Abstract: The present study was carried out to determine the seroprevalence of antibodies to C. burnetii in cattle and sheep in the east of Turkey. Serum samples collected randomly from 92 cattle and 92 sheep were examined by ELISA (Vircell-SL, Spain) to detect IgG antibodies against C. burnetii phase II antigen. Seropositivity was observed in 16.3% of the cattle and in 5.4% of the sheep. Coxiellosis has an important seropositivity in both cattle and sheep and it can cause serious health problem in humans living in Eastern Turkey.
INTRODUCTION
Q fever (query fever), a zoonosis caused by obligate intracellular microorganism Coxiella burnetii, is endemic throughout the world with the exception of Antarctica and possibly New Zealand, occurring in diverse geographic regions and climatic zones and infects arthropods, birds, pets, domestic and wild mammals, as well as humans (Woldehiwet, 2004; Rodolakis, 2006).
Natural reservoirs are more than 40 species of ticks, which remain infected life-long and which transmit C. burnetii transovarially and free-living vertebrates. Tick feces may contain large amounts (up to 1010 infectious units g-1) of C. burnetii (Norlander, 2000).
The main sources of environmental contamination and infection of humans are parturient ruminants, dogs and cats infected with C. burnetii (Woldehiwet, 2004). The main route of C. burnetii infection is by inhalation of contaminated aerosols or dusts containing the microorganism shed from infected animals. The source of human infection is often unidentified, although sheep and goats are more frequently involved in the disease cycle than other animal species. Infected animals shed highly stable bacteria in urine, feces, milk and through placental and birth fluids (Marrie, 2003).
Ccoxiella burnetii is very stable in the environment, resisting to elevated temperature, desiccation, osmotic shock, ultra-violet light and disinfectants (Arricau-Bouvery and Rodolakis, 2005). Oral transmission, by ingestion of contaminated raw milk or dairy products could lead to seroconversion in few cases to Q fever (Cutler et al., 2002; McQuiston et al., 2002; Rodolakis, 2006).
Until recently, the agent has been regarded as economically unimportant for domestic livestock. However, C. burnetii has been detected in a number of cases of abortion in different parts of the world (Waldham et al., 1978; Palmer et al., 1983; Raju et al., 1988; Zeman et al., 1989) including Turkey. As a result, the role of C. burnetii in reproductive disorders of livestock has been questioned (Cetinkaya et al., 2000). Although, the disease may cause agalactia (Kelly et al., 1993) and infertility in addition to sporadic abortions in heavily infected animals, it usually does not cause obvious clinical signs in ruminant populations.
In animals, C. burnetii can induce pneumonia as well as abortion, stillbirth and delivery of weak lambs, calves or kids, which are the most frequent clinical signs of the disease. In the majority of the cases, abortion occurs at the end of gestation without specific clinical signs until abortion is imminent, as observed with brucellosis or chlamydiosis. Aborted fetuses appear normal but infected placentas exhibit intercotyledonary fibrous thickening and discolored exudates, which are not specific to Q fever. A severe inflammatory response is observed in the myometrium and the stroma adjacent to the placentomal area during gestation in goats (Abe et al., 2001). From time to time clinical signs mentioned above have seen in the farm animals in this region. Taken into consideration of these clinical signs, the possibility of Q fever incidence in the region were therefore investigated serologically.
Isolation of C. burnetii is not performed for routine diagnosis in veterinary medicine, because the cultivation of the agent is a laborious and hazardous process (Rodolakis, 2006).
The diagnosis of Q fever remains difficult and epidemiological studies are often based only on serological investigations. These include Complement Fixation Test (CFT), Indirect Flourescent Antibody Test (IFAT), Capillary Agglutination (CA) test, indirect immunoflourescence assay (IFA), microagglutination test (MA), ELISA and PCR. Routine diagnosis of Q fever in aborted animals is usually established by examination of fixed impressions or smears prepared from the placenta stained by the Stamp, Gimenez or Machiavello methods, associated with serological tests. Because farm animals are the major source of the infection for people, it is important to determine the prevalence of coxiellosis in these species. The present study was carried out to determine the prevalence of coxiellosis in cattle and sheep in the eastern part of Turkey, using ELISA.
MATERIALS AND METHODS
Sample Collection
A total of 184 blood samples (92 samples each of cattle and sheep) were
collected by a simple random sampling method from 16 herds and flocks as well,
representing 16 locations (Van, Baskale, Caldıran, Catak, Edremit, Ercis,
Gevas, Gürpınar, Muradiye, Ozalp, Bitlis, Adilcevaz, Ahlat, Tatvan,
Mus, Dogubayezıt) in the eastern part of Turkey (Fig. 1)
during 2006-2008 years. The herds and flocks were selected randomly.
| Fig. 1: | The sites of blood samples collection at the Eastern part of Turkey |
No criteria such as size of herd or flock were used in the selection of the herds and flocks. All animals were adult females and males. The 5-7 mL blood were collected from flocks were selected randomly. No criteria such as size of herd or flock were used in the selection of the jugular vein aseptically using Vacutainer® tubes containing separator gel by qualified personnel and kept on ice while being transferred to the Laboratory of Microbiology and Clinical Microbiology, Medical Faculty at Yüzüncü Yil University, where serum was separated by centrifugation (3000 rpm for 10 min) and stored frozen at -20°C until required.
