Indian Journal of Experimental Biology

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VOLUME 48

NUMBER 9

SEPTEMBER 2010

CODEN: IJEB (A6) 48 (9) - 861–950 (2010)

ISSN: 0019-5189 (Print); 0975-1009 (Online)

 

CONTENTS

 

Papers

 

Herbs and herbal constituents active against snake bite

865

      Antony Gomes, Rinku Das, Sumana Sarkhel, Roshnara Mishra,
Sanghamitra Mukherjee, Shamik Bhattacharya & Aparna Gomes

 

 

 

T11 target structure exerts effector function by activating immune cells in CNS against glioma where cytokine modulation provide favorable microenvironment

879

      Anirban Ghosh, Malabika Bhattacharya, Pallab Sarkar, Sagar Acharya &
Swapna Chaudhuri

 

 

 

Evaluation of immunogenic potential of 75kDa and 56kDa proteins of Newcastle
disease virus (NDV)

889

      Payal Arora, B D Lakhchaura & S K Garg

 

 

 

Antioxidant and hepatoprotective effect of the ethyl acetate extract of Enicostemma axillare (Lam). Raynal against CCl4-induced liver injury in rats

896

      Jaishree V, Shrishailappa Badami & Praveen Thaggikuppe Krishnamurthy

 

 

 

Hepatoprotective properties of Caesalpinia sappan Linn. heartwood on carbon tetrachloride induced toxicity

905

      V Sathya Srilakshmi, P Vijayan, P Vasanth Raj, S A Dhanaraj &
H Raghu Chandrashekhar

 

 

 

Therapeutic effect of ethanolic extract of Hygrophila spinosa T. Anders on
gentamicin-inducted nephrotoxicity in rats

911

      K J Bibu, A D Joy & K A Mercey

 

 

 

Cardioprotection by Inula racemosa Hook in experimental model of myocardial ischemic reperfusion injury

918

      Shreesh Ojha, Mukesh Nandave, Santosh Kumari & Dharamvir S Arya

 

 

 

Antinociceptive activity of chronic administration of different extracts of
Terminalia bellerica Roxb. and Terminalia chebula Retz. fruits

925

      Sarabjit Kaur & R K Jaggi

 

 

 

In vitro effect of some India honeys on Staphylococcus aureus from wounds

931

      Sunita D Deshpande & Kirti S Kulkarni

 

 

Optimization of medium for lipase production by Acinetobacter haemolyticus from healthy human skin

936

      Shweta Jagtap, Sharad Gore, Supriya Yavankar, Karishma Pardesi & Balu Chopade

 

 

 

Poly-b-hydroxybutyrate production by Pseudomonas sp. RZS 1 under aerobic and
semi-aerobic condition

942

      R Z Sayyed & N S Gangurde

 

 

 

Notes

 

Comparative evaluation of PCR in Ziehl-Neelsen stained smears and PCR in tissues for diagnosis of Mycobacterium avium subsp. paratuberculosis

948

      A C Coelho, M L Pinto, A Miranda, A M Coelho, M A Pires & M Matos

 

 

 

Announcement

 

1st National Conference on Animal, Microbial, Plant Toxins & Snakebite Management (AMPTOX 2010)

864

 

 

 

 

 

 

——————————

 

 

 

 

 

Announcement

 

 

1st National Conference on Animal, Microbial, Plant Toxins &
Snakebite Management (AMPTOX 2010)

11 and 12 December 2010, KPC Medical College & Hospital, Kolkata

 

Jointly organised by the Indian Institute of Chemical Biology (IICB), CSIR, Kolkata and KPC Medical College & Hospital, Kolkata, the conference will cover following areas: (i) Animal toxins, (ii) Microbial toxins, (iii) Plant toxins, (iv) Toxins miscellaneous, (v) Snakebite management, (vi) Antivenom/antidotes, and (vii) Environmental issues and natural toxins. For further details, please contact, Dr. Aparna Gomes, Organizing Secretary, AMPTOX2010, Drug Development Diagnostics and Biotechnology Division, Indian Institute of Chemical Biology (IICB), CSIR, 4, Raja S.C. Mullick Road. Kolkata 700 032, India. Phone : +91-98311 85589; +91-94331 39031; E-mail: amptox2010@gmail.com; website : www.amptox2010.org

