Molecular investigation of Coxiella burnetii in aborted fetus of small ruminants in southeast Iran

Abstract

The domestic animal, known as a main reservoir of Coxiella burnetii, is susceptible to the occurrence of coxiellosis, which can lead to abortions in domestic animals, causing significant economic damage and posing risks to human health. Therefore, the purpose of this study is to investigate C. burnetii as the causative agent of Q fever in abortion samples of small ruminants in southeastern Iran. This study was conducted between 2020 and 2021 in Zarand city, located in Kerman province (southeast Iran). In this study, 50 abomasum swab samples of aborted sheep and goat fetuses were collected and analyzed using molecular methods to identify C. burnetii. The results revealed that 26% (n: 13) of the collected abortion samples were infected with C. burnetii. Among the positive samples, two (50%) belonged to goat abortion samples while 11 (23.9%) belonged to sheep abortion samples. This study demonstrates that C. burnetii is one of the causes of abortion in small ruminants in southeastern Iran. It is recommended to pay more attention to C. burnetii in domestic animals due to its significant economic impact on livestock and its potential implication for human health in Iran.

Introduction

Coxiella burnetii, the causative agent of Q fever in humans, is an obligate intracellular gram-negative bacterium that was first described in Australia in 1937. Q fever is reported worldwide except in New Zealand [1]. Sheep, goats, and cows have been proposed as the main reservoirs of this disease. However in recent years, the reservoirs have expanded and include a wide range of animals such as domestic and wild mammals, birds, reptiles, marine mammals, and ticks. The wide range of reservoirs and the unique resistance of this pathogen to environmental factors have made tracking the source of infection a great challenge [2].

A very high dose of C. burnetii is excreted into the environment through the birth products of infected domestic animals. Among other ways to shedding of this bacteria in to the environment, it can refer to the excretion of urine, feces, and milk from infected animals. C. burnetii infection in livestock is usually asymptomatic [3], except during pregnancy, it leads to reproductive disorders such as stillbirth, weak offspring, abortion, uterine infections, and infertility [4]. Humans primarily acquire the bacteria through the inhalation of contaminated aerosols [5]. In more than 60% of cases, primary infection with C. burnetii in humans is asymptomatic, however in 40% of people, acute Q fever appears as a self-limiting febrile illness with a wide range of non-specific symptoms. In severe cases, it can lead to pneumonia or hepatitis. In about 5% of individuals who have experienced acute Q fever, chronic Q fever may occur months to a years later. In most cases, chronic Q fever manifests as fatal endocarditis if left untreated [6].

Coxiellosis in animals mostly affects the reproductive organs. Based on laboratory studies, it seems that after coxiellosis in animals, the trophoblast cells of the allantochorion show more severe inflammatory symptoms, followed by infection of the remaining trophoblast cells [7]. Despite these complications, the most significant clinical manifestations in pregnant small ruminants are abortion and stillbirth. Abortion in small ruminants often occurs without clinical symptoms and at the end of pregnancy [8]. In small ruminants, the placental tissue exhibits purulent lesions, with the cotyledon areas being mainly thickened [9]. The birth products of infected domestic ruminants can transmit the infection to the environment and affecting other animals and the human population, thus posing a serious threat [10].

Q fever is an endemic disease in Iran, and human cases of this disease have been identified so far [11, 12]. Based on the results of two studies in the northwest and southeast of Iran, the prevalence of acute Q fever among febrile patients was reported as 13.8% and 35.2%, respectively [13, 14]. According to a study on patients with infective endocarditis, 30.7% of patients were reported positive for chronic Q fever endocarditis [15]. Additionaly, a study conducted in Iran reported a seroprevalence of Q fever at approximately 68% in people exposed to occupational contact with this disease (such as slaughterhouse workers) [16]. The prevalence of this bacterium in animal populations has also been investigated. According to the review study, the prevalence of C. burnetii in cow, sheep, and goat milk samples has been reported as 15%, 3.7%, and 7.8%, respectively [17]. In Iran, studies have also been conducted on ticks of domestic animals as potencial vectors of this disease. In one study, the prevalence of C. burnetii in domestic animals and their ticks in western Iran was reported as 0.8% and 14%, respectively [18].

Although several seroepidemiological studies have been conducted in Iran to investigate Q fever in humans, livestock, and the contamination of animal milk and ticks, there is limited information regarding abortion in domestic ruminants and its relationship with Q fever. The aim of this study is to investigate Q fever in aborted samples of small ruminants.

Materials and methods

Study area

This study was conducted in Zarand city, Kerman province, between 2020–2021. Zarand city is located in the northeast of Kerman province, and most of the people are engaged in agriculture and animal husbandry. The city has a population of about 60 thousand people. The climate of Zarand city is semi-desert and has relatively hot summers and cold winters. In the mountains, the climate is moderate in summer and cold in winter (Fig. 1).

Fig. 1.

