MOLECULAR DETECTION AND IDENTIFICATION OF COXIELLA BURNETII IN ABORTED SHEEP AND GOATS IN SULAIMANI PROVINCE, KURDISTAN-IRAQ

Assiut University web-site: www.aun.edu.eg

MOLECULAR DETECTION AND IDENTIFICATION OF COXIELLA BURNETII IN ABORTED SHEEP AND GOATS IN SULAIMANI PROVINCE, KURDISTAN-IRAQ

FATTAH MARIF HARDI ¹; HANA SHERZAD RAUF ¹; SHAKHAWAN LATIF MAHMOOD ¹; REBWAR BAHIR AHMAD ¹; BASIM ABDULWAHID ALI ¹

AND MOHAMED OMAR BABA SHEIKH ²

¹ College of Veterinary Medicine, University of Sulaimani, New Sulaimani, Street 27, Sulaymaniyah,

 Kurdistan Region, Northern Iraq

² Directorate of Veterinary in Sulaimani, Salim Street, Sulaimaniyah, Kurdistan Region, Iraq

Received: 31 December 2019;     Accepted: 31 January 2020

INTRODUCTION

Q fever is a zoonotic disease in humans and animals affecting a wide range of hosts. The causative agent, Coxiella burnetii, is a Gram-negative obligate intracellular bacterium and is known for its high tenacity and infectivity present worldwide (Heinzen, et al., 1999), (McCaul & Williams, 1981), (Aitken et al., 1987).  

Reservoirs of C. burnetii include many wild and domesticated mammals, birds and ticks (Raoult, et al., 2005). Ticks are considered to be the natural primary reservoirs of C. burnetii responsible for the spread of the infection in wild animals and for transmission to domestic animals (Norlander, 2000). In animals, C. burnetii infection does not usually provoke severe symptoms. However, in cattle it has

Corresponding author: Dr. FATTAH MARIF HARDI

E-mail address: hardi.marif@univsul.edu.iq

Present address: College of Veterinary Medicine, University of Sulaimani, New Sulaimani, Street 27, Sulaymaniyah, Kurdistan Region, Northern Iraq

been associated with infertility and in small ruminants (goats and sheep)   the   infection   can   result  in  late abortions. Increased abortion rates in infected caprine herds have been described, with up to 90% abortions in pregnant animals (Van den Brom, van Engelen, Roest, van der Hoek, & Vellema, 2015). Infected females shed a huge amount of bacteria in birth products and in urine, feces, and milk. This shedding can persist for several months in vaginal mucus, feces, and milk (Rodolakis, 2009). The massive shedding of C. burnetii during such abortions makes sheep and goats the main reservoirs responsible for infection of humans (Parisi et al., 2006).

C. burnetii is most often transmitted to humans by inhalation of an aerosol that has been contaminated with parturient products, urine, and feces of the infected animals (Parisi et al., 2006). Main clinical presentations may be as flu- like illness or a febrile pneumonia or hepatitis (Arricau-Bouvery & Rodolakis, 2005), (Kampschreur et al., 2012), While, in few cases, the disease progresses to a chronic stage characterized by endocarditis or vascular infection (Kampschreur et al., 2012), (Raoult et al., 2005), (Anderson et al., 2013). Polymerase Chain Reaction (PCR) assays are commonly used to directly detect C. burnetii in biological materials such as placentas, genital swabs, feces or milk samples, and reveal the existence of ongoing infections associated with bacterial shedding (Norlander, 2000). PCR assays provide a valuable approach that is sensitive, easy to perform, and safe for laboratory personnel and it holds the promise of timely diagnosis, since it should be positive before antibodies are detectable (Berri, et al., 2003).

Figure 1: Area of the research, samples were taken from different districts of Sulaimani province, Kurdistan- Iraq.

MATERIALS AND METHODS

Sample collection

This study was carried out from February to June 2019.Blood and fecal samples were collected from 90 sheep and 90 goats.Blood (5 ml) was collected from the jugular veins of the aborted small ruminants using disposable needles (18 gauges) and 10ml syringes. Blood samples were then stored at room temperature for one hour to allow clotting. After centrifugation (1,500 x g, 10 minutes) the serum samples were ready for DNA extraction.