 

 

Author Index

Acharya Sagar

879

Arora Payal

889

Arya Dharamvir S

918

 

 

Badami Shrishailappa

896

Bhattacharya Malabika

879

Bhattacharya Shamik

865

Bibu K J

911

 

 

Chandrashekhar H Raghu

905

Chaudhuri Swapna

879

Chopade Balu

936

Coelho A C

948

Coelho A M

948

 

 

Das Rinku

865

Deshpande Sunita D

931

Dhanaraj S A

905

 

 

Gangurde N S

942

Garg S K

889

Ghosh Anirban

879

Gomes Antony

865

Gomes Aparna

865

Gore Sharad

936

 

 

Jaggi R K

925

Jagtap Shweta

936

Jaishree V

896

Joy A D

911

 

 

Kaur Sarabjit

925

Krishnamurthy Praveen
 Thaggikuppe

 

896

Kulkarni Kirti S

931

 

 

Lakhchaura B D

889

 

 

Matos M

948

Mercey K A

911

Miranda A

948

Mishra Roshnara

865

Mukherjee Sanghamitra

865

 

 

Nandave Mukesh

918

 

 

Ojha Shreesh

918

 

 

Pardesi Karishma

936

Pinto M L

948

Pires M A

948

 

 

Raj P Vasanth

905

 

 

Santosh Kumari

918

Sarkar Pallab

879

Sarkhel Sumana

865

Sayyed R Z

942

Srilakshmi V Sathya

905

 

 

Vijayan P

905

 

 

Yavankar Supriya

936

 

 

Keyword Index

Alternative medicines

865

Acinetobacter haemolyticus

936

Antibacterial activity

931

Antinociceptive

925

Antioxidant

896

 

 

Caesalpinia sappan

905

Cardioprotection

918

Cytokines

879

 

 

Embryonated chicken eggs

889

Enicostemma axillare

896

Ethanol extract

925

 

 

Free radical scavenging

911

 

 

Gentamicin

911

Gentianaceae

896

 

 

Heartwood

905

Hepatocytes

905

Hepatoprotective

905

Herbal compound

865

Homeostasis

879

Honey sensitivity

931

Human skin

936

Hygrophila spinosa

911

 

 

Indian honeys

931

Inula racemosa

918

Ischemia-reperfusion

918

 

 

Lipase

936

Liver protection

896

Lymphocytes

879

 

 

Medium optimization

936

Microglia

879

Multi drug resistance

931

Mycobacterium avium subsp.
paratuberculosis

 

948

Myocardial infarction

918

 

 

New castle disease virus

889

 

 

PCR

948

Poly-b-hydroxybutyrate
 fermentation

 

942

Pro-inflammatory

879

Pseudomonas sp.

942

 

 

rRNA (16s) typing

942

 

 

Snake bite

865

Snake bite treatment

865

Snake venom

865

Staphylococcus aureus

931

Statistical analysis

936

 

 

T11TS

879

Terminalia bellerica

925

Terminalia chebula

925

Therapeutic

911

Tissues

948

 

 

Viral proteins

889

 

 

Ziehl-Neelsen

948

 

Correspondent author has been indicated by * sign

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 865-878

 

 

 

Review Article

 

 

 

Herbs and herbal constituents active against snake bite

Antony Gomes1*, Rinku Das1, Sumana Sarkhel1, Roshnara Mishra1, Sanghamitra Mukherjee1,
Shamik Bhattacharya2 & Aparna Gomes2

1Laboratory of Toxinology & Experimental Pharmacodynamics, Department of Physiology, University of Calcutta,
92 A P. C. Road, Kolkata 700 009, India

2Drug Development/Diagnostics & Biotechnology Division, Indian Institute of Chemical Biology,

4, Raja S. C. Mullick Road, Kolkata 700 032, India

Snake bite, a major socio-medical problem of south east asian countries is still depending on the usage of antisera as the one and only source of treatment, which has its own limitations.  In India, mostly in rural areas, health centres are inadequate and the snake bite victims mostly depend on traditional healers and herbal antidotes, as an alternative treatment. The present review has been focussed on the varied folk and traditional herbs and their antisnake venom compounds, which might be a stepping stone in establishing the future therapy against snake bite treatment and management.

