The first identification of rotavirus infection in wild boars in Iraqi marshes using molecular technique

Introduction

Rotaviruses recognized as common pathogens affecting animals and birds. The rotavirus belongs to Rioviridae. Rota genome have eleven segments. The item has been unwrapped and classified depending on vp6 into eight categoreis from A to G (Johne et al., 2024; Estes and Greenberg, 2013) [11,6] . Virus target site is GIT particularly the interic cells (He wt al., 2003; Liu et al., 2013) [13] . In Turkey they record rotavirus infectivity in Turkish sheep to be infected in ages between 2-3 years. Old aged pigs get higher chances for Rotavirus B infections; however, younger get infection almost due to rotavirus C. High detection of rotavirus B and C indicate the major of this virus as a important etiological factor of diarrhea in swine. Rota virus recorded as sporadic cases in Iraq are in cattle, sheep, some birds, dogs, and rodents specially rat. (Marthaler, 2014; Aljabory et al., 2019) [14,1] . In Iraq rotavirus detected in camels and cattle (Saif, 1999; Amimo et al., 2013; Hassan and Kashash, 2017) [18,2,7] . Signs include profuse enteritis, gastritis and diarrhea. Atrophied villi also seen so lead to marked decrease of rejection and pumping and losses of fluid and important electrolytes inside bowel cavity leading to sever dehydration and death that results in effective economic losses over all areas in the world (Tazipori et al., 1980; Shaw et al., 1989; Hassan et al., 2014) [21,19,8] . Wild boars represent a major factor affect epidemiology of rota virus infection in other mammals due to helping spread between animals in European countries (Massei et al., 2015) [15] . Researchers record wild boar to be a significant vector in transmission of this infection. We design our research to investigate possibility of infection in wild boars and the risk that may exist and the role of transmission of rotavirus among cattle and human because wild boars live in marked numbers depending on areas of cattle breeding.

Methods

Collection of samples

Twenty-four intestinal contents samples of wild boars from ileac area of the intestine were collected. Animals were in dissimilar age and sexes following hunted by the hunters for the period of December 2020 to April 2021. Boars were grassed in areas of buffaloes breeding and hunted because they harm buffaloes, cattle, sheep and even human properties. Intestinal contents were located in disinfected cold containers and preserved at -20 ° C till examined.

Primers

Primers used in Molecular examination assay were depending on sequences database, proved by NCBI, rotavirus Avp6 with a length of product 416bp and begin at 699 bp and stopped at 1114bp. Primer extent R= 20 and F= 20, Table . 1

RNA Extraction

Extraction of RNA was done from the supernatant of intestinal content solution prepared in dilution of 1:4 in Diethylpirocarbonate water (DEPC), then centrifuged the solution in cold centrifuge 5000 rpm for 15 minutes. The entire RNA content was extracted from rotavirus-positive stool samples using the QIAamp Viral RNA Collection Kit (QIAGEN, USA), following the manufacturer's protocol.

RT-PCR to detect the rotavirus group

Five microliters of RNA extract were added to 3.5 microliters of dimethyl sulfoxide and mixed in a microcentrifuge tube, followed by heating at 95°C for five minutes and then immediately chilled on ice. Complementary DNA (cDNA) synthesis was performed using SuperScript™ III reverse transcriptase (Invitrogen). Subsequent steps were carried out according to Ibrahim et al., 2025. The PCR products were then run on a 1.2% agarose gel containing 0.5 µg/mL ethidium bromide and visualized using the BIORAD UV gel

