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.

Anatomy of the Female Koala Reproductive Tract.

The koala (Phascolarctos cinereus), while being an iconic Australian marsupial, has recently been listed as endangered. To establish an improved understanding of normal reproductive anatomy, this paper brings together unpublished research which has approached the topic from two perspectives: (1) the establishment of an artificial insemination program, and (2) the definition of Chlamydia spp.-derived histopathological changes of the female koala urogenital system. Based on the presentation and histological processing of over 70 opportunistic specimens, recovered from wildlife hospitals in Southeast Queensland (Australia), we describe the gross and microanatomy of the koala ovary, oviduct, uteri, vaginal complex, and urogenital sinus during the interestrous, proliferative, and luteal phases of the reproductive cycle.

Concurrently mapping quantitative trait loci associations from multiple subspecies within hybrid populations.

Many of the world's agriculturally important plant and animal populations consist of hybrids of subspecies. Cattle in tropical and sub-tropical regions for example, originate from two subspecies, Bos taurus indicus (Bos indicus) and Bos taurus taurus (Bos taurus). Methods to derive the underlying genetic architecture for these two subspecies are essential to develop accurate genomic predictions in these hybrid populations. We propose a novel method to achieve this. First, we use haplotypes to assign SNP alleles to ancestral subspecies of origin in a multi-breed and multi-subspecies population. Then we use a BayesR framework to allow SNP alleles originating from the different subspecies differing effects. Applying this method in a composite population of B. indicus and B. taurus hybrids, our results show that there are underlying genomic differences between the two subspecies, and these effects are not identified in multi-breed genomic evaluations that do not account for subspecies of origin effects. The method slightly improved the accuracy of genomic prediction. More significantly, by allocating SNP alleles to ancestral subspecies of origin, we were able to identify four SNP with high posterior probabilities of inclusion that have not been previously associated with cattle fertility and were close to genes associated with fertility in other species. These results show that haplotypes can be used to trace subspecies of origin through the genome of this hybrid population and, in conjunction with our novel Bayesian analysis, subspecies SNP allele allocation can be used to increase the accuracy of QTL association mapping in genetically diverse populations.

Frontline perspectives on adoption and non-adoption of care management tools for high-risk patients in primary care.

BACKGROUND: Population health management tools (PHMTs) embedded within electronic health records (EHR) could improve management of high-risk patients and reduce costs associated with potentially avoidable emergency department visits or hospitalizations. Adoption of PHMTs across the Veterans Health Administration (VA) has been variable and previous research suggests that understaffed primary care (PC) teams might not be using the tools. METHODS: We conducted a retrospective content analysis of open-text responses (n = 1804) from the VA's 2018 national primary care personnel survey to, 1) identify system-level and individual-level factors associated with why clinicians are not using the tools, and 2) to document clinicians' recommendations to improve tool adoption. RESULTS: We found three themes pertaining to low adoption and/or tool use: 1) IT burden and administrative tasks (e.g., manually mailing letters to patients), 2) staffing shortages (e.g., nurses covering multiple teams), and 3) no training or difficulty using the tools (e.g., not knowing how to access the tools or use the data). Frontline clinician recommendations included automating some tasks, reconfiguring team roles to shift administrative work away from providers and nurses, consolidating PHMTs into a centralized, easily accessible repository, and providing training. CONCLUSIONS: Healthcare system-level factors (staffing) and individual-level factors (lack of training) can limit adoption of PHMTs that could be useful for reducing costs and improving patient outcomes. Future research, including qualitative interviews with clinicians who use/don't use the tools, could help develop interventions to address barriers to adoption. IMPLICATIONS: Shifting more administrative tasks to clerical staff would free up clinician time for population health management but may not be possible for understaffed PC teams. Additionally, healthcare systems may be able to increase PHMT use by making them more easily accessible through the electronic health record and providing training in their use.

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.

Sequence level genome-wide associations for bull production and fertility traits in tropically adapted bulls.

