Abstract
Coxiella burnetii is a causative agent of Q fever which is highly
infectious zoonotic disease. C. burnetii has become one of the
most important causes of abortion in livestock, which can lead to
widespread abortions in these animals. There are very limited studies on
the prevalence of C. burnetii infection in cases of animal
abortion in Iran. The aim of this study was to investigate C.
burnetii in livestock abortion samples in Iran. Abortion samples from
cattle, sheep and goat were collected from different parts of Iran and
were tested using Real-time PCR targeting IS1111 gene of C.
burnetii. In this study, 36 samples (24.66%) of the 146 collected
samples were positive for C. burnetii. The prevalence of C.
burnetii was 21.78% (20 of 94 samples) in sheep samples. Also, 10 of
46 cattle samples (21.74%) were positive. All six goat abortion samples
were positive for C.
burnetii. The findings of the study demonstrate that C. burnetiihave important role in livestock abortions in Iran and more attention
should be paid to Q fever by the health care system and veterinary
organization.
Keywords : Coxiella burnetii , Q Fever, Abortion, Iran,
Livestock.
Introduction
Coxiella
burnetii is a small coccobacillus Gram-negative and obligate
intracellular bacterium. C.
burnetii is causative agent of Q fever which is highly infectious
zoonotic disease. Based on 16SrRNA sequence analysis,C. burnetii belonged to the
Gama-subdivision of Proteobacteria within the Legionellales order
and family Coxiellaceae(Eldin et al., 2017).
Q fever occurs mainly in two forms of acute and chronic in human. Acute
Q fever is mainly a self-limiting, febrile illness and flu-like disease
that is asymptomatic in 60% of cases (Million and Raoult, 2015).
Persistence of C. burnetii infection in humans (in acute and
asymptomatic cases) can lead to the chronic form of Q fever. Chronic Q
fever is extremely serious and dangerous. Endocarditis is the main
clinical manifestation of the chronic Q fever that leads to death in
over 65% of the patients, if untreated(Angelakis and Raoult, 2010;
Eldin et al., 2017).
Livestock are the main reservoirs of C. burnetii. Q fever in
cattle, sheep, and goats is usually asymptomatic but in some cases is
associated with pneumonia and reproductive disorders, including
abortion, stillbirth, endometritis, and infertility(Guatteo et al.,
2011). The main route of transmission to humans is through inhalation of
aerosols and dust particles contaminated with C. burnetii, which
are excreted by infected animals through milk, feces, urine, placenta,
fetal fluids, and vaginal discharge(Eldin et al., 2017; Guatteo et al.,
2011). People at risk of the disease include farmers, veterinarians,
butchers, slaughterhouse workers, farmers, dairy factory workers,
laboratory staff, and people in contact with domestic animals,
especially during livestock calving(Million and Raoult, 2015). Very easy
and fast release capability by aerosol, survival in harsh environmental
conditions, very low infectious doses, as well as the high infectivity
of the bacterium have madeC. burnetii a very serious
biological threat to military personnel and civilians(Roest et al.,
2013; Van Schaik et al., 2013).
In recent years, C.
burnetii has become one of the most important causes of abortion in
livestock’s, which can lead to widespread abortions in
animals(Rodolakis, 2009). Also, abortion and the accompanying excretory
fluids are one of the main causes of the spread of infection in the
environment, which can lead to widespread infection among animals and
human populations(Angelakis and Raoult, 2010). There is a very high dose
of C. burnetii in amniotic
fluid and the placenta during childbirth infected animals. Also,
infected aerosols from animal abortions can be transmitted up to several
kilometers from the site of the abortion by wind(Tissot-Dupont et al.,
2004). Therefore, the role ofC. burnetii in cases of
animal abortion is of great importance both in terms of epidemiology and
in the transmission of the disease to humans.
