Diagnostic Studies on Gastroenteritis in Conjunction with the Anatomical and Histological Studies on the Gastrointestinal Tract of the Egyptian Tortoise (Testudo kleinmanni)

| Digestive problem is one of the most important complain of desert tortoises. A total of twenty four Egyptian tortoises (Testudo kleinmanni) were examined for clinical, anatomical andhistological studies. Gastroenteritis in our study was mainly occurred from parasitic infestations; large numbers of Oxyurid adult worms, small sized larvae and large number of Oxyurid eggs. In addition, protozoal infection included the presence of Nyctotherius and Plantadium species. Marked anorexia and greenish watery diarrhea were the main clinical signs. Also, significant increase in relative eosinophilic count was recorded. Marked significant decrease was recorded for serum calcium level leading to areas of discoloration at the carapace. Anatomical studies were applied to manage the Plastron celiotomy for intestinal impactions. In this study, we compared anatomically the healthy and unhealthy gastrointestinal tracts. Normal anatomical positions of the GIT were changed as, in case of severe gastroenteritis and accumulation of gases, the cecum was displaced to the left side, and the ascending colon reached the right side of the coelomic cavity. Also, the stomach was displaced more laterally to the left side in the diseased GIT. There was no external demarcation between jejunum and ileum. Densely backed mucous glandular tissue was found in the esophageal mucosa. No evidence for sub mucosal Brunner’s glands of duodenum were observed. More abundant gut associated lymphoid tissue was recorded in cecal region samples. Microscopic examination of intestinal/duodenal samples revealed many intra epithelial parasitic stages all over intestinal villi accompanied with underlying lamina propria or intra epithelial eosinophilic infiltrations.


MAtErIAlS And MEthodS
For the clinical examinations fourteen tortoises of different age and sexes were examined in different pet clinics. Hematological, serum biochemical and fecal examinations were applied on the blood and fecal samples respectively. Parasites were classified on the basis of key described by (Gango, 2006). Statistical analysis was applied using student T-test. P-values less or equal 0.05 considered significant.
The anatomical study was applied on five Egyptian tortoises (Testudo kleinmanni). Those tortoises were immediately administered dead at the hospital of faculty of veterinary medicine The shell was opened then the coelomic cavity was opened by careful reflection of either the carapace or the plastron, so that the digestive tract was viewed and dissected. All the fresh specimens were photographed by a camera 20.1 megapixels.
Statement of Animal Rights: This study has the approval number of Institutional Animal Care and Use Committee (IACUC) of the Faculty of Veterinary Medicine, Cairo University for using life animal in this research Vet CU01102020222.
For microscopic investigations, five tortoises were used. The turtles were anesthetized with xylazine 1mg/Kg/IM and ketamine 20mg/Kg/IM in forelimb muscles, and then they euthanized by means of an injection of sodium thiopental at 2.5% in the lethal doses of 60mg/Kg/ IVin jugular vein. Tissue samples were flushed and fixed in 10% neutral buffered formalin for 72 hrs. Samples were trimmed, processed in serial grades of ethanol, cleared in xylene, and then samples were infiltrated and embedded into Paraplast wax tissue embedding media. 4μn thick tissue sections were cut by rotatory microtome and mounted on glass slides from different anatomical regions samples. Tissue sections were stained by Hematoxylin and Eosin as a general morphological examination staining method (Bancroft and Gamble, 2008). All data and micrographs were obtained by Full HD microscopic camera operated by Leica application module (Leica Microsystems GmbH, Wetzlar, Germany). All standard procedures for samples fixation and staining were according to (Culling, 2013).

CliniCal examinaTion
Regarding clinical examination, diseased desert tortoises revealed dullness and depression with marked degree of dehydration showed by dry skin and increased skin tenting time in the skin of neck and limbs at the time of admission to clinic. By careful examination to the status of carapace and plastron, there were areas of discoloration at the carapace ( fig. 1/A) beside pores and cracks that were scattered on both carapace and plastron ( fig. 1/B). Marked anorexia was recorded as a main complain. During examination, diseased tortoises showed greenish watery diarrhea  (Table 1) of diseased tortoises, statistical analysis showed significant decrease (P≤0.001) in PCV and relative lymphocytic count. This decrease was also significant (P≤0.01) for both hemoglobin concentration and RBCs count. Significant (P≤0.001) increase was detected in both WBCs count and relative eosinophilic count. All the results were compared with apparent healthy group. In terms of serum biochemical examination (Table 2) among diseased tortoises, statistical analysis showed significant (P≤0.01) decrease in total protein, albumin and

Advances in Animal and Veterinary Sciences
April 2021 | Volume 9 | Issue 4 | Page 502 glucose levels. Marked significant decrease (P≤0.001) was recorded for serum calcium level while both potassium and phosphorus levels showed mild significant (P≤0.05) decrease. All the results were compared with apparent healthy group.