BACKGROUND: The genetics of male fertility is complex and not fully understood. Male subfertility can adversely affect the economics of livestock production. For example, inadvertently mating bulls with poor fertility can result in reduced annual liveweight production and suboptimal husbandry management. Fertility traits, such as scrotal circumference and semen quality are commonly used to select bulls before mating and can be targeted in genomic studies. In this study, we conducted genome-wide association analyses using sequence-level data targeting seven bull production and fertility traits measured in a multi-breed population of 6,422 tropically adapted bulls. The beef bull production and fertility traits included body weight (Weight), body condition score (CS), scrotal circumference (SC), sheath score (Sheath), percentage of normal spermatozoa (PNS), percentage of spermatozoa with mid-piece abnormalities (MP) and percentage of spermatozoa with proximal droplets (PD). RESULTS: After quality control, 13,398,171 polymorphisms were tested for their associations with each trait in a mixed-model approach, fitting a multi-breed genomic relationship matrix. A Bonferroni genome-wide significance threshold of 5 × 10- 8 was imposed. This effort led to identifying genetic variants and candidate genes underpinning bull fertility and production traits. Genetic variants in Bos taurus autosome (BTA) 5 were associated with SC, Sheath, PNS, PD and MP. Whereas chromosome X was significant for SC, PNS, and PD. The traits we studied are highly polygenic and had significant results across the genome (BTA 1, 2, 4, 6, 7, 8, 11, 12, 14, 16, 18, 19, 23, 28, and 29). We also highlighted potential high-impact variants and candidate genes associated with Scrotal Circumference (SC) and Sheath Score (Sheath), which warrants further investigation in future studies. CONCLUSION: The work presented here is a step closer to identifying molecular mechanisms that underpin bull fertility and production. Our work also emphasises the importance of including the X chromosome in genomic analyses. Future research aims to investigate potential causative variants and genes in downstream analyses.

The Development of Cutaneous Lesions in Tropically Adapted Beef Cattle Is Associated with Hypersensitive Immune Response to Buffalo Fly Antigens.

This study investigated the role of cattle immune responses in the pathogenesis of buffalo fly (Haematobia irritans exigua) (BF) lesions. Brangus steers phenotyped for lesion development were divided into three groups: high lesion susceptibility (HL), low lesion susceptibility (LL) and no lesions (NL), based on lesion severity scores. Each steer was injected intradermally with different concentrations of BF, Onchocerca gibsoni (Og), and Musca domestica (Md) antigens. At 1 h post-injection, wheal areas at BF injection sites were found to be significantly larger in HL than NL cattle, but there were no significant differences (p < 0.05) found between either the HL or NL cattle and LL cattle. At 24, 48, and 72 h post-injection, the skinfold thickness response to both BF and Md antigens was significantly greater in the HL group than the NL group. However, skin thickness was significantly greater for the BF antigens than the Md antigens (p < 0.05). There were no significant differences found between the LL and NL animals in response to the BF antigens at any time, and no significant differences were determined between any of the lesion groups in response to the Og antigens. Histological examination of skin sections taken from the BF antigen injection sites in HL cattle at 72 h post-injection revealed necrosis of the epidermis and superficial dermis, along with severe eosinophilic inflammation. This study suggests that differences in the hypersensitivity to BF antigens underlie differences amongst the cattle in their susceptibility to the development of BF lesions, and breeding for immune-related biomarkers may assist in selecting more BF lesion-resistant cattle.

Development and application of a new liver pathology recording system for use in cattle abattoirs.

Liver disease in beef cattle has a significant global economic impact on feedlot and abattoir industries due to reduced animal performance, carcase yield, and carcase quality. This study aimed to create a post-mortem data collection tool which could be deployed at chain speed on an abattoir floor, as well as to evaluate pathological findings in both normal and condemned livers from an Australian beef cattle population. The first 1006 livers were used to formulate a user-friendly, high-throughput liver grading tool for use in an abattoir setting and to evaluate the histological features of common liver abnormalities. Subsequently, over 11,000 livers from a Southeast Queensland abattoir were analyzed. The most observed defects in condemned livers were liver abscessation, fibrosis, adhesions, and liver fluke, with histological features similar to previous reports. Bacterial culture was performed in 29 cases of liver abscessation, revealing a different balance of flora to that reported internationally. This study has developed an easy to use, efficient data collection tool that enables rapid, highly detailed assessment of large numbers of beef cattle livers at slaughter. This tool will allow thorough investigation into the effect of liver disease on beef production, in both industry and research contexts.

Investigation and Management of an Outbreak of Lead Intoxication in an Extensively Managed Beef Herd.

