Abstract

The aim of the present investigation is to enhance the bioethanol production from agricultural feed stocks through fermentation process. To bring out facts, experiments were conducted on the biochemical analysis of Mahua flower (Madhuca indica) for its suitability as a raw material and optimizations of fermentation conditions using response surface methodology. The results of fermentation processes on bioethanol productions were compared and tabulated. From the data, it was observed that Saccharomyces cerevisiae-3190 NCIM is high bioethanol tolerant, acid resistant, and is able to produce high yields of bioethanol in high gravity medium. The optimum conditions such as pH 4.9, temperature 31.43 0C, agitation 117.28 RPM, and ammonium sulphate (NH4)2SO4) of 0.629 mg.l-1 were obtained on bioethanol production with statistical optimizations. The yeast strain could produce 150.562g.l-1 and 195.284 g.l-1 with the substrate concentration of 360 g.l-1 and 409 g.l-1 after 48 hours by medium-I and medium-II respectively.

Key Words

Bioethanol, Fermentation, RSM, Bioethanol Method, Gas Chromatography.

Introduction

Ethanol is an ancient organic solvent which next to the water and is named as “Bioethanol”, after its production through fermentation process using microorganisms. Conversion of sugars into ethanol is one of the earliest anaerobic organic reactions employed by humanity. Several authors reported that the attention has been devoted to the conversion of various substrates such as molasses, sugar cane, sorghum, potato, cassava, cashew apple juice, fruit juices and corn, wheat, pearl millet, rice to fuel bioethanol using bacterial and yeast cells. The industrial production of ethanol and commercial use of yeasts started at the end of the 19th century after their identification and isolation by Louis Pasteur. In 1908, Henry Ford has designed a fuel with a mixture of Gasoline and Alcohol and further it was referred as “The fuel of the future” in the year 1925 (Ravindra, 2007). Ethanol (C2 H5 OH) is pure in color, volatile flammable and non toxic. Worldwide fuel prices are rising due to increase in demand by the population (Dake, et al, (2010). It has a molecular weight of 46.07, melting point of -115 0C, boiling point of 78 0C and specific gravity of 0.79 GM/ml at 20 0C. The oxygenated bio-fuels like biodiesel and bioethanol are an effective substitute for renewable fuels and reduce particulate matter from in-use diesel vehicles (Krishna swamy, et al, 2012).

Substrate Mahua flower (Madhuca indica) belongs to the family Sapotaceae. It is a medium sized to large deciduous tree, usually with a short bole and rounded crown and found throughout the greater part of India up to an altitude of 1200m. It is usually found in mixed deciduous forest, usually of dry type, and grows on rocky and sandy soil and flourishes on the Deccan plateau. It is common throughout the deciduous forest in central India, Madhya Pradesh, Maharashtra, Gujarat, Orissa, Chota Nagpur, and Andhra Pradesh (Wealth of India, 1962). Mahua flower contains total fermentable sugars of 731.343µg/ml, Moisture content of 17 %, Reducing sugars of 18%, Protein of 4.6 mg and Fat of 0.5 %. A gram of glucose can be converted to 0.511 grams of ethanol (Maiorella, et al, 1981). The stoichiometric glucose conversion to bioethanol in the presence of yeast cells can be represented as follows:

Yeast cells

C6H12O6 (180 g.mol) →→→→ 2C2 H5 OH (92 g.mol) + 2CO2 (88 g.mole)

Optimization of medium constituents by laboratory method is a single–dimensional search involving change of one variable while fixing the others at a certain level is laborious and time consuming, especially when the number of variables is in large. Therefore, an alternative and potential method in microbial system is the function of statistical methods. Hence, the present optimization studies were carried out with Box-Wilson (Box and Wilson, 1951) response surface methodology using software Statistica8.

Materials and Methods

Microorganisms

Yeast and Bacterial strains such as Saccharomyces cerevisiae-3190 NCIM, S.cerevisiae-171 MTCC, K.thermotolerance-30 MTCC, S.cerevisiae-3288 NCIM, K.marxianus-1389 MTCC, Z.mobilis-92 MTCC, E.coli and S.cerevisiae-463 MTCC, which are obtained from National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratories, Pune and (MTCC) Microbial Type Culture Collection, Chandigarh, India) cultures were tested for bioethanol production.

Methods

Total sugars in Mahua flowers extract (MFE) estimated by Anthrone method (Yemm and Willis, 1954), Moisture content by Association of Official Agricultural Chemists (AOAC, 2000), Method No: 930.15, reducing sugars by Miller method (1959), protein by Lowry’s method (1951), and Fat by American association of cereal chemists (AACC, Method: 30-25, 1983). Determination of total cell count by American public health association (APHA Method, 1967). Estimation of total viable cell count was determined by methylene blue reagent (Bonara and Mares (1982).

Preparation of bacterial culture medium

Bacterial culture medium was prepared using beef extract of 3 g.l-1; peptone, 5 g.l-1; sodium chloride of 8 g.l-1 and 15 grams of Agar were added to 1000 ml of distilled water in 2 litre Erlenmeyer flask. The pH of the medium was adjusted to 7.5 using 1 N HCL and 1 N NaOH with the aid of pH Meter (Systronics). Then, the medium was allowed to sterilize at 121 0C for 30 minutes. Then, 10 ml of sterilized medium was aseptically transferred to the culture tubes and rotated at 450 for 25 minutes to develop agar slopes. After the solidification completed, one loopfull of original culture of bacterial strains were aseptically streaked on agar slopes and tightly capped with non-adsorbent cotton. Then, these agar slants were incubated at 25 0C for 48 hours of growth period.

Preparation of yeast culture medium

The yeast culture medium was prepared in 2 litre Erlenmeyer flasks containing glucose, yeast extract, malt extract and peptone (GYMP) in 1 litre of distilled water and pH was adjusted to 6 using 1 N HCL and 1 N NaOH .The medium was autoclaved at 1210C for about 30 minutes.

After autoclave was completed, 10 ml of medium was aseptically transferred to petri plates and 30 ml tubes. Then, the original cultures of yeast were aseptically inoculated with loop on agar slopes. Using this medium composition, cultures were incubated at 300C for 48 hours. For every 30 days, yeast culture was freshly prepared for maintaining cell viability and the total experiments were carried out with freshly prepared nutrient agar medium and nutrient broth medium.

Response surface methodology

Response surface methodology (RSM) is one of the suitable methods for identifying the effect of individual variables and optimizing the conditions for a multivariable system efficiently. Multiple regression and correlation analysis are used as tools to assess the effects of two or more independent factors on the dependent variables (Ratnam, et al., 2003). The central composite design (CCD) with 3k factorial design was applied for the optimization of fermentation conditions on bioethanol production. Three different levels such as low concentration level (-1), Middle level (0) and High level (+1) and three independent variables represented as X1, X2, X3 and dependent variable is the production of bioethanol (g.l-1) were applied to CCD. Recently, many statistical experimental design methods have been employed in bioprocess optimization. Two axial points on the axis of each design variable at a distance of α from the design centre were applied. Thus the total number of design points in central composite design (CCD) consisting of k variables are given as follows

N=2k+2k+no

γi = β̥+β1×1+ β2×2+ β2×3+ β11 x12+ β22 x22+ β33×32+ β12x1x2+ β13x1x3+β23x2x3

Where, Yi is Predicted Response; X1, X2 and X3 are Independent Variables; β̥ is Offset term; β1, β2, β3 are Linear effects; β11, β22 and β33 are Squared effects and β12, β13, β23 are Interaction terms.

Fermentation with 5 litre bioreactor

Bioreactor (B-Lite, Sartorious Private Limited, Mumbai, India) is batch scale bioreactor that can ferment up to 5 Litres of fermentative medium. In the present study, 2 litres of Mahua fermentative medium was used for bioethanol production under controlled conditions like pH, temperature, and agitation. During the batch fermentation process, bioreactor was sterilized at 121 0C for 15 minutes.

Bioethanol determination

Total content of bioethanol in fermented sample were determined by gas chromatograph (GC). Gas chromatography method for bioethanol production was developed based on the dilutions used in British pharmacopeia (2007).

Bioethanol (%)=( Peak Area of Biothanol)/( Peak Area of n-Butanol) x (Wt of Std)/(Std Volume ) x (Sample Volume)/(wt.of sample) x Std Potency

Therefore,

Peak Area of Bioethanol = peak hight of chromatogram of bioethanol

Peak Area of n- Butanol = peak hight of chromatogram of n-butanol

Sample Volume = sample dilution

Wt of Std = weight of the standard

Std Volume = standard volume

Wt of sample = weight of sample

Std Potency = standard concentration of bioethanol

Results & Discussion

During the process of fermentations, screening of various microorganisms was carried on bioethanol productions. The microorganisms such asSaccharomyces cerevisiae-171 MTCC, Kluyveromyces thermotolerance-30 MTCC, S.cerevisiae-3288 NCIM, S.cerevisiae-3190 NCIM,K.marxianus-1389 MTCC, Zymomonas mobilis-92 MTCC, Escherichia coli and S.cerevisiae-463 produced bioethanol concentrations of 36.437g.l^-1, 35.573 g.l^-1, 41.241 g.l^-1, 43.865 g.l^-1, 40.531 g.l^-1, 39.657 g.l^-1, 37.559 g.l^-1, and 41.327 g.l^-1 respectively. Amongst, S.cerevisiae-3190(NCIM) produced maximum bioethanol yield was 43.865 g.l^-1 after 48 hours of fermentation than other yeast and bacterial cells. From the results, it was found that the yeast strain S.cerevisiae-3190 (NCIM) is efficient and used throughout the optimizations of fermentative conditions on bioethanol productions. The result shown in figure: 1.The initial standardization of fermentative conditions such as physico-chemical and nutritional factors were carried out using S.cerevisiae-3190 (NCIM) with 5l bioreactor (B-Lite Sartorious limited, Mumbai).

The maximum bioethanol yields obtained by the standard optimizations of fermentative conditions (Medium-I) were 98.14 g.l^-1 at 400 g.l^-1 of substrate concentration , 103.15 g.l^-1 at 30 ⁰C of temperature, 108.69g.l^-1 at pH 5, 110.63 g.l^-1 at 120 RPM of agitation 111.19 g.l^-1 at 8v/v of inoculum volume, 112.98g.l^-1 at 0.6 g.l^-1 of ammonium sulphate (NH₄)₂SO₄, 110.189 g.l^-1 at 0.5 g.l^-1 of copper chloride (CuCl₂), 116.64 g.l^-1 at 0.06 g.l^-1 of manganese chloride (MnCl₂.4H₂O), 118.0 g.l^-1 at 0.4 g.l^-1 of magnesium chloride (MgCl_2.6H₂O), 114.75 g.l^-1 at 50 mg.l^-1 of zinc sulphate (ZnSO₄.7H₂ O), 115.68 g.l^-1 at 24 mg.l^-1 of biotin, 114.36 g.l^-1 at 0.150 of g.l^-1 of proline, 119.342 g.l^-1 at 5.0 g.l^-1 sodium di hydrogen phosphate (NaH₂PO₄), 117.653 g.l^-1 at 5 g.l^-1 of ethylene di-amine tetraacetic acid (EDTA), 116.981 g.l^-1 2.0 g.l^-1 of potassium phosphate (K₂HPO₄), 115.947 g.l^-1 at 0.06 g.l^-1 of calcium chloride (CaCl₂), 118.635 g.l^-1 at 80 mg.l^-1 of cobalt chloride (CoCl₂), 102.721 g.l^-1 at 0.5 g.l^-1, oxygen (O₂) at 0.3 mg.l^-1of ferrous sulphate (Fe₂(SO₄)₃.H₂O), 93.641 g.l^-1 at 0.10 g.l^-1 of sodium chloride (NaCl₂), 100.634 g.l^-1 at 3.0 g.l^-1 of peptone, 114.735 g.l^-1 at 2.5 g.l^-1 of urea and 118.462 g.l^-1 at 1.5 g.l^-1 of yeast extract. Under these optimum concentrations of medium-I, bioethanol yield obtained was 150.562g.l^-1at the fermentation efficiency of 3.1367g.l^-1.h^-1 and the percentage of yield of bioethanol was 73.66 %. Elena Patrascu, et al., (2009) who have been reported that some selected yeast strains can produce bioethanol yields up to 15 % or higher. The viable yeast cells were found to be 98 % after 48 hours of fermentation time. The experimental results are shown in figure. no. 2.

