Risk factors associated with self-reported Q fever in Australian wildlife rehabilitators: Findings from an online survey.

Australian wildlife rehabilitators (AWR) are at increased risk of developing Q fever, a serious zoonotic disease caused by the intracellular bacterium Coxiella burnetii. Previous studies have suggested that Australian wildlife may be a potential C. burnetii infection source for humans. However, a recent serological survey of AWR found no association between C. burnetii exposure and direct contact with any wildlife species. To further explore the potential risk that wildlife may pose, this study aimed to identify associations between self-reported Q fever in AWR and risk factors for exposure to C. burnetii. An online cross-sectional survey was implemented in 2018 targeting AWR nationwide. Risk factors for self-reported Q fever were determined using multivariable logistic regression. Medically diagnosed Q fever was self-reported in 4.5% (13/287) of unvaccinated respondents. Rehabilitators who self-reported medically diagnosed Q fever were significantly more likely to: primarily rehabilitate wildlife at a veterinary clinic (OR 17.87, 95% CI: 3.09-110.92), have domestic ruminants residing on the property where they rehabilitate wildlife (OR 11.75, 95% CI: 2.91-57.42), have been educated at a High School/Technical and Further Education level (OR 10.29, 95% CI: 2.13-84.03) and be aged >50 years (OR 6.61, 95% CI: 1.60-38.35). No association was found between self-reported Q fever and direct contact with wildlife. These findings support previous work suggesting that AWR are at increased risk of C. burnetii infection and may develop Q fever potentially via exposure to traditional infection sources including livestock, other domestic animals, or contaminated environments, in association with their rehabilitation practices and lifestyle. Although Q fever vaccination is recommended for AWR, vaccine uptake is low in this population. Future studies should aim to determine the level of Q fever awareness and identify barriers to Q fever vaccination in this at-risk group. The difficulty in accessing the AWR population also highlights the need for a national centralized AWR database.

Factors associated with Q fever vaccination in Australian wildlife rehabilitators.

Australian wildlife rehabilitators (AWR) are at risk of contracting Q fever, a serious zoonotic disease caused by Coxiella burnetii. Despite Australian government recommendations for AWR to receive Q fever vaccination (QFV), and the availability of a safe and effective vaccine in Australia, shortfalls in vaccine uptake have been observed in AWR. This study aimed to determine factors associated with QFV status and describe AWR attitudes and potential barriers towards QFV. Data were obtained from a nationwide, online, cross-sectional survey of AWR undertaken in 2018. Approximately-three quarters (200/265; 75.5 %) of those that had heard of Q fever were also aware of the Q fever vaccine, and of those, 25.5 % (51/200) were vaccinated. Barriers to QFV, among unvaccinated respondents who had also heard of Q fever and the vaccine (149/200; 74.5 %), included concerns regarding the safety, efficacy, and importance of the Q fever vaccine. Complacency toward vaccination, convenience of vaccination, and a lack of Q fever knowledge were also notable barriers. Only 27.7 % (41/148) of respondents reported having had vaccination recommended to them. Multivariable logistic regression identified that vaccinated AWR were more likely to be aged ≤ 50 years (OR 4.51, 95 % CI: 2.14-10.11), have had a university level education (OR 2.78, 95 % CI: 1.39-5.73), have resided in New South Wales/Australian Capital Territory and Queensland than in other Australian jurisdictions (OR 2.9, 95 % CI: 1.10-8.83 and OR 4.82, 95 % CI: 1.64-16.00 respectively) and have attended an animal birth (OR 2.14, 95 % CI: 1.02-4.73). Knowledge gaps regarding Q fever and QFV in AWR demonstrated the need for interventions to raise the awareness of the potential health consequences of C. burnetii exposure and Q fever prevention. Education programs to allow AWR to develop an informed perspective of Q fever and QFV, coupled with improvements in vaccine affordability and the implementation of programs to enhance accessibility, may also increase vaccine uptake.