Fifteen hundred 12−15-month-old tropically adapted heifers inadvertently grazed a paddock which had a refuse dump in it containing burnt out vehicle batteries. The cattle grazed this paddock for approximately seven days. Subsequently these cattle were managed as two cohorts (cull and potential replacement breeding animals). Deaths commenced in the cull heifer group approximately 18 days after initial exposure to the refuse dump during relocation to a feedlot. Mortalities continued for 12 days, with other heifers showing clinical signs of marked central nervous system dysfunction requiring euthanasia. Necropsy of several clinically affected cattle plus blood sampling for lead analysis confirmed a diagnosis of lead intoxication. The crude mortality rate in the cull heifers was 6.6% (n = 685). Following confirmation of the diagnosis most of the potential replacement heifers (second cohort) were also relocated to the feedlot. The estimated crude mortality rate in this cohort was 5.8% (n = 815). All possible lead intoxication deaths occurred within 34 days of initial exposure, and apparently after day 16 at the feedlot no further heifers showed any clinical signs which could be attributed to lead intoxication. Longitudinal monitoring of blood lead concentrations was used to identify cattle suitable for slaughter. Overall, 70% of heifers initially blood sampled (n = 1408) had no detectable lead in their blood, however 16% had markedly elevated blood lead concentrations (> 0.7µmol/L) which persisted, and 2% had above the maximum normal threshold 1.5 years later. These latter cattle were subsequently euthanized, and necropsy revealed that visible pieces of lead were still present in the reticulum of several animals. At no time did any of these heifers with persistently high blood lead concentrations show clinical signs of lead intoxication.

Multi-breed genomic predictions and functional variants for fertility of tropical bulls.

Worldwide, most beef breeding herds are naturally mated. As such, the ability to identify and select fertile bulls is critically important for both productivity and genetic improvement. Here, we collected ten fertility-related phenotypes for 6,063 bulls from six tropically adapted breeds. Phenotypes were comprised of four bull conformation traits and six traits directly related to the quality of the bull's semen. We also generated high-density DNA genotypes for all the animals. In total, 680,758 single nucleotide polymorphism (SNP) genotypes were analyzed. The genomic correlation of the same trait observed in different breeds was positive for scrotal circumference and sheath score on most breed comparisons, but close to zero for the percentage of normal sperm, suggesting a divergent genetic background for this trait. We confirmed the importance of a breed being present in the reference population to the generation of accurate genomic estimated breeding values (GEBV) in an across-breed validation scenario. Average GEBV accuracies varied from 0.19 to 0.44 when the breed was not included in the reference population. The range improved to 0.28 to 0.59 when the breed was in the reference population. Variants associated with the gene HDAC4, six genes from the spermatogenesis-associated (SPATA) family of proteins, and 29 transcription factors were identified as candidate genes. Collectively these results enable very early in-life selection for bull fertility traits, supporting genetic improvement strategies currently taking place within tropical beef production systems. This study also improves our understanding of the molecular basis of male fertility in mammals.

Promoting Whole Health and Well-Being at Home: Veteran and Provider Perspectives on the Impact of Tele-Whole Health Services.

Background: The Veterans Health Administration (VA) is undergoing a transformation in how healthcare is organized and provided. This transformation to a Whole Health System of Care encompasses the integration of complementary and integrative health services, education, and Whole Health coaching to develop Veterans' self-care skills. During the COVID-19 pandemic, these services were provided via telehealth (tele-WH). Objective: We sought to understand Veteran and provider perspectives on how tele-WH impacts Veteran engagement in Whole Health-aligned services and the impact on their well-being. Methods: Semi-structured interviews were conducted with 51 providers who delivered tele-WH at 10 VA Medical Centers (VAMCs) and 19 Veterans receiving tele-WH at 6 VAMCs. Participants were asked about their experiences with tele-WH, what they perceived to be the impact of tele-WH on Veterans, and their preferences moving forward. Interviews were transcribed, and a content analysis was performed using a rapid approach. Results: We identified 3 major themes that describe the perceived impact of tele-WH on Veterans. These include: (1) increased use of Whole Health-aligned services; (2) deeper engagement with Whole-Health aligned services; and (3) improvements in social, psychological, and physical well-being. Conclusion: Tele-WH is perceived to be a strong complement to in-person services and is a promising mechanism for improving engagement with Whole Health-aligned services and promoting Veteran well-being. Future research is needed to measure outcomes identified in this study and to support more equitable access to telehealth for all.

A novel transdermal ketoprofen formulation for analgesia in cattle.

