Journal of Life Sciences Volume 5 Number (5)

(1)

David Publishing Company www.davidpublishing.com P u b l i s h i n g Dav i d

Journal of Life Sciences

(2)

Publication Information

Journal of Life Sciences is published monthly in hard copy (ISSN 1934-7391) and online (ISSN 1934-7405) by David Publishing Company located at 1840 Industrial Drive, Suite 160, Libertyville, Illinois 60048, USA.

Aims and Scope

Journal of Life Sciences, a monthly professional academic journal, covers all sorts of researches on molecular biology, microbiology, botany, zoology, genetics, bioengineering, ecology, cytobiology, biochemistry, and biophysics, as well as other issues related to life sciences.

Editorial Board Members

Dr. Stefan Hershberger (USA), Dr. Suiyun Chen (China), Dr. Farzana Perveen (Pakistan), Dr. Francisco Torrens (Spain), Dr. Filipa João (Portugal), Dr. Masahiro Yoshida (Japan), Dr. Reyhan Erdogan (Turkey), Dr. Grzegorz Żurek (Poland), Dr. Ali Izadpanah (Canada), Dr. Barbara Wiewióra (Poland), Dr. Valery Lyubimov (Russia), Dr. Amanda de Moraes Narcizo (Brasil), Dr. Marinus Frederik Willem te Pas (The Netherlands), Dr. Anthony Luke Byrne (Australia), Dr. Xingjun Li (China), Dr. Stefania Staibano (Italy), Dr. Wenle Xia (USA), Hamed Khalilvandi-Behroozyar (Iran).

Manuscripts and correspondence are invited for publication. You can submit your papers via Web Submission, or E-mail to [email protected] or [email protected]. Submission guidelines and Web Submission system are available at http://www.davidpublishing.com.

Editorial Office

1840 Industrial Drive, Suite 160, Libertyville, Illinois 60048 Tel: 1-847-281-9826, Fax: 1-847-281-9855

E-mail:[email protected], [email protected]

Copyright©2011 by David Publishing Company and individual contributors. All rights reserved. David Publishing Company holds the exclusive copyright of all the contents of this journal. In accordance with the international convention, no part of this journal may be reproduced or transmitted by any media or publishing organs (including various websites) without the written permission of the copyright holder. Otherwise, any conduct would be considered as the violation of the copyright. The contents of this journal are available for any citation. However, all the citations should be clearly indicated with the title of this journal, serial number and the name of the author.

Abstracted / Indexed in

Database of EBSCO, Massachusetts, USA Cambridge Scientific Abstracts (CSA), USA

Chinese Database of CEPS, American Federal Computer Library Center (OCLC), USA Ulrich’s Periodicals Directory, USA

Chinese Scientific Journals Database, VIP Corporation, Chongqing, China Summon Serials Solutions

Subscription Information

Price (per year): Print $420, Online $300, Print and Online $560

David Publishing Company

1840 Industrial Drive, Suite 160, Libertyville, Illinois 60048 Tel: 1-847-281-9826, Fax: 1-847-281-9855

E-mail: [email protected]

Dav id Publishing Company

w ww.davidpublis hing.com

Pu b li sh i ng

Dav i d

(3)

J LS

Journal of Life Sciences

Volume 5, Number 5, May 2011 (Serial Number 37)

Research Papers

327 Macronutrient Composition and Digestibility of Extruded and Fermented Soya Protein Products

Anthony Ojokoh and Yimin Wei

332 Hormonal (Thyroxin, Cortisol) and Immunological (Leucocytes) Responses to Cistern Size and

Heat Stress in Tunisia

Rim Ben Younes, Moez Ayadi, Taha Najar, Margherita Caccamo, Iris Schadt and Moncef Ben M’Rad

339 Occurrence of Extended-Spectrum β-lactamase Producing Enterobacteriaceae (ESBLPE) among

Primary School Pupil in Obafemi-Owode, Nigeria

Akinduti Paul Akinniyi, Akinbo John Adeolu, Adenuga W. Funmilayo, Ejilude Oluwaseun, Umahoin Kingsley Omokhudu and Ogunbileje John Olusegun

344 G×E Interaction Effects on Yield of Twenty-five Genotypes of Bread Wheat (Triticum Aestivum L.)

during 2009 Winter in Zimbabwe Tegwe Soko and Ephrame Havazvidi

352 Mutated Clones of Caladium Humboldtii ‘Phraya Savet’ from in vitro Culture and Occurrence of

Variants from Somatic Hybridization between Two Caladium Species

Chockpisit Thepsithar, Aree Thongpukdee, Rungniran Sugaram and Usanisa Somkanae

360 Media Appraisal for Somatic Embryogenesis of Elite Inbred Lines of Maize

Inuwa Shehu Usman, Shehu Garki Ado and Ng Shou Yong

364 Circadian Rhythm of Root’s Apical Meristem Mitosis Cells of Soybean

Margarita Kozak

369 Life History of the Lenkoran Capoeta Capoeta Gracilis (Keyserling, 1981) in the Atrak River,

Northern Iran

Rahman Patimar, Abdol-Jalil Hajili Davaji and Aisoltan Jorjani

376 Effect of Pork Meat pH on Iron Release from Heme Molecule during Cooking

Monica Bergamaschi and Angela Pizza

381 A Survey on Morphological Traits of Basset Hound Dogs Raised in Italy

Francesca Cecchi, Giovanna Carlini, Elena Ciani, Assunta Bramante and Roberta Ciampolini

387 Using Morphological Markers to Assess Variations between and within Cultivated and

Non-cultivated Provenances of Moringa Oleifera Lam. in Tanzania

Mariam Godwin Mgendi, Agnes Morris Nyomora and Mkabwa Katambo Manoko

393 Evaluation of New Insecticide (Proteus 170 O - TEQ) for the Control of the Brown Cocoa Mirid

(Sahlbergella Singularis) in Nigeria

Evarestus Uche Asogwa, Feyisara Abiodun Okelana, Idongesit Umanah Mokwunye, Joseph Chucks Anikwe and Theophilos Chinyere Nkasiobi Ndubuaku

400 Research on the Inert Dust against Phosphine Resistance of Cryptolestes Ferrugineus

(4)

(5)

Journal of Life Sciences 5 (2011) 327-331

Macronutrient Composition and Digestibility of Extruded

and Fermented Soya Protein Products

Anthony Ojokoh and Yimin Wei

Institute of Agro-Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100094, China

Received: March 26, 2010 / Accepted: June 01, 2010 / Published: May 30, 2011.

Abstract: The macronutrient composition and in vitro protein digestibility of extruded fermented and unfermented extruded soya protein products (low, medium and high moisture), raw and fermented soya meal and soya kernel were studied. The protein content (g/100g soya dry weight) ranged from 38.20 to 62.98 with the highest content in the high moisture extruded protein product fermented with 5 mL inoculum of Bacillus natto. Contents of carbohydrates ranged from 14.77 to 29.08 while those of crude fibre, fat and ash were generally low. Fermentation better improved protein digestibility in the raw soya meal and kernel than in the unfermented extruded and extruded fermented products. SDS-PAGE electrophoresis revealed some degradation of the protein sub units of fermented samples.

Key words: Fermentation, extrusion, macronutrient, soya protein products, digestibility.

1. Introduction

Extrusion is one of the most common industrial processes used to make snacks and it is among the most

versatile technological processes for making food products. Extrusion technology has many advantages,

including its versatility, high productivity, low cost and the ability to produce unique product shapes and high product quality [1-3].

Extrusion-cooking is one of the most efficient and versatile food processing technologies that can be used

to produce pre-cooked and dehydrated foods. A major technological advantage of extrusion is that the product is simultaneously cooked and dried, resulting in

low-moisture shelf stable extrudates. This reduces the cost of post-extrusion drying and guarantees an

improved shelf-life of the product without the need for cooling or refrigeration. The pre-cooked extrudate requires only reconstitution in warm water and this

Anthony Ojokoh, Ph.D., research fields: industrial fermentation and biotechnology. E-mail: [email protected].

Corresponding author: Yimin Wei, Ph.D., research fields: agro-food processing and food safety. E-mail: weiyimin36@ hotmail.com.

means reduced cooking time and energy [4].

The reduction in protein digestibility of soya kernel

in humans and animals is caused by antinutritional factors such as phytic acid and trypsin inhibitor that bind to enzymes in the digestive tract and thus inhibit

utilization of proteins. This adverse effect can be overcome by fermentation, germination [5, 6] or

extrusion [7].

Improved protein digestibility in food is due to degradation of complex storage proteins by

endogenous and microbial proteases during fermentation whereas extrusion and other forms of

cooking improve digestibility by solubilization, gelatinization and formation of maltodextrins [8].

