A cross-sectional survey of risk factors for the presence of Coxiella burnetii in Australian commercial dairy goat farms.

The largest Australian farm-based outbreak of Q fever originated from a dairy goat herd. We surveyed commercial dairy goat farms across Australia by testing bulk tank milk (BTM) samples using a commercial indirect enzyme-linked immunosorbent assay and two quantitative polymerase chain reactions (PCRs). Of the 66 commercial dairy goat herds on record, managers from 61 herds were contacted and 49 provided BTM samples. Five of the surveyed herds were positive on at least one of the diagnostic tests, thus herd-level apparent prevalence was 10% (95% confidence interval [CI] 4 to 22). True prevalence was estimated to be 3% (95% credible interval: 0 to 18). Herd managers completed a questionnaire on herd management, biosecurity and hygiene practices and risk factors were investigated using multivariable logistic regression. Herds with >900 milking does (the upper quartile) were more likely to be Coxiella burnetii positive (odds ratio = 6.75; 95% CI 1.65 to 27.7) compared with farms with ≤900 milking does. The odds of BTM positivity increased by a factor of 2.53 (95% CI 1.51 to 4.22) for each order of magnitude increase in the number of goats per acre. C. burnetii was not detected in samples from the majority of the Australian dairy goat herds suggesting there is an opportunity to protect the industry and contain this disease with strengthened biosecurity practices. Intensification appeared associated with an increased risk of positivity. Further investigation is required to discriminate the practices associated with an increased risk of introduction to disease-free herds, from practices associated with maintenance of C. burnetii infection in infected dairy goat herds.

Q fever seroprevalence in Australia suggests one in twenty people have been exposed.

Q fever (caused by Coxiella burnetii) is thought to have an almost world-wide distribution, but few countries have conducted national serosurveys. We measured Q fever seroprevalence using residual sera from diagnostic laboratories across Australia. Individuals aged 1-79 years in 2012-2013 were sampled to be proportional to the population distribution by region, distance from metropolitan areas and gender. A 1/50 serum dilution was tested for the Phase II IgG antibody against C. burnetii by indirect immunofluorescence. We calculated crude seroprevalence estimates by age group and gender, as well as age standardised national and metropolitan/non-metropolitan seroprevalence estimates. Of 2785 sera, 99 tested positive. Age standardised seroprevalence was 5.6% (95% confidence interval (CI 4.5%-6.8%), and similar in metropolitan (5.5%; 95% CI 4.1%-6.9%) and non-metropolitan regions (6.0%; 95%CI 4.0%-8.0%). More males were seropositive (6.9%; 95% CI 5.2%-8.6%) than females (4.2%; 95% CI 2.9%-5.5%) with peak seroprevalence at 50-59 years (9.2%; 95% CI 5.2%-13.3%). Q fever seroprevalence for Australia was higher than expected (especially in metropolitan regions) and higher than estimates from the Netherlands (2.4%; pre-outbreak) and US (3.1%), but lower than for Northern Ireland (12.8%). Robust country-specific seroprevalence estimates, with detailed exposure data, are required to better understand who is at risk and the need for preventive measures.

The natural history of acute Q fever: a prospective Australian cohort.

BACKGROUND: A detailed description of the natural history of acute Q fever, caused by infection with Coxiella burnetii, AIM: : To significantly increase understanding of the illness. DESIGN: Subjects with provisional acute Q fever (n = 115) were recruited from primary care in rural Australia, and followed prospectively by interview and blood collection including for serological confirmation. A nested series of subjects with prolonged illness (cases), and those without (controls), were investigated in detail. METHODS: Total phase I and phase II anti-C. burnetii antibodies were detected by complement fixation test; and IgG, IgM and IgA phase I and phase II titres by immunofluorescence. Flow cytometric analysis was conducted to enumerate circulating T cells subsets, B cells, monocytes and natural killer cells. RESULTS: Serological testing confirmed acute Q fever in 73 subjects (63%). The acute illness featured fever, headache, sweats, fatigue and anorexia; and varied widely in severity, causing an average of 8 days in bed and 15 days out of work or other role in the first month of illness. The illness course varied from 2 days to greater than a year. No cases of chronic, localized Q fever infection, such as endocarditis, were identified. Neither severe nor prolonged illness were associated with persistence of C. burnetii DNA, altered patterns of C. burnetii-specific IgG, IgM or IgA antibody production, or altered leucocyte subsets. CONCLUSIONS: The severity of acute Q fever alone predicted prolonged duration. Further studies are warranted to better understand the pathophysiology of prolonged illness after acute Q fever.

One Health approach to controlling a Q fever outbreak on an Australian goat farm.

