Variation in Physiological Biomarkers with Different Clostridium perfringens Isolate Infections in Balkhi Sheep

Mumtaz Ali Khan1, Sher Bahadar Khan2, Shakoor Ahmad2, Irshad Ahmad3, Kashif Prince1*, Ghazunfar Rashid1, Mahboob Ali1, Imdad Ullah Khan4, Asad Ullah5, Naimat Ullah4, Muhammad Shoaib2 and Said Sajjad Ali Shah2 1University of Veterinary and Animal Sciences, Lahore 2Department of Animal Health, The University of Agriculture, Peshawar 3Instutute of Basic Medical Sciences, Khyber Medical University, Peshawar 4Gomal College of Veterinary Sciences, Gomal University, D.I. Khan 5College of Veterinary Sciences and Animal Husbandry, Abdul Wali Khan University, Mardan Article Information Received 28 April 2018 Revised 22 March 2019 Accepted 19 June 2019 Available online 12 December 2019


S mall ruminants face various health challenges including
Clostridium perfringens (CP) induced enterotoxemia. The sickness is primarily grouped into five types (A to E) based on distribution of four major toxins i.e. alpha, beta, epsilon and iota (Uzal et al., 2014). Type A causes yellow lamb disease, type B lamb dysentery; type C struck, type D pulpy kidney and type E enterotoxemia respectively (Uzal and Songer., 2008). There are three types of enterotoxemia per acute, acute and chronic forms (Fernandez and Uzal., 2003). The syndrome is a common and endemic illness in goats and sheep in Pakistan (Khan et al., 2008; O n l i n e F i r s t A r t i c l e (Ma et al., 2011). CP type D disease is characterized by changes in hematobiochemical parameters i.e. RBC, PCV, WBC, platelets, serum creatinine, blood glucose and urea levels (Nasir et al., 2013).

Geographical area
This project was carried out in Khyber Pakhtunkhwa province, Pakistan from January to December 2016 in two different topographic regions; Swat (hilly) and Mardan (plain) districts. The Animal Ethical Committee approved this project via Ref. No. DAS/5121, dated: March 9, (2016).

Sample collection
The samples were obtained from cases brought to veterinary hospitals through convenient sampling. A total of 184 fecal samples were obtained from enterotoxaemia suspected Balkhi sheep. From which seven blood samples were collected from infected with each type of CP and twenty on from healthy as reference for biomarkers through convenient purposive sampling technique. These samples were collected from 6-12 months age Balkhi sheep having similar size and area. Healthy were only included which produced no growth on tryptose sulfite cycloserine agar media.

Bacteriological examination
Samples were collected with rectal swabs; these were placed in sterile air tight bottles and sent to the laboratory under 4°C (Nayel et al., 2013). The fecal samples were inoculated on tryptose sulfite cycloserine agar media (HiMedia Laboratories pvt Ltd. India) and incubated for 24 hours at 37°C in anaerobic jar with CO 2 packs (Oxoid Ltd). Initially the isolates were identified through colony morphology and Gram staining; followed by biochemical tests (Remel RapID ANA II test kit, USA). The isolates were quantified on blood agar to sort out pathogenic isolates from field cases of CP related enterotoxaemia (Philippeau et al., 2003).

DNA extraction
Before DNA extraction, isolates were inoculated in Robertson cooked meat medium and incubated at 37°C for 12 h in shaking incubator. DNA was extracted according to the protocol of DNA extraction kit (GeneAll, South Korea). Obtained DNA was quantified using NanoDrop (Nano Drop, 2000, Thermo-Scientifics, Wilmington, DE 19810 USA) and stored at -20 °C.

