**5. Laboratory diagnosis**

384 A Bird's-Eye View of Veterinary Medicine

(Prusakowski, 2001), for example experiencing up to 2 years in human faeces, to 6 years in the ticks' feces, 2-3 years in the water, 1 year in the fur, 1 month in meat, and 1-2 months in dairy products (Little, 1999; Maurin & Raoult, 1999). Physical factors and especially pasteurisation kills germs in 15 seconds, while ultraviolet radiation is not an effective way to

Prevention of *Coxiella burnetii* shedding in cattle is critical to control the spread of the pathogen between animals and from animals to humans. A herd or a flock should be considered clinically infected with Q fever when abortion or stillbirths have occurred, positive PCR results confirming the presence of *Coxiella burnetii* on specimens from affected animals, positive results in the serology test. In cattle herds, the occurrence of a series of abortion: for herds less than 100 animals 2 abortion or more in the month or 3 abortions in the year; for herds more than 100 animals more than 4% of cows with abortions in the year,

Proceedings in the environment of healthy animals include a serological test of animals fur, quarantine of imported animals, the prohibition on animal breeding farms from areas where infection occurred, the serological tests of crossing animals, the fight against ticks and rodents. If Q fever occurs, the following activities should be performed: the district veterinarian must notify diagnostic laboratory indicating a positive result, sporadic cases should be eliminated, the animals should be selected to serologically positive and negative in the case of numerous infections in the herd, the dynamics of antibodies should be studied in animals showing the presence of antibodies, disinfection of rooms should be performed, farrowing should be separated, placenta and the stillborn foetuses should be protected after

The newest study made in Denmark reported an increased risk of becoming infected with *Coxiella burnetii* for herds where farmers had bought cattle from other herds. Therefore, as a general rule, the purchase of animals should be avoided as much is possible, even if tests before introduction are implemented (EFSA Panel on Animal Health and Welfare

Prevention of *Coxiella burnetii* shedding in cattle or sheep and goat is critical to control the spread the pathogen. Q fever vaccination of animals is not widely used because it is protective and safe only for animals that are uninfected at the time of vaccination. Moreover, extensive evaluation of the protective effect of such a vaccination on animal and human health compared to the cost of such prophylactic measures is lacking. There are two types of vaccines. The first one contains formalin-killed whole-cell vaccine preparations (WCV) and the second one - chloroform methanol-extracted bacterial residue (CMR). A WCV from the Henzerling strain (Q vax, CSL Limited, Parkville, Victoria, Australian) has been commercially available and used from human vaccination in Australia since 1989 (Arricau-Bouvery & Rodolaski, 2005). Among 942 no immune abattoir workers in Australia who were vaccinated, no Q fever case was diagnosed during 18 month whereas 34 cases were recorded among 1,349 unvaccinated controls (Marmion et al., 1984). This vaccination is very useful in the protection of exposed populations such as veterinarians or breeders of animals. A

is the major warning sing to be taken into account (Sidi-Boumedine et al., 2010).

each delivery, the direction of Q fever should be examined in newborn animals.

get rid of this microbe.

**4. Prevention** 

(AHAW), 2010).

The diagnosis of Q fever remains difficult and epidemiological studies. Regarding the diagnosis of Q fever, several tools are available for direct and indirect detection of C*oxiella burnetii*. The studies are often based on the serological investigations. Although, the serological methods are useful. They do not allow for the identification of *Coxiella burnetii* shedding animals. Some infected animals can be seropositive without sheding *Coxiella burnetii*, and the others can shed the bacteria and remain seronegative, what is of great concern and could have an important impact both on animal and public health. Moreover, the serological test can distinguish vaccinated and naturally infected animals. The complement fixation test (CFT) was proposed as an alternative test for international trade by the Office International des Epizooties (OIE). Additionally, alternative tests such as enzymelinked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) can be used for diagnosis Q fever within a local context, and it can also be used during importing and exporting animals following bilateral agreements. At present, there are three ELISA commercial kits for diagnosis Q fever in domestic ruminants whereas commercial kits using the IFA for veterinary investigation are not available. The diagnosis of Q fever is based on the serological test and the immunofluorescence assay. The ELISA and IFA tests are more sensitive than the CFT for detection of antibody response in animals or humans with acute Q fever but not for the detection of antibody response in humans with chronic Q fever and in aborting cows (Sidi-Boumedine et al., 2010). The ELISA and IFA detect antibody in acutephase sera, and they are quite efficient in binding IgM (these antibodies predominante in the acute stage), in contrast to the CF test. Recent infections are difficult to demonstrate serologically by the CFT, because antibodies detected by this test can persist longer than acute stage. However, chronic and persistent infections of *Coxiella burnetii* are difficult to diagnose by the ELISA, because this test is more accurate in the acute stage of Q fever, when the IgM predominate in the response (Jóźwik et al., 2007). Cross-reactions are the biggest source of false negative results during interpreting serological results. Cross-reactions have been described between *Coxiella burnetii* and *Legionella pneumophila* (Deyer et al., 1988; Finidori et al., 1992)*, Legionella micdadei* (Musso & Raoult, 1997), and *Bartonella quintana* and *Bartonella henselae* (La Scola & Raoult, 1996).