It is also well established that extrusion

thermomechanically denatures and reorients proteins in foods, leading to changes in digestibility and levels

of amino acids [8-10]. However, the magnitude of starched protein transformation due to extrusion is a function of pre and post processing operations, their

(6)

Macronutrient Composition and Digestibility of Extruded and Fermented Soya Protein Products

328

of either fermentation or extrusion on starch and

protein digestibility.

In this study, the combined effects of extrusion and fermentation on the macronutrient composition

and invitro protein digestibility of soya protein products were determined. Comparisons were also

made with (a) unfermented extruded soya protein products and (b) fermented and unfermented soya meal and kernel.

2. Materials and Methods

2.1 Preparation of Soya Samples and Fermentation

Extruded soya protein samples, soya meal and soya kernel were obtained from the Food and Technology Laboratory of the Institute of Agro-Food Science and

Technology, Beijing. The soya kernels were cleaned to remove broken seeds, dust and other extraneous

materials and then soaked in water at room temperature (25 ± 2 ℃) for 20 h while soya meal and extruded soya protein products with moisture contents of 20 (low),

32.5 (medium) and 45% (high) were soaked in water for 45 mins and drained thereafter. The samples were

inoculated immediately after steaming with 5, 10 and 15 mL of inoculum size of Bacillus natto previously isolated from a commercial natto product (natto

produced from large soya bean cultivar) and allowed to ferment at 37 ℃ for 48 h.

2.2 Macronutrient Estimation

Moisture content was determined by direct oven

drying method; the loss in weight after oven-drying was expressed as % moisture content [11]. Crude

protein was estimated from the total nitrogen (TN) determined by the micro- Kjeldahl method by multiplying the TN by a factor of 6.25. Crude fat was

determined by using the soxhlet extraction method using petroleum ether as the solvent [11]. Ash was

measured gravimentrically after ashing at 550 ℃ to constant weight. Carbohydrate was determined by the anthrone method according to Plummer [12].

2.3 Protein Digestibility

In vitro soluble protein (IVSP) digestibility was

determined by adding 200 mg sample to a 100 mL Erlenmeyer flask containing 35 mL 0.1 M sodium citrate tribasic dihydrate (pH 2.0) with pepsin (1.5 g

pepsin/L, Sigma P-7012; activity 2650 units/mg protein). The mixture was incubated for 2 h in a

shaking water bath at 37 ℃ and then centrifuged at 10,000 rpm for 15 min. The supernatant was decanted

and the residue was washed, dried at 80 ℃ and

analyzed for nitrogen content. Digestibility was calculated by subtracting residue nitrogen from total

nitrogen, dividing by total nitrogen multiplied by 100.

2.4 Nitrogen Solubility Index (NSI)

Nitrogen solubility index was determined by weighing 1 g sample into 50 mL centrifuge tube and

dispersed in 20 mL distilled water. The dispersion was mechanically shaken for 1 h, centrifuged at 10,000 rpm for 15 min and the supernatant collected. The residue

was resuspended and centrifuged twice in 10 mL distilled water. The combined supernatants were

analysed for soluble nitrogen by the Kjeldhal method. Nitrogen solubility index (NSI) was reported as soluble nitrogen expressed as a percentage of total protein.

2.5 SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 12.5% resolving gel and 50% stacking gel according to

the method of Wang and Fan [13]. Protein solutions extracted with phosphate buffer containing 8 M urea

and 0.1 M 2-ME were diluted with 5 x sample dissolution, and then heated in boiling water bath for 5 min. After centrifugation at 1,000 rpm for 10 min, a total of 10 μL solution was loaded into each lane and electrically separated. The gels were strained for 30 min with CBB

(7)

329

were analyzed by using Fluro Chem FC2 Imaging

System (Alpha Innotech Cooperation, USA) and the molecular weight of each subunit was quantified. Low molecular weight markers (Amersham Biosciences,

UK) used were rabbit phophorylase b (97.4 kDa), bovive serum albumin (66.2 kDa), rabbit actin (43.0 kDa),

bovine carbonic anhydrase (31.0 kDa), trypsin inhibitor (20.1 kDa) and hen egg white lysozyme (14.4 kDa).

2.6 Statistical Analysis

Analysis of variance (anova) of the samples, which

was performed with mean ± S.D. values, was compared at 5% significant level using Ducan’s multiple-range tests.

3. Results and Discussion

3.1 Macronutrient Composition

The macronutrient profile (g/100g) of soya samples is shown in Tables 1 and 2. There was significant (P < 0.05) increase in the protein content of the fermented

samples over unfermented samples. In this study, the

protein content varied among samples fermented with

different inoculum sizes with the highest (62.98 g/100g)

occuring in the high moisture extruded soya sample

fermented with 5 mL of Bacillusnatto.

Reade and Gregory [14] reported that autolysis is likely to increase with initial inoculum due to

disproportionate amount of nutrient and lower

conversion efficiency. At lower inoculum level, cells

are larger, especially when competition for available

nutrients was minimal [15]. Similar findings have been

reported by Ojokoh and Uzeh [16] in production of S.

cerevisiae biomass in papaya extract medium.

Table 1 Proximate composition of soya samples fermented with different inoculum sizes of Bacillus natto (mean ± SD*).

Samples Protein Crude fibre Ash Fat Carbohydrate

fermented with 5 mL inoculum

LMESPP 57.32 ± 0.18 1.49 ± 0.02 7.03 ± 0.17 0.51 ± 0.06 20.21 ± 0.40 MMESPP 57.61 ± 0.24 1.55 ± 0.04 7.27 ± 0.06 0.40 ± 0.00 20.12 ± 0.31 HMESPP 62.98 ± 0.49 1.28 ± 0.01 6.73 ± 0.03 0.45 ± 0.01 16.64 ± 0.18 SM 57.91 ± 0.91 2.06 ± 0.00 7.34 ± 0.15 0.16 ± 0.05 14.93 ± 0.16 SK 43.22 ± 0.58 1.58 ± 0.02 4.68 ± 0.16 21.33 ± 0.12 19.76 ± 0.24

LMESPP 56.84 ± 0.17 1.60 ± 0.01 7.05 ± 0.15 0.42 ± 0.02 21.44 ± 0.24 MMESPP 56.50 ± 0.21 1.45 ± 0.00 6.94 ± 0.19 0.34 ± 0.01 21.94 ± 0.81 HMESPP 57.91 ± 0.24 2.59 ± 0.04 6.78 ± 0.06 0.35 ± 0.05 18.56 ± 0.46 SM 58.04 ± 0.40 2.55 ± 0.01 7.52 ± 0.03 0.21 ± 0.00 14.77 ± 0.24 SK 43.74 ± 0.49 2.29 ± 0.02 4.57 ± 0.15 21.22 ± 0.07 19.31 ± 0.50

LMESPP 55.42 ± 0.91 1.12 ± 0.05 6.34 ± 0.06 0.31 ± 0.04 24.49 ± 0.46 MMESPP 56.83 ± 0.93 1.97 ± 0.00 7.22 ± 0.22 0.37 ± 0.01 21.38 ± 0.74 HMESPP 57.49 ± 0.58 1.68 ± 0.04 6.73 ± 0.15 0.40 ± 0.00 21.56 ± 0.31 SM 56.30 ± 0.50 2.60 ± 0.02 7.48 ± 0.09 0.12 ± 0.02 16.42 ± 0.16 SK 41.16 ± 0.35 1.66 ± 0.01 5.03 ± 0.03 21.28 ± 0.10 22.03 ± 0.42 LMESPP: Low Moisture Extruded Soy Protein Product; MMESPP: Medium Moisture Extruded Soy Protein Product; HMESPP: High Moisture Extruded Soy Protein Product; SM: Soy Meal; SK: Soy Kernel. * Values represent means of triplicate determinations. The same with Table 2.

Table 2 Proximate composition of unfermented soya samples (mean ± SD*).

Samples Protein Crude fibre Ash Fat Carbohydrate

LMESPP 53.86 ± 0.13 1.43 ± 0.01 6.71 ± 0.19 1.75 ± 0.06 27.04 ± 0.18

MMESPP 52.29 ± 0.16 0.53 ± 0.01 6.43 ± 0.07 1.35 ± 0.04 28.58 ± 0.81

HMESPP 53.47 ± 0.13 1.56 ± 0.00 6.34 ± 0.06 1.31 ± 0.02 29.08 ± 0.29

SM 52.20 ± 0.22 1.88 ± 0.04 6.57 ± 0.04 0.16 ± 0.00 25.84 ± 0.24

(8)

330

The increase in protein content after fermentation

was due to a decrease in carbon ratio in the total mass,

resulting in redistribution of nutrient percentages.

Microorganisms utilize carbohydrates as an energy source and produce carbon dioxide as a by-product.

This causes the nitrogen in the fermented samples to be

concentrated and thus the proportion of protein in the

total mass increases. The lower protein content in the

extruded samples compared to the extruded fermented

samples was possibly due to participation of amino

acids in Maillard reactions. The fat and crude fibre

contents were generally low while there was no significant difference in the ash content.