Advances in Animal and Veterinary Sciences
April 2021 | Volume 9 | Issue 4 | Page 503

dIScuSSIon
The clinical presentation among diseased desert tortoises included dullness, anorexia, lethargy, dehydration and weight loss. All these deteriorations were resulted from the heavy infestation with intestinal parasites. These findings were in the same context of (Pasmans et al., 2008;Martinez-Silvestre, 2011). Also, Loukopoulos et al. (2007) added that nematode infection may result in deleterious effects on the intestinal absorptive capacity that leads to severe weight loss and death.
Numerous pores and cracks found on the carapace and plastron were attributed to severe calcium deficiency and metabolic bone disease made by parasites. Fecal examination applied at the time of admission revealed Oxyurid species eggs, adult male and female worms. These results were in agreement with (Shanker et al., 2015;Hallinger et al., 2018).
Hematological examination in diseased tortoises revealed significant decrease for PCV, hemoglobin and RBCs count which come in the same findings of (Diaz-Figueroa, 2005;Ali et al., 2018). Relative lymphocytic count showed significant decrease in proportion to increase of relative eosinophilic and heterophilic count during acute phase of the disease. Significant increase in WBCs count in the present study was explained by severe gastro-enteritis and parasitic damage of GIT mucosa.
In the term of serum biochemical examination in diseased tortoises, the present results showed significant decrease in total protein, albumin and glucose levels. These findings were similar to some extent that of Martinez-Silvestre, (2011). who recorded marked decrease to such levels but without obvious significance. The present study revealed significant decrease in serum calcium level in diseased tortoises. This result explained as Balantidium spp. may interfere with intestinal calcium absorptive capacity which may resulted in metabolic bone disease, cracked and porous carapace and plastron (Pasmans et al., 2008). Mild significant decrease was recorded for serum potassium and phosphorus levels. These findings may result from severe diarrhea and heavy worm infestation. Decreased phosphorus level comes in the line with calcium deficiency due to Balantidium spp. infection.
In the present anatomical study the esophagus was short, dorsal to the trachea at midline then moved toward the left side to reach the cardia of stomach. Also, it has small longitudinal folds without papillae, In accordance with (Wyneken, 2011). Per-contra, Barboza (1995) stated that in the herbivorous wild tortoise Xerobates agassizii, the esophagus has Cornified esophageal epithelia. In addition to Melo et al. (2019) revealed that the green turtle (Chelonia mydas) esophagus has thin and conical keratinized dermal papillae.
Gastro esophageal sphincter was wide and conical in shape with small longitudinal folds internally without papillae, similar to observations of, Magalhães et al. (2012) cited that the sea turtles Gastro esophageal sphincter was devoid of papillae internally which demarcated the stomach.
Similar to the observations of Mansoori et al. (2018) as they mentioned that the stomach was J in shape, Per-contra Barboza, (1995) stated that the stomach was L-shaped, also McArthur et al. (2004) stated that it was fusiform. In accordance with Taylor et al. (1996) stomach was situated ventrally to the left lobe of the liver. In our study the healthy stomach mucosa has large corrugated longitudinal folds, conversely, Mansoori et al. (2018) documented that the mucosal folds were absent cranially and caudally at the stomach.
In the present study as well as Taylor et al. (1996) mentioned that the small intestine was convoluted. There was

