DETECTION AND ISOLATION OF ROTA (GROUP A) AND CORONA (GROUP 2) VIRUSES FROM NOMADIC CAMELS WITH SPECIAL REFERENCE TO PATHOLOGICAL ALTERATIONS

Fig. 1: FAT, Intracytoplasmic perinuclear fluorescence in the positive enterocyte.

Fig. 2: Camel rota virus infected Vero cells reveals positive haemadsorption.

Fig. 3: Duodenum of camel calf infected with Rota virus showing intense infiltration of mononuclear inflammatory cells and moderate neutrophilic infiltration in the lamina propria    (H&E; X100).

Fig. 4: Jejunum of camel calf infected with Corona virus showing mononuclear inflammatory cells and moderate edema in the lamina propria (H&E; X100).

Fig. 5: Ileum of camel calf infected with Corona virus showing severe congestion of the blood capillaries and wide dilatation of the lymphatic in some villi (H&E; X400).

Fig. 6: Ileum of camel calf infected with Rota virus and Corona virus showing destruction and necrosis of the intestinal villi with mononuclear inflammatory cell infiltration (H&E; X100).

Discussion

In this study, FAT has been used to stain intestinal cells from infected camels, where positive rota and corona virus antigens were detected, although, the staining results (positive/negative) tend to lack detail on intensity/distribution of staining. Although the immunofluorescence (IF) has been used to stain cells or cell debris excreted in stools from viral infected calves; the problem of non-specific fluorescence has prevented its widespread use on stools, but this can be overcome if the virus antigen is first separated by immune precipitation prior to staining (WHO, 1980). However, this method is laborious and time-consuming when done on a large scale. A simpler method is needed to make detection of rota or corona viruses in camel feaces more acceptable. The ELISA is reproducible, rapid and quantitative method for the detection of corona virus, rota virus and reovirus antigens particularly suited for the large-scale testing of fecal samples, the prevalence of rota and corona virus antigens was higher by ELISA than by EM. (Yolken et al., 1977 and Dea and Tijssen, 1988).

In this work, an indirect antigen capture ELISA employing monoclonal antibodies (MAbs) to BRoV and BCoV as the capture antibodies was used to detect BRoV and BCoV in the camel fecal suspensions because of their relative simplicity and speed. The results obtained revealed that rota virus or corona virus antigens or both; were detected, in 44.01%, 23.14% and 10.91% of the total examined fecal specimens respectively. These results like to some extent the frequency of bovine GARV (11 to 19.4%), BCoV (14 to 39%) (Alfieri et al., 2006; Takiuchi et al., 2006 and Oliveira Filho et al., 2007) and the bovine GARV and BCoV co-infection in (15.9%) recorded in cattle herds (Fernandes et al., 2009).

Our positive Ic-ELISA revealed 53.07%, 34.61% and 17.69% were from diarrheic, 34.37%, 12.55% and 3.12% from contact for rota, corona or both virus antigens respectively and 28.57%, from healthy camels for rota virus only. A high frequency of viruses associated were with that diarrheic camels, as previously described for prevalence of rota virus (26.98%) and Corona virus (3.17%) infection in diarrheic calves in Iran (Mayameei et al., 2009). In addition, rota virus infection were the most common causes of diarrhea in camel calves from 6 to 12-months-old (Wernery, 1999 and Wernery and Kaaden, 2002), while corona virus alone or mixed with rota virus was at 1 to 2 years old. Anyhow; bovine corona virus was the principal virus particle present in samples of winter dysenteric adult cattle (Saif et al., 1988), whereas chronically infected adult animals are often a source of the corona virus infection (Clark, 1993).

Interestingly, in the current study; healthy camels had a score for rota virus antigen detection. The exact reason for this finding is unknown. However Since the initial description of the clinical signs of rota virus-induced diarrhea in calves, much has been learned about the epidemiology of rota virus infection (Mebus et al., 1969). Rota viruses can be found in fecal samples from normal healthy animal or in animals exhibiting clinical disease of varied severity (Dea, et al., 1985). The source of the rota or corona viral infection is uncertain in the diarrheic or healthy camels present case. The fact that other contact animals species in the herd had a history of diarrhea supports the concept that there were either acquired or exchange of rota or corona viral infection among camels and the other animal species. Additionally, the possible interspecies corona virus infection may have implication for the cohusbandry of individuals of the family Camelidae with individuals of other families such as Bovidae, Cervidae, and Equidae (Wűnschmann   et al., 2002). The findings of the present study are supported by a recent report in which a corona virus antigenically related to BCoV was identified in a wild ruminants (Tsunemitsu et al., 1995) elk calves (Majhdi et al., 1997), foal (Davis et al., 2000 and Elizabeth et al., 2000) llamas and alpacas (Cebra et al., 2003), While the group A rota virus was identified to be a common intestinal infection in animals and has been demonstrated as a cause of diarrhea in mice, calves, piglets, foals, young rabbits, deer, pronghorn antelope, chickens, turkeys, goats, kittens, a chimpanzee, and a gorilla. There is also evidence that it can infect other animals, as shown by virus isolation studies in monkeys and dogs (WHO, 1980 and Ijaz et al., 1989).    

