Ong Pek Geck, Frederick Adzitey, Raja Arief Deli, Nurul Huda and Gulam Rusul Rahmat Ali

Veterinary Wrld, EISSN: 2231-0916 Available at www.veterinarywrld.rg/vl.7/july-2014/7.pdf RESEARCH ARTICLE Open Access Micrbial quality f culled chicken layers in Penang, Malaysia 1 Ong Pek Geck, Frederick Adzitey, Raja Arief Deli, Nurul Huda and Gulam Rusul Rahmat Ali 1. Fd Technlgy Prgramme, Schl f Industrial Technlgy, Universiti Sains Malaysia, USM 11800, Penang, Malaysia; 2. Animal Science Department, Faculty f Agriculture, University fr Develpment Studies, Bx TL 1882, Tamale, Ghana Crrespnding authr: Gulam Rusul Rahmat Ali, email: gulam@usm.my, Tel: +60122103046 Fax: +6046573678. OPG: peggygeck@htmail.cm, FA: adzitey@yah.c.uk, RAD: deckwan@gmail.cm, NH: nrlhd@usm.my, GRRA: gulam@usm.my Received: 02-04-2014, Revised: 05-05-2014, Accepted: 05-06-2014, Published nline: 11-07-2014 di: 10.14202/vetwrld.2014. 478-482 Hw t cite this article: Geck OP, Adzitey F, Deli RA, Huda N and Ali GRR (2014) Micrbial quality f culled chicken layers in Penang, Malaysia, Veterinary Wrld 7(7): 478-482. Intrductin Culled chicken layers r spent hens can be defined as the identificatin and remval f nn-laying, unprductive r lw prducing hens frm a laying flck. Due t the little value f culled chicken layers, they are usually slaughtered at the end f their prductive lives. Hens can supply eggs fr tw t three years befre being regarded as spent hens, but a depressin f egg prices can shrten this time [1]. Pullets prduced mre eggs and utilized their feed mre efficiently than spent layers althugh eggs frm spent layers were heavier than that f pullets [2]. Fr the egg industry, culled chicken layers are cnsidered as ne f the by-prducts and these birds have lw price [3]. Owing t the frmatin f large amunts f heat stable cllagen caused by aging, the muscles f culled chicken layers becme tugh and unfavurable. These muscles have lw functinal prperties and reduce its usefulness in whle meat fd as well as its market value [4]. The tugh texture f culled chicken layer meat causes them t be less ppular amng cnsumers. Hwever, the demand fr spent hens can increase during festive ccasins amng culled chicken layer 1,2 1 1 1 Abstract Aim: T determine the micrbial quality f culled chicken layers in Penang, Malaysia. Materials and Methds: Samples were btained frm three layer farms (designated as Farm A, Farm B and Farm C). A ttal f 67 culled chicken layer samples cnsisting f egg wash water, chicken carcass rinse, drinking water, claca swab, feed and faeces were examined fr enterbacteriaceae, ttal and faecal clifrms, and Escherichia cli using the prcedures in the bacterilgical analytical manual. 3 7 2 Results: The ttal plate cunt fr bacteria ranged frm 2.7 x 10 cfuml t 1.8 x 10 cfug (Farm A), <1.0 x 10 cfuml t 1.7 x 6 2 6 2 10 cfug (Farm B) and <1.0 x 10 cfuml t 3.1 x 10 cfug (Farm C). Enterbacteriaceae cunt ranged frm <1.0 x 10 cfuml 6 2 6 2 6 t 3.5 x 10 cfug (Farm A), <1.0 x 10 cfuml /cfug t 1.2 x 10 cfug (Farm B) and <1.0 x 10 cfuml /cfug t 2.8 x 10 cfug (Farm C). Ttal and faecal clifrms ranged frm <3.0 t >1100.0 Mst prbable number (MPN)ml / MPNg fr Farm A, B and C. Similarly, E. cli cunt fr all the three farms ranged frm <3.0 t >1100.0 MPNml /MPNg. E. cli cunts were very lw fr mst f the samples examined except chicken carcass and faeces. Cnclusin: In general, Farm A had higher bacterial cunt, fllwed by Farm C and Farm B. This wrk gives an indicatin that pathgenic fdbrne pathgens such as