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MOLECULAR DETECTION OF SALMONELLA AND E. COLI MICROORGANISMS AMONG DAIRY FARMS WITH DETECTION OF VIRULENCE AND ANTIBIOTICS RESISTANCE GENES

GIHAN MOHAMED OMER MOHAMED HASSAN ¹

and HASSAN EL-SAYED MOHAMED FARAG ²

¹ Assistance Researcher of Microbiology Department, Port-Said Lab Animal Health Research Institute Dokki –Giza.

² Chief Researcher of Food Hygiene Department, Port-Said Lab Animal Health Research Institute Dokki –Giza.

Received: 31 March 2019;     Accepted: 30 April 2019

INTRODUCTION

Although there have been increases in the modern and advanced methods of care for livestock, small herds in different localities were still found especially in the developing and underdeveloped country whereas the growth of animals in conditions of overcrowding often enhanced the appearance of bacterial and others infectious disease (Godinho and Carvalho, 2013) that affect the animals health and their productivity, resulting in large economic losses. Bacteria can occur in milk through, colonization in the teat canal or infected udder (clinical and subclinical mastitis), milker (manual as

Corresponding author: Dr. Gihan Mohamed Omer Mohamed

E-mail address: dr.gehanomer@yahoo.com     

Present address: Assistance Researcher of Microbiology Department, Port-Said Lab Animal Health Research Institute Dokki –Giza.

well as automated), extraneous dirt, milk utensils and unclean processing water (Hayes et al., 2001). Salmonellae and E. coli are the most economically important pathogens (Achá et al., 2004) affecting dairy cattle and calf.

Salmonella is an enteric pathogen found in the intestinal tract of animals and excreted in feces and spread in water, soil, plant surface, animal fecesand dairy farms(Halimi et al., 2014). The severity of infection and symptoms varies depending on the host species and serovars and ranging from severe disease to asymptomatic (Coburn et al., 2007). Although cattle are considered a major reservoir for infections with S. Typhimurium (Nastasi et al., 1993) whereSalmonella have been isolated from the feces of healthy cattle and considered a normal or transient member of the gastrointestinal microbial population (Callaway et al., 2005). Salmonellosis manifestations include fever, anorexia, diarrhea, dehydration, abortion, decreased milk production, depressed mentation, pneumonia, septic arthritis, meningitis, gangrene of distal extremities and sudden death (Mohler and House, 2009).

Salmonella produce a variety of putative virulence determinants including haemaglutinins, adhesion, invasions, fimbriae exotoxin and endotoxins (Lee et al., 1996). The invA gene of Salmonella contains sequence unique and recognized as an international standard for detection of Salmonella genus (Malorny et al., 2003) and considered a potential diagnostic for all known serovars of Salmonella (Jamshidi et al., 2008). While Salmonella enterotoxin (stn) is a putative virulence factor responsible for enterotoxic activity (Chopra et al., 1999) and bcfC coding for bacterial fimbriae, involved in surface adhesion and gut colonization (Barrow et al., 2010).

On the other hand, E. coli are a large and diverse group of bacteria of the family Enterobacteriaceae commonly found in the lower intestine of a variety of warm-blooded animals including cattle and humans (CDC, 2011).

E. coli is an ideal indicator organism for fecal contamination in water (well water, river water, other contaminated surface waters, soil and plants) or in food (milk, meat, vegetables ect.) (Kaper et al., 2004) and this increase the possibility for presence of enteropathogenic or toxigenic E. coli (Pamela     et al., 2008).

The pathogenicity of E. coli is dependent on the regulation and interaction between a number of virulence factors, and it is affected by environmental conditions such as host species, host health status, interaction with other bacteria species (Clermont     et al., 2011).

E. coli pathovars, such as enteropathogenic E. coli (EPEC), Shiga-toxigenic E. coli (STEC), and enterohemorrhagic E. coli (EHEC), have been observed in dairy herds (Farrokh et al., 2013), milk (Van Kessel et al., 2011) and other dairy products (Solomakos et al., 2009), with a unique set of virulence and colonization factors encoded in the chromosome or in episomal structures (Rúgeles      et al., 2010).

Enteropathogenic E. coli (EPEC) strains belonged to a series of O antigenic groups including 12 serogroups such as O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158 (Hernandes et al., 2009).

Conformation of E. coli from other bacteria can detect by the housekeeping gene, phoA (The alkaline phosphastase gene) which present in all E. coli strains (Kong et al., 1995) and encodes for a hydrolase enzyme, responsible for removing phosphate groups from molecule (Chang et al., 1986).

TraT gene is one of the virulent factors of E. coli that have been shown to be located on conjugative plasmids. The TraT (conjugal transfer surface exclusive protein) gene is a major outer membrane protein (Moll et al., 1980) which reduces the susceptibility of bacteria to phagocytosis (Agüero    et al., 1984).

