Correlation-between-Virulence-Genes-Profile-of-Currently-Circulating-E-coli-Pathotypes

Diarrhea is a major problem in calves causing high mortality rates and high economic Impact. The multifactorial nature of neonatal calf diarrhea makes this condition hard to control effectively. Therefore, prevention and control of such condition must be based on a good understanding of those problem complexities during the calving period before disease outbreaks [15]. Due to high distribution of E. coli strains in infectious calf diarrhea, this work was planned for isolation, identification and molecular characterization of E. coli and detection of virulence gens that play a role in E. coli pathogenesis. In this study a total number of 161 calves of different ages in different four farms in Egypt were clinically examined for diarrhea as well as related human workers in these farms. The clinical examination showed that, a total 43 out of 161 calves (24 out of 60 from calves aged from 1 day to 1 month, 11 out of 55 calves aged more than 1month up to 3 month and 8 out of 64 calves aged more than 3 month up to 6 month) suffer from diarrhea which ranged from pasty to profuse 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 the different 4 farms representing prevalence rates 22%, 29%, 21% and 31% respectively as shown in Table 2 and Figure 1. Regarding to age, our finding revealed that the highest prevalence rate of diarrhea was reported in the youngest age of calve group and the prevalence rate decreased with old age. Our finding confirmed by Garcia et al., [16] who stated that calves are at greatest risk of diarrhea within the first month of life and the incidence of diarrhea decreases with age increase. on the contrary Olaogun et al., [17] concluded that higher percent of diarrhea observed in old calves than young ones in his study on the prevalence of diarrhea in calves in 12 farms in Oyo and Ogun states in South Western Nigeria and also reported that a total 120 out of 825 calve up to 6 month old (14.5%) showing signs of diarrhea. Different prevalence percent was investigated by Achá et al., [18] in 8 dairy farms in Mozambique at 4 occasions during 2 consecutive years. He found that a total 63 out of 1241 calves up to 6 months of age (5%) had signs of diarrhea, two farms had an overall higher prevalence (13% and 21%) of diarrhea.

A total number of 43 fresh fecal samples were collected directly under aseptic condition from the rectum of diseased calves (out of 161clinicaly examined calves) as well as 18 stool swab samples from human workers then transferred to the microbiological laboratory for bacteriological and molecular examination. The colonial and morphological isolation revealed that, 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 and all isolates were Gram negative, motile, non- sporulated, medium sized bacilli. On the other hand the biochemical identification using API 20E identification system revealed that 23 isolates out of 35 suspected calves’ isolates and 9 out of 10 suspected human isolates representing recovery rates 66% and 90% respectively proved to be E. coli as shown in Table 3. All 23 calves and 9 human isolates were genotypically identified as E. coli by amplification of universal 16s rRNA gene giving the prospected PCR product of 585 bp as confirmed by Tonu et al., [13] as shown in Figure 2. Higher percent of E. coli were isolated by Elseedy et al., [19] who concluded that out of 127 collected fecal samples from diarrheic calves, 119 (93.7%) bacterial isolates were recovered, including 23 (18.1%) Salmonella serovars and 96 (75.6%) were E. coli strains. On contrary, a lower E. coli isolation percent (10%) was isolated by Olaogun et al., [17] from three farms in Oyo and Ogun States, Nigeria and 45% by Paul et al., [20] in some selected areas of Bangladesh. Also among 84 diarrheic calves samples, 30 (35.71%) E. coli were isolated by Masud et al., [21]. The difference in the prevalence of E. coli were explained by Cho and Yoon [15] who concluded its relation to geographical location of the farms, farm management practices and herd size.

Regarding to age, the highest isolation rate of E. coli were recorded in group I that aged from 1 day to 1 month by 77% where 20 out of 24 samples were finally identified as E. coli followed by 18% in group II that aged more than 1 month up to 3 month where 2 samples out 11 were identified as E. coli. Regarding group III that aged more than 3 month up to 6 month, 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. Anwarullah et al., [12] reported that the highest isolation rate of E. coli was recorded in old age group than young one representing 1.33%, 4%, 9.33% in similar groups respectively.

