Clostridium perfringens type A beta2 toxin in elephant (Elephas maximus indicus) and pygmy hog (Sus salvanius) with haemorrhagic enteritis in Assam, India

African Journal of Microbiology Research Vol.(2) pp. 196-201, August, 2008 Available online http://www.academicjournals.org/ajmr ISSN 1996-0808 ©2008 Academic Journals Full Length Research Paper Clostridium perfringens type A beta2 toxin in elephant (Elephas maximus indicus) and pygmy hog (Sus salvanius) with haemorrhagic enteritis in Assam, India Arunava Das1, Yahya Mazumder2*, Biman K. Dutta3, Bibek R. Shome4, Komal M. Bujarbaruah5 and Gauri D. Sharma6 1 Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam, Tamil Nadu, India. 2 Department of Biotechnology, Nagarjuna College of Engineering and Technology, Bangalore, India. 3 Department of Ecology and Environmental Science, Assam University, Silchar, Assam, India. 4 Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bangalore, Karnataka, India. 5 Division of Animal Science, Indian Council of Agricultural Research, Krishi Bhawan, New Delhi, India. 6 Department of Life Science, Assam University, Silchar, Assam, India. Accepted 30 June, 2008 This paper reported the investigation of haemorrhagic enteritis in female elephant (Elephas maximus indicus) and pygmy hog (Sus salvanius) at the Assam State Zoo, Guwahati, Assam, India. An eight year old female elephant and two and half year old female pygmy hog developed haemorrhagic enteritis of unknown cause maintained at the zoo died within four days. Bacteriological investigation revealed that the causative agent Clostridium perfringens was associated with the disease. Erythromycin, clindamycin and metronidazole were effective, however, ampicillin or penicillin G was more effective and probably the drug of choice for C. perfringens associated haemorrhagic enteritis. Isolates derived from elephant harboured four plasmids (4.1, 14.4, 38.8 and 48.2 kb), while that from pygmy hog carried two plasmids (42.8 and 51.9 kb). PCR analysis of C. perfringens isolates revealed presence of alpha toxin gene (cpa) and beta2 toxin gene (cpb2). None of the isolates were positive for beta, epsilon, iota and enterotoxin genes. The sequence analysis of partial cpa gene showed 98.6 to 100% homology among the isolates studied. The study confirmed the involvement of beta2 toxin producing C. perfringens type A associated with the haemorrhagic enteritis. Key words: Clostridium perfringens, haemorrhagic enteritis. INTRODUCTION The Indian elephant (Elephas maximus indicus) and pygmy hog (Sus salvanius) are considered among the endangered mammals in the world. The International Union for the Conservation of Nature and Natural resources, Morges, Switzerland has accorded the Indian elephant with criteria: EN A1 cd and pygmy hog with Criteria: A1c, B1+2cd, E as critically endangered. The Clostridial enteritis in captive pygmy hog until now appeared to be rare; however it has been reported in captive African elephants (Bacciarini et al., 2001). Clostridium perfringens is ubiquitous, gram positive anaerobe *Corresponding author. E-mail: [email protected]. anaerobe commonly associated with the enteric diseases in humans, domestic animals and wildlife (Songer, 1996). Depending on the production of four major toxins (alpha, beta, iota and epsilon), strains of C. perfringens can be classified into five genotypes A to E (Songer and Meer, 1996). Along with four major toxins, enterotoxin and beta2 toxin produced by types of C. perfringens also considered as important toxins for enteric diseases (Smedley III et al., 2004). There are reports of beta2-toxigenic C. perfringens type A associated with enterocolitis in horses (Herholz et al., 1999), ulcerative enteritis in African elephant (Bacciarini et al., 2001) and enterotoxaemia in goat (Dray, 2004). Differentiation between different strains of C. perfringens from the clinical cases is not trivial. Therefore, PCR has been used to detect the Das et al. used to detect the presence of toxin genes and to identify the specific strains of C. perfringens (Yamagishi et al., 1997). Marks and Kather, (2003) reported that ampicillin, erythromycin, metronidazole and tetracycline are very effective antibiotics for the treatment of C. perfringens associated diarrhoea. Eisgruber et al., (1996) reported that strains of C. perfringens isolated from different diseased