Bacterial burden and molecular characterization of Coxiella burnetii in shedding pregnant and postpartum ewes from Saint Kitts.

This study aimed to evaluate the bacterial burden and perform molecular characterization of Coxiella burnetii during shedding in pregnant (vaginal, mucus and feces) and postpartum (vaginal mucus, feces and milk) ewes from Saint Kitts. Positive IS1111 DNA (n=250) for C. burnetii samples from pregnant (n=87) and postpartum (n=74) Barbados Blackbelly ewes in a previous investigation were used for this study. Vaginal mucus (n=118), feces (n=100), and milk (n=32) positive IS1111 C. burnetii-DNA were analysed by real time qPCR (icd gene). For molecular characterization of C. burnetii, selected (n=10) IS1111 qPCR positive samples were sequenced for fragments of the IS1111 element and the 16 S rRNA gene. nBLAST, phylogenetic and haplotype analyses were performed. Vaginal mucus, feces and milk had estimated equal amounts of bacterial DNA (icd copies), and super spreaders were detected within the fecal samples. C. burnetii haplotypes had moderate to high diversity, were ubiquitous worldwide and similar to previously described in ruminants and ticks and humans.

Ultraviolet C inactivation of Coxiella burnetii for production of a structurally preserved whole cell vaccine antigen.

Q fever, a worldwide-occurring zoonotic disease, can cause economic losses for public and veterinary health systems. Vaccines are not yet available worldwide and currently under development. In this regard, it is important to produce a whole cell antigen, with preserved structural and antigenic properties and free of chemical modifications. Thus, inactivation of Coxiella burnetii with ultraviolet light C (UVC) was evaluated. C. burnetii Nine Mile phase I (NMI) and phase II (NMII) were exposed to decreasing intensities in a time-dependent manner and viability was tested by rescue cultivation in axenic medium or cell culture. Effects on the cell structure were visualized by transmission electron microscopy and antigenicity of UVC-treated NMI was studied by immunization of rabbits. NMI and NMII were inactivated at UVC intensities of 250 µW/cm2 for 5 min or 100 µW/cm2 for 20 min. Reactivation by DNA repair was considered to be unlikely. No morphological changes were observed directly after UVC inactivation by transmission electron microscopy, but severe swelling and membrane degradation of bacteria with increasing severity occurred after 24 and 48 h. Immunization of rabbits resulted in a pronounced antibody response. UVC inactivation of C. burnetii resulted in a structural preserved, safe whole cell antigen and might be useful as antigen for diagnostic purposes or as vaccine candidate.

Susceptibility, Immunity, and Persistent Infection Drive Endemic Cycles of Coxiellosis on Dairy Farms.

