Introduction
Abortion is one of the major problems for the livestock industry that affects the reproductive and productive performance of small ruminants. The causes of abortion can be categorized into non-infectious causes (genetic and physical factors, nutritional and metabolic problems, heat stress, and toxic agents) and infectious agents [Reference Givens and Marley1]. The major infectious agents associated with abortion are bacteria (such as Brucella spp., Campylobacter spp., Chlamydia abortus (C. abortus), Coxiella burnetii (C. burnetii), and Listeria monocytogenes (L. monocytogenes)), viruses (such as pestiviruses, bluetongue virus, Schmallenberg virus, and Cache valley virus (CVV)), and parasites (such as Neospora caninum and Toxoplasma gondii) [Reference Givens and Marley1, Reference Borel2]. Although non-infectious causes are related to lower abortion rates, infectious agents cause severe abortion outbreaks [Reference Givens and Marley1]. Infectious agents are also significant threats to public health since most of the abortifacient agents are considered zoonotic [Reference Givens and Marley1, Reference Borel2]. Zoonotic potencies of abortifacient agents such as C. abortus, C. burnetii, L. monocytogenes, and CVV have been described [Reference Stein and Raoult3–Reference Turin6].
Chlamydia abortus and C. burnetii are obligate intracellular zoonotic Gram-negative bacteria and have been associated with reproductive problems in livestock and pregnant women [Reference Givens and Marley1, Reference Stein and Raoult3, Reference Turin6]. Previous studies in pregnant women have found that C. abortus infection occurs in two cases per year in the UK [Reference Turin6], whereas C. burnetii infection occurs in at least one in every 540 pregnancies in Southern France [Reference Stein and Raoult3]. The prevalence of C. abortus and C. burnetii among sheep and goat flocks ranged from 2.0% to 43.3% [Reference Masala7–Reference Alzuguren11] and 2.0% to 49.0% [Reference Alzuguren11–Reference Esmaeili14], respectively. Ramo et al. [Reference Ramo15] reported that C. abortus and C. burnetii were identified in approximately 75% of ovine and caprine abortions in Spain. Furthermore, previous studies have also found that C. abortus and C. burnetii are the main abortive agents in small ruminants in Algeria [Reference Haif16], Ethiopia [Reference Alemayehu17], Hungary [Reference Kreizinger18], and Iran [Reference Esmaeili19]. In Türkiye, the proportion of abortive episodes potentially related to C. abortus was 7.7% in sheep and 14.3% in goats [Reference Kalender8, Reference Güler20], whereas the detection rate of C. burnetii was 6.1% in sheep and 8.5% in goats foetal samples analyzed between 2019 and 2020 [Reference Ozgen13].
Listeria monocytogenes is a motile, facultative intracellular Gram-positive bacterium. Various clinical manifestations can be seen in infected animals such as encephalitis, keratitis, uveitis, septicaemia, late gestation abortion, and stillbirth, whereas it can cause septicaemia, meningitis, miscarriage, and stillbirth in humans [Reference Dhama5]. L. monocytogenes-related foetal losses have been reported in pregnant women in Denmark, Iran, and the USA [Reference Smith21, Reference Silk22, Reference Ahmadi23]. The epidemiological studies revealed that L. monocytogenes have been reported as a cause of abortion among small ruminants in Austria (25%) [Reference Wagner24], Iraq (20.3%) [Reference Mohammad, AlFarwachi and Rasheed25], Denmark (8.3%) [Reference Agerholm26], and India (2.8%) [Reference Shoukat27]. Although the prevalence of L. monocytogenes in caprine foetuses in Türkiye is unknown, limited numbers of studies have investigated the role of L. monocytogenes in ovine foetuses in Türkiye, and it has been reported that the detection rate of L. monocytogenes in ovine foetuses ranged from 2.8% to 13.3% [Reference Akca28, Reference Gulaydin29].
Cache valley virus is a neurotropic arbovirus that can cause congenital defects, stillbirth, and spontaneous abortion in small ruminants [Reference Givens and Marley1, Reference Harvey30]. Furthermore, CVV infection has been associated with encephalitis and meningitis in humans [Reference Nguyen4]. CVV infection has not yet been reported in Türkiye. However, serological and molecular evidence of CVV infection in small ruminants has been reported in a few countries such as the USA [Reference Harvey30], Canada [Reference Uehlinger31], and Mexico [Reference Blitvich32].
