Introduction
Diseases causing diarrhea are one of the major causes of deaths in low and middle income countries and responsible for high mortality rate in young calves resulting in economic losses. Several studies concluded to the high distribution of Escherichia coli (E. coli) strains in infectious calf diarrhea. STEC causes human gastrointestinal illnesses with diverse clinical spectra. So this study was planned for isolation, identification and molecular characterization of the currently circulating E. coli between calves and related workers in Egypt and to determine the role of virulence genes and pathotypes of E. coli in diarrhea in both calves and humans.
Material and methods
A total of 161 Holsteins calves with varying ages in four different farms in Egypt were examined clinically for diarrhea as well as related human workers in these farms. 43 fresh fecal samples were collected from diarrheic calves as well as 18 stool swab samples from workers then transferred to microbiological laboratory for bacteriological and molecular examination.
Results
The prevalence rate of E. coli was 53% among diarrheic calf samples and the highest isolation rate was 77% among Group I (age <month) and decreased with age. Meanwhile isolation rate in human samples was 50% (9 out of 18). Regarding virulence genes, VT1, VT2 and eaeA virulence gens were successfully amplified in 1, 7 and 7 out of 23 calf isolates respectively. On the other hand human isolates showed only positive reaction with VT1 and VT2 were recovered from 5 and 4 out of 9 isolates while eaeA gave no positive reaction.
Conclusion
Depending on the virulence gene profiling of E. coli isolates, there was 8 out 23 animal isolates were Shiga Toxin producing E. coli (STEC) representing 35% of total animal isolates and 7 out of 23 animal isolates were Enteropathogenic E. coli (EPEC)representing 30% of total animal isolates. Meanwhile, 100% of the human isolates were STEC.
Calf diarrhea; E. coli; Human diarrhea; Virulence genes
Ethics statement
The collection of feces samples had been approved by the owner of the farm. Conduct animal experiments in accordance with laboratory regulations. This study was approved by the Ethics Committee of the New Valley University.
Study design
A cross-sectional study was carried out to investigate the prevalence of E. coli causing diarrhea in calves and humans by isolation, identification and molecular study and comparison of virulence genes.
Study animals
A total of 161 Holsteins calves with varying ages in four different farms in Egypt were examined clinically for diarrhea as well as all human workers in these farms. The calves were divided into three groups according to their age. Group I aged from 1 day to 1 month including 60 calves (20 clinical samples were collected from farm 1, 8 from farm 2, 15 from farm 3 and 17 clinical samples were collected from farm 4), Group II aged more than 1 month up to 3 month including 55 calves (8 clinical samples were collected from farm 1, 4 from farm 2, 16 from farm 3 and 27 clinical samples were collected from farm 4) meanwhile Group III aged more than 3 month up to 6 month including 46 calves (4 clinical samples were collected from farm 1, 5 from farm 2, 11 from farm 3 and 26 clinical samples were collected from farm 4).
Collection of samples
A total number of 43 fresh fecal samples were collected directly under aseptic condition from the rectum of 43 diarrheic calves (out of 161clinicaly examined calves) suffer from diarrhea which ranged from pasty to watery feces, varying degree of dehydration, off food and weakness (Figure 1) using sterile rectal swabs and 18 stool swab samples from workers [11]. Samples were properly identified and kept in sterilized containers and preserved on ice, transferred to the microbiology Laboratory in The Central Laboratory for Evaluation of Veterinary Biologics (CLEVB) in Abbassia, Cairo for bacteriological examination and molecular examination.
Figure 1: Showed clinical signs and diarrhea in suffered calves.
Isolation and identification of coli
Colonial morphology
All samples were diluted in phosphate buffered saline to be cultured on MacConkey agar (Oxoid; CM0115) and Eosin methylene blue (oxide; CM 69) and incubated at 37°C for 18 - 24hrs for primary isolation of E. coli.
Microscopic examination
Smears from freshly growing suspected colonies were stained with Gram stain and examined microscopically.
Motility
Motility was assured by growing and spreading of the pure colonies by stabbing in semisolid agar.
Biochemical identification
Pure cultures were examined biochemically by using API 20E identification system according to [12] following the procedures of kit manual.
Genotypic identification
DNA extraction
DNA templates were prepared using Isolate PrestoTM Mini gDNA Bacteria kit (Geneaid cat# GBB101).
Primer used
Specific primer for 16s rRNA gene, (Table 1), was used for molecular identification of E. coli isolates.
Genes |
Primer Sequence |
Product |
Reference |
16s rRNA |
F5-GACCTCGGTTTAGTTCACAGA-3 |
585 bp |
Tonu et al., [13] |
R5-CACACGCTGACGCTGACCA-3 |
|||
VT1 |
F5-CGCTCTGCAATAGGTACTCC-3 |
256 bp |
OIE [14] |
R5-CGCTGTTGTACCTGGAAAGG-3 |
|||
VT2 |
F5-TCCATGACAACGGACAGCAG-3 |
185 bp |
|
R5-GCTTCTGCTGTGACAGTGAC-3 |
|||
eaeA |
F5-GCTTAGTGCTGGTTTAGGATTG-3 |
618 bp |
|
R5-CCAGTGAACTACCGTCAAAG-3 |
5μl of genomic DNA, 12.5μl of dream taq green master mix (Thermoscientific #K1081), 1μl of each primer (50 pmole) and 5.5μl of deionized water were added to 0.5ml microfuge tubes. The amplification reactions were performed under following conditions: 94°C for 4 min, then 29 cycles each at 94°C for 90 sec, 62°C for 90 sec and 72°C for 2 min.; lastly 72°C for 10 min [13].
