Veterinary World, EISSN: 2231-0916 RESEARCH ARTICLE Available at www.veterinaryworld.org/Vol.8/January-2015/24.pdf Open Access Morphological and histological identification of Paramphistomum cervi (Trematoda: Paramiphistoma) in the rumen of infected sheep Vijayata Choudhary1, J. J. Hasnani1, Mukesh K. Khyalia2, Sunanda Pandey2, Vandip D. Chauhan1, Suchit S. Pandya1 and P. V. Patel1 1. Department of Veterinary Parasitology, College of Veterinary Science & Animal Husbandry, Anand Agricultural University, Anand - 388 001, Gujarat, India; 2. Department of Veterinary Pathology, College of Veterinary Science & Animal Husbandry, Anand Agricultural University, Anand - 388 001, Gujarat, India. Corresponding author: Vijayata Choudhary, e-mail: [email protected], JJH: [email protected], MKK: [email protected], SP: [email protected], VDC: [email protected], SSP: [email protected], PVP: [email protected] Received: 03-11-2014, Revised: 19-12-2014, Accepted: 25-12-2014, Published online: 30-01-2015 doi: 10.14202/vetworld.2015.125-129. How to cite this article: Choudhary V, Hasnani JJ, Khyalia MK, Pandey S, Chauhan VD, Pandya SS, Patel PV (2015) Morphological and histological identification of Paramphistomum cervi (Trematoda: Paramiphistoma) in rumen of infected sheep, Veterinary World, 8(1): 125-129. Abstract Aim: This study was undertaken to identify Paramphistomum cervi on the basis of its morphology and histology to be the common cause of paramphistomosis in infected sheep and its differentiation from other similar Paramphistomes in Gujarat. Materials and Methods: Adult rumen flukes were recovered from the rumen of naturally infected sheep slaughtered in various abattoirs in Gujarat. Some adult flukes were flattened and stained in Borax carmine, and some were sectioned in the median sagittal plane and histological slides of the flukes were prepared for detailed morphological and histological studies. Result: Microscopic pictures of the parasite used in identification define the similarity in the morphology and histology of the anterior sucker, pharynx, esophagus, genital atrium, posterior sucker (acetabulum) and testes to the P. cervi. Conclusion: It can be concluded that the most common species found in sheep infected with Paramphistomosis is P. cervi on the basis of its histo-morphological appearance in Gujarat. Keywords: abattoirs, histology, morphology, paramphistomosis, posterior sucker, rumen fluke, sheep. Introduction and Lymnaea bulimoides) as intermediate host and Paramphistomum spp. are Platyhelminth (flat- mammalian as definitive host [11,12]. worm) parasites (Platyhelminthes: Trematoda: P. cervi are plug feeders [9] and cause serious Digenea) responsible for Paramphistomosis i.e. gas- disease by burying themselves into the sub mucosa trointestinal parasitic disease in domesticated ani- of the duodenum and feeding on the epithelial cells mals, which causes heavy economic losses [1] to the of the Brunner’s gland resulting in anorexia, profuse fetid diarrhea, drop in plasma protein concentration livestock industry to the tune of several thousand and anemia, which weaken the host [13]. Mature crores of rupees annually [2]. It has been a neglected Paramphistomes are also responsible for rumenitis, trematode infectious disease in ruminants, but has irregular rumination, un-thriftiness, lower nutrition recently come out as a significant cause of produc- conversion and loss of body condition, resulting in tivity loss [3-7]. Distribution of Paramphistomosis considerable economic loss [9]. is worldwide [8], but the highest prevalence has The species identification is still neglected as the been accounted in tropical and subtropical regions, various species of the family Paramphistomatidae are particularly in Africa, Asia, Australia, and Eastern difficult to be detected through a systematic point of Europe [1,9]. view, and most of the reports do not quote the main It is caused by specific species of the parasite one [14]. depending on the regions [3]. The common rumen The rumen fluke, P. cervi has economic impor- fluke, Paramphistomum cervi is considered to be one tance in ruminants but presently no reliable methods of the most important species of Paramphistomes [2]. are available to identify and differentiate this parasite Adult flukes are located in the rumen of ruminants from other Paramphistomes. The present study rep- and immature flukes in the small intestine mainly in resents the morphological and histological identifi- the duodenum [1,10]. P. cervi has di-heteroxenous life cation of P. cervi using Borax carmine and H and E cycle, which involve snail (Bulinus spp., Glyptanisus staining methods, in order to provide a base for future gilberti, Indoplanorbis exustus, Planorbis planorbis molecular studies. Copyright: The authors. This article is an open access article licensed The present study on morphological and histo- under the terms of the Creative Commons Attributin License (http:// logical identification of P. cervi was designed to fulfill creative commons.org/licenses/by/2.0) which permits unrestricted use, distribution and reproduction in any medium, provided the the need for molecular understanding of this econom- work is properly cited. ically important parasite. Veterinary World, EISSN: 2231-0916 125 Available at www.veterinaryworld.org/Vol.8/January-2015/24.pdf Materials and Methods oral and ventral suckers, testes and ovary. The species Collection of adult parasites were identified according to the criteria outlined by The rumen of 350 sheep between 1 and 2 years Yamaguti [18], Eduardo [19] and Sey [20]. of age was inspected for the presence of rumen Results flukes during slaughter at various local abattoirs of Anand, Ahmedabad and Vadodara District in Gujarat It is very difficult to identify and differentiate state, of which 17 sheep were found positive for the species of amphistomes on the basis of its mor- Paramphistomes. Infected part of the rumen was phology due to significantly less variation in size and brought to the laboratory of Department of Veterinary shape of parasite and its internal organs, which var- Parasitology, where it was washed thoroughly. After ies even in mature and immature worms of same spp. washing, adult flukes were carefully picked up with Morphology cannot be very well appreciated in the the help of forceps from the mucosa of the rumen of stereo microscope, but can be confirmed well by the naturally infected sheep and then washed 3-4 times histological examination of the parasite. with phosphate buffer saline. After that, samples were Morphological identification preserved in 10% buffered formalin until being pro- P. cervi is reported in several studies conducted cessed for morphological and histological studies. The on ruminants [18-20]. Morphological identification samples were carefully labeled with proper details. of P. cervi was carried out on the basis of size and Morphological identification shape of fluke and position of anterior and posterior Rumen flukes were preliminarily identified sucker (acetabulum). In the present study, most of the under microscope using low power magnification and species were of P. cervi, which were found mainly in then slides were prepared for detailed morphologi- the rumen and were light pink in color with a sucker cal studies and identification. The collected flukes at the tip of the cone (Figure-1a) and another sucker were placed on petridish and observed through stereo ventrally at the posterior end. The body of P. cervi microscope to appreciate the morphology. Final iden- was pear-shaped, slightly concave ventrally (coni- tification of Paramphistomum spp. was done based on cal) and convex dorsally. The worm measures about morphology of flukes; its shape, anterior sucker, pos- 3-8 mm in length and 1.5-3.0 mm in width at anterior terior sucker (acetabulum), terminal genitalium and end of posterior third or at its junction with middle tegumental papillae, following the standard guide- third. Mouth was terminal, funnel-shaped, widened lines given by Urquhart et al. [15]. Parasites were posteriorly. Caeca were wide, pursued a serpentine processed for whole mounting and stained by Borax carmine according to the procedure given by Singh and Srivastava [16]. Out of all recovered adult flukes, 10 flukes were randomly picked from each infected sheep and were washed in water. After washing, flukes were flat- tened between two glass slides and fixed in Bouin’s fluid at room temperature for 24 h. After that, these specimens were washed in water and then stained a b for 24 h in 0.5% Borax carmine and subsequently destained in 1% hydrochloric acid until the pink color was observed. The acid was thoroughly washed out from all specimens with water. Thereafter, the spec- imens were washed with tap water and subsequently dehydrated through 50-100% alcohol for 1 h each, and cleared by xylene for 30 min. The cleared specimens were mounted using DPX and covered with a cover- c d slip. The mounted slides were allowed to the air dry and observed under the light microscope. Histological identification The species thus identified was further con- firmed by histological identification of 10 formalin preserved flukes from each sample, which was pro- cessed by paraffin embedding method and stained by e f H and E stain as per Luna [17]. 10 longitudinal and Figure-1: Whole mounted parasite stained with borax median sagittal sections, each 6-10 μ thick were cut carmine showing visceral organs: (a) Anterior sucker by a Leica RM2125 microtome. The prepared