Ketoprofen is registered in many countries for injectable administration in cattle. Because it is soluble in a wide range of excipients, development of a novel transdermal (TD) ketoprofen formulation was pursued to provide a convenient and pain-free route of administration in cattle. One hundred and six excipient combinations were screened using in vitro techniques (Franz diffusion cells), with a 20%(w/v) ketoprofen formulation dissolved in a combination of 45%:45%(v/v) ethanol and isopropyl myristate (IPM) and 10%(v/v) eucalyptus oil achieving maximal penetration of ketoprofen through bovine skin. A bioavailability study was then conducted using a randomized cross-over design (n = 12), including IV, IM (both 3 mg/kg) and TD (10 mg/kg) ketoprofen formulations administered with a one-week washout period between administrations. The IV and IM formulation pharmacokinetic results were as expected. The CMAX , Tmax and AUC0-Last were significantly higher (arithmetic mean ± SD) after TD administration (20.0 ± 6.5 µg/ml, 115 ± 17 min and 3940 ± 1324 µg*min/ml, respectively), compared to IM (11.0 ± 4.0 µg/ml, 74 ± 43 min and 2376 ± 738 µg*min/ml, respectively), although there were no significant differences for T½ß . However, dose corrected values CMAX and AUCinf were significantly higher for IM compared to TD. The arithmetic mean bioavailability (F) of the transdermal formulation was 50%. The plasma concentration of the TD formulation at a dose of 10 mg/kg was similar to the IM formulation at 3 mg/kg by 30 min post-dosing with an arithmetic mean ± SD of 7.97 ± 4.38 vs. 8.02 ± 3.55 µg/ml, respectively. The TD formulation was generally well tolerated by cattle, although some local irritation along the site of application was noted after 12 h of exposure during the bioavailability study. Results indicate that this novel TD formulation provides a substantial improvement in administration convenience, may improve animal welfare and end-user safety through needle-free administration, and achieves similar plasma pharmacokinetics to the IM product when administered at 10 mg/kg.

Role of Staphylococcus agnetis and Staphylococcus hyicus in the Pathogenesis of Buffalo Fly Skin Lesions in Cattle.

Buffalo flies (Haematobia irritans exigua) are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds. Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas, most commonly on the medial canthus of the eye, along the lateral and ventral neck, and on the abdomen of cattle. For closely related horn flies (Haematobia irritans irritans), Staphylococcus aureus has been suggested as a contributing factor in the development of lesions. To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions, swabs were taken from lesions and normal skin, and bacteria were also isolated from surface washings of buffalo flies and surface-sterilized homogenized flies. Bacterial identification was conducted by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and strain typing by repetitive sequence-based PCR (rep-PCR) and DNA sequencing to determine species similarity and virulence factors. Of 50 bacterial isolates collected from lesions, 38 were identified as Staphylococcus agnetis and 12 as Staphylococcus hyicus, whereas four isolates from normal skin were S. hyicus and one was Mammaliicoccus sciuri. Of the Staphylococcus isolates isolated from buffalo flies, five were identified as S. agnetis and three as S. hyicus. Fifty percent of the buffalo fly isolates had rep-PCR genotypic patterns identical to those of the lesion isolates. Genome sequencing of 16 S. agnetis and four S. hyicus isolates revealed closely similar virulence factor profiles, with all isolates possessing exfoliative toxin A and C genes. The findings from this study suggest the involvement of S. agnetis and S. hyicus in buffalo fly lesion pathogenesis. This should be taken into account in the development of effective treatment and control strategies for lesions. IMPORTANCE Skin lesions in cattle associated with feeding by Haematobia fly species are a significant welfare issue in Australia, North and South America, and Europe. The development of these lesions has been attributed to a number of causal factors, but the exact etiology and pathogenesis were unclear. This study characterized Staphylococcus agnetis and Staphylococcus hyicus strains from cattle skin lesions and in vector flies and demonstrated their role in the pathogenesis of these lesions. These findings will aid the development of targeted and more effective treatment and control strategies for lesions associated with fly infestation in cattle.

Detection and distribution of Stephanofilaria sp. in buffalo flies and buffalo fly lesions in north Australian beef cattle.