FimH is a mannose-specific adhesion located on the tip of type 1 fimbriae of E. coli that is responsible for mediating shear-enhanced bacterial adhesion and invasive properties of E. coli (Chassaing et al., 2011).

Antimicrobial resisitance has emerged in the past few years as a major problem in human and vetrinary medicine(Lanz et al., 2003)due to the wide spread use and misuse of antimicrobials in farms animals(Suojala et al., 2011). Also uses of antibiotic as growth promotion give raise to antimicrobial resistance in farms animals(Philips   et al., 2004). The resistance can occur between and within bacteria through mutation of genes and horizontal gene transfer (Buller et al., 2014).Thus antimicribial resistance strains can increase the treatment cost and period of treatment (Sawant        et al., 2007). Therefore, identification of resistance genes of bacteria seems to be so essential in reduction of treatment costs (Suojala et al., 2011).

GyrA (A subunit) is essential for epithelial invasion (Galan and Curtis, 1989), found predominantly in bacteria and composed of a single polypeptide, as in most eukaryotes. GyrA has two functional domains: N-terminal responsible for the breaking- and rejoining function and C-terminal that can bind DNA non-specifically (Huang, 1996).

Thus the aim of the current study was carried out for molecular detection of Salmonella and E. coli in different types of samples in dairy farms with detection of some virulence and resistance genes of the isolated strain. Also genes sequences of some strains were determined.

MATERIALS AND METHODS

1-Sample collection:

A total of 500 samples, 100 of each milk (pooling from 1000 lactating cows), feed, swabs from milk tanks, drinking tanks swabs and dairy cows fecal swabs samples (pooling from 1000 lactating cows) were collected from small herds of apparently healthy or subclinical dairy cattle in El-Kabotti and Bahr El-Baker zone at Port-Said Governorates during the period from September to December 2018. Each positive pooling samples were re-examined one by one.

2-Samples preparation, homogenation and pre-enrichment:

2-1: Milk samples:

Preparation of teats and udder for milk collection was done according to Cabral et al., 2015. Each milk sample was collected aseptically in clean, sterilized, marked and identified sterilized bottle and Keep in the refrigerator or on ice at 4^oC until microbiological examination. Under aseptic condition homogenation of milk samples with sterile buffered peptone water (BPW) and incubated at 34^oC- 38^oC for 18 h ± 3 h  according toISO 6887-1:2017 andISO 6887-5:2017.

2-2: Feed samples:

Aseptically collection of feedsamples and kept in refrigerator until bacteriological examination. Homogenation of grinding feed with sterile BPW and incubation was done according to ISO 6887-1:2017 and ISO 6887-4:2017.

2-3: Swabs from milking equipment (milk tanks):

According to WHO/FAO, 1994 milk tanks swabs were taken under aseptic condition and kept at 4^oC until bacteriological examination. Preparation of 1:10 and incubation at 34^oC- 38^oC for 18 h ± 3 h was done according toISO 6887-1:(2017).

2-4: Drinking tanks swabs:

Under aseptic condition drinking tanks swabs were collected and kept at 4^oC until bacteriological examinationaccording toWHO/FAO, 1994.Preparation of 1:10, homogenation and incubation at 34^oC- 38^oC for 18 h ± 3 h (ISO 6887-1:2017).

2-5: Dairy cows fecal swabs:

Fecal swabs were collected and kept at 4^oC until bacteriological examination according to WHO/FAO, (1994). Prepare a 1:10 dilution, homogenateand incubation at 34^oC- 38^oC for 18 h ± 3 h(ISO 6887-1:2017.

3- Isolation of microorganisms:

3-1: Isolation Salmonella species:

From each culture, 0.1 ml of pre-enrichment broth was added to 10 ml Rappaport-Vassiliadis broth with soya then incubated at 41.5^oC ± 1 ^o C for 24 hr ± 3h. and 1 ml from the culture of the same sample was added to 10 ml Muller-Kauffmann Tetrathionate/ novobiocin broth and incubated at 37 ^o C ± 1 ^o C for 24 hr ± 3 h. Then a loopful from the enriched broth was streaked onto the each surface of Xylose Lysine Deoxycholate agar plates and Brilliant Green agar plates then incubated at 37^oC±1^oC for 24 h ± 3 h  according to ISO 6887-1:(2017).

3-2: Isolation E. coli:

A loopful of the homogenate (pre-enriched culture were added to Lauryl sulphate tryptose broth (LST) test tube and incubated at 35°C ± 0.5°C. A loopful of each positive cultured tube (turbid and gas production) was transferred to tube of E. coli medium, (EC) and incubated at 44.5°C for 48 ± 3 h examined each 24 ± 2 h for gas production. A loopful from positive culture of EC broth was streak on L-EMB agar plate and incubates for 18-24 h at 35°C ± 0.5°C according to FDA's, (2017).

