Assiut University web-site: www.aun.edu.eg

FACTORS AFFECTING CALF ENTERITIS INFECTION CAUSED BY SALMONELLAE AND ESCHERICHIA COLI

MONA, M. EL-AZZOUNY; AHMED M. ELHADY and SALLY, H. ABOU- KHADRA

Microbiology Unit, Animal Health Research Institute, Zagazig Branch. Animal Health Research Institute (AHRI), Agriculture Research Center (ARC), Egypt.

Received: 31 March 2020;     Accepted: 30 April 2020

ABSTRACT

Calf diarrhea is a major economic concern in bovine industry all around the world. Therefore, the present study was designed to investigate seasonal and age variations in the prevalence of E. coli and Salmonella species in diarrheic calves and their virulence genes, pathotypes, serogroups and antibiogram. Bacteriological examination of 120 fecal samples collected from diarrheic calves less than three months of age in dry and rainy seasons showed that, 71 (59.5%) and 36 (30%) were positive for E. coli and Salmonella species, respectively while 26 (21.66%) had mixed E. coli and Salmonella species infection. The most prevalent E. coli serogroups were O111 and O26 and Salmonella serovars were S. typhimurium and S. enteritidis. Isolation frequency of E. coli was significantly higher than Salmonella species isolated from diarrheic calves in different ages and seasons (P value=0.004). E. coli and Salmonella species was statistically significant higher in rainy season than dry season. PCR investigation of six virulence determinants among the MDR E. coli isolates revealed that fimH and iss, were the most prevalent (100%), followed by sxt2 (90%), sxt1 (80%), hylA (60% ) and eaeA (40%). All examined multiple drug resisitant (MDR) Salmonella species isolates harbored both invA and sopB virulence genes. E. coli and Salmonella species isolates showed a high sensitivity rate for amikacin (100%), and ciprofloxacin (78.8% and 77.7%), respectively but showed resistance against amoxicillin and tetracycline. The most commonly detected resistance gene was tetA gene. Finally, strict management and hygienic measures should be taken in rearing neonatal calves especially in rainy seasons.

Keywords: Neonatal calf diarrhea - E.coli- Salmonellae - virulence and resistance genes.

Corresponding author: MONA, M. EL-AZZOUNY

E-mail address: monael3zzouny@yahoo.com

Present address: Microbiology Unit, Animal Health Research Institute, Zagazig Branch. Animal Health Research Institute (AHRI), Agriculture Research Center (ARC), Egypt.

INTRODUCTION

Calf diarrhea (CD) is one of the most common problems in young animals, causing huge economic and productivity losses to bovine industry worldwide (Cho and Yoon, 2014). In Egypt, neonatal calf diarrhea (NCD) continues to be the 1st cause of calf mortality, which ranges between 27.4% and 55% of the total deaths in young calves (Younis et al., 2009). The economic losses occur not only from mortality but also from other costs including treatment, diagnostics, labor intervention and decreased number of herd as well as subsequent chronic ill thrift and impaired growth performance (Bazeley, 2003). NCD is a multifactorial syndrome including pathogen (infectious NCD) as well as non-infectious factors related to the animal (immunological and nutritional status), the environment or the management (Izzo et al., 2011). Because of the multifactorial nature of NCD, it is difficult to be controlled effectively (Cho and Yoon, 2014). The number of cases of diarrhoea is normally higher during the rainy seasons, from October/November to March than during the dry seasons, from March to October Achá et al. (2004).

In spite of the complex etiology of calf diarrhea, the involvement of bacterial pathogens is still responsible for more than 50% cases of diarrhea in neonatal calves (Kumar et al., 2012) Among bacteria, enterotoxigenic Escherichia coli (ETEC) and Salmonellae are the most economically important pathogens (Achá et al., 2004), also other bacteria have been found as causes of enteric disease and NCD, e.g. Clostridium species (Cho et al., 2010). Identification of the possible causative agent in outbreaks of diarrhea is important to allow targeted

