Mark Edward Westman

Escherichia coli ( E. coli ) is a common cause of bovine mastitis, and rapid detection from milk is important for timely disease management. However, molecular assays such as colourimetric loop-mediated isothermal amplification (LAMP) are often limited by laboratory-based DNA extraction. This study evaluated a simplified HotSHOT (HS) DNA preparation method for extracting E. coli DNA from bovine milk and assessed its compatibility with colourimetric LAMP and conventional PCR. Pasteurised bovine milk spiked with E. coli was used for analytical evaluation, and culture‑positive clinical milk samples were tested to assess diagnostic performance. The HS method was compared with five commercial DNA extraction kits and a direct boiling method without centrifugation. Performance was assessed using limit of detection (LOD) expressed as colony-forming units per millilitre. Using the HS method, the LOD was 7.9 × 10⁴ CFU/mL for LAMP and 7.9 × 10³ CFU/mL for PCR. PCR performance of the HS method was comparable to the QIAamp and Norgen commercial kits, while LAMP sensitivity was ten- to a hundred-fold lower than the best kit-based methods. Direct boiling showed markedly reduced sensitivity, with an LOD of 7.9 × 10⁸ CFU/mL. Among 17 culture-confirmed clinical mastitis milk samples positive for E. coli , LAMP detected 15 (88.2%) and PCR detected all 17 (100%). These results show that simplified DNA preparation can support molecular detection of E. coli in bovine milk and provide a practical basis for future development of field-deployable diagnostics in resource-constrained settings.

Feral pigs ( Sus scrofa ) are important reservoirs for zoonotic and livestock pathogens in Australia, but robust information on the spatial distribution of pathogens in pig populations remains limited. We analysed serological data from a systematic, state‐wide surveillance program to investigate spatial heterogeneity in exposure to Brucella suis and pathogenic leptospires in feral pigs across New South Wales (NSW). Samples were collected from 2548 feral pigs at 81 locations between 2022 and 2026 and tested for antibodies to B. suis , L. interrogans serovars Pomona (hereafter Pomona) and Hardjo, L. borgpetersenii serovar Tarassovi and Coxiella burnetii . Bayesian spatial models were used to predict the probability of B. suis and Pomona seropositivity across the state in relation to feral pig relative abundance, generating spatial risk surfaces directly relevant to animal and human health management. Survey‐wide adjusted seroprevalence was 0.16 for B. suis , 0.52 for Pomona, 0.06 for Hardjo and 0.05 for Tarassovi. Brucella suis exposure was concentrated in the north‐eastern quadrant of NSW and strongly positively associated with local feral pig density (derived from expert opinion), whereas exposure to Pomona was widespread but locally variable. Seroprevalences of Hardjo and Tarassovi were spatially restricted, and antibodies to C. burnetii were rarely detected. By combining structured state‐wide sampling with spatial modelling, this study provides probabilistic inference on pathogen exposure. The results demonstrate how systematic surveillance can provide actionable spatial information for One Health risk management, prioritising risk‐based surveillance, assessing confidence in pathogen absence and supporting management responses for endemic, emerging, or exotic animal diseases.

Point‐of‐care (PoC) antibody (Ab) testing forms an integral component of feline immunodeficiency virus (FIV) diagnosis in both domestic and non‐domestic felids, with test kits designed for use in domestic cats frequently extrapolated for use in their wild counterparts. The SNAP® FIV/FeLV Combo lateral flow ELISA is commonly used to assign FIV infection status in wild and captive African lions ( Panthera leo ). The aim of this study was to investigate the performance of four other commercially available PoC FIV antibody test kits in African lions held in Australian zoos, when compared to SNAP® as a quasi‐reference test. To first validate the use of SNAP®, a subset of samples from SNAP® FIV‐positive ( n = 3) and SNAP® FIV‐negative ( n = 23) lions were evaluated with Western blotting, with perfect agreement demonstrated (Cohen's kappa coefficient = 1). Forty‐eight serum samples from SNAP® FIV‐positive ( n = 16) and SNAP® FIV‐negative ( n = 32) lions were then assessed with Anigen Rapid®, Witness®, VetScan® Rapid and SensPERT® FIV Ab tests. No seropositivity was observed in FIV‐infected lion sera using Anigen Rapid® or Witness® (Se 0%, Sp 100%), which utilise domestic cat FIV (FIV‐Fca) transmembrane glycoprotein gp40 as their capture antigen. In contrast, VetScan® Rapid, which employs FIV‐Fca capsid protein p24 as its capture antigen, demonstrated excellent performance (Se 100%, Sp 96.9%). The presence of p24 as a capture antigen did not guarantee the performance of an assay, as SensPERT®, which also features p24 as its capture antigen, was significantly less sensitive than VetScan® Rapid (Se 25%, Sp 100%; p < 0.001). Our findings have important implications for captive threatened felids, where timely, accurate diagnosis and careful management are critical for minimising retroviral transmission. We recommend using SNAP® or VetScan® Rapid FIV Ab tests for accurate diagnosis of FIV infection in African lions; however, the original VetScan® Rapid FIV test has been unavailable in Australia since 2019.