4- Identification of microorganisms:

4-1: Biochemical identification:

4-1-1: Biochemical identification of Salmonella species:

Presumptive colony with a characteristic morphology of typical Salmonella species were subjected to biochemical identification according to ISO 6579-1: (2017).

4-1-2: Biochemical identification of E. coli:

The suspected typical colonies of E. coli on L-EMB media was conducted to Gram's staining, oxidase and catalase tests. Then the colonies were subjected to various biochemical tests (Hitchins et al., 2001).

4-2: Serological identification of the isolates:

All biochemically identified Salmonella species and E. coli isolates were subjected to serologically identification.

4-2-1: Serological identification of Salmonella isolates:

Pure and primary culture plate of Salmonella species isolates were serotyped by slide agglutination test depending upon white-Kauffman-Le Minor scheme according to Grimont and Weill, 2007.

4-2-2: Serological identification of E. coli isolates:

Pure and primary culture plate of E. coli was agglutinated by slide agglutination test based on the presence of three principal surface antigens, O-antigens, flagellar H-antigens, and capsular K- antigens according to Ørskov and Ørskov (1984).

5-Antibiotic susceptibility testing:

All confirmed Salmonella serovars and E. coli serotypes were conducted to the antimicrobial susceptibility testing using the agar disk diffusion method and the interpretation of the results according to CLSI, (2013). All isolates were tested for susceptibility to 10 different antimicrobials agents as follows: ceftroiaxon (CRO) 30 μg; erythromycin (E) 15 μg; gentamicin (CN) 10μg; lioncomycin (MY) 10 μg; oxolinic acid (OA) 2 μg; oxytetracycline (OT) 30μg; penicillin G (P) 10 I.U; streptomycin (S) 10 μg; trimethoprim + sulphamethoxazole (SXT) (1.25 + 23.75) μg and vancomycin (VA) 30 μg.

6-Molecular study:

6-1: Conformation of Salmonella spp. and E. coli and their virulence and antibiotics resistance genes:

6-1-1: DNA extraction:

DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56°C for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer.

6-1-2:  Oligonucleotide Primer:

Primers used were supplied from Metabion (Germany)are listed in table (1).

Table 1: Primers sequences, target genes, amplicon sizes and cycling conditions.

6-1-3:  PCR amplification:

Primers were utilized in 25 µl reactions containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentrations, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an applied biosystem 2720 thermal cycler.

6-1-4: Analysis of the PCR Products:

The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 20 µl of the products was loaded in each gel slot. Gelpilot 100 bp (Qiagen, Germany, GmbH) and gene ruler 100 bp ladder (Fermentas, Germany) were used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

6-1-5: DNA Sequence:

PCR products were purified using QIAquick PCR Product extraction kit. (Qiagen, Valencia). Big dye Terminator V3.1 cycle sequencing kit (Perkin-Elmer) was used for the sequence reaction and then it was purified using Centrisep spin column. DNA sequences were obtained by Applied Biosystems 3130 genetic analyzer (HITACHI, Japan), A BLAST® analysis (Basic Local Alignment Search Tool) (Altschul et al., 1990) was initially performed to establish sequence identity to Gene Bank accessions.

6-1-6: Phylogenetic analysis:

The phylogenetic tree was created by the Meg Align module of Laser gene DNA Star (Thompson et al., 1994) and Phylogenetic analyses was done using maximum likelihood, neighbor joining and maximum parsimony in MEGA6 (Tamura et al., 2013).

RESULTS

Table 2: Prevalence of the isolated Salmonella species and E. coli isolated from the examined samples.

Table 3: Prevalence of Salmonella serotyping (n=9) isolated from the examined samples.

Table 4: Prevalence of E. coli serotyping (n=14) isolated from the examined samples.

Table 5: Antimicrobial susceptibility pattern of Salmonella serovar (n=9) and E. coli (n= 14) recovered from the examined samples.

Table 6: Prevalence of confirmatory genes among Salmonella (n=9) and E. coli (n= 14) strains isolated from the examined samples

Figure (1): Agarose gel electrophoresis of PCR prod­ucts after amplification of: 1- invA gene for Salmonella strains, MWM-molec­ular weight marker (100 – 600 bp DNA ladder), control (Positive, Negative) and different strains of Salmonella species. (invA gene products at 284 bp).

Figure (2): Agarose gel electrophoresis of PCR prod­ucts after amplification of: 1- phoA gene for E. coli strains, MWM-molec­ular weight marker (100 – 1000 bp DNA ladder), control (Positive, Negative) and different strains of E. coli. (phoA gene products at 720 bp).

Table 7: Prevalence of some virulence genes among Salmonella strains (n=9) isolated from the examined samples.

Figure (3): Agarose gel electrophoresis of PCR prod­ucts after amplification of: 1- stn gene for salmonella strains, MWM-molec­ular weight marker (100 – 1000 bp DNA ladder), control (Positive, Negative) and different strains of Salmonella (stn gene products at 617 bp).