3.2 Protein Digestibility

In vitro protein digestibility is a measure of soluble

proteins digested under conditions of the pepsin assay.

In vitro protein digestibilities increased after

fermentation because of partial degradation of complex

storage proteins by endogenous and microbial

proteolytic enzymes into soluble products [5, 17]. Protein digestibilities increased more when samples

were unfermented or extruded and fermented than in

the extruded samples (Table 3). Extrusion is a

high-temperature short-time treatment that improves

protein digestibility via denaturation which exposes

enzyme access sites. An increase in protein

digestibility was reported in fermented and extruded uji from maize-finger millet [4].

3.3 Nitrogen Solubility Index (NSI)

The extent of protein denaturation is assessed by the

solubility of protein in water and is measured as NSI

[18]. The increase in NSI in the fermented and extruded

fermented samples (Table 4) may be attributed to the

proteolytic activity of endogenous and microbial

enzymes. In contrast, the NSI of unfermented extruded

samples decreased, indicating polymerization,

cross-linking and reorientation of the native proteins to form new fibrous structures [10, 19, 20]. Extrusion

Table 3 Protein digestibility of soya samples.

Samples Protein digestibility (mean ± SD*)

FLMESPP 87.78 ± 0.09

FLMESPP: Fermented Low Moisture Extruded Soy Protein Product; FMMESPP: Fermented Medium Moisture Extruded Soy Protein Product; FHMESPP: Fermented High Moisture Extruded Soy Protein Product; FSM: Fermented Soy Meal; FSK: Fermented Soy Kernel; ULMESPP: Unfermented Low Moisture Extruded Soy Protein Product; UMMESPP: Unfermented Medium Moisture Extruded Soy Protein Product; UHMESPP: Unfermented High Moisture Extruded Soy Protein Product; USM: Unfermented Soy Meal; USK: Unfermented Soy Kernel. * Values represent means of triplicate determinations. The same with Table 4 and Fig. 1.

Table 4 Nitrogen solubility index of soya samples.

Samples NSI (mean ± SD*)

denatures proteins by opening up their quaternary and

tertiary structures [21].

3.4 SDS-PAGE Electrophoresis

The result of SDS-PAGE electrophoresis of soya samples is shown in Fig. 1. The two major components

in soya bean protein 11S with molecular weight of 38.99 kDa and 7S with molecular weight of 83.80,

(9)

331

Fig. 1 SDS-PAGE electrophoresis of soya samples. 1. USK; 2. USM; 3. ULMESP; 4. UHMESPP; 5. FSK; 6. FSM; 7. FLMESPP; 8. FMMESPP; 9. FHMESPP; 10. UMMESPP.

due to microbial degradation during fermentation. Modification of protein SDS-PAGE profile during

hydrolysis may improve the ability of protein to move quickly to interface [22].

4. Conclusion

The protein content, protein digestibility and

nitrogen solubility index of the soya protein products improved after fermentation resulting in better nutritional value and more effective utilization of the

products. SDS-PAGE electrophoresis indicated that the protein sub units of the fermented samples were

affected by fermentation.

Acknowledgments

These investigations were supported by the Chinese Academy of Agricultural Sciences.

References

[1] N. Singh, A.C. Smith, N.D. Frame, Effect of process variables and glycerol monostearate on extrusion of maize grits using two sizes of extruder, Journal of Food Engineering 35 (1998) 91-109.

[2] N. Singh, K. Kaur, B. Singh, K.S. Sekhon, Effects of phosphate salts on extrusion behaviour of rice, Food Chemistry 64 (1999) 481-488.

[3] H. Koksel, G.H. Ryu, A. Basman, Effects of variables on the properties of waxy hulless barley extrudates, Nahrung 48 (1) (2004) 19-24.

[4] C. Onyango, H. Neotzold, T. Bley, Proximate composition and digestibility of fermented and extruded uji from maize-finger millet blend, Lebensmitted Wissenschaft and Technologie 37 (8) (2004) 827-832.

[5] N. Khetarpaul, B.M. Chauhan, Effect of germination and fermentation on in vitro starch and protein digestibility of pearl millet, Journal of Food Science 55 (3) (1990) 883-884. [6] W. Lorri, U. Svanberg, Lactic fermented gruels with

improved in vitro protein digestibility, International Journal of Food Science and Nutrition 44 (1993) 29-36. [7] K.M. Dahlin, K.J. Lorenz, Carbohydrate digestibility of

laboratory extruded cereal grains, Cereal Chemistry 70 (3) (1993) 329-333.

[8] I. Bjork, N.G. Asp, The effect of extrusion cooking on nutritional value - A literature review, Journal of Food Engineering 2 (1983) 281-308.

[9] G. Della Valle, L. Quillien, J. Gueguen, Relationships between processing conditions andstarch andprotein modifica tions during extrusion-cooking of pea flour, Journal of the Science of Food and Agriculture 64 (1994) 509-517.

[10] M.O. Iwe, D.J. Van Zuilichem, P.O. Ngoddy, Amino acid and protein dispersibility index (PDI) of mixtures of extruded soy and sweet potato flours, Lebensmittel Wissenschaft und Technologie 34 (2001) 71-75.

[11] Association of Official Analytical Chemists, Official Methods of Analysis, Washington DC, 1990.

[12] D.T. Plummer, An Introduction to Practical Biochemistry, McGraw Hill, New York, 1971, pp. 112-113.

[13] J.Z. Wang, M. Fan, Handbook of Protein Technique, Science in China Publication, Beijing, 2000, pp. 77-110. [14] A.E. Reade, K.E. Gregory, High temperature protein

enriched feed from cassava fungi, Applied Microbiology 30 (1975) 897-907.

[15] A.E. Chikwendu, Microbial treatment of cassava whey and single cell protein production, M.Sc. Thesis, Uni. of Benin, Benin City, Nigeria, 1987, p. 162.

[16] A.O. Ojokoh, R.E. Uzeh, Production of Saccharomyces cerevisiae biomass in papaya extract medium, African Journal of Biotechnology 4 (11) (2005) 1281-1284. [17] U.D. Chavan, J.K. Chavan, S.S. Kadam, Effect of

fermentation on soluble proteins and in vitro protein digestibility of sorghum, green gram andsorghum-green gram blends, Journal of Food Science 53 (5) (1988) 1574-1575.

[18] M.E. Camire, in: R. Guy (Ed.), Extrusion Cooking: Technologies and Applications, CRC Press, Wood Head Publishing Co., Cambridge, England, 2001, pp. 109-129. [19] L.A.M. Pelembe, C. Erasmus, J.R.N. Taylor, Development

of a protein-rich composite sorghum–cowpea instant porridge by extrusion cooking process, Lebensmittel Wissenschaft und Technologie 35 (2002) 120-127. [20] D.W. Stanley, Extrusion cooking, in: C. Mercier, P. Linko,

J.M. Harper (Eds.), American Association of Cereal Chemists, St. Paul, MN, 1989, pp. 321-341.

(10)

Hormonal (Thyroxin, Cortisol) and Immunological

(Leucocytes) Responses to Cistern Size and Heat Stress

in Tunisia

Rim Ben Younes1, Moez Ayadi2, Taha Najar1, Margherita Caccamo3, Iris Schadt3 and Moncef Ben M’Rad1

1. Ressources Animales, Halieutiques et Technologie Alimentaire, Institut National Agronomique de Tunisie, Tunis 1082, Tunisie

2. Production Animale, Institut Supérieur de Biologie Appliquée de Médenine, Médenine 4100, Tunisie

3. CoRFiLaC, Regione Siciliana, Ragusa 97100, Italy

Received: December 15, 2010 / Accepted: January 25, 2011 / Published: May 30, 2011.

Abstract: This study was designed to determine the effects of heat stress on plasma leucocytes, Thyroxin (T4) and cortisol concentrations in dairy cows with small and large cistern under hot climate. This experiment was carried out in 2006, in North Tunisia, using a randomized block design per udder cistern size, using 60 Holstein cows. Cows were classified according to udder cistern size by ultrasonography as large-cisterned (44 ± 13 cm²; LC) and small-cisterned (21 ± 8 cm²; SC). The experiment was carried out in two different periods: spring (Apr. 5 (D1)) and summer (July 19 (D2), Aug. 19 (D3) and Sept. 19 (D4)). On each test day, temperature and relative humidity data were registered hourly and cows’ blood was sampled from the jugular vein to determine serum concentrates of cortisol and T4. Leucocytes (lymphocytes, eosinophils, neutrophils and monocytes) were counted differentially, and percentages of lymphocytes relative to total counted cells were calculated. Mean temperature-humidity index (THI) values were 62 ± 2, 79 ± 2, 84 ± 2, and 77 ± 1 in D1, D2, D3, and D4, respectively. Lymphocyte incidence relative to total cell counts and T4 concentrations were affected by test day (P < 0.001). Lymphocytes (%) were significantly less in hotter months. During summer, T4 concentration at D2 (87.4 nmol/L) was higher relative to concentrations at D3 (42.8 nmol/L) and D4 (53.5 nmol/L). T4 concentrations were higher (P < 0.01) in SC cows (67.7 ± 0.1 nmol/L) compared to LC cows (52.7 ± 0.1 nmol/L). Cortisol concentration was effected neither by test day nor by cistern size. However, the decrease of lymphocyte concentration during summer compared to spring could be considered as an evidence of the suppression of cows’ immune system under heat stress.