Statistical optimizations of fermentative conditions (Medium-II) were conducted in order to enhance the bioethanol yields with S.cerevisiae-3190 (NCIM) response surface methodology (RSM) with software Statistica8. The Three factors at a time was applied using central composite design and eight central composite designs were developed based on the standard optimization of fermentative factors of medium-I. The maximum bioethanol concentrations were produced with statistical optimum fermentative conditions: the experiments on fermentation conditions optimum with substrate concentration of 409.916 g.l^-1, temperature of 31.43 ⁰C and pH of 4.9 of central composite design-I produced bioethanol yield was 113.570 g.l^-1. The surface plot bioethanol production was shown in figure no. 3. The optimum concentrations of central composite design-II produced bioethanol yield was 121.878 g.l^-1 with inoculum volume of 9.000 v/v, agitation of 117.28 RPM and inoculum age of 53.66 hours (figure.no. 4).

The statistical optimization of fermentation conditions with ammonium sulphate (NH₄)₂SO₄) of 0.629 mg.l^-1, copper chloride (CuCl₂) of 0.522 mg.l^-1 and manganese chloride (MnCl₂.4H₂O) of 0.061 mg.l^-1 produced bioethanol yield was 128.763 g.l^-1 (figure. no. 5). The optimum concentrations of central composite design-IV with magnesium chloride (MgCl_2.6H₂O) of 0.430 g.l^-1, zinc sulphate (ZnSO₄.7H₂ O) of 54.021 mg.l^-1, biotin of 22.453 mg.l^-1 produced bioethanol yield was 131.281 g.l^-1(figure. no. 6).

Figure no.1: Screening of microorganisms

Figure no.2: Bioethanol production with medium-I

Figure no.3: statistical optimizations of substrate concentration and temperature with pH was kept constant on Bioethanol production.

Figure no.4: statistical optimizations of inoculum volume and inoculum age with agitation was kept constant on Bioethanol production.

Figure no.5: statistical optimizations of copper chloride and ammonium sulphate with manganese was kept constant on Bioethanol production.

Figure no.6: Figure no.7: statistical optimizations of Zinc and magnesium with biotin was kept constant on Bioethanol production.

Figure no.8: statistical optimizations of EDTA and phosphorous with proline was kept constant on bioethanol production.

Figure no.9: statistical optimizations of oxygen and ferrous sulphate with cobalt was kept constant on bioethanol production.

Figure no.10: statistical optimizations of peptone and yeast extract with urea was kept constant on bioethanol production.

Table No: 11. Statistical optimum fermentation conditions of central composite designs on Bioethanol productions

Figure no.12: bioethanol production with medium-II

Conclusion

From the data of experiments, it can be concluded that the yeast strain Saccharomyces cerevisiae-3190 is resistant to acidic conditions, osmotic stresses by very high gravity medium and high ethanol tolerant. Mahua flower (Madhuca indica) was proved as a suitable substrate for bioethanol production. One million tonns of Mahua flower could produce 3, 48,303 Litres of bioethanol. When bioethanol used as blend in petrol, thus replace 3,48,303 Litres of petrol in India. To meet the required fuel quantity, bioethanol production through fermentation process in industrial scale is the only alternative method to avoid fuel crisis. The statistical optimizations of fermentation conditions for bioethanol productions with Mahua flower by response surface methodology could be feasible economic bioprocess.

References

1. American association of cereal chemists, (AACC), (1983), Method: 30-25, The association: St. Paul, Minnesota, United States of America, P1.
2. American public health association (APHA), (1967), “Compendium of methods for microbiological examination of food”, Lin. Speck. MI. (Ed). Washington (DC).
3. Association of official agricultural chemists, (AOAC), (2000), Official methods of analysis. 17th Ed. Gaithersburg, MD, Method: 930.15.
4. Bonara, A. and Mares, D. (1982). “A simple colorimetric method for detecting cell viability in culture of eukaryotic micro organisms”. Current Microbiology, Volume-7, 217- 222.
5. Box G.E.P and Wilson K.B., (1951). “On the experimental attainment of optimum conditions”. Journal of the Royal Statistical Society, 13:1-45.
6. British Pharmacopeia., (2007), “Monograph of ethanol: Analysis of volatile impurities”. p: 628.
7. Dake.M.S, Amarapurkar, S.V, Salunkhe, M.L and Kamble, S.R., (2010), “Production of alcohol by Saccharomyces sp. using natural carbohydrate sources”, Advanced Biotech. Volume-10 Issue: 06, p: 37-41.
8. Elena Patrascu, Gabriela Rapeanu, and Traian Hopulele., (2009), “Current approaches to efficient biotechnological production of ethanol”, Innovative Romanian Food Biotechnology, Volume-4, Issue of March, p.1-11.
9. Eshtiaghi, M.N, Yoswathana, N, Kuldiloke. J and Ebadi. A.G., (2012), “Preliminary study for bioconversion of water hyacinth (Eichhornia crassipes) to ethanol”. African Journal of Biotechnology, Volume-11(21), pp. 4921-4928.
10. Krishnaswamy. T, and Shenbaga Vinayaga Moorthi. N., (2012), “Performance evaluation of diesel engine with oxygenated bio-fuel blends”, Journal of Engineering and Applied Sciences, Volume-7, No: 1, p: 10-14.
11. Lowry, O.H, Rosinbrough, N.I, Farr, A.L, and Randall, R.J., (1951). “Protein measurement with the folin phenol reagent”, Journal of Biology and Chemistry, 193, 265-275.
12. Maiorella. B.L, Wilke. C.R, and Blanch. H.W., (1981). “Alcohol Production and Recovery”. Advanced Biochemical Engineering. 20: 43.
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14. Ratnam. B.V.V, Rao. MN, Rao. M.D, Rao. S. S, and Ayyanna. C., (2003), “Optimization of fermentation conditions for the production of ethanol from sago starch using Response Surface Methodology”, World Journal of Microbiology & Biotechnology, 19: 523–526.
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16. Ravindra. P, Anuj kumar chandel, Chan. E.S, Ravinder Rudravaram, Lakshmi Narasu. M and Venkateswar Rao. L., (2007). “Economics of environmental impact of bioethanol production technologies: an appraisal”. Biotechnology and Molecular Biology Review. Volume-2 (1), 014-032.
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Abstract

Sputum decontamination by modified Petroff’s method is routinely used by many laboratories for isolation of Mycobacteria in solid culture. With the introduction of line probe assay (LPA) for MDR TB diagnosis, our laboratory adopted the recommended NALC–NaOH decontamination procedure for sputum processing. Out of 284 and 1039 specimens processed by modified Petroff’s and NALC-NaOH, significantly high percentage (P=0.004) of contamination was observed by the latter method. In the initial period, the contamination reached 19.5% with the NALC-NaOH method and reduced with time. Laboratories adopting new decontamination methods may be careful during the initial months for avoiding the loss of specimen due to contamination while adopting LPA.

Key Words

Decontamination, LPA, Sputum

Introduction

Tuberculosis remains to be one of the leading causes of death in the world today and 80% active cases are found in 22 low and middle income countries [1]. In programmatic condition the diagnosis still relies on microscopical observations of smear, a method having low sensitivity with paucibacillary specimens [2]. Due to the emergence of multi drug resistant (MDR) and extensively drug resistant (XDR) Tuberculosis (TB), countries are scaling up with establishment of culture and drug susceptibility testing laboratories and also adopting newer molecular based techniques like line probe assay (LPA)  for its early detection. For processing of sputum specimen, mostly contaminated with oral bacteria and environmental fungi, modified Petroff’s method with 4% NaOH is being used by many laboratories attempting to isolate mycobacteria. The introduction of new technology like LPA also necessitated the shifting of decontamination procedure from modified Petroff’s to adoption of N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) method for decontamination. During the transitional phase of adoption of newer decontamination method, that may result in higher contamination rates for culture. Hence laboratories may have to develop a strategy to overcome the transition for getting a good yield of Mycobacterium tuberculosis in culture.

Materials and Methods

This study was conducted in RMRC, Bhubaneswar from January to September 2014. Sputum specimens were collected from suspected pulmonary TB patients those who were attending outpatient department of Capital Hospital, Bhubaneswar. Samples were transported to RMRC, Bhubaneswar after the Laboratory Technician of Capital hospital made slides for Acid Fast Bacilli (AFB) microscopy by Ziehl-Neelsen (ZN) staining.  Written informed consent was obtained from all patients who have participated in this study. The sputum specimens were processed at National Reference Laboratory of Regional Medical Research Centre, Bhubaneswar for microscopy and isolation of M. tuberculosis.  Duplicate smears were made and stained by both ZN and auramin staining methods and graded following International Union of Tuberculosis and Lung Diseases (IUTLD) guidelines.

Sputum specimens were processed by modified Petroff’s method up to May 2014 after which processed by NALC-NaOH method [3] due to introduction of Line Probe Assay for detection of MDR TB in the laboratory. In brief, for modified Petroff’s method, 3-5 ml of sputum was homogenized for 15 minutes in a shaker using an equal volume of 4% sodium hydroxide. After centrifugation at 3,000 rpm for 15 min, the deposit was neutralized with 45 ml of sterile distilled water.

The samples were again centrifuged and from the sediment, a loop full was inoculated in to two slants of Lowenstein-Jensen (LJ) and to one slant of LJ containing para-nitro benzoic acid (PNB). For NALC-NaOH method, 3-5 ml of sputum sample, an equal volume of NALC-NaOH citrate reagent was added and the tube was vortexed briefly. Following 15 min of incubation at room temperature, the volume was made to 50 ml with 0.067 M phosphate buffer (pH 6.8) and the contents were mixed by inversion. Bacteria were sedimented by centrifugation at 3,000 x for 15 min. The supernatant was discarded after centrifugation at 3,000 x g for 15 min, and the pellet was resuspended in 1 ml of phosphate buffer. From the pellet a loop full was inoculated like the modified Petroff’s method. The culture slants were incubated at 37°C. All slopes slants were observed for occurrence of growth daily for first week and then at weekly intervals for 8 weeks. The isolates were identified by following tests: rate of growth, optimum temperature of growth, colony morphology, pigmentation, growth in PNB, catalase test and niacin test which confirmed that all isolates were M.tuberculosis. The Ethical Committee of RMRC, Bhubaneswar approved the study protocol.

Results

Out of 40 and 179 positive smear specimens processed by modified Petroff’s and NALC-NaOH method, isolation of M. tuberculosis was successful in 82.5 and 86.0 percent specimens respectively (Table 1). Out of 244 and 860 negative smear specimens, 7.0 and 8.4 percent specimens were found positive respectively by modified Petroff’s and NALC-NaOH method (Table 1). The rate of contamination between the two methods was similar in positive smear specimens. While a significant proportion of negative smear specimens were found contaminated with NALC-NaOH method (P=0.001).

The overall contamination rate observed was also significantly higher (P=0.004) in NALC-NaOH method (8.3% vs 3.5%). The month wise contamination rate was between 0 and 8.5 percent with modified Petroff’s method, while contamination increased up to 19.5% in the initial month with NALC-NaOH method (Table 2).

