Screening for Coxiella Burnetii in Dairy Cattle Herds in Poland.

Introduction: The intracellular bacterium Coxiella burnetii is the aetiological agent of Q fever, a zoonosis affecting many animal species worldwide. Cattle and small ruminants are considered the major reservoirs of the bacteria and they shed it through multiple routes. Material and Methods: A total of 2,180 sera samples from 801 cattle herds in all Polish voivodeships were tested by ELISA for the presence of specific antibodies. Milk samples were obtained from seropositive cows in 133 herds as part of a separate study. The milk samples were examined by ELISA and real-time PCR tests. Results: Seroprevalence at the animal level was 7.06% and true positive seroprevalence was 6.0% (95% confidence interval (CI) 1.1-9.4). Seroprevalence at the herd level was estimated at 11.1% and true positive seroprevalence was 10.5% (95% CI 3.2-15.8). Shedding of the pathogen in milk was detected by real-time PCR in 33 out of 133 tested herds (24.81%, 95% CI 17.74-33.04%) and the presence of C. burnetii antibodies was confirmed in 85 of them (63.9%, 95% CI 55.13-72.05%). The highest level of conformity between ELISA and real-time PCR results was obtained for bulk tank milk samples. Conclusion: Coxiella burnetii infections are quite common in cattle herds across the country, which emphasises the crucial roles of surveillance and adequate biosecurity measures in the prevention and limitation of Q fever spread in Poland.

Experimental inoculation of chicken broilers with C. gallinacea strain 15-56/1.

Chlamydia gallinacea is one of the new Chlamydia species, encountered predominantly in birds and occasionally in cattle, and its dissemination, pathogenicity and zoonotic potential have not yet been fully elucidated. Until now, no case of clinical infection has been described in poultry, but the number of studies is limited. This study was conducted to evaluate the course of infection and the impact on production parameters in chicken broilers inoculated with the strain 15-56/1 isolated from a Polish flock. The presence of C. gallinacea was confirmed in oropharyngeal and cloacal swabs by real-time PCR from the fifth day post inoculation (dpi). Pathogen DNA was also detected in many internal organs of inoculated chickens. All infected animals remained asymptomatic during the entire experimental period, although statistical analyses showed that broilers in the experimental group exhibited significantly lower body weight gains and feed conversion ratios than animals in the control group. These data indicate that subclinical C. gallinacea infection in broilers may lead to financial losses for poultry farmers.

Molecular detection of Coxiella burnetii in small ruminants and genotyping of specimens collected from goats in Poland.

BACKGROUND: Coxiella burnetii is the etiological agent of Q fever, a zoonosis affecting many animal species including sheep and goats. The aims of this study were to evaluate the shedding of Coxiella burnetii in small ruminant herds and to identify the pathogen's genotypes and sequence types (STs) using multiple-locus variable number tandem repeat analysis (MLVA) and multispacer sequence typing (MST) methods. RESULTS: Overall, 165 samples from 43 herds of goats and 9 flocks of sheep were collected including bulk tank milk (BTM), individual milk samples, vaginal swabs, tissue sections from stillborn kids, feces and placentas. These were tested by real-time PCR targeting the IS1111 element. C. burnetii infection was confirmed in 51.16% of the herds of goats and 22.2% of the flocks of sheep. Six out of nine samples originating from goats were successfully genotyped using the MLVA method. The presence was confirmed of two widely distributed MLVA genotypes (I and J) and genotype PL1 previously reported only in cattle. Only one sequence type (ST61) was identified; however, the majority of specimens represented partial STs and some of them may belong to ST61. Other partial STs could possibly be ST74. CONCLUSION: This study confirmed the relatively common occurrence of Coxiella burnetii in small ruminant herds in Poland. Interestingly, all genotyped samples represent cattle-associated MLVA genotypes.

Occurrence of Coxiella burnetii in Polish dairy cattle herds based on serological and PCR tests.

The aim of the research was to assess the prevalence of antibodies to Coxiella burnetii in dairy cattle herds in Poland and to compare the results of real-time PCR and ELISA tests performed on bulk tank milk (BTM) samples. In total, 2635 serum samples collected from 969 dairy cattle herds from all provinces were tested using ELISA. Additionally, BTM specimens from 101 herds were analysed by ELISA and real-time PCR targeting IS1111 element. Presence of anti-C. burnetii antibodies was confirmed in 25.39% of serum samples in 237 herds (24.46%) and the herd-level seroprevalence in Voivodeships varied from 2.5% to 61.4%. Moreover, 46 (45.5%) of analysed bulk tank milk samples gave postive result in ELISA and microbial DNA was detected in 40 (39.6%) of tested herds. The comparative analysis of ELISA and real-time PCR results obtained for BTM samples using the chi-square test showed statistically significant relationship between results of both methods.

Evaluation of the Possibility of C. Burnetii Transmission by the Alimentary Route in a Guinea Pig Model.

