Diyala Journal of Agricultural Sciences, 10(Special issue): 366-372, 2018 Hassoon et al. 633 Diyala Univ., 2018 ,Scientific Conf., College of Vet. Med. st1 http://www.agriculmag.uodiyala.edu.iq/ BACTERIOLOGICAL STUDY ON OVINE RAW MILK IN BAQUBAH CITY Shaymaa Jabbar Hassoon1,2 Noor Their Talib1 Mohammed Abed Mahmood1 Heba Ibrahim Abaas 1 Zainab Fadle Rahman1 Heba Ayad Ismail1 1College of Vet. Med., Diyala University, Iraq. 2Corresponding author: sheimaajabbar@gmail.com ABSTRACT This study aimed to detect the behavior of bacterial pathogens in raw apparently normal milk. For this purpose 85 milk samples were collected from 85 ewes in different areas in Baqubah city. Study was carried from the period 1/4/2017 to 15/8/2017. The results showed that only 73 samples showed growth of more than one species of bacteria, while 12 samples gave negative bacterial growth on different media. The isolated bacteria were identified by studying cultural properties on different media, Gram staining and biochemical tests. Bacterial isolates included Escherichia coli (64.7%), Staphylococcus aureus (45.8%), Pseudomonas aeruginosa (32.9%), Streptococcus spp. (24.7%), Klebsiella spp. (22.3%), Proteus mirabilis (18.8%), bacillus spp (11.7%), and serratia marcesences (2.3%). The S. aureua and E. coli were the most predominant bacterial isolates. The results of antibiotic sensitivity test showed variable results in different isolates (Ampicilin, tetracyclin, Amoxcilin, Gentamicin and Vancomycin). Key wards: Raw milk, Baqubah city, antibiotics susceptibility. INTRODUCTION Milk is an important source of nutrients to humans and nursing animals and it is a source of food that is widely available, inexpensive and safe if boiled or pasteurized. Two main factors contribute to the microbiological quality of milk: (a) the existence of organisms in secreted milk (pre harvest) and (b) the contamination of milk at the time of collection, processing, packaging, transport, distribution and storage (post-harvest). If pathogenic bacteria are among the contaminants, the product will pose a food safety threat to consumers Although milk was once considered to be an important vehicle for foodborne diseases, but processes such as pasteurization and ultra-high-temperature treatments (UHT) have and are being used to produce safe and clean milk (LeJeune et al., 2009). The consumption of raw milk poses a risk to public health via its potential contamination with pathogenic microorganism as Brucellaand Mycobacteria Spp. Milk and milk products may carry toxic metabolites of different pathogenic Diyala Journal of Agricultural Sciences, 10(Special issue): 366-372, 2018 Hassoon et al. 633 Diyala Univ., 2018 ,Scientific Conf., College of Vet. Med. st1 http://www.agriculmag.uodiyala.edu.iq/ organisms growing in it. Ingestion of such products contaminated with these metabolites cause food poisoning for consumers. On the other hand the ingestion of viable pathogenic bacteria along with the food product leads to food borne infection (Aneja et al., 2002). In recent years, there has been a growing interest in local food production and consumption, and consumers are looking for foodstuffs that have undergone the least processing. As a result, there is an increased tendency to consume raw milk (Claeys et al., 2013). Milk has a complex biochemical constituent and its high water activity and nutritional value serves as a good medium for growth and multiplication of many types of microorganisms when suitable conditions exists (4) .This study aimed to identify bacteria present in sheep`s raw milk in Baqubah city and study its susceptibility to certain antibiotic. MATERIALS AND METHODS Samples A total of 85 sheep`s raw milk samples were collected from different areas in Baqubah city in the period from the period 1/4/2017 to 15/8/2017. Processing was carried out immediately following delivery to the laboratory collection. The collected milk samples were immediately