Bovine bacterial mastitis remains a major challenge for the dairy industry, and timely identification of causative bacteria is essential for effective disease management. This study compared six regions of the 16S rRNA gene for PCR high-resolution melt (HRM) curve analysis to identify the most suitable target for species-level differentiation of mastitis-associated bacteria. A total of 547 clinical bovine milk samples from cows with clinical mastitis were cultured, and representative isolates of 18 bacterial species were selected for comparative analysis. Species identity was confirmed using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) prior to molecular analysis. All six primer pair combinations were tested across the 18 species panel, and discriminatory performance was assessed using a genotype confidence percentage (GCP)-based classification model that allowed for objective interpretation without visual inspection. Among the six target regions, the 203 bp amplicon generated by primer pair F1R1, spanning the V1-to-V2 region and part of V3, showed the strongest overall discriminatory performance with no cross-classification observed. Sequencing of F1R1 amplicons confirmed species identity and showed concordance with HRM-based classification. Phylogenetic analysis of sequences from this region showed clustering broadly consistent with established taxonomic relationships. Proof-of-concept testing showed that the workflow could also be applied to DNA extracted directly from selected milk samples without prior bacterial isolation. Overall, this approach provides a rapid, cost-effective, and high-throughput method for preliminary discrimination of mastitis-associated bacteria.

Bovine mastitis caused by Streptococcus uberis (S. uberis) remains a major challenge of dairy production system worldwide. This study aimed to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay and a conventional PCR assay targeting the 16S rRNA gene for detection of S. uberis in bovine milk. Both assays were evaluated with a simplified HotSHOT (HS) DNA preparation method that can be completed in under 10 min and was compared with a commercial DNA extraction kit before LAMP and PCR testing. LAMP reactions using HS-extracted DNA required an extended incubation time of 30 min, compared with the standard 60 min, to account for the crude DNA preparation. Using tenfold serial dilutions of purified DNA in nuclease-free water, the limit of detection was 1.84 × 10⁻⁴ ng/µL for LAMP and 1.84 × 10⁻⁵ ng/µL for PCR. In pasteurised milk spiked with S. uberis DNA and extracted by HS, the limit of detection was 10 ng/mL for LAMP and 1 ng/mL for PCR. Using the commercial kit, both methods achieved 1 ng/mL. Among 17 culture-confirmed clinical milk samples extracted by HS, LAMP detected S. uberis in 14 samples (82.35%; 95% CI: 0.56 to 0.96), while PCR detected 15 samples (88.24%; 95% CI: 0.64 to 0.99), with no significant difference between methods (McNemar’s test, p > 0.05). Both LAMP and PCR showed 100% analytical specificity against 10 non-target bacterial species under the conditions tested. These findings support HS combined with colorimetric LAMP as a practical diagnostic workflow that warrants further validation under field conditions.