outbreaks possess a high molecular weight identical plasmid which is useful for the strain differenttiation and characterization of C. perfringens isolates. The involvement of C. perfringens in African elephant causing ulcerative enteritis has been reported earlier but probably rare reports or unnoticed from pygmy hog. This study reports the occurrence of fatal haemorrhagic enteritis due to C. perfringens type A in Indian elephant and pygmy hog. MATERIALS AND METHODS History of animals and sample collection One female elephant of age eight year and one female pygmy hog of age two and half year at the Assam State Zoo, Guwahati, Assam, India, suddenly developed off feed, weakness, poor growth, anorexia, pyrexia and prolonged episodes of watery followed by bloody diarrhoea. The atmospheric temperature and humidity was recorded between 32 - 39oC and 80 - 90% respectively with heavy rainfall during the month of March. Both the animals were isolated and unresponsive to the treatment with 5 mg tetracycline (Himedia, Mumbai) per kg of body weight parentarily for three days. On fourth day, the animals were died. In both cases, postmortem was performed within two to four hours duration after death. Body surfaces were examined for any injuries or wound. All the internal organs were thoroughly examined and any macroscopic and gross lesions observed were recorded. The small and large intestinal contents were aseptically collected during the postmortem examination for microbiological investigation. Samples from feed and water used for the animals were also collected aseptically for microbiological investigation. Bacterial isolation and biochemical tests Both aerobic and anaerobic bacterial isolations were attempted. For aerobic isolation, collected samples were first inoculated in nutrient broth and incubated at 37oC for 4 h. Soon after, inoculum from each tube were transferred to freshly prepared sterile nutrient agar plates and incubated at 37oC for 24 h. Bacterial colonies were purified and identified as per standard bacteriological procedures (Holt et al., 1994). For anaerobic isolation, samples were inoculated in Robertson’s cooked meat (RCM) broth medium supplemented with glucose, hemin and vitamin K (Himedia, Mumbai), overlaid with neutral oil and incubated at 370C for 48 h. The inoculums from the RCM media were seeded onto 10% goat blood agar and incubated anaerobically with an anaerobic gas-pack system (BBL Microbiology Systems Cockeysville, Md. [Div. Becton Dickinson and Co.]) for 24 h at 37oC. Bacterial colonies were purified based on the size, shape, color and patterns of haemolysis on blood agar and were subjected to Gram’s and malachite spore staining. The isolates were identified based on the litmus milk test, gelatinase, deoxyribonuclease (DNase), lecithinase and fermentation of glucose and lactose (Holt et al., 1994). 197 Antimicrobial susceptibility test Antimicrobial susceptibility test was performed by agar dilution method (Sutter et al., 1975) using 10 commercially available antibiotics. The antimicrobial agents used were amoxicillin, ampicillin, clindamycin, doxycycline, erythromycin, metronidazole, penicillin G, cephalexin and tetracycline from Hi media, (Mumbai); and cefazaflur from GSK, (Bangalore). Sets of three laked sheep blood agar plates containing no antibiotic were inoculated before each series of antibiotic-containing plates and incubated anaerobically, 10% CO2 in air and aerobically to serve as growth and contamination controls. Data were deleted if poor or no growth occurred in anaerobic growth control plate or if any contamination was evident. Detection of plasmid One milliliter of bacterial culture grown overnight anaerobically in brain heart infusion broth at 37oC was used for the extraction of plasmid DNA by alkali lysis method (Birnboim and Doly, 1979). The plasmid DNA was finally dissolved in 35 µl of TE-RNase (1 mg/ml in 10 mM tris-hydrochloric acid and 1 mM ethylenediamine tetraacetic acid [pH 8.0]) solution, electrophoresed in 0.7% agarose dissolved in 1X TAE (tris-acetate-EDTA; pH 8.0) buffer and stained with 0.4 µg/ml ethidium bromide. The molecular weight of the plasmids was determined by comparing with known DNA ladder ( DNA / Hind III digest; GENEI, Bangalore). The separated plasmid DNA bands were visualized and photographed in gel doc system (Image Master® VDS, Pharmacia