Journal of Veterinary Parasitology, 26(1) 2012 : 77-79. SHORT ... College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University,.
Journal of Veterinary Parasitology, 26(1) 2012 : 77-79
SHORT COMMUNICATION
Enzyme histochemistry of Eimeria tenella infected caeca of chicks: A preliminary study A. Pangasa, L.D. Singla, Neelam Bansal1 and P.D. Juyal Department of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, India Abstract Enzyme histochemical investigation of different tissue stages (schizogonic and gametogonic) in the life cycle of E. tenella were undertaken in the caeca of chicks for the demonstration of phosphatases (AKPase, ACPase and ATPase) and oxidoreductases (SDH, LDH, NADH and MDH). Strong activity of NADH, ACPase, AKPase, LDH and MDH was found in schizonts, while gametocytes were having moderate to strong activity of ACPase, AKPase, LDH and MDH. Other enzymes (ATPase and SDH) showed weak activity. The variation of enzymatic activity in the caeca of E. tenella infected birds can be correlated with the normal physiological activity of caeca. Key words: Chicks, Eimeria tenella, Histochemistry, Oxidoreductases, Phosphatases.
Introduction
Materials and Methods
The apicomplexon protozoan parasite, Eimeria tenella, infects the caecal epithelial cells of the domestic chicken and cause caecal coccidiosis. Annual loss due to coccidiosis in India has been found to be Rs. 1.14 billion (approx.) (Bera et al., 2010). This parasite has a complex life cycle involving both asexual and sexual multiplication. The parasites multiplies by mechanisms yet unknown within the caecal epithelial cells. Majority of the cytochemical investigations carried out in the different stages of life cycle of coccidia are mainly concerned with the presence and distribution of non-enzymatic substances such as carbohydrates, proteins, lipids and nucleic acids (Michael and Hodges, 1973). Studies related to the presence and distribution of enzymes in different stages of coccidia are very limited (Gill and Ray, 1954; Andrews et al., 1990; Smith et al., 1994; Johnston and Fernando, 1997). Various enzymes from Eimeria species of the fowl have been identified in a variety of ways, usually by in vitro biochemical reactions, electrophoresis or histochemistry (Williams, 1999). Present study was conducted on the enzyme histochemical investigation to know activity of certain enzymes in tissue stages (schizontal and gametogonic) in the life cycle of E. tenella.
Day old male broiler chicks (IBL 80) of a single breeder flock and almost of similar body weight were procured and reared in wire cages. The feed procured for the chicks was without any anticoccidial additives. Feed and water was given ad libitum to chicks. Single line strain of E. tenella previously isolated from the birds died of caecal coccidiosis and confirmed by post mortem was used for propagation of E. tenella. The 7 day old broiler chicks were experimentally infected with 1 X 105 sporulated oocysts of E. tenella orally. Three birds each were scarified by cervical dislocation at the atlanto-occipital joint at 12 h, 24 h, 48 h and 3, 4, 5, 6, 7, 8, 9 day post infection (DPI) for evaluating enzyme activity during asexual and sexual phase of infection. Ten micrometer (10µm) thick sections were obtained at 20ºC with the cryostat microtome from the fresh caecal tissue collected from infected birds. These sections were incubated in various substrates for the demonstration of phosphatases and oxidoreductases. The alkaline phosphatase (AKPase) and acid phosphatase (ACPase) were demonstrated by coupling azodye method (Barka and Anderson, 1963), adenosine triphosphatase (ATPase) by calcium method (Chayen et al., 1969), succinic dehydrogenase (SDH), lactate dehydrogenase (LDH) and reduced nicotinamide adenine dinucleotide diaphorase (NADH-diaphorase) and malate dehydrogenase (MDH) by standard method of bound enzyme (Pearse, 1972).
1
Department of Veterinary Anatomy
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Pangasa / J. Vet. Parasitol., 26(1), 2012 Table 1. Histoenzymatic acitivity in the caeca of chicks infected with Eimeria tenella ––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
S.No
Enzyme
Schizonts
Gametocytes
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
A (i) (ii) (iii) B. (i) (ii) (iii) (iv)
Phosphatases Alkaline Phosphatase (AKPase) Acid Phosphatase (ACPase) Adenosine triphosphatase (ATPase) Oxidoreductases Succinic dehydrogenase (SDH) Lactate dehydrogenase (LDH) Reduced Nicotinamide adenine dinucleotide diaphorase (NADH-diaphorase) Malate dehydrogenase (MDH)
+++ +++ +
++/+++ ++/+++ +
+ +++ +++ +++
+ ++/+++ ++ ++/+++
––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––
+++ = strong activity, ++ = moderate activity, + = weak activity
Fig. 1. LDH: strong activity in schizonts
Fig. 2. MDH: Strong activity in schizonts
Results and Discussion
Weak SDH reaction in the infected caeca may reflect changes in mitochondrial activity during differentiation of stage of life cycle of E. tenella. Strong LDH activity may be correlated with different glycolytic pathways in the developing schizonts and gametocytes. The variation of NADH and MDH enzymes indicated the metabolic activity during differentiation and growth of schizonts, gemetocytes and oocysts. This preliminary study can prove to be a good biological tool, as these enzymes can act as a target site for many drugs and can help us in exploring the blockage of enzymatic activity during testing of a new drug.
The results of the activity of various enzymes against schizogonic and gametogonic stages in the life cycle of E. tenella are depicted in Table 1. In this preliminary study, during the initial phase of the infection (up to 48 hours of infection) much differential recognizable activity of different enzymes was not observed. However, once schizonts were formed on day three we were able to differentiate activity of different enzymes into strong, moderate and week. Strong activity of NADH, ACPase, AKPase, LDH (Fig. 1) and MDH (Fig.2.) was found in schizonts from day three to six post-infection. Gametocytes were having moderate to strong activity of ACPase, AKPase, LDH and MDH from day 6th post infection onwards. Other enzymes (ATPase and SDH) showed weak activity. LDH is one of the best-known enzyme markers for the coccidia of fowl (Shirley, 1989) and has been detected in great abundance in both E. tenella and E. maxima. The variation of enzymatic activity in the caeca of E. tenella infected birds was correlated with the normal physiological activity of caeca. It was concluded that strong ACPase and AKPase activity in the schizonts and gametocytes of E. tenella may be associated with the cellular differentiation at sexual and asexual stages of life cycle. J. Vet. Parasitol. 200-0064-480/$2.50 © IAAVP, India
References Andrews, R., O’Donoghue, P., Adams, M. and Prowse, S., 1990. Enzyme markers for the genetic characterization of avian Eimeria spp. Parasitol. Res., 76: 627-629. Barka, T. and Anderson, P.J., 1963. Histochemistry: Theory, Practice and Bibliography. Harper and Row Publishers Inc., New York, pp 237-316. Bera, A.K., Bhattacharya, D., Pan, D., Dhara, A., Kumar, S. and Das, S.K., 2010. Evaluation of economic losses due to coccidiosis poultry industry in India. Agrl. Econo. Res. Rev., 23: 91-96.
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Pangasa / J. Vet. Parasitol., 26(1), 2012 Pearse, A.G.E., 1972. Histochemistry: Theoretical and Applied. 3rd Edn. Churchill Livingstone, London.
Chayen, J., Bitensky,L. Butcher, R.G. and Poulter, L.W. 1969. A Guide to Practical Histrochemistry. Oliver and Boyd, Edinburg, England, pp. 83-174.
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