Figure (4): Agarose gel electrophoresis of PCR prod­ucts after amplification of:  1- bcfC gene for salmonella strains, MWM-molec­ular weight marker (100 – 1000 bp DNA ladder), control (Positive, Negative) and different strains of Salmonella (bcfC gene products at 467 bp).

Table 8:Prevalence of some virulence genes among E. coli strains (n=14) isolated from the examined samples.

Figure (7): Agarose gel electrophoresis of PCR prod­ucts after amplification of: 1- ampC gene for salmonella strains, MWM-molec­ular weight marker (100 – 1000 bp DNA ladder), control (Positive, Negative) and different strains of Salmonella (ampC gene products at 550 bp)

Figure (8): Agarose gel electrophoresis of PCR prod­ucts after amplification of: 1- mphA gene for salmonella strains, MWM-molec­ular weight marker (100 – 1000 bp DNA ladder), control (Positive, Negative) and different strains of Salmonella (mphA gene products at 403 bp).

Figure (9): Agarose gel electrophoresis of PCR prod­ucts after amplification of: 1- aacC gene for salmonella strains, MWM-molec­ular weight marker (100 – 1000 bp DNA ladder), control (Positive, Negative) and different strains of Salmonella (aacC gene products at 448 bp).

Table 10:Prevalence of some resistance genes among E. coli strains (n=14) isolated from the examined samples.

Figure (15): Phylogenic diversity tree for stn gene amino acids sequence of S. Typhimurium GH3 (sample 3) isolated from milk of cattle and S. Typhimurium GH9 (sample 9) isolated from fecal swabs of cattle with 23 of the most similar stn gene amino acid sequences from Gene bank.

Figure (16): Phylogenic diversity tree for bcfC gene amino acids sequence of S. Typhimurium GH3 sample 3) isolated from milk of cattle and S. Typhimurium GH9 (sample 9) isolated from fecal swabs of cattle with 24 of the most similar bcfC gene amino acid sequences from Gene bank.

Figure (17): Phylogenic diversity tree for TraT gene amino acids sequence of E. coli O26 GH12 (sample 12) isolated from fecal swab of cattle and E. coli O26 GH13 (sample 13) isolated from fecal swab of cattle with 24 of the most similar TraT gene amino acid sequences from Gene bank.

Figure (18): Phylogenic diversity tree for fimH gene amino acids sequence of E. coli O26 GH12 (sample 12) isolated from fecal swab of cattle and E. coli O26 GH13 (sample 13) isolated from fecal swab of cattle with 24 of the most similar fimH gene amino acid sequences from Gene bank.

Table 11:Nucleotide change in gyrA gene of two isolates of S. Typhimurium.

Table (12):Nucleotide change in gyrA gene of two isolates of E. coli.

Figure (19): Agarose gel electrophoresis of PCR prod­ucts after amplification of: 1- gyrA gene for S. Typhimurium (No. 2 and 4) and E. coli O26 (No. 3 and 11) strains, MWM-molec­ular weight marker (100 – 600 bp DNA ladder), control (Positive, Negative) and gyrA gene products at 344 bp).

Figure (20): Phylogenic diversity tree for gyrA gene amino acids sequence of S. Typhimurium GH2 (sample 2) isolated from feed sample and S. Typhimurium GH4 (sample 4) isolated from fecal swab of cattle with 23 of the most similar gyrA gene amino acid sequences from Gene bank.

Figure (21): Phylogenic diversity tree for gyrA gene amino acids sequence of E. coli O26GH3 (sample 3) isolated from fecal swab of cattle and E. coli O26 GH11 (sample 11) isolated from feed sample with 23 of the most similar gyrA gene amino acid sequences from Gene bank.

DISCUSSION

For long term milk production with hygienic measures, dairy cattle should be in a good health condition (Godinho and Carvalho, 2013).

The results in Table (2) showed that the isolated Salmonella species and E. coli were found with a percentage of 1.8% and 2.8% in the total examined samples respectively. On the other hand Salmonella species in each of the examined milk, feed, milk tanks swabs, drinking tanks swabs and fecal swabs samples were found with an incidence of 1%, 1%, 1%, 1% and 5%, respectively, while that of E. coli were 2%, 2%, 1%, 1% and 8%, respectively. These results were approximately agreed with the result recorded by Halimi et al. (2014)and Warnick et al. (2003)who found the incidence of Salmonella spp. was 1.5% and 1.1%, respectively, meanwhile lower than that recorded by each of Wells et al. (2001)who found that salmonella species were isolated from fecal and milk samples with an incidence 5.4% and 21.1%, respectively, that of El-Gedawy et al. (2014) who found that the incidence of Salmonella spp. in bulk tank milk and milking equipment were 9% and 6%, respectively and that of Sotohy and Khalifa (2018) who found that the incidence of the isolated Salmonella species from dairy farms was 3.2%. The lower incidence may be attributed to the sample may contain other organisms that may compete with Salmonella(Karns et al., 2005). On the other hand the results were higher than that recorded by Halimi et al. (2014) who found that no Salmonella species recovered from water, feed, milk filers, and milk fed to calves. The variation between our results and that of other author's may be referred to the differences in the survival of Salmonella spp. in water, soil and pasture depending up on the differing serovars, dose rates and environmental conditions whereas Salmonella spp. can survive for up to 20 weeks in soil and water(Guan and Holley, 2003). In case of E. coli, our results were lower than that recorded by Abd El- Tawab et al. (2017) who found that the incidence of E. coli in milk collected from different localities in Egypt was 6.2% and this may be attributed to the variation in samples types whereas our samples from apparently healthy while the other sample from mastitic milk and that recorded byMaity et al. (2010) who found the incidence of E. coli in fecal sample was 27.91% and the potential EPEC was 21.66%.