Fecal (3g) was obtained and placed in sterile plastic tubes, transported to the laboratory at 4°C and subsequently preserved at -20°C.

Sample preparation and DNA extraction   

The fecal samples were vortexed in phosphate buffered saline (1 mL 0.1 M PBS of pH 7), then genomic DNA was extracted from fecal and serum specimens using a DNA extraction kit (Genaild, Co, Korea) according to the manufacturer's instructions.

Oligonucleotide primer

A PCR protocol targeting IS1111repetitive transposon-like region of C. burnetii was used for the detection of C. burnetii DNA in feces and serum. For the PCR amplification primers Trans-1 (5’-TAT GTA TCC ACC GTA GCC AGT C-3’) and Trans-2 (5’-CCCAAC AAC ACC TCC TTA TTC-3’) were used (Hernychova et al., 2008). The length of the genome target for amplification was expected to be (687) bp (table 1).

Table 1: Sequences of primers and PCR conditions

Uniplex Polymerase Chain Reaction

C. burnetii was amplified by using PCR Premix (2X). This kit provides a complete system for fast, high yield and reliable single tube PCR (Genet-bio, Korea). The reactions were carried out in 0.2 ml PCR tube based on the following specifications: 10 μL supreme script PCR premix, 5.μL DNA, 1.μL forward (10 pmol), 1.μL reverse primers (10 pmol), and 3 μL ultra-pure water to make up a final volume of 20.μL. The conventional PCR machine (Hercuvan, USA) was programmed as followed: initial denaturation at 95 °C for 10 min followed by 40 cycles of 95 °C for 30 s; annealing at 58 °C for 35s, and extension at 72 °C for 40 s and a final extension at  72 °C for 10-min. Five-microliters of each amplified DNA sample were loaded on to a 1% agarose gel stained with a safe dye (Eurx-poland) on preparation. Electrophoresis was>performed at 100V.for 50 minutes.

Sequencing the PCR products

In order to confirm the PCR results, sequencing method was depended, 25 μl of amplified PCR product of two positive samples with both direction primers were sent for sequencing using Sanger sequencing method (Macrogen, South Korea).The results were submitted in NCBI/ GenBank with accession number (MK994501 & MK994502).

Phylogenetic tree and sequence analysis

Phylogenetic trees were constructed based on partial sequence IS1111A transposase gene 26 strain of C. burentii. The sequence homology and multiple sequences alignment at the nucleotide and amino acid level was performed by the CLUSTALW program (Thompson, et al., 1994), the phylogenetic tree was constructed by MEGA.X program employing the neighbor-joining (NJ) method (Kimura, 1980), (Kumar, et al., 2018).

RESULTS

Positive amplification was obtained, using the primers which amplify the repetitive transposon-like regions of C. burnetii, from 10 out of 180 blood and faecal samples with 5.55% investigated in the present study. None of the blood samples collected from aborted sheep and goats revealed positive amplification for C. burnetii DNA. Shedding of C. burnetii by aborted small ruminants was found only in 10 out of 60 faecal samples (16.66%) investigated. Six out of 48 faecal samples (12.5%) and 5 out of 12 faecal samples (41.66%) were positive in aborted sheep and goats respectively (table 2).

Table 2: PCR results of examined blood and fecal samples in aborted sheep and goats according to places in Sulaimani province, Kurdistan-Iraq.

Figure 2:  lane 1: 100 bp DNA ladder, +ve: positive control, -ve: negative control, lanes (2-5): an example of positive samples.