Key words: Heat stress, cistern size, cortisol, thyroxin, lymphocytes.

1. Introduction

Tunisia is characterized, during summer months, by persistent intense hot temperature. In Tunisia, heat stress usually begins in June and lasts through September [1]. Moreover, dairy cattle are sensitive to high ambient temperatures (AT) and relative humidity (RH). These conditions compromise the ability of lactating cows to dissipate heat which induces heat stress. As a result, the cow activates physiological mechanisms for coping with the heat stress. The

Corresponding author: Rim Ben Younes, research field: production animals. E-mail: [email protected].

temperature-humidity index (THI) is widely used as an index to estimate the intensity of heat stress on dairy cows. Heat stress causes changes in the homeostasis status of the animals and has been quantified through measurements of rectal temperature (RT), respiratory rate (RR), plasma thyroxin (T4) and cortisol concentrations [2].

(11)

Hormonal (Thyroxin, Cortisol) and Immunological (Leucocytes) Responses to Cistern Size and Heat Stress in Tunisia

333

of utmost importance in the heat adaptation process, allowing the adjustment of metabolic rates in favour of the body heat balance. Similarly, the secretion of cortisol stimulates physiological adjustments that enable an animal to cope with heat stress [6]. Besides, cortisol depresses the activity of the immune system and lowers its resistance to diseases. Kamwanja et al. [7] have reported a slight decrease in lymphocyte population in heat stressed cows.

High-yielding dairy cows are the most sensitive to heat stress because they produce more heat than

low-yielding dairy cows [8]. It has been shown that

animals with large cisterns are more efficient producers of milk yield than little cistern’s cows [9-11]. As a consequence, cistern size could be related to heat stress, it becomes important to know hormonal and immunological response to heat stress and their effect on cistern size in dairy cows. The present study was designed to determine the effects of heat stress on plasma leucocytes, T4 and cortisol concentrations in dairy cows with small and large cistern under significant climate changes.

2. Materials and Methods

2.1 Cows, Measurements and Sampling

The study was carried out in 2006 at the OTD Ghezala Farm, Mateur (North Tunisia), which is

located at 37°3′ North latitude and 9°39′ East

longitudes.

Sixty multiparous lactating Holstein Friesian dairy cows (499 ± 19.7 kg BW, 170 ± 15 DIM and 18 ± 5.7 l/d milk yield) were used. Cows were housed in free

stalls with concrete surfaces and bedded with hay.

Farm management and diet composition was typical for the region with forage ratio of 63%, 53%, 59%, and 54% per Apr. 5 (D1), July 19 (D2), Aug. 19 (D3) and Sept. 19 (D4), respectively, on dry matter (DM) basis. The concentrate (8 kg/cow/day) was fed in five equal meals daily. Food and water were available ad libitum. Ingredients and chemical composition of diets fed to animals during the experiment are reported in Table 1.

Diets were defined including ingredients commonly used in Northern Tunisia. Crude protein and neutral detergent fiber content in diets ranged from 13.8 to 15.8% and 39.6 to 42.4% (on dry matter basis), respectively.

All cows were machine-milked in a herringbone parlor (Alpha Laval, The Netherlands) twice daily (2×) at 04:00 a.m. and 04:00 p.m. Routine milking included udder and teat cleaning as well as teat dipping in an iodine solution (Iodine, Veto Lab, Tunisia).

At the beginning of the study in summer, cows were classified according to udder cistern size as

large-cisterned (44 ± 13 cm2; LC) and small-cisterned

(21 ± 8 cm2; SC) (Table 1). Cisternal area was

measured according to the methodology described by Ayadi et al. [11]. Udder scans for the right front and rear quarters were performed in duplicate 8 to 10 h after the a.m. milking by using a real time B-mode ultrasonograph (Ultra Sound Scanner B7v; Noveko Echograph Inc., Quebec, Canada) equipped with a multi-frequency linear probes (7.5-2.6 MHz 2 dB

power; 80° scanning angle, 0.5 mm axial and 1.5 mm

lateral resolution).

The first sampling was carried out during spring (D1) when mean daily THI value was 62 ± 1.9. Further samplings were conducted during summer (D2, D3 and D4), when mean daily THI were 79 ± 2.3, 84 ± 2, and 77 ± 0.5, respectively. At each test day, rectal temperatures (RT) and respiratory rates (RR) were recorded. The RT was measured using a medical digital

thermometer (precision ± 0.01 ℃). The RR

(12)

334

Table 1 Ingredients and chemical composition of the total mixed ration diet (on dry matter basis) and means and standard deviation of production performance of cows during the experiment.

Item Apr. (D1) July (D2) Aug. (D3) Sept. (D4)

Feed ingredient %

Oat silage - 31.7 37.6 -

Corn silage 17.3 - - -

Alfalfa forage 9.2 13.9 13.9 13.26

Oat hay 6.7 7.3 7.8 -

Tritical ground green forage 18.0 - - -

Bersim green forage 12.0 - - -

Corn green forage - - - 24.1

Sorghum green forage - - - 17.0

Corn grain grind 11.0 11.9 13.4

Soybean meal 7.5 7.6 8.2 9.3

Barley grain 9.1 9.2 9.9 11.2

Wheat bran 7.2 7.3 7.9 8.9

Mineral 2.0 2.0 2.2 2.5

Corn grain - 9.1 - -

Calcium phosphate - 0.5 - -

Sodium bicarbonate - 0.3 0.4 0.4

Chemical composition

DM% 29.2 33.4 31.0 28.2

CP, % of DM 15.8 13.8 14.0 15.1

NDF, % of DM 40.8 39.6 42.4 41.9

NDF forages, % of DM 80.3 74.8 79.3 76.4

Starch, % of DM 18.5 23.4 19.1 21.2

NEL,Mcal/kg 1.5 1.5 1.5 1.4

Animals LC SC LC SC LC SC

Cows (n) 60 30 30 30 30 30 30

Age (m) 81 (20) 83 (20) 65 (25) 84 (20) 66 (25) 8 (20) 67 (25)

DIM (d) 80 (15) 170 (15) 169 (16) 200 (15) 199 (16) 230 (15) 229 (16)

Under Cisterns Size (cm2) - 44 (13) 21 (8) - - - -

Milk yield (L/d) 18 (8) 20 (5.2) 16 (5.8) 17 (6.6) 15 (5.8) 15 (5.4) 13(6) ( ) = Standard deviation; LC = Large cistern, SC = Small cistern.

2.2 Laboratory Analysis

As soon as blood was collected, smears were prepared using wrights- Giesma method (Fisher Scientific Company) for differential leucocytes profile.

Total leucocytes number was determined for each smears blood by light microscope. Every test day, collected blood was centrifuged at 3,000 rpm for 15 minutes and sera were collected and frozen in vials at

-20 ℃. Blood plasma was analyzed for total thyroxin

using RIA kit (Immunotech, IM 1447). The total

cortisol concentration was determined using the 125I

RIA kit (IM 1841).

2.3 Data Analysis

To estimate the effect of test day on RT, RR, T4, cortisol and leucocytes a mixed model was used:

Yijk = μ + ci + tj + (c × t)ij + ak + eijk

where yijk is the measured values of RT, RR,

(13)

335

thyroxin, and cortisol; μ is the model mean value, ci

fixed effect of cistern size, tj fixed effect of test day, (c × t)ij interaction cistern size-test day, ak effect of animal and eijk is the residual error. Observations within cows were considered random as repeated measurements. Eosinophil, monocyte, thyroxin and cortisol were transformed using the logarithmic link function, since the distributions of data were not normal.

All analyses were conducted using Jmp V 8.0.1 (SAS Institute Inc., 2004). Differences were

considered significant at P < 0.05.

3. Results and Discussion

According to Armstrong [13]and Johnson [14], heat

stress is considered absent when THI ≤ 72, mild when

THI is between 73 and 77, moderate when THI is between 78 and 88 and severe when THI > 88. In the present study, recorded THI in D1 of 62 (± 1.9) indicated absence of heat stress. As expected, heat stress occurred during D2, D3 and, D4 with THI values of 79 (± 2.3), 84 (± 2), and 77 (± 0.5), respectively (Table 2).