Study area and number of abortion samples

Sample collection

During the calving seasons between 2020–2021, following the reports of abortion cases in sheep and goat herds, 50 abortion samples were collected from 46 sheep and 4 goats. After the autopsy of the abortion samples, a sterile swab was used to sample the abomasum contents and transfer them to sterile microtubes. To keep the samples moist, 200 µl of physiological salin were added to the microtubes, and the samples were transferred to the microbiology laboratory of the Shahid Bahonar University of Veterinary Medicine in Kerman and kept at -20 ºC until DNA extraction.

DNA extraction

DNA was extracted from abomasum swab samples using the Tissue Genomic DNA Extraction Kit (Pars Company, Iran), following the manufacturer's instructions. The extracted DNA kept at -20 ºC until molecular examination.

Detection of C. burnetii

The extracted DNA from abomasum swab samples was screened for C. burnetii by a Real-time PCR with specific primers for the IS1111 gene (Table 1). The reaction volume for Real-time PCR was 25 μl and contained 5 μl of commercial master mix (Solis Bio Dyne 5 × Hot FIREPOL Eva Green qPCR Mix- NO ROX, New Zealand), 300 nmol of forward primer (5′-AAAACGGATAAAAAGAGTCTGTGGTT-3′), 300 nmol reverse primer (5′-CCACACAAGCGCGATTCAT-3′) [19], 1.5 μl of template DNA and distilled water to reach the final volume. C. burnetii strain Nine Mile RSA493 and distilled water were used as positive and negative controls. The amplification was performed using the LightCycler 96-GenEx (Roch-Germany). The temperature program included an activation step at 95°C for 15 min, follow by 40 cycles in three steps (95°C 15 s, 60°C 20 s, and 72°C 20 s), and a melting step at 87°C.

Table 1.

Relationship between infection with Coxiella burnetii and factors of age, history of previous abortion and number of parturitions

		No. Negative (%)	No. Positive (%)	OR* (95%CI)	P value
Animal Species	Goat	2 (50.0)	2 (50.0)	0.31 (0.04–2.50)	0.28
	Sheep	35 (76.1)	11 (23.9)
History of previous abortion	No	30 (71.4)	12 (28.6)	0.36 (0.04–3.22)	0.66
	Yes	7 (87.5)	1 (12.5)
Age	< 3	9 (60.0)	6 (40.0)	0.38 (0.10–1.41)	0.17
	≥ 3	28 (80.0)	7 (20.0)
Parturitions (times)	≤ 2	31 (75.6)	10 (24.4)	1.55 (0.33–7.37)	0.68
	≥ 3	6 (66.7)	3 (33.3)

^*; odds ratio

Statistical method

The data were analyzed using SPSS software version 16 (SPSS Inc., Chicago, IL, USA). Descriptive analysis was performed, and frequencies, percentages, minimum, maximum, and mean (standard deviation) were reported. Age and times of pregnancy were converted into categorical variables using median. Fisher^’s exact test was used to assess whether a significant association exists between categorical variables and infection. Additionally the Mann–Whitney U test was used to assess the distribution of age and times of pregnancy between positive and negative animals. A p-value of < 0.05 was considered statistically significant.

Results

In this study, a total of 50 abomasum swab samples from aborted fetuses of small ruminants (sheep and goats) were examined out of the 7776 heads in the statistical population. Among these samples, 46 were collected from aborted fetuses of sheep, and 4 were collected from goats. The average age of the small ruminants was 2.92 years, with a minimum of 1 year and a maximum of 6 years. All of the animals had at least one pregnancy history, with a maximum of four times and a median of two times. Among the animals, 16% of them had a history of previous abortion, while the remaining animals had not experienced any history of abortion during their number of births. The average age of sheep and goats that had experienced abortion was 2.8 and 3.7, respectively. The average birth frequency was 1.9 in sheep and 2.5 in goats. Half of the goats and 13% sheep had a history of previous abortion.

According to the molecular test results, 13 (26%) out of the 50 abortion samples collected from small ruminants were infected with C. burnetii. Two out of four goat samples (50%), and 11 out of 46 sheep samples (23.9%) were reported positive. The average age of small ruminants that tested positive for C. burnetii in the abortion samples was 2.69 years (4.5 years in goats and 2.36 years in sheep). Among the positive samples, one sample belonged to a goat that had a history of previous abortion, while the remaining positive samples (28.6%) were from animals without a history of abortion. There was no statistical relative observed between C. burnetii infection with age, time of pregnancy, species, and history of abortion. Additionally, there was no statistical difference between infected and non-infected animals in terms of age and time of pregnancy (Table 1).

Discussion

In this study, Q fever was identified in abomasum swab samples of aborted fetuses of small ruminants. The results of the study indicated that 26% of abortion samples tested positive for C. burnetii. Given the pathogenic consequences of animal abortion for both humans and animals, as well as the potential economic losses, it is crucial to identify and prevent the occurrence of this infection. It appears that in Iran, the role of C. burnetii as an infectious bacterial agent in the small ruminant’s abortion has been neglected, and there is little information available in this regard. In a previous study conducted in Iran on abortion samples of small ruminants, 2% of the samples tested positive for C. burnetii [20]. Another recent study in Iran, the prevalence of C. burnetii in abortion samples of domestic animals was reported as 24.7%. Interestingly, in our study, the majority of positive samples were detected in aborted sheep fetuses (24%), whereas in the aforementioned study, the highest prevalence was observed in aborted goat fetuses (21.3%) [21]. It should be noted that in our study, only 4 samples of goats were examined, two of which were reported positive, and this does not make comparison possible.