Coxiella (C.) burnetii, a zoonotic bacterium, is prevalent in dairy farms. Some cows develop a persistent infection and shed C. burnetii into milk and occasionally by amniotic fluid at calving. Serological diagnosis of Q fever in humans is performed by phase (Ph)-specific antibody tests; PhII antibodies usually indicate an acute infection, while the development of a chronic infection is characterised by elevated PhI antibody titres. Phase-specific tests have now been established for diagnosis of coxiellosis in cattle. Additionally, an interferon-γ (IFN-γ) recall assay has been implemented to assess cellular immunity to C. burnetii in cattle. Milk samples from all lactating cows (n = 2718) of 49 Bavarian dairy farms were collected through a convenience sample and analysed for phase-specific antibodies. Antibody profiles were evaluated by age. Based on the seropositivity of first-lactation cows, three distinct herd profiles were observed: an 'acute' state of herd infection was characterised by a PhI-/PhII+ pattern. The detection of PhI antibodies (PhI+/PhII+) characterised the 'chronic' state, and seronegative results defined the 'silent' state of herd infection. If antibodies had not been detected in multiparous cows, the herd was considered as probably free of coxiellosis. The analysed cattle herds were noted to have an 'acute' (n = 12, 24.5%), 'chronic' (n = 18, 36.8%), or 'silent' state of herd infection (n = 16, 32.6%). Only three farms (6.1%) were classified as 'free' of C. burnetii. The detection of these herd states over a time period of 4 years in one farm indicated that the described states occur in a cyclical manner. Frequently, a wave-like profile was seen, i.e., a circumscribed seronegative age group was flanked by seropositive age groups. In seronegative animals, IFN-γ reactivity was demonstrated. Seroconversion after vaccination was observed by day 7 post-vaccination in chronically infected herds, whereas in the case of silent infection, it started by day 14. These data indicated a pre-existing immunity in seronegative animals in chronically infected herds. Additionally, IFN-γ reactivity was detected in seronegative calves (>3 months) and heifers from chronically infected farms compared to a negative farm. An infection prior to 3 months of age resulted in cellular immunity in the absence of detectable antibodies. An infection around calving would explain this. The aforementioned circumscribed seronegative age groups are, therefore, explained by an infection early in life during active shedding at calving. Based on these results, an endemic cycle of coxiellosis is proposed: Susceptible young heifers get infected by persistently infected cows. Subsequently, shedding of C. burnetii at calving results in infection and then in cellular immunity in offspring. When these calves enter the cow herd two years later, a maximum of herd immunity is achieved, shedding ceases, and new susceptible animals are raised. In an acutely infected dairy farm, the PhI+/PhII+ serological pattern prevailed in second-lactation cows. In this study, stored sera collected since birth were analysed retrospectively. From the earliest seroconversion, the peak of seroconversion took about 33 months. These data suggested a slow spread of infection within herds. The classification of dairy cow herds is a promising basis for further analysis of the clinical impact of coxiellosis.

Interdisciplinary studies on Coxiella burnetii: From molecular to cellular, to host, to one health research.

The Q-GAPS (Q fever GermAn interdisciplinary Program for reSearch) consortium was launched in 2017 as a German consortium of more than 20 scientists with exceptional expertise, competence, and substantial knowledge in the field of the Q fever pathogen Coxiella (C.) burnetii. C. burnetii exemplifies as a zoonotic pathogen the challenges of zoonotic disease control and prophylaxis in human, animal, and environmental settings in a One Health approach. An interdisciplinary approach to studying the pathogen is essential to address unresolved questions about the epidemiology, immunology, pathogenesis, surveillance, and control of C. burnetii. In more than five years, Q-GAPS has provided new insights into pathogenicity and interaction with host defense mechanisms. The consortium has also investigated vaccine efficacy and application in animal reservoirs and identified expanded phenotypic and genotypic characteristics of C. burnetii and their epidemiological significance. In addition, conceptual principles for controlling, surveilling, and preventing zoonotic Q fever infections were developed and prepared for specific target groups. All findings have been continuously integrated into a Web-based, interactive, freely accessible knowledge and information platform (www.q-gaps.de), which also contains Q fever guidelines to support public health institutions in controlling and preventing Q fever. In this review, we will summarize our results and show an example of how an interdisciplinary consortium provides knowledge and better tools to control a zoonotic pathogen at the national level.

Corrigendum: Sero-epidemiological study of zoonotic bacterial abortifacient agents in small ruminants.

[This corrects the article DOI: 10.3389/fvets.2023.1195274.].

White-Toothed Shrews (Genus Crocidura): Potential Reservoirs for Zoonotic Leptospira spp. and Arthropod-Borne Pathogens?

Three species of white-toothed shrews of the order Eulipotyphla are present in central Europe: the bicolored (Crocidura leucodon), greater (Crocidura russula) and lesser (Crocidura suaveolens) white-toothed shrews. Their precise distribution in Germany is ill-defined and little is known about them as reservoirs for zoonotic pathogens (Leptospira spp., Coxiella burnetii, Brucella spp., Anaplasma phagocytophilum, Babesia spp., Neoehrlichia mikurensis and Bartonella spp.). We investigated 372 Crocidura spp. from Germany (n = 341), Austria (n = 18), Luxembourg (n = 2) and Slovakia (n = 11). West European hedgehogs (Erinaceus europaeus) were added to compare the presence of pathogens in co-occurring insectivores. Crocidura russula were distributed mainly in western and C. suaveolens mainly in north-eastern Germany. Crocidura leucodon occurred in overlapping ranges with the other shrews. Leptospira spp. DNA was detected in 28/227 C. russula and 2/78 C. leucodon samples. Further characterization revealed that Leptospira kirschneri had a sequence type (ST) 100. Neoehrlichia mikurensis DNA was detected in spleen tissue from 2/213 C. russula samples. Hedgehogs carried DNA from L. kirschneri (ST 100), L. interrogans (ST 24), A. phagocytophilum and two Bartonella species. This study improves the knowledge of the current distribution of Crocidura shrews and identifies C. russula as carrier of Leptospira kirschneri. However, shrews seem to play little-to-no role in the circulation of the arthropod-borne pathogens investigated.

Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever.

Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis-aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti-microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1-/- mice caused increased C. burnetii burden, weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1-/- mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1-/- mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1-derived itaconate is a key factor in the macrophage-mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever.

Seroprevalence and Molecular Evidence of Coxiella burnetii in Dromedary Camels of Pakistan.

Coxiellosis is a zoonosis in animals caused by Coxiella burnetii. A cross-sectional study was conducted on 920 (591 female and 329 male) randomly selected camels (Camelus dromedarius) of different age groups from 13 districts representative of the three different ecological zones in the Province Punjab, Pakistan to determine the prevalence and associated risk factors of coxiellosis. The blood samples were collected and tested for anti-C. burnetti antibodies using indirect multispecies ELISA. Real-time PCR was used for the detection of C. burnetii DNA to determine the prevalence in heparinized blood pools. Out of 920 investigated camels, anti-C. burnetii antibodies were detected in 288 samples (31.3%) (95% CI: 28.3-34.4%). The highest (78.6%) and lowest (1.8%) seroprevalence were detected in Rahimyar Khan (southern Punjab) and in Jhang (central Punjab), respectively. Potential risk factors associated with seropositivity of the Q fever in camels included desert area (42.5%; OR = 2.78, 95% CI 1.12-3.21) summer season (35.7%; OR = 2.3, 95% CI: 1.31-3.2), sex (female) (39.1; OR = 2.35, 95% CI: 1.34-2.98), tick infestation (51.3%;OR = 2.81, 95% CI: 1.34-3.02), age (>10 years; 46.4%; OR = 1.56, 95% CI: 0.33-2.05) and herd size (38.5%; OR = 1.21, 95% CI: 0.76-1.54). Coxiella burnetii DNA was amplified in 12 (20%) and 1 (10%) of 60 ELISA-negative and 10 suspected camels, respectively. DNA could not be detected in ELISA positive blood pools. This study emphasizes the seroprevalence and associated risk factors of coxiellosis as well as its potential to spill over to animals and humans in contact with these camel herds.

Sero-Epidemiology of Coxiella burnetii Infection in Small Ruminants in the Eastern Region of Punjab, Pakistan.

The aim of this study was to investigate the seroprevalence of Q fever in sheep and goats in Kasur, Okara, and Pakpattan in the Punjab of Pakistan. Q fever is a widely reported zoonotic disease caused by Coxiella (C.) burnetii. The main reservoirs are small ruminants that excrete the bacteria in birth by-products in high numbers. Thus, the bacteria can also be detected in the air and the dust of livestock farms. The infection is often asymptomatic in ruminants, but it can lead to reproductive disorders. This cross-sectional study found that a significant number (n = 43; 11.3%) of 300 randomly selected small ruminants of nine tehsils were seropositive using a commercially available ELISA. Seroprevalence was significantly higher in goats (17.1%) than in sheep (4.9%). Binary logistic regression analysis proved that species, age, and breed have a significant effect on the prevalence of Q fever. Tick infestation, contact with animal fomites, contact with other animals, production system, and health status of an animal had a significant impact on the prevalence of Q fever. These findings on Q fever in animals can be used to improve the visibility of this zoonotic disease. These findings will help local health authorities to focus on the origin of the problem and facilitate applying preventive measures to the affected livestock farms.

Serological Prevalence of and Risk Factors for Coxiella burnetti Infection in Women of Punjab Province, Pakistan.