cooled and transported to the Microbiology of Laboratory at the College of Veterinary Medicine, Diyala University for bacteriological analysis. Bacteriological study Culture media used for isolation and purification of bacteria included: Nutrient agar (NA), Blood agar, MacConkey agar, Mannitol salt agar (MSA), Salmonella –Shigella (SS) agar medium and Brain heart infusion agar (BHI), Media were prepared according to the manufacturer's instructions. Inoculated media were incubated aerobically at 37 ºC for 24 hours. All the isolates were stored in brain heart infusion broth with 15% glycerol at deep freeze until further use. Different colony types were seen in the initial cultures, each colony type was sub cultured on sheep blood agar for purification. The bacterial isolates were identified according to colony morphology on different media, biochemical characters and Gram`s reaction for Gram`s negative rods. The biochemical tests included: oxidase, IMVC test (indol production, methyl red, vogas-proskauer and citrate utilization), TSI (triple sugar iron), urease production, gelatein liquefaction, hemolysis on sheep blood agar and different carbohydrates utilization. For staphylococcus Spp., hemolysis on sheep blood agar, Catalase and Coagulas tests were applied (Sneath et al, 1986). Diyala Journal of Agricultural Sciences, 10(Special issue): 366-372, 2018 Hassoon et al. 633 Diyala Univ., 2018 ,Scientific Conf., College of Vet. Med. st1 http://www.agriculmag.uodiyala.edu.iq/ Antibiotics susceptibility Antimicrobial susceptibility tests was determined by the disc diffusion technique on Muller Hinton agar according to (Bauer et al., 1996). Results were recorded as sensitive or resistant, based on the diameter of inhibition zone of each antimicrobial disc used. RESULTS AND DISCUSSION Bacterial growth was obtained from 73/85 (85.88%) of milk sample. Most samples showed growth of more than one species of bacteria on different media. Bacterial isolates included Escherichia coli (64.7%), Staphylococcus aureus (45.8%), Pseudomonas aeruginosa (32.9%), Streptococcus spp. (24.7%), Klebsiella spp. (22.3%), Proteus mirabilis (18.8%), Bacillus spp. (11.7%), and serratia marcesences (2.3%) table 1. Table 1. Bacterial species which isolated from ewes raw milk and its Percentage Bacterial species No. Percentage (%) Escherichia coli 55 64.7 Staphylococcus aureus 39 45.8 Pseudomonas aeruginosa 28 32.9 Streptococcus spp 21 24.7 Klebsiella spp. 19 22.3 Proteus mirabilis 16 18.8 bacillus spp 10 11.7 serratia marcesences 2 2.3 These results are in agreement with (Melese et al., 2016), who reported 84.1% of milk samples contained at least one bacterium including E. coli, Salmonella sp., Staph. aureus, and Proteus Sp., with isolation rates of 70 (58 %), 4 (3.3%), 29 (24.2%), and 9 (7.5%), respectively and also it agreed with (Abbas, 2011) that isolated different types of bacteria from 100 milk samples collected from Abu–Ghraib district in Baghdad, including Staphylococcus aureus, (%10) Streptococcus group D (%51), Escherichia coli (%10) Klebsiella Sp. (%1). The increased incidence of E. coli may be attributed fecal contamination of samples E. coli opportunistic bacteria, when they move to location other than their natural habitat, may become pathogenic (Azher, 2013). Presence of other bacterial species may be due to several factors (Sligh and Timbury, 1994), including their being a cause of subclinical mastitis as staph. aureas which is well known as udder pathogen (Tenhagen et al., 2009). A number of special Diyala Journal of Agricultural Sciences, 10(Special issue): 366-372, 2018 Hassoon et al. 633 Diyala Univ., 2018 ,Scientific Conf., College of Vet. Med. st1 http://www.agriculmag.uodiyala.edu.iq/ kind of Serratia can act as a reason of mastitis, including S. marcescens, S. rubidaea and S. liquefaciens (Hogan, 1997 ; Asmaa, 2017). The antibiotics susceptibility test reveal that S. aureua was highly resistance to