Evaluation of three immunological assays to mitigate the risk of transboundary spread of Coxiella burnetii by alpacas.

Coxiella burnetii causes coxiellosis in animals and Q fever in humans, a potentially debilitating zoonotic disease commonly transmitted through domestic ruminants. To prevent transboundary spread of C. burnetii, animals may be tested prior to export. In alpacas, this process is complicated by the lack of scientific evidence for C. burnetii infection in the species, and the unique composition of camelid antibodies, which may cause false-positive results in assays developed for ruminants. We evaluated a complement fixation test (CFT; currently recommended for alpacas in New Zealand), an enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence assay (IFA). Positive analytical control samples were generated through vaccination of alpacas with a human Q fever vaccine, whereas negative analytical control samples were sourced from New Zealand (deemed free of C. burnetii). Immunological assays were conducted on 131 alpaca sera submitted for export testing. Test characteristics (sensitivity, specificity, positive and negative predictive values) for CFT, ELISA and IFA were determined using Bayesian latent class analysis. Due to anticomplementary activity, 37 (28.2%) of the CFT results were inconclusive, making CFT unsuitable for routine use. Of the remaining 94 samples, 10.6%, 0% and 7.4% were positive for C. burnetii antibodies based on CFT, ELISA and IFA, respectively, yielding estimated sensitivities of 58%, 26% and 78%, and estimated specificities of 95%, 98% and 95%, with the estimates for sensitivity being imprecise, as evidenced by wide 95% credible intervals. Positive predictive values were similar across assays, albeit very low at the estimated seroprevalence of 5%. Our results indicate that, of the tests available, IFA appears to be the most appropriate for use in alpacas. Higher sensitivity of antibody detection, use of antigen detection assays and availability of samples from individuals with evidence of infection could provide additional insight into the risk of transboundary spread of C. burnetii by alpacas.

Renal Crest Proliferative Lesions in Cats with Chronic Kidney Disease.

In a histopathological study of the renal crest (RC) of kidneys of cats with chronic kidney disease (CKD), 58/90 (64%) had epithelial proliferation. Of these, 33 cats had hyperplasia of the collecting duct (CD) epithelium (CDH) alone, eight had hyperplasia of the urothelium covering the RC (RCUH), of which one had concurrent abaxial renal pelvic urothelial hyperplasia (UH), and eight had both CDH and RCUH. CDH or RCUH were present in five cats with marked dysplasia of the CD epithelium (CDD) and four cats with invasive carcinomas, which also had epithelial dysplasia. All nine cats with marked dysplasia or neoplasia of the RC also had substantially altered RC contours due to focal haemorrhage, papillary necrosis or fibrosis. Three of the carcinomas had a strong desmoplastic response. In control cats, both urothelial (RC and renal pelvis) and tubular (CD and distal tubular) cells were immunopositive for cytokeratin (CK; AE1/AE3), tubular epithelial cells were positive for vimentin (Vim) and aquaporin 2 (Aq2), while urothelial cells were positive for p63. PAX8 immunolabelling was difficult to validate. CD and UH labelling was similar to control tissue. While urothelial dysplasia had the same immunolabelling pattern as UH and control tissue, CDD was generally immunonegative for Aq2. As immunolabelling of the four carcinomas did not distinguish between tubular and urothelial origin, with three positive for both Vim and p63, all were broadly designated as RC carcinomas. Overall, proliferative epithelial lesions are common in cats with CKD and form a continuum from simple hyperplasia to neoplasia of the urothelium or CD of the RC.

Serological Evidence of Exposure to Spotted Fever Group and Typhus Group Rickettsiae in Australian Wildlife Rehabilitators.