Determining the cause of abortion in ruminants is difficult due to the complex aetiology of abortion. Previous studies conducted in Türkiye have reported the presence of zoonotic abortifacient agents in goats and sheep [Reference Güler20, Reference Ural33, Reference Günaydın34]. Despite the previous studies that demonstrated the presence of zoonotic abortifacient agents among small ruminants, the prevalence of the zoonotic abortifacient agents is largely unknown at the national level in Türkiye because previous studies were mostly conducted in a small area or a limited number of flocks. Furthermore, many studies on major infectious agents causing abortions in small ruminants in Türkiye rely on serological tests only [Reference Ural33, Reference Karagul, Malal and Akar35, Reference Kaya and Öztürk36]. This can be misleading to determine the actual cause of abortion. Identifying the aetiologic agent of abortion has important implications for developing effective flock health strategies, and also for the prevention and control of zoonotic diseases [Reference Haif16, Reference Alemayehu17]. Therefore, the current study aimed to investigate the prevalence of important zoonotic abortifacient agents in small ruminants in Türkiye.
Methods
Study area
Türkiye has seven geographical regions. The present study was performed in three different geographical regions of Türkiye where livestock production is one of the main sources of income, including the Mediterranean region (Isparta, Burdur, and Antalya Provinces), the Aegean region (Afyonkarahisar Province), and the Central Anatolian region (Niğde, Aksaray, Karaman, and Konya Provinces) (Figure 1). These regions had 5 012 677 sheep and 1 714 788 goats. The small ruminant industry in surveyed regions is dominated by small-scale family flocks (n = 10–50) and settled village flocks (n = 100–500). White Karaman and Dağliç breeds of sheep and Hair goat breed of goats were common in surveyed regions.
Clinical samples
A total of 1 226 foetuses (1 144 sheep and 82 goats) were submitted to the Veterinary Control Institute (Konya, Türkiye) during 2012–2017 from different flocks (n = 1 226) with abortion histories. These investigated flocks constitute about 0.005% of all the sheep and goat flocks in surveyed regions. Aborted foetuses were submitted within 24 h after abortion. Unfortunately, the placenta samples were not submitted to the institute in most of the abortion cases. Therefore, only aborted foetuses were used to determine the cause of abortion.
To avoid cross-contamination, a necropsy was done using sterile surgical instruments under biosafety guidelines. During the necropsy, foetal tissues were obtained from foetuses with gestational age < 3 months, while organ samples, including lymph nodes, liver, lung, kidney, intestine, and spleen were obtained from foetuses with gestational age 3 to 5 months. Each animal’s samples were put in sterile 50 ml falcon tubes and were kept at −85 °C until analysis.
Furthermore, a questionnaire was administered to farmers to obtain information related to abortion cases, the clinical signs, the number of pregnant animals on the flock, the number of animals that had been aborted, and the date of abortion.
Nucleic acid extraction
Pooled tissue/organ samples of each foetus (30–60 mg) were placed into sterile DNAse/RNAse-free microcentrifuge tubes (2 ml) containing phosphate-buffered saline (400 μl) and homogenized using the TissueRuptor (Qiagen, Germany). Total nucleic acids were extracted from the supernatants of the centrifuged foetal tissue homogenates (5 000 g for 5 min at 4 °C) using the QIAamp Cador Pathogen Mini Kit (Qiagen, Germany), according to protocols reported by the kit manufacturers, and were stored at −85 °C until analysis.
Molecular identification of abortifacient agents
Samples were tested for the presence of C. abortus, C. burnetii, L. monocytogenes, and CVV by molecular detection methods. For C. abortus, C. burnetii, and L. monocytogenes, real-time PCR assays reported by Pantchev et al. [Reference Pantchev37], Klee et al. [Reference Klee38], and Rossmanith et al. [Reference Rossmanith39] were used, respectively. Furthermore, one-step real-time duplex RT-PCR was performed using CVV G1 glycoprotein-specific probes and primers described by Wang et al. [Reference Wang40]. Negative control (sterile nuclease-free water) was used to verify the absence of cross-contamination during the analyzes, whereas positive controls were used to ensure that results were reliable.
Statistical analysis
The differences in positivity between species (sheep and goats) were assessed by Fisher’s exact test, whereas one-way ANOVA with Tukey post-test was used to assess the relationship between positivity and provinces and years of origin. The statistical analyzes were performed by using the GraphPad Prism software (San Diego, CA, USA), and a p-value of ≤ 0.05 was considered statistically significant.
