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Molecular diagnosis and therapeutic management of black quarter--a clinical study of 21 bovines.

Introduction

Black quarter is a highly fatal infectious disease principally affecting cattle in India caused by Clostridium chauvoei. The spores of Clostridium chauvoei after getting ingested along with fodder, feed reaches the muscular tissue via blood stream and on development of hypoxia, germinate, multiply and produce potent toxins (Useh et al., 2003, Radostits et al., 2007). One of the major potent toxins i.e. alpha toxin is involved mainly in pathogenicity of disease (Hang'ombe et al., 2006).

The alpha toxin is reported as hemolytic, necrotic and histotoxic (Quinn et al., 2005). The primers designed to amplify Clostridium chauvoei alpha toxin gene has been used in present study for detection of Clostridium chauvoei capable of producing alpha toxin along with published primers for molecular diagnosis of Black quarter.

The presumptive diagnosis of blackleg is achieved by clinical observations and routine diagnosis is done by isolation and identification of bacteria, by traditional time consuming method. For confirmation and identification of bacteria molecular diagnostics are being widely used with specificity and sensitivity.

The present study was carried out to isolate the bacteria from suspected cases of Black quarter from swab and tissues by conventional methods and detection of alpha toxin producing Clostridium chauvoei by amplification of Ccta gene for diagnosis.

Materials and methods

Animals

Twenty one clinical cases of Deoni and Red Kandhari cattle and Marathwadi buffaloes, ageing 3 months to 3 years, suspected for black quarter, reported during 2010-2013 formed the material for the present study.

Collection of sample

A total of 21 samples (Emphysematous exudates and muscle piece) from cattle and buffaloes suspected for black quarter were collected aseptically and were processed for isolation and identification of Clostridium chauvoei by molecular and traditional method.

Isolation and identification of organism

The samples were inoculated in Reinforced Clostridial Medium (RCM) broth and incubated at 37OC for 24 hours. Tubes containing growth were streaked on Clostridial Anaerobic Agar Medium and incubated in anaerobic jar with anaerobic gas pack (Hi-media). Gram's staining and spore staining were used to observe microscopic characteristics of bacteria and morphological and cultural characteristics.

DNA extraction using bacterial isolates

Field isolates were subjected to DNA extraction using Qiagen DNA Extraction kit. After treatment with Proteinase K and using DNA Extraction kit (QIAamp spin column--QIAGEN, Hilden, Germany) and centrifugation at 6000 x g (8000rpm), as per protocol.

Quantitation of DNA

After extraction of DNA directly from isolates, the DNA was quantitated using Nanodrop 2000.

Amplification of universal oligonucleotide sequence and alpha toxin gene (Ccta)

The primer sequences used in the present study are:

Published primer used in the present study, universal i.e. CC-193F AGCTTCGCATG GAGCAGTA CC-16SR TCTTCGGAGACAGGA TGA (Uzal et al.,2003).

Primers set designed using PRIMER 3 for Ccta gene were used in the present study. A positive control of known vaccine standard strain of Clostridium chauvoei was used.

Designed Primers i.e., Ccta-F AGCTTCGCATG GAGCAGTA and Ccta-R TCTTCGGAGACA GGATGA were used for amplification of alpha toxin gene of Clostridium chauvoei.

PCR Reaction and electrophoresis

The reaction of 25ul using AmpliTaq Gold premix was set. The amplification was performed using non-gradient thermocycler for 30 cycles, with denaturation at 94[degrees]C for 60 sec, annealing at 61[degrees]C for 45 sec and extension at 72[degrees]C for 30 sec followed by a final extension at 72[degrees]C for 6 min. On amplification, the products were analysed for presence of a single band of desired molecular weight on 1.5% agarose gel in presence of ethidium bromide (0.5 pg/ml) and documented using gel documentation system.

Blast

Sequences obtained from PCR products were analysed by converting it into FASTA format and BLAST and percent homology was recorded.