Virulence gene detection
Recovered pathogenic isolates were used to detect major virulence genes including VT1, VT2 and eaeA genes. Specific primers shown in Table 1.
Polymerase chain reaction
5μl of genomic DNA, 12.5μl of dream taq green master mix (Thermoscientific #K1081), 1μl of each primer (50 pmole) and 5.5μl of deionized water were added to 0.5ml microfuge tubes. The amplification reactions were performed under following conditions: 94°C for 2 min, then 25 cycles each at 94°C for 60 sec, 62°C for 90 sec and 72°C for 2 min.; lastly 72°C for 5 min [14].
Prevalence of diarrhea in examined calves
Clinical examination of examined calves showed that, a total 43 out of 161 calves (24 out of 60 from group I calves, 11 out of 55 group II calves and 8 out of 64 group III calves) suffer from diarrhea which ranged from pasty to watery feces, varying degree of dehydration, off food and weakness representing prevalence rates 40%, 20% and 17% respectively. Regarding the prevalence of diarrhea in the farms, it was found that 7 out of 32, 5 out of 17, 9 out of 40 and 22 out of 70 calves suffering from diarrhea from farm 1, 2, 3 and 4 respectively representing prevalence rates 22%, 29% , 21% and 31% respectively as shown in Table 2 and Figure 1.
Ages group
|
Total Ex |
Farm (1) |
Farm (2) |
Farm (3) |
Farm (4) |
Total D |
Age Prev |
||||
Ex |
D |
Ex |
D |
Ex |
D |
Ex |
D |
||||
Group I |
60 |
20 |
4 |
8 |
4 |
15 |
5 |
17 |
11 |
24 |
40 |
Group II |
55 |
8 |
2 |
4 |
1 |
16 |
3 |
27 |
5 |
11 |
20 |
Group III |
46 |
4 |
1 |
5 |
0 |
11 |
1 |
26 |
6 |
8 |
17 |
Total |
161 |
32 |
7 |
17 |
5 |
42 |
9 |
70 |
22 |
43 |
27 |
Farms Prev |
22% |
29% |
21% |
31% |
Table 2: Prevalence of diarrhea in examined calves related to age and farms.
Ex: Examined; D: Diseased; Prev: Prevalence
Isolation and identification of E-coli
Colonial morphology
Only 35 out of 43 isolates recovered from diarrheic calves while 10 out of 18 isolates recovered from human workers showed pink-colored smooth colonies on macConkey agar and produced distinct, clear greenish metallic sheen over EMB. All isolates were Gram negative, motile, non-sporulated and medium sized bacilli.
Biochemical identification
By using API 20E identification system, the suspected E. coli isolates were 23 out of 35 calves isolates while 9 out of 10 human isolates representing recovery rates 66% and 90% respectively. The biochemical reactions were classified into five groups as shown in Table 3. The first group includes 9 out of 23 calve isolates and 5 out of 10 human isolates and gave positive reaction with ONPG, ADH, LDC, ODC, TDA, IND, GLU, MAN, SOR, RHA, SAC, MEL and ARA tests and negative reaction with CIT, H2S, URE, VIP, GEL, INO and AMY tests. The second group includes 5 out of 23 calve isolates and 4 out of 10 human isolates and gave positive reaction with ONPG, LDC, ODC, TDA, IND, GLU, MAN, SOR, RHA, SAC, MEL and ARA tests and negative reaction with ADH, CIT, H2S, URE, VIP, GEL, INO and AMY tests. The third one includes 4 out of 23 calve isolates and gave positive reaction with ONPG, ADH, LDC, ODC, TDA, IND, GLU, MAN, SOR, RHA, MEL and ARA tests and negative reaction with CIT, H2S, URE, VIP, GEL, INO, SAC and AMY tests. The fourth one includes 3 out of 23 calve isolates gave positive reaction with ONPG, ADH, LDC, TDA, IND, GLU, MAN, SOR, RHA, SAC, MEL and ARA tests and negative reaction with ODC, CIT, H2S, URE, VIP, GEL, INO and AMY tests. The last 5th group includes 2 out of 23 calve isolates and gave positive reaction with ONPG, LDC, TDA, IND, GLU, MAN, SOR, RHA, SAC, MEL and ARA tests and negative reaction with ADH, ODC, CIT, H2S, URE, VIP, GEL, INO and AMY tests.