At high environmental temperatures cows attempted to restore their thermal balance. In this study, heat

stress altered (P < 0.001) RT and RR (Table 2). Rectal

temperature increased from D1 with 38.5 ℃ to D3 with

39.6 ℃. Recorded RT in D4 (38.6 ℃) was not different

(P > 0.05) from D1. The decrease of RT in D4

compared to D2 and D3 could be explained, at least in part, by the lower THI values in D4 compared to D2 and D3 and by the fact that cows in D4 were adapted to high temperatures. Respiratory rates increased from D1 with 55.7 breaths/min to D3 with 90.6 breaths/min. Measured RR in D4 (65.1 breaths/min) was not

different (P > 0.05) from D2. THI values in D2 and D4

were alike. When THI increased from 62 to 79, RT and

RR raised by 0.4 ℃ and 14 breaths/min, respectively.

In particular, RT and RR raised by 0.2 ℃ and 5

breaths/min respectively per increase of THI unit.

Cistern size did not affect RT and RR (P > 0.05).

Least square means and standard deviation values

for total T4 and cortisol concentrations are presented in Table 2. During summer, T4 concentration at D2 (87.4 nmol/L) was higher relative to concentrations at D3 (42.8 nmol/L) and D4 (53.5 nmol/L). Thyroid hormones are of utmost importance in the heat adaptation process, allowing the adjustment of the metabolic rates to favor body heat balance. Lu [15] reported reduced metabolism in cattle under heat stress, which was associated with reduced T4 concentration. Working with lactating cows, Johnson et al. [16] found a decline in triiodothyronine (T3) and T4 in response to heat stress. They attributed this decline in attempts, by the cow, to reduce metabolic heat production. Thompson [17] concluded that adaptation to high temperatures is followed by an increase of body temperature and a decreased thyroid activity. Thyroxin

concentrations were significantly higher (P < 0.05) in

cows with small cisterns (67.7 nmol/L) compared to cows with large cisterns (52.7 nmol/L). Ayadi et al. [11] reported a positive correlation of cistern size to milk production. High yielding dairy cows might need to reduce body heat production to a large extent relative to low producing cows [8], because they produce more heat than low yielding cows. However, T4 concentrations are also negatively correlated to milk production [18], and response in T4 to cistern size could be explained by both, different susceptibility to heat stress and different milk production.

In the present study, cortisol concentrations of 25.0, 37.3, 28.9, and 31.0 nmol/L in D1, D2, D3 and D4, respectively, were neither affected by test day nor by

cistern size (P > 0.05). These results are in accordance

(14)

336

Table 2 Heat stress and cistern size (LC: Large cistern, SC: Small cistern) effects on rectal temperature (RT), respiration rates (RR), plasma thyroxin (T4) and cortisol concentrations and Leucocytes percentages.

Item

Test Day (TD) Cistern Size _Effect

Apr. 5 (D1) July 19 (D2) Aug. 19 (D3) Sept. 19 (D4) LC _SC

TD Cis TD × Cis LSM1 SE2 LSM SE LSM SE LSM SE LSM SE LSM SE

Environment

Ambient temperature (℃) 16.9 1.3 30.6 2.4 33.5 2.8 29.0 0.7 - - - - - - -

Relative humidity (%) 76.4 5.5 52.1 6.9 59.1 10.8 51.7 3.2 - - - - - - -

THI3 62.0 1.9 79.0 2.3 84.0 2.0 77.0 0.5 - - - - - - -

Animal response

Rectal temperature (℃) 38.5c 0.1 38.9b 0.1 39.6a 0.1 38.6c 0.1 38.8a 0.1 39.0a 0.1 *** ns ns Respiratory rate (breaths/min) 55.7c 3.3 70.0b 2.8 90.6a 2.7 65.1b 2.7 68.4a 2.4 72.3a 2.5 *** ns ns Heart rates (beats/min) 97.7a 2.3 80.3c 2.0 86.2b 1.9 84.5bc 1.9 84.5a 1.6 89.9b 1.6 *** * ns

Hormones

Thyroxin (T4) (nmol/L) - - 87.4a 0.1 42.8b 0.1 53.5b 0.1 52.7a 0.7 67.7b 0.1 *** ** ns Cortisol (nmol/L) 25.0a 0.3 37.3a 0.1 28.9a 0.1 31.0a 0.1 28.4a 0.1 32.3a 0.1 ns ns ns

Leucocytes

Lymphocyte (%) 73.6a 2.7 64.7b 1.1 65.6b 1.3 60.4c 1.1 65.4a 1.2 66.8a 1.2 *** ns ns Neutrophil (%) 23.4ab 2.7 20.0b 1.0 22.8b 1.3 26.8a 1.1 23.9a 1.2 22.6a 1.2 *** ns ns Eosinophil (%) 1.2c_{2.2 11.5}a_0.9 _4.7c_1.1_8.2b_0.9_6.3a _{1.0 6.5}a_{0.9 *** ns ns}

Monocyte (%) 1.8c 1.0 3.8b 0.4 5.1a 0.5 4.6ab 0.4 3.5a 0.4 4.1a 0.4 * ns ns *** P < 0.001; ** P < 0.01; * P < 0.05; ns = not significant (P > 0.05); Cis = Cistern;

a,b,c

Means within row per Test day and Cistern size not sharing the same superscript are significantly different; 1

LSM = Least square means; 2SE = Standard error; 3THI (Temperature Humidity Index) = 1.8 × T – (T – 14.3) × (100 – H) / 100 + 32.

cortisol concentration.

High variability in response of cortisol to heat stress reported in literature, as well as the lack of response in the present study, might be explained at least in part, by the fact that other parameters than heat stress might be able to alter cortisol levels. As well, stress in general can alter cortisol concentration. Other stress factors occurred in this trial might have influenced individual cow responses, such as the experiment procedure itself (e.g. blood sampling) could have influenced cortisol response of cows [22].

Lymphocytes, neutrophils, eosinophils and monocytes, relative to total number of leucocytes, were

affected by period (P < 0.001). Percentage of

lymphocytes decreased from D1 (73.6%) to D4 (60.4%), whereas in D2 and D3 were intermediate, with 64.7% and 65.6%, respectively.

Percentage of leucocytes types did not vary

according to cistern size (P > 0.05). The lymphocytes

count depression during summer suggested decrease of the immune system activity. Both factors, the duration of exposure, and the severity of heat stress, decrease immune activity [23]. In the present study THI values in D4 were lower relative to D2 and D3, but lymphocytes were lower in D4 compared to D2 and D3. These results are in accordance with Kamwanja [7]. This study reported that in vitro exposure of bovine

lymphocytes to an ambient temperature of 45 ℃ for 3

hours decreased the number of viable cells. However, our results are conflicting with those studies where no

effect [24]or an improvement [25, 26] were reported.

Lee et al. [26] showed that high ambient temperature caused leucocytosis in cattle. The percentage of white blood cells types varied at high ambient temperatures. In the present study, Neutrophils (%) least square means in D1, D2, D3, and D4, respectively, were as

follows (superscripts differ by P < 0.05): 23.4ab, 20.0b,

(15)

337

D1, D2, D3, and D4, respectively, were as follows: 1.2c,

11.5a, 4.7c, and 8.2b. Moncytes (%)least square means

in D1, D2, D3, and D4, respectively, were as follows:

1.8c, 3.8b, 5.1a, and 4.6ab. Brouček et al. [27] found a

decrease in neutrophil and eosinophil and an increase in lymphocytes and monocytes. In our study, an increase in neutrophil count from D2 and D3 to D4 and a fall in lymphocyte count from D1 to D4 occurred. Lee et al. [26] noted a tendency of eosinophil count increase in dairy cows exposed to high temperature. In the present study, eosinophil (%) was highest at D2 when THI was highest. Differences observed in the present study relative to literature could be in part attributed to the fact that we were not able to measure absolute numbers of leucocytes.

Lacetera et al. [28] indicated that the depression of the immune system activity observed in hot environment may have an impact on the occurrence of

diseases. Smith et al. [29]and Morse et al.[30] reported

an increase in mastitis infections at high environmental temperatures. High temperature measured during summer in Tunisia could be responsible for the decrease of the cow resistance to diseases. In order to contain economic losses of dairy farming during summer, hygienic conditions and milking procedures should be attended.

4. Conclusion

In the present study T4 levels in dairy cows decreased under prolonged exposure at high ambient temperatures and large cisterned cows had lower T4 levels compared to small cisterns. Cortisol concentrations might not be a sensible parameter to detect heat stress if other stress factors are not controlled.

Reduced lymphocyte (%) measured in the present study during the hot period suggest major attention on Tunisian dairy farms especially during summer months.

Management strategies are needed to minimize heat stress and restores the physiological responses to

normal values. More emphasis should be placed on the influence of heat stress on the immune system of dairy cows. Moreover, additional studies might also determine whether or not it is possible to establish upper critical values of THI above which the immunological functions of dairy cows start to change.