Rickettsioses are arthropod-borne zoonotic diseases, several of which occur in Australia. This study aimed to assess the exposure levels and risk factors for Rickettsia spp. among Australian wildlife rehabilitators (AWRs) using serology, PCR and a questionnaire. Antibody titres against Spotted Fever Group (SFG), Typhus Group (TG) and Scrub Typhus Group (STG) antigens were determined using an immunofluorescence assay. PCR targeting the gltA gene was performed on DNA extracts from whole blood and serum. Logistic regression was used to identify risk factors associated with seropositivity. Of the 27 (22.1%; 27/122) seropositive participants all were seropositive for SFG, with 5/27 (4.1%) also positive for TG. Of the 27 positive sera, 14.8% (4/27) were further classified as exposure to R. australis, 3.7% (1/27) to R. honei, 3.7% (1/27) to R. felis and 77.8% (21/27) were classified as 'indeterminate'-most of which (85.7%; 18/21) were indeterminate R. australis/R. honei exposures. Rickettsia DNA was not detected in whole blood or serum. Rehabilitators were more likely to be seropositive if more than one household member rehabilitated wildlife, were older than 50 years or had occupational animal contact. These findings suggest that AWRs are at increased risk of contracting Rickettsia-related illnesses, however the source of the increased seropositivity remains unclear.

Coxiella burnetii seroprevalence and Q fever in Australian wildlife rehabilitators.

Coxiella burnetii is the causative bacterium of the zoonotic disease Q fever, which is recognised as a public health concern globally. Macropods have been suggested as a potential source of C. burnetii infection for humans. The aim of this cross-sectional study was to determine the prevalence of C. burnetii exposure in a cohort of Australian wildlife rehabilitators (AWRs) and assess Q fever disease and vaccination status within this population. Blood samples were collected from adult participants attending the Australian Wildlife Rehabilitation Conference in Sydney in July 2018. Participants completed a questionnaire at the time of blood collection. Antibody titres (IgG, IgA and IgM) against phase I and phase II C. burnetii antigens as determined by immunofluorescence assay, revealed that of the unvaccinated participants, 6.1% (9/147) had evidence of exposure to C. burnetii. Of the total participants, 8.1% (13/160) had received Q fever vaccination, four of whom remained seropositive at the time of blood collection. Participants reporting occupational contact with ruminants, were eight times more likely to have been vaccinated against Q fever, than those reporting no occupational animal contact (OR 8.1; 95% CI 1.85-45.08). Three AWRs (2%) reported having had medically diagnosed Q fever, two of whom remained seropositive at the time of blood collection. Despite the lack of association between macropod contacts and C. burnetii seropositivity in this cohort, these findings suggest that AWRs are approximately twice as likely to be exposed to C. burnetii, compared with the general Australian population. This provides support for the recommendation of Q fever vaccination for this potentially 'at-risk' population. The role of macropods in human Q fever disease remains unclear, and further research into C. burnetii infection in macropods including: infection rate and transmission cycles between vectors, macropods as reservoirs, other animals and humans is required.

Cat fleas (Ctenocephalides felis clade 'Sydney') are dominant fleas on dogs and cats in New South Wales, Australia: Presence of flea-borne Rickettsia felis, Bartonella spp. but absence of Coxiella burnetii DNA.

The cat flea (Ctenocephalides felis) is the most common flea species parasitising both domestic cats and dogs globally. Fleas are known vectors of zoonotic pathogens such as vector-borne Rickettsia spp. and Bartonella spp. and could theoretically transmit Coxiella burnetii, the causative agent of Q fever. A total of 107 fleas were collected from 21 cats and 14 dogs in veterinary clinics, a feline rescue organisation and a grooming salon in New South Wales, Australia, to undergo PCR detection of Bartonella spp., Rickettsia spp. and C. burnetii DNA. Morphological identification confirmed that the cat flea (C. felis) is the most common flea in New South Wales, Australia, with only a single stick fast flea, Echidnophaga gallinacea recorded. The examined fleas (n = 35) at the cox1 locus revealed five closely related C. felis haplotypes (inter-haplotype distance < 0.5%). Multiplex TaqMan qPCR targeting the gltA (Rickettsia spp.) and ssrA (Bartonella spp.) genes was positive in 22.9% (95% CI: 11.8-39.3%) and 11.4% (95% CI: 3.9-26.6%) of samples, respectively. None of the DNA isolated from fleas was positive on TaqMan qPCRs targeting the C. burnetii IS1111, Com1 and htpAB genes. Co-infection of C. felis with Bartonella henselae and Bartonella clarridgeiae was demonstrated using gltA and ssrA Illumina next-generation amplicon sequencing. These findings reinforce the importance of flea control on domestic dogs and cats to effectively control the transmission of Rickettsia felis and Bartonella spp. The flea, however, is unlikely to be a vector of C. burnetii between companion animals and humans.