Confirmation of Clostridium perfringens by PCR
Genotyping of CP was performed through PCR, targeting four major toxins (α, β, ε, and ι), as described by Greco et al., (2005) in Table I. Briefly, the reaction was performed in a thermocycler (BIO RAD T 100) in 25μL volume, in micro amplification tubes (PCR tubes) containing 2XAmpmaster TMTaq (Gene All Biotechnology CO. Ltd) 10μL, forward and reverse primers 1.5μL each, template DNA 5μL and DNA free distilled water 7µL. The amplification reactions consisted of 35 cycles, 5 minutes initial denaturation followed by 35 cycles; consisting of denaturation at 94 0 C for 60 seconds, annealing at (α=60 0 C, β=64 0 C, ε=53.4 0 C, ί=61 0 C) for 30 seconds, extension at 72 0 C for 60 seconds and final extension at 72 0 C 5 for minutes. The PCR products (5 μL) along with DNA ladder of 1Kb (Genesta TM ) and optimized positive and negative control were run on 1.5% agarose gel for electrophoresis. Ethidium bromide (1.5μg/mL) was used for staining the gel before being photographed under UV light.

Hematobiochemical analysis
Six milli Liter blood samples each from infected and healthy Balkhi sheep were collected from the jugular vein using disposable syringes. The blood was transferred to two sterile vacutainers, one containing heparin. Total RBC, WBC, platelets count, hemoglobin levels, and PCV were analyzed through hematology analyzer (Beckman Coulter, USA) from heparin added blood. Heparin free coagulated blood was centrifuged at 1500 rpm for 20 minutes to collect serum for biochemical analysis. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured with spectrophotometer by using their test kits (Bio-Diagnostics, Cairo, Egypt) (Reitman and Frankel, 1975). Serum creatinine and urea were also measured by using their appropriate diagnostic kits (Human, Germany). Blood glucose level was measured directly on glucose strip (codefree TM , Korea) and read with a Glucometer before the addition of anticoagulant.

Statistical analysis
Descriptive analysis was used to represent all parameters as mean ± Standard Error. Two tail t-test at the level of 95% confidence interval (P<0.05) was used for hematobiochemical comparisons between diseased and healthy animals through SPSS 21.0 version statistical program.

Clinical parameters
Clinically infected sheep showed the signs of abdominal pain, mild to severe (blood-tinged to bloody) yellow pasty diarrhea, frothy salivation and varying degree of nervous signs. Clostridium perfringens infected sheep O n l i n e  TGC TAA TGTTAC TGC CGT TGA TAG-3  5-TGC TAA TGTTAC TGC CGT TGA TAG  were dehydrated with pale mucous membranes and their mean body temperature was 40.1 ± 0.21 0 C.

Bacteriological parameters
Growth of CP was identified as small black colonies on tryptose sulfite cycloserine agar media (HiMedia Laboratories pvt Ltd. India); identity was confirmed by Gram staining and biochemical testing panel results with test kit (remel-RapID ANA II system test kit, USA). Colony counts were quantified on blood agar and colony countsmore than 104-107 CFU/g were considered, as pathogenic. Balkhi sheep were only considered healthy when no growth was obtained on tryptose sulfite cycloserine agar media (Hi Media Laboratories pvt. Ltd. India) after 24 hours incubation at 37°C in anaerobic jar with CO 2 packs. Out of 184 suspected sheep,107 (58.15%) were identified infected with CP. Genotyping of 107 strains from infected sheep indicated 57(53.27%) infected with CP type A, 11 (10.28%) with type B and 39 (36.44%) with type D by conventional PCR however no case of type C and type E were found (Table I).

Hematobiochemical parameters
Mean hemoglobin levels and erythrocyte counts (RBC) decreased while the white blood cells (WBC), packed cell volume (PCV), platelet counts and total bilirubin increased significantly (P<0.05) in CP type A infected sheep (Tables III and IV). In CP type B and type D infected sheep mean erythrocytes count (RBC) and hemoglobin levels decreased while white blood cells (WBC), packed cell volume (PCV), platelets count, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, serum creatinine, blood glucose and urea increased significantly (P<0.05). Fluctuations in mean hemoglobin level, erythrocyte counts (RBC), packed cell volume (PCV) and total bilirubin were beyond the normal limits while others were within normal ranges in CP type A infected sheep. Alanine Aminotransferase (ALT), total bilirubin, serum creatinine, blood urea and glucose were beyond the normal limits while others were within normal ranges in CP type B and type D infected sheep (Tables III and IV).