Biotech, Sweden). Detection of toxin genes by polymerase chain reaction To study the virulence of C. perfringens, isolates were tested to detect the alpha toxin gene (cpa), beta toxin gene (cpb), epsilon toxin gene (etx), iota toxin gene (iA), enterotoxin gene (cpe) and beta2 toxin gene (cpb2) by PCR assay (Songer and Meer, 1996). Freshly grown bacterial colonies from solid media plates were suspended in 200 µl of Milli-Q water in a microcentrifuge tube, gently vortexed and boiled for 10 min in a water bath. Supernatant after centrifugation at 10000 g for 5 min was used as a template DNA. The amplification was carried out in 25 µl reaction volume containing 12.5 µl of 2× PCR master mix (Promega, USA) containing 4 mM magnesium chloride, 0.4 mM of deoxynucleotide triphosphates (dNTPs), 0.5U of Taq DNA polymerase, 150 mM trishydrochlroric acid, pH 8.5 (Promega, USA), 0.5 µM primers and 2.5 µl of template DNA. The PCR reactions were performed in iCycler (BioRad, USA). After initial denaturation at 94oC for 4 min, the amplification cycle had denaturation, annealing and extension at 94oC, 55 and 72oC for 1 min each respectively. Final extension was done at 72oC for 10 min. The specific forward and reverse primer pairs used in this study were commercially synthesized from GENSET (Table 1). C. perfringens type A positive for cpa and cpb2 genes isolated from atypical blackleg in cattle was used as positive control (Shome et al., 2006) and C. septicum strain negative for cpa and cpb2 genes was used as negative control. The PCR amplicons (5 µl) were electrophoresed in 1.5% agarose gel in TAE (tris-acetic acid-EDTA, pH 8) buffer, stained with ethidium bromide and observed in gel doc system (Pharmacia Biotech, Sweden). Sequencing of PCR amplified product Four PCR amplified products of partial cpa gene of C. perfringens were purified using QIA quick® PCR purification kit (QIAGEN, USA) and sequenced in an automated DNA sequencer (Microsynth, Switzerland and Genei, Bangalore, India). Partial sequence obtain- 198 Afr. J. Microbiol. Res. Table 1. Details of primers used in polymerase chain reaction for the detection of toxin genes from C. perfringens. Toxin / toxin genes Alpha toxin / cpa Beta toxin / cpb Epsilon toxin / etx Iota toxin / iA Beta2 toxin / cpb2 Enterotoxin / cpe Primer sequences / / For 5 -gctaatgttactgccgttga-3 / / Rev 5 -cctctgatacatcgtgtaag-3 / / For 5 -gcgaatatgctgaatcatcta-3 / / Rev 5 -gcaggaacattagtatatcttc-3 / / For 5 -gcggtgatatccatctattc-3 / / Rev 5 -ccacttacttgtcctactaac-3 / / For 5 -actactctcagacaagacag-3 / / Rev 5 -ctttccttctattactatacg-3 / Primer Concentration / For 5 -agattttaaatatgatcctaacc-3 / / Rev 5 -caatacccttcaccaaatactc-3 / / For 5 -ggagatggttggatattagg-3 / / Rev 5 -ggaccagcagttgtagata-3 Amplicon size (bp) 324 Reference Titball et al., (1989) 180 Hunter et al., (1993) 655 Hunter et al., (1992) 446 Perelle et al., (1993) 567 Gibert et al., (1997) 233 Czeczulin et al., (1993) 0.5 µM For: Forward primer; Rev: Reverse primer; bp: Base pair. obtained were submitted to BLAST analysis (Altschul et al., 1990) to determine the similarities to other sequences available in GenBank. The cpa gene sequences derived from the C. perfringens isolated from elephant were deposited into the GenBank under accession numbers DQ838706 and DQ838707, while sequences derived from pygmy hog were deposited under accession numbers DQ838708 and DQ838709. The cpa sequences up to 292 bases were aligned with the corresponding sequences of four selected C. perfringens isolated from bovine enterotoxaemia from USA (DQ184079), healthy chicken from Denmark (AF477009), soil from Japan (NC_003366) and strain C57-1 from China (AY823400) available in the GenBank by using ClustalW algorithm of MegAlign program (DNASTAR, Lasergene, USA). The phylogenetic analysis of the sequences and 97 deduced amino acids residues were also analysed by using same programme. periphery with cremator edges. Gram staining showed gram-positive rods, while malachite green staining showed sub-terminal oval endospores. The isolates produced stormy fermentation, acidity, reduction and coagulation in litmus milk and liquefied gelatin. Isolates also showed both DNase and lecithinase activities and fermented glucose and lactose. Upon the detailed bacteriological investigation, four isolates (two from elephant and two from pygmy hog) were identified as Clostridium perfringens. No