preventative measures, such as vaccination, and identification of possible risk factors or sources of infection (Izzo et al., 2011). Salmonella enterica colonizes the digestive  system of both adult cattle and calves, but the infection is more common and often causes severe symptoms in 10-day to 3- month-old calves (Fossler et al., 2005). On the other hand, E. coli K99+ causes a watery diarrhea, dehydration, and weakness in 1- to 4-dayold newborn calves (Acres et al., 1977). Younger age and low colostrum feeding calves were significantly associated with E. coli isolation (Ashenafi and Tesfaye, 2016). Diarrhea due to Salmonella infection is watery and mucoid with the presence of blood and fibrin (Fossler et al., 2005). Salmonella pathogenicity island SPI-1 and SPI-5 including invA and  sopB genes are known to influence the type III secretion system, and are mainly responsible for Salmonella induced diarrhea in calves (Treuer and Haydel, 2011).

Infected cattle and carriers can serve as source of infection for other animals or even human through food-borne routes or direct contact and so the determination of Salmonella strains in fecal samples is not only important for the diagnosis of salmonellosis, but also essential to identify the carriers (Warnick et al., 2003).

E. coli classified into enterohemorrhagic (EHEC), enterotoxigenic (ETEC), necrotoxigenic (NTEC), enteroinvasive (EIEC), enteropathogenic (EPEC) and attaching and effacing E. coli (AEEC) pathotypes Kaper et al. (2004). The diarrhea in calves is  commonly caused by enterotoxigenic Escherichia coli (ETEC), the most common type of colibacillosis in neonatal calves (Nagy

and Fekete, 2005). Two of the more prominent virulence factors identified for ETEC strains are (i) expression of fimbrial (pili) antigens that enables the bacteria to adhere to and to colonize the luminal surface of the small bowel and

(ii) elaboration of one or more enterotoxins that influence the intestinal secretion of fluids (Holland, 1990). Unlike ETEC, Enteropathogenic E. coli (EPEC) strains do not produce toxins, but habor an outer membrane protein, intimin, which mediates the intimate attachment of bacteria to the enterocyte, causing typical attaching and effacing (A/E) intestinal lesions (Law, 2000). One feature EHEC and EPEC have in common is the causation of intestinal epithelial lesions known as A/E (Moxley and Smith, 2010). EHEC strains are a subset of STEC strains (Nguyen et al., 2011).

The most common cause of NCD is ETEC strains that produce the K99 (F5) adhesion antigen (E. coli K99+) and heat-stable (STa or STb) and/or heat- labile (LT1 or LT2) enterotoxins (Kaper et al., 2004). Another virulence factors including Shiga toxin 1 (stx1) and Shiga toxin 2 (stx2), the protein intimin (eae) and                                      enterohaemolysin        or enterohaemorrhagic E. coli haemolysin (ehly) which are related to the pathogenesis of STEC strains (Law, 2000).

Furthermore, it appears that E. coli, particularly serogroups O26, O111and O118 are virulent to cattle, particularly young calves (Wieler et al., 1998).  E. coli O26 has been isolated from clinical cases of hemorrhagic diarrhea in young and neonatal patients (Pearson et al., 1999).

Antimicrobials are commonly used in treatment of diseased calves either orally or parentally. Some commonly used antimicrobials as sulbactam, ampicillin, neomycin, cephalosporin, tetracycline and sulphonamide- trimethoprim mixture have been used (Kumar et al., 2010). However, in the last decade or so the development of drug resistance and the presence of multi drug resistant pathogenic bacteria are major problems in treating bacterial diseases (Anita et al., 2013).

Therefore, the present study was planned to assess seasonal and age variations in the prevalence and serogroup distribution of E. coli and Salmonella species in fecal sample of diarrheic calves and investigate their susceptibility to commonly used antimicrobials.

MATERIAL AND METHODS

Sampling and bacteriological examination.

A total of 120 fecal samples from diarrheic calves were collected randomly from September 2018 to October 2019. Seasons of samples collection (rainy and dry) and age of diarrheic calves (one week to 3 months) were recorded during sampling. Fecal samples were taken using sterile rectal swabs. All samples were transferred in an ice box to the laboratory and cultured in the same day or stored at 4ºC and cultured within 3 days.

