SEROPREVALENCE OF COXIELLA BURNETII IN CATTLE IN THE BELGRADE EPIZOOTIOLOGICAL AREA

: Q-fever is antropozoonosis which is caused by Coxiella burnetii, obligate intracellular pathogen. The most significant characteristics of this pathogen are resistance and stability in the environment, possibility of aerosol dissemination, and very low infective dose. C. burnetii can infect domestic and wild animals, rodents, birds and ticks. Q fever in animals is generally asymptomatic, although it can lead to reproductive disorders during pregnancy. The main route of infection in humans is inhalation of contaminated aerosol and dust. Serological studies have shown the presence of antibodies to C. burnetii in the serum samples of cattle in Belgrade epizootiological area. Seroprevalence of 18% was found in farm bred cattle, while it was only 1.5% in individual breeding. In farm bred cows that have suffered abortion prevalence was 49%, and only 1.9% in individual breeding. The overall results indicate that the circulation of this pathogen in cattle, in Belgrade epizootiological area, poses a health risk, not only to the cattle, but also to the humans, especially persons working with animals. Q fever control programs most often recommend serological research and vaccination of animals. Accordingly, it is necessary to define a strategy for the implementation of biosecurity measures and preventive measures against Q fever. antibody, inhalation.


Introduction
Q fever is one of the most well-known anthropozoonoses, present worldwide, with the exception of New Zealand (Angelakis and Raoult, 2010). The disease has also been recorded in Serbia, both in humans and in various species of animals. It was established for the first time in humans in 1937, in Australia (Derrick, 1937). It was clinically manifested by fever, and due to ignorance about working in the laboratory in accordance with the biosafety level standard 3 (OIE, 2018).
C. burnetii is a highly virulent bacterium (Shrestha, 2020). Research has shown that 1-10 viable bacteria (Sawyer et al., 1987), and even one bacterium, is sufficient to cause infection (Waag, 2007). Q fever used to be rare and regionally limited. At the beginning of the 21st century, the disease has spread as a re-emergent zoonosis in many European countries (Gwida et al., 2012;Dijkstra et al., 2012: Pandit et al., 2016. It is assumed that the spread of the disease is due to increased virulence of the pathogen, changes in the clinical picture, application of more reliable tests in diagnostics, as well as changes in epidemiological characteristics (Aricau-Bouvery and Rodolakis, 2005).
C. burnetii can infect a variety of animal species, including domestic animals such as cattle, sheep, goats, dogs, cats, also rodents, wildlife, reptiles, birds, fish, and ticks (Angelakis and Raoult, 2010;Gwida et al., 2012). Ticks play a significant role as reservoirs of pathogens, but also as vectors in the transmission of pathogens, especially from wild to domestic animals (Cantas et al., 2011). In animals, Q fever is mostly asymptomatic, although reproductive disorders such as abortion, stillbirth, placental abruption, and foetal underdevelopment may occur during pregnancy (Gwida et al., 2012;Pexara et al., 2018).
By monitoring of the epidemiological data, it was concluded that humans are most often infected by inhalation of contaminated aerosols and dust particles containing bacteria from infected animals. Most often, this contamination occurs during the birth of infected animals through products such as placenta, amniotic fluid, colostrum, etc. (Maurin and Raoult, 1999;Vidić et al., 2008;Angelakis and Raoult, 2010). Consumption of contaminated raw milk and dairy products is also considered to be a potential source of infection for humans (Boboš et al., 2013;Radinović et al., 2014;Pexara et al., 2018). For people who come in contact with animals, such as veterinarians, livestock breeders, slaughterhouse staff, but also laboratory workers, Q fever is considered an occupational disease (Maurin and Raoul, 1999;OIE, 2018). According to the data of the City Institute for Public Health of Belgrade, in the period from 1984 to 2018, 78 cases of Q fever in humans were recorded in the area of the city (https://www.zdravlje.org.rs/).
The aim of this study was to determine the seroprevalence of C. burnetii in cattle in the Belgrade epizootiological area, whether they come from dairy farms or individual households. Based on the obtained research results, it will be possible to assess the epizootiological situation, and accordingly develop Q fever control programs.