Molecular detection of Coxiella burnetii in raw meat intended for pet consumption.

The discovery of antibodies against Coxiella burnetii in cattery-confined breeding cats indicating prior or current exposure (Shapiro et al., 2015) prompted an investigation into possible sources of infection. One hypothesis was that raw meat diets containing reservoir species may provide a source of C. burnetii transmission. The aim of this pilot study was to determine whether C. burnetii DNA was present in raw meat sold exclusively for companion animal consumption. The sample population consisted of raw meat packages (n = 58) of primarily kangaroo origin, with three to four aliquots (50-120 mg) randomly selected from each package. Genomic DNA was extracted from whole tissue in each of these aliquots using a modified protocol. Three quantitative PCR assays were used for the detection of C. burnetii targeting the IS1111 gene, the heat shock operon htpAB and the C. burnetii outer membrane protein-coding gene, com1. Coxiella burnetii DNA was detected in 25/58 samples (43%) using the IS1111, htpAB and/or com1 PCR assays and confirmed by DNA sequencing. All samples amplifying a product in the com1 assay also amplified a product in the htpAB and IS1111 assays. A total of 17/58 (29%) packets were positive with all three genes, 4/58 (7%) were positive with two genes (IS1111 and htpAB) and 4/58 (7%) were positive with the IS1111 gene only. Coxiella burnetii DNA was five times more likely to be found in offal than skeletal muscle meat samples. All meat samples in which C. burnetii DNA was found were from kangaroo tissues, while samples labelled as non-kangaroo meat (n = 4) were negative. Multi-locus variable number of tandem repeat analysis (MLVA) identified three different genotypes of C. burnetii that have all been identified previously from Australian human clinical Q fever cases. Further investigations are required to determine the potential role of certain raw meats in the transmission of C. burnetii to cats and humans.

New insights on the epidemiology of Coxiella burnetii in pet dogs and cats from New South Wales, Australia.

Q fever is considered one of the most important zoonoses in Australia. Whilst ruminants are the primary reservoirs for Coxiella burnetii, and the major source of human infection, human cases have also been reported following contact with pet dogs and cats. This study aimed to estimate the prevalence of seropositivity to, and bacterial shedding of, C. burnetii by pet dogs and cats in a region with a high human Q fever incidence and explore risk factors for C. burnetii exposure. Samples (serum, whole blood, reproductive tissue, reproductive swabs) and questionnaires (completed by the pet's owner) were collected from dogs and cats from eight communities across remote New South Wales (NSW), Australia. Overall 86/330 dogs (26.1%, 95% CI 21.3-30.8%) and 19/145 cats (13.1%, 95% CI 7.6-18.6%) were seropositive to C. burnetii. Seroprevalence varied significantly between communities and was highest in communities within 150 km of a 2015 human Q fever outbreak. Feeding raw kangaroo was identified as a risk factor for seropositivity (adjusted OR 3.37, 95% CI 1.21-9.43). Coxiella burnetii DNA was not detected from any dog or cat whole blood, reproductive tissue or vaginal/preputial swab using qPCR targeting the IS1111 and com1 genes. Our findings suggest that companion animals are frequently exposed to C. burnetii in western NSW. Geographical variation in C. burnetii seroprevalence amongst companion animals - which corresponds with a human Q fever outbreak - suggests a shared environmental source of infection is likely with important consequences for public and animal health. The lack of detection of C. burnetii DNA from healthy companion animals suggests that pet dogs and cats are not an important reservoir for human Q fever infection outside a narrow periparturient window.

