Com1 Gene Research Articles

Absence of Coxiella burnetii in kangaroo ticks (Amblyomma triguttatum) from a high seroprevalence population of eastern grey kangaroos

Q fever, caused by Coxiella burnetii, is an important zoonotic and public health concern worldwide. Kangaroos are thought to be a likely wildlife reservoir for C. burnetii in Australia and the kangaroo tick (Amblyomma triguttatum) has often been considered a vector. In this descriptive study of ticks collected from a population of eastern grey kangaroos (Macropus giganteus) with a high serological (84 %) and molecular (65 %) prevalence of C. burnetii in northern New South Wales, a total of 72 A. triguttatum ticks were tested by PCRs targeting the IS1111, htpAB, and com1 genes of the Coxiella genome. Despite the remarkably high prevalence of coxiellosis in the host population, none of the ticks were positive for Coxiella. This finding suggests that the kangaroo tick may not play a significant role in the transmission dynamics of C. burnetii in this particular host population.

Acute Q fever in individuals with acute febrile illness & exposure to farm animals: Clinical manifestations & diagnostic approaches.

Background & objectives Q fever is an important zoonotic disease affecting humans as well as animals. The objective of this study was to assess the burden of Q fever in individuals with acute febrile illness, particularly those in close contact with animals. Various diagnostic methods were also evaluated in addition to clinical examination analysis and associated risk factors. Methods Individuals presenting with acute febrile illness who had animal exposure were enrolled (n=92) in this study. Serum samples were tested using IgG and IgM phase 2 enzyme linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). The PCR targeting the com1 and IS1111 genes was performed on blood samples. PCR amplicons were sequenced and phylogenetically analysed. Demographic data, symptoms, and risk factors were collected through a structured questionnaire. Results Among individuals with acute febrile illness, 34.7 per cent (32 out of 92) were found to be infected with Coxiella burnetii. PCR exhibited the highest sensitivity among the diagnostic methods employed. The most common clinical manifestations included headache, chills, arthralgia, and fatigue. Individuals engaged in daily livestock-rearing activities were found to be at an increased risk of infection. Interpretation & conclusions Q fever is underdiagnosed due to its varied clinical presentations, diagnostic complexities, and lack of awareness. This study underscores the importance of regular screening for Q fever in individuals with acute febrile illness, particularly those with animal exposure. Early diagnosis and increased awareness among healthcare professionals are essential for the timely management and prevention of chronic complications associated with Q fever.

Tissue distribution of Coxiella burnetii and antibody responses in macropods co-grazing with livestock in Queensland, Australia.

Coxiella burnetii, the causative agent of Q fever, is a zoonotic bacteria of global public health significance. The organism has a complex, diverse, and relatively poorly understood animal reservoir but there is increasing evidence that macropods play some part in the epidemiology of Q fever in Australia. The aim of this cross-sectional survey was to estimate the animal- and tissue-level prevalence of coxiellosis amongst eastern grey (Macropus giganteus) and red (Osphranter rufus) kangaroos co-grazing with domestic cattle in a Q fever endemic area in Queensland. Serum, faeces and tissue samples from a range of organs were collected from 50 kangaroos. A total of 537 tissue samples were tested by real-time PCR, of which 99 specimens from 42 kangaroos (84% of animals, 95% confidence interval [CI], 71% to 93%) were positive for the C. burnetii IS1111 gene when tested in duplicate. Twenty of these specimens from 16 kangaroos (32%, 95% CI 20% to 47%) were also positive for the com1 or htpAB genes. Serum antibodies were present in 24 (57%, 95% CI 41% to 72%) of the PCR positive animals. There was no statistically significant difference in PCR positivity between organs and no single sample type consistently identified C. burnetii positive kangaroos. The results from this study identify a high apparent prevalence of C. burnetii amongst macropods in the study area, albeit seemingly with an inconsistent distribution within tissues and in relatively small quantities, often verging on the limits of detection. We recommend Q fever surveillance in macropods should involve a combination of serosurveys and molecular testing to increase chances of detection in a population, noting that a range of tissues would likely need to be sampled to confirm the diagnosis in a suspect positive animal.