References

[1] R. Bouraoui, A. Majdoub, M. Djemali, Study of heat stress effect in the Tunisian conditions, through the calculation of the temperature humidity index, in: Book of Abstracts, EAAP-50th Annual Meeting, Zurich, Aug. 22-26, 1999, p. 217. (in French)

[2] F. Ferreira, M.F.A. Pires, M.L. Martinez, Physiologic parameters of crossare cattle subjected to heat stress, Arquivo Brasileiro Medicina Veterinaria e Zootecnia 58 (2006) 732-738. (in Portuguese)

[3] A.A.M. Habeeb, I.F.M. Marai, T.H. Kamal, Heat stress, in: C. Phillips, D. Pigginns (Eds.), Farm Animals and the Environment, CAB International, Wallingford, UK, 1992, pp. 27-47.

[4] A. Magdub, H.D. Johnson, R.L. Belyea, Effect of environmental heat and dietary fiber on thyroid physiology of lactating cows, J. Dairy Sci. 65 (1982) 2323-2331.

[5] D.K. Beede, R.J. Collier, Potential nutritional strategies for intensively managed cattle during heat stress, J. Anim. Sci. 62 (1986) 543-550.

[6] G.I. Christison, H.D. Johnson, Cortisol turnover in heat stressed cows, J. Anim. Sci. 35 (1972) 1005-1010. [7] L.A. Kamwanja, C.C. Chase, J.A. Gutierrez, V. Guerriero,

T.A. Olson, A.C. Hammond, et al., Responses of bovine lymphocytes to heat shock as modified by breed and antioxidant Status, J. Anim. Sci. 72 (1994) 438-444. [8] H. Barash, N. Silanikov, A. Shamay, E. Ezrat,

Internationships among ambient temperature, day length, and milk yield in dairy cows under a Mediterranean climate, J. Anim. Sci. 84 (2001) 2314-2320.

[9] K. Stelwagen, C.H. Knight, V.C. Farr, S.R. Davis, C.G. Prosser, T.B. McFadden, Continuous versus single drainage of milk from the bovine mammary gland during a 24 hour period, Experimental Physiology 81 (1996) 141-149.

(16)

338

[11] M. Ayadi, G. Caja, X. Such, C.H. Knight, Use of ultrasonography to estimate cistern size and milk storage at different milking intervals in the udder of dairy cows, J. Dairy Res 70 (2003) 1-7.

[12] H.H. Kibler, Thermal effects of various temperature-humidity combinations on Holstein cattle as measured by eight physiological responses University of Missouri, Agricultural Experiment Station, Research Bulletin 862 (1964) 1-42.

[13] D.V. Armstrong, Heat stress interaction with shade and cooling, J. Dairy Sci. 77 (1994) 2044-2050

[14] H.D. Johnson, Bioclimate effects on growth, reproduction and milk production, in: H.D. Johnson (Ed.), Bioclimatology and the Adaptation of Livestock, Elsevier, Amsterdam, 1987, pp. 33-57.

[15] C.D. Lu, Effect of heat stress on goat production, Small. Rum Res. 2 (1989 ) 151-162.

[16] H.D. Johnson, P.S. Katti, L. Hahn, M.D. Shanklin, Short-term heat acclimation effects on hormonal profile of lactating cows, in: Research Bulletin University of Missouri Columbia, 1988, p. 1061.

[17] G.E. Thompson, Review of the progress of dairy science climatic physiology of cattle, J. Dairy Res. 40 (1973) 441-473.

[18] T. Tiirats, Thyroxine, triiodothyronine and reverse-triiodothyronine concentrations in blood plasma in relation to lactational stage, milk yield, energy and dietary protein intake in Estonian dairy cows, Act Vet Scand 38 (1997) 339-348.

[19] H.D. Johnson, R. Li, W. Manalu, K.J. Spencer-Johnson, B.A. Becker, R.J. Collier, et al., Effects of somatotropin on milk yield and physiological responses during summer farm and hot laboratory conditions, J. Dairy Sci. 74 (1991) 1250-1262.

[20] F. Elvinger, R.P. Natzke, P.J. Hansen, Interactions of heat stress and bovine somatotropin affecting physiology and immunology of lactating cows, J. Dairy Sci. 75 (1992) 449-462.

[21] A. Correa-Calderon, D. Armstrong, D. Ray, S. DeNise, M. Enns, C. Howison, Thermoregulatory responses of Holstein and Brown Swiss heat-stressed dairy cows to two different cooling systems, Int. J. Biometeorol. 48 (2004) 142-148.

[22] A, Fonss, L. Munksgaard, Automatic blood sampling in dairy cows, Computers and Electronics in Agriculture 64 (2008) 27-33.

[23] K.W. Kelley, Immunobiology of domestic animals as affected by hot and cold weather, in: Proceedings of the Second International Livestock Environment Symposium, ASAE Publ. No. 3-82, St. Joseph, Michigan, 1982, pp. 470-483.

[24] N. Lacetera, U. Bernabucci, B. Ronchi, D. Scalia, A. Nardone, Moderate summer heat stress does not modify immunological parameters of Holstein dairy cows, Int. J. Biome. 46 (2002) 33-37.

[25] F. Soper, C.C. Muscoplat, D.W. Johnson, In vitro

stimulation of bovine peripheral blood lymphocytes: Analysis of variation of lymphocyte blastogenic response in normal dairy cattle, Am J Vet Res 39 (1978) 1039-1042. [26] J.A Lee, J.D. Roussel, J.F. Beatty, Effect of temperature season on adrenal cortical function, blood cell profile and milk production, J. Dairy Sci. 59 (1976) 104-108. [27] J. Brouček, M. Kovalčiková, K. Kovalčik, P. Fľak,

Reaction haematological indicators of dairy cows to high temperature, Poľnohospodárstvo 30 (1984) 163-172. (in Czech)

[28] N. Lacetera, U. Bernabucci, D. Scalia, B. Ronchi, G. Kuzminsky, A. Nardone, Lymphocyte functions in dairy cows in hot environment, Int. J. Biome 50 (2005) 105-110. [29] K.L. Smith, D.A. Todhunter, P.S. Schoenberger,

Environmental mastitis, cause, prevalence, prevention, J. Dairy Sci. 68 (1985) 1531-1553.

(17)

Occurrence of Extended-Spectrum

β

-lactamase

Producing Enterobacteriaceae (ESBLPE) among

Primary School Pupil in Obafemi-Owode, Nigeria

Akinduti Paul Akinniyi1, Akinbo John Adeolu2, Adenuga W. Funmilayo3, Ejilude Oluwaseun4, Umahoin

Kingsley Omokhudu5 and Ogunbileje John Olusegun6

1. Department of Medical Microbiology, Olabisi Onabanjo University, Ago-Iwoye, Ogun State 102107, Nigeria

2. Department of Pathology, Federal Medical Center, Abeokuta, Ogun State 102107, Nigeria

3. Department of Community Medicine, Federal Medical Center, Abeokuta, Ogun State 102107, Nigeria

4. Microbiology Laboratory, Sacred Heart Hospital, Abeokuta, Ogun State 102107, Nigeria

5. Department of Chemical Pathology & Immunology, Olabisi Onabano University, Ago-Iwoye, Ogun State 102107, Nigeria

6. Department of Chemical Pathology, University of Ibadan, Ibadan, Ogun State 102107, Nigeria

Received: March 31, 2010 / Accepted: June 21, 2010 / Published: May 30, 2011.

Abstract: Occurrence of extended-β-lactamase producing enterobacteriaceae (ESBLPE), which has reduced the antibacterial efficacy and potency of many 3rd generation cephalosporins, was investigated among the primary school pupils. 88 primary school pupils in Obafemi-Owode Local Government , Southwestern Nigeria, including 49 males (55.7%) and 39 females (44.3%) (mean age 12 ± 3) were screened for ESBLPE isolates with exclusion criterion of antimicrobial use in the preceding 2 weeks either as therapy for gastro-intestinal complication or prophylaxis. ESBLPE detected include 4.5% of Eschericia coli, 2.3% of Enterobacter cloaca, 0% Proteus mirabilis, 2.3% Pseudomonas aeruginosa, 1.1% Staphylococcus aureus and 4.5% of Klebsiellaoxytoca. 10 (76.9%) of ESBLPE isolates were resistant to disc of cefuroxime (30 μg), 8 (61.5%) susceptible to amoxicillin/clavulanic (20/10 μg) and low susceptibility of 7 (53.8%) was recorded for ceftazidime (30 μg). 0% susceptibility was recorded for the ESBLPE isolates to cefuroxime MIC > 8 μg/mL and ampicillin MIC > 8 μg/ mL while E. coli and E. cloca each show 50.0% and P. aeruginosa and K. oxytoca show 100.0% and 75.0% susceptibility to augmentin (MIC ≤ 8). This study has shown a 14.7% proportion of the pupil to harbour ESBLPE from enteric source with increased resistant to most new generation cefuroximes. Therefore, transfer of virulent and antibiotic resistant ESBLPE could be aided by sharing feeding materials while fecal-oral route of transmission cannot be ruled out as hygiene level is very low thereby increasing emergence of virulent resistant enteric strains leading to treatment failure.