Our study was conducted in one of the cities of Kerman province. In previous studies, the prevalence of C. burnetii in animal abortion samples in this province was reported as 86.8% in sheep and 8.82% in goats. However, in our study, the majority of the positive samples were detected in abortion samples of sheep. In the same study, the prevalence of C. burnetii in swab samples of aborted embryos from sheep and goats was reported as 15.47% and 20.43%, respectively [22]. According to other studies, the prevalence of C. burnetii in sheep abortion samples in different regions of Iran has been reported as 17.3% in Mashhad city and 16.6% in Sistan and Baluchistan province [23, 24]. Based on these studies, it appears that sheep primarily excrete this bacteria through their feces and vaginal secretions, while goats often excrete the bacteria through milk, feces, and vaginal secretions. However, the highest excretion of bacteria in goats occurs during childbirth and lactation [25]. Abomasum swab samples from aborted fetuses can contain vaginal and placental fluids and other fluids at birth, so it is superior to other samples for identifying this bacteria [26].

It seems that C. burnetii is highly contagious among livestock herds, and if it is not detected in time, the abortion rate among herds can vary between 10–60% [27]. According to a systematic review in Iran, the seroprevalence of Q fever antibodies in goat and sheep herds was reported as 93.42% and 96.07%, respectively [28]. Other studies conducted in various part of the world have investigated the prevalence of C. burnetii in animal abortion samples. For instance, in Turkey, the prevalence of C. burnetii in animal abortion samples (cows, sheep, and goats) ranges from 2–11% [29, 30], while in Italy, it is 18.9% [31]. In Cyprus, the prevalence is reported as 37% [32], in Egypt as 0.9% [33], and in England as 25–11% [34, 35]. As mentioned earlier, small ruminants also excrete this bacteria into the environment through their milk. In Iran, studies on the milk of animals with a history of abortion have also been conducted. Based on the results of this study, the prevalence of C. burnetii in milk samples was reported as 34.9% [36]. Also, in another study in Iran in 2018, 16.6% of goat milk samples and 7.6% of sheep milk samples were infected with C. burnetii. Also, based on the results of this study, the amount of contamination in the samples collected in the summer season (25%) was significantly higher than in other seasons [37]. Considering the data from these studies, including ours, it can be concluded that the incidence of coxiellosis among domestic animals in Iran is significant.

In our study, the majority of positive samples belonged to aborted sheep fetuses. Although the number of samples collected from goats it is very low, both in terms of absolute or relative values, however, half of the abomasum swab samples from aborted goat fetuses were reported positive for this pathogen. This finding is consistent with the results of other studies where the majority of positive samples were found in goats. Examining the prevalence of Q fever in domestic animals during calving seasons through studies can help establish the relationship between C. burnetii and abortion. In a study conducted in Iran on animal abortion samples, the prevalence of Q fever was reported to be 74.38% in spring, while in winter, the prevalence was 61.6% [26]. It is important to note that the samples in our study were collected during the breeding season. In Iran, based on the studies, the highest level of sexual activity and fertility of domestic animals is from the beginning of June to near the middle of October, and the animals are fertile once a year on average [38]. Based on the studies conducted, infection by C. burnetii in livestock is mostly asymptomatic. Consequently, greater attention needs to be directed towards human outbreaks of Q fever. For instance, a study in Turkey has confirmed the link between a human outbreak of Q fever and animal contamination [39]. One limitations of this study is the small number of samples used. Additionally, the study was conducted in only one province, which does not cover a significant geographical area to investigate the relationship between abortion in livestock and Q fever. In this study, the number of samples collected from goats was also very low. Considering the significant potential for goats to transmit the bacteria through vaginal secretions, it is crucial to pay more attention to goat herds. Furthermore, C. burnetii is highly resistant to environmental conditions, making it difficult to control once it has been transmitted to the environment. Therefore, given the endemic nature of Q fever in Iran, comprehensive investigations should be conducted to prevent the spread of the disease.

Conclusion

Q fever is an endemic disease in Iran. Given that one of the primary means of releasing this bacterium into the environment is through the birth waste of infected animals, greater emphasis should be placed on this disease, particularly among individuals who have occupational contact with livestock. Furthermore, the occurrence of coxiellosis in livestock has an impact on their reproduction as it was observed based on the results of this study, and can lead to significant economic losses for livestock farmers.

Authors’ contribution

Conceptualization: SE and MKh. Methodology: SE, and MKh. Validation: SE, and MJ. Investigation: RB. Resources: SE, and MKh. Data Curation: ML, RB, and MJ. Writing – Original Draft Preparation: RB, ML, and SE. Writing – Review & Editing: MKh, and MJ. Supervision: SE; Project Administration: SE.

Funding

Shahid Bahonar University of Kerman.

Data availability

Data supporting the findings of this study are available within the article and can be obtained from the corresponding author upon request.

Declarations

Conflict of interest

The authors declare no competing of interests.