Background: Coxiella burnetii, the etiological agent of Q (query) fever, provokes abortions in ruminants and is suspected to cause adverse pregnancy outcomes in women. Infection of pregnant women is linked with high mortality and morbidity of the fetus and the mother is at high risk to acquire chronic Q fever. This research was conducted to evaluate the prevalence of Q fever in women and to detect associated risk factors in four districts of Punjab Province, Pakistan. Methods: A total of 297 blood samples were obtained from 147 pregnant and 150 non-pregnant women of the districts Okara, Jhang, Chiniot and Faisalabad of Punjab, Pakistan. Data related to risk factors and demographic parameters were collected using a questionnaire. Serum samples were screened for phase I and phase II specific IgG antibodies for antigens of phase I and phase II using ELISA tests. Univariate and binary regression were used to analyze important risk factors of Q fever. Results: Twenty-five serum samples (8.4%) were found seropositive for Q fever. Seventeen women were positive for Phase-I and twenty-one were positive for phase-II antibodies. Highest and statistically significant (p < 0.05) seroprevalence of 17.1% was observed in Faisalabad. Age, urbanicity, living status, pregnancy status, abortion history, occupation, and consumption of tap water were positively correlated (p < 0.05) with Q fever, while being aged, urbanity, low income, contact with animals and consumption of tap water was identified as potential risk factors. Conclusions: Q fever is prevalent in women of Pakistan. There is a need for an awareness program about the importance of C. burnetii infections and prevention strategies in women during pregnancy to minimize adverse pregnancy outcomes.

Zoonotic bacteria in clinically healthy goats in petting zoo settings of zoological gardens in Germany.

Goats and other small ruminants are frequently used as contact animals in petting zoo settings of zoological gardens. However, they are capable to carry a broad spectrum of zoonotic pathogens without clinical signs. In this study, we analysed the presence of different zoonotic pathogens in 300 clinically healthy goats from 14 zoological gardens in Germany. Rectal and nasal swabs were investigated with a series of cultural and molecular techniques. In addition, vaginal swabs of the 230 female goats were investigated for the presence of Coxiella burnetii by real-time PCR. Antibodies against C. burnetii were tested in milk and serum by ELISA. Campylobacter spp. were found in 22.7%, Shiga-toxigenic Escherichia coli in 20.0% and Arcobacter spp. were found in 1.7% of the tested 300 goats after culture from rectal swabs and subsequent PCR. One sample contained an Escherichia fergusonii isolate with a blaCTX-M-1 -encoded extended-spectrum beta-lactamase phenotype. Neither Yersinia spp. nor Salmonella spp. were found. Nasal swabs of 20.7% of the goats yielded Staphylococcus aureus including one mecC-positive methicillin-resistant isolate. Neither Yersinia spp. nor Salmonella spp. were found, and none of the 230 vaginal swabs was positive for C. burnetii. Attempts to detect dermatophytes failed. In conclusion, a possible risk of transmission of zoonotic bacteria from goats in petting zoos to visitors should be considered. Appropriate information and facilities for hand washing and disinfection should be provided in all zoological gardens using goats as contact animals due to the regular presence of zoonotic bacteria in the collection.

Research Trends and Hotspots of Q Fever Research: A Bibliometric Analysis 1990-2019.

Q fever is a worldwide distributed zoonosis caused by Coxiella burnetii, a Gram-negative bacterium. Despite existence of large amount of research data on the developments related to Q fever, no bibliometric analysis of this subject is available to our knowledge. Bibliometric studies are an essential resource to track scholarly trends and research output in a subject. This study is aimed at reporting a bibliometric analysis of publications related to Q fever (2,840 articles published in the period 1990-2019) retrieved from Science Citation Index Expanded, an online database of Clarivate Analytics Web of Science Core Collection. Data was retrieved using keywords "Q fever" or "Coxiella burnetii" in title, abstract, and author keywords to describe important research indicators such as the kind and language of articles, the most important publications, research journals and categories, authors, institutions, and the countries having the most significant contribution to this subject. Finally, the emerging areas in field of diagnosis, host range, and clinical presentation were identified. Word cluster analysis of research related to Q fever revealed that major focus of research has been on zoonosis, seroprevalence, laboratory diagnosis (mainly using ELISA and PCR), clinical manifestations (abortion and endocarditis), vectors (ticks), and hosts (sheep, goat, and cattle). This bibliometric study is intended to visualize the existing research landscape and future trends in Q fever to assist in future knowledge exchange and research collaborations.

