can generate useful demographic data for FRD in urban and rural settings which can be useful to understand the epidemiology of rabies and its control.

Utilising Group-Size and Home-Range Characteristics of Free-Roaming Dogs (FRD) to Guide Mass Vaccination Campaigns against Rabies in India.

Adequate vaccination coverage of free roaming dogs (FRD) against canine rabies is not achieved primarily due to difficulties in administering parenteral vaccinations to this population. One factor associated with this difficulty is the tendency of FRD to form groups, which increases their aggressive behavior, resulting in a significant risk of dog-bites for the vaccinators. This study investigated factors that influenced FRD forming groups and their home-ranges, using data obtained from photographic capture-recapture/sight-resight surveys conducted in rural Shirsuphal (584 sightings) and urban Panchkula (3208 sightings), India. In the rural site, older dogs (OR 0.5, 95% CI 0.2-0.9, p = 0.03) and FRD sighted within 20 m of garbage sites (OR 0.6, 95% CI 0.4-0.9, p = 0.02) were less likely to be in groups. The number of dogs sighted with an FRD decreased with increased resight-probability of that dog (ß= -1.0, p < 0.001). The rural FRD with smaller home-ranges were more likely to be sighted alone (OR 2.3, 95% CI 1.0-95, p = 0.04) than those with larger home-ranges. In the urban site, females (OR 1.3, 95% CI 1.1-1.5, p = 0.002) and older dogs (OR 1.5, 95% CI 1.1-2.1, p = 0.07) were more likely to be found in groups, and groups of dogs were more likely to be seen within 20 meters of garbage sites (OR 1.7, 95% CI 1.5-2.0, p < 0.001). The distribution of urban FRD sighted alone, in pairs, triads, and in packs of ≥4 dogs were not random in the administrative (p = 0.02), and the two industrial (p = 0.03 & 0.01) survey tracks of the urban site, implying stable groups. The resighting probability of a dog (ß = 0.3, p < 0.0001) and presence of garbage within 20 m (ß = 0.2, p < 0.0001) in the urban site increased the likelihood of sighting a FRD with other dogs. It is concluded that data on the resighting probability, presence of garbage points, and home-ranges can be utilised to guide the selection of parenteral or oral rabies vaccination to achieve a population vaccination coverage of 70% to break the transmission cycle of rabies virus in FRD in India.

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.

Knowledge, attitudes and practices (KAP) towards rabies and free roaming dogs (FRD) in Panchkula district of north India: A cross-sectional study of urban residents.

Canine rabies is endemic in urban India. A questionnaire was administered to 204 residents of the urbanised municipality of Panchkula in north India to assess the influence of gender, age, family size, social status and dog ownership, over the knowledge, attitudes and practices (KAP) towards rabies control and free-roaming dogs (FRD) in their locality. Bivariate analyses revealed significant knowledge gaps regarding crucial information on the control and transmission of rabies. Multivariable logistic regression models found that the respondents with a high/middle socio-economic status were likely to be more knowledgeable than those from low socio-economic levels (OR 3.03, 95%CI 1.5-6.0, p = 0.001). Households with children ≤14 years of age were likely to be lacking in knowledge about rabies compared to households with older or no children (OR 0.5, 95%CI 0.3-0.9, p = 0.04). The attitudes and practices of the respondents towards rabies control was positive in households with a high/middle socio-economic status (OR 3.4, 95%CI 1.7-7.2, p = 0.0008) but poor in older (≥ 35 years) participants (OR 0.4, 95%CI 0.2-0.7, p = 0.001). It is concluded that rabies awareness campaigns should be developed and conducted to target sectors of the urban community such as those belonging to lower socio-economic sections and schools to improve the residents' knowledge and practices towards rabies. Educating dog owners about sterilising their pets is also recommended to alter the attitudes of the residents towards FRD population control.

Knowledge, attitudes and practices (KAP) towards rabies and free-roaming dogs (FRD) in Shirsuphal village in western India: A community based cross-sectional study.