A recent outbreak of Q fever was linked to an intensive goat and sheep dairy farm in Victoria, Australia, 2012-2014. Seventeen employees and one family member were confirmed with Q fever over a 28-month period, including two culture-positive cases. The outbreak investigation and management involved a One Health approach with representation from human, animal, environmental and public health. Seroprevalence in non-pregnant milking goats was 15% [95% confidence interval (CI) 7-27]; active infection was confirmed by positive quantitative PCR on several animal specimens. Genotyping of Coxiella burnetii DNA obtained from goat and human specimens was identical by two typing methods. A number of farming practices probably contributed to the outbreak, with similar precipitating factors to the Netherlands outbreak, 2007-2012. Compared to workers in a high-efficiency particulate arrestance (HEPA) filtered factory, administrative staff in an unfiltered adjoining office and those regularly handling goats and kids had 5·49 (95% CI 1·29-23·4) and 5·65 (95% CI 1·09-29·3) times the risk of infection, respectively; suggesting factory workers were protected from windborne spread of organisms. Reduction in the incidence of human cases was achieved through an intensive human vaccination programme plus environmental and biosecurity interventions. Subsequent non-occupational acquisition of Q fever in the spouse of an employee, indicates that infection remains endemic in the goat herd, and remains a challenge to manage without source control.

An analysis of Q fever patients 6 years after an outbreak in Newport, Wales, UK.

BACKGROUND: A cohort of 211 factory workers was exposed to a point source of Q fever in 2002. A total of 38 cases and 14 controls took part in a follow-up study 6 years after the outbreak. AIM: To compare Q fever serology, the presence of viable Coxiella burnetii, its DNA and fatigue between patients and controls. DESIGN: Laboratory case study. METHODS: Q fever serology was by microimmunofluroescence. Viable C. burnetii was detected by VERO cell culture and SCID mice inoculation with patient blood samples. Coxiella burnetii DNA was detected by qPCR (com1 gene) on patients' PBMC and on VERO cultures after 6 weeks incubation. Fatigue was measured by the Chalder Fatigue Scale. RESULT: At 6 years after the outbreak, 7 of the 38 patients had become seronegative and 4 of the 14 of the controls had become seropositive for Q fever. None of the patient/control peripheral blood mononuclear cells (PBMC) contained viable C. burnetii by VERO cell culture or by SCID mouse inoculation (death or splenomegaly) and none contained C. burnetii DNA by qPCR. CONCLUSION: Six years after acute Q fever, some patients had become seronegative but none contained viable C. burnetii or its DNA in their PBMC.

A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay.

AIMS: To determine the optimal DNA extraction method for the detection of Coxiella burnetii including the small-cell variant (SCV) by real-time PCR (qPCR) in clinical samples. METHODS AND RESULTS: A duplex qPCR detecting two Coxiella burnetii gene targets (com1 and IS1111a genes) was developed. Each target in this PCR had a sensitivity of one copy number per reaction. DNA extraction methods were compared on spiked negative samples and included a silica column kit, a chloroform separation prior to a silica column method and a chloroform/phenol separation and DNA precipitation method. CONCLUSIONS: The silica column extraction method was more efficient at recovering C. burnetii DNA, from large-cell and small-cell variants, than a chloroform or chloroform/phenol method. The silica column method was useful on spiked human samples including serum, buffy coat and bone marrow samples. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrated that a simple column kit method is efficient to use for the detection of C. burnetii in clinical samples including the SCV.

Serological prevalence study of exposure of cats and dogs in Launceston, Tasmania, Australia to spotted fever group rickettsiae.

A sero-epidemiological study of cats and dogs in the Launceston area of Tasmania, Australia was undertaken to determine the prevalence of antibodies to spotted fever group (SFG) rickettsiae. Results showed that 59% of cats and 57% of dogs were positive for antibodies, but there was no correlation between the animal's health and seropositivity at the time of testing, suggesting that rickettsial exposure is unrelated to ill-health in these two species of domestic animals.

Markers of exposure to spotted fever rickettsiae in patients with chronic illness, including fatigue, in two Australian populations.