Serological results showed in Table (3 and 4) revealed that Salmonella species were serotyped as S. Typhimurium, S. Enteritidis and S. Saintipaul and found with a percentage of 66.67%, 22.22% and 11.11% from the total isolated Salmonella species, respectively. S. Typhimurium was the predominant serotype and found in milk, feed, milk tanks swabs and the fecal swabs samples with an incidence of 16.67%, 16.67% and 50% respectively. The serotyped S. Enteritidis was found in drinking tanks and fecal samples with an incidence 50% and 50% respectively, while S. Saintipaul was found with an incidence of 100% in the fecal samples only. The results of the isolated S. Saintpaul in our results were lower than that recorded by Sotohy and Khalifa (2018) who isolates 2 strain of S. Saintpaul, one from air 4% and one from manure 2.9%. S. Typhimurium was the predominates serotypes found with an incidence 66.67% and this attributed to the ability of S. Typhimurium can survive up to 28 weeks on pasture(Josland, 1951). E. coli isolates were serotyped to O26, O119, O125, O126 and O127 with an incidence 35.71%, 14.28%, 28.60%, 7.14% and 14.28%, respectively. This serotyped E. coli considered members of the enteropathogenic E. coli and this agree with the classification performed by WHO, (1987). The most predominant serotyped were O26 which found with an incidence of 80% and 20% in the fecal and feed samples respectively, followed by O125 which present with a percentage 25% for each of milk, feed, drinking tanks and fecal samples. The incidence of O119 was 50% for each of the examined milk tanks and fecal samples, while O127 was typed with an incidence of 50% for each of feed and fecal samples. The lowest incidence was O126 which present in the fecal samples only with an incidence of 100%. Our results of E. coli serotyping were differed than results recorded by Sayed, (2014) who found that E. coli isolates were 18 strains (17.82%) from 101 clinical mastitic milk samples of cows and serotyped to nine different serogroups; O111:H4 (3), O127:H6 (3), O26 (2), O126 (2), O119:H6 (1), O114:H21 (1), O55:H7 (1), O44:H18 (1), O124 (1) and (3) untyped. Also differed than that of Abd El-Tawab et al. (2017)who found that E. coli serotypes were 15 typed as O27, O146, O125, O126, O111, O20 and O157 and 2 untyped. This variation was attributed the difference in in their natural reservoir (Foley et al., 2008).

By agar disk diffusion method, antibiotic sensitivity test were applied against the isolated Salmonella (n=9) and E. coli (n= 14) strains and recorded in Table (5). The results revealed that Salmonella and E. coli strains have led to development of resistance to antimicrobial agents which originally effective against the examined microbes. Salmonella strains showed 100% resistance against ceftroiaxon; gentamicin; lioncomycin and vancomycin. Also Salmonella strains showed a resistance against oxolinic acid; penicillin G and streptomycin; with an incidence 88.89%, 77.78% and 88.89%, respectively. While the incidence of resistance Salmonella strains against erythromycin; oxytetracycline; trimethoprim + sulphamethoxazole were 55.56%, 33.33% and 22.22%, respectively. Our results showed multi-drugs resistance as recorded by Halimi et al. (2014)andagree with the results recorded byTamba et al. (2016)who found that all isolates of Salmonella showed 100% resistance to lincomycin. Also our resistance results of Salmonella species against oxytetracycline were lower than that recorded by Halimi et al. (2014) who found that 94.74% of salmonella species were resistance to oxytetracycline and that ofTamba et al. (2016)who recorded that 85.71% of Salmonella species were resistance to erythromycin. On the other hand, our results were higher than that of Mohamed et al. (2011) who found that 14.3% of Salmonella strains were resistant to gentamycin, that ofWells et al. (2001)who recorded that 0.1% of Salmonella strains were resistant to gentamycin.The difference between our results and the results recorded by Halimi et al. (2014) come back to the differences between farms in the frequency of usages, widespread and inappropriate usage of oxytetracycline in dairy operations and dairy farms and association with fecal shedding.