Table 1: Isolation of M. tuberculosis from sputum samples following different methods of decontamination

Table 2: Sputum processing month wise for culture on LJ

ND: Not done LJ: Lowenstein-Jensen

Acknowledgement

We thank the State TB Officer, Odisha and District Tuberculosis Officer, Bhubaneswar who permitted the collection of sputum samples for this study. The technical help of Mr Sisir Kumar Barik, TA, Ms Lucy Parija, M Sc dissertation student and Laboratory Technicians of NRL, Bhubaneswar has been acknowledged. This work was supported by the Indian Council of Medical Research under an extramural research grant which is acknowledged.

References

1. Lawn SD, Zumla AI. Tuberculosis. Lancet, 2001; 378:57-72. doi: 10.1016/S0140-6736(10)62173-3
2. Allen BW, Mitchison DA. Counts of viable tubercle bacilli in sputum related to smear and culture grading. Med Lab Sci, 1992; 49:94-98.
3. Kent PT, Kubica GPW. Public Health Mycobacteriology. A guide for the level III Centres for Disease Control, Atlanta; 1985.
4. Buijtels PC, Petit PL. Comparison of NaOH-N-acetyl cysteine and sulfuric acid decontamination methods for recovery of mycobacteria from clinical specimens. J Microbiol Methods, 2005; 62:83-88. doi:10.1016/j.mimet.2005.01.010
5. Grandjean L, Martin L, Gilman RH et al. Tuberculosis diagnosis and multidrug resistance testing by direct sputum culture in selective broth without decontamination or centrifugation. J Clin Microbiol 2001; 46:2339-44.doi: 10.1128/JCM.02476-07
6. Chatterjee M, Bhattacharya S, Karak K, Dastidar SG. Effects of different methods of decontamination for successful cultivation ofMycobacterium tuberculosis. Ind J Med Res, 2013; 138:541-48.

Cope of Interest 

None of There

How to cite this article

Dasarathi Das*,Prakasini Satapathy, Biswanath Murmu. “Shifting from modified Petroff’s to NALC-NaOH method for processing of sputum specimens for solid culture.” Microbioz Journals, Journal of Microbiology and Biomedical Research 2.2 (2016).

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Acceptance Date    :  June,28,2016

Publication Date    :  Aug,20,2016

Mode of Access      : Open access

Authors                 :¹Abimiku, R.H.,^*1Ngwai, Y.B., ¹Nkene, I.H., ²Tatfeng, Y.M.

Affiliation               :  ¹Microbiology Unit, Department of Biological Sciences, Nasarawa State University, P.M.B.1022, Keffi,          Nigeria                    ;  ²Molecular Biology Unit, Department of Medical Laboratory Sciences, Niger Delta    University, P.M.B.                                              071, Wilberforce Island,Bayelsa State, Nigeria

Country                : Nigeria

Contact                 : rejoiceafrica40@gmail.com

Abstract

Escherichia coli (E. coli) is among the pathogens that cause diarrhea both in developed and developing countries. Molecular detection of pathotypes of E. coli causing diarrhea in Keffi, Nigeria was carried out. Fifty (50) stool samples were collected from patients attending two health facilities in Keffi, who are suspected to have diarrhea. E. coli was isolated from the stool samples using standard microbiological methods. Molecular detection of genes encoding virulence factors of the different pathotypes of diarrhegenic E. coli(DEC) namely: enteroaggregative E. coli(EAEC), enteroinvasive E.coli (EIEC), enterohemorrhagic E.coli(EHEC), enteropathogenicE. coli (EPEC), and enterotoxigenic E.coli (ETEC) was carried out using multiplex Polymerase Chain Reaction (mPCR) method. All the samples collected harbored E. coli. The order of detection frequency of DEC was: EAEC (22.0%) > EIEC and ETEC (2.0%) > EHEC and EPEC (0.0%).

The order of detection frequency of EAEC in relation to age (in years) was: 6-10 (50.0%) > 11-15 (40.0%) > 16-20 (20.0%) > 0-5 (17.2%). The frequency of detection of ETEC was 3.4% in age group 0-5 yr and EIEC was 20.0% in age group 11-15 yr. The order of detection frequency of EAEC was high in female (36.4%) than male (10.7%) respectively. The EIEC was detected only in male (2.0%) and ETEC was detected only in female (2.0%). The frequency of detection of EAEC in relation to age and gender of diarrheic patients was statistically insignificant (P > 0.05). Enteroaggregative E.coli was the most commonly detected DEC pathotypes; and EHEC and EPEC were not detected. A further study on the molecular detection of pathotypes of DEC in other parts of Nasarawa State, Nigeria is on-going

Key Words

Diarrheic patients, Escherichia coli, Keffi, Pathotypes.

Introduction

Escherichia coli (E. coli) are the predominant facultative anaerobe and commensal microbiota in the mammalian gastrointestinal gut, yet some strains can cause severe diarrhea illnesses in humans [1, 2]. Gastrointestinal colonization occurs within hours of life and the bacteria will remain in a symbiotic relationship with the host [3, 4]. It remains harmlessly, confined to the intestinal lumen; but in the debilitated or immunosuppressed hosts, or when gastrointestinal barriers are violated, even normal ‘‘non-pathogenic” strains of E.coli can cause infection [5]

Diarrheagenic Escherichia coli (DEC) is important intestinal pathogens causing a wide variety of gastrointestinal diseases, particularly among children in developing countries [6].Six pathotypes of DEC strains are frequently detected worldwide; and described based on the genes mediating the virulence factors associated with the diarrheal diseases caused by them: EnteropathogenicE.coli (EPEC), Enterotoxigenic E.coli (ETEC), Shiga toxin-producing E.coli (STEC) or Enterohaemorrhagic E. coli (EHEC), EnteroaggregativeE. coli (EAEC), Enteroinvasive E.coli (EIEC) and diffusely adherent E. coli (DAEC) [7, 6].Person-to-person transmission and consumption of foods and drinking water contaminated with human waste are the main modes of acquiring DEC infections [6].

The detection of DEC strains is difficult since these strains cannot be easily distinguished from the normal faecal flora using conventional phenotypic methods. Molecular identification and classification of DEC is established by the presence or absence of one or more specific virulence genes, which are absent in the commensal E. coli [1].

In Nigeria, there are reports on the isolation of DEC pathotypes in different parts of the country [8, 9, 7, 4]. There is, however, no study from Keffi on the DEC pathotypes causing disease amongst residents. This study is the first report in that direction.

Materials and Methods

Study Area and Sample Collection

This study was carried out in Keffi metropolis. A total of fifty (50) stool samples were collected from patients with cases of diarrhea attending General Hospital, Keffi and Federal Medical Centre, Keffi using sterile containers and transported to Federal Medical Centre Laboratory, Keffi, for analyses. Ethical clearance was obtained from the Ethical Committees of the hospitals. The consent of each patient was obtained before sample collection.

Isolation and Identification of Escherichia coli

Escherichia coli were isolated and identified from the stool of diarrheic patients using standard cultural, microscopical and biochemical methods [10]. Loopful of the stool was streaked on Mac Conkey agar (MCA: Biotec Laboratories Ltd, Ipswich, United Kingdom) and incubated at 37^oC for 24 h. Pink colonies from MCA were further streaked on Eosine Methylene Blue Agar (EMB: Biotec Laboratories Ltd, Ipswich, United Kingdom) and incubated at 37^oC for another 24 h. Colonies with greenish metallic sheen characteristics on EMB were identified and confirmed by microscopy (Gram staining) and some minimal biochemical tests for E. coli identification namely:indole, methyl red, Voges-Proskauer and citrate (IMViC). The isolates were stored in the refrigerator at 4°C on Nutrient agar (NA: Merck KGaA, Darmstadt, Germany) slants and reactivated by sub-culturing on MCA and used in experiments.

Multiplex Polymerase Chain Reaction (mPCR) for Diarrheagenic Escherichia coli

The DNA of the E. coli isolates was extracted using a method earlier described by [11]. Briefly, 3 pure colonies of E. coli isolates from MCA plate was inoculated into 5 ml of sterile Luria-Bertani Broth (InqabaBiotec, South Africa) in a bijou bottle and incubated at 37^oC for 7 h. The 7-hLuria-Bertani Broth culture was transferred into a sterile Eppendorf tube and boiled at 100^oC for 20 mins and the culture was centrifuged at 10,000rpm for 10 mins to pellet the cell debris. The supernatant was used for mPCR.

The mPCR assay of DNA extracted from E. coli isolates was carried out following a method described by [12].The DNA templates were subjected to multiplex PCR with specific primers for the detection of the following virulence markers: eaeA(structural gene for intimin of EHEC and EPEC),bfpA(structural gene for the bundle-forming pilus of EPEC), vt1 and/or vt2 (Shiga toxins 1 and 2 of EHEC), eltBand/or estA(enterotoxins of ETEC), ial(invasion-associated locus of the invasion plasmid found in EIEC and Shigella) and pCVD (the nucleotide sequence of the EcoRI-PstI DNA fragment of pCVD432 of EAEC).

The mPCRs were performed with a 25 μl reaction mixture containing 5 μl of template DNA, 0.2 μl of 18x PCR buffer II, 1.6 μl of a 1.25 mM mixture of deoxynucleoside triphosphates, 1.6 μl of 25 mM MgCl₂, 0.1 μl of 5 U of AmpliTaq Gold DNA polymerase (Inqababiotec, South Africa) per μl and a 0.2 μM concentration of each primer except primer VT1 (all primers were from InqabaBiotec^tm, South Africa), which was used at a concentration of 0.4 μM. The thermocycling conditions with a Gene Amp PCR System 9700 (AB Applied Biosystem) were as follows: 95°C for 5 min, 94°C for 20 sec., 55°C for 30 sec., and 72°C for 30 sec. for 30 cycles, with a final 7min extension at 72°C as described by [11].

The agarose gel electrophoresis for detection of amplified genes for different DEC pathotypes in the E. coli isolates was carried out following a method described by [11]. Briefly, 10μl of PCR products stained with ethidium bromide was loaded into 1.0% (wt/vol) agarose gel wells with a molecular marker (1-kb DNA ladder) run concurrently at 120 mV for 30 min. The DNA bands were visualized and photographed under UV light at 595nm using an ultraviolet trans-illuminator.

The data obtained from this study were analyzed using chi-square test by the use of Smith Statistical Package (SSP) version 2.8 and the significance was determined at 5 % probability (that is P= 0.05).

Table 1: Primers and aplicon size for different pathotypes of Escherichia coli 

LT= Enterotoxigenic E.coli(ETEC); ST= Enterotoxigenic E. coli (ETEC); VT= Enter hemorrhagic E.coli (EHEC); Eae= Enter hemorrhagic E.coli(EHEC); SHIG= Enteroinvasive E. coli (EIEC); BfpA= EnteropathogenicE.coli(EPEC); EA= EnteroaggregativeE. coli (EAEC).

The primers were products of InqabaBiotec, South Africa as shown in Table 1.

The reference diarrhegenic and non-diarrhegenic. E.coli strains and their target genes are shown in Table 2.

Table 2 Reference diarrhegenic and non-diarrheagenic Escherichia coli strains for multiplex Polymerase Chain reaction

ETEC= Enterotoxigenic E.coli; EHEC= Enterohemorrhagic E. coli; EPEC= Enteropathogenic E.coli; EIEC= EnteroinvasiveE. coli; and AEC= Enteroaggregative E.coli.

Plate 1: Multiplex PCR amplification of reference strains of diarrheagenic Escherichia coli from pure cultures (Lane 1, E. coli ATCC 11775; lane 2, EAEC 97R; lane 3, EIEC ATCC 43893; lane 4, EPEC ATCC 43887; lane 5, EHEC ATCC 43889; lane 6, EHEC ATCC 43890; lane 7, ETEC ATCC 35401; lane M, marker (1-kb DNA ladder; Gibco/BRL). Numbers on the right are in base pairs).