E. coli can be classified into six pathogroups based on virulence scheme: Enterotoxigenic E. coli (ETEC), Shiga Toxin-producing E. coli, Enteropathogenic E. coli, Enteroinvasive E. coli, Enteroaggregative E. coli and enterohaemorrhagic E. coli [22]. Although there are a wide range of different virulence factors that may play a role in the pathogenesis of E. coli, the present study investigated the presence of only 3 virulence genes encoding putative virulence factors and the obtained 23 isolates of E. coli were screened for the presence of certain Virulence factors, shiga toxin production (VT1 and VT2 genes), attaching and effacing character (eae A gene) and it was found that VT1, VT2 and eaeA gens were successfully amplified in 1, 7 and 7 out of 23 calve isolates giving rise the prospected PCR products of 256, 185 and 618 bp respectively as described 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. Regarding the correlation between virulence genes in the isolated E. coli, it was found that, there is only one calf isolate carry both VT1 and eaeA virulence genes while there are 4 calves isolates carry both VT2 and eaeA virulence genes. Meanwhile in human isolates there is no isolates carry more than one virulence gene.

Depending on the virulence gene profiling of E. coli isolates, 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. The overall prevalence of STEC in all samples and farms was 53%. Different prevalence of STEC among calves was studied and was 20% by Shaw et al., [23] 37.5% by Fukushima and Seki [24] 12.14% by Tahamtan et al., [25] 17.7% by Irshad et al., [26] and 22.7% by Kohansal and Asad [27].

Dastmalchi and Ayremlou [28] reported that, prevalence was 19.6% among diarrheic calves, 23.1% carried VT1 gene, 26.92% possessed VT2 gene while 13 isolates (50%) gave positive amplicon for both VT1 and VT2 genes. Shahrani et al., [29] and Akter et al., [30] concluded that VT1 is the most prevalent than VT2. Prevalence of 51% among calves was obtained by Wang et al., [31] which was closely contact to our finding. Higher prevalence 83% among diarrheic calves was obtained by Pervez et al., [32]. The highest STEC prevalence was detected in age under one month as shown in Tables 4 and 5. Similar results was observed by Cobbold and Esmarchelier [33], they concluded that calves as young as 48 to 72h old excrete STEC. Humans can become infected with STEC by ingesting contaminated food or water or by transmission from infected animals or humans [34]. Prevalence of STEC infection in human was 15% by Salmanzadeh-Ahrabi et al., [35], 2.3% by Rajendran et al., [36], 1.7% by van Duynhoven et al., [37], 7% was reported in patients with diarrhoea in Morogoro, Tanzania in 2006 by Raji et al., [38] and 56% by Matussek et al., [39]. The overall prevalence of virulence-associated gene VT1 only, VT2 only, VT1and VT2 and eaeA were 10.7%, 20.8%, 68.5%, 3.9%, respectively as reported by Wang et al., [31]. Pervez et al., [32] reported that 10% of the isolates of calves were positive for VT1 gene while eaeA not detected. The VT1 gene was detected more frequently in calves than in adult animals [40].

Some factors that contribute to the presence and spread of STEC in a herd are the management practices, stress, diet, population density, geographic region and season [41]. Contact with feces of cattle, direct contact with the animals or their environment and consumption of contaminated beef, milk, dairy products, water, unpasteurized apple juices and vegetables are possible routes for STEC human exposure and disease [42]. So, measures to prevent direct contact with animal fecal material in the environment include the wearing of protective clothing, increased hand washing and targeted education of the population at risk regarding possible sources of STEC infection.

So, from the previously mentioned results, we discerned that there is a correlation between E. coli pathotypes isolated from animals and contact humans but, further study is needed and will be done to study the sequence analysis of amplified genes to discuss deeply the direct relation between human and animal pathotypes. Other studies may be recommended to cover other different virulence genes of different E. coli pathotypes like Enterotoxigenic (ETEC), Enteroaggregative (EAEC) and enteroinvasive E. coli.

Also, our finding can concluded to the risk of increasing the prevalence of pathogenic E. coli increase with the decrease in ages of susceptible calves, control and management of the diarrhea in calves should referenced to the actual situation of the exact farm regarding antibiotic therapy, autovaccination of calves and housing parameters. Cross infection of pathogenic E. coli between infected animals and contact workers is highly possible. Biosafety and biosecurity measures should be implemented to minimize the risk of calves' infection and cross infection between human and animals.