E. cli, Salmnella, Citrbacter, Enterbacter, Klebsiella, Shigella andyersinia spp. may be present in culled chicken layers and cnsequently pse the risk f causing fd pisning r utbreaks. Keywrds: clifrms, culled chicken layers, E. cli, enterbacteriaceae, ttal plate cunt. Cpyright: The authrs. This article is an pen access article licensed under the terms f the Creative Cmmns Attributin License (http://creativecmmns.rg/licenses/by/2.0) which permits unrestricted use, distributin and reprductin in any medium, prvided the wrk is prperly cited. cnsumers. Ariff [5] reprted that the lack f briler chickens supply in the market t fulfill cnsumers demand as well the tremendus increase in the price f briler chickens during festivals frced many peple t pt fr spent hens instead f brilers. Furthermre, if meats frm culled chicken layers are prcessed prperly they can be gd surce f nutrients especially prtein and amin acids [4, 6]. Culled chicken layers are highly enriched with mega-3 fatty acid and high in myfibrillar prtein, making them suitable raw materials fr surimi-based prducts [7]. Spent hens have been reprted t be gd alternative fr surimi-based prduct because their price is cnsiderably cheap and their use ensures the imprvement and utilizatin f waste/ underutilized resurces [4]. Culled chicken layers have been used largely in the preparatin f chicken sups and emulsified chicken prducts such as frankfurter and blgna. They have als been used in canned prducts including sups, sauces, stews, and gravies [8]. Micrrganisms including Escherichia cli, Salmnella spp., Campylbacter spp., Shigella spp., Yersinia spp. and many mre may be habured in the intestines f animals, and can crss cntaminate carcasses and meat prducts [98]. They are usually excreted int the envirnment via faeces, and can cntaminate eggs, water, feed, sil, fds etc. As a Veterinary Wrld, EISSN: 2231-0916 478

Available at www.veterinarywrld.rg/vl.7/july-2014/7.pdf Table: Ttal plate cunt f bacteria bserved in culled chicken layers 3 5 3 5 4 5 Egg wash water 2.7 x 10 2.6 x 10 cfuml < 2.5 x 10 1.9 x 10 cfuml 9.2 x 10 2.0 x 10 cfuml 5 5 5 5 5 5 Chicken carcass rinse 2.6 x 10 2.9 x 10 cfuml 2.0 x 10 2.5 x 10 cfuml 2.1 x 10 2.3 x 10 cfuml 4 4 3 3 Drinking water 1.4 x 10 1.5 x 10 cfuml < 2.5 x 10 cfuml < 2.5 x 10 cfuml 5 2 2 Clacal swab 2.0 x 10 cfuml <1.0 x 10 cfuml < 1.0 x 10 cfuml 4 4 3 3 4 Feed 2.6 x 10 2.9 x 10 cfug < 2.5 x 10 cfug 2.9 x 10 3.1 x 10 cfug 7 6 6 6 6 Faeces 1.8 x 10 cfug 1.4 x 10 1.7 x 10 cfug 2.9 x 10 3.1 x 10 cfug cnsequence, humans can cntract them frm the cnsumptin f cntaminated fd r cntact with cntaminated surces. In Malaysia, culled chicken layers are cnsumed especially amng individuals that prefer tugh chicken meat. Determining the micrbilgical status f culled chicken layers is essential because it will give an indicatin f the risk invlved in cntracting fdbrne pathgens frm the cnsumptin f their meats and eggs. Therefre, this study was carried ut t determine the micrbial lad f culled chicken layers in three farms in Penang, Malaysia. Materials and Methds Lcatin, duratin and data cllectin: This study examined culled chicken layers and their envirnmental samples btained frm three different layer farms in Penang, Malaysia between Nvember 2011 and April 2012 fr their micrbial quality. Samples frm culled chicken layers were egg wash water, chicken carcass rinse, faeces and claca swab, while envirnmental samples were litter, feed and drinking water. These samples were stred under 4 C during transprtatin