Results
Questionnaire survey results
According to flock owners’ reports, sampled flocks had not been vaccinated against investigated diseases, and animals exhibited no distinct symptoms, except for abortion. Abortions mostly occurred during the last trimester of gestation. The rate of abortion in C. abortus, C. burnetii, and L. monocytogenes positive flocks ranged from 8.6% to 46.5%, 7.2% to 10.5%, and 6.3% to 9.7%, respectively.
Detection of abortifacient agents
Chlamydia abortus DNA was detected in 276 (22.5%) of the 1 226 aborted foetuses with 23.1% (264/1144) positive ovine foetuses and 14.6% (12/82) positive caprine foetuses. There was no significant difference in the positivity of C. abortus between ovine and caprine foetuses (p = 0.099). The highest number of C. abortus-positive foetuses was detected in 2016 (41.3%) (p = 0.006), followed by 2015 (28.3%) (Table 1). The number of C. abortus-positive foetuses (88/277, 31.8%) was significantly higher in Konya Province than in Isparta and Antalya Provinces (p = 0.03). Aksaray Province had the second highest rate, with 30.9% (29/94), followed by 27.4% (20/73) in Afyonkarahisar, 23.1% (9/39) in Karaman, 20.8% (59/284) in Niğde, 19.2% (10/52) in Burdur, 15.3% (23/150) in Isparta, and 14.8% (38/257) in Antalya Province.
Source: CVV = Cache valley virus, N = number of tested samples, P = number of positive samples, % = prevalence rate, and any fractional part greater than or equal to 0.5 was rounded up to the next whole number.
Coxiella burnetii positivity was significantly higher in caprine foetuses (8.5%, 7/82) than in ovine foetuses (2.8%, 32/1144) (p = 0.012). The number of C. burnetii-positive foetuses (5.1%, 2/39) was higher in Karaman Province than in other provinces, without significant differences. Konya Province had the second highest rate, with 5.0% (14/277), followed by 3.5% (10/284) in Niğde, 3.2% (3/94) in Aksaray, 2.7% (2/73) in Afyonkarahisar, 2.0% (3/150) in Isparta, and 1.9% (5/257) in Antalya Province. However, C. burnetii was not detected in samples from Burdur Province (0/52).
Listeria monocytogenes DNA was detected in 28 (2.4%) and 2 (2.4%) of the 1 144 ovine, and 82 caprine foetuses, respectively. There was no significant difference in the positivity of L. monocytogenes between ovine and caprine foetuses (p = 1.000). The highest L. monocytogenes-positive foetuses were detected in Aksaray Province (5/94, 5.3%), without significant differences, followed by 3.6% (10/277) in Konya, 2.7% (2/73) in Afyonkarahisar, 2.7% (7/257) in Antalya, 2.6% (1/39) in Karaman, and 1.8% (5/284) in Niğde Province. However, L. monocytogenes was not detected in samples from Burdur Province (0/52) and Isparta Province (0/152).
CVV RNA was not detected in ovine and caprine foetuses.
In this study, although the predominant mixed infection was C. abortus and C. burnetii (sheep, n = 4; goat, n = 1), mixed infection of C. abortus and L. monocytogenes (sheep, n = 1), and C. burnetii and L. monocytogenes (sheep, n = 1) were also found.
Discussion
Abortions in small ruminants have important impacts on livestock production, and non-infectious and infectious agents can cause abortion in ruminants [Reference Borel2]. Many abortifacient agents in small ruminants pose a serious health threat to humans [Reference Givens and Marley1]. The exact prevalence of common zoonotic and abortifacient agents in Türkiye is still unclear because the diagnostic testing of abortifacient agents in small ruminants in Türkiye relies mostly on serological tests. Therefore, in this study prevalence of major zoonotic and abortifacient agents was investigated. To the best of my knowledge, this study is the longest and most comprehensive study that investigated the prevalence of zoonotic agents in ovine and caprine foetuses.
WOAH recommended the molecular diagnostic assays to detect the aetiologic agents of disease [Reference Cullinane and Garvey41]. Therefore, molecular diagnostic assays were used for the detection of C. abortus, C. burnetii, L. monocytogenes, and CVV in aborted foetuses.