Also the resistance of E. coli strain against ceftroiaxon; lioncomycin; oxolinic acid; penicillin G and vancomycin were highly resistance and found with incidence 100 %, 100 %, 100 %, 100 % and 85.71%, respectively, while the resistance against erythromycin; gentamicin; oxytetracycline; streptomycin and trimethoprim + sulphamethoxazole were 21.42 %, 21.42 %, 35.71%, 50.00% and 14.30 %, respectively. Our results showed multi-drugs resistance as recorded byAbd El- Tawab et al. (2017) and Yassin et al. (2017).On the other hand our results varied with the results recorded by other authors whereas my results were lower than that of Abd El- Tawab et al. (2017) who found that the incidence of resistance against gentamicin; oxytetracycline and streptomycin were 30%, 70% and 100%, respectively, while a higher than the resistance against penicillin G (80%) recorded by Abd El- Tawab et al. (2017)and also higher than that recorded byYassin et al. (2017) who found that the incidence of resistance against gentamicin; streptomycin; ceftroiaxon and trimethoprim + sulphamethoxazole were 8.2%, 18.0%, 4.9% and 18.0%, respectively. The variation in the incidence of resistance against antibacterial between our results and the results of other authors were referred to the variation between the use and misuse of antimicrobials in farm animals (Sawant et al., 2007).

All serotyped Salmonella (No. =9) and E. coli (No. =14) were conducted for molecular characterization by conventional PCR Table (6) and Fig. (1and2). Firstly, confirmation of Salmonella and E. coli applied by detection of invA gene (at 284 bp) and phoA gene (at 720 bp) which were found with an incidence 100% and 100%, respectively. These results agree with results reported by Sotohy and Khalifa (2018) who found that invA (at284 bp) virulence and conformity gene was found in all isolated Salmonella strains. In this study invA gene was used as confirmatory genes for genus Salmonella due to the invA gene of Salmonella species contains unique sequences to this genus and has been proved to be a suitable PCR target with potential diagnostic application (Jamshidi et al., 2008). invA gene is recognized as an international standard for detection of Salmonella genus (Malorny et al., 2003). On the other hand phoA genes was selected to confirm the detection of E. coli strains and this results agree with result recorded by Kong et al. (1995); Kong et al. (1999) and Yu and Thong, (2009)who performed the confirmation of E. coli by detection of phoA gene at 720 bp. which present in all E. coli strains.

The results in Table (7) and Fig. (1, 3 and 4) showed that the incidences of each of the studied invA (at 284 bp), stn (at 617 bp) and bcfC (at 467 bp) virulence genes were detected in 100% of each of the isolated S. Typhimurium, S. Enteritidis and S. Saintipaul). The incidence of each of invA gene, stn gene and bcfC gene in all isolated Salmonella species were 66.67%, 22.22% and 11.11%, for S. Typhimurium, S. Enteritidis andS. Saintipaul respectively. Our results showed that the virulence invA, stn gene and bcfC gene were detected in all of the isolated salmonella strains and this was disagree with results recorded bySotohy and Khalifa (2018) who found only invA (at 284 bp) virulent gene was detected in S. Saintipaul. This variation was attributed the difference in their natural reservoir (Foley et al., 2008).

Our results of stn gene were higher than that of Maysa and Abd-Elall (2015) who found that stn were detected in S. Typhimurium and S. Enteritidis with incidence of 78.9% and 75%, respectively. Our results of bcfC were agree with the results recorded byMaysa and Abd-Elall (2015) who found that bcfC was detected in 100% of S. Enteritidis, while higher than that of Maysa and Abd-Elall (2015) who found bcfC in 88.9% of the isolated S. Typhimurium. The variation between our results and results recorded by other authors were regarded to widely distribution of the microorganisms among animals, humans and environment and some diversity in distribution could be explained by serovar specificity of virulence plasmid (Heithoff et al., 1997 and Rotger and Casadesus, 1999).

The studied phoA (at 720 bp), TraT (at 307 bp) and fimH (at 508 bp) virulence genes of the isolated E. coli present in Table (2) and Fig. (2, 5 and 6) were detected in 100% of each the isolated E. coli serotypes. Each of phoA gene, TraT gene and fimH gene were detected in all serotyped E. coli with incidences 35.71%, 14.28%, 28.57%, 7.14% and 14.28%, for O26, O119, O125, O126 and O127 respectively. The phoA gene(at 720 bp) was detected in E. coli strains and this result agrees with result recorded byHu et al. (2011) and Alnahass et al. (2016).