Coxiella burnetii seroprevalence in unvaccinated veterinary workers in Australia: Evidence to support Q fever vaccination.

Q fever (caused by Coxiella burnetii) is a serious zoonotic disease that occurs almost worldwide. Occupational contact with animals increases the risk of exposure, and Q fever vaccination is recommended for veterinary workers in Australia. This study aimed to investigate C. burnetii seroprevalence among unvaccinated veterinary workers in Australia and determine factors associated with a positive serological result. During 2014 and 2015, convenience sampling at veterinary conferences and workplace vaccination clinics was undertaken. Participants completed a questionnaire and provided a blood sample for C. burnetii serology. Participants were predominantly veterinarians (77%), but veterinary support staff, animal scientists, and administration workers also participated. Blood samples (n = 192) were analysed by an immunofluorescence assay and considered positive where the phase I or phase II IgG titre was ≥1/50. Seroprevalence was 19% (36/192; 95% CI 14%-25%). A positive serological result was significantly associated with (a) working in outer regional/remote areas (odds ratio [OR] 6.2; 95% CI 1.9-20.8; reference = major cities; p = .009) and (b) having spent more than 50% of total career working with ruminants (OR 4.8; 95% CI 1.7-13.5; reference = <15% of career; p = .025). These findings confirm an increased risk of exposure to C. burnetii compared to the general population, providing new evidence to support Q fever vaccination of veterinary workers in Australia.

Validation of an indirect immunofluorescence assay (IFA) for the detection of IgG antibodies against Coxiella burnetii in bovine serum.

There is limited knowledge of the true prevalence and distribution of coxiellosis in dairy and beef cattle populations in Australia. For this to occur, apparent prevalence estimates need to be reliably adjusted, accounting for diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the test used. However, there are few tests available with known diagnostic specifications suitable to inform screening and surveillance activities in the Australian context. We initially modified and optimised a human indirect immunofluorescence assay (IFA) test for the detection of IgG antibodies against phase I and/or phase II Coxiella burnetii in bovine sera and determined an optimal screening dilution cut-off to be 1:160. Direct comparison of the modified IFA with the commercial IDEXX enzyme-linked immunosorbent assay (ELISA) kit (Q Fever Ab Test IDEXX Laboratories, United States of America) was performed by testing 458 serum samples from four distinct cattle populations across the east coast of Australia and New Zealand. Cross classified test results were then analysed using Bayesian latent class modelling, to validate the tests in the absence of a gold standard reference test. Results from this analysis indicate that the IFA, at a 1:160 serum dilution, has an estimated DSe of 73.6% (95% Credible Interval (CrI) 61.1, 85.9) and DSp of 98.2% (95% CrI 95.1, 99.7). The commercial IDEXX ELISA kit was found to have a higher DSe of 87.9% (95% CrI 73.9, 96.4) and similar DSp of 97.7% (95% CrI 93.2, 99.7). Evaluation of the diagnostic performance of the IFA and ELISA methods, specifically for use in cattle will enable more accurate interpretation of prevalence estimates of C. burnetii exposure to be reported for cattle in Australia and other countries.

Frequency of Adverse Events Following Q Fever Immunisation in Young Adults.