Although most field strains of BCoV grow poorly in cell culture and fail to produce CPE until after blind passage, primary calf kidney (PCK) and Vero cells have permitted primary isolation of virus (Linda  et al., 1988)while; BRV were isolated from fecal specimens in Vero cells (Sato et al., 1997). In the present study, isolation of camel corona virus in Vero cells were completely failed where no CPE or Ic- ELISA viral detection; until after 5 blind passages; were obtained, whereas camel group A rota virus was isolated in Vero cells and identified by Ic-ELISA.where CPE were manifested on days 3-5 on the 2^nd-5^th passages as rounding, elongation, and granulation of cells. This characteristic CPE goes hand in hand with the growth characteristics in MDBK cells of a calf rotavirus isolated in Northern Ireland (McNulty et al., 1977) and those recorded in MA104 cells during detection and isolation of group A rota virus from camel calves in Sudan which were identified by ELISA and EM (Ali et al., 2004). This finding may attributed to the presence of proteolytic enzymes. The ability of pancreatic proteolytic enzymes to enhance rota virus infectivity in both permissive and semipermissive cell lines was recorded, the degree of enhancement observed was dependent upon the initial multiplicity of infection and the host cell line employed, Vero cells showed that trypsin treatment allowed multiple rounds of rota virus replication to occur in these cells (Graham and Estes, 1980).

In present work, camel rota virus infected Vero cells were positive for haemadsorption assay. This finding strongly pointed to the relation of this isolated virus to BRoV as among the rota viruses for which the presence of a haemagglutinin has been demonstrated are rota calf diarrhoea virus (Spence et al., 1976 & 1978 and Inaba et al., 1977).

In titration of the camel rota virus isolate, a greatest titer observed was equal to 10^5.4 TCID₅₀/ml, thus confirm the hypothesis of high viral concentration, what may contribute for more knowledge of CRV antigenic properties and consequently; seroneutralization assay in microplates may be used in the future to detect and titerate the camel rota virus antibodies in camel flocks.

The histopathological changes associated with rota virus infection were observed in villous atrophy and flattening of the epithelial cells. These changes were consistent with the lesion previously described in natural or experimental rota virus infections in bovine calve, piglets and mice (Mebus et al., 1971, Carpio et al., 1981, and Jos et al., 2003). In many domestic animals, the features of the infection, the intestinal involvement and the pathogenesis are similar to bovines, even though considerable serotype differences exist among animal rota viruses of different geno-groups (Wani et al., 2004andFukai et al., 2006). After entry through oral route, rota viruses have been found to replicate in the mature villous epithelial cells (enterocytes). For their efficient colonization and infectivity, viruses need their outer capsid to be cleaved and removed, which is facilitated by intestinal proteases like chymotrypsin (Ramig, 2004 and Desselberger et al., 2005). The small and large intestine infected by corona virus had multifocal mucosal ulceration and various degrees of mucosal to submucosal edema with moderate accumulation of lymphocytes. This is in agreement with previously record in bovine calves (Mebus et al., 1973, Mebus et al., 1975 and Sharpee et al., 1976). On the other hand,Wűnschmann et al. (2002) recorded that pathological changes were probably marked in the colon and cecum than the parts of small intestine in camel calves and mixed nonhemolytic coliforms and nonhemolytic Streptococcus sp. were cultured from colon. In addition, the corona virus infection may have predisposed the animal to the putative fatal clostridial infection.

In conclusion; the results of the present study refer to the significant role of rota virus and corona virus or both in the enteric infection of camels in Egypt. In addition, the study refers the antigenic similarities between bovine and camel rota and corona viruses and suggest that camel may be a source for rota or corona viruses that infect domestic ruminants or vice versa. However, Molecular studies should warranted to clear-up the detailed pathogenesis and strains variance.

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