In this study, abortion-related pathogens were detected in 28.1% (345/1226) of the cases. However, an agent could not be detected in 71.9% of the cases, which may either be related to other infectious agents that were not examined in this study or non-infectious factors such as genetic factors, toxins, stress, nutritional, and hormonal problems [Reference Esmaeili14].
In this study, the detection rate of C. abortus in ovine foetuses (23.1%) was higher than that reported in previous studies from Türkiye (range from 3.5% to 7.7%) [Reference Kalender8, Reference Malal and Turkyilmaz10, Reference Güler20], Iran (12.3%) [Reference Esmaeili14], and Italy (2%) [Reference Masala7], but was lower than detection rates from Spain (38.3%) [Reference Alzuguren11] and Algeria (43.3%) [Reference Merdja9]. Possible explanations for this discrepancy may be the number of sampled flocks and animals, the difference in sampling technique, the diagnostic technique used, and the lack of strategies to prevent and control C. abortus infection. There is no control programme for C. abortus infection at the national level in Türkiye. Furthermore, the higher detection rate of C. abortus in ovine foetuses observed in this study can be explained by the flock management practices. The introduction of animals with unknown health status is considered a significant risk factor for C. abortus infection [Reference Esmaeili19]. However, in the study area, it was common to introduce purchased animals from flocks with unknown health status to the flock without any testing or quarantine. This could be one of the factors associated with the higher detection rate of C. abortus in this study. Another reason for the higher detection rate of C. abortus in this study could be the mineral nutrition deficiencies. Tejedor-Junco MT et al. [Reference Tejedor-Junco42] reported that proper nutrition is a protective factor against C. abortus infection. In most flocks in the studied regions, the system of sheep management is extensive. The extensive management system is more likely to lead to mineral nutrition deficiencies. Mineral nutrition deficiencies can cause an impaired immune response and loss of host resistance to infection [Reference Morris and Drewe43].
In this study, the detection rate of C. abortus in caprine foetuses (14.6%) is in agreement with a previous report from Eastern Türkiye that approximated detection rate of 14.3% [Reference Kalender8], but was lower than that detected (21.4%) in a previous study from the Marmara region of Türkiye [Reference Malal and Turkyilmaz10]. This result can be explained by the fact that goat farming in the Marmara region is generally under intensive and semi-intensive production systems, which facilitates the spread of C. abortus infection. It has been reported that the disease spreads faster when animals are overcrowded, as the contact between healthy and infected animals increases in intensive and semi-intensive flocks [Reference Merdja9].
This study shows no significant differences in the positivity of C. abortus between ovine and caprine foetuses (p = 0.099), indicating that both sheep and goats are equally susceptible to the disease. This finding is in agreement with the results of a previous study from Spain [Reference Alzuguren11].
In this study, the highest C. abortus rate was recorded in Konya Province (31.8%). In Konya Province, the main sheep breed is Merino, which lambs year-round. Lambing throughout the year can cause the pathogen to circulate continuously within the flock, which can be a constant source of infection [Reference Wolf44]. Furthermore, the average flock size (greater than 152 sheep) in Konya Province was larger than other sheep populations in other studied provinces. A significant positive association between C. abortus seropositivity and larger flock size has been reported [Reference Fayez45]. Sheep overcrowding in large size flocks can have an impact on animal welfare and hygiene, which increases the risk of C. abortus transmission [Reference Alemayehu17, Reference Tejedor-Junco42].
In this study, the detection rate of C. abortus increased from 2012 to 2016, and it tended to decline in 2017 (Table 1). Possible explanations for this discrepancy may be the increasing knowledge of C. abortus infection and awareness of good hygienic measures among farmers.
In this study, the detection rate of C. burnetii in ovine foetuses (2.8%) is in agreement with previous studies from Türkiye that reported C. burnetii detection rate in ovine foetuses ranges between 2.0% and 2.9% [Reference Kılıç12, Reference Kilicoglu46], but the detection rate in this study was lower than that observed in previous studies that reported detection rate ranges from 6.1% to 49.0% in ovine foetuses [Reference Alzuguren11, Reference Ozgen13, Reference Esmaeili14, Reference Günaydın34]. Possible explanations for this discrepancy may be the difference in husbandry practices, and the sample type. According to the farmers’ report, pregnant animals were separated from the rest of the flock during parturition, placentas, aborted foetuses, and uterine fluids were removed by burning. The transmission of the disease in the flock can be controlled by implementing these measures [Reference Kreizinger18]. Furthermore, in this study, only, foetal tissue samples were used to detect C. burnetii DNA. However, it has been reported that placental tissue is more useful for the detection of C. burnetii nucleic acids [Reference Alzuguren11].