Sequence and phylogenetic tree analysis

Nucleotide and amino acid identity of different countries of partial sequence IS1111A transposase gene were compared with other fourteen C. burentii strains. Field sequences strain exhibited identities ranging from 96.82 to 99.84%, and the highest similarity with (Iran, Brazil, Iraq and China) strain with identity rate of 99.84%, and lowest identity with Algeria strain with identity 96.82%. A comparative analysis of the two field isolates exhibited a 100% identity to each other. Interestingly, these isolates presented very limit amino acid diversity from other country strain except Nigeria and Namibia strain ranging from 1.51-20.0%. The partial sequence IS1111A transposase gene of both field sequences were aligned and compared with the reference strains for sequence analysis (Fig. 3), transposaese protein revealed limited variation between them (only one or two amino acid changed) except Namibia and Negeria strain due to different origin (Fig. 3).  In phylogenic tree is shown in (Fig. 4), the two sequence of field isolate clustered together and make grouped with Brazil, Portugal and china strain.

Figure 3: Two fields isolate sequences alignment with differences reference. Multiple sequences alignment of the amino acid of transpoanes gene region two fields isolates Coxiella burentii with fourteen reference strain in different countries.

Figure 4: Neighbor-joining (NJ) phylogenetic tree generated using the nucleotide sequences for the partial sequence transpaneas gene of coxiella burentii spp.

DISCUSSION

The amplification of transposase gene (IS1111) allowed for the sensitivity of the assay to be increased, because this is a multi-copy gene (7-110 copies) (Klee et al., 2006). DNA sequences generated in the present study confirm that C. burnetii is circulating in goats and sheep from some herds of Sulaimani province. A few studies were conducted on C. burnetii in Iraq. An outbreak of Q fever occurred with high morbidity in U.S. marines located in Iraq (Faix et al., 2008).

The circulation of C. burnetii has been reported in AL- Diwaniayh city of Iraq in 2017. The current study constitutes the second attempt to genotype C. burnetii strains in Iraq. Certainly much more work needs to be done and many more samples need to be tested in order to record as many different genotypes as possible, as well as, to cover most of the country territory. Furthermore, the collection of such data and their comparison with data deposited in international databases will help towards both the continuing of the active surveillance and strain genotyping of the pathogen.

Q fever cases have been reported from some countries neighboring Iraq, such as Turkey and Iran. Results of a serosurvey undertaken on 42 sheep flocks in Turkey showed that 20% of sheep were seropositive (Kennerman, et al., 2010). Serologic evidences indicate people and animals in Iran are exposed to C. burnetii (Khalili & Sakhaee, 2009), (Khalili, et al., 2010).

C. burnetii has already been detected using different PCR methods in blood samples of infected camels in Iran (Schoffelen et al., 2014).The sequence results analysis revealed 99% identity with Iranian, Brazilian and china strains. This indicates that the source of the bacteria in Iraq is Iran, Brazil and China which entered the country by importing meat and animal products from these countries.

Limited variation between field sequences, past Iraqi strain, and other countries is an indicator for stability of transposase gene (Fig. 3). In phylogenetic tree the sequences of current study make cluster with other Iraqi strain, this result epidemiologically exhibit that Coxiella burnetii has the same source in Iraq (fig. 4). The main route of shedding by ovine was found to be the faeces and vaginal mucus, while these routes were rare in bovine herds. Caprines were found to shed the organism via vaginal discharges, faeces and milk (Rodolakis et al., 2007).

 Shedding of C. burnetii in goats via faeces lasted for 2-5 weeks. In the present study the absence of C. burnetii DNA from the serum samples of sheep and goats could be attributed to the fact that the organism in these animal species is shed primarily via vaginal mucus and faeces, and this probably confirms that milk and blood are not the preferred routes of discharge for C. burnetii in sheep and goats (Rodolakis et al., 2007).

CONCLUSION

The present study reports the first molecular detection of C. burnetii in sheep and goat in Sulaimani province. Further studies are necessary to characterize the genotype of C. burnetii and to identify the potential risk of transmission between human and animals regarding the public health issue of Q fever.

ACKNOWLEDGEMENT

Authors are thankful to the Sulaimani Veterinary Diagnostic Laboratory (SVDL) for providing facilities to carry out this research word.

CONFLICT OF INTEREST

The authors disclose no conflict of interest.

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