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 879-888

 

 

T11 target structure exerts effector function by activating immune cells in CNS against glioma where cytokine modulation provide favorable microenvironment

Anirban Ghosh1, Malabika Bhattacharya2, Pallab Sarkar, Sagar Acharya & Swapna Chaudhuri*

Department of Laboratory Medicine, School of Tropical Medicine, 108, CR Avenue, Kolkata 700 073, India

Received 1 January 2010; revised 28 April 2010

Glycoprotein T 11 target structure (T11TS), derived from sheep erythrocyte membrane, directly interacts with T cells to activate them to enter in the brain. When untreated, glioma exerts an immune-suppressive environment in its vicinity by secreting prostaglandin E2 (PGE2), IL-10, tumor growth factor β, gangliosides etc. to dampen the immune attack. But exogenous administration of T11TS reverses the situation to pro-inflammatory immune active state by expressing enhanced IL-12 and tumor necrosis factor α (TNF-α) production and suppression of IL-4 and IL-10 levels. The T11TS activated lymphocytic accumulation along the capillary endothelium in brain and their penetration in the matrix was evident from histological sections. IL-6 with TNF-α facilitates leukocyte migration to glioma site to exert cytotoxic effector function. Brain infiltrated lymphocytes offer cytotoxic proximity to neoplastic glial cells, which lead them to apoptosis. In the Th1 dominated microenvironment microglial cells was found with enhanced phagocytic functions. Initially infiltrated lymphocytes with microglia showed increased production of TNF-α, interferon γ (IFN-γ) to facilitate their effector actions. Repeated dosing of T11TS shows glioma abrogation in rat model, but also a resurgence of anti-inflammatory cytokine environment found with increased IL-4, IL-10 and decreased IL-12, IL-6, TNF-α. This is a unique homeostatic regulation of total immune system after T11TS mediated carnage of glioma. The resultant balance of cytokines between interacting glioma cells, T cells and microglia in T11TS induced condition determines the success of its immunotherapeutic effect in glioma.

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 889-895

 

 

 

 

Evaluation of immunogenic potential of 75kDa and 56kDa proteins of newcastle disease virus (NDV)

Payal Arora1*, B D Lakhchaura2 & S K Garg3

1,3Department of Microbiology, 2Department of Animal Biotechnology, G. B. Pant University of Agriculture and Technology,
College of Veterinary Sciences Pantnagar 263 145, India

Received 17 March 2010; revised 13 May 2010

The R2B strain of virus of new castle disease virus (NDV) was propagated in 9-11 day old embryonated chicken eggs via allantoic cavity route and after seven serial passages virus was purified from allantoic fluid. Purified virus was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis which yielded six major polypeptides ranging from 38-200 kDa. Protein fractions, corresponding to 75 and 56kDa, resembling haemagglutinin-neuraminidase (HN) and fusion (F) proteins were used to ascertain their immunization potential. Immunization of viral proteins was compared with the whole virus vaccine. Among different group of birds, highest haemagglutination inhibition (HI) and enzyme linked immunosorbent assay (ELISA) titers were obtained in birds immunized with whole virus vaccine followed by viral proteins, 75 and 56kDa in combination which was comparable with birds immunized with 56kDa protein alone. Despite lower values of HI and ELISA titers elicited by viral subunits in immunized birds, when challenged with virulent NDV strain, protection accorded by viral proteins in combination (75 +56kDa) or 56kDa alone was comparable with whole virus vaccine.

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 896-904

 

 

Antioxidant and hepatoprotective effect of the ethyl acetate extract of Enicostemma axillare (Lam). Raynal against CCl4-induced liver injury in rats

Jaishree V*, Shrishailappa Badami & Praveen Thaggikuppe Krishnamurthy

Department of Pharmaceutical Chemistry, J. S. S. College of Pharmacy, Ootacamund 643 001, India

Received 16 June 2009; revised 21 April 2010

Enicostemma axillare is used in Indian traditional medicine as a liver tonic. Its ethyl acetate extract has shown potent in vitro antioxidant activity and found to contain 7.26% of a bitter secoiridoid glycoside, swertiamarin. Hence, in the present study the ethyl acetate extract was screened for hepatoprotective and antioxidant properties against CCl4 induced hepatic injury in rats. The hepatoprotection was assessed in terms of reduction in histological damage and changes in serum enzymes and metabolites. The pretreatment with the extract at 100 and 200 mg/kg body weight doses given orally for eight days prior to CCl4 caused significant restoration of altered biochemical changes due to CCl4 towards the normal in serum, liver and kidney. The extract treatment at 200 mg/kg body weight was found to be more potent than the standard silymarin at 100 mg/kg body weight in reversing most of the biochemical parameters. Histopathological studies complemented the results of biochemical estimations in providing a proof of hepatoprotective and antioxidant actions of the extract. The study provides a support to the ethnomedical use of E. axillare in India.