C. perfringens was isolated from the feed or water used for the animals. RESULTS The susceptibility test of 10 commercially available antibiotics against the four isolates of C. perfringens used in this study is shown in Table 2. All isolates were inhibited by 0.5 µg/ml or less of ampicillin and 0.5 U/ml or less of penicillin G, 1.0 µg/ml or less of erythromycin and 2.0 µg/ml or less of any of the clindamycin and metronidazole tested. Only 77, 42, 43, 52 and 33% isolates were inhibitted by 2.0µg/ml or less of any of the amoxicillin, cefazaflur, cephalexin, doxycycline and tetracycline respecttively tested. All the isolates harboured multiple plasmids. Two isolate derived from elephant harboured four plasmids (4.1, 14.4, 38.8 and 48.2 kb), while the remaining two isolates from pygmy hog carried two plasmids (42.8b and 51.9 kb). None of the isolates carried identical or common plasmids. Postmortem findings The postmortem examination of elephant and pygmy hog showed diffused haemorrhagic and necrotic enteritis, lesions and edema in both small and large intestines. Discrete areas of necrosis were present on the walls of the caecum and colon. Rest of the organ was found apparently healthy. Isolation and identification of C. perfringens On aerobic isolation, mixed colonies of both Gram positive and Gram negative bacteria were found, which did not lead to any conclusive evidence. However, on anaerobic culture process, pure colonies showing alpha and beta haemolysis on goat blood agar was clearly observed. The bacterial colonies originated from the intestinal contents of elephant were observed to be flat with irregular thin border. However, colonies that originnated from pygmy hog were having radially striated Antimicrobial susceptibility and plasmid profiling PCR assay and sequence analysis In PCR analysis, out of six virulence genes of C. perfringens screened only alpha toxin gene (cpa) of 324 bp fragment and beta2 toxin gene (cpb2) of 567bp Das et al. 199 Table 2. Activity of ten antibiotics against four isolates of C. perfringens derived from haemorrhagic enteritis of elephant and pygmy hog. Name of antibiotic Cumulative percentage susceptible to indicated concentration (µg/ml) 0.1 0.5 1.0 2.0 4.0 8.0 16.0 32.0 64.0 128.0 32 42 65 77 82 87 92 96 100 38 100 18 28 38 42 53 64 73 86 94 100 22 27 33 43 66 77 95 100 31 49 79 100 14 27 42 52 68 75 81 100 32 55 100 28 44 84 100 39 100 11 19 26 33 41 47 58 76 89 100 Amoxicillin Ampicillin Cefazaflur Cephalexin Clindamycin Doxycycline Erythromycin Metronidazole *Penicillin G Tetracyclin * concentration in U/ml M P N 1 2 3 4 324bp gene. The partial cpa gene sequences from four Indian field isolates in BLAST showed similarity values greater than 99% to the published database sequences of C. perfringens. The comparative alignment study of partial cpa gene sequences of four Indian field isolates and three reference sequences derived from bovine (USA), chicken (Denmark) and strain C57-1 (China) respectively showed no nucleotide substitution in compared to majority (Figure 2). However, the reference sequence originated from soil (Japan) had four nucleotide substitutions at positions 105A→G, 156G→A, 229C→T and 255G→A respectively. The alignment of 97 deduced amino acids revealed no change in proteins. The phylogenetic tree constructed with partial cpa gene sequences of C. perfringens revealed 98.6 to 100% sequence homology among the isolates irrespective of different source of origin and geographical distribution (Figure 3). 567bp DISCUSSION 3000bp 500bp 300bp 100bp M P N (a) 1 2 3 4 3000bp 500bp 100bp (b) Figure 1. Detection of alpha and beta2 toxin genes of C. perfringens by PCR. (a) isolates positive for 324bp fragment of alpha toxin gene; (b) isolates positive for 567bp fragment of beta2 toxin gene. Lane P: positive control; lane N: negative control; lanes 1 to 4: field isolates positive for toxin genes; lane M: 100bp marker DNA. fragment were detected in all the isolates (Figure 1). None of the isolates were positive for any of the cpb, etx, iA and cpe toxin genes. PCR assay thus revealed that the field isolates originated from elephant and pygmy hog were C. perfringens type A with an additional cpb2 toxin Reports on Clostridial infection of elephant and pygmy hogs are rare due to limited population of these mammals. Of these animals, the pygmy hog is only captive population