Molecular Typing of Burkholderia mallei Isolates from Equids with Glanders, India.

We collected 10 Burkholderia mallei isolates from equids in 9 districts in India during glanders outbreaks in 2013-2016. Multilocus variable-number tandem-repeat analysis showed 7 outbreak area-related genotypes. The study highlights the utility of this analysis for epidemiologically tracing of specific B. mallei isolates during outbreaks.

The Prevalence of Coxiella burnetii in Hard Ticks in Europe and Their Role in Q Fever Transmission Revisited-A Systematic Review.

The zoonosis Q fever is caused by the obligate intracellular bacterium Coxiella burnetii. Besides the main transmission route via inhalation of contaminated aerosols, ticks are discussed as vectors since the first isolation of the pathogen from a Dermacentor andersonii tick. The rare detection of C. burnetii in ticks and the difficult differentiation of C. burnetii from Coxiella-like endosymbionts (CLEs) are questioning the relevance of ticks in the epidemiology of Q fever. In this review, literature databases were systematically searched for recent prevalence studies concerning C. burnetii in ticks in Europe and experimental studies evaluating the vector competence of tick species. A total of 72 prevalence studies were included and evaluated regarding DNA detection methods and collection methods, country, and tested tick species. Specimens of more than 25 different tick species were collected in 23 European countries. Overall, an average prevalence of 4.8% was determined. However, in half of the studies, no Coxiella-DNA was detected. In Southern European countries, a significantly higher prevalence was observed, possibly related to the abundance of different tick species here, namely Hyalomma spp. and Rhipicephalus spp. In comparison, a similar proportion of studies used ticks sampled by flagging and dragging or tick collection from animals, under 30% of the total tick samples derived from the latter. There was no significant difference in the various target genes used for the molecular test. In most of the studies, no distinction was made between C. burnetii and CLEs. The application of specific detection methods and the confirmation of positive results are crucial to determine the role of ticks in Q fever transmission. Only two studies were available, which assessed the vector competence of ticks for C. burnetii in the last 20 years, demonstrating the need for further research.

Inactivation Kinetics of Coxiella burnetii During High-Temperature Short-Time Pasteurization of Milk.

The Gram-negative, obligate intracellular bacterium Coxiella burnetii is the causative organism of the zoonosis Q fever and is known for its resistance toward various intra- and extracellular stressors. Infected ruminants such as cattle, sheep, and goats can shed the pathogen in their milk. Pasteurization of raw milk was introduced for the inactivation of C. burnetii and other milk-borne pathogens. Legal regulations for the pasteurization of milk are mostly based on recommendations of the Codex Alimentarius. As described there, C. burnetii is considered as the most heat-resistant non-spore-forming bacterial pathogen in milk and has to be reduced by at least 5 log10-steps during the pasteurization process. However, the corresponding inactivation data for C. burnetii originate from experiments performed more than 60 years ago. Recent scientific findings and the technological progress of modern pasteurization equipment indicate that C. burnetii is potentially more effectively inactivated during pasteurization than demanded in the Codex Alimentarius. In the present study, ultra-high heat-treated milk was inoculated with different C. burnetii field isolates and subsequently heat-treated in a pilot-plant pasteurizer. Kinetic inactivation data in terms of D- and z-values were determined and used for the calculation of heat-dependent log reduction. With regard to the mandatory 5 log10-step reduction of the pathogen, the efficacy of the established heat treatment regime was confirmed, and, in addition, a reduction of the pasteurization temperature seems feasible.

Evaluation of the Diagnostic Potential of Recombinant Coxiella burnetii Com1 in an ELISA for the Diagnosis of Q Fever in Sheep, Goats and Cattle.