Results

Isolation rates of diarrhegenic pathotypes of Escherichia coli

Pink colonies from MCA that grew with green metallic sheen on EMB and which were Gram-negative rod, indole- positive, methyl red- positive, Voges-Proskauer- negative and citrate- negative were taken as E. coli. All the stool samples contained E. coli.

The isolation rates of the DECs from the stool of the diarrheic patients are as given in Table 3. Of the 50 E. coli isolated, 11 (22.0%) was EAEC; 1 (2.0%) was ETEC; 1 (2.0 %) EIEC; and EHEC and EPEC were not detected.

Table 3: Isolation rates of different pathotypes of diarrhegenic Escherichia coli

ETEC= Enterotoxigenic E.coli; EHEC= Enterohemorrhagic E. coli; EPEC= Enteropathogenic E. coli; EIEC= Enteroinvasive E. coli; and  EAEC= Enteroaggregative E. coli.P= 0.0535; χ²= 5.8500

Fig. 1 Isolation rate of different pathotypes of diarrhegenic Escherichia coli in relation to age.

ETEC= Enterotoxigenic E.coli; EHEC= Enterohemorrhagic E. coli; EPEC= EnteropathogenicE. coli; EIEC= EnteroinvasiveE. coli; and EAEC= EnteroaggregativeE. coli.P= 0.9209; χ2= 0.4908.

The order of isolation frequency of EAEC in relation to age group was 5-10 (50.0%) > 10-15 (40.0%) > 15-20 (20.0%) and 0-5 (17.2%) respectively as given in Table 3. The frequency of isolation of EIEC was 20.0% in age group 10-15 and 0.0% was observed in other age groups as given in Table 3. For ETEC, the frequency of isolation was 2.9 % in age group 0-5 and was not observed in other age groups as shown in Table 3. However, the frequency of isolation of EAEC in relation to gender was high in female (36.4%) than male (10.7%) respectively as shown in Fig 1.

Table 4: Isolation rate of different pathotypes of coli in relation to gender

P= 0.2495; χ2=1.3258

ETEC= Enterotoxigenic E.coli; EHEC= Enterohemorrhagic E. coli;EPEC= EnteropathogenicE.coli; EIEC= EnteroinvasiveE. coli; and EAEC= Enteroaggregative E.coli.

The isolation frequency of ETEC was 2.2% in female and none was observed in male. The frequency of isolation for EIEC was 2.8% in male and 0.0% in female respectively as shown in Table 4.

Plate 2: DNA Ladder for Escherichia coli isolates from stools of diarrheic patients in Keffi, Nigeria (L= Lane, L1= negative; amplification; L2-L4, L5-L9 and L12-L13= pCVD (EAEC); L11= ial (EIEC); L10= eltB ETEC; M= 100bp

Discussion

Diarrheagenic Escherichia coli (DEC) are important intestinal pathogens causing a wide variety of gastrointestinal diseases, particularly among children in developing countries [6]. Six pathogenic diarrheagenic Escherichia coli (DEC strains) are frequently detected worldwide [11].

These pathotypes have been described based on the genes mediating the virulence factors associated with the diarrheal diseases caused by them: EnteropathogenicE. coli (EPEC), EnterotoxigenicE. coli (ETEC), Shiga toxin-producing E. coli (STEC) or Enterohemorrhagic E.coli (EHEC), Enteroaggregative E.coli (EAEC) and Enteroinvasive E.coli (EIEC) [7, 6].

The isolation of E. coli in all (100%) the stool samples is in agreement with studies reported by [7, 13, 8]; and it confirms the fact that E. coli is a common bacteria isolated in stool of human [14]. This high detection E. coli may be due to improper personal hygiene.

The high frequency of detection of EAEC observed in this study was not surprising and in agreement with another study reported elsewhere [11].EAEC was previously reported to be endemic in southern Nigeria as well as in sub-Saharan Africa [8]. So, our observation on the occurrence of EAEC in Keffi concurs with what was reported in South-western Nigeria and elsewhere especially in the sub-Saharan Africa [15].The frequency of detection of EAEC in this study is higher than that reported by [4]; but the detection of ETEC and EIEC was low as study reported by[4] and [11]. The very low frequency of detection of diarrheagenic E.coli obtained in this study is in close agreement with the study conducted and reported by [16] who reported a prevalence of E. coli O157: H7 in children with diarrhoea as 5.4% in Zaria, Nigeria. Also, [17] and [18], reported a prevalence of 5% EHEC O157:H7 in humans, in Lagos, Nigeria. But it is in contrast with the study conducted by [9], who reported 19.6% prevalence of diarrheagenic E.coli in a study conducted in South Eastern Nigeria.

An incidence higher than 40% has been reported in Bangladesh by [19]. Also, we observed that EHEC and EPEC were not detected from this study although other studies in other part of the country reported low frequency of detection of EHEC and EPEC [4]. This is also in total disagreement with studies carried out in South-East Nigeria, which reported that EPEC was the most isolated of all DEC pathotypes followed by EAEC, ETEC, EIEC and EHEC in that order [9]. Outbreaks and sporadic cases of EHEC have been reported in developed countries of North America, Japan, Europe and even Australia [20].

However, there have been few reports of sporadic EHEC in African countries. Three large EHEC outbreaks were previously reported in Swaziland, Central African Republic and the Came room [20, 21]; but some authors criticized the methodology used in those studies as being nonspecific or insensitive [8]. Despite this, our findings tend to align with the earlier observation that EPEC and EHEC may be rare after all [9].The subjects employed in this study may be infected by other pathogens other than diarrheagenic E.coli since there are different pathogens that can cause diarrhoea in children and adults, including Rotavirus, Salmonella spp., Shigella spp., Campylobacterjejuni, Entamoeba histolytica, and Giardia lamblia[9].

The detection frequency of EAEC in respect to age was high in age groups of 6-10 and 11-15 then 16-20 year. This observation is different from a study reported in Hanoi, Vietnam by [11]. Although the frequency of detection of EAEC gene of E. coli isolates in respect to age of patients was statistically insignificant. The EAEC genes were not detected in age > 21 years and this however disagrees with other studies reported by [19, 4, 9]. Though, we observed that age may not be a factor for prevalence of EAEC. From this study we also observed that EIEC and ETEC were detected in only age groups of 11-15 and 0-5, respectively. This seems to disagree with a study conducted by [9]. Though, EIEC is also reported by researcher across all ages [8, 20, 21].This study also revealed that, the order of frequency of detection of EAEC was high in female than male. The EIEC was detected only in male and ETEC was detected only in female. The frequency of detection of EAEC in relation to age and gender of diarrheic patients was statistically insignificant. This is also in agreement with studies carried out in South-East Nigeria [8, 9]. This may be an advantage in Keffi, because the isolation rate of different pathotypes of diarrheagenic E.coli have been reported to be vary in different geographical areas [8].

Conclusion 

Enteroaggregative E.coli (EAEC), Enteroinvasive E.coli (EIEC), and Enterotoxigenic E.coli (ETEC) were the only pathotypes detected; and EAEC frequency was highest at 22%. Age and gender may not necessarily be a factor for prevalence of EAEC gene from E. coli causing diarrhea.

Acknowledgement

We are grateful to staff of Microbiology Laboratory at Federal Medical Centre, Keffi and General Hospital, Keffi for the use of their facility for sample collection and technical support during the study; and to the Department of Medical Laboratory Sciences, Niger Delta University, Bayelsa State for the multiplex PCR analysis.

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How to cite this article

Abimiku, R.H.,*Ngwai, Y.B., Nkene, I.H., Tatfeng, Y. M. (2016). Molecular detection of diarrheagenic pathotypes of Escherichia coli from diarrheic patients in Keffi, Nigeria. Microbioz Journals,Journal of Microbiology and Biomedical Research, 2(3), 1-to.

The post Molecular detection of diarrheagenic pathotypes of Escherichia coli from diarrheic patients in Keffi, Nigeria appeared first on Microbioz Journals.

Chlamydophilial infections leading to outbreaks of avian chlamydophilosis in domestic as well as companion birds have been reported worldwide, which reflects in economic loss to the owner and potential zoonotic transmission to humans. Study on the occurrence and prevalence ofChlamydophila psittaci in birds at live bird markets in Kaduna metropolis, Nigeria was carried out using Immuno Comb enzyme linked immunosorbent assay (IC ELISA) and modified Gimenez staining technique (mGS). Sera from 64 local chickens, 43 ducks, 64 pigeons, 64 broilers, 64 exotic layers, 48 turkeys and 64 guinea fowls were examined. Using the IC ELISA, 41.46% (85) and 34.95% (72) from thebirds sampled from Central and Waff Road markets tested positive for C. psittaci, with an overall seroprevalence of 38.00%. There was no significant difference (P>0.05) in the prevalence of the infection from birdssampled in Central and Waff Road markets. Among various types of birds examined, the highest seroprevalence was recorded in pigeons 67.19% (43), duck 51.16% (22) and guinea fowl 46.88% (30) while the least seroprevalencewere recorded in exotic layers 18.75% (12) and broilers 15.63% (10). Nevertheless, there was significant difference (P<0.05) in prevalence of the infection among the various types of birds examined. The prevalence of the infection by mGS showed 4.10% (9) and 3.40% (7) from Central and Waff Road markets, with the highest prevalence in local chickens 6.25% (4) and 0.0% (0) in turkey. Statistical analysis by Kappa statistics indicate a slight agreement between the two tests. The results of this study confirmed our suspicion about the presence of C. psittaci in birds at live bird markets in Kaduna metropolis. Thus, routine screening of birds in abattoirs, quarantine stations, farms and enlightenment of the general public on avian chlamydophilosis control should be advocated.

Key Words

Birds, ELISA, mGS, Chlamydophila psittaci, zoonotic.

Introduction

Chlamydophila psittaci is an obligate, intra­cellular zoonotic bacterium, found in a wide range of birds, including companion, domestic and wild birds worldwide (Harkinezhad et al., 2009). It is the causative agent of psittacosis, also known as orni­thosis, parrot fever, or avian chlamydophilosis (AC) (Van Droogenbroeck et al., 2009).Several serovars of C. psittaci have been identified, using monoclonal anti­bodies, these correspond to 7 genotypes, seven of which (A-F, and E/B) are endemic to domestic poultry and other birds and can be transmitted to humans causing psittacosis or parrot fever (Geens et al., 2005).Earliest descriptions of epidemics of this disease in late nineteenth century Europe, incriminated imported Argentinean parrots as the vector of this malady.However,parrots may have been unduly maligned, as most cases of Avian chlamydophilosis (AC) may well be unconnected with parrots (Meyer, 1965), but also associated with wild birds and poultry species such as pigeons, ducks, geese, turkeys as well as domestic fowls (Vanrompayet al., 2008).

The importance of poultry as a source of human infection became more evident following outbreaks in humans in the 1950s, due to contact with infected turkeys (Meyer, 1965).This refutes the early belief that C. psittaci infections only occur in psittacine birds.Among poultry; turkeys, ducks and pigeons are most often affected while infection of chickens are infrequent (Rawdonet al., 2010). In pigeons, infection is very wide spread especially as a carrier state without clinical symptoms (Pavlack et al., 2000).

Avian chlamydophilosis is an occupational hazard with commercial pet birds’ handlers, hobbyists, veterinarians as well as poultry workers exposed to the greatest risk (Fengaet al., 2007). Outbreaks commonly associated with ducks and turkeys farms and abattoirs have been described (Bennedsen and Filskov, 2000). Transmission to humans is common during handling and slaughter of birds (Dickx et al., 2010). Recent studies have indicated that the disease has been on the increase in several parts of the world (Yang et al., 2007).