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 905-910

 

 

 

Hepatoprotective properties of Caesalpinia sappan Linn. heartwood on carbon tetrachloride induced toxicity

V Sathya Srilakshmi1, P Vijayan1, P Vasanth Raj2, S A Dhanaraj1 & H Raghu Chandrashekhar2*

1 Department of Pharmaceutical Biotechnology, J S S College of Pharmacy, Ootacamund 643 001, India.

2 Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences,
Manipal University Manipal 576 104, India.

Received 16 December 2008; revised 30 March 2010

Aim of the study was to investigate the methanol and aqueous extracts of heartwood of C. sappan for its hepatoprotective activity against CCl4 induced toxicity in freshly isolated rat hepatocytes and animals. Freshly isolated rat hepatocytes were exposed to CCl4 (1%) along with/without various concentrations of methanolic and aqueous extract of
C. sappan (1000-800 µg/ml) and the levels of selected liver enzymes were estimated. Antihepatotoxic effect of methanolic extract was observed in freshly isolated rat hepatocytes at concentrations 1000-800 µg/ml and was found to be similar to that of standard drug silymarin. Wistar strain albino rat model was used for the investigation of in vivo hepatoprotective properties of aqueous and methanolic extract of C. sappan (100 and 200 mg/kg body weight). Liver damage was induced by ip administration of CCl4 (30%) suspended in olive oil (1 ml/kg body weight). Both the tested extracts showed potent hepatoprotective activity at 200 mg/kg body weight test dose which was comparable with that of the standard silymarin used in similar test dose. The methanolic and aqueous extract was able to restore the biochemical levels to normal which were altered due to CCl4 intoxication in freshly isolated rat hepatocytes and also in animals.

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 911-917

 

 

Therapeutic effect of ethanolic extract of Hygrophila spinosa T. Anders
on gentamicin-induced nephrotoxicity in rats

K J Bibu*, A D Joy & K A Mercey#

Department of Pharmacology and Toxicology, #Department of Statistics

Faculty of Veterinary and Animal Sciences, Mannuthy, Thrissur 680 651, India

Received 3 December 2009; revised 10 May 2010

Therapeutic effect of ethanolic extract of Hygrophila spinosa in gentamicin-induced nephrotoxic model of kidney injury in male Sprague-Dawley rats was studied. Rats were administered with gentamicin at a dose of 80 mg/kg intraperitoneally (ip) to induce nephrotoxicity. Kidney function was assessed by measuring serum creatinine and urea. Kidney superoxide dismutase, lipid peroxidation, catalase and reduced glutathione were also measured in control and treated rats.
H. spinosa extract showed free radical scavenging activities at doses of 50 and 250 mg/kg with a predominant activity at
250 mg/kg. The ethanolic extract also caused a reduction in serum creatinine and urea levels. Histopathological studies were conducted to confirm the therapeutic action of the plant extract. The results demonstrated that the ethanolic extract of whole plant of H. spinosa evinced the therapeutic effect and inhibited gentamicin-induced proximal tubular necrosis.

 

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 918-924

 

 

Cardioprotection by Inula racemosa Hook in experimental model of myocardial ischemic reperfusion injury

Shreesh Ojha, Mukesh Nandave, Santosh Kumaria & Dharamvir S Arya*

Cardiovascular Laboratory, Department of Pharmacology, All India Institute of Medical Sciences, New Delhi 110 029, India

aDivision of Plant Physiology, Indian Agriculture Research Institute, New Delhi 110 029, India