Coxiella burnetii is the causative agent of Q fever, a zoonosis infecting domestic ruminants and humans. Currently used routine diagnostic tools offer limited sensitivity and specificity and symptomless infected animals may be missed. Therefore, diagnostic tools of higher sensitivity and specificity must be developed. For this purpose, the C. burnetii outer membrane protein Com1 was cloned and expressed in Escherichia coli. The His-tagged recombinant protein was purified and used in an indirect enzyme-linked immunosorbent assay (ELISA). Assay performance was tested with more than 400 positive and negative sera from sheep, goats and cattle from 36 locations. Calculation of sensitivity and specificity was undertaken using receiver operating characteristic (ROC) curves. The sensitivities and specificities for sheep were 85% and 68% (optical density at 450nm, OD450 cut-off value 0.32), for goats 94% and 77% (OD450 cut-off value 0.23) and for cattle 71% and 70% (OD450 cut-off value 0.18), respectively. These results correspond to excellent, outstanding and acceptable discrimination of positive and negative sera. In summary, recombinant Com1 can provide a basis for more sensitive and specific diagnostic tools in veterinary medicine.

Performance Analysis of Anaplasma Antibody Competitive ELISA Using the ROC Curve for Screening of Anaplasmosis in Camel Populations in Egypt.

Anaplasmosis is a tick-born and potential zoonotic disease caused by Anaplasma (A.) phagocytophilum, A. ovis, A. platys and A. capra. Anaplasma marginale affecting bovines and camels causing significant economic losses. Camels as an integral part of the socio-economic lifestyle of nomads in semi-arid to arid ecosystems are prone to suffer from subclinical Anaplasma infections. This study aimed to determine the performance and adaptation of commercial competitive Anaplasma ELISA (cELISA) as a tool for screening the seroprevalence of anaplasmosis whitin the camel populations in Egypt. This study was based on the serological investigation of 437 camel sera collected between 2015 and 2016 during a Q fever prevalence study in Egypt using commercially available cELISA for the detection of antibodies specific for Anaplasma in bovine serum. The receiver operating characteristic (ROC) curve, an analysis method for optimizing cutoff values in cELISAs, was used to estimate the sensitivity and specificity using 76 true as serological positive (n = 7) and negative (n = 60) for Anaplasma antibodies. ROC curve analysis was done for 7 true positive and 60 true negative bovine samples and 7 true positive and 29 true negative camel samples serum. Real time PCR and/or conventional PCR was applied to confirm Anaplasma spp. specific-DNA in camel serum as an indication of a true positive and true negative for ROC analysis. Chi square analysis was performed to estimate the association between risk factors and anaplasmosis in camels. The cutoff value was determined as 0.42 (p value ≤ 0.001). Data simulation with randomly generated values revealed a cutoff value of 0.417 (p ≤ 0.001) with resulting 58.1% Se and 97.8% Sp. Seven true positive and 29 true negative camel serum samples was confirmed by PCR. Using the estimated cut off, the seroprevalence in the Nile Valley and Delta and the Eastern Desert domain was 47.4% and 46.4%, respectively. The potential risk factors as domains and origin of animals were less significantly associated with the prevalence of anaplasmosis (domains: χ(2) = 41.8, p value ≤ 0.001 and origin: χ(2) = 42.56, p value ≤ 0.001). Raising awareness especially for veterinarians and animal owners will significantly contribute to the best understanding of anaplasmosis in camels in Egypt. Alternative (in silico) validation techniques and preliminary prevalence studies are mandatory towards the control of neglected anaplasmosis in the camel population.

Uptake and fecal excretion of Coxiella burnetii by Ixodes ricinus and Dermacentor marginatus ticks.

