Point-of-need mastitis pathogen biosensing in bovine milk: From academic sample preparation novelty to industry prototype field testing.

Bovine mastitis is an inflammation of the mammary gland, and it is the most common infectious disease in dairy cattle. Mastitis reduces milk yield and quality, costing dairy farmers millions of dollars each year. The aim of this study was to develop a point-of-need test for identifying mastitis pathogens that is field portable, cost-effective and can be used with minimal training. Using a proprietary polymer-based milk sample preparation method to rapidly extract pathogen DNA in milk samples, we demonstrated quantitative Polymerase Chain Reaction (qPCR) assays for six common bovine bacterial mastitis pathogens: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Mycoplasma bovis and Escherichia coli. We also implemented this sample preparation method on a prototype point-of-need system in a proof-of-concept field trial to evaluate user experience. Importantly, the protype system enabled a sample-to-result turnaround time of within 70 min to quantitatively detect all six target pathogens. The key advantage of our point-of-need prototype system is being culture-independent yet providing automated milk sample preparation for molecular identification of key mastitis pathogens by non-expert users. Our point-of-need prototype system showed a good correlation to laboratory-based qPCR for target pathogen detection outcomes, thus potentially removing the need for milk samples to be transported off-site for laboratory testing. Above all, we successfully achieved our objective of developing a point-of-need biosensor technology for mastitis and increased its readiness level with industry partners towards technology commercialization.

Bacterial culture and susceptibility test results for clinical mastitis samples from Australia's subtropical dairy region.

This study aimed to identify the pathogens isolated from the milk of cows with clinical mastitis in the subtropical region of Australia and to determine the antimicrobial susceptibility of these bacteria. Thirty dairy herds in the subtropical dairy region were asked to submit milk samples for the first 5 cases of clinical mastitis each month for 12 mo. Samples underwent aerobic culture, and isolates were identified via MALDI-TOF mass spectrometry. Antimicrobial susceptibility was determined for Escherichia coli, Enterococcus spp., Streptococcus agalactiae, Streptococcus uberis, Streptococcus dysgalactiae, Staphylococcus aureus, and non-aureus staphylococci and mammaliicocci (NASM). Between March 2021 and July 2022, 1,230 milk samples were collected. A positive culture result was recorded for 812 (66%) of the milk samples; from these samples, 909 isolates were obtained, including 49 isolates where no identification was possible. The remaining samples were classified as having no growth (16.8%) or as being contaminated (17.2%). The most common isolates with a MALDI-TOF diagnosis (n = 909) were Strep. uberis (23.6%), followed by the NASM group (15.0%). Farms enrolled in the study were in 3 distinct locations within the subtropical dairy region: North Queensland, Southeast Queensland, and Northern New South Wales. Some variation in isolate prevalence occurred between these 3 locations. We found lower odds of a sample being positive for E. coli in North Queensland (odds ratio [OR]: 0.25; 95% confidence interval [CI]: 0.07-0.87) and higher odds in Southeast Queensland (OR: 4.01; 95% CI: 1.96-8.20) compared with the reference, Northern New South Wales. We further found higher odds of Strep. dysgalactiae in North Queensland (OR: 5.69; 95% CI: 1.85-17.54) and Southeast Queensland compared with Northern New South Wales (OR: 3.99; 95% CI: 1.73-9.22). Although some seasonal patterns were observed, season was not significant for any of the analyzed isolates. Farm-level differences in pathogen profiles were obvious. Overall, clinical mastitis pathogens had low levels of resistance to the antimicrobials tested. This research demonstrates that Strep. uberis and the NASM bacterial group are the most common pathogens causing clinical mastitis in the subtropical dairy region. It highlights the importance of understanding pathogenic causes of mastitis at the farm and regional level for targeted control and therapy.

Bacterial culture and antimicrobial susceptibility results from bovine milk samples submitted to four veterinary diagnostic laboratories in Australia from 2015 to 2019.