Occurrence of Coxiellosis in ruminants and its associated risk factors

Coxiellosis in animals is caused by the zoonotic pathogen, Coxiella burnetii. Although the disease is of public health importance it remains underdiagnosed and underreported. The cross- sectional study was aimed to estimate the occurrence of the disease in livestock of study area and also to identify the risk factors associated with the disease in animals. Blood, serum, and vaginal swabs samples were collected from 200 ruminants (cattle, sheep, and goats), across various farms in Karnataka, India. These samples were then screened using ELISA and PCR (com1 and IS1111). A questionnaire was administered to the farm owners to collect the risk factor-related information. About 5.26 % cattle, 12.3 % sheep, and 12.5 % goats were positive by ELISA. By PCR, 9.47 % cattle, 9.3 % sheep, and 10 % goats were positive. Overall, the occurrence of 14.73 %, 18.46 % and 17.5 % was estimated in cattle, sheep and goat, respectively. PCR targeting the IS1111 gene detected higher number of samples as positive as compared to the com1 gene PCR. Higher number of vaginal swab samples were detected as positive as compared to blood. History of reproductive disorders (OR: 4.30; 95 %CI:1.95- 9.46), abortion (OR: 30.94; 95 %CI:6.30- 151.84) and repeat breeding (OR:11.36; 95 %CI:4.16- 30.99) were significantly associated with coxiellosis (p < 0.005). Multivariable analysis by logistic regression model analysis suggested retained abortion, repeat breeding and rearing of animal in semi-intensive system as factors significantly associated with the infection. Cultural identification of the PCR positive samples were cultured using embryonated egg propagation and cell culture techniques and positivity was confirmed in six samples. Phylogenetic analysis of the com1 and IS1111 gene revealed clustering based on similar geographic locations. The study estimated the occurrence of the disease in the study area and identified the potential risk factors.

A Real-time Quantitative Loop Mediated Isothermal Amplification Assay Targeting com1 Gene of Coxiella burnetii

Background &amp; Objectives: The present study designed a loop mediated isothermal amplification (LAMP) method to rapidly detect Coxiella burnetii (C. burnetii) and develop a sensitive Real-time quantitative LAMP (Q-LAMP) assay to quantify Q fever. Materials &amp; Methods: Primers were specifically designed for use in targeting the com1 conserved gene of C. burnetii to carry out LAMP detection of the agent causing Q fever. After obtaining the LAMP reaction, the sensitivity of the method was assessed by preparing a serial ten-fold dilution of a plasmid carrying com1 gene. Results: The assay’s sensitivity for the visual detection of changes in turbidity or turbidimeter and electrophoresis of agarose gel were 0.4 fg and 0.04 fg, respectively. Hence, the lower limit of detection was 120 and 12 copies of the gene detected in 60 min. The results of this study were indicative of the simplicity, rapidness, sensitivity, and specificity of the LAMP assay for C. burnetii detection and a probable improvement of the diagnostic procedure used in clinical laboratories. Conclusion: The assay specificity was assessed using Coxiella genomic DNA and a panel of Gram-negative and Gram-positive bacteria. The LAMP assay was shown to be highly specific for detection of Coxiella without any observable amplification products from non-Coxiella organisms.

Molecular Detection of Coxiella burnetii by Nested PCR Method in Cattle and Buffalo Raw Milk, Urmia Region, Iran

Background: Coxiella burnetii, a pleomorphic coccobacillus with a Gram-negative cell wall and the cause of query (Q) fever. Amongst animals, farm animals, goats, and sheep are the main reservoirs of Q fever. Methods: This study was conducted to outline the presence of C. burnetii in raw milk received from farm animals and buffalo in all 12 months of 2020 within the Urmia region, northwest Iran. A total of 600 milk samples were received from 3 regions by registering the animals' ages. DNA extraction from milk samples was performed. Results: The nested-polymerase chain reaction (PCR) was efficient in the detection of C. burnetii based on the transposable com1 gene. The results showed that 12.33% (95% CI: 9.9 - 15%) of the total samples (12.66% buffalo and 12% in cattle raw milk) were positive for C. burnetii DNA. The prevalence of C. burnetii in raw milk samples was considerably higher in summer (12.66%, P &lt; 0.05, 95% CI: 9.3 - 17%). In addition, the superiority of C. burnetii in livestock milk drastically varied (P &lt; 0.05) amongst age groups. However, it was not significant in buffalo milk samples. Conclusions: The farm animals and buffalo population in Urmia may be taken into consideration as an important parameter in the epidemiology of Q fever.