Q fever is a zoonosis of concern in many countries. Vaccination is the most effective means of prevention, and since 1989, Australia has had a licensed Q fever vaccine, Q-VAX®. This vaccine was also used in the Netherlands in 2011 following the largest recorded Q fever outbreak globally. There is a paucity of available data regarding adverse events following immunisation (AEFI) for young adult females. Such data are important for informing future vaccination recommendations both within Australia and internationally. This study collected Q fever vaccine (Q-VAX®) AEFI data in veterinary and animal science students at Australian universities. Students were enrolled at the time of vaccination and were emailed a link to an online AEFI survey one week later. Of the 60% (499/827) that responded, 85% were female and the median age was 18 years. Local injection site reactions (ISRs) occurred in 98% (95%; CI 96⁻99%) of respondents, of which 30% (95% CI 24⁻32%) were severe. Systemic AEFI occurred in 60% (95%; CI 55⁻64%) of respondents within the seven days following immunisation. Medical attention was sought by 19/499 (3.8%) respondents, of whom one sought treatment at a hospital emergency department. Females were more likely than males to experience any local ISR (odds ratio [OR] 9.3; 95% CI 2.5⁻33.8; p < 0.001), ISRs of greater severity (OR 2.5; 95% CI 1.5⁻4.2; p < 0.001), and any systemic AEFI (OR 1.9; 95% CI 1.1⁻3.1; p = 0.016). These safety data suggest that a high frequency of adverse events following immunisation should be expected in young adults, particularly females. However, the consequences of Q fever disease are potentially far more debilitating.

Willingness of veterinarians in Australia to recommend Q fever vaccination in veterinary personnel: Implications for workplace health and safety compliance.

Q fever vaccine uptake among veterinary nurses in Australia is low, suggesting veterinarians are not recommending the vaccination to veterinary personnel. This study aimed to determine the willingness of veterinarians to recommend Q fever vaccination to veterinary personnel and to identify factors influencing Q fever vaccine uptake by veterinary nurses in Australia. An online cross sectional survey targeted veterinarians and veterinary nurses in Australia in 2014. Responses were analysed using multivariable logistic regression. Factors significantly (p<0.05) associated with a willingness to recommend the vaccination, expressed by 35% (95% CI 31-38%) of veterinarians (n = 828), were (1) being very concerned for colleagues regarding Coxiella burnetii (OR 4.73), (2) disagreeing the vaccine is harmful (OR 3.80), (3) high Q fever knowledge (OR 2.27), (4) working within small animal practice (OR 1.67), (5) disagreeing the vaccine is expensive (OR 1.55), and (6) age, with veterinarians under 39 years most likely to recommend vaccination. Of the veterinary nursing cohort who reported a known Q fever vaccination status (n = 688), 29% (95% CI 26-33%) had sought vaccination. This was significantly (p<0.05) associated with (1) agreeing the vaccine is important (OR 8.34), (2) moderate/high Q fever knowledge (OR 5.51), (3) working in Queensland (OR 4.00), (4) working within livestock/mixed animal practice (OR 3.24), (5) disagreeing the vaccine is expensive (OR 1.86), (6) strong reliance on work culture for biosecurity information (OR 2.5), (7) perceiving personal exposure to Coxiella burnetii to be at least low/moderate (OR 2.14), and (8) both agreeing the vaccine is safe and working within a corporate practice structure (OR 4.28). The study identified the need for veterinarians to take greater responsibility for workplace health and safety promotion, and calls for better education of veterinary personnel to raise awareness of the potential for occupational exposure to C. burnetii and improve the perception of the Q fever vaccine as being important, safe and cost-effective.

One health in our backyard: Design and evaluation of an experiential learning experience for veterinary medical students.