In recent years, seroepidemiological studies in animal and human
populations in Iran have shown that Q fever is an endemic disease in
Iran(Mobarez et al., 2017). However, clinical cases of Q fever are
rarely diagnosed and reported by the health system in Iran(Mobarez et
al., 2017). On the other hand, there are very limited studies on the
prevalence of C. burnetii in cases of animal abortion in Iran.
Also, conducting various studies and showing the importance of the
disease can cause more sensitivity of the health system and clinical
physicians to Q fever in Iran. The aim of this study was to molecular
investigation of C. burnetii in livestock abortion samples in
Iran.
Methods
2.1. Sample collection: This study was conducted during
2017-2018 with the cooperation of clinical veterinarians as well as the
Iranian Veterinary Organization. Livestock aborted samples were
collected from different province of Iran and samples included spleen
and liver of aborted fetuses, and
abortion fluids and cotyledons of
aborted animals (cattle, sheep and goat). Sampling was performed in
accordance with biological safety conditions. Samples were frozen
immediately after obtaining and were transported to the laboratory by
maintaining the cold chain. All laboratory works were done under B2
Biological Safety Cabinet.
2.2. Ethical Considerations: This study was approved by the
Ethics Committee for Biomedical Research of Tarbiat Modares University
(Ethic Code: IR.TMU.REC.1395.510). The Ethics Committee for Biomedical
Research of Tarbiat Modares University approved the consent procedure,
the proposal and protocol of this study.
2.3. DNA extraction: Genomic DNA was isolated using the Roche
High Pure PCR Template Preparation Kit (Roche, Germany), according to
the manufacturer’s instruction. For tissues samples, 50 mg from each
sample used for DNA extraction.
Also, 200 μL of abortion fluids was used for DNA extraction. All
extracted DNA were stored at -20 ° C until molecular test.
2.4. Real-time Polymerase Chain Reaction (qPCR): Real-time PCR
was performed using specific primers and probe sequences targeting
IS1111 gene of C. burnetii . Real-time PCR reactions were
performed using the following reaction mixture: 10 μL of 2x RealQ Plus
Master Mix for Probe (Ampliqon, Denmark), 900 nM forward primer
(AAAACGGATAAAAAGAGTCTGTGGTT), 900 nM reverse primer
(CCACACAAGCGCGATTCAT), 200 nM probe (6-FAM-AAAGCACTCATTGAGCGCCGCG-TAMRA)
and 4 μL of DNA template(Schneeberger et al., 2010). Real-time performed
on the Corbett 6000 Rotor-Gene system (Corbett, Victoria, Australia),
with a final volume of 20 µL for each reaction. The PCR amplification
program were 10 minutes at 95°C, followed by 45 cycles of 15 s at 94°C
and 60 s at 60°C. DNA of Nine Mile strain (RSA 493), was used as a
positive control and double distilled water was used as a negative
control. Results were generated with performed using Rotor-Gene® Q 2.3.5
software (QIAGEN). Samples showing cycle threshold (Ct) values of 36 or
lower for C. burnetii IS1111 qPCR assays were considered
positive.
Results
A total of 146 samples of abortions were collected from different parts
of Iran, of which 94 samples were from sheep’s abortions, 46 samples
were from cattle abortions, and 6 samples were from goats’ abortions.
In total, 36 samples (24.66%) of the 146 collected samples were
positive for C. burnetii using Real-time PCR. The prevalence ofC. burnetii was 21.78% (20
of 94 samples) in aborted sheep samples.
Highest prevalence of C.
burnetii was detected in Tehran (54.54%), Mazandaran (50%),
West-Azarbaijan (40%) and Hamadan (38.46%) provinces, respectively
(Table 1).
10 of 46 cattle aborted samples (21.74%) were positive for C. burnetii.
Highest prevalence of C. burnetii was detected in Tehran
(24.32%), and Alborz (16.67%) provinces, respectively (Table 2).
All six goats’ aborted samples were positive for C. burnetii . Two
and four of samples were collected from West-Azerbaijan and Tehran
provinces, respectively.