and analyzed immediately upn arrival at the Fd Micrbilgy Labratry f the Schl f Industrial Technlgy, Universiti Sains Malaysia, Penang. Samples cllected were analyzed fr ttal aerbic bacteria, enterbacteriaceae, clifrms, faecal clifrms and E. cli. Enumeratin and cnfirmatin f bacteria grups: Enumeratin and cnfirmatin f micrbes was dne using slightly mdified methd in the Bacterilgical Analytical Manual f the Fd and Drug Administratin (BAM-FDA) [19, 20]. Egg wash water (10 ml), chicken carcass rinse (10 ml), drinking water (10 ml), feed (10 g), faeces (10 g) and claca swab (1 swab) were transferred int 90 ml, 90 ml, 90 ml, 90 ml, 90 ml, and 10 ml f 0.1% saline bacterilgical peptne water (SBPW), respectively and hmgenized t serve as the 10 dilutin factr. Subsequently, serial dilutins -6 were made frm the 10 t 10 in 9 ml SBPW. -6 Ttal aerbic plate cunt: Serial dilutins (10 t 10 ) were spread plated n plate cunt agar (PCA). Plates were incubated at 37 C fr 48 ± 2 h under aerbic cnditin. After 48 hurs f incubatin, the number f clnies (25-250) frmed n the plates were cunted and the cfug r cfuml was calculated based n the guidelines given in the BAM-FDA. Enterbacteriaceae: Similarly, serial dilutins (10 t -6 10 ) were spread plated n vilet red bile agar (VRBG) plates. Plates were incubated at 37 C fr 18 h t 24 h under aerbic cnditin. Cunting and calculatin f cfug r cfuml was dne as in ttal aerbic plate cunt. Ttal clifrm, faecal clifrms and E. cli: The 3 tube mst prbable number (MPN) methd f BAM-FDA was used. One ml f aliqut frm dilutin factrs 10, -2-3 10 and 10 fr each sample was transferred int 3 lauryl sulphate tryptse (LST) cntaining Durham tube. Inculated LST tubes were incubated at 37 C fr 24 h t 48 h. Psitive LST tubes were indicated by gas prductin in the Durham tubes and turbidity f brths. Psitive LST tubes were further transferred int brilliant green lactse bile (BGLB) and EC brths cntaining Durham tubes. Inculated BGLB brths were incubated at 37 C fr 48 h ± 2 h fr the cnfirmatin f clifrms. Inculated EC brths were incubated at 45 C fr 24 h t 48 h fr the enumeratin f ttal faecal clifrms. A lpful f cultures frm psitive EC tubes were streaked nt Levine-EMB agar (L-EMB) plates and incubated at 37 C fr 18 h t 24 h fr the enumeratin f E. cli. Presumptive E. cli clnies which appeared as dark centred and flat, with r withut metallic sheen were purified n PCA. Purified E. cli islates were subjected t recmmended bichemical test (IMViC test) accrding t BAM- FDA[20]. Results and Discussin The results btained fr ttal aerbic bacteria, enterbacteriaceae, ttal clifrm, faecal clifrms and E. cli cunts are presented in Tables 1, 2, 3, 4, and 5, respectively. Frm Table, samples cllected frm Farm B namely egg wash water, chicken carcass rinse, feed and faeces had lwer ttal aerbic plate cunt as cmpared t the same samples cllected frm Farm A and C except drinking water and clacal swab. Drinking water and clacal swab frm Farm B exhibited the same ttal aerbic plate cunts with that f drinking water and clacal swab frm Farm C. Their 3 ttal plate cunts were <2.5 x 10 cfuml (drinking 2 water) and <1.0 x 10 cfuml (clacal swab). On the ther hand, samples cllected frm Farm A ntably, chicken carcass rinse, drinking water, clacal swab, feed and faeces generally shwed higher ttal aerbic plate cunts. Drinking water frm Farm B and C, faeces frm FarmAand B, and clacal swab frm Farm A, B and C shwed n range fr their ttal aerbic plate Veterinary Wrld, EISSN: 2231-0916 479