BACKGROUND: New educational approaches are needed to improve student understanding of the wider sociological and ecological determinants of health as well as professional responsibilities in related areas. Field trips allow students to observe interaction between plant, animal and human communities, making them an ideal tool for teaching One Health concepts. METHODS: Veterinary medical students participated in a field trip to a local parklands area, frequented by humans, dogs, horses, and wildlife. Students rotated through 5 learning activities ('stations') that focused on: (1) response to exotic animal disease incursion (equine influenza); (2) impact of cultures and belief systems on professional practice; (3) management of dangerous dogs; (4) land use change, biodiversity and emerging infectious disease; and (5) management of environmentally-acquired zoonoses (botulism). Intended learning outcomes were for students to: evaluate the various roles and responsibilities of veterinarians in society; compare the benefits and risks associated with human-animal and animal-animal interactions; and evaluate the contributions made by various professionals in safeguarding the health and welfare of animals, humans and the environment. Following the field trip, students participated in a debrief exercise and completed an online survey on their experiences. RESULTS: Feedback from students collected in 2016/2017 (n = 211) was overwhelmingly positive. The learning experience at each station was rated as 4 ('Good') or 5 ('Very Good') out of 5 by 82-96% of students. Responses to closed- and open-ended questions - as well as outputs generated in the debrief session - indicated that students achieved the learning outcomes. Overall, 94% of students agreed or strongly agreed that they had a better understanding of One Health because of the field trip. CONCLUSIONS: Field trips to local parklands are effective in promoting learning about One Health and can be incorporated into the core curriculum to maximize student exposure at relatively low cost.

Q fever: A rural disease with potential urban consequences

BACKGROUND: Q fever often presents as an undifferentiated febrile illness. Cases occur throughout Australia, with higher rates occurring in northern New South Wales and southern Queensland. OBJECTIVE: This article aims to provide clinicians with an overview of Q fever, and covers epidemiology, clinical features, laboratory diagnosis, sequelae, management and prevention. DISCUSSION: In Australia, Q fever is the most commonly reported zoonotic disease. Presentation includes fever, rigors, chills, headache, extreme fatigue, drenching sweats, weight loss, arthralgia and myalgia, often in conjunction with abnormal liver function tests. These features make it indistinguishable from many other febrile illnesses. Exposure occurs through contact with livestock and other animals. Coxiella bacteria can survive in dust, where infection may result from inhalation. Laboratory diagnosis is made by serology or polymerase chain reaction. An effective vaccine is available for adults (aged >15 years), but can only be administered after a rigorous pre-vaccination assessment to exclude prior exposure to Coxiella burnetii, requiring a detailed medical history, skin test and serology.

A review of mycoplasma diagnostics in cattle.

Mycoplasma species have a global distribution causing serious diseases in cattle worldwide including mastitis, arthritis, pneumonia, otitis media and reproductive disorders. Mycoplasma species are typically highly contagious, are capable of causing severe disease, and are difficult infections to resolve requiring rapid and accurate diagnosis to prevent and control disease outbreaks. This review discusses the development and use of different diagnostic methods to identify Mycoplasma species relevant to cattle, with a particular focus on Mycoplasma bovis. Traditionally, the identification and diagnosis of mycoplasma has been performed via microbial culture. More recently, the use of polymerase chain reaction to detect Mycoplasma species from various bovine samples has increased. Polymerase chain reaction has a higher efficiency, specificity, and sensitivity for laboratory diagnosis when compared with conventional culture-based methods. Several tools are now available for typing Mycoplasma spp. isolates, allowing for genetic characterization in disease outbreak investigations. Serological diagnosis through the use of indirect ELISA allows the detection of antimycoplasma antibodies in sera and milk, with their use demonstrated on individual animal samples as well as BTM samples. While each testing method has strengths and limitations, their combined use provides complementary information, which when interpreted in conjunction with clinical signs and herd history, facilitates pathogen detection, and characterization of the disease status of cattle populations.

Vector-borne and zoonotic diseases of dogs in North-west New South Wales and the Northern Territory, Australia.