Our results of the detection of TraT gene were higher than that recorded by each of Ashraf et al. (2018) who found that the incidence of TraT in the isolated E. coli was 66%, that of Nemeth et al. (1991)who found that the incidence of TraT in the isolated E. coli from mastitic milk and milk filler samples were 43% and 40%, respectively and that recorded by Mahmoud et al. (2015)who found that the incidence of TraT gene was 25%. Our results of fimH gene agree with the results recorded byFernandes et al. (2011) and Abd El-Tawab et al. (2017)who found that the incidence of fimH in all strains of the isolated E. coli were 100%. The incidence of fimH in O125 and O126 were agreed with the results recorded by Abd El- Tawab et al. (2017). Our results were higher than the results recorded byBronzato et al. (2017)who detected fimH with incidence 77.7% in isolated E. coli strain. This variation may be due to difference in the percentage of dispersion of microorganism in the dairy farm environment and horizontal gene transfer (Madsen et al., 2012).

The detected resistance genes in Table (9) and Fig. (7, 8 and 9) showed that each ampC gene (at 550 bp) and aacC gene (at 448 bp) were detected in 100% of each of the isolated S. Typhimurium, S. Enteritidis andS. Saintipaul, while mphA gene (at 403 bp) was detected in each of S. Typhimurium, S. Enteritidis andS. Saintipaul, with an incidence 50%, 50% and 100% respectively. The incidences of each ampC gene and aacC gene in all isolated Salmonella species were 66.67%, 22.22% and 11.11%, for S. Typhimurium, S. Enteritidis andS. Saintipaul respectively, while mphA gene was detected in all Salmonella strain with an incidence 55.56%. Our result for resistance mphA gene (at 403 bp) for the isolated Salmonella strain werehigher than that recorded byWang et al. (2017) who detected the mphA gene with a percentage of 48.39% of resistant Salmonella isolates and that of Abdel Aziz et al. (2018)who found that themphA resistance gene cassette was detected in 41.7% of isolated salmonella showed multidrug resistance. Also our results of ampC gene (at 550 bp) was higher than that recorded by Zhao et al. (2008) who found that ampC resistance gene was detected in Salmonella species isolated from ground turkey meat and chicken breast with a percentage of 46.67% and 11.11%, respectively. Also higher than that cited byPublic Health Agency of Canada (2007) whereas ampC resistance gene was detected in approximately 30% of salmonella isolates in 2003 and the prevalence was gradually increased to approximately 48% in the second quarter of 2005. The results of aacC gene (at 448 bp) was higher than that of Randall et al. (2004) and Lynne et al. (2008)who recorded that aacC was detected in 71.43 % and 42.90% (3) isolates, respectively.

While the detected resistance genes of E. coli in Table (10) and Fig. (10, 11, 12, 13 and 14) showed that blaTEm gene (at 516 bp) and ampC gene (at 550 bp) resistance genes were found in 100% of each the isolated E. coli serotypes while that of mphA gene (at 403 bp), Aada1 gene (at 484 bp) and aacC gene (at 448 bp) resistance genes were found in 21.43%, 50.00% and 21.43%, respectively. Each of blaTEm gene and ampC gene resistance genes were detected in each of O26, O119, O125, O126 and O127 with an incidence 35.71%, 14.28%, 28.57%, 7.14% and 14.28%, respectively, while mphA gene was detected only in O125 with incidence of 75%. Also the results revealed that Aada1 resistance gene was detected in O26, O119, O125 and O127 with an incidence of 60%, 50%, 50% and 50%, respectively. Meanwhile aacC gene was found with incidence of 20%, 50% and 50% in O26, O119 and O127 respectively. Our results agree with results recorded by Hussein et al. (2008)who found mphA resistance genes in a percentage 100% of the isolated E. coli. Meanwhile the results of the resistanceblaTEm gene (at 516 bp), ampC gene (at 550 bp)and Aada1 genes (at 484 bp) were higher than that recorded byAshraf et al. (2018)who found that the blaTEm, ampC and Aada1 genes were detected with a percentage of 4% and 26% and 12% of the isolated E. coli respectively. Also higher than that of Hinthong et al. (2017)who detectedblaTEm, Aada1 and aacC gene with a percentage of 61.3%, 3.3% and 4.9%, respectively and that ofWassef et al. (2014)who detected the ampC gene in the isolated E. coli with a percentage 66.7%. The variation between results may regards to geographical discrepancy in ampC β-lactamase types Pai et al. (2004). In general the variation between results was regarded to the dissemination of strains carrying resistance genes for antimicrobials whereas the antimicrobial drugs as aminoglycosides, beta-lactams, tetracycline chloramphenicol, sulfonamides, and trimethoprim has been acquired by E. coli strains from other microorganisms (Lietzau et al., 2006).

In Fig. (15 and 16), DNA Sequence was initially performed to establish sequence identity to Gene Bank accessions.S. Typhimurium GH3 and S. Typhimurium GH9 were selected to study the similarity of virulence stn gene andbcfC gene with other types in Gene bank, while in Fig. (17 and 18), E. coli O26 GH12 and E. coli O26 GH13 were selected to study the similarity of virulence fimH gene and TraT gene with other types in Gene bank. Sequence alignments using the NCBI BLASTP program showed that of S. Typhimurium GH3 virulence stn had high genetic similarity (99.4%) of S. Enteritidis with accession-numbers: Cp018659.1 _S._ Enteritidis_93-0639 & Cp018640.1_S._ Enteritidis_70-1605 & Cp018661.1_S._ Enteritidis_ 95-0621 & Cp025554.1_S._ Enteritidis_ATCC_ BAA-708, while S. Typhimurium GH9 virulence stn had height genetic similarity with LS483489.1_S_ Poona_NCTC4840 and CP019201.1_S_ Muenster _420_CFSAN001201.

