This study was carried out in order to isolate and diagnosis of common bacteria from bovine mastitis in Baghdad city which have the ability to produce biofilm and detect its molecular composition. Twenty five milk samples were collected from different regions in Baghdad city from udders of cows suffering from clinical and subclinical mastitis.Then cultured on blood agar. Gram staining was done to differentiate between bacteria gram positive which cultured on Mannitol Salt Agar and Nutrient Agar while gram negative bacteria were cultured on MacConkey agar and Eosin Methylene Blue agar. All bacterial isolates were subjected to different biochemical tests, API 20 E System, API Staph System and RapIDTMONE System kit to confirm the diagnosis.Christensen tube method was used to detect the ability of the diagnostic bacterial isolates to produce biofilm. Specific forward and reverse primers were designed according to a program from NCBI-Genbank  for Staphylococcus aureus (Genbank code: gb |KR265472.1|), for Escherichia coli : gb|JQ781567.1| and for Klebsiella pneumoniae gb|KT944736.1|, To study  the sequence of these genes  after amplification of 16srRNA genes  by using PCR was appeared because it is sensitive and highly specific assay serve as suitable molecular diagnostic tool for detection and compare these genes sequencing with references strains.Results showed that 8 (32%) out of 25 milk samples were positive for Staphylococcus , 6 (24%) out of  25  samples were positive for Klebsiella pneumonia . and 11(44%) were positive for E.coli . The results showed that 22 (88%) isolates out of 25 milk have the ability to produce biofilm . Genetic identities results showed that Staphylococcus aureus and Klebsiella pneumonia isolates gave 99% matching and resembling the reference strains while Escherichia coli isolates identity showed resemble match of 100% with the reference strain

Detection of pathogenic bacteria, such as Listeria  monocytogenes, in food is crucial for safeguarding public health in Iraq.  Forty five samples of frozen meat (15 samples of each of minced red meat, chicken, and fish) were collected from different markets in Baghdad city. Molecular (RT-PCR) and culturing (conventional microbiological examination) methods were used to determine the level of contamination of L. monocytogenes in these types of meat. For the culturing method, TSYEB broth was used as an enrichment medium, whereas BALCAM medium (HiMedia) with the listeria selective supplement FD061 was used as a selective medium, for the isolation and identification of this bacterium. The isolates were confirmed microscopically and biochemically. The results of the culturing method showed that the total number of the isolates of L. monocytogenes was 14/45 (31.1%). The incidence of this bacterium was high in fish (11/15, 73.3%), while it was low in the other two types of meat.  2/15 (13.3%) in red meat and 1/15 (6.7 %) in chicken. Molecular detection of each sample of the bacteria was performed using RT-PCR technique after preparing the Genomic DNA extraction of these samples according to the protocol provided by ReliaPrep™ Blood gDNA Miniprep System kit (Promega, USA). The PCR primers and the hybridization probe ((Macrogen, Korea) were used to target the inlA gene sequence (specific for L. monocytogenes). The results of the RT-PCR assay showed that 10/45 (22.2%) of the samples were positive for L. monocytogenes, which was detected only in fish samples ((10/15, 66.7%), while not found in minced red meat and chicken. However, our results showed differences when compared to other previous works because there were many studies found that the highest contamination rate was in red meat products. We conclude that the PCR kit used for the detection of L monocytogenes appears to give accurate results in the diagnoses of this bacterium in meat products and in comparison with the other routine diagnosis methods in the laboratory, which included culturing and doing biochemical tests which last for approximately 7 days, the RT-PCR technique was able to confirm the findings within 48 hours.

This study was conducted to detect the ability of E.coli isolated from diarrhea to produce biofilm and protection against diseases cause by these bacteria compared with whole cell sonicated antigen. One hundred two fecal samples (52 fecal samples from cows, calves and 50 fecal samples from sheep,goat) were collected   from College of veterinary medicine-university of Baghdad, College of Agriculture-university of Baghdad, Dora zone and Abu-Ghraib zone. Samples were cultured on MacConkey and Eosin Methylene Blue agar and after purification of cultured bacteria, biochemical tests, API 20 E System and RapIDTM ONE System kit were done.  Results showed that  91 out of  102 fecal samples have the characteristics belong to E.coli ,the ability of these isolates to produce biofilm were detected and the results showed that 38 out of 49 E.coli  isolates from fecal samples of cow  produce biofilm (77.55%)and 39 out of 42 E.coli  isolates from fecal samples of sheep produce biofilm (92.85%) with different thickness ranged between (0.2-2)mm, while 11 isolates from 49 fecal samples of cow and calves and 39 isolates from 42 fecal samples of sheep had not  produce biofilm. The results showed that protein concentration of biofilm was 92 mg/ml for one isolate and 70 mg/ ml for the whole sonicated antigen of the bacteria that produce biofilm. Three types of bacterial antigens were prepared as follow; Whole cell sonicated antigen (WCA) of biofilm producer E.coli, biofilm extract antigen (biA) with protein concentration 3.5 mg/ml and biofilm extract antigen (biA) with protein concentration 14 mg/ml, then these antigens were injected in 50 healthy White BALB mice. Results  showed that survival time of animals in the immunized group with biofilm antigen of high protein concentration (14 mg/ml)  was longer (652.8 hrs) than  animals immunized with whole sonicated antigen of protein concentration 3.5 mg/ml antigen (378 hrs ) and than the group with biofilm antigen of protein concentration 3.5 mg/ml (513.6 hrs)   , heavy bacterial isolation were recorded in the internal organs of the immunized infected animals at (12-48) hours post infection, while moderate bacterial isolation at day 30 post infection. Histopathological examination showed large abscess which caused adhesion of liver with stomach and spleen with stomach and pancreas surround by dense cellular fibrous tissue, the result showed that all animals of control positive group,5 animals of group injected with(WCA) produce biofilm,3 animals of group injected with (biA) 3.5 mg/ml and 1 animal of group injected with (biA) 14 mg/ml were died during 12-48 hours post infection, acute suppurative reaction were seen in internal organs of animals died at 12-24 hours post infection, granulomatous lesions were seen in most internal organs of animals in group 2 , 3 and 4 at day 30 post challenge, while mild inflammatory reaction was recorded in most internal organs of  group 4 at day 30 post challenge.