ampicillin, intermediate to amoxicillin and gentamycin, highly sensitive to tetracycline as showed in table 2 E. coli showed sensitivity to ampicillin, vancomycin and resistance to amoxicillin, P. aeruginosa sensitive to tetracycline, and intermediate to vancomycin and Amoxicillin while was resistant to ampicillin, the Bacillus spp was sensitive to tetracycline, Amoxicillin, gentamycin and ampicillin, The other species included Klebsiella spp. were resist to Amoxicillin and vancomycin while they were sensitivity to tetracycline. Streptococcus spp. showed sensitivity to gentamycin and amoxicillin but resistant to ampicillin figure (1, 2, 3 and 4). Bacteria detected in ovine raw milks may be present as saprophytes opportunistic pathogen under conditions of stress may result in disease, (Bukar et al., 2007). Table 2. Results of Antibiotic Susceptibility Test of All bacteria species Isolated from milk Ampicillin Amoxicillin Gentamicin Vancomycine Tetracycline Bacteria 4 52 52 53 44 Staph. 63 44 44 63 45 Bacillus. 44 3 04 63 63 E.coli 3 63 40 63 63 Strept. 3 54 03 53 44 Pseudomonase 64 3 3 3 63 Klebsiella 3 3 65 3 53 Serratia Figure 1. show Antibiotic Susceptibility test of bacillus to different antibiotic Diyala Journal of Agricultural Sciences, 10(Special issue): 366-372, 2018 Hassoon et al. 634 Diyala Univ., 2018 ,Scientific Conf., College of Vet. Med. st1 http://www.agriculmag.uodiyala.edu.iq/ Figure 2. show Antibiotic Susceptibility test of Klebseilla to different antibiotic Figure (3) show Antibiotic Susceptibility test of streptococcus to different antibiotic . Diyala Journal of Agricultural Sciences, 10(Special issue): 366-372, 2018 Hassoon et al. 630 Diyala Univ., 2018 ,Scientific Conf., College of Vet. Med. st1 http://www.agriculmag.uodiyala.edu.iq/ Figure 4. show Antibiotic Susceptibility test of Pseudomonas to different antibiotic REFRENCES Abbas, M. S. 2011. Isolation of bacteria from milk and study the sensitivity of bacterial isolates to antibiotic and medical plants. Al-Anbar Journal of Veternary Science, 4(2): 7-14. Aneja, R. P., B. N. Muthur, R. C. Chandan, A. K. Banerejee. 2002. Technology of Indian milk products. New Delhi: Dairy Indian Yearbook. p. 183-96. Asmaa, H. A., Ban N. Nadhom and Hind H. Al-Ammiri. 2017. Isolation and identification of Serratia marcescens from Bovine Mastitis infections in Iraq and their susceptibility to antibiotics. Journal of Entomology and Zoology Studies, 5(2): 489-492. Azher S. K. 2013. Study of drugs sensitivity aginst bacterial isolated from patients with urinary tract infection in Baqubah City. Diayala journal for pure science. 9(3). Bauer, A. W., W. M. Kirby, J. C. Sherris and M. Jurck. 1996. 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Journal of Dairy Research. 76(2): 179-87. بعقوبة مدينة في االغنام حليبل بكتيريلوجية دراسة 2محمد عبد محمود 2نور ثاير طالب 1،2شيماء جبار حسون 2هبة اياد اسماعيل 2زينب فاضل رحيم 2هبة ابراهيم عباس كلية الطب البيطري، جامعة ديالى، العراق 0 sheimaajabbar@gmail.comالمسؤول عن النشر: 5 المستخلص عينة 32تم جمع لذا ،وجود المسببات المرضية في الحليب الظاهر طبيعياللكشف عن هدفت الدراسة وحتى 0/4/5403للمدة من اجريت الدراسة .مختلفة في مناطق بعقوبة أماكننعجة من 32حليب من عينة 05عينة فقط اظهرت نمو اكثر من نوع واحد من البكتريا بينما 36. اظهرت النتائج ان 02/3/5403 تم التعرف على البكتريا المعزولة من خالل دراسة سالبة النمو على اوساط زرعية مختلفة. اظهرت نتائج وشملت العزالت الفحوصات البايوكيمياوية، صبغة كرام وو ،الخصائص الزرعية على االوساط المختلفة ( %65.3)الزوائف الزنجارية ( و%42.3والمكورات العنقودية ) ،(%34البكتيرية االشريشيا القولونية ) البكتريا العصوية و (%03.3المتقلبات الرائعة )و( %55.6( والكليبسيال )%54.3المكورات المسبحية )و اكثر العزالت . كانت المكورات العنقودية واالشريشيا القولونية(%5.6والسريشيا ميرسيسين )( 00.3%) ختلفة في عزالت مختلفة )امبيسيلين . واظهرت نتائج حساسية المضادات الحيوية نتائج مالبكتيرية انتشارا اموكسيلين جنتامايسين وفانكومايسين(. وتتراسايكلين و .بعقوبة مدينة ، الحيوية للمضادات الحساسية ،الخام الحليب: الكلمات المفتاحية