In this study, the detection rate of C. burnetii in caprine foetuses (8.5%) is in agreement with a previous report from Türkiye [Reference Ozgen13], but was lower than the 40.0% reported by Günaydin et al. [Reference Günaydın34]. This variation could be related to the number of sampled animals (82 in this study vs. 5 in Günaydin et al. [Reference Günaydın34]), the sample type (foetal tissues and organ samples in this study vs. abomasal contents in Günaydin et al. [Reference Günaydın34]), the diagnostic technique used (real-time PCR in this study vs. conventional PCR in Günaydin et al. [Reference Günaydın34]), the hygiene conditions, and the presence of tick species which are capable of transmitting C. burnetii. Ticks are believed to be the vectors for C. burnetii transmission to animals, and C. burnetii is most commonly present in Ixodes, Racicephalus, Dermacentor, and Haemaphysalis genera [Reference Kilicoglu46, Reference Rahravani47]. The tick species, Hyalomma marginatum, Hyalomma anoliticum excavatum, Hyalomma detritum, and Boophilus annulatus belonging to the Haemaphysalis and Ixodes genera, that can transmit C. burnetii to animals have been detected in the Black Sea region of Türkiye [Reference Kilicoglu46], where a higher detection rate (40.0%) of C. burnetii was reported by Günaydin et al. [Reference Günaydın34].
In this study, the highest C. burnetii detection rate (7/82, 8.5%) was observed in caprine foetuses. This finding is in agreement with previous studies that reported C. burnetii-related abortions are more common in goats than in sheep [Reference Ozgen13, Reference Günaydın34]. However, detection rates of C. burnetii in sheep and goats may not be due to differences in susceptibility to the infection. Because the precise time of the entry of C. burnetii onto the flock or the route of transmission was not known in the investigated flocks.
In this study, the highest C. burnetii rate was recorded in Karaman Province (2/39, 5.1%). This result can be explained by the flock type. Sheep and goats are often raised together in mixed flocks in Karaman Province. The higher prevalence of C. burnetii in mixed flocks has also been reported in Germany and Italy [Reference Wolf44, Reference Rizzo48]. It has been reported that keeping sheep and goats in the same flock may increase animal density and increase the chances of contact between animals, thus facilitating cross-transmission between sheep and goats [Reference Alemayehu17].
Unfortunately, there is no control programme against C. burnetii infection in Türkiye, and this leads to the persistence of the infection (Table 1).
The detection rate of L. monocytogenes (2.4%) in this study is in agreement with a previous study that reported the detection rate of L. monocytogenes in ovine foetuses was 2.8% in Türkiye [Reference Gulaydin29]. However, a lower detection rate was found in this study compared with previous studies that reported the detection rate of L. monocytogenes rates in ovine foetuses ranged from 9.0% to 13.3% [Reference Akca28, Reference Sharma49]. Furthermore, the detection rate of L. monocytogenes in caprine foetuses (2.4%) was lower than that reported in previous studies that L. monocytogenes rates in aborted caprine foetuses ranged from 8.0% to 16.2% [Reference Sharma49, Reference Kim, Kim and Kim50]. Feed quality and storage, farm management practices, animal health, and hygienic conditions are all possible explanations for this discrepancy. L. monocytogenes is commonly found in poorly fermented silage, and there is a causal relationship between the prevalence of listeriosis in ruminants and the consumption of poorly fermented silage [Reference Nightingale51]. Akca et al. [Reference Akca28] reported that the high-detection rate of L. monocytogenes in ovine foetuses in their study may be due to the consumption of low-quality silage feed. In the regions being studied, the traditional method of raising sheep and goats involves extensive farming, and the animals’ nutrition is largely dependent on meadows and pastures. Nightingale KK et al. [Reference Nightingale51] reported that access to pasture is a protective factor against L. monocytogenes infection. Furthermore, in this study sampled animals exhibited no distinct symptoms, except for abortion. However, Kim et al. [Reference Kim, Kim and Kim50] collected aborted foetuses from the flocks that had neurological symptoms associated with listeriosis. This might be the explanation for the higher detection rate of L. monocytogenes in caprine foetuses in their study.