Avian chlamydophilosis can be economically devastating to producers/farmers and a serious public health problem. It has the potential to cause severe human disease or even death and produces avian respiratory infections which pose a serious threat to industrial poultry production, causing significant economic losses in the US, Europe and China (Laroucau et al., 2009).

Poultry production is a major economic activity in Kaduna State and Nigeria as a country. It contributes significantly to the family income especially in peri-urban and poor rural communities (Sonaiya et al., 2000). It provides the affordable source of protein needed to meet the recommended 0.8g/kg daily protein intake in the country (Shane and Neil, 2006).

There is also intense co-habitation of birds with human both in the cities and in rural areas,where many Nigerians live in close contact with poultry and poultry products on farms, live bird markets, poultry slaughter houses and processing plants, homes, shops, restaurants and during transportation of poultry to markets and other locations with little or no regard for adequate biosecurity (Abdu, 2010). With paucity of information in the veterinary as well as human health services on the epidemiology and public health significance of this disease in Nigeria, there is a need to carry out such a study.The aim of this study is to investigate the presence of C. psittaci in poultry at live bird markets in Kaduna metropolis using serology and histochemical staining techniques.

Materials and Methods

Study Area

The research was conducted in Kaduna State, Nigeria, which is located within the semi-arid sub humid regions of North Central Zone of Nigeria. It lays between longitudes E006.5^o – E008.6^o (East of Greenwich Meridian) and latitude N09.2^o – N11.3^o (North of the equator) (FRN, 2009).

Sources of Samples

Two live birds markets (Central and Waff Road markets) were randomly selected, and a total of 411 blood samples were collected from birds at slaughter. This was comprised of 205 birds [32 broilers, 32 exotic layers, 32 local chickens, 32 pigeons 32 guinea fowls, 25 turkeys, and 20 ducks] from central market and 206 (32 broilers, 32 exotic layers, 32 local chickens, 32 pigeons 32 guinea fowls 23, turkeys and 23 ducks) from Waff-Road. The sampling was done over a period of 8 weeks (February to May, 2015), with one visit to each market per week. Sampling was conducted by consent based on pre-sensitization conducted in each market prior to sampling. For the histochemical staining, tissue samples consisting of liver and spleens were aseptically harvested from the same birds sampled above identified by tagging with multiple different colour threads at slaughter.

Blood Sample Collection

For the serological analysis, apparently healthy birds were selected at slaughter and about 2ml of blood were collected and transferred into sterile sample tubes. It was then allowed to stand at room temperature before transporting to the laboratory. In the laboratory, sera were separated by centrifuging the whole blood at 1000g for 10 minutes (WOAH, 2008), then the resultant sera were dispensed into well labeled sterile serum bottles and stored at -20℃ (NVSL, 2006).

Tissue Sample Collection

All samples were collected under aseptic conditions to prevent contamination or cross-contamination. Sampling materials (i.e. forceps, polythene bags and scissors) were made sterile by wrapping in aluminium foil and sterilized in hot air oven (160 ℃) for 1 hour before embarking to sampling site. The harvested tissues were transferred into well labeled sterile polythene bags, and transported immediately together with the blood samples to the Bacterial Zoonoses Laboratory of the Department of Veterinary Public Health Preventive Medicine, Ahmadu Bello University Zaria in ice packed boxes for analysis.

Detection of C. psittaci Antibodies Using Immuno Comb ELISA Test Kit

The ELISA test Kit for Avian C. psittaci(ImmunoComb^® (IC), Biogal, Kibbutz Galed, Israel) is based on a rapid competitive enzyme – linked immunosorbent assay technique that detects immunoglobulin G – antibodies (1gG) against C. psittaciin avian species. Serum samples collected during this study were screened for the presence of antibodies to C. psittaci. C. psittaci IgG level was determined by comparing each specimen’s colour to the positive control intensity. A test that yielded colour reaction exceeding that of the negative control was considered positive while that comparable to the negative control were considered negative.

Detection of C. psittaci Inclusion Bodies in Impression Smears of Liver and Spleen by Modified Gimenez (PVK) Stain

The stain kit comprised 10ml ofsolution 1: (450.0 ml distilled H₂O and 5.0 ml phenol added to 2.5g basic fuchsin and 50ml of 95% ethanol, incubated at 37°C for 48 hours, filtered and stored in dark at room temperature), 12.5ml of solution 2: (11.65g Sodium phosphate dibasic; 2.47g ; Sodium phosphate monobasic made to 1.0 litre distilled H₂O),the staining solution; solution 3: (20.0 ml solution 1 and 25.0 ml of solution 2) let to stand for 10 minutes filtered and used, solution 4;(0.2 g fast green; 100.0 ml distilled water and 0.2 ml glacial acetic acid), and the counterstain; solution 4 (20.0 ml of solution 4 and 50.0 ml distilled water) (OIE, 2012).

Touch impression smear of cut surface of liver and spleen were made on a sterile glass slides and fixed in methanol for 4 minutes. The fixed slides were covered with the staining solution (solution3) then washed after 10 minutes at room temperature. It was counterstained with solution 4, for approximately 2 minutes and then rinsed in tap water, air dried and examined under light microscope at 40X magnification (Andersen and Vanrompay, 2003).

Data Analysis

Data collected at the end of this study were reduced to contingency tables and Statistical Package for Social Science (SPSS), Version 16.0 (SPSS Inc., Chicago, IL, USA) was used to determine Chi-square test.

Results

Seroprevalence of C. psittaci Specific Antibodies

Out of the 205 birds sampled from Central market, 85 (41.46%) were tested positive for C. psittaci while 72 (34.95%) out of the 206 birds sampled from Waff-road market were positive for C. psittaci, with an overall seroprevalence of 38% (Table 1). Although high rate of the infection was recorded in Central market, there was no statistically significant difference (P=0.174) in occurrence of infection between the two locations (Table 1).

During the study, 7 different types of birds were sampled. Sera from 64 local chickens, 64 exotic layers, 64 broilers, 48 turkeys 64 guinea fowls 64 pigeons and 43 ducks were examined. Our investigation detected Cp psittaci specific antibodies in; 10 (15.63%) broilers, 43 (67.19%) pigeons, 22 (51.16%) ducks, 30 (46.88%) guinea fowls, 12 (18.75%) exotic layers, 23 (35.94%) local chickens and 17 (35.42%) turkeys respectively. The highest seroprevalence was recorded in pigeon; 67.9%, followed by guinea fowl with seroprevalence of 46.88%, with the least in broiler having 15.63% seroprevalence (table 2). Statistically, there was a significant difference (P=0.0001) in rate of occurrence of infection among various types of birds examined (Table 2).

Prevalence of C.psittaci Inclusions in Birds by Modified Gimenez (PVK) Stain.

During the study period, liver and spleen were harvested from the same birds sampled for serology at slaughter. C. psittaci inclusion bodies were detected in 16 (3.90%) of the 411 birds examined. Inclusion bodies were detected in 9 (4.40%) of 205 birds examined from central market, and 7 (3.40%) in Waff road, with overall prevalence of 3.90% (Table 3).There was no statistical significance in the prevalence rate of the infection among types of birds examined in the two markets (Table 3).

Among the various types of birds examined, mGS detected C. psittaci inclusion bodies in; 1 (1.56%) broiler, 1 (2.33%) duck, 4 (6.25%) pigeons, 3 (4.69%) guinea fowls, 4 (6.25%) local chickens, 3 (4.69%) exotic layers and 0 (0.0%) in turkey. Statistical analysis indicated no statistical significance in occurrence of the infection between the two markets and among types of birds examined (Table 3 and 4).

IC ELISA test recorded seroprevalence of 38.20% while prevalence rate detected by mGS was 4.90%. Statistical analysis by Kappa statistics indicated a slight agreement between the two tests. The slight agreement indicated that the two tests may detect the infection at a varying rate, though it is pertinent to mention that the two tests are testing for different pathophysiologic parameters (antibody and inclusion bodies) of the infection which are not always accompanied by one another.

Discussion

In this investigation, the overall seroprevalence of C. psittaci infection in birds examined by IC ELISA test in Central and Waff road markets in Kaduna metropolis was 38.20%. It is lower than the 45.34% reported by Bendheim et al. (1993) in Israel, and higher than that obtained by Maluping and colleagues (2007), in the Philippines where seroprevalence of 25% was obtained from a sample population of 36 birds using the same test methods.

Using a different test method; RIDASCREEN ELISA^® test in China, Yang et al. (2007) reported a significantly high seroprevalence of 77.8% from a sample population of 150 broilers, 210 ducks and 165 laying hens. In Bosnia and Hezergovinia, Residbegovic et al. (2006) reported a seroprevalence of 27.6% from sample population of 58 intensive breeding chicken, free-living pigeons and parrots using indirect immunoflourescent test. Thus, the seroprevalence of the infection appear to vary from one country to another. These variations in seroprevalence of positive findings, our study inclusive, could be attributed to difference in test assays, sampling protocols and variation in susceptibility of different species of birds to C. psittaci infection, as well as ecological and geographical diversity. The high variability in seroprevalence of the disease, makes accurate estimation of the transmission of the pathogen entirely impossible (Schnebell, 2004).

In our investigation by mGS, C. psittaci inclusions were not detected in every bird examined especially in turkey where inclusions were not detected in either liver or spleen of the birds examined. This could be interpreted as; either the infection in these birds is in its localized phase and has not yet gone systemic, or the organs were not infected with the organism. It could also be that the organisms were not present in sufficient numbers to be captured by the stain or the smears were thick.

Among the various type of birds examined, it is obviously evident that the highest percentage of seropositivity (46%) was found in pigeons. This result corroborates with those obtained by various workers across the globe. Dovč (1993) in Slovenia reported 66.7% serological positive free-living pigeons and 59.2% in feral pigeons using IIF test. Also using the same test Residbegovic et al. (2006) reported the presence of specific IgG antibodies against C. psittaci in 42.1% of 38 pigeons sera tested. Pavlack et al. (2000), using CF test found 49.2% serologically positive pigeon from city area in Bosnia and Herzegovina.

In most of these surveys, complement fixation test was used, while indirect immunofluorescence and enzyme linked immunosorbent assay tests were employed less frequently, but led to detection of higher percentages of seropositivity (Magnnino et al., 2009).

It is pertinent to mention here that pigeons are extremely adaptable, which enables them to accept breeding places that are unnatural to them e.g. trees or over running ventilation systems. They are gregarious in nature, gathering in swarms in streets, squares, parks, worship places, markets, milling points etc., and do not commute on risky flights for natural food supply but rather search their immediate environment.

The characteristic behavior of these birds aforementioned, facilitate zoonotic transmission of C. psittaci infection to humans.

In Nigeria, a lot of people live in close proximity with these birds both in rural and urban communities. It is a common finding especially in the northern part of the country where these birds are commonly kept, to see houses, roof tops littered with excreta from these birds especially in the rural communities. Thus, the finding of higher seroprevalence of C. psittaci antibody in pigeon in the study area is of a significant public health concern judging from the close interaction between pigeons and humans, since zoonotic transmission of the infection especially via aerososlized infected bird’s droppings could easily occur.

Based on different locations studied, our investigation showed that central market recorded higher occurrence (41.46%) rate of the infection than Waff road market (34.95%). This could be attributed to the fact that the later; a private owned facility offers a better sanitary condition compare to the former, where stocking capacity of the plant is not strictly observed and hygienic condition compromised. It was also observed that cages that housed birds in the former are overcrowded and closely situated, thus facilitating easy spread of the infection. In this market domestic birds as well as wild birds are housed together in dirty cages littered with bird’s faeces obviously due to space constrains. This environmental stressor could induce increase faecal excretion of the bacterium from infected birds there by contributing to high occurrence of the organism. The detection of C. psittaciin these processing plants especially in central market is of public health significance since employees of these markets most especially evisceration workers and the general public could be at a high risk of contacting the infection.