DISCUSSION
Clostridium perfringens is the normal occupant of the gastrointestinal tract in animals and people but turns pathogenic when gastrointestinal tract condition disturbed by sudden changes in feed, eating regimen or stasis in digestive system. The number of CP increases exponencially and production of distinctive toxins is increased many folds (Uzal, 1996). The sickness is associated with elevated amounts of toxins in the digestive system, with death of the animal or acute form of disease (Vaikosen and Ikhatua, 2005). Present study demonstrated 58.15% pathogenic isolates of CP from enterotoxemia suspected sheep. Out of them, 57 (53.27%) were CP type A, no. (10.28%) type B and no. (36.44%) type D, types C and E were not diagnosed. Itodo et al. (1986) depicted the pervasiveness of CP writes A (22.05%), type B (4.0%) and type D (15.75%) in their examination in Nigeria.   (Itodo et al., 1986;Efuntoye and Adetosoye, 2003). It is a typical view that CP types B, C, and D have major part of enterotoxaemia, while CP type A alone is not included or has extremely constrained impact. As of late, a few analysts have given an account of the significance of CP type A (Manteca et al., 2001). Greco et al. (2005) revealed that CP types A and D were recognized, 84% and 16% by PCR, in 87 sheep and 15 kids. Results of our study have indicated pronounced anemia because of diminished erythrocyte check and decreased hemoglobin level while increase in leukocyte count, platelets count, and packed cell volume expanded in all CP type A, B and D infection in sheep when compared with sound sheep. Comparative discoveries were accounted for by Hassanein et al. (2017) in Egypt. In another study Nasir et al. (2013) reported similar findings following the experimental CP type D disease in sheep. Alpha toxin is phospholyphase having hemolytic and necrotizing while epsilon is lethal in nature (Quinn et al., 2004;Fatmawati et al., 2013). Comparable perceptions were noted by Smith (1975) in his investigation demonstrating the rise of acid soluble phosphate are because of alpha toxin. Alpha toxin hydrolyzes the phospholipids in the cell film of RBC's, causing its lysis. Our findings were supported by the findings of Ombe et al. (2006) who detailed that the presence of toxin restricting receptors on the surface of erythrocytes of different species is related with hemolysis. The alpha toxin likewise causes the lysis of leukocytes and platelets. This may be because of a similar mechanism, like RBC hemolysis. The increase in white platelets is considered as a guide in diagnosing the enterotoxemia in living organisms (Smith and Sherman, 1994;Dennis and Bryant, 2002), same results are obtained in present study. Clostridium chauvoei hemolysins receptors exist on RBSs surfaces in different species, examined by Ombe et al. (2006) additionally supported our findings. Increase in WBCs and platelets can help as supportive in the diagnosis of enterotoxaemia (Smith and Sherman, 1994;Dennis and Bryan, 2002). Increase in packed cell volume was because of lack of hydration which was noted in all infected sheep.
In our results mean AST, ALT and bilirubin increased significantly (p<0.05) in CP type A infected sheep while the mean AST, ALT add up to bilirubin, serum creatinine, blood glucose and urea levels increased significantly O n l i n e  Mylashiro et al. (2007) showed that epsilon toxin produces renal and liver damages, gastroenteritis, and systemic hemorrhage in various organs upheld our results of expanded liver and kidneys enzymes are because of the harms created in these organs. Epsilon toxin of CP causes harms in the kidneys (Miyakawa et al., 2007;El-Ghareib and Amer, 2009;ElSify et al., 2016). Clostridium perfringens causes renal damage, which brings about serum creatinine and blood urea increase. The activity of CP toxin on kidneys particularly the epsilon toxin has likewise been exhibited by different researchers (Miyakawa et al., 2007;Heba et al., 2009).

CONCLUSION
All in all, the hematological and biochemical parameters varied significantly in all CP type A, B and D. however. the vast majority of these stayed within the normal range. Mean RBC counts, hemoglobin level, PCV and total bilirubin fluctuated beyond the normal limits in CP type A infected sheep while liver enzyme, total bilirubin, serum creatinine, blood urea and glucose levels fluctuated beyond the normal limits in CP type B and D infected sheep.

O n l i n e F i r s t A r t i c l e O n l i n e F i r s t A r t i c l e
Clostridium perfringens Isolate Variation in Balkhi Sheep