Test Procedure
Serum samples were tested for C. burnetii antibodies using ELISA
test and Labsystems iEMS Reader MicroELISA system (Finland) was used. Commercial
ELISA kit (Vircell SL®, Spain) was used to detect IgG antibodies
against C. burnetii phase II antigen. The test was carried out according
to the instructions of the manufacturer. Four wells (two for the cut off serum
and one each for the negative and positive sera) in each ELISA plate were used
as control. The mean OD for the cutoff serum and antibody index was calculated
with the following formula:
Samples were interpreted as: samples with indexes <9.0 were considered as not having IgG antibodies; samples with equovocal results (AI = 9.0-11.0) being retested for confirmation and samples with indexes >11 were considered as having IgG antibodies against C. burnetii.
RESULTS AND DISCUSSION
Coxiella burnetii seropositivity reactions were obtained 10.9% of the sera tested from all animals (20 of 184). The highest percentage of positive animals was for cattle (%16.3) (Table 1).
The interest for Q fever is increasing worldwide as indicated by the rising number of reviews published (Arricau Bouvery and Rodolakis, 2005) even in countries where its incidence is supposed to be very low. Indeed, the disease is considered as a re-emerging zoonosis in many countries. This could be due to the evolution of its epidemiology, or of the agent, which could become more virulent, to modifications of its clinical signs, to an improvement of the sensitivity of diagnostic tests, or because practitioners are better informed and look for it more often (Arricau Bouvery and Rodolakis, 2005).
Past investigation confirmed that some infections occur frequently in farmers, where the contact with cattle and sheep is associated with Q fever (Coleman, 2000). Salinas-Melendez et al. (2002) showed that exposure to C. burnetii is common in animals in the state of Nuevo Leon Mx. The risk of Q fever on people who work with domestic animals is related to contact with farm environment rather that any specific animal exposure (Thomas et al., 1995).
Infection of livestock with C. burnetii often goes unnoticed. Indeed,
sporadic abortion of up to 5% is considered normal and does not prompt investigation.
Furthermore, the lack of overt clinical signs exhibited among infected livestock
make diagnosis a challenge (Cutler et al., 2007).
The zoonosis must be considered as a truly global problem, both in terms of
their distribution and the measures required for their control.
| Table 1: | Serological status of cattle and sheep against C. burnetii |
Q fever is a zoonosis that may occur worldwide, in all geographic and climatic zones. Coxiella burnetii is a highly infectious agent that is widespread among livestock around the world (Arricau Bouvery and Rodolakis, 2005). Its resistance to chemical and physical agents is exceptional and enables the organism to maintain itself in nature without an arthropod vector and makes air-borne dissemination of infection possible (Little, 1983).
The distribution of Boophilus ticks and a high seroprevalence in animals suggest that these ticks play a role in the transmission and maintenance of C. burnetii infection in cattle (Schutt et al., 1976). The feces of ticks infected are heavily contaminated with the microorganisms, which remain viable for long period of time and therefore may be a potential source of infection for man and animals (Peacock et al., 1983). Such infected feces may become powdered and windborne, thereby infecting the upper respiratory tract of man and animals (Salmon et al., 1982). Coxiella burnetii was detected in 5 of 10 dust samples from a barn housing dairy cattle by the PCR (Yanase, 1998).
Because of the polymorphism of the clinical picture and because the diagnosis is based exclusively on serology, the prevalence of C. burnetii infection among animals is largely unknown (Rey et al., 2000). The studies were mostly depend on serological investigation, worldwide and in Turkey as well (Kalender, 2001).
The ELISA test is more sensitive than the CF test and allows for testing a greater number of animals and flocks (Rodolakis, 2006). ELISA and microimmunoflourescence (MIF) tests give similar results with cow sera, but the ELISA test is more sensitive than the MIF test with goat and sheep sera (Arricau-Bouvery et al., 2003).
Similar study was performed in Turkey and in Elazıg region. Cetinkaya et al. (2000) examined 416 cattle and 411 sheep sera by indirect fluorescent antibody test (IFAT) to determine the prevalence of Q fever. Seropositivity was observed 5.8% in cattle and 10.5% in sheep. In an another study (Ozdemir et al., 1999) reported C. burnetii seropositivity in 33.8% of pregnant and 26.75% of aborted animals in Elazig and its vicinity using indirect flourescence antibody test. On the other hand, Kalender (2001) reported seropositivity in 38.59% of aborted sheep and 11% in sheep given healthy birth.
