PCR- BASED DETECTION OF Helicobacter SPECIES IN Camelus dromedarius

الکشف القائم على بی سی ار لأنواع هیلیکوباکتر فی الجمل العربى.

احمد إبراهیم یوسف⁽¹⁾ ، احمد فاروق عفیفى⁽²⁾ ، محمد السید عنانى  ⁽³⁾ ، سعید حامد عبادى⁽⁴⁾ ، ایمن حامد محمود⁽⁵⁾

1-  أستاذ مساعد الصحة – الإمراض المشترکة کلیة الطب البیطری – جامعة قناة السویس.

2-  مدیریه الطب البیطری بالاسماعیلیه.

3- أستاذ المیکروبیولوجى کلیة الطب البیطری – جامعة قناة السویس.

4-  أستاذ المیکروبیولوجى والمناعه عمید کلیه الطب -  جامعة السویس.

5-  أستاذ البیوتکنولوجى – بمعهد بحوث الصحة لحیوانیة بالدقى.

العدوى بانواع الهیلیکوباکتر لها أهمیة وبائیة وبالإضافة الى امکانیة انتقالها من الحیوان للإنسان. وقد ورد أن الأنواع المختلفة من الهیلیکوباکتر تم تعبئتها فی البشر والحیوانات. و کان الهدف من هذه الدراسة هو الکشف عن انواع الهیلیکوباکتر فی عینات البراز التی تم جمعها من الإبل باستخدام البیولوجیه الجزیئیه بى سی ار التی تستهدف جین.16S rRNA  وکان اجمالى اعداد الإبل 32 جمل من تسع مزارع للإبل صغیرة موزعة فی أماکن مختلفة بمحافظة الإسماعیلیة ، مصر. أظهرت النتائج أن 2 (22.22٪) من أصل تسع مزارع من الإبل کانت إیجابیة بالنسبة لأنواع الهیلیکوباکتر. باجمالى 4 (12.5٪) من 32 عینه من عینات الإبل کانت إیجابیة  لانواع الهیلیکوباکتر. و فی الختام ، أظهرت هذه الدراسة التقریر الأول للکشف عن العدوى بالأنواع هیلیکوباکتر من الجمل العربى. و هناک الحاجة لمزید من الدراسات لتحدید الإصابات بالأنواع الهیلیکوباکتر فی الإبل ولتوضیح الجوانب الوبائیة وتحدید الأنواع ، وتوصیف الجینوم التى تصیب الإبل من انواع الهیلیکوباکتر.

PCR- BASED DETECTION OF Helicobacter SPECIES IN   Camelus dromedarius

Youssef, A.I. ¹ ; A.F. Afifi ² ;  M.E. Enany ³ ; S.H. Abbadi ⁴

and A.H. Mahmoud ⁵

¹Animal Hygiene and Zoonoses, Faculty of Veterinary Medicine, Suez Canal University, Egypt.

²Veterinarian, Directorate of Veterinary Medicine, Ismailia, Egypt.

³Microbiology department (Bacteriology), Faculty of Veterinary Medicine, Suez Canal University, Egypt.

⁴Microbiology Department (Bacteriology), Faculty of Medicine, Suez University, Egypt.

⁵Biotechnology department, Animal Health Research Institute, Giza, Cairo

Key Words:Helicobacter, Camelus dromedaries, PCR, 16rRNA.

ABSTRACT:

Helicobacter spp. infections are of epidemiological and zoonotic impacts. Different species of Helicobacter spp have been reported among humans and animals. The aim of this study was to detect Helicobacter spp. in fecal samples collected from camels using conventional PCR assays targeting 16S rRNA gene. A total of 32 camels were collected from nine of the small camel farms distributed in different localities in Ismailia governorate, Egypt. Results revealed that camel samples from 2 (22.22%) out of nine of camel farms was positive for Helicobacter species. A total of 4 (12.5%) out of 32 camels were positive for Helicobacter spp. In conclusion, this study showed the first report of detection of Helicobacter species infections of Camelus dromedaries. Further studies are need for Helicobacter species infections in camels to clarify the epidemiological aspects and species identification, genome characterization of Helicobacter spp infecting camels.

