Eimeria Stiedae: Infection Rate and Molecular Characterization by Nested PCR in Rabbits from Minoufiya Governorate, Egypt
Total Page：16
File Type：pdf, Size：1020Kb
AbouLaila et al. EVMSPJ 2020; 16:34-49 Eimeria stiedae: Infection rate and molecular characterization by nested PCR in rabbits from Minoufiya Governorate, Egypt Mahmoud AbouLaila1,*, Abstract: Anis Zaid2, Tamer Eimeria stiedae infects the epithelium of bile ducts of the liver and Roshdey3, Tamer Allam4, causes economic losses for the rabbit industry. In this study, we aimed 5 Ahmed Elkhatam to study the infection rate and histopathology and to characterize 1Department of Parasitology, Faculty Eimeria stiedae from Ashmoun and Sadat City, Minoufiya governorate, of Veterinary Medicine, Damanhour Egypt by a nested polymerase chain reaction. Specific PCR and University, Damanhour22511, El- nested PCR primers to the ITS-2 gene of Eimeria stiedae were used. Behera, Egypt 2Department of Pathology, Faculty The infection rate was 12.5%. The infection rate among localities was of Veterinary Medicine, University of 19% for Ashmoun and 7% for Sadat. The infection rate among sex Sadat City, Sadat City 32511, groups was 12.35% for females and 13.33% for males. The infection Minoufiya, Egypt 3Department of Molecular Biology, rate among age groups was 11.11% for >1year and 15.38% for <1year Genetic Engineering and age groups. The infection rate was 8.75 for autumn, 18.33% for winter, Biotechnology Research Institute, 12% for spring, and 10% for summer. The locality had a significant University of Sadat City, Sadat City effect on the infection rate (P< 0.0378), while the sex, age, and season 32511, Minoufiya, Egypt 4Department of Clinical Pathology, did not significantly affect it. The histopathological lesions were Faculty of Veterinary Medicine, identical to hepatic coccidiosis caused by E. stiedae and its stages University of Sadat City, Sadat City were observed. There was hydropic degeneration in hepatocytes. The 32511, Minoufiya, Egypt infection significantly increased ALT, AST, ALP, GGT, total bilirubin, 5Department of Parasitology, Faculty of Veterinary Medicine, University of direct bilirubin, indirect bilirubin, and urea while decreased PCV, Hb, Sadat City, Sadat City 32511, RBCS, MCH, MCHC, and lymphocytes. The PCR and nested PCR Minoufiya, Egypt amplified the expected bands of the ITS-2 gene. The PCR and nested Corresponding author: PCR products were sequenced. The sequences had high identity Dr. Mahmoud AbouLaila percent with E. stiedae isolates from China (LY) and Egypt (BSU-1). E-mail: [email protected] Nested PCR of the ITS-2 gene might be useful in the molecular diagnosis of E. stiedae in rabbits. Keywords: Eimeria stiedae; Infection rate; Nested PCR; ITS-2; Hematology; Biochemical parameters; Histopathology; Minoufiya; Egypt INTRODUCTION rabbit can produce about 80 kilos (kg) of meat annually, i.e. 2900-3000% of its weight The Phoenicians first encountered the of the meat. Rabbit meat is highly nutritious, rabbit in Spain around 1000 B.C. Rabbit juicy, cholesterol-free, and high in calcium, farming has tremendous potential in vitamins, and minerals. Rabbits rising from a developing countries improving food safety backyard give smallholder farmers additional and quality. Because of the short income and increase the food of provincial pregnancy and pronounced prolificacy, the and urban individuals (FAO, 2001). For rabbits are extremely productive. A female household output, rabbits suit well high labor 34 AbouLaila et al. EVMSPJ 2020; 16:34-49 and investment costs. Rabbits are Australia (Stodart, 1971), Portugal (Silva et herbivores and do not fight for food with al., 2015), Poland (Nosal et al., 2006), Brazil humans. World rabbit production amounts (de Almeida et al., 2006), Kenya (Okumu et to over 1 million tons per year from which al., 2014), and Egypt (El-Shahawi et al., Egypt delivered 69,600 tonnes (FAO, 2012). Complement fixation test was applied 2001). Animal diseases are of the general for diagnosis in a former study (Rose, 1961). fields of restrictions on widespread rabbit Recently, molecular tools were utilized for its farming (FAO, 2001). Protozoan parasites identification in some studies (Faraj, 2017; are from these restrictions. Hassan et al., 2016; Ütük et al., 2015; Yan Eimeria stiedae is an important protozoan et al., 2013). In this research, we assessed of rabbits. It infects the liver and the utilization of a nested PCR targeting the reproduces in the epithelial lining of the ITS-2 gene for characterization of Eimeria bile ducts (Bangoura and Daugschies, stiedae in rabbits from Minoufiya, Egypt. 