Results and Discussion

Rotavirus infection considered to be an important viral disease causing gastroenteritis and diarrhea in cattle, buffaloes, sheep and human. It considered as being a common disease (El-Sadek et al., 2019) [5] . The infection with rota virus was diagnosed in Brazilian pigs with infectivity (23.6%) by using real time Polymerase Chain Reaction and gel electrophoresis. This outcome were out of 288 animal examined and show (34/288). A percentage of (11, 8%) were positive for rota antigen (29/34) with no diarrhea in pigs with percentage of 85.3% in Southern of Mozambique (Mediei et al., 2011; Boene et al., 2019) [16,3] . In Thailand researchers record that ages infected not older than 3 months was 24 out of 132 (18.2%). They infected with type A rotavirus (18). The results gained in Iraq represenes the earliest resting on southren Iraqi wild boars that well thought-out be common over countries in the world. Results in current work concerning molecular diagnosis were obtained by means of electrophoresis of real time-PCR and shown in (Fig. 1). The current study agreed with various researches (El-Sadek et al., 2019) [5] who recored that in Egypt the infection was (17%) 22 out of 82 of infect cattle with type A. In Thailand about 21.6% (11/51) in pigs infected (Bulgin et al., 1988) [4] , and in Japan 4 out of 90 wid boars (4.4%) infection were recorded (Tuanthap et al., 2018) [20] . The positive outcomes that recorded in our study were high in January, we recorded 4 positive cases, then in February and March at 1 positive in each month as shown in Table [17,22] . who indicate winter is the max out spread of the infection, which support our conclusion. We suggest that wild boars in Iraq take part in the epidemiology of infection in calves, calf buffalo and also human because they survive near area of cattle breeding, this was mentioned by several field workers (Tzipori et al., 1980; Shaw et al., 1989) [21,19] .

Acknowledgement

AcknowledgementsI would like to express my sincere thanks to the staff members of the Zoonotic Diseases Research Unit at the College of Veterinary Medicine, University of Baghdad, for their valuable support and assistance. I also extend my gratitude to the laboratory technicians who helped with sampling and laboratory analyses.