Type of samples |
No of Samples |
API 20E RESULTS |
No. of Recovered isolates |
Recovery rate |
|||||||||||||||||||||
Calve isolates |
35 |
ONPG |
ADH |
LDC |
ODC |
CIT |
H2S |
URE |
TDA |
IND |
VP |
GEL |
GLU |
MAN |
INO |
SOR |
RHA |
SAC |
MEL |
AMY |
ARA |
OX |
|||
+ |
+ |
+ |
+ |
- |
- |
- |
+ |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
+ |
+ |
- |
+ |
- |
9 |
23 |
66% |
||
+ |
- |
+ |
+ |
- |
- |
- |
+ |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
+ |
+ |
- |
+ |
- |
5 |
||||
+ |
+ |
+ |
+ |
- |
- |
- |
+ |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
- |
+ |
- |
+ |
- |
4 |
||||
+ |
+ |
+ |
- |
- |
- |
- |
+ |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
+ |
+ |
- |
+ |
- |
3 |
||||
+ |
- |
+ |
- |
- |
- |
- |
+ |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
+ |
+ |
- |
+ |
- |
2 |
||||
Human isolates |
10 |
+ |
+ |
+ |
+ |
- |
- |
- |
+ |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
+ |
+ |
- |
+ |
- |
5 |
9 |
90% |
+ |
- |
+ |
+ |
- |
- |
- |
+ |
+ |
- |
- |
+ |
+ |
- |
+ |
+ |
+ |
+ |
- |
+ |
- |
4 |
||||
Total |
45 |
5 biochemical reactions |
32 |
Table 3: Identification of E coli isolates using API 20E identification system.
Genotypic identification
All 23 calves isolates and 9 human isolates were identified as E. coli using 16s rRNA gene primer giving a PCR product at the prospected size of 585 bp [13] as shown in Figure 2.
Figure 2: Results of PCR using specific primer of 16S rRNA gene of E. coli (A) Samples taken from diarrheic calves. (B) Samples taken from diarrheic workers.
coli isolation rates according to age of calves
Regarding to age, the highest isolation rate of E. coli were recorded in group I by 77% where 20 out of 24 samples were finally identified as E. coli followed by 18% in group II where 2 samples out 11 were identified as E. coli. Regarding group III, it recorded 13% representing the lowest isolation rate where only 1 sample out of 8 was identified as E. coli. Meanwhile in human samples E. coli was recovered from 50% of the collected samples where 9 out of 18 samples were identified as E. coli as shown in Table 4.
Age group |
Diseased |
Identified E. coli |
% |
Group I |
24 |
20 |
77 |
Group II |
11 |
2 |
18 |
Group III |
8 |
1 |
13 |
Total |
43 |
23 |
53 |
Humans |
18 |
9 |
50 |
Table 4: Percent of isolation of E. coli according to age of calves.
Isolated coli virulence genes profiling
The virulence genes profile of E. coli isolates was studied and it was found that VT1, VT2 and eaeA gens were successfully amplified in 1, 7 and 7 out of 23 calf isolates giving rise the prospected PCR products of 256, 185 and 618 bp respectively as mentioned in OIE [14]. Also only VT1 and VT2 in 5 and 4 out of 9 human isolates respectively while eaeA gave no positive reaction with the human isolates, as shown in Table 5 and Figures 3-5. Regarding the correlation between virulence genes in the isolated E. coli, it was found that, there is only one animal isolate carry both VT1 and eaeA virulence genes while there are 4 animal isolates carry both VT2 and eaeA virulence genes. Meanwhile in human isolates there is no isolates carry more than one virulence gene.
Species
|
Total isolates
|
Virulence gene |
Pathotypes |
|||
Vt1 |
Vt2 |
eaeA |
STEC |
EPEC |
||
Calves
|
23 |
1 |
7 |
7 |
8 35% |
7 30% |
Humans
|
9 |
5 |
4 |
0 |
9 100% |
0 |
Total |
32 |
6 |
11 |
7 |
17 53% |
7 22% |
Percent |
Table 5: Studying the virulence gene profile of E. coli isolated from diarrheic calves and human.
Figure 3: Results of PCR using specific primer of VT1 gene of E. coli (A) Samples taken from diarrheic calves. (B) Samples taken from diarrheic workers.
Figure 4: Results of PCR using specific primer of VT2 gene of E. coli (A) Samples taken from diarrheic calves. (B) Samples taken from diarrheic workers.
Figure 5: Results of PCR using specific primer of eaeA gene of E. coli (A) Samples taken from diarrheic calves. (B) Samples taken from diarrheic workers.
Isolated coli pathotyping
Depending on the virulence gene profiling of E. coli isolates as shown in Table 5, it is clear that, there was 8 out 23 animal isolates were STEC representing 35% of total animal isolates and 7 out of 23 animal isolates were EPEC representing 30% of total animal isolates. Meanwhile, 100% of the human isolates were STEC.
Citation: Gamal FE, Diab MS, Gadallah FM, Awad N, Soliman EM, et al. (2019) Correlation between Virulence Genes Profile of Currently Circulating E. coli Pathotypes Isolated from Diarrheic Calves and Humans. J Vaccines Res Vaccin 5: 009.
Copyright: © 2019 Fatma Elzhraa Gamal, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.