INTRODUCTION

Following the discovery of H. pylori in humans, Helicobacter species have been detected in pets (Buczolits, et al. 2003). Helicobacter species have been reported in animals of wild and domestic mammals of different dietary habits such as, dogs, cats, mice, swine, cattle, and sheep (Solnick 2003). Helicobacter species identified in animals cannot be distinguished microscopically when they are examined in histopathological sections in gastric tissue by light microscopy (Henry, et al. 1987). Helicobacter species have been considered a potential reservoir of zoonosis (Fox 2002, Priestnall, et al. 2004).However, the role of these species in the pathogenesis of gastric disease in companion animals remains unknown (Amorim, et al. 2015).

Numerous detection methods for the presence of Helicobacter species have been used for diagnosis. Each one of them has been associated with advantages and disadvantages. Till date, majority of the microbiological laboratories in the world are not equipped and trained to isolate such fastidious bacterium. Therefore, PCR methods to detect H. pylori’s DNA in clinical samples provide a valuable tool for the identification of Helicobacter spp. with high sensitivity and specificity rates when compared with traditional methods (Chong, et al. 1996;Patel, et al. 2014 and Makristathis, et al. 2019 ).  Identification based on the 16SrDNA gene remains the most frequently used method of detection (Dewhirst, et al. 2005)..

Camels are implicated to be a reservoir in many infectious diseases including parasitic, bacterial, and viral diseases. Some of camel diseases are zoonotic and poses a great public health concern. However, there is a lake of information about Helicobacter infections in camels.

This study was designed to detect Helicobacter spp. infection among Camelus dromedaries.

MATERIAL AND METHODS

Sample collection:

A total number of 32 fecal samples were collected from 9 camels from private farms located at different localities in Ismailia governorate. All the samples were collected from apparently healthy animals. The Farms were distributed in 4 centers from Ismailia governorate which were Ismailia center (n=3), Al Tal AlKabir (n=1), Faid (n=1), and the Al Kantarahgharb (n=4) (fig 1). One hundred gram of fresh fecal sample was collected in sterile containers.

Fig (1): Map showing the geographical distribution of the camel farms at Ismailia Governorate, Egypt. The Farms were distributed in 4 centers from Ismailia governorate which where Ismailia center (n=3), Al Tal AlKabir (n=1), Faid (n=1), and the Al Kantarahgharb (n=4).

DNA extraction from fecal samples

Bacterial DNA was extracted from fecal samples using QIA amp DNA stool Mini Kit (QIAGEN) as per manufacturer’s instructions.

PCR reactions of Helicobacter species:

Reactions of PCR were performed using Emerald Amp GT PCR master mix kit (Takara). Oligonucleotides primers used to amplify targeted genes of Helicobacter spp were ordered from (Metabion, Germany). The oligonucleotides sequences of the primers targeting16S rRNA of Helicobacter spp. were ordered as following: forward primers 5’--AAG GAT GAA GCT TCT AGC TTG CTA-3’, and reverse primers 5’-GTG CTT ATT CGT GAG ATA CCG TCA T-3 for amplification of 398 bp according to (Germani, et al. 1997). Each PCR reaction consisted of a total of 25μl contained 12.5 μl of master mix, 1 μl of each primer (20pmol) each, 5μlof DNA template and PCR grade water up to 25μl PCR grade water. The cycling conditions consisted of one cycle of initial denaturation at 94 ˚C for 5 min followed by 35 cycles consisted of denaturation at 94 ˚C for 30s, Annealing  of ‎54˚C for ‎40 sec.‎, and extension at 72 for 30 sec. The final extension was one cycle at 72 ˚C for 7 minutes. The PCR reactions were electrophoresed after loading on 1% agarose gel with a final concentration of 0.1-0.5 μg/ml Ethidium bromide solution. The DNA molecular weight marker was used in gel electrophoresis using 100 bp ladder (Gel Pilot QIAGEN, USA). The gel was photographed by a gel documentation system (Alpha Innotech), and the data was analyzed through computer software.

RESULTS

Prevalence of Helicobacter spp. and sub-species among samples livestock animals by PCR

Results revealed that camel samples from 2 (22.22%) out of nine of camel farms was positive for Helicobacter species.. A total of 4 (12.5%) out of 32 camels were positive for Helicobacter spp. one of 2 camel sample (50%) from Ismailia center and 3 (75%) of 4 camel sample from Qantarah center were positive for Helicobacter spp. (Table 1 and Fig 2).

Table 1: Distribution of samples collection sites and the detection rates of Helicobacter species among farms and camels:

Fig (2): Agarose gel electrophoresis of PCR performed on Helicobacter spp. strains for the detection of 16rRNA gene amplification product with 398bp. Lanes (1, 3) were positive, Lanes (2) were negative; L indicated DNA ladder (100bp ~ 600bp) and (Pos) showed control positive  and (Neg) showed control negative.