Received 11 February 2010; revised 6 April 2010

To evaluate the cardioprotective potential of Inula racemosa in myocardial ischemic-reperfusion injury, Wistar male albino rats were randomly divided into four groups. The group I and II animals were administered saline orally {(sham, ischemia- reperfusion (I-R) control group)} and animals of group III and group IV received I. racemosa extract (100 mg/kg) for 30 days. On the 30th day, animals of I-R control and I. racemosa treated groups were underwent 45 min of ligation of left anterior descending coronary artery and were thereafter re-perfused for 60 min. In the I-R control group, a significant decrease of mean arterial pressure (MAP), heart rate (HR), contractility, (+)LVdP/dt and relaxation, (-)LVdP/dt and an increase of left ventricular end diastolic pressure (LVEDP) were observed. Subsequent to haemodynamic impairment and left ventricular contractile dysfunction, a significant decline was observed in endogenous myocardial antioxidants; superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and reduced glutathione (GSH). Increased lipid peroxidation characterized by malonaldialdehyde (MDA) formation along with depletion of cardiomyocytes specific enzymes, creatine phosphokinase-MB (CK-MB) isoenzyme and lactate dehydrogenase (LDH) in I-R control group compared to sham group revealed I-R injury of heart. However, treatment with I. racemosa significantly restored the myocardial antioxidant status evidenced by increased SOD, CAT, GPx and GSH and prevented leakage of cardio-specific enzymes; CK-MB and LDH and favorably modulated the altered MAP, HR, (+)LVdP/dt, (-)LVdP/dt and LVEDP as compared to I-R control. Furthermore, I-R induced lipid peroxidation was significantly inhibited by I. racemosa treatment. These beneficial cardioprotective effects translated into significant improvement in cardiac function. In conclusion, our study has demonstrated that the cardioprotective effect of I. racemosa likely resulted to improved antioxidant status, haemodynamic and left ventricular contractile function subsequent to suppression of oxidative stress.

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 925-930

 

 

Antinociceptive activity of chronic administration of different extracts of Terminalia bellerica Roxb. and Terminalia chebula Retz. fruits

Sarabjit Kaur* & R K Jaggi

University Institute of Pharmaceutical Sciences, Punjab University, Chandigarh.

Received 4 September 2009, revised 20 May 2010

The petroleum ether (PE), chloroform (CH), ethanol (ETH) and water extracts of Terminalia bellerica and T. chebula fruits were evaluated for their analgesic activity using the tail immersion model in mice. The ethanolic extracts of both the plants exhibited analgesic response at 200,400 and 800mg/kg. The studies were further carried for 15 days to evaluate the effect of these extracts in chronic pain and maximum analgesic response was observed on 14th day in both the plants. Phytochemical investigation of ethanolic extract of the fruits of Terminalia bellerica and T. chebula revealed the presence of saponins, triterpenoids, carbohydrates, tannins and proteins. The results indicate that fruits of T. bellerica and T. chebula could be considered as potential candidate for bioactivity-guided isolation of natural analgesic agents used in the management of chronic pain.

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 931-935

 

 

In vitro effect of some Indian honeys on Staphylococcus aureus from wounds

Sunita D Deshpande* & Kirti S Kulkarni

Department of Microbiology,T.N.M.C & B.Y.L. Nair Hospital, Mumbai Central 400 008, India

Received 4 September 2009; revised 3 May 2010

Staphylococcus aureus is the most frequently isolated pathogen from wounds with multiple resistances to antibiotics. Honey has been demonstrated and reported to be effective antibacterial agent on Gram positive and Gram negative organisms. Hence, the present study was conducted to evaluate the in vitro antibacterial effect of Indian honeys on Staphylococcus aureus obtained from wounds. A total of 123 Staphylococcus aureus isolates along with ATCC 25923 were categorized as sensitive, multi drug resistant (MDR) and non-MDR strains. Out of total nine Indian honeys (three each of unifloral, multifloral and branded marketed honey) used, three unifloral and three multifloral honey samples showed antibacterial activity against all the organisms tested by Agar diffusion method but not the branded marketed honeys. The MIC values of all honey samples for all studied Staphylococcus aureus isolates ranged between 5-15% (v/v). Unifloral honey samples showed higher antibacterial activity than multifloral honey. The single sample of Jambhul honey showed the highest activity. Thus, Indian honeys were found to be effective for their antimicrobial activity on sensitive, non-MDR, MDR and ATCC strains of S. aureus.