A 5-year retrospective study was conducted to describe the mastitis-causing organisms isolated from bovine milk samples submitted to four veterinary diagnostic laboratories in Australia. The aim of this study was to identify temporal, geographical, and seasonal patterns of occurrence for the organisms and report the in vitro susceptibility of the most common mastitis-causing pathogens. In total, 22,102 milk samples were submitted between 2015 and 2019. The results were reported as positive growth for at least one significant organism (n = 11,407; 51.6%), no growth (n = 5,782; 26.2%), and mixed/contaminated growth (n = 4,913; 22.2%). Culture results for no growth, gram-negative bacteria, and eukaryotic organisms were combined for each region, and they were accounted for between 23 and 46% of submissions. These results represent a subset of mastitis cases for which the antibiotic treatment may not be warranted. A total of 11,907 isolates were cultured from 11,407 milk samples. The most common isolated organisms were Streptococcus uberis [41.3%; 95% confidence interval (CI): 40.4-42.1%] and Staphylococcus aureus (23.6%; 95% CI: 22.8-24.3%). For S. uberis and S. aureus, there was an association between a positive culture result and the dairy region. All regions except for the Sub-tropical Dairy region were more likely to culture S. uberis compared to the reference, Dairy NSW (P < 0.001). Similarly, for S. aureus, a positive culture result was more likely in all other dairy regions compared to Dairy NSW (P < 0.001). The LISA cluster analysis identified differences between High-High (hotspot) postcodes for S. aureus and S. uberis throughout all the analyzed dairy regions. These results highlight the need for further investigations into specific risk factors, such as environmental factors and herd-level predictors, which may have influenced the observed regional variations. Common mastitis-causing pathogens showed overall good susceptibility to a range of antimicrobials used in the treatment of mastitis. On-going surveillance of mastitis-causing pathogens and their antimicrobial susceptibilities will facilitate targeted mastitis control and treatment programs.

Identification and Prevalence of a Gammaherpesvirus in Free-Ranging Northern Brown Bandicoots (Isoodon macrourus).

Herpesviruses have been reported in several Australian marsupial species, with an overt, sometimes fatal disease described in macropods. Our study identifies a gammaherpesvirus in northern brown bandicoots (Isoodon macrourus) and provides virus prevalence data for bandicoots in southeast Queensland, Australia. Herpesvirus DNA was detected using pan-Herpesviridae family primers in a nested PCR format. Samples from 35 northern brown bandicoots were screened, including whole blood (n=29), oropharyngeal swabs (n=34), urine (n=22), and feces (n=23). Combining all sample types, herpesvirus DNA was detected at a total prevalence of 51% (18/35). Whole blood and oropharyngeal swabs proved to be the optimal samples for detection of this virus, with prevalences of 34% and 38%, respectively. Herpesvirus DNA was detected in 4.5% (1/22) of urine samples and not at all in fecal samples. Detection of herpesvirus was more likely in males than females. Animals were trapped at eight different locations, and at all but one location at least one herpesvirus positive animal was detected. This study indicates a high prevalence of the virus within northern brown bandicoot populations in southeast Queensland. Further research is required to understand the clinical manifestations, if any, of herpesvirus infection in this species and how this may affect populations in the face of stressors such as land clearing and habitat fragmentation.

CLINICAL PATHOLOGY OF CAPTIVE ENDANGERED MAHOGANY GLIDERS (PETAURUS GRACILIS) IN RESPONSE TO ENVIRONMENTAL CHANGE.

The impetus of this study was the imperative to establish blood biomarker values for clinically healthy mahogany gliders (Petaurus gracilis) in order to monitor the health status of eight captive individuals during their movement to a new facility. The study established ranges for 18 hematologic and 21 biochemical blood biomarkers for healthy individuals in a captive environment. The reported values are consistent with those published for other Australian glider and possum species. No statistically significant differences were found between the sexes, but significant age effects were observed. Specifically, subadult animals reported significantly higher total white cell counts, lymphocyte counts, alkaline phosphatase, creatine kinase, and glucose and chloride levels, compared to adult animals. Although there were no clinically significant changes in blood biomarkers associated with the relocation, many of the hematologic and biochemical biomarkers demonstrated the expected changes associated with the physiological stress of relocation. Specifically, triglycerides, glucose, globulins, creatinine kinase, aspartate transferase (AST), total protein, urea, phosphorous, potassium, sodium, chloride, neutrophils, and hematocrit showed changes with the large environmental change. The majority of the blood biomarkers returned to baseline levels 5 wk postrelocation, with all but one aged animal showing no signs of chronic health derangements following the relocation. The abnormal blood biomarker profiles of two geriatric individuals, one male diagnosed with pericloacal and adrenal gland tumors at the beginning of the study, and one female diagnosed with chronic urinary tract infections and suspected bone marrow disease following the relocation, are presented. The findings of this study inform the health monitoring of native gliders in captivity, rehabilitation, and clinical research scenarios. These findings also provide useful baseline data to aid in the health assessment of captive-bred individuals during their reintroduction into free-living populations.