BACKGROUND: Some investigators believe that a proportion of chronically unwell patients, many with fatigue, have an underlying rickettsial disease. AIM: To investigate the prevalence of markers of rickettsial infection in patients with chronic illnesses. DESIGN: Observational study. METHODS: A 526 patient cohort with chronic illnesses from Melbourne, Australia and 400 control patients from Newcastle, Australia were assessed using serology, culture and PCR for the detection of rickettsiae. Rickettsial serology was performed on another cohort of 581 chronically unwell patients (and 34 non-fatigued patients from the same practice) from Adelaide, Australia. RESULTS: Of the Melbourne patient cohort, 14/526 (3%) were real-time PCR positive for rickettsial DNA compared to none of the 400 control patients (P < 0.001). Of these 14 patients, Rickettsia honei strain 'marmionii' was detected in 5 and isolated from 2. Rickettsaemia was seasonal, with more in winter (8/145; P < 0.03) and less in spring (0/143; P < 0.03). Positive rickettsial serology titres of >or=1:256 were seen in 206 (39%) patients. Of the Adelaide patient cohort, 238/581 (41%) had positive rickettsial antibodies titres. Of the 34 control sera, 5 (15%) were serologically positive (P < 0.002). Both Melbourne and Adelaide patient cohorts had significantly higher seropositivity than the Newcastle control cohort (3/399; P < 0.0001). CONCLUSION: In patients with chronic illness, rickettsial DNA in peripheral blood and/or rickettsial seropositivity may represent exposure to rickettsiae or underlying rickettsial diseases. It is not known whether the presence of rickettsiae is causally related to the patients' chronic illnesses, or reactivation of a latent rickettsial infection.

Rickettsia felis: molecular characterization of a new member of the spotted fever group.

In this report, placement of Rickettsia felis in the spotted fever group (SFG) rather than the typhus group (TG) of Rickettsia is proposed. The organism, which was first observed in cat fleas (Ctenocephalides felis) by electron microscopy, has not yet been reported to have been cultivated reproducibly, thereby limiting the standard rickettsial typing by serological means. To overcome this challenge, several genes were selected as targets to be utilized for the classification of R. felis. DNA from cat fleas naturally infected with R. felis was amplified by PCR utilizing primer sets specific for the 190 kDa surface antigen (rOmpA) and 17 kDa antigen genes. The entire 5,513 bp rompA gene was sequenced, characterized and found to have several unique features when compared to the rompA genes of other Rickettsia. Phylogenetic analysis of the partial sequence of the 17 kDa antigen gene indicated that R. felis is less divergent from the SFG rickettsiae than from the TG rickettsiae. The data corroborate results from previous reports that analysed the citrate synthase, 16S rRNA, rompB (135 kDa surface antigen), metK, ftsY, polA and dnaE genes that placed R. felis as a member of the SFG. The organism is passed trans-stadially and transovarially, and infection in the cat flea has been observed in the midgut, tracheal matrix, muscle, hypodermis, ovaries and testes.

The rickettsial outer-membrane protein A and B genes of Rickettsia australis, the most divergent rickettsia of the spotted fever group.

The genes for rickettsial outer-membrane protein A (rOmpA), a distinguishing feature of spotted fever group (SFG) rickettsiae, and rOmpB, a genus-specific protein, were identified and sequenced in Rickettsia australis. The amino acid sequences of domains I, III and IV of the R. australis rOmpA share close homology with those of rOmpA of other SFG rickettsiae, but the repeat region (domain II) is dramatically different from that of other known SFG rOmpA. R. australis rOmpB is more similar to rOmpB of other SFG rickettsiae than to that of typhus group rickettsiae.

Rickettsia honei sp. nov., the aetiological agent of Flinders Island spotted fever in Australia.

The name Rickettsia honei, strain RBT, has been proposed for a unique spotted fever group (SFG) agent which is pathogenic for humans. This agent has previously been compared to the other SFG agents and was shown to be distinct in protein structure by SDS-PAGE and by immunoblotting. Genetic comparisons of the 16S rRNA, rompA, gltA and the 17 kDa antigen genes with the other SFG rickettsiae confirmed the phylogenetic distance between R. honei and the previously described species. Genetically, Rickettsia honei is more closely related to the Thai tick typhus (TT-118) rickettsia than to any other member of the SFG. Indeed, it is proposed that TT-118 is a strain of R. honei which was previously isolated in Thailand. These results elucidate the presence of a unique SFG rickettsial species in Australasia.

Protein characterization of Australian spotted fever group rickettsiae and monoclonal antibody typing of Rickettsia honei.

Rickettsial proteins rOmp A and rOmp B exist in both Rickettsia australis and Rickettsia honei but differ in molecular weight and antigenicity; in addition, they produce distinct immunogenic responses and appear to be to conformationally dependent antigens. Species-specific monoclonal antibodies for other spotted fever group rickettsial species did not react with R. honei. A PCR product of the repeat region of the rOmp A gene from R. honei was amplified and calculated to contain 11 repeat units.

Genetic variation in Australian spotted fever group rickettsiae.

