BIOCHEMICAL AND ELECTRON MICROSCOPIC CHANGES INDUCED BY GIARDIA IN EXPERIMENTALLY INFECTED LAMBS

The present study was conducted to evaluate the effect of Giardia on the biochemical serum constituents of experimentally infected lambs, in addition to studying the observed alterations accompanying Giardia infection in the intestinal mucosa using scanning electron microscopy. Twenty lambs were allotted into two equal groups, Group A ( non infected) was kept as control negative and Group B (infected) was orally inoculated by 10 Giardia cysts. The biochemical changes were assessed in both groups on 7. 14, 21 and 30 days post inoculation (dpi). The study disclosed that Giardia induced a significant drop in the levels of serum electrolytes (chloride,sodium, potassium and calcium), blood glucose, different enzymes (lipase, amylase and alkaline phosphatase). Furthermore, the levels of urea, liver enzymes (alanine aminotransferase and aspartate aminotransferase), inflammatory marker (C-reactive protein) and oxidative stress marker (malondialdehyde) were elevated, but nitric oxide was declined by 30 dpi. The scanning electron microscopy of the intestinal mucosa of the infected lambs revealed a notable alteration which was fully explained. All the presented results interpret the pathophysiological effect of Giardia which adversely affects the health status of lambs.


INTRODUCTION
Giardia sp. is the most common and important intestinal parasite and it is a frequent common cause of diarrheal diseases through the world. It localized and multiplies in the small intestines of human and domestic animals [1,2]. In ruminant, Giardia causes impairment in feed conversion efficiency which adversely affects the health status and causes enormous economic losses to livestock industry, greatly affects reproductive performance and causes significant production losses [3,4]. Giardiasis outbreak in sheep mostly showed no clinical sign or mortality, but Giardia-infected lambs had malabsorption, decreased weight gain, and reduced feed efficiency [5]. The pathophysiological changes associated with Giardia infection include: malabsorption of electrolytes and nutrients, intestinal microvillus atrophy, loss of epithelial barrier function due to the produced ulceration and infarction , increased permeability and narrowing of the lumen [6,7]. The Changes in the sero-biochemical constituents and the hypothesis of decreased activity of the defense system for protecting tissues from the free radical damage associated with Giardia infection in lambs need further investigation. This was explained in the current study by detecting the alteration in the sero-biochemical parameters, electrolytes and digestive enzymes and by assessing

MATERIALS AND METHODS
Cysts. Giardia cysts were collected from naturally infected sheep. They were identified after lugol's iodine and Giemsa staining. All the collected cysts were liquefied in distilled water and stored at 4 o c . Concentration of fecal specimens was practiced by zinc sulfate floatation technique, followed by twice washing of the concentrated cysts by centrifugation at 1000 rpm for 10 minutes. Lastly, the supernatant was decanted and the sediment was resuspended in a saline solution. Haemocytometer was used to quantify the density of the cysts inoculum. The final inoculum were adjusted to 10 4 Cysts/ lamb. Animal groups. Lambs used in this study were of both sexes and aged 6-8 months. They were kept in wellventilated bin cages and provided with standard food and water. Twenty lambs were equally allocated into two groups; group A ( non-infected group) and Group B (infected group). The lambs were kept in acclimatized conditions for two weeks. During this period, the feces of the lambs were examined daily by direct wet smear and flotation techniques to exclude the existence of any parasites before the experiment.
Ethical approval. Lambs in this work were permitted by the local committee of the Faculty of Veterinary Medicine, Benha University and according to the guidelines of the National Institute of Health in Egypt.

Giardia cyst inoculation.
Each lamb in group B was inoculated orally by a single feeding of Giardia cysts (10 4 Cysts/ lamb).

Cysts detection.
Because of the intermittent shedding of Giardia cyst is, the lambs feces were examined 3 times daily for ten days till appearance of Giardia cysts and establishment of the infection among all lambs.
Serum collection. Ten ml of blood was collected from the jugular vein of each lamb in group A and B into non heparinized tubes. The blood samples were left to stand for 30-60 minutes at room temperature for clotting. The clotted blood were centrifuged at 3,000 rpm for 10 min for serum separation, all the sera were stored at −20°C till further analysis [8].
Biochemical assessments. Estimation of biochemical changes was done by analyzing the serum samples of the two groups for chloride, sodium, potassium, calcium, creatinine (mg/dl), urea , alanine aminotransferase (ALT), aspartate aminotransferase (AST), C-reactive protein(CRP), glucose, amylase, lipase, alkaline phosphatase (ALP), malondialdehyde (MDA) and nitric oxide (NO) using a high performance liquid chromatography method (HPLC, Sykam 1125 pump system, Germany) [9][10][11]. The changes in the biochemical parameters were assessed on 7 th , 14 th , 21 th ,30 th dpi by comparing the mean values ± SE of lambs in A and B groups.