Simultaneous detection of 7 important Rickettsiales pathogens by TaqMan-probe quantitative real-time PCR

Objective: To establish and optimize a TaqMan-probe quantitative real-time PCR (qPCR) assay for the detection of 7 important Rickettsiales pathogens and simultaneous identification of the infection types. Methods: Based on the ompB gene of Rickettsia prowazekii, Rickettsia mooseri and spotted fever group rickettsiae, the groEL gene of Orientia tsutsugamushi, the 16S rRNA of Ehrlichia chaffeensis, the gltA gene of Anaplasma phagocytophilum and the com1 gene of Coxiella burnetii, we synthesized primers and TaqMan-probes and optimized the reaction system and reaction process to same solution. The sensitivity, specificity and reproducibility of this assay were evaluated and the assay was used for the detection of simulated and actual samples. Results: The Ct value of the standard curves of the 7 pathogens showed a good linear relationship with the number of DNA copies (all R2 >0.990 0), the minimum detection limit was 10 copies/μl, showing good specificity. In the 96 tick nucleic acid extracts, Coxiella burnetii was detected in 1 sampleand spotted fever group Rickettsiae was detected in 3 samples. In the 80 blood samples from patients with undefined febrile illness, Orientia tsutsugamushi was detected in 1 sample and spotted fever group rickettsiae was detected in 2 samples. Conclusions: In this study, based on the established TaqMan-probe qPCR assay, the reaction system and reaction condition of the 7 important pathogens of Rickettsiales were optimized to the same solution. This method overcomes the shortcomings of using different reaction systems and reaction conditions for different pathogens, which can precisely identify the species of 7 important pathogens of Rickettsiales in clinical sample detections and is important for the infection type identification and laboratory detection time reduction to facilitate precise treatment of the patients.

Molecular and serological investigation of Coxiella burnetii in goats with reproductive disorders

One of the important yet neglected diseases in goats that affect the reproductive system is Q fever, caused by the Gram-negative, obligate intracellular pathogen, Coxiella burnetii. Molecular techniques cannot be used solely to detect the presence of this pathogen, due to the shedding pattern of the organism. So, both molecular and antibody detection assays were used to know the actual burden of the disease. Although many countries and states have reported Q fever in independent studies, the actual scenario of C. burnetii in Kerala is unknown. In the present study milk, vaginal swabs and blood samples were collected randomly from 150 goats with reproductive disorders in Thrissur, Palakkad and Malappuram districts. The presence of antibodies (7.99 per cent) against C. burnetii revealed the presence of seropositive goats in Kerala. None of the samples were positive by PCR targeting the Com1 gene and IS1111 genes.

MLVA and com1 genotyping of Coxiella burnetii in farmed ruminants in Great Britain

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, has been shown to be endemic in Great Britain, but information on the prevailing genomic lineages or Genomic Groups (GGs) of Coxiella burnetii is limited. The aim of this study was to genotype C. burnetii isolates from infected farmed ruminants by Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) and identify their associated Genomic Group. A total of 51 Coxiella-containing abortion samples from farmed ruminants (sheep, goats, and cattle), which were collected in Great Britain during 2013–2018, were included in the study, 34 of which returned a C. burnetii MLVA genotype. All bovine samples (n = 18), 5/7 of the ovine samples, and 3/9 of the caprine samples belonged to an MLVA cluster which we could link to the MST20 genotype of GG III, whereas 6/9 of the caprine samples and 2/7 of the ovine samples belonged to MLVA clusters which we could link to the MST33 or MST32 genotypes of GG II (7 vs 1 sample(s), respectively). We also noted that the Coxiella-specific com1 gene contained unique mutations that could genomotype isolates, i.e. assign them to a Genomic Group. In conclusion, both goats and sheep in Great Britain (from 2014 onward) were found to carry the same MLVA genotypes (MST33-like; GG II) that were linked to a human Q fever outbreak in the Netherlands. This knowledge in combination with the usage of genotyping/genomotyping methods should prove useful in future surveillance programs and in the management of outbreaks.

Genetic diversity of wild rodents and detection of Coxiella burnetii, the causative agent of Q fever, in Saudi Arabia.

Throughout history, wildlife has been regarded as a major source of infectious diseases. Rodentia, the most speciose order of mammals, whose members are recognised hosts of more than 60 zoonotic diseases, represent a potential threat to human health. Recently, epidemiological data from Saudi Arabia indicated an actual growth in the number of emerging and/or re-emerging cases of several zoonoses. However, there is a lack of studies focusing on the molecular taxonomy of rodents and the pathogens they may harbour in this region. In this study, the first molecular characterisation of six rodent taxa in this region is provided, based on partial Cyt B and 16S genes. The data confirm the spread of rodent-associated C. burnetii strains in Jazan, southwestern Saudi Arabia. The PCR targeting IS111, the multi-copy transposase gene, revealed 17.5% (36/205) positive samples, whereas the second nested PCR, targeting the single-copy Com1 gene, revealed 16.6% (34/205) positive samples. Phylogenetic and network analyses indicated the presence of four haplotypes of C. burnetii within the studied localities. One major haplotype (H-2) was observed in all rodent species and from 18 localities. The infection rates of C. burnetii among studied species, localities and habitats were not significantly different (>0.05). Our results facilitate the assessment of the health risk associated with rodents and the development of strategies to control the increasing impacts of Q fever.