available in Assam, India. This report now gives a good insight into the susceptibility of these animals to Clostridial disease. The role of C. perfringens in present study was made clear by isolation of the organism alone under anaerobic conditions, without any other species of Clostridia. Further, aerobic isolation had not yielded any dominant bacterial type suggestive of enteric infection. The result of antibiotic susceptibility test clearly revealed that ampicillin or penicillin G appeared to be the drug of choice in C. perfringens associated with haemorrhagic enteritis. However, other antibiotics like erythromycin, clindamycin and metronidazole can also be very useful 200 Afr. J. Microbiol. Res. Figure 2. Nucleotide sequence polymorphic sites of 292 bp fragment of cpa gene of C. perfringens. The ‘dots’ and ‘shade’ represent the residues that match the consensus exactly and that differ from the consensus respectively. Accession numbers: AF477009 = chicken, Denmark; AY823400 = strain C57-1, China; DQ184079 = bovine, USA; NC_003366 = soil, Japan; DQ838706 to DQ838707 = elephant, India; DQ838708 to DQ838709 = pygmy hog, India. Figure 3. Phylogenetic tree analysis based on the nucleotide sequence of partial alpha toxin gene of C. perfringens. for the treatment of C. perfringens associated diarrhoea. This result agrees with the earlier finding reported by Marks and Kather, (2003). But tetracyclines, cefazaflur and even amoxicillin cannot be depended upon and useful in C. perfringens infections (Sutter and Finegold, 1976). In plasmid profiling, the isolates showed multiple plasmids of molecular weight ranging from 4.1 to 51.9 kb. None of the isolates in this study shared identical or common plasmid which was previously reported to be common in strains of C. perfringens associated diseases (Eisgruber et al., 1996). In PCR, only cpa and cpb2 genes were detected which revealed that all the isolates used in this study were C. perfringens type A. The presence of pathogenic cpb2 positive strains of C. perfringens type A in occurrence of ulcerative enteritis in typhlocolitis in horses (Herholz et al., 1999) and African elephant (Bacciarini et al., 2001) have been reported earlier. The analysis of C. perfringens toxin genes by PCR, replacing elaborate animal experiments is extremely useful for diagnostic purpose (Songer and Meer, 1996; Bacciarini et al., 2001). There was no nucleotide substitution within the 292bp region of cpa gene among the Indian field isolates of C. perfringens from elephant and pygmy hog and among three reference sequences from bovine, chicken and strain C57-1 from USA, Denmark and China respectively. Hence, all were placed in a single cluster in the phylogram. However, the four nucleotide substitution at position 105 (adenine to guanine), 156 (guanine to adenine), 229 (cytocine to thiamine) and 255 (guanine to adenine) observed only in isolate derived from soil from Japan also resulted in no amino acid residue change. In general, all the nucleotide substitutions occurred in third nucleotide base of codons which is predominantly silent (Rooney et al., 2006). The phylogenetic analysis also revealed that the partial cpa gene of C. perfringens used in this study is highly conserved among isolates irrespective of different sources of isolation and geographical distribution. The role of the cpa gene in the pathogenesis of haemorhagic enteritis is still not clear because C. perfringens type A strains possessing cpa gene are also frequently isolated from healthy animals. However, among types A to E of C. perfringens, type A isolates carrying cpb2 have been implicated to gastrointestinal diseases in various animals (Bueschel et al., 2003). The detection of cpb2 gene in C. perfringens type A isolated from diarrheic and absence of that in healthy piglet has been reported (Klaasen et al., 1999). Keeping all the above, our findings Das et al. clearly revealed that C. perfringens type A containing an additional cpb2 gene might play a significant role in occurrence of haemorrhagic enteritis in elephant and pygmy hog in Assam. However, the potential synergies of both cpa and cpb2 gene in association with the disease is required to be understood, therefore further studies have to be conducted to understand the molecular pathogennesis of haemorrhagic enteritis. 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