BACKGROUND: The bacterium Coxiella burnetii is the etiological agent of Q fever and is mainly transmitted via inhalation of infectious aerosols. DNA of C. burnetii is frequently detected in ticks, but the role of ticks as vectors in the epidemiology of this agent is still controversial. In this study, Ixodes ricinus and Dermacentor marginatus adults as well as I. ricinus nymphs were fed on blood spiked with C. burnetii in order to study the fate of the bacterium within putative tick vectors. METHODS: Blood-feeding experiments were performed in vitro in silicone-membrane based feeding units. The uptake, fecal excretion and transstadial transmission of C. burnetii was examined by quantitative real-time PCR as well as cultivation of feces and crushed tick filtrates in L-929 mouse fibroblast cells and cell-free culture medium. RESULTS: Ticks successfully fed in the feeding system with engorgement rates ranging from 29% (D. marginatus) to 64% (I. ricinus adults). Coxiella burnetii DNA was detected in the feces of both tick species during and after feeding on blood containing 105 or 106 genomic equivalents per ml blood (GE/ml), but not when fed on blood containing only 104 GE/ml. Isolation and cultivation demonstrated the infectivity of C. burnetii in shed feces. In 25% of the I. ricinus nymphs feeding on inoculated blood, a transstadial transmission to the adult stage was detected. Females that molted from nymphs fed on inoculated blood excreted C. burnetii of up to 106 genomic equivalents per mg of feces. CONCLUSIONS: These findings show that transstadial transmission of C. burnetii occurs in I. ricinus and confirm that I. ricinus is a potential vector for Q fever. Transmission from both tick species might occur by inhalation of feces containing high amounts of viable C. burnetii rather than via tick bites.

Seroprevalence and Molecular Detection of Bovine Anaplasmosis in Egypt.

Bovine anaplasmosis is a tick-borne disease with zoonotic potential, caused by the obligate intracellular bacterium Anaplasma marginale. The disease is distributed worldwide in tropical and subtropical regions. The economic losses from anaplasmosis in animals is of significant importance because it causes severe morbidity and mortality in cattle. Recovered animals may become persistent carriers. Epidemiological information on the actual status of bovine anaplasmosis in Egypt is scarce. Thus, this study aimed to determine anti-Anaplasma antibody and DNA in serum samples using ELISA and PCR, respectively. In total, 758 bovine sera were collected from cattle farms located in 24 Egyptian governorates in 2015 to 2016. Sera were analyzed with the commercially available 'Anaplasma antibody competitive ELISA v2' kit and 'AmpliTest Anaplasma/Ehrlichia spp. real time TaqMan TM PCR. Anaplasma spp. antibodies were detected in 140 (18.5%) (CI: 15.8-21.4%) of the investigated sera by ELISA, and Anaplasma/Ehrlichia-DNA was detected in 40 (5.3%) (CI: 3.8-7.1%) of the positive sera by real time PCR. Co-detection of both Anaplasma spp. and Coxiella burnetii-specific antibodies was proven in 30 (4%) of the investigated sera. The results of this work confirm the significant prevalence of bovine anaplasmosis in Egypt. Raising awareness in decision makers of the public health, veterinarians and animal owners is required to reduce the spread of infection.

Serological and Molecular Investigation of Coxiella burnetii in Small Ruminants and Ticks in Punjab, Pakistan.

Coxiellosis is a zoonotic disease caused by the obligate intracellular bacterium Coxiella burnetii affecting the productive and reproductive capabilities of animals. This study was conducted to gain insight into the seroprevalence of coxiellosis in small ruminants in seven farms of the Punjab, Pakistan. Potential risk factors were assessed. In total, 1000 serum samples (500 from sheep and 500 from goats) and 163 ticks were collected from the ruminants. All these 163 ticks were merged into 55 pools (29 pools for ticks from sheep and 26 pools for ticks from goat). Serum samples were investigated using an indirect ELISA and PCR. Coxiella burnetii DNA was detected in 29 pooled seropositive samples and 11 pooled ticks by real-time qPCR. Serological analysis revealed a prevalence of 15.6% and 15.0% in sheep and goats, respectively. A significant association was found between seropositivity and different variables like district, lactational status, reproductive status, body condition and reproductive disorders. Univariate analysis showed that detection of C. burnetii DNA in tick pools was significantly associated with the presence of ticks on sheep and goats. However, a non-significant association was found for the prevalence of C. burnetii DNA in serum pools. Hence, C. burnetii infection is prevalent in small ruminants and ticks maintained at livestock farms in Punjab, Pakistan.