First Molecular Detection of &lt;i&gt;Coxiella burnetii&lt;/i&gt; in Beef Cattle in West Java, Indonesia

Coxiella burnetii is a bacterial pathogen that causes Q fever, which is widespread worldwide. Livestock such as cattle, goats, and sheep are the main sources of C. burnetii infection. C. burnetii infection causes abortion in livestock, resulting in economic damage. Q fever is a zoonotic disease and a potential public health hazard. To date, little is known about C. burnetii infection in livestock in Indonesia. The objective of this study was to screen the genome of C. burnetii bacteria in beef cattle in West Java, Indonesia. Organ tissue samples were collected from cattle slaughtered in slaughterhouses in West Java. C. burnetii genome was detected in cattle samples obtained from three sampling areas using nested PCR, targeting the com1 gene of C. burnetii. Sequencing analysis of the 16S rRNA gene revealed that the amplicons showed 99.9% nucleotide identity to the C. burnetii strains: Heizberg, 1843, 2574, 701CbB1, and 14160-001. Our results indicate that C. burnetii infection occurs in Indonesian beef cattle and highlight the risk of exposure to C. burnetii infection in humans.

Understanding the zoonotic pathogen, Coxiella burnetii in Australian fur seal breeding colonies through environmental DNA and genotyping

Context Coxiella burnetii is suspected as a novel pathogen contributing to decreased pup production in Australian fur seals (Arctocephalus pusillus doriferus). It has recently been described from a single breeding colony in Bass Strait, has previously been associated with two decreasing populations of northern hemisphere pinnipeds and is a known reproductive pathogen. Data around its disease ecology in marine mammals are sparse. Aims To determine whether environmental DNA (eDNA) can be used to survey for C. burnetii in Australian fur seal breeding colonies. To determine whether C. burnetii in Australian fur seals is the same genotype as terrestrial Australian C. burnetii. Methods Soil samples were collected from Kanowna Island and Seal Rocks. Placental samples were collected from Kanowna Island. Soil was evaluated for eDNA using a quantitative polymerase chain reaction (qPCR) for com1 gene. Placental samples were evaluated with com1, htpAB and IS1111 markers. Multiple-locus variable number of tandem repeats analysis for three microsatellite loci (ms-24, ms-28 and ms-33) was used to determine relatedness to Australian C. burnetii genotypes. Key results eDNA results varied between pre-and post-pupping at Seal Rocks. When targeting the com1 gene, the post-pupping prevalence at Kanowna Island and Seal Rocks was 59.6% and 90%, respectively. eDNA PCR inhibition of samples was low at 1.9%. There was very poor, sporadic to absent IS1111 amplification in placental samples. The com1 and htpAB qPCRs had an overall prevalence across placental samples of 39.2% and 56.7% respectively. In 90.1% of placental samples (n = 11), the ms-28 locus amplified. Neither ms-24 nor ms-33 amplified. Conclusions eDNA is an effective tool to survey Australian fur seal breeding colonies in the post-pupping period for C. burnetii. The prevalence appears to be much higher in the Seal Rocks colony than in the Kanowna Island colony. It appears that this is not a terrestrial Australian genotype but rather closely related to genotypes detected in marine mammals in the northern hemisphere. Implications This research significantly expands our ability to survey for C. burnetii in Australian fur seals and other marine mammals. It highlights knowledge gaps in our understanding of the disease ecology and phylogeny of C. burnetii in marine mammals.