2018). It causes high profitable losses for rabbit production. Clinical signs include MATERIALS AND METHODS anorexia, depression, brown watery 1. Animals diarrhea, emaciation, harsh hair coating, droopy and swollen abdomen with Two hundred rabbits were collected, 100 progressive weakness, jaundice, and from Sadat City and 100 from Ashmoun. The death (Pakandl, 2013). Clinical signs only age of collected rabbits was arranged into appear on the young rabbits and may lead two groups135 rabbits were more than one to their death while the adults are usually year and 65 were less than one year. The carriers (Okerman, 1988). The lesions sex groups included 170 females and 30 typically bound to liver and swollen bile males. Animals were examined in the period ducts (Al-Naimi et al., 2012). from April 2019-Aprile 2020. Macroscopically, the liver is enlarged and 2. Fecal analysis has numerous small white nodules on its Feces from all collected animals were surface (Wang and Tsai, 1991). The examined by the flotation technique (Foreyt, histopathological changes comprise 1989). hyperplasia and hypertrophy of the bile duct epithelium with the developing stages 3. Parasite of E. stiedae (Darzi et al., 2007). Eimeria stiedae was collected from infected Eimeria stiedae infection was recorded in rabbits from Ashmoun and Sadat City rabbits from different geographical areas districts, Minoufiya governorate, Egypt. either clinically or microscopically using Oocysts were collected from the fecal parasitological or histopathological samples from Sadat City as delineated by techniques such as in Taiwan (Wang and Foreyt (1989). Oocysts collected from liver Tsai, 1991), Iraq (Al-Naimi et al., 2012), samples from Ashmoun by macerating the Kingdom Saudi Arabia (Abdel-Baki and liver and filtration to detain out the coarser Al-Quraishy, 2013; Al-Mathal, 2008; particles of the minced tissues by passing the Toula and Ramadan, 1998), Iran (Tehrani mixture through several sieves followed by et al., 2013), India (Darzi et al., 2007; centrifugation (Rose, 1961). Oocysts were Singla et al., 2000; Sivajothi et al., 2016), sporulated using potassium dichromate 2.5% 35 AbouLaila et al. EVMSPJ 2020; 16:34-49 (MAFF, 1977). total protein (TP), albumin (Alb), blood urea 4. Histopathology (U), creatinine (Cr), total bilirubin (TB), direct Five liver tissue samples with lesions were bilirubin (DB), indirect bilirubin (IB) were collected from positive animals in 10% measured spectrophotometrically using neutral buffered formalin (NBF) for histopathological examination. Thin Spinreact diagnostic kits (Spain) and sections were prepared from liver tissues following the manufacturer's instructions. and stained with H and E (Bancroft et al., 1996). 7. DNA extraction 5. Blood samples: Oocysts were broken by sonication (Hassan Twenty Blood samples were collected from et al., 2016). DNA was extracted using G- TM the rabbits either infected or non-infected spin Total DNA extraction kit (iNtRON, with E. stiedae in tubes enclosing EDTA Seoul, Korea). DNA was quantified using for measuring hematological parameters. NanoDrop 2000c (Thermo Scientific, USA). Other blood samples were collected in 8. Polymerase chain reaction (PCR), plain centrifuge tubes and serum samples and Nested Polymerase chain reaction were separated and stored at -20°C until (nPCR) evaluated for the selected biochemical The PCR and nested PCR were performed parameters. consistent with previous studies (AbouLaila 6.Hematological and biochemical et al., 2010a,b) with some modifications. A analysis 25 μL reaction volume contains Dream TaqTM Green PCR Master Mix (2X) (Thermo Fisher Hematological variables included packed Scientific Inc., California, USA), 120 ng of cell volume (PCV), hemoglobin DNA, 50 μM of each primer, and nuclease- concentration (Hb), red blood cell counts free water. The PCR reaction was completed utilizing forward 5ʹ - (RBCs), Mean corpuscular volume (MCV), GCAACGGCGTGCAGGGTC TA-3ʹ and Mean corpuscular hemoglobin (MCH), reverse 5ʹ - CACTACTACTCTACCTTCCGC- ʹ mean corpuscular hemoglobin 3 primers (Table 2) for ITS-2 gene of Eimeria stiedae, which were deliberate concentration (MCHC), total leukocyte dependent on ITS-2 gene sequences in 18S count (TLC) and differential leukocyte rRNA, ITS-1, 5.8S rRNA, ITS-2, and 28S count were performed according to the rRNA region gene bank accession numbers JQ328190 and KU886239 and targeting a routine blood procedures adopted by sequence of 393 bp from 692 to 1084 and Feldman et al. (2000). Activities of Serum from 504 to 896 bp, respectively. The TM enzymes included alanine SimpliAmp Thermal Cycler (Applied Biosystems, Singapore) was used to conduct aminotransferase (ALT), aspartate the PCR reaction. The PCR conditions aminotransferase (AST), alkaline comprised: denaturation at 95 ° C for 3 phosphatase (ALP) and Gamma-glutamyl minutes and 35 cycles of denaturation at 95 ° transferase (GGT) and concentrations of C for 30 seconds, annealing at 58 ° C for 30 36 AbouLaila et al. EVMSPJ 2020; 16:34-49 seconds, extension at 72 ° C for 30 (JX406874),