Buffalo flies (Haematobia irritans exigua) are ectoparasites of major animal health and production concern in north Australian beef herds. Skin lesions associated with buffalo fly infestation, cause hide damage and welfare issues and are manifested as dermatitis or ulcerated areas found most commonly near the medial canthus of the eye, along the lateral and ventral neck and on the abdomen. Buffalo flies can transmit a nematode, Stephanofilaria sp., which has been considered the main aetiological agent for buffalo fly lesions, but the role of nematodes in the development of the lesions has not been defined. To investigate the geographical distribution of Stephanofilaria, swabs were taken from the surface of lesions and buffalo flies were netted from the backs of beef cattle from 20 properties located in northern, central and southern Queensland. Swabs and buffalo flies were then tested for the presence of Stephanofilaria by qPCR. In northern and central Queensland, all properties except one, tested positive for the presence of Stephanofilaria in either buffalo flies or swabs, or in both. The infection rate varied amongst sites ranging from 0% to 100% in lesions and 0-34% in female buffalo flies. No nematodes were found in male buffalo flies. In contrast, none of the 66 lesion swabs or 1220 buffalo flies collected from southern Queensland tested positive for Stephanofilaria infection despite the frequent occurrence of lesions in the herds from which samples were collected. These findings suggest that infection with Stephanofilaria, although frequently detected, is not essential for the development of buffalo fly lesions and other factors may contribute to the initiation of lesions. This study also confirmed the potential for using surface swabs as a quicker and less invasive means of sampling lesions than dermal biopsies when testing for the presence of Stephanofilaria by qPCR, but further studies will be required to estimate the sensitivity of this technique. Understanding the pathogenesis of buffalo fly lesions will aid the development of optimal treatment and control strategies.

The Unrecognized Role of VA Call Center and Primary Care Clerical Staff in Assisting Patients with Obtaining Needed Care.

BACKGROUND: VA clerks, or medical support assistants (MSAs), are a critical part of patients' primary care (PC) experiences and are often the first points of contact between Veterans and the healthcare system. Despite the important role they might play in assisting Veterans with accessing care, research is lacking on the specific tasks they perform and what training and preparation they receive to perform their roles. OBJECTIVE: Our primary aim in this study was to document MSA perceptions of their roles, the tasks they undertake helping Veterans with accessing healthcare, and additional training they may need to optimally perform their role. DESIGN: Thematic analysis of semi-structured qualitative interviews with VA call center and PC MSAs (n=29) collected as part of in-person site visits from August to October 2019. PARTICIPANTS: MSAs at administrative call centers and primary care clinics in one large VA regional network representing 8 healthcare systems serving nearly 1.5 million Veterans. KEY RESULTS: We identified three key findings from the interviews: (1) MSAs perform tasks in addition to scheduling that help Veterans obtain needed care; (2) MSAs may not be fully prepared for their roles as first points of contact; and (3) low status and lack of recognition of the important and complex tasks performed by MSAs contribute to high turnover. CONCLUSIONS: As healthcare systems continue expanding virtual access, the roles of administrative call center and PC MSAs as first points of contact will be increasingly important for shaping patient experiences. Our research suggests that MSAs may need better training and preparation for the roles they perform assisting Veterans with accessing care, coupled with an intentional approach by healthcare systems to address MSAs' concerns about recognition/compensation. Future research should explore the potential for enhanced MSA customer service training to improve the Veteran patient experience.

Pathology and pathogenesis of cutaneous lesions in beef cattle associated with buffalo fly infestation.

Haematobia irritans exigua, commonly known as buffalo fly, is the major hematophagous ectoparasite of north Australian cattle herds. Lesions associated with buffalo fly infestation are generally alopecic, hyperkeratotic, or scab encrusted wounds with variable hemorrhagic ulceration. Buffalo flies can transmit a filarial nematode, Stephanofilaria sp., which has been implicated in the pathogenesis of buffalo fly lesions, but Stephanofilaria infection has not been detected in all lesions suggesting that other causal factors may be involved. This study characterized the pathology of buffalo fly lesions to identify the role of Stephanofilaria in lesion development, as well as to identify other potential agents. Lesion biopsies were collected from north and south Queensland and tested for the presence of Stephanofilaria by qPCR. Each lesion was scored grossly (0-4) for hemorrhage, ulceration, exudation, and alopecia. Lesions were also scored microscopically (0-4) for epidermal and dermal damage and inflammatory characters. Stephanofilaria infection was detected in 31% of lesion biopsies. Grossly, Stephanofilaria-infected lesions had significantly larger lesion area and higher scores for alopecia and hyperkeratosis than lesions where no nematodes were found (P < 0.05). Histologically, epidermal, dermal, and adnexal damage was significantly higher in Stephanofilaria infected lesions than lesions without nematodes. Eosinophils, macrophages, and lymphocytes were significantly more abundant in Stephanofilaria positive lesions as compared to negative lesions. This study also noted bacterial infection with colonies of coccoid bacteria, observed in skin sections from 19 lesions. Grossly, lesions with bacterial infection had significantly higher ulceration scores compared to Stephanofilaria positive lesions, and histologically epidermal disruption was significantly greater in bacteria-infected lesions. We found no evidence of bacteria or Stephanofilaria infection in 49% of the lesions assessed and tissue damage patterns and eosinophilic inflammation suggested hypersensitivity to buffalo fly feeding as a possible cause of these lesions. These findings suggest that although the presence of Stephanofilaria infection may increase the severity of lesion pathology, it is not essential for lesion development. These outcomes also suggest a potential role of bacteria and hypersensitivity in pathogenesis of some lesion. A better understanding of buffalo fly lesion etiology will contribute to the optimal treatment and control programmes.