In our study, apparently healthy birds with no evidence of clinical disease suggestive of C. psittaci infection were examined; however, the organism was detected both serologically and histochemically in these birds. This finding is in agreement with the observation of Gaede et al. (2008), which revealed that genetically related non-classified Chlamydophila spp were found in symptomless chickens during an outbreak of clinical psittacosis.

Thus it could be deduced from our finding that a balanced co-existence is the most common form of chlamydial infection in the study area. This phenomenon has been observed not only in birds, but also in many other animal species (Schachter, 1995). However, this well-balanced host-parasite relationship could be of public health significance, since alteration of this association by environmental factors or human activities could change the latency status of C. psittaci infected birds, predisposing them to clinical disease and possibly zoonotic transmission of the infection to humans. Thus this finding indicated that clinical status is not a reliable standard for diagnosis of C. psittaci infection in birds.

Conclusion

In conclusion, integrated and improved control strategies and measures should be implemented to prevent and control the occurrence of the infection in birds which serve as important source of infection to humans.

Table 1 Seroprevalence of C. psittaci specific antibodies in birds at live bird markets in Kaduna Metropolis.

X² = 1.846 df = 1 p = 0.174

Table 2. Seroprevalence of C. psittaci at live bird markets in Kaduna metropolis based on poulry type sampled.

X2 = 52.23 df = 6 p<0.0001

Table 3. Prevalence of C. psittaci inclusion bodies detected based on location

X² = 0.270 df 1 p = 0.603

Table 4. Prevalence of Chlamydophila psittaci inclusion detected based on poultry species examined based on inclusion bodies

X² = 5.272 df = 6 p = 0.509

Table 5. Comparison of IC ELISA and mGS diagnostic tests

K = 0.123 p = 0.029

Acknowledgement

We wish to acknowledge the markets officials and laboratory staff during sampling and laboratory analyses for their unfeigned support.

References

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How to cite this article

Jatau, G.A., Reuben, C.R., Chukwu, O.O.C. and Bdliya, M. I. (2016).Serological and Histochemical Detection of Chlamydophila psittaci in Poultry at Live Bird Markets in Kaduna Metropolis, Nigeria. Microbioz Journals, Journal of Microbiology and Biomedical Research, 2(4).

The post Serological and Histochemical Detection of Chlamydophila psittaci in Poultry at Live Bird Markets in Kaduna Metropolis, Nigeria appeared first on Microbioz Journals.

Abstract

Root rot of moth bean (Vigna aconitifolia (Jacq.) Marechal, caused by Macrophomina phaseolina is quite prevalent in the moth growing areas of Rajasthan and Uttar Pradesh state. The pathogen infects the moth plant at all ages and it results in a huge loss. The present study was undertaken to study the development of necrosis and occurrence of sclerotia and pycnidiospores in order to study the pathogenic variability of M. phaseolina.and to determine the morphological and pathogenic variability and their correlation with the age ofvigna plant. The isolates showed variation in mycelia growth and sporulation. Variability is the very basis of survival of the pathogen and It was observed that the sclerotia were produced in collar regions of 15,30,45 and 60 day old plants but symptoms were found to be more prevalent at maturity stage as compared to initial, seedling and flowering stages. Data reported here indicates that the sclerotia contribute to death of infected plants..It was observed that maximum disease incidence in plants occurs at maturity stage and susceptibility of plants to Macrophomina increased with age.

Key Words

Macrophomina phaseolina, root rot, moth bean, sclerotia.

Introduction

Moth bean (Vigna aconitifolia(Jacq.) Marechal,is one of the important crops extensively grown in India mainly as a rain fed crop in kharif season primarily in the state of U.P. and Rajasthan besides other states on a limited scale. It belongs to the family ‘Leguminosae’.(Jain and Mehra,1980; Banerjee et al,1983;Pant,1990; Stephan,1994; Chaturvedi.D.,2009).

Macrophomina phaseolina(Tassi.) Goid. Rhizoctonia bataticola(Taub.) Butl..causes number of diseases in legumes.M.phaseolina is universally distributed, fungi with high competitive ability,high lethal viability and a wide host range that makes the fungus economically important. Complex disease syndromes associated with this fungus are root rot,seed rot,seedling blight and charcol rot.(Khare et al.,1973;Nene,1977;Sharma and Gupta,1981;Singh and Srivastava,1989;Rout and Ingle,1989;Pal,1998;Lodha 1998;Rathore,2000.)

Number of fungal diseases of moth bean which resulted in heavy losses of the crop was reported from various part of the country and the pathogen being both seed and soil borne may attack the plant at any growth stage, however maximum damage reported to be caused at seedling and seed maturing stage of the crop. (Aghnihotri et al., 1987; Byadgi and Hedge, 1988; Rathore et al, 1999, Lakhey and Shakya; 2007).The extent of yield loss depends on the stage at which the plants are affected. As in case of M.phaseolina it was observed plant was found to be Maximum diseased at Harvesting and Post Harvesting stages. Little work has been done on the pathogenic variability of M.phaseolina,and its correlation with plant age stages. Hence, these studies were undertaken.

Materials and Methods

Pathogen Isolates:

Root rot affected plant samples were collected from moth growing fields.M.phaseolina(Tassi.)Goid. was isolated from there samples and single spores of each isolate were transferred to sabarouds agar medium and incubated as 28±1ºC.

Cultural and Morphological Variation:

Mycelial bits of 5 mm.diameter was resumed from 7 day old culture of each isolate, inoculated on Sabarouds agar and incubated at 28±2ºC for 7 days. The morphological and cultural characteristics of colony were assessed for mycelia growth, sporulation, and sclerotia size.

Standardization of inoculation technique for pathogenic variation Studies :

Three methods of inoculation viz., Wound method, Soil drenching with inoculums and soil infestation were conducted.

In Wound Method,Moth bean (Jacq.)Marechal seedlings were planted in four wooden boxes (3 planted in each and was maintained on PDA (Potato Dextrose Agar).Plants were wounded above the soil line with a sterile scalpel. A small portion of mycelium from a 7 day old moth bean broth culture grown at 25+3°C was placed into the wound cut of half of the plants. Then all wounds were covered with petroleum jelly (Vaseline) to prevent drying. After 14 days, lesions observations were made, then plants (according to their age) were cut off at the soil line, and observations on necrosis were made. (Upadhyay and Pandey, 2000).

The collar regions and roots were examined under a dissecting microscope.Four wooden boxes with three plants in each were wound-inoculated with Macrophomina phaseolina as described above. Four boxes (three plants in each) wounded, but non-inoculated, plants served as controls.

In soil drenching method, seedlings of 8 days old were used. A spore suspension was poured around the root zone in the soil by disturbing the root zone.

In soil infestation method, the fungus was grown on autoclaved sorghum seeds for 8-10 days. The inoculums was mixed with soil and left for 7 days. Then moth seeds were sown in captan solution (0.2%) for 2 hours and then sown.

When individual plants first expressed symptoms (7-14 days after inoculation).They were sectioned with a rotary microtome and stained with safranin.Sections were examined under a light microscope for the presence of M.phaseolina mycelium,Sclerotia,Pycnidiospores.(Dhingra and Sinclair,1975.;Singh and Mehrotra,1982,Mukherjee et al.,1983;Singh and Sandhu,1994).

Results

The isolates showed great variability in colony characteristics such as mycelia growth, Sporulation..

Standarization of inoculation techniques for pathogenic variations:

Among the three methods of inoculation,soil infestation and soil drenching were superior in showing root rot symptoms on inoculated plants.

Pathogenic Variability Studies:

Significant difference in virulence was observed in isolates.Isolates of latter stage produced more sporulation (++++) in the medium Similarly Desai et al.2003 also suggested that highly virulent isolates produced abundant sporulation.

All wound inoculated plants developed dark brown lesions about the point of inoculation.Sclerotia, Pycnidiospores and mycelium ofM.phaseolina were found in all plants inoculated(Fig.1) but very less sclerotia were found in 15 days old plants and No sclerotia were found in 10 days old plants. No discoloration developed on controls

All inoculated plants starts showing symptoms in 14 days as lesions enlarged. The youngest plants (15 days old) wilted and show 3.68% disease incidence within 7-8 days and the oldest plant (60 days old) shows 90.5% disease incidence after inoculation. The average lesions size at the time of rotting increased with increase in plant age and were related to collar region size.

Table 1 revealed the influence of plant age on its susceptibility to rot. It was observed that 60 days old plants (Mature Stage) bearing mature pod were more susceptible to disease than younger plants (15 days). Maximum disease incidence in plants occurs at maturity then seedling and flowering stages. Similar studies were done by Cheng and Tu, 1972. He found that the susceptibility of plants toMacrophomina increased with age.

Discussion

In this study, age of plants played a significant role in disease infection. It was observed that the younger plants at 10 and 15 days were less susceptible to infection for all the inoculations methods than plants at 60 days.This agree with Agrios (2005),who reported that plant age is important in disease infection.He also reported that plants in their reactions (susceptibility or resistance)to disease depends largerly on age and for instance infections caused by phytium;damping off and root rots ,downy mildews, bacterial blight and viral infections, The host plants are susceptible during the growth period and become resistant during the adult period but that’s exception with moth bean. So depending on the particular plant-pathogen combination, the greater the no. of pathogen propagules within or near fields of host plants, the more inoculums reaches the host.

The histopathology and isolation of the fungus from collar region portions suggested that the fungus mycelium grows rapidly and is associated with discoloration.The symptoms revealed dark browning and shredding of stem, numerous black microsclerotia were observed in the root and collar regions of infected plants.Similar symptoms were recorded by Vallijos(1988).Percentage disease observed 90.5% at 60 days crop age and 3.68% at younger stages. Maximum disease incidence in plants occurs at maturity than at younger stages. This may be the effect of physiological state of plants. (Sheldrake,1978).

Root rot development in moth bean plants is probably due to tissue disintegration,caused by enzymes and toxin activity.In plants(30-60 days)old,Sclerotia may contribute to symptom development,assuming that they occur in naturally infected plants. Also, viable sclerotia of the fungus in moth bean plant debris may be a source of inoculum for stored seed or for introducing the pathogen into non-infested fields.(Banerjee et al,1982;Pareek and Jain,1997;Upadhyay and Pandey,2000).

Conclusion

These studies demonstrate the role and association of plant age in the causation,development and spread of root rot diseases of moth bean.The reaction of the pathogen against the plant age help us to draw conclusion that Macrophomina phaseolina was found to be more wild at maturity stage.The pathogen infects the moth plant at all ages but heavy loss observed at maturity stage in comparison to other stages.

Acknowledgement

It is a great oppurtunity for me to write about”Effect of plant age upon development of necrosis and occurrence of sclerotia, pycnidiospores in moth bean infected with Macrophomina phaseolina”.The research paper is made possible through the help and support of my well wishers! First and foremost,I would like to thank God for his unconditional guidance and wisdom as i make my research!I owe a debt of gratitude to Esteemed Professor L.C. Sharmaji,(Ex faculty AU)Rajasthan for the vision and forsight which inspired me to conceive this work.Highly grateful for providing me with material and links that I could not possibly have discoverd my own!Thank you so much Sir!I would like to thank my Guide Dr. Savita Pareek,Ex Professor,BU)Rajasthan for her support and encouragment for giving us this research.I am highly indebted and thoroughly grateful to Professor Vinay Sharma(Dean and HOD,BU)Rajasthan for his suggestions,his precious and kind advice.Sir, I do not think I can ever repay the debt I owe you!Finally I sincerely thanks to my Parents,My Husband, my family who provide the financial support and cooperation.Once again,thanks everyone who collaborated in producing this work !