DISCUSSION

   PCR is a powerful method known for its high sensitivity and specificity, and can detect low numbers of Helicobacter spp. present in different types of clinical specimens (Germani, Dauga et al. 1997, Kabir 2001).Because of the difficulty in growing the bacteria in culture, PCR are considered the gold standard in Helicobacter diagnostics. (Dewhirst, et al. 2005, Poynter et al. 2009).

In our study, using the PCR method, we identified the presence of Helicobacter spp. in feces samples of camels. The detection of Helicobacter spp DNA in two camel farms from different localities indicated the spread of infection among the camels. Camels were reared in area in close contact to humans and other animals. Thus, there is a possibility that camels could be one of the reservoirs of human infections by Helicobacter species. The findings of this study have been supported by many previous studies of detection of Helocbacters among animals. Animals are infected with Helicobacter species that generally named helicobacter-like organisms (De Groote, et al. 2005). Helicobacter non-pylori species could be infect both human and animals their probable transmission in humans as reported. These animals might serve as a reservoir for the transmission of pathogenic microorganisms to human contacts(De Witte, et al. 2018). Humans have been reported to be infected by at least 11 different Helicobacter spp., most of which are common or potentially pathogenic, and most of which are likely zoonotic infections transmitted from contact with animals. H. heilmannii infections clearly appear able to pass regularly between species. Contact with cats, dogs, cattle and pigs have all been associated with the infection of humans by H. heilmannii (Meining, et al. 1998). H. pyloriwas previously isolated from sheep milk and its zoonotic transmission has been proposed (Dore, et al. 1999). H. pylori DNA has been detected among environmental ‎samples such as ‎tap water, well water, field soil, flies, and cow feces (Sasaki, et al. 1999).

In conclusion, this is the first report of detection of Helicobacter species infections in Camelus dromedaries. Further studies are need for Helicobacter species identification, genome sequencing and phylogenetic analysis of Helicobacter spp infecting camels. Epidemiological investigations of Helicobacter infections in camels and studying its zoonotic potentials are also warranted.

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الکشف القائم على بی سی ار لأنواع هیلیکوباکتر فی الجمل العربى.

احمد إبراهیم یوسف⁽¹⁾ ، احمد فاروق عفیفى⁽²⁾ ، محمد السید عنانى  ⁽³⁾ ، سعید حامد عبادى⁽⁴⁾ ، ایمن حامد محمود⁽⁵⁾

1-  أستاذ مساعد الصحة – الإمراض المشترکة کلیة الطب البیطری – جامعة قناة السویس.

2-  مدیریه الطب البیطری بالاسماعیلیه.

3- أستاذ المیکروبیولوجى کلیة الطب البیطری – جامعة قناة السویس.

4-  أستاذ المیکروبیولوجى والمناعه عمید کلیه الطب -  جامعة السویس.

5-  أستاذ البیوتکنولوجى – بمعهد بحوث الصحة لحیوانیة بالدقى.

العدوى بانواع الهیلیکوباکتر لها أهمیة وبائیة وبالإضافة الى امکانیة انتقالها من الحیوان للإنسان. وقد ورد أن الأنواع المختلفة من الهیلیکوباکتر تم تعبئتها فی البشر والحیوانات. و کان الهدف من هذه الدراسة هو الکشف عن انواع الهیلیکوباکتر فی عینات البراز التی تم جمعها من الإبل باستخدام البیولوجیه الجزیئیه بى سی ار التی تستهدف جین.16S rRNA  وکان اجمالى اعداد الإبل 32 جمل من تسع مزارع للإبل صغیرة موزعة فی أماکن مختلفة بمحافظة الإسماعیلیة ، مصر. أظهرت النتائج أن 2 (22.22٪) من أصل تسع مزارع من الإبل کانت إیجابیة بالنسبة لأنواع الهیلیکوباکتر. باجمالى 4 (12.5٪) من 32 عینه من عینات الإبل کانت إیجابیة  لانواع الهیلیکوباکتر. و فی الختام ، أظهرت هذه الدراسة التقریر الأول للکشف عن العدوى بالأنواع هیلیکوباکتر من الجمل العربى. و هناک الحاجة لمزید من الدراسات لتحدید الإصابات بالأنواع الهیلیکوباکتر فی الإبل ولتوضیح الجوانب الوبائیة وتحدید الأنواع ، وتوصیف الجینوم التى تصیب الإبل من انواع الهیلیکوباکتر.