Genotyping of Rotavirus in Neonatal Calves with Acute Gastroenteritis in Iraq

Globally, Rotavirus is the common major etiologic agents of diarrhea in infant, young children and neonatal calves. It is very important to early diagnose the disease for effective treatment. The objective of this study was to determine the prevalence, molecular characteristics, and the effect of rotavirus strains for severe gastroenteritis in neonatal calves in five Iraqi governorates (Al-Qadissiya, Babel, Kerbala, Missan, Wassit). A total of 125-stool specimens were examined, it have been collected from calves form the period between November 2015 to March 2016. The ages were ranging from 6 to 60 weeks. The specimens were examined using Chromatographic Immunoassay, enzyme-linked immunosorbent assay (ELISA) and Polymerase-chain reaction (PCR). Our results gave us 67 (53.6%) positive by chromatographic immunoassay, 45 (36%) positive by ELISA and 32 (25.6%) positive by PCR. Genotyping were analyzed by multiplex PCR. Genotype combination G1P[8] was (30%) followed by G1P[4] (20%), G3P[4] (20%), G2P[4] (10%), G2P[8] (10%) and G9P[4] (10%). Such information will not only aid in seeking advocacy for introducing rotavirus vaccine in national immunization program in Iraq, but will also help in the evaluation of the efficacy of these vaccines in relation to the rotavirus genotyping circulation.


Introduction
The wheel-like (Latin rota = wheel) particles of rotavirus were first determined as a human pathogen in 1973 by Bishop Ruth [1].When characteristic particles were noticed in the cytoplasm of duodenal epithelial cells got from young children severing from acute diarrhea [2].Rotavirus is a 65 -70 nm RNA virus of the family Reoviridae, icosahedral, with segmented double-stranded, it is classified into seven serogroups A -G; Group A subtypes 1, 2, 3, 4 constitute the main human pathogens and Group B -E infects mainly animals and birds, the rotaviruses own 3 protein shells surrounding the genome, this triple layered structure have capsomeres look like the spokes of a wheel radiating from the inner to the outer capsid [3].Viral pathogens account for approximately 70% of episodes of acute infectious diarrhea in children, and rotavirus is the most commonly implicated virus, Group A rotaviruses are responsible for 30% -60% of all cases of severe watery diarrhea in young children and animals [2].Diarrhea caused by rotavirus could not be recognized clinically because the clinical symptoms (diarrhea, vomiting, fever, and dehydration) are not entirely associated with rotavirus infection [4].The most commonly used tests in diagnosing rotavirus infections are electron microscopy, latex agglutination test, and enzyme-linked immunosorbent assay (ELISA), polyacrylamide gel electrophoresis, and immune chromatographic and polymerase chain reaction (PCR) tests [5].Since there are many combinations of P and G genotypes in bovine group A rotavirus (BRV), research into the genotyping of BRV is very important for preventive veterinary medicine and, more specifically, for the development of a vaccine.It is also important from the point of view of ecology and public health, because interspecies transmission from cattle to humans and from humans to cattle have been reported.Particularly rotavirus P [11], G10 strains, which are commonly found in cattle had frequently been associated with asymptomatic neonatal infections in Tamil Nadu, India [6].
The aim of the present study was to identify the genotype distribution of bovine rotavirus in five governorates in Iraq and to know the vaccine isolates depending to the genotyping of rotavirus.

Materials and Methods
A total of 125 stool samples obtained From November 2015 to March 2016, from neonatal calves with acute gastroenteritis were randomly collected at 5 Iraqi governorates, for each governorates 25 samples have been taken (Babel, Kerbala, Missan, Qadissiya, and Wassit).10 gm of yellow to white liquid with fatty droplet feces were examined under microscope and collected directly into sterile disposable plastic containers by rectal stimulation then stored in a cool box and transported to the laboratory, where each sample was added to specimen collection tube with extraction buffer that used in chromatographic immunoassay.The feces samples were centrifuged for 5 minute to remove particulates at 3000 rpm.Thereafter, the supernatants were stored at −20˚C until the assay day.
The chromatographic immunoassay performed to the first method that we used to detect the rotavirus in stool samples (ABON Biopharm Co., Ltd., Hangzhou, China).The qualitative Rotavirus assay ELISA was performed to the second method, which detect the rotavirus (RV) antigen (Ag), the ELISA was carried out using Cusabio kit (Cusabio Biotech Co., Ltd, China).Chromatographic immunoassay and ELISA were performed according to the manufacturers' instructions.While the PCR and Genotyping were evaluated by using (Accu-Power PCR Premix of Bioneer Corporation) from Republic of Korea.The primers as it shown in (Table 1 and Table 2) and thermo-cycler conditions were performed according to the manufacturers' instructions that we used in RT-PCR and Genotyping for both G and P were performed according to World Health Organization manual [7].

Results
The present study showed the relationship among three diagnosis tests for detecting rotavirus serotype in Iraq.Chromatographic immunoassay revealed 55 (44%) The highest positive results were in Missan 60% (15 of 25 samples); however the lowest incidence was in AlQadissiya 28% (7 of 25 samples) by using chromatographic immunoassay.Table 1.The primers and their sequences that are specific for human rotavirus genotyping.P-type-specific oligonucleotide Primers.World Health Organization [7].Nevertheless, ELISA test revealed 53 (42%) positive samples.The highest positive result was recorded in Missan 60% (15 of 25 samples), and the lowest in AlQadissiya 20% (5 of 25 samples).In this study, PCR technique was used for detection the two outer layer protein's VP7 and VP4.All animals samples, which had been tested using Chromatographic and ELISA, were tested by using PCR 47 (38%) were positive.The higher percent was in Missan 48% (12 of 25 samples) and the lowest was in AlQadissiya 28% (7 of 25 samples) as it shown in Table 3.

Discussion
Chromatographic Immunoassay and ELISA are the straightforward and effortless standard methods for detection of rotavirus.These methods, however, require low cost equipment and simple experience, which is available in many laboratories.Some researchers for detecting rotavirus infection have used ELISA and PCR [8].Our study suggested that chromatographic immunoassay is a possible method for examining stool samples that have been collected from animal with suspected diarrhea that be infected by rotavirus.
This study suggested that chromatographic immunoassay was screening test which can be performed without need of trained personnel or expensive equipment also it can read with naked eye, making it easy to perform in every laboratory.The positive results that have been obtained by ELISA method were approximately similar to the results by using chromatographic method, which is probably responsible to the reported superior accuracy of the method.By chromatographic immunoassay 55 (44%) were positive samples, and by ELISA revealed 53

Figure 1 .
Figure 1.The presence of G and P of animal samples in the five governorates.

Table 2 .
The primers and their sequences that are specific for human rotavirus genotyping.G-type-specific oligonucleotide Primers.

Table 3 .
Comparison of the results that have been obtained from three testing method for rotavirus in 125 bovine fecal samples.