897Environmental contamination of Coxiella burnetii in and around an endemically infected goat farm

Abstract Background Coxiella burnetii is the cause of Q fever, a zoonotic disease spread by aerosol transmission. This study investigated C. burnetii environmental contamination in and around an endemically infected, intensively managed dairy goat farm in Victoria, Australia. Methods Dust, soil and water were collected in and around kidding pens. Samplings were collected before, during and after each kidding season. Soil was sampled along a 500 m transect from the main kidding pen in the predominant wind direction to assess the risk of C. burnetii spread from the main farm shed as a point source. DNA extraction and quantitative PCRs targeting the IS1111 and com1 genes were performed. Analyses are ongoing to describe the change in the frequency of C. burnetii positive environmental samples as a function of distance from the main farm shed. Results Dust inside the kidding pen contained C. burnetii DNA at all time points, with higher loads during the kidding seasons. Soil samples were positive for both PCR targets and were evenly dispersed within close ranges (500 m) from the main farm shed. Only those water samples taken from close to the main farm shed were positive. Conclusions C. burnetii was readily found in dust and soil in and around a farm shed where coxiellosis was endemic. Further environmental sampling will allow us to estimate the distance over which C. burnetii contamination occurs around a known point source. Key messages Results of this study will provide information critical for estimating Q fever risk around livestock facilities.

Coxiella burnetii in cattle and their human contacts in a gaushala (cattle shelter) from India and its partial com1 gene sequence-based phylogenetic analysis

Q fever caused by Coxiella burnetii is an important zoonosis and has great public health significance. A total of 905 clinical samples from 387 cattle [serum (n = 387); vaginal swabs (n = 387); milk (n = 131)] and 59 serum samples from humans were collected from gaushala (cattle shelter) and screened for anti-C. burnetii IgG antibodies in the sera using an indirect-ELISA kit. Further, the samples were tested for C. burnetii DNA employing TaqMan real-time and conventional PCR assays targeting the com1 gene. In ELISA, 9.56% and 6.78% of animal and human sera samples were positive for anti-C. burnetii antibodies, respectively. Upon pathogen detection, 3.87% sera, 1.81% vaginal swabs, and 6.87% milk samples from cattle tested positive in TaqMan real-time PCR and 1.55% sera, 0.52% vaginal swabs, and 3.05% milk samples were found positive in conventional PCR. In humans, one serum sample was positive in both the PCR assays. The PCR positive samples (n = 12) were partially sequenced and the phylogenetic tree was constructed using com1 gene sequences (n = 42) from a different host and geographical areas. The study highlights infection of cattle and their human contacts in gaushala and identifies relationships between strains identified in the gaushala and those in other parts of the globe.

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.

Apparent prevalence and risk factors of coxiellosis (Q fever) among dairy herds in India.

Coxiella burnetii is a highly infectious zoonotic pathogen infecting wide range of mammals, including humans. In the present study, a total of 711 blood samples from bovines [cattle (n = 543) and buffaloes (n = 168)] from eight farms at different geographical locations in India were screened for C. burnetii targeting the IS1111 and the com1 genes. The anti-C. burnetii antibodies in serum samples were detected using indirect-ELISA kits. Also, a total of 21 parameters pertaining to animal health and farm management were identified to assess their role as possible risk factors for coxiellosis among the targeted farms. The apparent prevalence (positive for PCR and/or ELISA) for coxiellosis was reported to be 24.5% in cattle and 8.9% in buffaloes. In cattle, the detection rate of C. burnetii employing the IS1111 gene (8.5%) was found to be significantly higher (p<0.05) as compared to the com1 (6.5%) gene. The seropositivity by ELISA was higher among cattle (17.7%) than in buffaloes (8.3%). Further, on univariable analysis of risk factors, species (cattle) (OR:3.31; 95%CI:1.88–5.82), inadequate floor spacing (OR:1.64; 95%CI:1.10–2.43), mastitis (OR:2.35, 95%CI:1.45–3.81) and reproductive disorders (OR:2.54; 95%CI:1.67–3.85) were significantly (p<0.05) having high odds for coxiellosis. The multivariable logistic regression analysis of the animal level risk factors revealed that species and age were found to be significantly associated with coxiellosis. However, since the number of screened farms is limited; further research is needed with a higher number of animals to confirm the farm level odds ratio of risk factors. Quarantine and biosecurity measures including farm hygiene operations were observed to be inadequate and also the lack of awareness about coxiellosis among the farm workers. In absence of vaccination program for coxiellosis in India, robust surveillance, farm biosecurity measures and the awareness for the disease among risk groups can play an important role in the disease prevention and subsequent transmission of the pathogen.

Host-induced silencing of the Colletotrichum gloeosporioides conidial morphology 1 gene (CgCOM1) confers resistance against Anthracnose disease in chilli and tomato.