Testing of NKA expression by mobile real time PCR is an efficient indicator of smoltification status of farmed Atlantic salmon.

Assessment of seawater readiness of freshwater salmon smolts is a crucial husbandry step with economic implications in salmon aquaculture but current methods rely on delayed centralised enzymic activity measurement. The efficiency of a qRT-PCR assay for sodium potassium ATPase (NKA) α1a mRNA was tested in a 3-year study on 19 hatcheries across Scotland incorporating environmental factors such as temperature and metal contamination. The NKA qRT-PCR assay was transferred to a mobile laboratory and on-site testing was carried out at 3 hatchery sites. For the first two years standard enzymatic and gene expression assays had similar success rates in detecting smoltification (NKA activity 60%, qRT-PCR 57%). In the third year, all but one site were determined as sea water ready by qRT-PCR but only at 4 by enzymatic testing. On site testing with mobile qRT-PCR was successfully performed on four farm sites. Altogether, high sensitivity was shown for the in lab (98.9%, SE 0.24) and mobile (93.43%, SE 0.119) assays when tested using a quantitative RNA standard. Some indication for obscured smoltification assay results due to environmental increased heavy metal contamination was observed. Our results prove it is possible to test a smoltification marker on site and provide results on the day of testing during the smolt period allowing for informed decisions on seawater transfer.

Development and Validation of Novel PCR Assays for the Diagnosis of Bovine Stephanofilariasis and Detection of Stephanofilaria sp. Nematodes in Vector Flies.

BACKGROUND: Stephanofilaria spp. nematodes are associated with cutaneous lesions in cattle and other livestock and mammalian wildlife species. In Australia, Haematobia irritans exigua, commonly known as buffalo fly (BF) transmits a well-described but presently unnamed species of Stephanofilaria, which has been speculatively implicated in the aetiology of BF lesions. The sensitivity of current techniques for detecting Stephanofilaria spp. in skin lesions and vector species is low, and there is no genomic sequence for any member of the genus Stephanofilaria currently available in sequence databases. METHODS: To develop molecular assays for the detection of the Australian Stephanofilaria sp., skin biopsies were collected from freshly slaughtered cattle with typical lesions near the medial canthus. Adult nematodes and microfilariae were isolated from the biopsies using a saline recovery technique. The nematodes were morphologically identified as Stephanofilaria sp. by scanning electron microscopy. DNA was extracted and the internal transcribed spacer 2 (ITS2) region of rDNA, and the cytochrome c oxidase subunit 1 (cox1) region of mtDNA was amplified and sequenced. Stephanofilaria sp. specific polymerase chain reaction (PCR) and qPCR assays (SYBR Green® and TaqMan™) were developed and optimised from the novel ITS2 sequence obtained. The specificity of each assay was confirmed by testing against nematode species Onchocerca gibsoni and Dirofilaria immitis, as well as host (bovine) and BF DNA. RESULTS: Scanning electron microscopy of the anterior and posterior ends of isolated nematodes confirmed Stephanofilaria sp. A phylogenetic analysis of the cox1 sequence demonstrated that this species is most closely related to Thelazia callipaeda, a parasitic nematode that is a common cause of thelaziasis (or eyeworm infestation) in humans, dogs, and cats. Both conventional and qPCR assays specifically amplified DNA from Stephanofilaria sp. Conventional PCR, TaqMan™, and SYBR Green® assays were shown to detect 1 ng, 1 pg, and 100 fg of Stephanofilaria DNA, respectively. Both qPCR assays detected DNA from single Stephanofilaria microfilaria. CONCLUSION: Molecular diagnostic assays developed in this study showed high specificity and sensitivity for Stephanofilaria sp. DNA. The availability of an accurate and sensitive PCR assay for Stephanofilaria will assist in determining its role in the pathogenesis of cattle skin lesions, as well as in understanding its epidemiological dynamics. This assay may also have application for use in epidemiological studies with other species of Stephanofilaria, most particularly closely related S. stilesi, but this will require confirmation.