Isolation and identification of E. coli and Salmonella species among the collected samples were confirmed based on their morphology, cultural and biochemical tests using standard bacteriological procedures described by Quinn et al. (2002) and (ISO 6579, 2002),

respectively. Serological identification of

E. coli and Salmonella species isolates was conducted at Serology Unit, Animal Health Research Institute, Dokki, Giza, Egypt using commercial antisera (Difco, Detroit, MI, USA) according to the manufacturer’s instructions.

Antimicrobial susceptibility testing.

In vitro susceptibility of all E.coli and Salmonella isolates to commonly used antimicrobial drugs was tested by the Kirby-Bauer standard agar disk diffusion technique as described earlier (Bauer et al., 1966), using Mueller Hinton agar and commercial  antibiotic       disks                           (Oxoid, Basingstoke, Hampshire, England, UK). The       tested                          antibiotics     and                               their concentrations         in     μg/disk                   were                as following:              amoxicillin             (AX;10), spectinimycin(SPT:100), amikacin(AK:30),     ceftriaxone                      (CTX; 30), colistin(CT:10), tetracycline (TE; 30),                sulfamethoxazole/trimethoprim

(SXT; 25), streptomycin (S; 10), gentamicin (CN; 10), and ciprofloxacin (CIP; 5). The inhibition zones, in millimeters, were measured in duplicate and scored as sensitive, intermediate, and resistant categories in accordance with the critical breakpoints recommended by the Clinical and Laboratory Standards Institute (CLSI, 2011).

Polymerase chain reaction procedure DNA extraction:

DNA extraction from bacterial samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations.

Oligonucleotide Primer:

Primers     used     were     supplied    from Metabion (Germany). Primer sequences,

amplicon size and references are listed in table (1).

PCR amplification:

Primers were utilized in a 25- µl reaction containing 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration,

4.5 µl of water, and 6 µl of DNA template. E. coli virulence genes (stx1, stx2, hlyA and eaeA) were performed in Multiplex DNA amplification reaction. The reaction was performed in an applied biosystem 2720 thermal cycler.

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) was 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.

Statistical analysis.

The SPSS (Statistical Package for the Social Sciences) software (Ver. 24) and Chi-square test were used to study the statistical relationship between the incidence of a bacterium in various ages, and seasons and also between the frequency of various virulence genes, antibiotic resistance genes and serogroups. P-value < 0.05 was considered statistically significant.

RESULTS

Occurrence of E. coli and Salmonella species among the examined samples.

Out of 120 fecal samples collected in both dry and rainy seasons from diarrheic calves between 1 week and 3 month of age, 71 (59.1%) and 36 (30%) were characterized as E. coli and Salmonella species, respectively. From examined samples 26 (21.66%) had mixed E. coli and Salmonella species infection as represented 11/60 (18.33%) in dry and 15/60 (25%) in rainy seasons). Prevalence of E. coli and Salmonella species in the tested samples were listed in (Table 2).

Effect of seasonal and age variations on E. coli and Salmonella species isolated from diarrheic calves.

The results of E. coli and Salmonella species prevalence in fecal swabs obtained from diarrheic calves showed that E. coli and Salmonella species significantly higher in rainy season 40/60(66.6%), 22/60(36.6%) than dry season 31/60(51.6%), 14/60 (23%),

respectively also, their isolation rate significantly different according to age of diarrheic calves.

E. coli isolates were significantly higher occurrence in diarrheic calves with age

Salmonellaspp. isolates were significantly lower occurrence in diarrheic calves with age

Totally, E. coli isolates were significantly higher than Salmonella species isolates in diarrheic calves with different age and seasons (p-value=0.004) (Table 2).

E. coli were serogrouped to O111, O26, O119, O124, O17 and O8. The most

common serogroups were O26 and O111 (35.2% and 32.3 %, respectively), followed by O124, O119, O17 and O8. There was different between the pathotypes of E.coli in different seasons as we found that in dry season the prevalence of EHEC and EPEC pathotypes were 64.5% and 35.4% while in rainy season EHEC, EIEC and ETEC pathotypes were 70%, 20%, and 10%,

respectively (Table 3).