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TABLE 1.Effect of crop age on root rot disease development in moth bean (Vigna aconitifolia )(Jacq.)Marechal.

Figures

Figure 1.

Photomicrographs of moth bean(Vigna aconitifolia)(Jacq.)Marechal.Collar region section, inoculated with M.phaseolina. Longituidnal section showing clear sclerotia, mycelium and pycnidiospores

Figure 2.

Photomicrographs of moth bean (Vigna aconitifolia)(Jacq.)Marechal.Showing Pycnidiospores

How to cite this article

Chaturvedi, D. (2017). Effect of plant age upon development of necrosis and occurrence of sclerotia, pycnidiospores in moth been infected with Macrophomina phaseolina (Tassi.) Goid. Microbioz Journals, Journal of Microbiology and Biomedical Research, 3(1).

The post Effect of plant age upon development of necrosis and occurrence of sclerotia, pycnidiospores in moth been infected with Macrophomina phaseolina (Tassi.) Goid. appeared first on Microbioz Journals.

Abstract

Isolation and characterization of three distinct amino acid producing bacteria from the cow dung (CD) suspensions under aerobic condition have been studied. Based on their morphological and biochemical characteristics, the isolates of white colour bacteria (WCB), red colour bacteria (RCB) and yellow colour bacteria (YCB) were identified to be Gram-positive, rod-shaped and non-motile microbes belonging to E. coli, Bacillus sp.1 and Bacillus sp.2, respectively. The growth conditions of the bacterials isolated were then studied in the laboratory and the corresponding pH and temperature ranges were determined. Results on the antibiogram profiles of the bacterial isolates showed that 80-90% of the bacteria were sensitive against the antibiotics tested during the study. Amino acid producing capability of the bacteria was finally assessed in molasses-based fermentation media, and the amino acids isolated and identified from the three types of bacteria using paper chromatography were cysteine, serine and methionine. The relevance of the findings in relation to the commercial production of amino acids of medicinal, agricultural and nutritional significance has been discussed.

Key Words

Cow dung, amino acids, antibiogram profile, paper chromatography, E. coli, Bacillus spp.

Introduction

Amino acids are the most important source of energy which is crucial for the metabolic activities and thus play important role in various physiological processes in all living organisms. In addition, these are the building blocks of proteins, constitute a major part of the body, involved in building cells and repairing tissues, and form antibodies to combat foreign bodies like bacteria and viruses (Shakoori et al., 2012). Amino acids have a wide variety of applications in many different ways; they are used as animal feed additives (Campbell, 2001), some are flavour enhancers while others are used for therapeutic and psychotherapeutic purposes (Akashui et al.,1979; Kinoshita, 1987; Hermann, 2003).

Cow dung (CD) is bovine excreta that contain a mixture of dung and urine, generally in the ratio of 3:1. It contains crude fibre, crude protein, cellulose and various types of minerals such as N, K, S, and traces of P, Fe, Co, Mg, P, Cl and Mn (Nene, 1999). CD micro-flora usually contains abundant number of bacilli, lactobacilli and cocci and some identified and unidentified fungi and yeasts (Muhammad and Amusa, 2003) that can play an important role in producing enzymes, amino acids and other biomolecules.

Teo and Teoh (2011) identified five distinct morphologically and physiologically bacterial isolates from CD where all the isolates produced protease, lipase and esterase lipase. In a couple of recent studies, researchers have produced clear evidence that CD is an excellent source of biogas production owing to its methanogenic bacteria (Gopinath et al., 2014) and CD is capable of releasing major amino acids (Chomini et al., 2015).

In several recent studies, CD has been shown to be a cheap and available bio-resource that harbours a diverse group of microorganisms which may be beneficial to humans due to their ability to produce a range of metabolites. Thus cellulase producing bacteria (Bai et al., 2012; Hong-li et al., 2015), methanogenic bacteria (Pradhan and Gireesh Babu, 2012), indole acetic acid and ammonia producing bacteria (Radha and Rao, 2014) have been isolated and identified from CD. Moreover, bacterial isolates from CD having enzymatic activities (Sharma and Singh, 2015; Vijayaraghavan et al., 2016), and CD microbes capable of bio-fuel production and combating environmental pollutants (Gupta et al., 2016) have been reported, which imply that CD could be harnessed for medicinal, agricultural and industrial usages. Keeping the aforesaid findings in mind, the present investigation was aimed at isolating, characterizing and identifying amino acid producing bacteria from CD of exotic Jersey (Australian) cows at Rajshahi, Bangladesh.

Materials and Methods

Collection of samples

The samples of the CD were collected from lactating Jersey cows of a dairy farm located at Gosh Para, Kazla, Rajshahi. Soon after collecting the CD sample in sterile polythene bags, they were transported to the Genetic Engineering Laboratory, Department of Zoology, Rajshahi University, for further processing and analyses.

Enrichment of culture for amino acid producing bacteria

The CD samples were suspended in individual 250 ml Erlenmeyer flaks each containing 100 ml of Luria-Bertani (LB) medium. Control flasks without an inoculum were also maintained for comparisons. The primary enrichment was incubated for two days at 30°C with shaking at 120 rpm on an orbital shaker. The cultures that were found turbid after a period from 0 to 2 days were used as inocula in subsequent experiments.

Microscopic examinations and identification of bacterial cells

For the identification of the bacteria, morphological characters, microscopic observations, growth characteristics, biochemical tests and antibiotic sensitivity tests were performed. The microorganisms were identified using Bergey’s Manual of Determinative Bacteriology(Holt et al., 2005).

Effects of temperature and pH on bacterial growth

Since both pH and temperature have been found to influence bacterial growth (Pradhan and Gireesh Babu, 2012; Radha and Rao, 2014), the present nutrient broth culture media (Hi-media, India) were adjusted to pH 5.0, 6.0, 7.0 and 8.0, and the incubation temperatures were varied at 25ºC, 30ºC, 37ºC and 40ºC to study growth characteristics of the bacterial isolates. Bacterial cell density of the nutrient broth was determined by measuring optical density at 660 nm with a photoelectric colorimeter (AE-11M, Erma Inc., Tokyo) following the procedures described by Mohanta et al. (2012).

Screening of bacterial isolates for amino acid production

Media formulation:For the production of amino acids, molasses-based fermentation media (MF media) were tested for the isolation of amino acid fermenting bacteria obtained from the CD samples. The ingredients for the 100 ml media were: KH₂PO₄ (0.05g), K₂HPO₄(0.05g), MgSO₄.7H₂O (0.025g), (NH₄)₂SO₄ (2.00g) and CaCO₃ (2.00g). Agricultural industrial waste (10g.100 ml^-1 cane molasses) was used in the media as carbon source. The pH of the media was kept within a range of 7.0-7.2. Sterilization was done through autoclaving the media at 121ºC at 15 lbs pressure for 15 min

Fermentation:A loopful of bacterial culture from a desired plate was inoculated into 250 ml Erlenmeyer flask containing 50 ml of the fermentation medium. The flask was incubated at 30^oC on a rotary shaker. The experiments for amino acid producing potential of these bacterial strains were carried out in a rotary shaker at 120 rpm for a maximum period of 96 hrs. During this period, 3 ml sample was monitored every 24 hrs. The harvest pH of each flask was also noted during the 96-hr incubation period.

Analysis and identification of the fermented amino acids

About 3 ml sample of fermented broth was centrifuged at 15000 rpm for 10 min in order to collect the cell-free broth. Qualitative analyses of amino acids produced by bacteria in this fermented and cell-free broth were performed as detailed below

Qualitative analysis using paper chromatography

Lederer and Laderer (1957) method was followed for paper chromatography, where 0.03M standard solutions of six amino acids viz., methionine, serine, leucine, proline, cysteine and glycine were prepared. Solvent made of n-butanol: acetic acid: water (at 4: 1: 1) was added in a chromatographic rectangular glass jar. Standard amino acids and samples, 50 μl each, were loaded on to the Whatmann I chromatographic papers (Desaga Nr. 2045). The chromatographic tank papers were irrigated vertically in the solvent system for few hrs until the solvent traveled the distance on filter paper up to a certain point. Then the papers were air dried and sprayed with 0.1% ninhydrin solution (0.1g.100 ml^-1 ethanol), and later dried at 60-80ºC for 10 min to get purple spots of the amino acids. The results were confirmed by comparing the retention factors (R_fvalues) of the samples with that of the standard amino acids. The R_f values were calculated by the following formula:

The distance was measured from the point where the amino acid was loaded to the point where solvent came to a halt.

Results

Isolation and identification of the bacteria

Bacteria were isolated by plating onto an agar solidified nutrient medium.

Among the three methods of inoculation,soil infestation and soil drenching were superior in showing root rot symptoms on inoculated plants.

The plates were incubated at 30°C for a day and bacterial colonies were found to grow on the medium. Results of microscopic analysis of bacterial cells and their colony morphological characteristics are presented in Tables 1 (a) and 1 (b) whereas the biochemical tests of the isolates are presented in Table 2. Preliminary characterization of the isolates according to their morphological and biochemical tests indicated that the bacterial strains belonged to E. coli, Bacillus sp. 1 and Bacillus sp. 2.

Antibiogram profile of the bacterial isolates

The antibiogram profile analyses of the three bacterial isolates revealed that 80-90% bacteria were sensitive whereas the remaining 10-20% bacteria were resistant against 10 antibiotics tested during the study (Table 3).

Effects of temperature and pH on bacterial growth

The growth of the bacterial isolates depended on pH and temperature. WCB and YCB showed optimum growth at pH 7 while the maximum growth of RCB was observed at pH 6 and the extreme pH (5.0 and 8.0) restricted for the growth of the bacteria (Figs.1, 2 and 3, respectively).

The optimum growth conditions of WCB and RCB, on the hand, were determined at 30ºC while YCB showed maximum growth at 25ºC (Figs. 4, 5 and 6, respectively).

It is apparent from the results that both the temperature and pH were important factors for the bacterial growth and so they will affect enzymatic reactions necessary for the production of amino acids from the CD suspensions.

Identification of amino acids

The maximum production of amino acids was obtained by the MF media used in the present experiment. The amino acids produced by the isolates in the media were identified by using paper chromatography followed by calculating their specific R_f values. Results presented in Table 4 and Fig. 7 demonstrated that the bacterial strains WCB, RCB and YCB produced the amino acids cysteine, serine and methionine, respectively.

Discussion

In the present study three distinct amino acid producing bacteria were isolated from the CD suspensions at 30°C under aerobic condition. Based on their morphological and biochemical characteristics, the isolates WCB, RCB and YCB were identified to be Gram-positive, rod-shaped and non-motile bacteria belonging to E. coli, Bacillus sp.1 and Bacillus sp.2, respectively. The optimum growth conditions of the bacterials isolated were then studied in the laboratory and the corresponding pH and temperature ranges were determined.

Results on the antibiogram profiles of the bacterial isolates showed that maximum of the bacteria were sensitive against the antibiotics tested during the study. The amino acid producing capability of the bacteria was finally assessed in molasses-based fermentation media, and the amino acids isolated and identified from the three types of bacteria using paper chromatography were cysteine, serine and methionine.

Antibiotic sensitivity tests of the present study slightly differ from those of Teo and Teoh (2011) who used Kirby-Bauer assay to evaluate susceptibilities of five isolates to 17 different types of antibiotics, where each individual isolate was resistant to at least 35% of the antibiotics tested. In a recent study, eight antimicrobial drugs were used to treat the susceptibility patterns of isolated bacteria from CD; among four isolates two showed resistance to penicillin and rest of the strains were sensitive to all the antibiotics (Sharma and Singh, 2015). This is in good agreement with those of the present results.