Key words: Extended-spectrum β-lactamase producing enterobacteriaceae, cephalosporin, pupil.

1. Introduction

Increasing emergence of extended-betalactamase enterobacteriaceae producer (ESBLPE) in recent years has reduced the antibacterial efficacy and potency of many cephalosporin and other antibiotics due to various resistance mechanisms. It is, however,

Corresponding author: Akinduti Paul Akinniyi, FMLSCN, M.Sc., research field: antibiotic resistance. E-mail: niyiakinduti @yahoo.com.

important to continuously monitor emerging

betalactamase resistance mechanisms on a routine basis mostly among the under-aged school pupil in

developing countries [1]. Originally K. pneumoniae

and E. coli were the most common ESBL-producing

bacteria worldwide. Although in recent years, ESBL

production amongst Proteus mirabilis and

AmpC-producing Enterobacteriaceae has become

(18)

Occurrence of Extended-Spectrum ß-lactamase Producing Enterobacteriaceae (ESBLPE) among Primary School Pupil in Obafemi-Owode, Nigeria

340

are contained on plasmids, horizontal gene transfer in many species of bacteria is as well becoming prevalent. ESBL production has rarely been

transferred to non-Enterobacteriaceae; but

ESBL-producing Pseudomonas aeruginosa and

Acinetobacterspp. have been reported in Europe [4-6].

Extended-spectrum β-lactamase-producing

Enterobacteriaceae (ESBLPE) were first described in

Europe in 1983 but in the subsequent 20 years, bacteria with this resistance mechanism have become increasingly important [7]. ESBLPE are not only resistant to penicillins and cephalosporins but are often also resistant to a wide range of other antibiotic classes (including fluoroquinolones, aminoglycosides, and trimethoprim/sulfamethoxazole) due to accumulation of other resistance genes [7-9]. This limits effective treatment options for enteritis and other gastrointestinal infections [10]. This study was undertaken to assess the occurrence and colonisation of ESBLPE in gastrointestinal tract of primary school pupils, who are more predisposed to unhygienic food materials and indiscriminate use of antibiotics which are peculiar to their demographic setting.

2. Materials and Methods

Study population: A total of 88 stool samples collected from primary school pupils in Obafemi-Owode Local Government, located at rain

forest belt, Southwestern Nigeria, latitude 7°10′49.46″

N and longitude 3°26′11.98″ E [11], were screened for

β-lactamase producing enterobacteria isolates. The

only exclusion criterion for participation in the study

wasthe use of antimicrobial agents in the preceeding 2

weeks either as therapy for gastro-intestinal complication or prophylaxis.

Isolation and serology test: Stool samples were collected into sterile universal container and stored at

4 ℃ until they were cultured on chocolate agar

containing 1 mg/L Ampicillin [12] and MacConkey agar with salt (Oxoid CM 516, UK) and each organisms was confirmed by colonial morphology,

Gram staining,oxidase, catalase tests, and the sugar

fermentation test according to Stoke protocol [13].

β-Lactamase test: All isolates were tested for

β-lactamase production by a starch-iodide paper

acidometric method as described by Odugbemi et al., 1977 [14].

ESBL Detection/Double Disc Synergy Test (DDST): The organisms were considered to be ESBLPE when the zone of inhibition around Cefuroxime and Ceftazidime (expanded-spectrum cephalosporin discs) showing a clear-cut increase towards the Augmentin disc. Distances between the discs were suitably adjusted between 16 and 20 mm for each strain in order to accurately detect the synergy [15, 16].

Conjugation: The isolate resistant to at least one extended-spectrum cephalosporin, which was positive by the DDST testing, was subjected to conjugation test as previously described [17]. Transconjugants growing in the selection plates were subjected to DDST to confirm the presence of ESBL genes and were examined for the co-transfer of other antibiotic resistance determinants present in the donor isolates.

Disc diffusion test: Antibiotic disc containing

ampicillin (10 µg), amoxicillin/clavulanic acid (20

µg/10µg), cefuroxime (30 µg), ceftazidime (30 µg), cefpodoxime (10 µg), cefpodoxime/clavulanic acid (10 µg/1µg), imipenem (10 µg), gentamicin (10 µg),

ciprofloxacin (5 µg), ofloxacin (5 μg), norfloxacin (10

μg), pefloxacin (10 μg), azithromycin (5 μg),

amoxicillin (10 μg), tetracycline (30 μg), (from Oxoid,

Hampshire, UK) were used by Kirby-Baure method [18] and inhibition zones were determined according to respective CLSI guidelines (Formerly NCCLS) on nutrient agar (Oxoid CM 543) [19].

Minimum inhibitory concentration (MIC): MICs of augmentin, cefuroxime, ceftazidime, ampicillin, gentamycin, imipenem, ofloxacin, pefloxacin, and ciprofloxacin were determined by broth dilution method. Two fold serial dilutions of antibiotics in 1%

(19)

341

incubated in 5% CO2 overnight. The end pointwas

read as the lowest concentration of antimicrobial agent

giving complete inhibition of growth by colour

changes to 0.25% phenol red [20]. Control strain of E.

coli ATCC 25532, S. aureus ATCC 25534 and P.

aeruginosa ATCC 25543, whose MICs were known,

were included as control in each test. The

antimicrobial susceptibility was judged by breakpoint

criteriadefined by the National Committee for Clinical

Laboratory Standards(NCCLS) [21].

Phenotypic disc confirmatory test: This test was performed as the disc diffusion test as recommended

by CLSI (formerly NCCLS). A ≥ 5 mm increase in

zone diameter for either cefuroxime tested in combination with clavulanic acid, versus its zone diameter when tested alone, confirmed an

ESBL-producing organism. E. coli (ATCC 25922)

and K. pneumoniae (ATCC 700603) were used as

negative and positive control reference strains, respectively.

Correlation of MIC and Disc inhibition zone: The correlation of the MIC to the zone of inhibition and the correlation coefficient were calculated by the

method of leastsquares, with the zone diameter as the

independent variable (x-axis) and the MICs as the dependent variable (y-axis).

3. Results

Surveillance for the occurrence of ESBLPE in Obafemi-Owode primary school pupils were based on their use of antimicrobial agents in the last two weeks among 49 males (55.7%) and 39 females (44.3%), which shows that ESBLPE (Table 1) was detected in

4.5% of E. coli, 2.3% of Enterobacter spp., 0% P.

mirabilis, 2.3% P. aeruginosa, 1.1% S. aureus and

4.5% of K. oxytoca among the pupil examined. By

disc diffusion in Table 2, 10 (76.9%) of ESBLPE were

resistant to cefuroxime (30 μg), 3 (23.1%) susceptible

to amoxicillin/clavulanic (20/10 μg) and low

susceptibility of 7 (53.8%) was recorded for

ceftazidime (30 μg). From Table 3, most isolates were

Table 1 Proportion of β-lactamase producing

Enterobacteriae (ESBLPE) isolates. Organism Ampicilin agar

growth susceptible isolates. The same as Table 2.

Table 2 Antibiotic disc susceptibility test for 13 ESBLPE isolates.

resistant to cefuroxime (MIC ≥ 8) and ampicillin

(MIC ≥ 8), 1 (25%) E. coli, 1 (50%) P. aeruginosa, 1

(25%) K. oxytoca and 1 (50%) E. aerogene had

reduced susceptibility (MIC ≥ 8) to ceftazidimie

respectively. P. aeruginosa and S. aureus were 100%

susceptible to Augmentin (MIC ≤ 8) while 3 (75%) K.

oxytoca, 2 (50%) E. coli and 1 (50%) E. aerogenes

were susceptible. The MIC and ESBL results were interpreted in accordance with NCCLS criteria (M 100-S 11). Fig. 1 shows percentage proportion of betalactamase and ESBLPE isolates.