Serotyping results revealed that Salmonella species belonged to 4 different serovars. S. typhimurium was accounted for 61.11% of total Salmonella isolates and others serotypes were 38.88% included: S. enteritidis (25%), S. infantis (11.1%) and S. essen (2.8%) as listed in table (3).

Antimicrobial sensitivity test among E. coli and Salmonella species isolates.

Antibiogram results illustrated the presence of multi-drug resistance in most of E. coli and Salmonella species isolates recovered from diarrheic calves against the ten used antibiotics. Interestingly, E. coli isolates were resistant to most of the tested antibiotics as amoxicillin, colistin, tetracycline, streptomycin, gentamycin sulfamethoxazole/trimethoprim         and spectinomycin with percentage of (100%, 100%, 83% ,  81,6%,  77.4%,  73.2%,and

67,6%, respectively) while all examined Salmonella species isolates exhibited absolute resistance (100%) to them except       tetracycline   (88.8%), streptomycin (80%) and gentamycin (69.4%). On the other hand amikacin and ciprofloxacin were found to be the effective drugs for E.coli and Salmonella that showed sensitivity rate 100% for amikacin and in case of ciprofloxacin (78.8% and 77.7%), respectively as listed

in table (4).

Virulence and resistant genes among

E. coli and Salmonella species isolates. Ten MDR E. coli isolates were examined by PCR to detect 6 virulence genes (fimH, iss, stx1, stx2, eaeA,, and hlyA) (Fig.  1-3) (Table 5).       All isolates were positive for both fimH and iss genes (100%). Amongst isolates that carried the stx genes, 8 (80%) were stx1, 9 (90%) were stx2 and 4(40%) were have both stx1/stx2.               Moreover,      hlyA                 gene  was detected in 60% of isolates. Only 4 E. coli isolates carried eaeA gene  (40%) that includes 3 and 1 isolates in dry season and rainy season, respectively. Our results illustrated that 40% of the analyzed E. coli isolates harbored all 6 examined virulence genes and 20% of isolates   had   at   least   three   virulence genes.

Screening the presence of tetA, aadB, aacC genes in the most MDR E. coli by PCR technique revealed that distribution of resistance genes among 10 phenotypically resistant E. coli were as the following: all examined isolates were positive for tetA genes with percentage of (100%) (Fig. 4) while 3 isolates were positive for aacC (30%) (Fig. 5) and aadB gene were not detected.

All examined ten MDR Salmonella species isolates had both invA and sopB virulence genes (100%) (Fig. 6 and 7). Interestingly, the results of tetA gene (100%) (Fig. 8) but, aadB and aacC genes were not detected.

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

Target

Amplified

Primary

Amplification (35 cycles)

Final

gene          Primers sequences

segment (bp)

denaturation

Secondary

denaturation       Annealing      Extension

extension        Reference

stx1

ACACTGGATGATC

TCAGTGG                   614                  95°C

CTGAATCCCCCTC                                  3 min.

95°C

20 sec

58˚C

40 sec.

72˚C

90 sec.

72˚C

5 min.

Dhanashree and Mallya, (2008)

Stx2

eaeA

hlyA

CATTATG

CCATGACAACGG ACAGCAGTT            779

CCTGTCAACTGAG CAGCACTTTG

GTGGCGAATACTG GCGAGACT             890

CCCCATTCTTTTTC ACCGTCG

ACGATGTGGTTTA

           TTCTGGA                    165

CTTCACGTGACCA TACATAT

95°C

3 min.

95°C 20 sec             58˚C 40 sec.

72˚C

90 sec.

72˚C

5 min.

Mazaheri et al., (2014)

Fratamico

et al., (1995)

iss

fimH

invA

sopB

aadB

aacC

tetA(A)

ATGTTATTTTCTG

          CCGCTCTG          CTATTGTGAGCAA TATACCC TGCAGAACGGAT AAGCCGTGG

GCAGTCACCTGCC CTCCGGTA

GTGAAATTATCGC CACGTTCGGGCAA

TCATCGCACCGTC AAAGGAACC

TCAGAAGTCGTCT AACCACTC

TACCGTCCTCATG CACACTC

GAGCGAAATCTGC CGCTCTGG

CTGTTACAACGGA CTGGCCGC

GGCGCGATCAAC GAATTTATCCGA

CCATTCGATGCCG AAGGAAACGAT

GGTTCACTCGAAC GACGTCA

CTGTCCGACAAGT TGCATGA

266                     94˚C

5 min.