Available at www.veterinarywrld.rg/vl.7/july-2014/7.pdf Table-2: Enterbacteriaceae cunt bserved in culled chicken layers and their envirnmental samples 2 Egg wash water 4 < 1.0 x 10 2.4 x 10 cfuml <1.0 x 10 2.9 x 10 cfuml < 2.5 x 10 cfuml 5 5 5 Chicken carcass rinse 2.3 x 10 2.6 x 10 cfuml 1.7 x 10 2.2 x 10 cfuml 1.7 x 10-2.0 x 10 cfuml 3 Drinking water < 2.5 x 10 cfuml <1.0 x 10 cfuml <1.0 x 10 cfuml 2 2 3 3 5 5 5 2 2 2 2 Clacal swab <1.0 x 10 cfuml <1.0 x 10 cfuml <1.0 x 10 cfuml 3 2 2 Feed <2.5 x 10 cfug <1.0 x 10 cfug <1.0 x 10 cfug 6 6 6 6 6 6 Faeces 3.4 x 10 3.5 x 10 cfug 1.1 x 10-1.2 x 10 cfug 2.3 x 10-2.8 x 10 cfug Table-3: MPN fr clifrm bserved in culled chicken layers Egg wash water < 3.0 - > 1100.0 MPNml < 3.0 120.0 MPNml 38.0 - > 1100.0 MPNml Chicken carcass rinse 210.0 - > 1100.0 MPNml 150.0 - > 1100.0 MPNml 160.0 - > 1100.0 MPNml Drinking water 240.0 MPNml < 3.0 MPNml < 3.0 3.6 MPNml Clacal swab < 3.0 23.0 MPNml < 3.0 MPNml < 3.0 11.0 MPNml Feed >1100.0 MPN g >1100.0 MPN g < 3.0 43.0 MPN g Faeces >1100.0 MPN g >1100.0 MPN g >1100.0 MPN g Table-4: MPN fr faecal clifrm bserved in culled chicken layers Egg wash water < 3.0 - > 1100.0 MPNml < 3.0 120.0 MPNml 3.0- > 1100.0 MPNml Chicken carcass rinse 120.0 - > 1100.0 MPNml 120.0 - > 1100.0 MPNml 160.0 - >1100.0 MPNml Drinking water 14.0 23.0 MPNml < 3.0 MPNml < 3.0 3.6 MPNml Clacal swab < 3.0 15.0 MPNml < 3.0 MPNml < 3.0 11.0 MPNml Feed 7.4 11.0 MPNg < 3.0 9.2 MPN g < 3.0 3.6 MPN g Faeces > 1100.0 MPN g 210.0 - > 1100.0 MPN g >1100.0 MPN g Table-5: MPN fr Escherichia cli bserved in culled chicken layers Egg wash water 9.2 21.0 MPNml < 3.0 7.4 MPNml < 3.0 9.2 MPNml Chicken carcass rinse 43.0 - > 1100.0 MPNml 20.0 460.0 MPNml 27.0 - > 1100.0 MPNml Drinking water 3.0-3.6 MPNml < 3.0 MPNml < 3.0 MPNml Clacal swab < 3.0 7.2 MPNml < 3.0 MPNml < 3.0 3.6 MPNml Feed 3.0 3.6 MPN g < 3.0 MPN/g < 3.0 MPN g Faeces 210.0 - >1100.0 MPN g 64.0 - >1100.0 MPN g 150.0 - >1100.0 MPN g cunt. This is because all triplicate tests fr these samples gave the same result. Frm Table-2, drinking water, clacal swab and feed frm Farm B and Farm C exhibited the same 2 enterbacteriaceae cunt f <1.0 x 10 cfuml fr 2 drinking water and clacal swabs, and <1.0 x 10 cfug fr feed. The enterbacteriaceae cunts fr drinking 3 3 water (<2.5 x 10 cfuml ) and feed (<2.5 x 10 cfug ) frm Farm A were fund t be the same. On the ther hand, egg wash water, chicken carcass rinse, drinking water, feed and faeces frm Farm A were bserved t have higher level f enterbacteriaceae as cmpared t the same samples cllected frm Farm B and C. It was als bserved that feed frm FarmA, Farm B and Farm C, clacal swab and drinking water frm FarmA, Farm B and Farm C, and egg wash water frm Farm C shwed n range fr enterbacteriaceae cunt as all cunts fr these samples gave the same results. Besides that, chicken carcass rinses were fund t have higher enterbacteriaceae cunts than egg wash water samples. In general, faeces frm Farm A, Farm B and Farm C have the highest enterbacteriaceae cunt as cmpared t ther samples (egg wash water, chicken carcass rinse, drinking water, clacal swab and feed). Faeces frm Farm A had the highest level f enterbacteriaceae. Members f the enterbacteriaceae such as E. cli, Salmnella spp., Enterbacteria spp., Klebsiella spp. and Yersinia spp. are cmmnly fund in the intestinal tract f animals, which are widely shed in faeces during defaecatin [21]. The result btained fr the mst prbable number (MPN) f ttal clifrm, faecal clifrms and E. cli is shwed in Table-3, 