Electron Microscopy
Two infected lambs were sacrificed at the end the experiment and small portions of theirduodenum, proximal part of the jejunum and the lower digestive tract were obtained to identify the changes associated with Giardia trophozoites and cysts. The specimens were kept in glutraldehyde (2.5%) at 4 o c for scanning electron microscope (SEM). They were then rinsed in distilled water and dehydrated in acetone alcohol series (25%, 50%, 75%, and

Clinical Symptoms
The prepatent period of Giardia ranged between 8-10 days in group B (infected group). The cysts shedding continued all through the period of the experimental study ( Fig. 1. a, b). The infected lambs suffered from decreased weight gain, loss of feed efficacy and diarrhea. The onset of diarrhea was on 14 th dpi and continued intermittently throughout the study period.

Biochemical Assessment
The

Electron Microscopy
In the infected lambs, scanning electron microscopy of the duodenum and the proximal part of the jejunum revealed varying degrees of destruction in the intestinal villi. Large number of pear shaped trophozoites measuring 12-18 X 7-10 µm were seen clasped in situ by their convex sucking discs and bulging above the brush border of the microvilli. Some intestinal villi showed absence of the microvillus border with their basic epithelium.
The connective tissue of the intestinal mucosa and submucosa showed fibrosis. (Fig. 1. d,e). Correspondingly, oval shaped Giardia cysts measuring 9-12 X 8-10 µm were noticed located in the lower digestive tract , without evident changes in the intestinal structure. (Fig 1. c).

DISCUSSION
In the current study, a recorded significant decrease in the electrolyte level ( CL, Na, K and Ca) was encountered among infected lambs as compared with the non-infected ones. This aforesaid results came in agreement with other reports [1,14,15]. Previous studies demonstrated that the reduced absorption of Na + is due to the loss of the absorptive power of the intestinal epithelium [16,17]. In contrast, Ayaz, et al. [18] reported that K and Na levels didn't significantly alter in animals infected with many endoparasites. Moreover, [19,20]   The recorded change in the electrolyte balance could be explained by the intestinal inflammation produced due to the activity of Giardia trophozoites Such inflammation and the consequential impairment in electrolytes secretion and absorption often lead to acid, base and electrolyte imbalance which may cause diverse complications in body [21]. This may also interpret the recorded cases of diarrhea which were noticed during the experiment.
The non-significant effect of Giardia on creatinine level was formerly revealed by Rosa, et al. [22]. Though, Giardia showed a significant decreasing effect on urea level as previously encountered by Ragbetli, et al. [1]. In our opinion, this elevation may be created as a result of the increase of nitrogen metabolism in Giardia infection.
The malnutrition effect of Giardia among infected lambs was associated with a low ALP level. Since, the hepatobiliary integrity is measured by the serum ALP [23] so the low ALP level accuses the indirect negative effect of Giardia on liver tissues and bile duct .Accordingly, this effect on the liver tissues could explain the recorded elevation of the liver enzymes ( ALT and AST) among infected lambs in the study.
Mostly, the low levels of amylase and lipase among infected lambs was in accordance with the result of Buret [6]. This outcome was explained by the drawback effect of Giardia trophozoites on the pancreatic function which inhibits pancreatic lipase and amylase activities [24]. The impairment of serum glucose level was also observed among infected lambs. This observation may reflect the malabsorption and maldigestion effects accompanying Giardia infection [25].
The significant elevation of CRP among infected than non infected lambs may be accredited to the damage produced by the attachment of Giardia trophozoites to the mucosa of the intestine which continuously stimulated the hepatocytes for elevating the synthesis of CRP as innate defense mechanism. Mainly, CRP cooperates with the raised mucosal phagocytic activities along with IgA which acts as a humoral defense mechanism against Giardia [26,27].
The high level of MDA among infected lambs is an indication of the oxidative stress produced in the infected tissues [28] due to Giardia. The decreased activity of the defense mechanism for protecting the tissues from the damage produced by Giardia might be one of the reasons of elevation the level of MDA among infected lambs [29].
As regard, the significant decrease in the level of serum NO among infected lambs was consistent with another report [1]. Nitric oxide formation has been encountered in many parasitic infections including Giardia [30,31]. It is considered as only part of an immunopathological series against infection [32]. The low NO level may be credited to the release of arginine deiminase and flavohemoglobins by Giardia trophozoites which consumes all the local arginine (precursor of nitric acid formation) [33].
The intetstinal changes scanned by the electron microscopy came in agreement with other reports on Giardia [7,34]. Such changes ensured that the colonization of the intestine with Giardia trophozoites caused diffuse destruction in the microvillus brush border which in turn resulted in maldigestion and malabsorption effects.
Accordingly, a combination of electrolyte hyper secretion and malabsorption appears to be accountable for accumulation of fluid in the intestine, which aggravate diarrhoea [35,36]. All the above mentioned alterations mainly had a drawback effect on the biochemical serum constituents specially electrolytes [21].

CONCLUSION
The study advanced our standing of the pathophysiological effect of Giardia on lambs. It induced a significant declining effect on serum electrolytes (CL, Na, K, and Ca), digestive enzymes (Lipase, amylase and ALP) and glucose. Likewise, it significantly elevated the level of urea, Liver enzymes (ALT, AST), libertaed inflammatory