508                  94˚C

5 min.

284                  94˚C

5 min.

517                  94˚C

5 min.

319                  94˚C

5 min.

448                  94˚C

5 min.

576                  94˚C

5 min.

94˚C

30 sec.

94˚C

30 sec.

94˚C

30 sec.

94˚C

30 sec.

94˚C

30 sec.

94˚C

30 sec.

94˚C

30 sec.

54˚C

30 sec.

50˚C

45 sec.

55˚C

30 sec.

58˚C

40 sec.

58˚C

40 sec.

60˚C

40 sec.

50˚C

40 sec.

72˚C

30 sec.

72˚C

45 sec.

72˚C

30 sec.

72˚C

45 sec.

72˚C

40 sec.

72˚C

45 sec.

72˚C

45 sec.

72˚C

7 min.

72˚C

10 min.

72˚C

7 min.

72˚C

10 min.

72˚C

10 min.

72˚C

10 min.

72˚C

10 min.

Yaguchi et

al., )2007)

Ghanbarpo ur and Salehi,

)2010)

Olivera et

al., )2003)

Huehn et

)al., 2010)

Frana et al.,

)2001)

Lynne et al.,

)2008)

Randall et al., (2004)

Table 2: Occurrence of E. coli and Salmonella spp. in diarrheic calves.

^* Significant differences in isolation rate according to seasons and ages diarrheic calves (P> 0.05).

^** Significant differences between isolation rate of E. coli and Salmonella species in diarrheic calves (P

<0.05).*** mean ± stander error

Table 3: serological identification of E. coli and salmonella species isolated from diarrheic calves in different seasons.

Isolates(n)

Season/ Age

Dry season                                                                 Rain season

E.coli (71)

EHEC

O111 (5)        EHEC

O111 (3)        EHEC

O111

(12)          EHEC(7)

O111 (3)

EPEC

(10)

O119 (7)

EPEC

(1)

O17

ETEC            O8          ETEC (1)            O8 (3)

Salmonella (36)

S.typhimurium (5)           S.typhimurium(6)

S.typhimurium (5)            S.typhimurium (6)

S. enteritidis(4)                S. enteritidis(5)

S.infantis (1)                   S.infantis (2)                  S.infantis (1)                      S. essen(1)

EHEC (Enterohemorrhagic Escherichia coli), EPEC (Enteropathogenic Escherichia coli), ETEC (enterotoxigenic Escherichia coli, EIEC (Enteroinvasive Escherichia coli).

Table 4: Antimicrobial susceptibility profiles of E.coli and Salmonella spp. isolates.

Table 5: Distribution of virulence and resistant genes of E. coli and Salmonella species from diarrheic calves.

* Significant differences according to seasons in presences of eaeA gene (P> 0.05).

** Significant differences according to seasons in presences of aacC gene (P> 0.05).

Figure 1: PCR amplicons of fimH gene of E.coli. Lane L: 100-bp ladder; lane Neg.: negative control; lane Pos.: positive control; lanes 1–10: positive isolates at 508 bp amplicons.

Figure 2: PCR amplicons of iss gene of E.coli. Lane L: 100-bp ladder; lane Neg.: negative control; lane Pos.: positive control; lanes 1–10: positive isolates at 266 bp amplicons.

Figure 3: Multiplex PCR of stx1 (614 bp), stx2 (779 bp), eaeA (890 bp) and hlyA (165 bp) virulence genes of E. coli. L:100-bp ladder; lane Neg.: negative control; lane Pos.: positive control

Figure 4: PCR amplicons of tetA(A) gene of E.coli.. Lane L: 100-bp ladder; lane Neg.: negative control; lane Pos.: positive control; lanes 1–10: positive E.coli isolates at 576 bp amplicons.

Figure 5: PCR amplicons of aacC gene of E.coli.. Lane L:100-bp ladder; lane Neg.: negative control; lane Pos.: positive control; lanes 6,8,10: positive E.coli isolates at 448 bp amplicons.