References

1. Amimo JO, Vlasova AN, Saif LJ. Detection and genetic diversity of porcine group A rotaviruses in swine fecal samples in Ohio: Predominance of the G9P [13] genotype in nursing piglets. Journal of Clinical Microbiology. 2013;51(4):1142-1151. https://doi.org/10.1128/JCM.03135-12
2. Boene SS, João E, Munlela B, Nabetse E, Cala A, Mapaco L, et al. Detection and molecular characterization of Rotavirus A antigen in fecal samples from diarrheic and non-diarrheic pigs in Maputo City and Province, southern Mozambique. International Journal of Infectious Diseases. 2019;79:70. https://doi.org/10.1016/j.ijid.2018.11.179
3. Bulgin MS, Ward AC, Barrett DP, Lane VM. Detection of rotavirus and coronavirus shedding in two beef cow herds in Idaho. Canadian Veterinary Journal. 1989;30(3):235-239.
4. El-Sadek E, Shahein M, Abdelwahab S, El Shahidy M. Investigation of Rota and Corona viruses as causative agents for diarrhea in Egyptian calves. Suez Canal Veterinary Medicine Journal (SCVMJ). 2019;24(2):257272. https://doi.org/10.21608/scvmj.2019.79238
5. Estes MK, Greenberg HB. Rotaviruses. In: Knipe DM, Howley PM, et al., editors. Fields Virology. 6th ed. Philadelphia (PA): Wolters Kluwer Health/Lippincott Williams & Wilkins; 2013. p. 1347-1401.
6. Hassan HA, Kshash QH. Molecular detection of Rotavirus type A in diarrheic calves of Mid-Euphrates governorates, Iraq. Iraqi Journal of Veterinary Medicine. 2017;41(2):48-53.
7. Hassan HA, Kshash QH, Mansur KA. Detection of bovine rotavirus in diarrheic calves by using rapid test in some Mid-Euphrates provinces. Al-Qadisiyah Journal of Veterinary Medicine. 2014.
8. He B, Yang F, Yang W, Zhang Y, Feng Y, Zhou J, et al. Characterization of a novel G3P[3] rotavirus isolated from a lesser horseshoe bat: A distant relative of feline/canine rotaviruses. Journal of Virology. 2013;87(22):12357-12366. https://doi.org/10.1128/JVI.02013-13
9. Ibrahim AHA, Noor MM, Al-Rubaiey MGA. Identification of rotavirus in human and calf buffalo using RT-PCR technique and rapid testing. International Journal of Veterinary Sciences and Animal Husbandry. 2025;10(8):297-299. https://doi.org/10.22271/veterinary.2025.v10.i8.4315
10. Johne R, Donato C, Bányai K. Rotavirus diversity, evolution, and lineage classification. Bacterial and Viral Bioinformatics Resource Center (BV-BRC). 2024. https://www.bv-brc.org/docs/downloads/subspeciesclassification-workshop/040824S2.4-Donato.pdf
11. Kumthip K, Khamrin P, Kongkaew A, Vachirachewin R, Malasao R, Ushijima H, et al. Molecular epidemiology and characterization of porcine adenoviruses in pigs with diarrhea in Thailand. Infection, Genetics and Evolution. 2019;67:73-77. https://doi.org/10.1016/j.meegid.2018.11.021
12. Liu F, Li G, Wen K, Wu S, Zhang Y, Bui T, et al. Lactobacillus rhamnosus GG on rotavirus-induced injury of ileal epithelium in gnotobiotic pigs. Journal of Pediatric Gastroenterology and Nutrition. 2013;57(6):770-778. https://doi.org/10.1097/MPG.0b013e3182a35616
13. Marthaler D. Widespread Rotavirus H in commercially raised pigs, United States. Emerging Infectious Diseases. 2014;20(7):1195-1198. https://doi.org/10.3201/eid2007.131680
14. Massei G, Kindberg J, Licoppe A, Gačić D, Šprem N, Kamler J, et al. Wild boar populations up, numbers of hunters down? A review of trends and implications for Europe. Pest Management Science. 2015;71(4):492-500. https://doi.org/10.1002/ps.3965
15. Médici KC, Barry AF, Alfieri AF, Alfieri AA. Porcine Rotavirus groups A, B, and C identified by polymerase chain reaction in a fecal sample collection with inconclusive results by polyacrylamide gel electrophoresis. Journal of Swine Health and Production. 2011;19(3):146-150.
16. Okadera K, Abe M, Ito N, Morikawa S, Yamasaki A, Masatani T, et al. Evidence of natural transmission of group A Rotavirus between domestic pigs and wild boars (Sus scrofa) in Japan. Infection, Genetics and Evolution. 2013;20:54-60. https://doi.org/10.1016/j.meegid.2013.08.002
17. Saif LJ. Enteric viral infections of pigs and strategies for induction of mucosal immunity. Advances in Veterinary Medicine. 1999;41:429-446.
18. Shaw DP, Morehouse LG, Solorzano RF. Rotavirus replication in colostrum-fed and colostrum-deprived pigs. American Journal of Veterinary Research. 1989;50:19661970.
19. Tuanthap S, Phupolphan C, Luengyosluechakul S, Duang-in U, Theamboonlers A, Vongpunsawad S, et al. Molecular characterization of porcine Rotavirus C in pigs with gastroenteritis in Thailand, 2011-2016. PeerJ Preprints. 2018;6:e3515v1. https://doi.org/10.7287/peerj.preprints.3515v1
20. Tzipori SR, Makin TJ, Smith ML. The clinical response of gnotobiotic calves, pigs and lambs to inoculation with human, calf, pig and foal Rotavirus isolates. Australian Journal of Experimental Biology and Medical Science. 1980;58:309-318.
21. Yu J, Lai S, Geng Q, Ye C, Zhang Z, Zheng Y, et al. Prevalence of Rotavirus and rapid changes in circulating Rotavirus strains among children with acute diarrhea in China, 2009-2015. Journal of Infection. 2019;78(1):6674. https://doi.org/10.1016/j.jinf.2018.09.002