In this study, the highest L. monocytogenes rate was recorded in Aksaray Province (5.3%, 5/94), whereas L. monocytogenes was not detected in samples from Burdur and Isparta Provinces. This variation could be related to the number of sampled animals and flocks, differences in animal population density in sampled provinces, and the strains of L. monocytogenes. The virulence potential of L. monocytogenes strains varies. While some of the L. monocytogenes strains are non-virulent and unable to cause infection, others are virulent and cause high mortality and morbidity rates [Reference Kim, Kim and Kim50]. In this study, the virulence characterization of L. monocytogenes strains circulating in the study area was not performed. However, the circulation of virulent L. monocytogenes strains in Aksaray Province has been reported [Reference Kevenk and Aras52]. This could explain the highest detection rate of L. monocytogenes in ovine and caprine foetuses in the Aksaray Province.
The detection rate of L. monocytogenes increased from 2013 to 2015, and it tended to decline in 2016 (Table 1). This observation can be explained by increased awareness and improvement of hygiene measures on flocks in response to the worsening situation with peste des petits ruminants (PPR) in the study area. PPR outbreaks were observed in the study area in 2015 (WAHIS database), and control measures, including quarantine and hygiene measures, were applied within the study area. Hygienic measures were found to be a significant tool in reducing the spread of L. monocytogenes on farms [Reference Nightingale51].
In this study, the predominant mixed infections were C. abortus and C. burnetii. This finding is similar to previous studies where C. burnetii and C. abortus coinfection were the most frequently diagnosed in ovine and caprine foetuses [Reference Alzuguren11, Reference Kreizinger18]. This result suggests that pathogen synergism could play a role as an abortigenic agent in some abortions associated with C. burnetii infection.
There is no report of CVV-associated cases in Türkiye. However, new arboviral diseases are emerging in new geographical regions as a result of climate change [Reference Uehlinger31]. Therefore, the presence of CVV was investigated in this study. In this study, CVV RNA was not also detected in aborted foetuses. CVV is transmitted by mosquitoes and biting midges, and it is widely distributed in North America [Reference Harvey30, Reference Uehlinger31, Reference Wang40]. Therefore, not detection of CVV in this study may be related to the climatic conditions and geographical distribution of vector populations involved in the transmission of CVV [Reference Wang40].
This study has some limitations. First, data was gathered through self-administered questionnaires, which could lead to bias in reporting. Second, being conducted in three different geographical regions of Türkiye, there are concerns regarding geographical variation, and the representativeness of obtained data for all regions of Türkiye is uncertain. Third, other infectious agents causing abortions in sheep and goats, such as Brucella spp., Campylobacter spp., Toxoplasma gondii, and Neospora caninum, were not investigated in this study due to budgetary limits. This limitation should be taken into account when interpreting the findings of the study. Finally, genotyping of the detected isolates was not performed. A future study should assess the genotypes of these isolates to provide more comprehensive data on C. abortus, C. burnetii, and L. monocytogenes epidemiology.
Conclusions
Chlamydia abortus was the most frequently detected abortifacient agent in the 1 226 abortion cases in the evaluation period, with a frequency of 22.5%. To a lesser extent, C. burnetii (3.3%), and L. monocytogenes (2.4%) were also detected in these abortion cases. The findings of the present study suggest that C. abortus, C. burnetii, and L. monocytogenes should be taken into consideration in abortion cases of small ruminants in the surveyed provinces. To implement effective control strategies against diseases that cause abortion in small ruminants, it is necessary to determine the infectious agents circulating in the field. Therefore, the results of this study should be taken into account in the development of control and protection strategies for abortion in small ruminants. Furthermore, these diseases have zoonotic potential, and therefore, farmers should take hygienic precautionary measures when handling aborted materials.
Data availability statement
The data presented in this study are available within the article.
Acknowledgements
I thank the staff of the Molecular Microbiology Laboratory (Veterinary Control Institute, Konya, Türkiye) for all the support.
Author contribution
Conceptualization, methodology, formal analysis, writing-review, and editing: M.Ş.
Competing interest
The author declares that there is no conflict of interest.
Ethics statement
This study was carried out with the permission of the General Directorate of Food and Control dated 27th December 2017 and numbered E.3335546. In this study, samples were collected during routine diagnostic evaluation and necropsies.