Figure 6: PCR amplicons of invA gene of salmonella. Lane L:100-bp ladder; lane Neg.: negative control; lane Pos.: positive control; lanes 1–8: positive salmonella isolates at 284 bp amplicons; lane 9-10: negative isolates

Figure 7: PCR amplicons of sopB gene of salmonella. Lane L:100-bp ladder; lane Neg.: negative control; lane Pos.: positive control; lanes 1–8: positive salmonella isolates at 517 bp amplicons

Figure 8: PCR amplicons of tetA(A) gene of salmonella. Lane L:100-bp ladder; lane Neg.: negative control; lane Pos.: positive control; lanes 1–8: positive salmonella species isolates at 576 bp amplicons

DISCUSSION

Neonatal calf diarrhea (NCD) remains as one of the most important problems encountered by livestock industry, causing great economic losses. Calves are at greatest risk of diarrhea within the first month of life and the incidence of

diarrhea decreases with age (Garcia  et al., 2000).

Herein, the prevalence of E. coli and Salmonella in neonatal diarrheic calf was 59.5% and 30%, respectively. From examined samples 18.33 % and 25% had mixed E. coli and Salmonella species

infection in both dry and rainy season, respectively.

The prevalence of E. coli in the current study had nearly coincided with the findings of Osman et al. (2013) (63.6%), and Hassan (2014) (50%), while higher than those of Azzam et al. (2006) (5.4%) and El-Shehedi et al. (2013) (35.83%), and lower than that obtained by Ibrahim, (1995) (100%). In comparison to other countries, the present results were similar to that mentioned by Hemashenpagam et al. (2009) in India (75%), while it was higher than those obtained by Joon and Kaura (1993) in India (23%), Viring et al. (1993) in Sweden (11.5%), Steiner et al. (1997) in Germany (42%).

E.coli isolation rate in rainy season was significant higher than dry season. Similar finding obtained by Masoud  et al. (2014) who found that prevalence of E.coli in samples collected in the winter had (54.92%), but in summer (2.53%). One possible explanation for the high prevalence of E. coli strains in calves in winter is that climatic variables such as heat, rain and thunderstorms, together with variable barometric pressure may have affected the autonomic nervous systems. These variables could affect immunity, thus making calves more susceptible to infections. Alternatively, Norheim et al. (1985) stated that the higher prevalence of E. coli strains in winter may be related to the fact that the mean serum IgG1 concentrations were low in winter born calves and increased during the spring and summer.

We found that in case of calves less than 2 month age had the highest incidence  of

E. coli (71%), while the 2-3 months age calves had the lowest incidence (38.6%) similarly    to    previous    mentioned  by

Masoud et al. (2014). Similarly, another study found that E. coli is the most common agent causing diarrhea in calves during the first week of life (Pereira et al., 2011 and Wagdy et al., 2016).

Serogrouping of E. coli isolates showed that six O-serogroups were identified; out of them O26 and O111 were the most prevalent groups (35.2% and 32.3 %, respectively) followed by O124, O119, O17 and O8. These findings were also nearly similar to that obtained by Wieler et al. (1998) who revealed that E. coli, particularly serogroups O26, O111 and O118 are virulent to cattle, particularly young calves. Also, Pearson et al. (1999) mentioned that E. coli O26 has been isolated from clinical cases of hemorrhagic diarrhea in young and neonatal patients. EHEC Pathotype was the most common pathogenic E.coli isolates (67.6%) obtained from examined diarrheic calves, while, higher than stated by Pereira et al. (2011) (38%). On contrary to previously recorded by Nataro and Kaper (1998) who found the most common cause of neonatal diarrhea is ETEC stains. Interestingly, we found that in dry season the prevalence of EHEC and EPEC pathotypes were 64.5% and 35.4%, respectively. EHEC, EIEC and ETEC pathotypes in rainy season were 70%, 20%, and 10%, respectively. The previous study by Nasr-Eldin et al. (2018) characterized pathogenic E.coli and the common detected pathogroups were EPEC and EHEC, ETEC and EIEC. Conversely, another study by Borriello et al. (2012) reported infrequent occurrences of ETEC strains.