Materials and Methods
Serological surveillance is carried out in many countries with the aim of assessing the prevalence of C. burnetii in domestic ruminants. The Rulebook on the Program of Animal Health Measures adopted annually by the Ministry of Agriculture, Forestry and Water Management of the Republic of Serbia, Veterinary Directorate, stipulates diagnostic tests in cases of abortion of domestic ruminants to Q fever. In accordance with that, the Scientific Veterinary Institute of Serbia conducts serological tests of cows that have had abortions. In addition, animals that had other reproductive disorders or were in circulation were also examined. The samples originated from commercial farms or individual agricultural households with extensive production, and are located in the epizootiological area of the city of Belgrade.
Blood samples for diagnostic tests were delivered during 2017, 2018 and 2019 to the Scientific Veterinary Institute of Serbia, Belgrade, by the competent veterinary stations. Blood sera were tested for the presence of antibodies against C. burnetii using a commercial ELISA test, ID Screen® Q Fever Indirect Multispecies/ ID Vet, Grabels, France. A total of 862 samples were tested, of which 226 originated from aborted cows. Serological tests were performed by stipulated methods performed in accordance with the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE, 2018), and the test results were interpreted according to the instructions of the diagnostic kit manufacturer.

Results and Discusion
Our three-year research included the examination of bovine serum for the presence of antibodies against C. burnetii in cattle from the epizootiological area of the city of Belgrade. The tested sera originated from cattle reared on three commercial farms and cattle from individual agricultural households. A total of 862 samples were analyzed, of which 145 (16.8%) showed the presence of antibodies to C. burnetii. Significantly higher seroprevalence was found in cattle from farms than in cattle from individual agricultural households. The prevalence on farms 1, 2 and 3 was 17.4%, 9.0% and 39.2%, respectively. The antibodies to C. burnetii were found in only one sample of a total of 65 tested samples from individual agricultural households. The results (number/percentage) are shown in Table 1. 226 samples of a total of 862 were from cows after abortion. Of those 226 sera, 175 were from farm animals and 51 were samples of animals from the individual sector. The seroprevalence of Q fever was 38% in cows after abortion. Out of 175 farm animals, 86 (49%) were positive and only 1 sample of 51 cows from individual sector. The results of the analysis (number/percentage) are shown in Table 2. The obtained results show that the highest number of positive animals was established during 2018, as a consequence of the epizootic incidence on Farm 1. We assume that the occurrence of the disease is related to the position of Farm 1, which geographically gravitates to the endemic area of the South Banat district. A milder form of the disease was found on Farm 3, also during 2018, with the remark that fewer blood samples were sampled from this farm. The prevalence of Q fever in the animals with abortion ranged from 47.3% to 66.6% on Farm 1, which was most exposed to the infection. At the same time, only 1 of 51 samples from individual rearing was serologically positive, which indicates a low prevalence, i.e. almost complete absence of Q fever in private sector. Previous research in Serbia in the area of Vojvodina showed that Q fever was found in 9.5% of herds (Vidić et al., 2008), while the percentage of infected heads in the population ranged from 5 to 80% .
The results of the study indicate a correlation between ruminant abortions with C. burnetii infection (Žutić et al., 2019). In Cyprus, 35% of aborted cows, 33% of sheep, and 50% of goats were positive for C. burnetii (Cantas et al., 2011). Lower percentages were found in Italy, where 11.6% of cows and 21.5% of sheep and goats that had aborted were positive for Q fever (Parisi et al., 2006). In France, 2,695 cows, 658 sheep, and 105 goats that had aborted, seroprevalence of C. burnetii was found in 36%, 55.7%, and 61% of the herds, respectively (Gache et al., 2017). In Latvia, seroprevalence was recorded in 13.4% of herds where abortions occurred in dairy cattle (Boroduske et al., 2017). In the Netherlands, 3,264 cases of Q fever in humans were reported during the 2007-2010 epidemic (Dijkstra et al., 2012). Research has shown that the epidemic appeared in a narrower area where dairy goat farms were located, where abortions occurred in waves. The cause of the epidemic is believed to have been the airborne transfer of contaminated dust particles from farms to densely populated areas.
Q fever is a very complex disease in both humans and animals, so control and eradication measures require a series of procedures over a long period of time. Q fever control programs most often recommend serological testing and vaccination of animals. Serological examination can identify positive herds and thus determine risk levels on farms and in the regions (Valčić et al., 2014). Rodents and ticks as reservoirs of pathogens should be systematically destroyed in natural habitats and on farms (Vidić et al., 2012;Vidić et al., 2014). By applying preventive and biosafety control measures, it is possible to reduce environmental contamination, and thus the risk to human and animal health.