Advances in Animal and Veterinary Sciences
April 2021 | Volume 9 | Issue 4 | Page 506 no external demarcation between jejunum and ileum as stated by Magalhães et al. (2012). Similar to the observations of Mansoori et al. (2018), the duodenum has two flexures. Also, Magalhães et al. (2012) documented that the small intestine internally had reticular folds. In accordance with McArthur et al. (2004) as they cited that the small intestine lies in the caudal coelomic cavity, On the other hand, Magalhães et al. (2012) cited that the small intestine had a constant diameter throughout its length and the duodenum was located medially in the coelomic cavity.
In the desert tortoise of our study, the healthy cecum was large sac like dilation; conversely, the cecum formed a small dilatation as mentioned by (Taylor et al., 1996). In accordance with Magalhães et al. (2012) that cecum and colon has saccular and narrow areas, in addition to uneven mucosal folds.
Similar to the observations of McArthur et al. (2004) the caecum situated at the right caudal region of the coelom. The rest of large intestine was the ascending, transverse and descending colons. The cloaca was the last part of the urogenital and digestive tracts (Divers, 2015). The cloacal mucosal had rectilinear folds which was in agreement with (Magalhães et al., 2012).
Histologically, similar to the observations of Barboza (1995), our study revealed that the esophagus had large number of mucus glands. Also, the esophageal region near the cardiac junction transformed into stratified layers of columnar epithelial cells alternated with goblet cells, which was in conjunction with Wyneken (2011) who cited that it contained secretory cells and with Perez-Tomas et al. (1990) who reported that the lower third of the esophagus of was lined by a stratified columnar epithelium composed of a superficial layer of columnar mucous cells, a middle layer of polyhedral cells, and a cuboidal cells forming the basal layer.
In accordance with Stevens and Hume (2004), our results showed that the esophageal outer muscular layers were arranged into inner circular and outer longitudinal smooth muscle fibers.
In addition, the results of the current study revealed that the stomach was lined with simple columnar secretory epithelium in the cardiac, fundic and the pyloric regions, and that the fundic gland was lined with the mucous cells without clear distinguish between the acidophilic oxyntic (parietal) cells and the basophilic peptic (chief ) cells. These findings were in consistence with the observation of Perez-Tomas et al. (1990) that revealed the mucous secretory columnar cells of the stomach lining epithelium, in addition to the lining of the fundic gland with mucous neck cells and oxyntic peptic cells.
Similar to observations of McArthur et al. (2004), the small intestine mucosa had simple columnar epithelium. On the other hand, Tlachia et al. (2014) mentioned that the small intestine mucosa had pseudo-stratified columnar cells with goblet cells. In addition, our study added that there was no evidence for sub mucosal Brunner's glands of duodenum. These findings come in line with the results of Perez-Tomas et al. (1990) exhibited the absence of Lieberkiihn crypts, Brunner glands, and Paneth cells in the small intestine. They also reported the columnar lining of the large intestine which was observed in our study alternated with abundant goblet cells.

concluSIonS
In our study the examined Egyptian tortoises (Testudo kleinmanni) suffering from Gastroenteritis were diagnosed as parasitic infestations with Oxyurid worms. In addition to Protozoal infection included the presence of Nyctotherius and Plantadium species. Marked anorexia and greenish watery diarrhea were the main clinical signs. A significant increase in relative eosinophilic count was recorded. Marked significant decrease was recorded for serum calcium level leading to areas of discoloration at the carapace. Normal anatomical positions of the GIT was changed as, in case of severe gastroenteritis and accumulation of gases, the cecum was displaced to the left side and the ascending colon reached the right side of coelomic cavity. Also the stomach was displaced more laterally to the left side in the diseased GIT. There was no external demarcation between jejunum and ileum. Densely backed mucous glandular tissue was found in the esophageal mucosa. No evidence for sub mucosal Brunner's glands of duodenum were observed. More abundant gut associated lymphoid tissue was recorded in cecal region samples. Microscopic examination of intestinal/duodenal samples revealed many intra epithelial parasitic stages all over intestinal villi accompanied with underlying lamina propria or intra epithelial eosinophilic infiltrations.

AuthorS contrIButIon
Reem Rashad Tahon, Yara Sayed Abouelela we studied the anatomical part as a whole. Hitham Abdel-Saeed: he made all the clinical, hematological, biochemical examinations as well as the statistical analysis. Mohamed Abdel-Razik Khattab, Zainab Sabry Othman Ahmed they studied the histological part as a whole.

conFlIct oF IntErESt
There is no conflict.