The prevalence of Salmonella in the present study (30%) was similar to those of other studies in Australia by Izzo et al. (2011) (23.8%), while higher than that

reported by Riad et al. (1998) (18.2%); Seleim et al. (2004) (17.5%); Youssef

and El-Haig, (2012) (18.66%); Haggag and Khaliel, (2002) (4%), and Younis et al. (2009) (4.09%). On contrary, much higher prevalence was reported by Moussa et al. (2010) (43.53%) and Akam et al. (2004) (66.6%).

In our study the differences in prevalence rates of Salmonella and E.coli isolated from diarrheic calves in comparison to the previous studies could be explained in the light of species and geographical locations and hygienic measures, and these factors significantly influence the prevalence of salmonellosis in calves (Younis et al., 2009).

The diarrheic calves in both dry and rainy season with age Salmonella species isolation rate (21%) than diarrheic calves with age 2-3 months (45.4%) that in accordance with finding of Wagdy et al. (2016) and Achá et al. (2004) whom stated that E. coli and Salmonella are the most common identified pathogens in scouring calves less than 2 months of age.

Of interest, S. typhimurium (ST) and S. enteritidis (SE) were the most prevalent serotypes with percentages 61.1% and 25%, respectively. This result supported by many previous reports in Egypt (Seleim et al., 2004; Younis et al., 2009; Moussa et al., 2010 and Youssef and El- Haig, 2012) and reports from the other countries by Smith-Palmer et al. (2003) in Scotland and Rothenbacher (1965) in the USA. On contrary to previous obtained by El-Seedy et al. (2016) who found that S. enteritidis (60.9%), S. typhimurium (30.4%).

Antimicrobial resistance of Salmonella and E.coli is particularly worrisome in

view of its potential to extend into the human food chain, posing a challenge to public health. The data from the present study showed widespread resistance of

E. coli and Salmonella species isolates against the tested antibiotics especially against amoxicillin and colistin (100%) followed                 by                                tetracycline                         (83% and 88.8%, respectively) that in consistence with                               previous                    related  studies       on antimicrobial resistance (De Verdier et al., 2012 and Call et al., 2008). On the other          hand,            they                 were    sensitive                                to amikacin        (100%)        followed        by ciprofloxacin                                                                            (78.8%,           77.7%, respectively) as reported previously by El-Seedy et al. (2016). Similar findings have been found by Pereira et al. (2011) who                 found              that                the   most     prevalent multidrug            resistant                     pattern                                  within    all isolates was     tetracycline-ampicillin- streptomycin-sulfamethoxazole- trimethop. Call et al. (2008) attributed the epidemiology of resistant E. coli in calves to be multifactorial, complex that influenced by co-selection due to linkage of resistance genes. Totally, most of isolates were resistant to at least 6 of the tested antimicrobial agents, making them MDR. This finding was in agreement with that reported by Pereira et al. (2011). This is not surprising in view of the high level of resistance observed for almost all the Salmonella and E.coli in this  study,    representing a significant disease burden in Egypt. This resistance may be attributed to indiscriminate use of antibiotics at recommended doses or at subtherapeutic doses as feed additives to promote growth, and as chemotherapeutic agents irrespective of etiological agents (Kumar et al., 2010 and Verma et al., 2007).

The use of PCR-based technology to identify virulence genes has become widely adopted to distinguish pathogenic

1. E.      coli strains from normal gut flora. Concerning E.coli, PCR was applied on 10 isolates representing all the recovered serogroups for screening some virulence genes, all E.coli isolates were harbored both fimH and iss genes. Similar prevalence has been found by De Verdier et al. (2012). Our findings contradicted  to that recorded by Wagdy et al. (2016) who found that iss gene was not detected in any isolates while fimH were positive in all isolates (100%). Nguyen et al. (2011) reported that 31.30% of E. coli strains of diarrheic calves had one of the fimbrial genes.

Amongst isolates that carried the stx genes: (80%) were stx1, (90%) were stx2 and (40%) were have both stx1/stx2. Similarly, high percentages (40% or more) of stx gene in E. coli strains (STEC) have been reported in Brazil by Salvadori et al. (2003) and in India by Arya et al. (2008). However, Osek et al. (2000) has reported a lower rate (less than 10%) of STEC in diarrheic calves. Only 4 E. coli isolates carried eaeA gene (40%) includes three isolates in dry season and one rainy season, respectively that higher than reported by Natalia et al. (2015) who found that eaeA gene was (12.8%).