Objectives: Vaccines have traditionally been administered to cats into the interscapular subcutaneous region (‘scruff’) for ease of restraining the cat while the vaccine is injected. Concern about feline injection-site sarcomas (FISSs) has prompted recommendations to use distal sites such as the hindlimb or tail, but evidence of adequate immune response at these sites is limited. This field study evaluated the antibody response to feline panleukopaenia virus (FPV) in kittens vaccinated with one of two commercial vaccines, injected at different sites. Methods: An inactivated (killed), adjuvanted pentavalent vaccine (Fel-O-Vax 5; Boehringer Ingelheim Animal Health Australia) was injected subcutaneously into the scruff, left distal hindlimb or proximal tail. A modified-live virus (attenuated [MLV]), non-adjuvanted trivalent vaccine (Feligen RCP; Virbac Animal Health Australia) was given into the scruff only. For the latter, an inactivated, adjuvanted monovalent feline leukaemia virus vaccine was administered concurrently into the contralateral scruff, left distal hindlimb or proximal tail. Only one FPV-containing vaccine was administered to each cat/kitten: either the inactivated vaccine given in one of the three different anatomical regions or the MLV vaccine given into the scruff only. Cats were re-vaccinated in the same site and using the same vaccine type. Kittens were randomly allocated into study groups according to order of recruitment. Kittens were sampled at four time points over 12 months (T0, T1, T2 and T12). Protective FPV antibody titres were determined by haemagglutination inhibition (HI; negative P = 0.006) and T12 ( P = 0.001). However, the proportion of kittens protected in each group (MLV vaccine vs inactivated vaccine) was the same. For most kittens (91%), a single vaccination was sufficient to produce protective antibody levels, irrespective of whether they were given the MLV (scruff) or inactivated vaccine (at any site). Age and sex had no effect on antibody titres after vaccination. Two kittens (2%) failed to achieve protective titres after two doses of vaccine and required re-vaccination between 16 and 18 weeks. Inexplicably, one (1%) adult cat lacked a protective antibody titre response at T12 despite earlier seroconversion. Conclusions and relevance: Both inactivated and MLV vaccines induce protective titres against FPV. Although most kittens aged above 7 weeks are protected 1 month after a single vaccination, some require re-vaccination to develop protection. Some kittens require a final vaccination at 16 weeks or older and a booster 6–12 months later to develop robust protective titres. Comparable FPV antibody responses are elicited when inactivated vaccines are administered into the scruff, left distal hindlimb or proximal tail. Notably in this study, the MLV vaccine tended to produce higher antibody titres. These results support evidence-based recommendations on vaccination site selection to mitigate FISS risk in jurisdictions where this condition is prevalent.

Objectives: This study aimed to investigate long-term (up to 6 years) post-adoption outcomes in rehomed shelter cats with confirmed feline coronavirus (FCoV) infection, including the development of feline infectious peritonitis (FIP) and gastrointestinal diseases. Methods: A cohort study was performed with a population of adopted cats from a shelter in Sydney, Australia. FCoV infection status was determined within 48 hours of shelter admission by faecal viral nucleic acid detection. Long-term health outcomes were determined via an online survey distributed by the shelter to 2641 cat owners two to six years after adoption. The relationship between FCoV infection and health outcomes was analysed using logistic regression analysis, including calculation of odds ratios for the occurrence and frequency of health outcomes based on the cat’s infection status. Results: A total of 882 cat owners responded to the survey (response rate = 34%). FCoV faecal test results were available for 767/882 (87%) cats with corresponding completed survey responses. The prevalence of FCoV infection in this response group was 20%. Two of 767 (0.3%) adopted cats were ultimately diagnosed with FIP after rehoming. Of the two adopted cats diagnosed with FIP, one had a negative FCoV result on shelter entry (0.2%, 1/611), and one cat had a positive result (0.6%, 1/156). Vomiting (68%, 486/719) and diarrhoea (39%, 262/680) were commonly observed in the adopted cats during the study period. Conclusions and relevance: There was no association between FCoV infection upon initial testing and subsequent long-term development of FIP or other post-adoption health conditions. The estimated incidence of FIP 2-6 years after confirmed FCoV infection (0.6%) was lower than previously reported.

Bovine bacterial mastitis is an inflammation of cows’ udders by bacterial infection, leading to significant economic, animal welfare and public health consequences. Conventional diagnostic tests are expensive, time‐consuming, and require specialised laboratories, making them unsuitable for on‐farm surveillance and timely interventions. As an alternative, loop‐mediated isothermal amplification (LAMP) has gained attention as a rapid, cost‐effective and sensitive molecular diagnostic. LAMP amplifies DNA at a constant temperature, offering a simple and efficient method for bacterial detection. This review compiles studies on LAMP assays developed to detect major mastitis‐causing bacteria. Thus, a total of 18 LAMP assays were identified using PRISMA guidelines. Colourimetric and turbidity‐based LAMP assays were most frequently used. Staphylococcus aureus , Streptococcus agalactiae , Streptococcus uberis and Escherichia coli were the most common bacteria detected. While LAMP demonstrates high sensitivity and specificity in laboratory settings, its efficacy in field conditions remains uncharacterised. Nevertheless, the simplicity of sample preparation, resistance to inhibitors, short turnaround time, ease of running and simple‐to‐interpret results make LAMP a promising method for on‐farm diagnosis. Overall, this review highlights the current state of LAMP developed for bovine mastitis and identifies gaps for future studies.