Other techniques used for Q fever diagnosis include dot immunobloting, Western blotting, indirect hemolysis test and radioimmunoassay. Western blotting has been reported to be sensitive and specific. The molecular masses of antigens detected vary from 10 to 100 kDa. Antibodies reacting with 50, 80, 160 kDa antigens are considered indicative of chronic Q fever (Blondeau et al, 1990). However Western blotting is very time consuming technique and the results obtained by this technique are not reproducible. Dot blotting and radioimmunobloting

Epidemiology, Zoonotic Aspect and Current Epidemiological Situation of Q Fever in Poland 387

The direct detection of *Coxiella burnetii* without using PCR or real-time PCR is very difficult because this bacteria does not grow on the standard bacteriological media, and isolation is time consuming, difficult and hazardous to perform. Moreover, the biosafety level 3 condition is required for cultivation of *Coxiella burnetii* in embryonated eggs, eukaryotic cell culture and laboratory animals. Although, *Coxiella burnetii* has been successfully isolated in guinea pig, mice and embryonated eggs, such techniques have been abandoned because they are more hazardous than in vitro cell cultures (Maurin & Raoult, 1999). *Coxiella burnetii* can infect embryonated eggs and multiply within the yolk sac. The growth and multiplication of the intracellular bacterium leads to death of the embryo, usually within 14 day postinoculation (Maurin & Raoult, 1999). The most often the Buffalo Green Monkey

Although, possessing a membrane similar to gram-negative bacterium is usually stainable by the Gram techniques. The Gimenze method is usually used to stain *Coxiella burnetii* in

(GMB) cells is use for culturing this microorganism.

Lines: M - marker

1 - positive control, 2 - negative control, 4 – positive sample, 3, 5 - 9 – negative sample

laboratory cultures or clinical specimens (Gimenez, 1964).

Fig. 2. Electrophoresis separation of amplification products.

are sensitive and specific technique but radioimmunoblotting can be performed only in radioactivity equipped laboratories (Cowley et al., 1992, Doller et al., 1984).

PCR is rapid and the most sensitive technique for direct diagnosis of *Coxiella burnetii* and identification of shedding animal. Several PCR diagnostic assays were developed to detect *Coxiella burnetii* DNA including conventional PCR, nested PCR and real-time PCR (Klee et al., 2006). The PCR is adapted to a wide range of samples. The results of conventional PCR are presented in figure 2. The development of real-time PCR technology has recently allowed the quantification of *Coxiella burnetii,* and commercial kits are now available. The most suitable sample use in the PCR are: a vaginal swab, a placenta swab focusing the zones of necrosis or cotyledons presenting lesions, the organs (spleen, lung, liver) or the stomach contents of the aborted foetus. Besides *Coxiella burnetii* is also detected in the shed in faeces and milk. For direct detection of *Coxiella burnetii*, sampling should be targeted at pregnant animals either giving birth normally or aborting. The sampling should be carried out as soon as after abortion or parturition because the shedding level of bacteria decrease very fast. *Coxiella burnetii* genes such as 16S rDNA, sodB, and gltA may be amplified. The threshold for quantitative PCR is not officially approved at the international level. A group of French experts has suggested that abortion in ruminants should be suggested to be caused by *Coxiella burnetii* when at least 104 bacteria per gram of placenta or vaginal swabs are detected. Testing tissues or stomach contents from aborted foetuses, the same group considered that a positive result by quantitative PCR is sufficient to diagnose Q fever as the origin of abortion. For pooled samples, the proposed threshold is 103 bacteria per pool (EFSA Panel on Animal Health and Welfare (AHAW), 2010). Conventional PCR is the most commonly used technique in diagnosis. This technique cannot be completely automated, due to the time between the amplification and revelation of the DNA. Moreover, conventional PCR does not distinguish animals shedding huge amounts of bacteria and those with only few bacteria. The real-time PCR is quantitative method. The fusing media that *Coxiella burnetii* was detected and identified by RT-PCR which, presented bacterial load greater than 104. This concentration reflects active infection by this bacterium. Considering the frequency of secretion accompanying normal parturition and the heavy bacterial load present in the placenta or vaginal mucus of aborting females, a definitive diagnosis of infection with *Coxiella burnetii* as the cause of abortion can only be made if bacterial tissue concentrations are greater than 104 bacteria per gram (Alsaleh et al., 2011).