While S. Typhimurium GH3 virulence bcfc showed a high percentage of genetic similarity (99.6%) with accession-numbers: Cp031359.1_S._Heidelberg_5 & Cp012349.1_S._Slotedijk_ATCC_15791 & Cp019186.1_S._Pomona_ATCC_10722.

On the other hand the E. coli O26 GH12 and E. coli O26 GH13 virulence TraT were agree with other E. coli with accession-numbers: X06915.1_E._coli_F and Cp014273.1_E._coli_K_12_C3026 with a percentage of 99.7% and 100% respectively. While E. coli O26 GH12 virulence fimH was highly genetic similarity with accession-numbers: Cp007592.1_E._coli_O157:H16_Saintai & Cp001368.1_E._coli_O157:H7_TW14359 & Cp001164.1_E_coli_O157:H7_EC4115 with a percentage 98.4% for each one. Also E. coli O26 GH13 virulence fimH had a highly genetic similarity with E. coli with accession-numbers: Cp034843.1_E_coli_L103-2, Cp034734.1_E_coli_ L53, Cp033092.1_E_coli_ATCC_117755, FJ865813.1_E_coli_Top1371, FJ865736.1_E _coli_TB154A and FJ865622.1_E_coli_ECOR33.

Sequence alignments of antimicrobial resistance gene as gyrA (at 344 bp) for both S. Typhimuriumand E. coli O 26 were performed by the NCBI BLASTP program after confirmation by PCR as showed in fig. (19). The main principle was the detection of the substitutions in terms of amino acid positions gyrA Ser 83, gyrA Asp 87 and gyrA Alar 179, which are located within the QRDR. The mutations induced a local conformation changes of the A subunits cause marked resistance to specific antibiotic as quinolones. On studied of the selected 2 strains of Salmonella, S. Typhimurium GH2 and S. Typhimurium GH4 in Table (11)and Fig.(20)showed that amino acid changes detected at amino acid 83 were Ser changed to phe results in one point mutant. Also the selected E._coli_ O26_ GH3 and E._coli_ O26_ GH13 in Table (12)and Fig.(21)had one point mutation at amino acid 83 whereas Ser changes into Leu. Our results agree with the results recorded by Yoshida et al. (1988)who found thatamino acid changes detected in amino acid 83and the point mutations in codon TCG at Ser 83and with the results recorded byNakamura et al. (1989)who found that Mutations in the gyrA and gyrB subunits of DNA gyrase play a major role in conferring a high level of resistance to fluoroquinolone in Gram-negative bacteria, such as E. coliwhile themutations in gyrA gene (at 344 bp) are more common in quinolone resistance of E. coli.

In this study some virulence genes of both Salmonella species and E. coli were detected but no records of symptoms within the examined dairy cattle herds, this referred to the pathogenicity is not dependent on one virulence factor but occurred due to the regulation and interaction between a numbers of virulence factors that affected by environmental conditions as host species, species stress, host health status, immune status of the individual, interaction with other bacteria, the infecting dose, the method of delivery of the organisms to the host. Therefore the examined dairy cattle herds considered carrier animals that shed and spread the Salmonella and    E. coli microorganisms in the feces, milk and/or environment after ingestion feed or water contaminated with feces from other carrier or infected animals (cross contamination). Also the detected resistance genes regards to a problem in the role of corrected treatments. Thus we concluded that strictly purchasing cattle from good source, with its life history from birth, vaccination, treatment and diseases history, not from dealers of unknown sources. Strict control of the environment of farms by preventing contacts between dairy cattle, calves and the other carrier species such as dogs, birds, cats, people, pig, feral cats and wild birds. Also prevent contacts between the different carrier species and feed, water and all equipment used in the production of milk especially feral cats and wild birds. Only good sources of feed and good sources of water were used in the farms. Awareness should be created among the dairy farmers on the transmission of various diseases from dairy environment to dairy cattle and the preventive measures used. Governorates should cite the supervision of veterinarian in farms is strictly. Strict hygienic measures were applied during the waste management and effluent control. Antimicrobial drugs should be used when needed with an accurate dose, in specific times and for specific cases during a certain period under the supervisions of veterinarian. Applied the recommendations and the hygienic measurement of HACCP, biosafety and biosecurity for dairy farms especially for water, soil, udder, unhygienic milking utensil, and Milkers’ hands to obtain a good health dairy cattle and calves and good hygienic safe milk for consumers.

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