4. Discussion

(20)

342

Table 3 MIC of all ESBLPE isolates. Antibiotic MIC reference

value μg/mL

Susceptible ESBLPE isolates (n/N (%))

E. coli P. mirabilis P. aeruginosa S. aureus K. oxytoca E. cloca

Imipenem ≤4 4/4 (100) 0/0 (0) 2/2 (100) 1/1 (100) 3/4 (75) 2/2 (100)

Azithromycin ≤8 3/4 (75) 0/0 (0) 2/2 (100) 1/1 (100) 2/4 (50) 2/2 (100) Ofloxacin ≤8 4/4 (100) 0/0 (0) 2/2 (100) 1/1 (100) 3/4 (75) 2/2 (100)

Gentamicin ≤8 1/4 (25) 0/0 (0) 1/2 (50) 1/1 (100) 1/4 (25) 1/2 (50)

Augmentin ≤8 2/4 (50) 0/0 (0) 2/2 (100) 1/1 (100) 3/4 (75) 1/2 (50)

Pefloxacin ≤8 4/4 (100) 0/0 (0) 2/2 (100) 1/1 (100) 4/4 (100) 2/2 (100)

Cefuroxime ≤8 0/4 (0) 0/0 (0) 0/2 (0) 0/1 (0) 0/4 (0) 0/2 (0)

Ceftazidime ≤8 1/4 (25) 0/0 (0) 1/2 (50) 0/1 (0) 1/4 (25) 1/2 (50)

Ampicillin ≤8 0/4 (0) 0/0 (0) 0/2 (0) 0/1 (0) 0/4 (0) 0/2 (0)

Teteracyclin ≤16 2/4 (50) 0/0 (0) 0/2 (0) 1/1 (100) 1/4 (25) 0/0 (0)

N = total number of the isolates; % = percentage susceptibility.

Fig. 1 Proportion of beta-lactamase and ESBLPE isolates. Correlation is significant at the 0.01 level (2-tailed).

such as severe diarrhoea, acute enteritis and paratyphoid diseases. Heseltine (2000) also reported that community-acquired strains possessing ESBLs might be selected from the existing gastrointestinal flora when it was exposed to broad-spectrum antimicrobial agents [22]. Kumar et al. reported 19.2%

of E. coli isolates and 21.2% of K. pneumoniae

isolates as ESBL producers [23] from enteric source. Previous use of various antibiotics was a well-recognised risk factor for colonisation and infection with an ESBLPE. Hospital- and community-based studies had found an association with previous use of penicillin, a second- or third-generation cephalosporin, or a fluoroquinolone

and ESBLPE infection [24]. Marked resistance of E.

coli, P. aeruginosa, P. mirabilis and S. aureus to

cefuroxime suggest a high predictive failure in the use of third-generation cephalosporins and inappropriate

use of these antibiotics might be contributing to the recent increase in ESBLPE in this locality where invalid prescription was made to further increase resistant strain [25].

The transfer of ESBLPE to non-colonised individuals in some primary schools via side-road food vendors are necessary transmission route of ESBLPE in these facilities. Thus the prevalence of

ESBLs among members of Enterobacteriaceae

constitutes a serious threat to the current beta-lactame therapy, leading to treatment failure and consequent escalation of costs. There is an urgent need to emphasize rational use of drugs to minimize the misuse of available antimicrobials.

In summary, transfer of virulent and antibiotic

resistant E. coli and other enteric isolates could be

aided by sharing feeding materials while fecal-oral route of transmission cannot be ruled out as hygiene level is very low. Indiscriminate prescription and use of beta-lactame drugs should be checked to prevent emergence of virulent resistant enteric strains.

Statements

Ethical consideration: Permission to carry out this study in public primary school in Obafemi-Owode, Local Government Area, Nigeria was obtained from Universal Basic Education Board, Ogun State, Nigeria. Ethical approval was given by the Health Research Committee of Federal Medical Centre, Abeokuta, Nigeria.

(21)

343

Neither financial nor personal relationships with other people or organisations inappropriately influence our work in any way. This study was self sponsor. No financial obligation was owned to any individual or establishment during or after the study.

References

[1] B. Simon, U. James, T. Susan, Extended-spectrum betalactamase producing enterobacteriacea at Middlemore Hospital, J. New Zealand Med. Assoc. 118 (1218) (2005) 61-70.

[2] C. Sang-Ho, E.L. Jung, P. Su Jin, C. Seong-Ho, L. Sang-Oh, J. Jin-Yong, et al., Emergence of antibiotic resistance during therapy for infections caused by

Enterobacteriaceae producing AmpC β-lactamase: Implications for antibiotic use, Antimicro Agents and Chemotherapy 52 (3) (2008) 995-1000.

[3] H. Knothe, P. Shah, V. Krcmery, Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens, Infection 11 (1983) 315-317.

[4] L. Poirel, E. Lebessi, M. Castro, Nosocomial outbreak of extended-spectrum beta-lactamase SHV-5-producing isolates of Pseudomonas aeruginosa in Athens, Greece, Antimicrob Agents Chemother 48 (2004) 2277-2279. [5] H. Vahaboglu, R. Ozturk, G. Aygun, Widespread

detection of PER-1-type extended-spectrum beta-lactamases among nosocomial Acinetobacter and

Pseudomonas aeruginosa isolates in Turkey: A

nationwide multicenter study, Antimicrob Agents Chemother 41 (1997) 2265-2269.

[6] V. Jarlier, M. Nicolas, G. Fournier, A. Philippon, Extended spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae: Hospital prevalence and susceptibility patterns, Rev. Infect. Dis. 10 (1988) 867-878.

[7] K. Guillaume, P. Isabelle, V. Sabine, C. Nathalie, B. Annie, G. Laurent, Extended-spectrum β-lactamase in

Enterobacteriaceae, LabLink, Institute of Environmental Science and Research Limited, 1994, pp. 1-6.

[8] S. Nathisuwan, D.S. Burgess, J.S. Lewis, ESBLs: Epidemiology, detection and treatment, Pharmacotherapy 21 (2001) 920-928.

[9] E. Sturenburg, D. Mack, Extended-spectrum beta-lactamases: Implications for the clinical microbiology laboratory, therapy and infection control, J. Infect. 47 (2003) 273-295.

[10] U. Chaudhary, R. Aggarwal, Extended spectrum beta lactamases: An emerging threat to clinical therapeutics, Indian J. Med. Microbiol. 22 (2004) 75-80.

[11] National Population Commission (NPC), National Census Sentinell, Nigeria, 2007.

[12] H.M. Palmer, J.P. Leeming, A.A. Turner, Multiplex polymerase chain reaction to differentiate β-lactamase plasmids of Neisseria gonorrhoeae, J. Antimicrob. Chemother. 45 (2000) 777-782.

[13] J.E. Stokes, G.L. Rigway, Clinical Bacteriology Ch 7, 5th ed., Edward Arnold Publisher, 1980.

[14] T.O. Odugbemi, S. Hafiz, M.G. McEntegart, Penicillase producing Neisseria gonorhoae: Detection by starch iodide paper techniquies, Br. Med. J. 11 (1977) 500. [15] K.S. Thomson, C.C. Sanders, Detection of

extended-spectrum beta-lactamases in members of the family Enterobacteriaceae: Comparison of the double-disk and three dimensional test, Antimicrob Agents Chemother 36 (1992) 1877-1882.

[16] E. Sturenburg, D. Mack, Extended-spectrum beta-lactamases: Implications for the Clinical Microbiology Laboratory, therapy and infection control, J. Infect. 47 (2003) 273-295.

[17] A. Petra, A. Ojan, D. Florian, M.H. Alexander, L.R. Manfred, M. Athanasios, Extended double disc synergy testing reveals a low prevalence of extended-spectrum ß-lactamases in Enterobacter spp. in Vienna, Austria. J. Antimicrobial Chemotherapy 59 (5) (2007) 854-859. [18] Clinical and Laboratory Standards Institute, Guidelines

by CLSI/NCCLS - CLSI Informational Supplement, Approved Standard M100-S15 Wayne, PA, 2005.

[19] Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing: 16th Informational Supplement M100-S16, Vol. 26, No. 3, 2006.

[20] P.A. Akinduti, J.O. Akinbo, O.A. Ejilude, M.I. Mannie-udoh, K.O. Umahoin, J.O. Ogunbileje, et al., Prevalence of enterohaemorrhagic E. coli 0157: H7 causing severe urinary tract infection, Abeokuta, Nigeria, J. of American Science 4 (2) (2007) 4-9.

[21] National Committee for Clinical Laboratory Standards, Performance Standards for Antimicrobial Susceptibility Testing: 5th Informational Supplement, Document M 100-55, Vol. 14, No. 16, 1994.

[22] P. Heseltine, Has resistance spread to the community?, Clin. Microbiol. Infect. 6 (2000) 11-16.

[23] M.S. Kumar, V. Lakshmi, R. Rajagopalan, Occurrence of extended spectrum beta lactamases among

Enterobacteriaceae spp. isolated at a tertiary care institute, Indian J. Med. Microbiol. 24 (2006) 208-211. [24] J. Rodriguez-Bano, M.D. Navarro, L. Romero,

Epidemiology and clinical features of infections caused by extended-spectrum beta-lactamase-producing E. coli

in nonhospitalised patients, J. Clin. Microbiol. 42 (2004) 1089-1094.

[25] I. Aibinu, T. Odugbemi, B.J. Mee, Extended-spectrum beta-lactamases in isolates of Klebsiella spp. and E. coli