In human, rapid nested PCR assay may be used for early diagnosis of acute Q fever, using serum as the template and Light-Cycler (Roche Diagnostic, Basel, Switzerland) as the thermal cycler, named the LightCycler nested PCR assay (LCN-PCR). In this techniques the two primer pairs had different hybridisation temperatures, reaction tubes were not opened during amplification. Moreover, it is very fast techniques because both amplification and reamplification were performed within 90 minutes. However, the presence of inhibitors in the serum and usually small amount of bacterial DNA limit the efficiency of PCR for this assay. For early diagnosis of acute Q fever, where LCN-PCR is used, the serology tests should be performed together with serology in the first 2 weeks of the diseases but they should be reserved for seronegative patients in the next 2 weeks and not used later than 4 weeks following onset, when serology is highly sensitive (Fournier & Raoult, 2003).

The direct detection of *Coxiella burnetii* without using PCR or real-time PCR is very difficult because this bacteria does not grow on the standard bacteriological media, and isolation is time consuming, difficult and hazardous to perform. Moreover, the biosafety level 3 condition is required for cultivation of *Coxiella burnetii* in embryonated eggs, eukaryotic cell culture and laboratory animals. Although, *Coxiella burnetii* has been successfully isolated in guinea pig, mice and embryonated eggs, such techniques have been abandoned because they are more hazardous than in vitro cell cultures (Maurin & Raoult, 1999). *Coxiella burnetii* can infect embryonated eggs and multiply within the yolk sac. The growth and multiplication of the intracellular bacterium leads to death of the embryo, usually within 14 day postinoculation (Maurin & Raoult, 1999). The most often the Buffalo Green Monkey (GMB) cells is use for culturing this microorganism.

Although, possessing a membrane similar to gram-negative bacterium is usually stainable by the Gram techniques. The Gimenze method is usually used to stain *Coxiella burnetii* in laboratory cultures or clinical specimens (Gimenez, 1964).

386 A Bird's-Eye View of Veterinary Medicine

are sensitive and specific technique but radioimmunoblotting can be performed only in

PCR is rapid and the most sensitive technique for direct diagnosis of *Coxiella burnetii* and identification of shedding animal. Several PCR diagnostic assays were developed to detect *Coxiella burnetii* DNA including conventional PCR, nested PCR and real-time PCR (Klee et al., 2006). The PCR is adapted to a wide range of samples. The results of conventional PCR are presented in figure 2. The development of real-time PCR technology has recently allowed the quantification of *Coxiella burnetii,* and commercial kits are now available. The most suitable sample use in the PCR are: a vaginal swab, a placenta swab focusing the zones of necrosis or cotyledons presenting lesions, the organs (spleen, lung, liver) or the stomach contents of the aborted foetus. Besides *Coxiella burnetii* is also detected in the shed in faeces and milk. For direct detection of *Coxiella burnetii*, sampling should be targeted at pregnant animals either giving birth normally or aborting. The sampling should be carried out as soon as after abortion or parturition because the shedding level of bacteria decrease very fast. *Coxiella burnetii* genes such as 16S rDNA, sodB, and gltA may be amplified. The threshold for quantitative PCR is not officially approved at the international level. A group of French experts has suggested that abortion in ruminants should be suggested to be caused by *Coxiella burnetii* when at least 104 bacteria per gram of placenta or vaginal swabs are detected. Testing tissues or stomach contents from aborted foetuses, the same group considered that a positive result by quantitative PCR is sufficient to diagnose Q fever as the origin of abortion. For pooled samples, the proposed threshold is 103 bacteria per pool (EFSA Panel on Animal Health and Welfare (AHAW), 2010). Conventional PCR is the most commonly used technique in diagnosis. This technique cannot be completely automated, due to the time between the amplification and revelation of the DNA. Moreover, conventional PCR does not distinguish animals shedding huge amounts of bacteria and those with only few bacteria. The real-time PCR is quantitative method. The fusing media that *Coxiella burnetii* was detected and identified by RT-PCR which, presented bacterial load greater than 104. This concentration reflects active infection by this bacterium. Considering the frequency of secretion accompanying normal parturition and the heavy bacterial load present in the placenta or vaginal mucus of aborting females, a definitive diagnosis of infection with *Coxiella burnetii* as the cause of abortion can only be made if bacterial tissue concentrations are greater than 104 bacteria