Host mediated silencing of COM1 gene of Colletotrichum gloeosporioides disables appressorial differentiation and effectively prevents the development of Anthracnose disease in chilli and tomato. Anthracnose disease is caused by the ascomycetes fungal species Colletotrichum, which is responsible for heavy yield losses in chilli and tomato worldwide. Conventionally, harmful pesticides are used to contain anthracnose disease with limited success. In this study, we assessed the potential of Host-Induced Gene Silencing (HIGS) approach to target theColletotrichumgloeosporioides COM1 (CgCOM1) developmental gene involved in the fungal conidial and appressorium formation, to restrict fungal infection in chilli and tomato fruits. For this study, we have developed stable transgenic lines of chilli and tomato expressing CgCOM1-RNAi construct employing Agrobacterium-mediated transformation. Transgenic plants were characterized by molecular and gene expression analyses. Production of specific CgCOM1 siRNA in transgenic chilli and tomato RNAi lines was confirmed by stem-loop RT-PCR. Fungal challenge assays on leaves and fruits showed that the transgenic lines were resistant to anthracnose disease-causing C. gloeosporioides in comparison to wild type and empty-vector control plants. RT-qPCR analyses in transgenic lines revealed extremely low abundance of CgCOM1 transcripts in the C. gloeosporioides infected tissues, indicating near complete silencing of CgCOM1 gene expression in the pathogen. Microscopic examination of the Cg-challenged leaves of chilli-CgCOM1i lines revealed highly suppressed conidial germination, germ tube development, appressoria formation and mycelial growth of C. gloeosporioides, resulting in reduced infection of plant tissues. These results demonstrated highly efficient use of HIGS in silencing the expression of essential fungal developmental genes to inhibit the growth of pathogenic fungi, thus providing a highly precise approach to arrest the spread of disease.

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.

Cloning and Expression of Com1 and OmpH Genes of Coxiella burnetii in Periplasmic Compartment of Escherichia coli with the Aim of Recombinant Subunit Vaccine Production.

Coxiella burnetiiis an obligate and gram-negative bacteria causing query fever (Q fever) disease, despite the importance of Q fever, there is no universal vaccine against this disease. Therefore, application of the recombinant subunit vaccines which use Com1 and OmpH as immunogenic proteins can be useful in this regard. To perform the current project, Com1 and OmpH genes were amplified by polymerase chain reaction (PCR) method, then, the PCR products were purified by DNA precipitation technique. In order to clone, first, both genes along with the pET-22b(+) vector were digested by NcoI and XhoI enzymes and then, Com1 and OmpH genes were ligated in linear vectors by T4 DNA ligase. The recombinant vectors were transformed in BL21 (DE3) strain of Escherichia coli and expression was induced by 1 mM Isopropyl &amp;beta;-D-1-thiogalactopyranoside. Expression of Com1 and OmpH was investigated using 12% Sodium dodecyl sulfate polyacrylamide gel electrophoresis. Finally, both proteins were purified by Ni-NTA columns and consequently confirmed by western blotting. The results of assessing 1% agarose gel showed that PCR amplification, DNA precipitation, and digestion of both genes were successfully performed.Theresults of colony PCRs and sequencing revealed that Com1 and OmpH were correctly cloned in pET-22b(+) vector. Finally, the results of expression, purification, and western blotting of both proteins showed thatBL21 (DE3) strain of Escherichia colicould be able to express Com1 and OmpH proteins. Based on the collected data, it seems that Escherichia coli as an affordable and simple host can be applied to express Com1 and OmpH genes. It should be mentioned that products of the present project can be examined as recombinant subunit vaccines against Q fever.

Molecular detection of Coxiella (Gammaproteobacteria: Coxiellaceae) in Argas persicus and Alveonasus canestrinii (Acari: Argasidae) from Iran

BackgroundCoxiella burnetii and non-C. burnetii bacteria or endosymbiotic Coxiella-like were reported in various tick species. We aimed to detect C. burnetii within soft tick species, Argas persicus and Alveonasus canestrinii. MethodsArgasid ticks were collected from different counties of Lorestan province, west of Iran. Partial fragments of 16S rRNA, IS1111 insertion sequence, com1, htpB, and icd genes related to Coxiella genus were sequenced. ResultsA partial 16S rRNA and com1 gene fragment as well as IS1111 was detected in four Ar. persicus and twelve Al. canestrinii pools. Moreover, partial htpB and icd gene was only detected in one pool of Ar. persicus. ConclusionsDetection of C. burnetii in tick samples was failed due to the occurrence of Coxiella-like endosymbionts and leads to misidentification. Thus, the house-keeping genes should be designated to distinguish C. burnetii within Coxiella-like endosymbionts.