BACKGROUND: Vector-borne diseases of dogs in Australian Aboriginal communities are relatively unexplored. These dogs represent a unique group with variable ecto- and endo-parasitic burdens, nutritional stresses and a general lack of veterinary intervention. We investigated haemoprotozoal and bacterial pathogen prevalences in relation to erythrocyte and platelet numbers in dogs from North-West New South Wales (N-W NSW) and the Northern Territory (NT; Central Australia). METHODS: Real-time PCR (qPCR) amplification of Anaplasma platys, Babesia vogeli, Mycoplasma haemocanis, Candidatus Mycoplasma haematoparvum and Bartonella spp., serological screening for Coxiella burnetii, and Bartonella spp. and haematological analyses were performed on dogs from the two cohorts (96 dogs in total). Brucella suis serology was determined additionally for the N-W NSW cohort. RESULTS: Anaplasma platys (n = 26 dogs), Babesia vogeli (n = 7), Candidatus Mycoplasma haematoparvum (n = 10 dogs), and Mycoplasma haemocanis (n = 14) were detected in the sample population (n = 96) using qPCR. There were significant associations between (i) A. platys and anaemia (OR 8.7, CI 2.4-31.7; P < 0.001), thrombocytopenia (OR 12.1, CI 3.4-43.2; P < 0.001) and breed (OR 16.1, CI 2.1-121.5; P = 0.007), and (ii) between B. vogeli and anaemia (OR 11.8, CI 2.3-61.6; P = 0.003). Neither protozoal nor bacterial DNA loads, estimated using qPCR, were positively correlated with anaemia or thrombocytopenia. Haemotropic mycoplasmas were not associated with any haematologic abnormality. Four dogs from the NT were seropositive for Coxiella burnetii, while no dogs were seropositive for Brucella suis or to a panel of Bartonella spp. antigens. Despite directed efforts, Bartonella DNA was not detected in blood from any of the cohorts studied. A sample of dogs from the NT recruited specifically for Bartonella α-proteobacteria growth medium enrichment blood culture were also Bartonella PCR negative. CONCLUSIONS: Vector-borne pathogens occur in dogs free ranging near Aboriginal communities, with higher detection rates in NT than N-W NSW. The preponderant haematologic abnormalities were anaemia and thrombocytopenia, likely attributable to A. platys and B. vogeli infections, but also probably affected by nutritional, parasitic, lactational and environmental stressors. The absence of Bartonella spp. is of importance to the Australian setting, and work needs to be extended to tropical coastal communities where fleas are present as well as ticks. Dogs living in and around Aboriginal communities may provide valuable sentinel information on disease infection status of human public health significance.

Comparison of culture and a multiplex probe PCR for identifying Mycoplasma species in bovine milk, semen and swab samples.

Mycoplasma spp. are a major cause of mastitis, arthritis and pneumonia in cattle, and have been associated with reproductive disorders in cows. While culture is the traditional method of identification the use of PCR has become more common. Several investigators have developed PCR protocols to detect M. bovis in milk, yet few studies have evaluated other sample types or other important Mycoplasma species. Therefore the objective of this study was to develop a multiplex PCR assay to detect M. bovis, M. californicum and M. bovigenitalium, and evaluate its analytical performance against traditional culture of bovine milk, semen and swab samples. The PCR specificity was determined and the limit of detection evaluated in spiked milk, semen and swabs. The PCR was then compared to culture on 474 field samples from individual milk, bulk tank milk (BTM), semen and swab (vaginal, preputial, nose and eye) samples. Specificity analysis produced appropriate amplification for all M. bovis, M. californicum and M. bovigenitalium isolates. Amplification was not seen for any of the other Mollicutes or eubacterial isolates. The limit of detection of the PCR was best in milk, followed by semen and swabs. When all three Mycoplasma species were present in a sample, the limit of detection increased. When comparing culture and PCR, overall there was no significant difference in the proportion of culture and PCR positive samples. Culture could detect significantly more positive swab samples. No significant differences were identified for semen, individual milk or BTM samples. PCR identified five samples with two species present. Culture followed by 16S-23S rRNA sequencing did not enable identification of more than one species. Therefore, the superior method for identification of M. bovis, M. californicum and M. bovigenitalium may be dependent on the sample type being analysed, and whether the identification of multiple target species is required.