INTRODUCTION: Q fever (coxiellosis) is an infectious disease of animals and humans, caused by.C. burnetii and widely distributed throughout the world. It is known that people and animals acquire the disease predominantly.via inhalation of infectious aerosols. The possibility of transmission of the pathogen by the alimentary route is still a matter of debate and remains controversial. Therefore the aim of this study was to fill the gaps in knowledge of oral transmission of.C. burnetii by conducting biological tests on the guinea pig model. MATERIAL AND METHODS: Guinea pigs, divided into five groups comprising a negative control and four experimental groups, received specified concentrations of.C. burnetii per os. To determine the presence of specific antibodies, blood samples were tested using CFT. Also, internal organs collected during necropsy were screened by a real-time PCR targeting I.1111. Additionally, histopathological evaluation of the tissues was performed. RESULTS: The presence of antibodies and pathogen DNA in caecum was confirmed in one guinea pig from experimental group IV..C. burnetii was also detected in testicular tissue collected from one animal of experimental group II. CONCLUSIONS: The presence of pathogen DNA in the testicular tissue indicates that infection spreads haematogenously. In the majority of experimental animals specific antibodies and genetic material of.C. burnetii were not detected. This fact suggests that development of infection depends on many factors, such as animal immune status.

Shedding and genetic diversity of Coxiella burnetii in Polish dairy cattle.

Q fever is a worldwide zoonotic disease reported in humans and many animal species including cattle. The aims of this study were to evaluate the prevalence of Coxiella (C.) burnetii shedding in Polish dairy cattle herds and to identify the pathogen's genotypes and sequence types (STs) using multiple-locus variable number tandem repeat analysis (MLVA) and multispacer sequence typing (MST) methods. The presence of C. burnetii DNA was detected using a commercial real-time PCR kit, targeting the IS1111 element. Overall, 1,439 samples from 279 herds were tested including: 897 individual milk specimens, 101 bulk tank milk samples, 409 genital tract swabs and 32 placentas. Furthermore, 30 consumer milk samples, including 10 from vending machines and 77 dairy products were also analyzed. C. burnetii shedding was confirmed in 31.54% of tested cattle herds as well as in 69.16% of consumer milk and dairy products. Among real-time PCR-positive samples, 49 specimens obtained from 49 cattle herds and 8 samples of purchased dairy products were selected for genotyping. Overall, five previously known MLVA genotypes (I, J, BG, BE, and NM) and three new ones (proposed as PL1, PL2, and PL3) were identified. Two MST sequence types were recorded: ST16 and a novel sequence (ST61). The new genotypes and sequence types need further research particularly into their pathogenicity to humans.

Dissemination and genetic diversity of chlamydial agents in Polish wildfowl: Isolation and molecular characterisation of avian Chlamydia abortus strains.

Wild birds are considered as a reservoir for avian chlamydiosis posing a potential infectious threat to domestic poultry and humans. Analysis of 894 cloacal or fecal swabs from free-living birds in Poland revealed an overall Chlamydiaceae prevalence of 14.8% (n = 132) with the highest prevalence noted in Anatidae (19.7%) and Corvidae (13.4%). Further testing conducted with species-specific real-time PCR showed that 65 samples (49.2%) were positive for C. psittaci whereas only one was positive for C. avium. To classify the non-identified chlamydial agents and to genotype the C. psittaci and C. avium-positive samples, specimens were subjected to ompA-PCR and sequencing (n = 83). The ompA-based NJ dendrogram revealed that only 23 out of 83 sequences were assigned to C. psittaci, in particular to four clades representing the previously described C. psittaci genotypes B, C, Mat116 and 1V. Whereas the 59 remaining sequences were assigned to two new clades named G1 and G2, each one including sequences recently obtained from chlamydiae detected in Swedish wetland birds. G1 (18 samples from Anatidae and Rallidae) grouped closely together with genotype 1V and in relative proximity to several C. abortus isolates, and G2 (41 samples from Anatidae and Corvidae) grouped closely to C. psittaci strains of the classical ABE cluster, Matt116 and M56. Finally, deep molecular analysis of four representative isolates of genotypes 1V, G1 and G2 based on 16S rRNA, IGS and partial 23S rRNA sequences as well as MLST clearly classify these isolates within the C. abortus species. Consequently, we propose an expansion of the C. abortus species to include not only the classical isolates of mammalian origin, but also avian isolates so far referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates.

Evaluation of qPCR and phase I and II antibodies for detection of Coxiella burnetii infection in cattle.

Diagnosis of Q fever in cattle is not easy due to the need to test the samples by both serological and molecular methods. Aim of this study was to evaluate qPCR, and phase I and II antibodies for detection of C. burnetii infection in cattle. A total of 187 bovine blood and vaginal swabs, and 97 milk samples, were tested. Limitations of serological tests were that the available indirect enzyme linked immunosorbent assay (iELISA) could lose positive results if antibody titres were low; or phase II antibodies were present. The highest level of correlation between iELISA and complement fixation test (CFT) was noted with the antigen specific phase I antibodies. Neither the mode of shedding nor its intensity correlated with phase I and II antibodies, but positive results in CFT mixed-phase and shedding in vaginal mucous did correlate, and showed the highest correlation. Antigenic diversity, and variability could be crucial in laboratory diagnosis of Q fever.