Interestingly, the hlyA gene was detected at high percent (60%) as it considered pathogenic virulence factor, supported our results Wieler et al. (1992) who mentioned that enterohemolysin (hlyA) gene can be used as a diagnostic marker because its presence is strongly correlated with Shiga toxin. Nataro and Kaper (1998) stated that the role of enterohemolysin hlyA gene in causing diarrhea in calves has not been demonstrated, but may be compliment the effects of Shiga toxin.

We found that the EPEC pathoytpe were expressed both sxt1 and sxt2 beside iss and fimH genes but most of the virulence profiles were compatible with EHEC strains and almost equally distributed in the both dry and rainy seasons. While, Natalia et al. (2015) found that most of the virulence profiles were compatible with ETEC strains.

Our results illustrated that 40% of the analyzed E. coli isolates harbored all 6 examined virulence genes and 20% of isolates had at least three virulence genes. The successful combinations of virulence factors have persisted to become specific E. coli pathotypes that are capable of causing disease in healthy individuals Kaper et al. (2004). Another study recorded that (30.1 %) were found to be positive for at least one of the virulence genes Natalia et al. (2015).

PCR was applied on 10 isolates of Salmonella species to determine the virulence invA gene that encodes a protein in the inner membrane of bacteria, which is responsible for invasion to the epithelial cells of the host. Malorny et al. (2003) mentioned that invA gene has been recognized as an international standard for detection of Salmonella. The results revealed that all tested isolates harbored invA gene (100%). These results were similar to those obtained by Soliman (2014). With respect to PCR screening of sopB gene in Salmonella species isolates found that all isolates had sopB gene which similar to results obtained by Rahman, (2006). This study revealed that there was association between virulence genes and antimicrobial resistance of the tested E. coli and Salmonella species isolates, similar to previous study by Wagdy et al. (2016).

Of interest, PCR screening for some resistant genes among phenotypically MDR E. coli isolates showed that all isolates were positive for tetA genes with percentage of (100%) while 3 isolates were positive for aacC (30%) and aadB gene not detected. In case of examined MDR Salmonella species, harbored tetA(A) gene (100%) but, aadB and aacC genes were not detected The prevalence of tetA gene was relatively in accordance   with    phenotypic antimicrobial susceptibility in E. coli isolates as mentioned before by Chopra and Roberts (2001). In case of examined MDR Salmonella species, aadB and aacC genes were not detected on contrary to previously study by Masoud et al. (2014) who found that genes encode resistance to streptomycin, sulfonamide, gentamicin, ampicillin and trimethoprim antibiotics, were the most common antibiotic resistance genes in  the diarrheic calves.

The discrepancies between genotypic and phenotypic antimicrobial sensitivity results for streptomycin and gentamicin in E.coli and Salmonella spp. were justified by the possibility of carrying other drug resistance genes or harboring some other extra-chromosomal genetic elements or antimicrobial resistance by other resistance mechanism as efflux pump (Jouini et al., 2009). The wide distribution of tetA gene across gram- negative genera, including Escherichia and Salmonella, indicates that it is highly likely that horizontal transfer of tetracycline resistance occurs (Chopra and Roberts, 2001).

CONCLUSION

This work provides updated information on the molecular characterization of MDR pathogenic E. coli and salmonella

spp. isolates obtained from diarrheic calves. In rainy season pathogenic E. coli and Salmonella species were higher isolation rate than dry season. Furthermore, EHEC (O26) and S. Typhimurium were most predominant serogroups. Almost of pathogenic isolates were resistances to commercial used antibiotics. Therefore, we advise for routine bacteriological screening of fecal samples from diarrheic calves followed by testing for antimicrobial susceptibility that are important to formulate a suitable treatment against E. coli and Salmonella species, to reduce or prevent the losses. Application of strict hygienic measures in rearing calves especially in rainy season and calves age less than 2 months to decrease infections.

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