In human, rapid nested PCR assay may be used for early diagnosis of acute Q fever, using serum as the template and Light-Cycler (Roche Diagnostic, Basel, Switzerland) as the thermal cycler, named the LightCycler nested PCR assay (LCN-PCR). In this techniques the two primer pairs had different hybridisation temperatures, reaction tubes were not opened during amplification. Moreover, it is very fast techniques because both amplification and reamplification were performed within 90 minutes. However, the presence of inhibitors in the serum and usually small amount of bacterial DNA limit the efficiency of PCR for this assay. For early diagnosis of acute Q fever, where LCN-PCR is used, the serology tests should be performed together with serology in the first 2 weeks of the diseases but they should be reserved for seronegative patients in the next 2 weeks and not used later than 4

weeks following onset, when serology is highly sensitive (Fournier & Raoult, 2003).

radioactivity equipped laboratories (Cowley et al., 1992, Doller et al., 1984).

per gram (Alsaleh et al., 2011).

Lines: M - marker


Fig. 2. Electrophoresis separation of amplification products.

Epidemiology, Zoonotic Aspect and Current Epidemiological Situation of Q Fever in Poland 389

Human infection with *Coxiella burnetii* have been reported from many countries on all continents, including Poland (Lutyński, 1956). The first outbreak of Q fever was diagnosed in Poland in 1956. Since then, few outbreaks of the disease both in humans and animals were recorded in the country. Several cases of human Q fever occurred in Poland, however it is really not known whether this relatively low number of cases of illness is caused by the facts of its occurrence, whether or not all cases are detected and diagnosed. Data on these cases in human are presented in Table 1. Moreover, in recent times at the turn of 2008-2009, seven outbreaks of the disease were detected in cattle. These cases have occurred in the region of south-eastern Poland. In total, the disease was confirmed in more than 340 heads of cattle, the disease has been confirmed in humans handling these animals and in their family members. The other studies carried out in eastern Poland present that cattle may play an important role in epidemiology of Q fever, in addition to sheep and goats which are

> **Duration of** *Coxiella burnetii* **shedding vaginal mucus feces milk**

> > lambing 8 days

**cow** not determined 14 days 13 months **goat** 14 days 20 days 52 days

Table 1. The longest observed duration of *Coxiella burnetii* excretion in ruminant vaginal mucus, milk and feces during the follow-up of naturally or experimentally infected herds

The European Community system for the monitoring and collecting information on zoonoses is established by Directive 2003/99/EC on the monitoring of zoonoses and zoonotic agents. According to this directive, reporting information on Q fever takes place on the basis of the epidemiological situation in the country, what means that the Member States should report the information whether those zoonotic agents are considered to be of importance in their country. For the reporting year 2006, 10 Member States reported information on Q fever in animals. Based on the Regulation of the Minister of Agriculture and Rural Development of 24 June 2010, monitoring tests in the direction of Q fever are conducted in Poland. In order to control Q fever, the blood samples from cattle or sheep and goats located in the different area of country, are tested annually. Studies conducted in the framework of this monitoring in 2010 showed the presence of seropositive units. The first study of blood serum is performed by the field laboratory. If the test result for antibodies to *Coxiella burnetii* is a positive sample, it is sent to the National Reference Laboratory (NRL) for re-implementation of the study. In 2010, the research review in the NRL of Q fever tested a total of 99 samples. The presence of antibodies was confirmed in 81 samples tested. Tests were conducted using the method of CFT. The samples which are confirmed as positive are also tested using real-time PCR. The positive results in this study allows the unambiguous

**6. Prevalence of O fever infection in Poland** 

considered the main reservoirs of *Coxiella burnetii* (Cisak, 2003).

**ewe** 71 days 8 days after

**species of animals** 

(Arricau-Bouvery & Rodolakis, 2005).

confirmation of a case of Q fever.

Fig. 3a. The results of immunofluorescence assay (Friedrich-Loeffler-Institute, Jena)

Fig. 3b. The results of immunofluorescence assay (Friedrich-Loeffler-Institute, Jena)