Treatment

The affected animals were treated with Amoxycillin+Dicloxacillin (Intamox-D (a)) @ 20mg/ kg b. wt., intravenously and locally for 3 days followed by intramuscular daily for next 6-8 days, intensive fluid therapy for first 24 hrs followed by maintenance therapy twice daily for next 3-5 days depending upon severity of toxaemia and anorexia, Dexamethasone (Dexona (b)) @ 0.5-1 mg/kg b.wt. iv/im and Chlorpheniramine maleate (Anistamin (a)/Zeet (b)) @ 20-100 mg im daily for 3 days, Meloxicam (Melonex (a)) @ 0.5mg/kg b. wt. im for 9-11 days, and B-complex (Tribivet (a)) @ 2-5 ml im for 5-6 days.

Results and discussion Clinical observations

The changing pattern of black quarter has been recorded in cattle at any age, but most cases of black quarter have been recorded in cattle and buffaloes between age group of 3 months 3 years. The site of emphysematous lesions again varied not fixed at hind quarter but generalized multilocational emphysematous lesions were recorded in both cattle and buffaloes (Fig 1-3). Other signs included variable degree of fever, dullness, lameness, recumbency, loss of appetite, pain in affected quarter and crepitating sound at the site of emphysematous swelling.

Bacterial Isolation

A total of 21 samples (emphysematous exudates and muscle piece) from cattle and buffalo suspected for black quarter were collected aseptically and subjected to bacterial isolation yielded 28 isolates including Clostridium chauvoei, Clostridum tertium and Clostridium sporogenes. (Fig. 4 and Fig. 5).

Isolation of bacteria from emphysematous muscle and exudates yielded Clostridium chauvoei, single bacteria in six cases whereas yielded mix isolates in 15 cases under study i.e., Clostridium chauvoei, Clostridium tertium, Clostridium sporogenes and Clostridium septicum, after 24-48 hours of incubation in RCM at 37[degrees]C.

Bacterial isolation using pre-heated RCM yielded pure culture because of killing of all the vegetative of growth of bacteria and contaminants from the surrounding gets killed and only spores remain intact, which germinates on cooling the RCM at room temperature followed by yield of pure culture of the Clostridium species.

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PCR Amplification

PCR amplification of Ccta gene yielded 206 bp product and 836 base pairs (bps) (universal) long on electrophoresis gel. This size of PCR product was expected for positive samples for Clostridium chauvoei. Similar amplification pattern were reported by Sasaki et al. (2001), Uzal et al. (2003) and Idrees et al. (2014) for universal primers. All the positive field isolates (confirmed biochemically) were subjected to PCR amplification using universal and Ccta gene primers and all samples yielded PCR product.

The PCR product was subjected to sequencing. The resulted sequence was subjected to BLAST. BLAST showed 99% homology/identity with Clostridium Ccta gene (Accession Number: JQ692583.1Accession number Clostridium chauvoei ATCC strain complete CD).

Treatment

Although it has been reported in the literature that treatment is not effective in black quarter, in this study 76.2% treated animals survived owing to intensive therapeutic management of shock in the initial stage and long term treatment of muscle sloughs. Penicillin is the traditional drug of choice. However in this study Amoxicillin in high doses was effectively used as bactericidal antibiotic. Institution of immediate and intensive fluid therapy at frequent intervals with repeated dosing of dexamethasone and administration of anti-histaminics in critically ailing patients during first 12-24 hours of treatment helped in reversal of life threatening shock state. Meloxicam was given as NSAID for relieving inflammation, local pain and pyrexia. In treated animals, appetite and water intake gradually improved, lameness slowly disappeared with reduction in swelling within 5-9 days. For complete recovery, it took 10-12 days to 3-4 months depending upon size of muscle slough. Earlier Mouli et al. (1995) were able to save 2 black quarter affected buffaloes with Penicillin and Oxytetracycline combination therapy. Subsequently, Bhikane et al. (1998) successfully treated a case of black quarter in 8 year old crossbred cow with Procaine Penicillin, Diclofenac sodium, Pheniramine maleate and Dextrose saline. However, Kachhwaha and Tanwar (2008) reported that all 14 black quarter affected cattle treated with Penicillin and Diclofenac died.