Furthermore, in a study carried out on 1593 cattle in the Marmara Region, Turkey, using ELISA, 8.04% was found to be positive to C. burnetii (Yurtalan, 2003). In addition, KiliC et al. (2005) reported seroprevalence of C. burnetii in 3% of sheep in Aydin and its vicinity.
In the region of Eastern Anatolia, Seyitoglu et al. (2006) detected seropositivity in 22.6% of cattle with a history of abortion and 5.6% in cattle given healthy birth, using ELISA test.
In Europe, there are several studies reporting the seroprevalence of Q fever using different diagnostic methods. In Italy, Capuano et al. (2001) detected seropositivity in 14.4% of cattle using IFAT. Masala et al. (2004) found that 38% of sheep and 47% of goats was seropositive using ELISA. Parisi et al. (2006) identified C. burnetii in 11.6% of cattle and 21.5% of sheep and goats using PCR. Cabassi (2006) detected seroprevalence of 44.9% in aborted cattle and 22% in healthy cattle using ELISA. In France, Rousset et al. (2007) found 88% seropositivity in aborted group and 60% in healthy group using ELISA in 8 goat flocks. Berri et al. (2007) also examined a goat flock and found that 30% seropositivity in aborted animals and 80% 6 weeks later after abortion in the same flock by ELISA. Tellez et al. (1989) used indirect immunofluorescence test, reported the seroprevalence of Q fever as 76.6% in goat and 17.7% in cattle in Madrid, Spain. Christoffersen (2007) investigated seroprevalence of Q fever in Denmark between 2004 and 2006 in cattle using ELISA and found seropositivity as 35 and 25%, respectively.
Furthermore, Wagner et al. (2005) tested a total of 744 blood samples (70 Styrian sheep and 674 goats) by CFT for antibodies to C. Burnetii in Vienna, Austria and found 1.5% seropositivity. Also, Lausevic (2001) investigated seroprevalence of Q fever in sheep in Yugoslavia using microagglutination and microimmuneflourescence and found 5.03% seropositivity.
In USA, studies with concern to Q fever are summarized by McQuiston and Childs (2002) and reported as 41.6% in goat, 16.5% in sheep and 3.4% in cattle. In addition, Deforgeand Cone (2006) in California, seroprevalence of Q fever in 268 bighorn sheep reported to be 10% using complement fixation test.
In Canada, Hatchette et al. (2002) examined Q fever seroprevalence between 1997 and 2000 and found an important increase in seropositivity from 3.1 to 23.5% using microimmuneflourescence test (MIF).
In Mexico, seroprevalence of Q fever in dairy cattle found to be 28%, beef cattle 10%, goat 35% and 40% in sheep (Salinas-Melendez et al., 2002).
In Africa, Reinthaler et al. (1988) found that 62.5% of sheep, 53% of goat and 40.4% of cattle using MAT in Southern Sudan, were seropositive. In Zimbabwe, Kelly et al. (1993) detected seropositivity to C. burnetii in 39% of cattle, 10% of goats using IFAT. Furthermore, in Chad, Schelling et al. (2003) found that 80% of camel, 13% goats, 11% sheep and 4% cattle using ELISA method, were seropositive. Nakoune et al. (2004) found 14.3% in cattle using IFAT in Central African Republic. In addition, Cekani et al. (2008) investigated Q fever seroprevalence in 1656 serum sample and found 9.8% in sheep and goat and 7.9% in cattle in Algeria using ELISA technique.
In other countries, seroprevalence of Q fever were; 46.6% in cattle, 28.1% in sheep and 23.5% in goats were observed in Japan using IFAT (Htwe et al., 1992). In Cyprus, Psaroulaki et al. (2006) found 48.2% in goats, 24% in cattle, 18.9% in sheep using IFAT. On the other hand, Loukaides et al. (2006) found 13.8% using same IFAT in Cyprus.
It is shown that seroprevalence of Q fever is very much changeable with regard to country and even provinces. These alterations is also changeable with regard to test and kit used. Studies regarding to Q fever seroprevalence in Turkey indicated that it varied between 5.6-22.6% in cattle and 3-38.59% in sheep.
The present study indicated that animal population in this region is determined to be infected with Q fever at 10.9%. Infection rate in cattle was higher (16.3%) In the seroprevalance studies with concern to cattle and sheep, some found seroprevalance higher in cattle (Htwe et al., 1992; Psaroulaki et al., 2006) some in sheep (Schelling et al., 2003; Cekani et al., 2008). In the present study, several reasons could play role for the high seroprevalence observed in cattle. One of the reason is the tick density in the farm which could be more compared to field and cattle are mainly kept in indoor conditions in comparison to sheep therefore, seroprevalence were higher in cattle. The disease morbidity is very high and pathognomonic symptoms of the disease do not occur. Therefore, challenge against this disease is quite difficult. Thus, required precautions with regard to animal discharges and vector ticks to be taken into consideration and effective treatment should be implied.