4 and 5 respectively. Samples cllected frm Farm B which include egg wash water, chicken carcass rinse, drinking water, and clacal swab generally had the lwest level f clifrms as cmpared t the same samples cllected frm Farm A and C. Farm A, hwever, was bserved t have the highest level f clifrm fr samples such as chicken carcass rinse, drinking water and clacal swab as cmpared t the same samples cllected frm Farm B and C. It was bserved that feed frm Farm A and B, and faeces frm FarmA, B and C have the same level f clifrm which was >1100 MPNg. Drinking water as Veterinary Wrld, EISSN: 2231-0916 480

well as the clacal swab cllected frm Farm B was fund t be least cntaminated with clifrms. Envirnmental and culled chicken layer samples thus drinking water, clacal swab and feed frm Farm A had higher level f faecal clifrms as cmpared t the same samples cllected frm Farm B and C. It was fund that the level f faecal clifrms in chicken carcass in Farm A was the same as in Farm B with their MPN ranging frm 120.0 t >1100 MPNml. Chicken carcass rinse frm Farm C was mst cntaminated with faecal clifrms with MPN number ranging frm 160.0 t >1100.0 MPNml. On the ther hand, egg wash water and sme f the envirnmental and culled chicken layer samples which include drinking water, clacal swab and feaces frm Farm B had the lwest level f faecal clifrms as cmpared t the same samples cllected frm FarmAand C. Egg wash water, chicken carcass rinse, drinking water, clacal swab, feed and faeces frm Farm A had the highest level f E. cli as cmpared t the same samples cllected frm Farm B and Farm C. Farm B was bserved t have the lwest level f E. cli fr egg wash water, chicken carcass rinse, clacal swab and faeces. The level f E. cli in drinking water and feed frm Farm B was fund t be the same as the drinking water and feed cllected frm Farm C which was <3 MPNml. The MPN fr E. cli in drinking water and feed frm Farm A was fund t be the same which was 3.0 t 3.6 MPNml fr drinking water and 3.0 t 3.6 MPNg fr feed. Overall, faeces frm all the three farms were fund t have the highest level f E. cli and the ther bacteria cmpared t egg wash water, chicken carcass rinse, drinking water, and clacal swab and feed samples. This is because E. cli and ther bacteria are usually fund in the intestines f humans and warmblded animals [22] and it is usually shed int the envirnment via faeces [9]. Chicken carcasses are als mstly cntaminated by bacteria during the remval f gastrintestinal tract [23]. Eggs in all the three farms were rlled nt a cnveyr after laying. This practice kept the eggs ff the flr all the time and thus reduces cntact with faeces and litter, which culd prbably reduce the chances f faecal cntaminatin f egg shells and cnsequently egg wash water. Cnclusin This wrk gives an verview f the micrbilgical quality (ttal plate cunt, enterbacteriaceae, ttal clifrms, faecal clifrms and E. cli) f culled chicken layers and their related samples cllected frm 3 farms in Penang, Malaysia. The micrbilgical cunts vary accrding t the 3 farms namely Farm A, Farm B and Farm C. Ttal plate cunt fr bacteria was highest in egg wash water (Farm C), chicken carcass rinse (Farm A), drinking water (Farm A), clacal swab (Farm A), feed (Farm A) and faeces (Farm A). Enterbacteriaceae cunts fr drinking water, clacal swab and feed samples cllected frm Farm A, Farm B Available at www.veterinarywrld.rg/vl.7/july-2014/7.pdf and C were lw, except fr faecal samples. 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