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 936-941

 

 

Optimization of medium for lipase production by Acinetobacter
haemolyticus
from healthy human skin

Shweta Jagtap1, Sharad Gore2, Supriya Yavankar1, Karishma Pardesi1 & Balu Chopade1, 3*

1Department of Microbiology, University of Pune 411 007, India.

2Department of Statistics, University of Pune 411 007, India.

3Institute of Bioinformatics and Biotechnology, University of Pune 411 007, India.

Received 25 November 2009, revised 18 May 2010

A lipase producing Acinetobacter haemolyticus TA106 was isolated from healthy human skin of tribal population. The maximum activity of 55 U/ml was observed after medium optimization using the “one variable at a time” and the statistical approaches. The optimal composition of the medium was determined as (% w/v or v/v): tryptone - 1, yeast extract - 0.5, sodium chloride-1, olive oil-1, Tween - 80 1, manganese sulphate - 5 mM, sucrose- 1, pH-7. It was found that maximum production occurred in late log phase i.e. after 72 h and at 200 rpm. From factorial design and statistical analysis, it was found that pH, temperature, salt, inoculum density and aeration significantly affected the lipase production. It was also noted that inoculum density of 3 % (v/v), sucrose (1% w/v) and manganese sulphate (5 mM) displayed maximum lipase activity of 55 U/ml by conventional as well as statistical method. Optimization studies also indicated the increase in specific activity from 0.2 U/mg to 6.7 U/mg.

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 942-947

 

 

Poly-β-hydroxybutyrate production by Pseudomonas sp. RZS 1 under aerobic and semi-aerobic condition

R Z SayyedÜ & N S Gangurde

PG Department of Microbiology, S. I. Patil Arts, G. B. Patel Science and S.T.S.K.V. S. Commerce College,
Shahada, Dist. Nandurbar 425409, India.

Received 13 November 2009; revised 5 May 2010

Pseudomonas sp. RZS1 was isolated from distillery effluent and identified based on phenotypic characters and 16s rRNA sequencing. It accumulated optimum amount (703.79 µg/mg of biomass) of poly-β-hydroxybutyrate (PHB) under aerobic process of fermentation and 75 µg/mg of biomass under the anaerobic process of fermentation. Aerobic fermentation yielded 9.3-fold more PHB than semi-aerobic fermentation. Acetone alcohol method proved to be the best suitable recovery method as it gave 703.79 µg PHB per mg of biomass with a percentage recovery yield of 70.37. It started to accumulate PHB at the end of lag phase (from 6 h of incubation). Optimum amount of PHB (20 µg/ml) was reported during early stationary phase (30 h of incubation). Extracted PHB showed two peaks, minor one at 248 nm and major one at 365 nm. IR spectra revealed the presence of functional groups characteristics of PHB.

 

 

 

Indian Journal of Experimental Biology

Vol. 48, September 2010, pp. 948-950

 

Notes

Comparative evaluation of PCR in Ziehl-Neelsen stained smears and PCR in tissues for diagnosis of Mycobacterium avium subsp. paratuberculosis

A C Coelho 1,2*, M L Pinto1,2, A Miranda1, A M Coelho3,
M A Pires1,2 & M Matos4

1Veterinary Microbiology Laboratory, Veterinary Medicine, Department of Veterinary Sciences,

2CECAV, University of Trás-os-Montes and Alto Douro (UTAD), 5001-801 Vila Real, Portugal

3Veterinary Services of the North, Division of Veterinary Intervention of Vila Real, Corgo Nucleus, 5000-421
Vila Real, Portugal

4Institute of Biotechnology and Bioengineering, Centre of Genetics and Biotechnology, University of Trás-os-Montes and Alto Douro (UTAD), 5001-801 Vila Real, Portugal

Received 3 December 2009; revised 8 April 2010

Thirty six tissues from sheep, previously diagnosed with paratuberculosis, were tested by PCR in positive Ziehl-Neelsen staining smears of tissues, and PCR in tissues targeting IS900 specific for Mycobacterium avium subsp. paratuberculosis. DNA amplification was achieved in 33.3% Ziehl-Neelsen smears, and in 61.1% tissue samples. Combination of both techniques found 66.7% samples as positive. Combination of techniques would, therefore, increase the sensitivity of diagnosis.