Cysteine production by bacterial isolates in different fermentation media has been documented by Ali et al. (2011). Moreover, Gopinath et al. (2014) isolated different bacterial species from CD and reported that it contained high amount of methanogenic bacteria that increased its efficiency of biogas production. Recently, Chomini et al. (2015) demonstrated that four major amino acids viz., threonine, proline, glycine and alanine were released from digested CD. However, the present findings, corroborate to Shakoori et al. (2012) whereB. cereus was found to produce cysteine and glutamic acid, E. coli produced valine, and cysteine and methionine were produced by B. anthracis.

The right conditions for the growth of bacteria are present in a cow’s stomach. These bacteria are able to produce all the essential amino acids, even though its feed may not contain a full set of amino acids (Majda, 2014). For examples, Fungsin et al. (2008) isolated 240 strains of lactic acid bacteria Lactobacillus salivarius, and Khan et al. (2011) diagnosed endospore producing Gram-positive cocci from samples of CD. Teo and Teoh (2011) isolated five morphologically and physiologically distinct isolates, two Gram-negative and three Gram-positive, from CD that produced enzymes such as protease, lipase and esterase lipase. Cellulase producing Bacillus subtilis (Bai et al., 2012) and methanogenic Bacillus sp. and Proteus sp. (Pradhan and Gireesh Babu, 2012)were identified using 16S rDNA sequencing and BLAST search. In addition, indole acetic acid and ammonia producing Bacillus spp. and Lysinibacillus xylanilyticus (Radha and Rao, 2014), cellulase producing Stenotrophomonas sp. and Bacillus cereus (Hong-li et al., 2015), and strains of Gram-positive cocci and gram-negative bacilli (Sharma and Singh, 2015) were identified from CD. Recently, Gupta et al. (2016) and Vijayaraghavan et al. (2016) utilized CD microbes for bio-fuel production and management of environmental pollutants, and fibrinolytic enzyme production, respectively. So far the bacterial genera isolated from the CD are concerned; the present findings are in line with most of the work cited above, particularly Bai et al. (2012), Pradhan and Gireesh Babu (2012); Radha and Roa (2014); Hong-li et al. (2015) and Vijayaraghavan et al. (2016).

Amino acids are of great nutritional importance in food. In general, the world at present is confronted with the serious problems of food and nutrition deficiencies. Increased demand of proteins has led the researchers to search for unconventionally sources of proteins and amino acids (Majda, 2014; Vijayaraghavan et al., 2016). One of such sources is the microbes, particularly bacteria present in the CD which are capable of producing amino acids in profuse quantities (Fungsin et al., 2008; Radha and Rao, 2014; Chomini et al., 2015). The results of the present study are quite encouraging in that the fermented bacterial suspensions of the CD could be utilized on commercial scale in the country, with special reference to their agricultural, medicinal and nutritional significance.

Conclusion

In summary, we conclude that the microbial fermentation process can be used for the production of amino acids at very economical rates by using locally isolated strains from natural sources. The natural source including CD (cowdung) which is rich in micro-flora can provide bacterial strains for production of amino acids by fermentation on commercial scale to meet the local demand in the country.

Acknowledgement

The authors are grateful to the Chairperson, Department of Zoology, University of Rajshahi, Bangladesh, for providing laboratory facilities, and to laboratory Attendants, for their technical assistance. This research did not enjoy any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

References

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25. Chomini MS, Ogbonna CIC, Falemara BC, Micah P (2015). Effect of co-digestion of cow dung and poultry manure on biogas yield, proximate and amino acid contents of their effluents. J. Agric. Vet. Sci. 8(11): 48-56.

How to cite this article

MFHAKS Moni Krishno Mohanta*, Mst. Sabia Aktar Mohua, M. Saiful Islam (2017) Isolation and characterization of amino acid producing bacteria from cow dung, Microbioz Journals, Journal of Microbiology and Biomedical Research 3 (2)

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Department of Health and sport, University of East London, London, United Kingdom,Current Address: Fabson close, Kagoro bye Express, Kaduna State, Nigeria,Telephone: +2348068423974

Abstract

Introduction:

Since the discovery of AIDS in 1980, the development of cancers has featured as a major problem in HIV patients. The extents of malignancies kept increasing among HIV infected population than the normal population. HAART widely became available in the 1990s, and has marked a notable decrease in the occurrence of diseases and death rate in HIV infected population but long-term use of it have been associated with increase cancer risk. Therefore, it will be of significance important to study the tumour promoting potentials of HAART Backbone (NNRTIs and NRTIs) leading to increase cancer risk in HIV infected population.

Method:

Cell differentiation potential was evaluated using U937 cells exposed to Nevirapine (NVP), Abacavir Sulphate (ABC) and Zidovudine (AZT) for 72hrs. Also, DNA breakage induced by NVP, ABC and AZT on HeLa cells at 40ug/ml and 50ug/ml using a modified FADU protocol was evaluated.

Results:

The highest transforming ability was recorded with AZT 50ug/ml as 98% of treated cells transformed into adherent cells after 72hrs exposure as compared to untreated cells. DNA breakage was not induced with NVP, ABC and AZT concentrations used on cells for 4hrs (40 and 50ug/ml).

Conclusion:

Overall, the research suggests that NVP, ABC and AZT have cell differentiation potential concluding that these drugs have tumour promoting potentials leading to increase cancer risk.

Keywords

Tumour promoting potential, DNA Damage, HAART classes, AIDs, Cancer

How to cite this article

Z Aisha*Microbioz Journals, Journal of Microbiology
and Biomedical Research 4 (3)

Article Details

Type of article

Original Artilcle

Date of publication

16/08/2018

Mode of access

Open Access

Correspondance

Department of Health and sport, University of East London, London, United Kingdom,Current Address: Fabson close, Kagoro bye Express, Kaduna State, Nigeria,Telephone: +2348068423974

Email ID: aishazakz@gmail.com

The post Tumour Promoting Potentials of HAART backbone (NNRTIs and NRTIs) leading to increase cancer risk among AIDs patients appeared first on Microbioz Journals.

Department of Environmental Science, Amar Singh College, J&K, India-190008

Abstract

A total of 36 fungal species were isolated during the study. The soil mycoflora was cultured using serial dilution and plate count method on PDA and Malt Extract Agar supplemented with streptomycin. The fungal density was recorded maximum during summers followed by spring and winter.

In summers the fungal density was recorded highest in descending order: Mixed wood forest of pine and oak (12.4×10⁴cfu g^-1), land put to non-agriculture use (11.8×10⁴ cfu g^-1), Silvo-pasture (8.2×10⁴cfu g^-1), agricultural field (8.0×10⁴cfu g^-1), floating garden (7.8×10⁴cfu g^-1). In spring the density remained more or less same in sites 1, 3, 6 and 7. In winters sampling could only be carried out in only three sites, density in these sites decreased appreciably due to drop in temperature.

Keywords

soil; fungus, density, soil

How to cite this article

S Habib Microbioz Journals, Journal of Microbiology and Biomedical Research 4 (2)

Article Details

Type of article 

Original Artilcle

Date of publication

21/06/2018

Mode of access

Open Access

Correspondance 

Shazia Habib

Department of Environmental Science, Amar Singh College, J&K, India-190008

Email ID:  habibshazia3185@gmail.com

The post Soil mycofloral communities across different land-use types appeared first on Microbioz Journals.

Department of Microbiology, SRM Medical college Hospital and Research Institute, Kattankulathur

V.Mangayarkarasi

Department of Microbiology, SRM Medical college Hospital and Research Institute, Kattankulathur

Abstract

Vancomycin resistant Enterococci (VRE) have recently emerged as nosocomial pathogen with intrinsic resistance to many antimicrobial agents making them difficult to treat. We investigated the prevalence of vancomycin resistance in Enterococci isolated in a tertiary health care set up.

Materials and Methods:

128 Enterococcal isolates form patients specimens were screened for vancomycin resistance. Screening for vancomycin resistance  was done by utilizing vancomycin screen agar. Vancomycin resistance was also confirmed phenoypically by determining the Minimum inhibitory concentration of both Vancomycin and teicoplanin  by broth microdilution method.

Results:

Vancomycin screen agar detected four resistant isolates of Enterococcus faecium (12.90%) which is inclusive of  an additional isolate which Kibybaeur disk difusion method failed to identify .

Interpretation and conclusion:

Being an emerging pathogen, VRE acts as a sensitive marker for measuring the effectiveness of

Infection control programme and the appropriate application of preventive measures. The study resulted in an increased awareness about VRE and implementation of control measures in the hospital to restrict spread of VRE

Keywords

Vancomycin screen Agar, Vancomycin, Teicoplanin, Drug resistance, MIC

How to cite this article

VM L.Triveda*Microbioz Journals, Journal of Microbiology and Biomedical Research 4 (2)

Article Details

Type of article

Original Artilcle

Date of publication

28/05/2018

Mode of access

Open Access

Correspondance 

L.Triveda

Department of Microbiology, SRM Medical college Hospital and Research Institute, Kattankulathur

Email ID:  drtriveda@gmail.com

The post Screening of VRE with special emphasis on the determination of MIC of Vancomycin and Teicoplanin for Enterococci appeared first on Microbioz Journals.

GITAM University, Visakhapatnam, Andhra Pradesh

Y Vimala 

GITAM University, Visakhapatnam, Andhra Pradesh

Abstract

Flacourtia jangomas is mostly observed in Southeast Asia and East Asia. Its origin is wild and from tropical Asia, especially India. The Common names of Flacourtia jangomas are Coffee plum, Indian plum, Manila cherry and Paniala. Traditional uses of Flacourtia jangomas are that the fruits are used to cure diarrhea and nausea. The leaves and bark are used in bleeding gums and aching teeth, and the bark infusion is gargled to alleviate hoarseness. Flacourtia jangomas fruits are traditionally used to cure diarrhea and nausea.

The leaves and bark are used in bleeding gums and aching teeth. Antibacterial activity of Ethyl acetate extract of the stem was determined by Agar diffusion method against Gram -positive bacteria viz., Staphylococcus aureus, Bacillus polymyxa, Bacillus megaterium, and Gram-negative bacteria viz., E.coli, Salmonella typhi, Pseudomonas aeruginosa and Vibrio cholera. The extracts shown zone of inhibition, ranging from 13 mm-28 mm. The extracts had the profound effect on Gram-negative bacteria with the highest zone of inhibition against Pseudomonas aeruginosashowing 28 mm followed by Vibrio cholerae with 20 mm. Antifungal activity was determined by Agar diffusion against four Fungal strains viz.,Aspergillus niger ,Aspergillus flavus, Trichoderma viridae and Neurospora crassa.

The extracts showed modearte inhibitory activity ranging from 2-15 mm. Antioxidant activity of Flacourtia jangomas stem extract was determined by Nitric oxide scavenging assay, the extract showed prominent antioxidant activity with Butylated hydroxy anisole (BHA) as a standard compound.

Keywords

Flacourtia jangomas, Antibacterial activity, Antifungal activity, Antioxidant activity.

How to cite this article

YV Gulnaaz sabri*(2018) Antimicrobial activity and Antioxidant activity of Flacourtia jangomas stem from Bihar, India, Microbioz Journals, Journal of Microbiology and Biomedical Research 4(1)

Article Details

Type of article 

Original Artilcle

Date of publication

31/03/2018

Mode of access

Open Access

Correspondance 

Gulnaaz Sabri 

Department of Microbiology and Food science and Technology, GITAM Institute of science, GITAM University, Visakhapatnam, Andhra Pradesh.

Email ID:  gulnaaz08@gmail.com

The post Antimicrobial activity and Antioxidant activity of Flacourtia jangomas stem from Bihar, India appeared first on Microbioz Journals.