Rickettsiae were isolated by cell culture of buffy coat blood from six patients with spotted fever from southeastern Australia and Flinders Island in Bass Strait. The isolates were genetically compared with two previous Rickettsia australis patient isolates. The genus-specific 17-kDA genes from the isolates were compared after DNA amplification and restriction fragment analysis of the amplified DNA. This comparison revealed that mainland rickettsial isolates from southeastern Australia were identical to two previous isolates of R. australis from northeastern Australia. Rickettsial isolates from Flinders Island were distinct from the mainland isolates. The 16S rRNA gene sequences from the isolates were determined and compared. The Flinders Island rickettsial agent was most closely related (0.3% structural divergence) to Rickettsia rickettsii, Rickettsia conorii, and Rickettsia slovaca. The Flinders Island rickettsial agent was 1.3 and 2.1% structurally divergent from R. australis and Rickettsia akari, respectively. The 16S rRNA gene sequence from the Flinders Island agent shows that this rickettsia is more closely related to the rickettsial spotted fever group than is R. australis. We conclude that there are two populations of spotted fever group rickettsiae in Australia and propose that the genetically distinct causative organism of Flinders Island spotted fever be designated Rickettsia honei. The extent of distribution and animal host reservoirs remain to be elucidated.

Severe perinephric hemorrhage after shockwave lithotripsy.

We report a case of a 69-year-old man who, after a second session of shockwave lithotripsy for multiple stones in the right kidney, showed symptoms of severe hemorrhage and flank pain unresponsive to analgesics, with the gradual development of extensive and serious perinephric hematoma. The bleeding necessitated nephrectomy. Unrecognized chronic pyelonephritis may have been a predisposing factor.

Spotted fever group rickettsial infection in south-eastern Australia: isolation of rickettsiae.

Flinders Island spotted fever (FISF), a spotted fever group (SFG) rickettsial disease first described in 1991, occurs in south-eastern Australia. The isolation of the aetiological agent is described for the first time having been obtained from the blood of two patients. An additional 22 cases are also reported. Of these patients four had positive initial serology, and 20 showed seroconversion (using Rickettsia australis as antigen). Acute phase blood specimens taken from seven patients caused neonatal mice to seroconvert to R. australis and a blood specimen from one of these patients (and one other) yielded rickettsiae. A field survey for possible reservoir and vector animals on Flinders Island, Tasmania and in Gippsland, Victoria (both in south-eastern Australia) yielded 217 vertebrates and 1445 invertebrate ectoparasites, mostly ticks. Ixodes cornuatus from humans and dogs in Gippsland produced seroconversion to SFG rickettsia when inoculated into mice but no invertebrate pools from Flinders Island produced seroconversion in mice. Haemolymph from an individual I. cornuatus removed from a human in Gippsland, yielded a SFG rickettsia on tissue culture. Sera from several species of native vertebrates, especially the bush rat, Rattus fuscipes, were positive for antibodies to SFG rickettsia.

Characterization and comparison of Australian human spotted fever group rickettsiae.

The microbiological and molecular characteristics of the rickettsiae isolated from humans with Queensland tick typhus (QTT) caused by Rickettsia australis and the recently described Flinders Island spotted fever (FISF) were compared. Clinically and serologically, the diseases are similar. Cell culture reveals differences in the plaque-forming abilities of the isolates. Characterization of the gene encoding the genus-specific 17-kDa antigen of R. australis revealed a unique nucleotide sequence unlike those of the FISF isolate and Rickettsia rickettsii. Southern blot analysis of rickettsial DNA from the isolates with a 17-kDa-antigen gene probe revealed the presence of this gene in all isolates but no difference in banding patterns. When a probe for the rRNA genes was used, clear differences in banding patterns of isolates from patients with QTT and FISF were revealed. Thus, the rickettsiae isolated from patients with FISF differ from those from patients with QTT and may represent a new rickettsial species.

Quantification of Rickettsia australis.

Several assay systems were compared for measuring the concentration of viable Rickettsia australis, including embryonated eggs, tissue cultures, and mouse inoculation. Direct rickettsial counts that included the enumeration of both viable and nonviable rickettsiae were used to obtain baseline values. Assays were conducted in parallel using serially diluted R. australis preparations to establish which assay displayed the greatest sensitivity and reproducibility. Overall, the plaque assay using buffalo green monkey kidney cells with centrifugation of the rickettsiae onto the monolayers was the most sensitive assay for detecting R. australis, while the embryonated egg assay and mouse lethality titrations were the least sensitive.

[Intratesticular hemorrhage mimicking a tumor]. / Hémorragie intratesticulaire imitant une tumeur.

The authors describe a case of painless swelling of the testis considered, both clinically and by echography, as a tumor, and which was submitted to an orchiectomy. The histopathological examination showed an extended, not organised, intratesticular haematoma. The review of the bibliography confirms the difficulties in the pre-operative diagnosis of testis tumours.