Conclusion

The findings of the study indicate that PCR can be best employed for diagnosis of Black quarter using Ccta gene . The PCR diagnosis is sensitive, specific and effective method in confirmatory diagnosis as compared to time consuming biochemical and sugar fermentation tests. Molecular diagnosis helps in early confirmation of outbreak and early treatment thereby helpful in effective treatment and recovery of animals.

Also, characterization of Ccta gene of Clostridium is helpful in epidemiological studies and designing effective vaccine and protective immunity against Black quarter in Deoni cattle and Marathwadi buffaloes. The treatment with Amoxicillin-Dicloxacillin with intensive supportive treatment for management of shock in initial stage and regular dressing of wound in cases of muscle slough proved effective in management of black quarter.

References

Bhikane, A.U., Kulkarni D.D. and Salunke, V.M. (1998). Successful treatment of black quarter in a crossbred cow. Indian Vet. J. 75 : 58-59.

Hang'ombe, B.M., Kohda, T. Mukamoto, M. and Kozaki, S. (2006). Purification and sensitivity of Clostridium chauvoei hemolysin to various erythrocytes. Comparative Immunology, Microbiology and Infectious Diseases, 29:263-68.

Idrees, Z.I., Chaudhary, M.Y. and Ashraf, K. (2014). Isolation and molecular detection of clostridium chauvoei alpha toxin gene from clinical cases of black quarter in cattle. The J. of Anim. Plant Sci. 24: 755-59.

Kachhwaha, S. and Tanwar, R.K. (2008). An investigation and management of blackleg in cattle. Intas Polivet 9 : 74-76.

Mouli, S.P.; Ratna Sree, P. and Prasannakumar, V. (1995). Blackleg in buffaloes and its medico-surgical treatment. Indian Vet J. 72: 500-02.

Quinn, P.J., Carter, M.A. Markey, B. and Carter, G.R. (2004). Clinical Veterinary Microbiology. 2nd Ed: Elsevier - Health Sciences Division. USA. pp: 191-208.

Radostitis, O.M., Gay, C.C., Hinchcliff, K.W. and Constable, P.D. (2007). Veterinary Medicine, 10th edn. WB Saunders Company Ltd. London. pp: 828-30.

Sasaki Y, Kojima, A., Uchida, I., Sekizaki, T., Ogikuba, Y., Kijima, M. and Tamura, Y. (2001). Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence. Vet. Microbiol. 78: 363-71.

Useh, N.M., Nok, A.J. and Esievo, K.A. (2003). Pathogenesis and pathology of blackleg in ruminants: the role of toxins and neuraminidase. A short review. Vet. Q. 25:155-59.

Uzal, F.A., Hugenholtz, P., Blackall, L.L., Petray, S., Moss, S. and Assis, R.A. (2003). PCR detection of Clostridium chauvoei in pure cultures and in formalinfixed, paraffin-embedded tissues. Vet. Microbiol. 91: 239-48.

(a)--Brand of Intas Animal Health, Ahmedabad

(b)--Brand of Alembic Ltd., Mumbai

A.V. Bhonsle (1), A.U. Bhikane (2), M.A. Khan (3), M.B. Kulkarni (4) and A.G. Karpe (5)

Department of Veterinary Microbiology

College of Veterinary and Animal Sciences

Maharashtra Animal and Fishery Sciences University (MAFSU)

Udgir--413517

Dist. Latur (Maharashtra)

(1.) Assistant Professor and Corresponding author. E-mail: ashokvbhonsle@hotmail.com

(2.) Associate Professor, Dept. of Veterinary Medicine

(3.) Assistant Professor, Department of Veterinary Pathology

(4.) Laboratory Technician

(5.) Dean
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Title Annotation:Clinical Article
Author:Bhonsle, A.V.; Bhikane, A.U.; Khan, M.A.; Kulkarni, M.B.; Karpe, A.G.
Publication:Intas Polivet
Article Type:Report
Date:Jul 1, 2015
Words:1775
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