The lack of awareness about dog-bite related rabies in the rural population of developing countries, including India, is a major impediment to controlling the incidence of disease in humans. A survey of 127 rural residents was undertaken in Shirsuphal village in western India using a structured questionnaire to assess the influence of demographic and pet/livestock owning characteristics on the knowledge, attitudes and practices of the respondents towards rabies and free roaming dogs (FRD). Multivariable logistic regression models were constructed and the knowledge of the rural residents of Shirsuphal village was found to be significantly influenced by family size (OR 2.1, 95%CI 1.0-4.6, p = 0.04) and poultry ownership (OR 2.3, 95%CI 1.1-4.9, p = 0.03), while their attitudes towards FRD was significantly influenced by age of the respondents (OR 2.6, 95% CI 1.2-5.8) and ownership of cattle/buffalo (OR 2.2, 95% CI 1.1-5.5). Although the knowledge score about rabies was high, a comprehensive understanding of the disease was lacking. Concerted efforts to widen the knowledge about rabies and promote healthier practices towards FRD are recommended.

Knowledge, attitudes and practices towards dog-bite related rabies in para-medical staff at rural primary health centres in Baramati, western India.

The lack of awareness regarding rabies amongst rural primary care health staff and their adverse practices towards the management of dog-bite wounds is a major contributor to the high incidence of rabies infection and subsequent human mortality in India. A Knowledge, Attitudes and Practices survey was carried out involving 54 nursing and non-nursing staff working in 18 rural Primary Health centres and sub-centres around Baramati town of Pune district in Western India. Multivariable logistic regression models were constructed to assess factors that influenced knowledge of rabies and practices towards management of dog-bite related wounds. The more experienced and better-educated workers were found to have a good awareness of rabies (OR 3.4, 95%CI 1.0-12.1) and good practices towards dog-bite wound management (OR 5.6, 95%CI 1.2-27.0). Surprisingly, non-nursing staff were significantly more knowledgeable about rabies (OR 3.5, 95%CI 1.0-12.3), but their practices towards dog-bite wound management were inadequate (OR 0.18, 95%CI 0.04-0.8) compared to the nursing staff. It is recommended that a mandatory training module for primary care health staff be developed and implemented to improve their knowledge regarding rabies and management of dog-bite wounds to reduce the incidence of human rabies in rural India.

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).

Mammary Gland Pathology Subsequent to Acute Infection with Strong versus Weak Biofilm Forming Staphylococcus aureus Bovine Mastitis Isolates: A Pilot Study Using Non-Invasive Mouse Mastitis Model.

BACKGROUND: Biofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model. METHODS: Two S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection. RESULTS: Histopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1ß and TNF-α inflammatory biomarkers were produced in infected mice, level of TNF-α produced was significantly higher (p<0.05) in mice inoculated with strong biofilm forming S. aureus than the weak biofilm forming strain. CONCLUSION: This finding suggests an important role of TNF-α in mammary gland pathology post-infection with strong biofilm-forming S. aureus in the acute mouse mastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage, with or without use of antimicrobials and/or anti-inflammatory compounds for the treatment of bovine mastitis.

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.

Comparative studies of the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine against bovine mastitis using non-invasive mouse mastitis as a model system.

This study was undertaken to compare the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine for the prevention of mastitis using the mouse as a model system. Mice immunized with formalin-killed whole cell vaccine of S. aureus residing in a biofilm when delivered via an intramammary route produced a cell mediated immune response. Mice immunized with this biofilm vaccine showed significant reductions in colonization by S. aureus in mammary glands, severity of clinical symptoms and tissue damage in mammary glands in comparison with the mice immunized with formalin-killed whole cells of planktonic S. aureus. The planktonic vaccine administered by a subcutaneous route produced a significantly higher humoral immune response (IgG1 and IgG) than the biofilm vaccine. However, considering the host response, tissue damage, the clinical severity and colonization of S. aureus in mammary glands, the biofilm vaccine performed better in immunogenicity and protective potential when administered by the intramammary route.