Frequency of k99, stx1, and stx2 Virulence Factors in Escherichia coli isolated from Diarrheic and Clinically Healthy Suckling Calves in Sistan and Baluchistan Province, Iran

1. Introduction

Despite the importance of neonatal calf diarrhea (NCD) as the most significant cause of morbidity and mortality in neonatal calves, the numbers of calves’ losses due to diarrhea are not declining (Younis et al., 2009). The effective control and prevention of NCD must be based on a proper understanding of those disease complexities because of its multifactorial nature (Cho and Yoon, 2014). The identification of the possible risk factors and delineation of the possible causative agent of diarrhea are important to allow targeted preventative measures, such as vaccination (Younis et al., 2009). Calf scours mainly caused by Escherichia coli (E. coli) and Salmonella in less than two months of age (Quinn et al., 2011). The frequency of causative agents of diarrhea varies by the geographical location of the farms, farm management practices, and herd size (Cho and Yoon, 2014).

Although most E. coli strains are inoffensive, pathogenic E. coli can make a range of diseases of zoonotic importance (Cho et al., 2010). Harmless E. coli may cause intestinal and extraintestinal diseases due to the translocation of the virulence genes by lateral gene transfer. Moreover, new pathotypes probably emerge resulting from the genetic recombination of some virulence genes (Cabal et al., 2016).

Enterotoxigenic E. coli (ETEC) and Shigatoxigenic E. coli (STEC) are two pathotypes that can produce intestinal disease in humans (Cabal et al., 2016). The ETEC has been reported as the causative agent of the traveler’s diarrhea in patients who traveled to developing countries (Cabal et al., 2016). It has been considered the most important economically and the most common cause of NCD among pathotypes of E. coli (Younis et al., 2009; Cho et al., 2010). This pathotype has many virulence factors. The expression of k99 fimbriae (k99 adhesion antigen) accounts for nearly all cases of ETEC (E. colik99+) infection was found in newborn calves (Younis et al., 2009). The first and key step of ETEC pathogenicity is the capability to be colonized in the host body by adhesive agents, such as k99 and K88 (Quinn et al., 2011).

The STEC infection, a zoonotic pathogen (Cabal et al., 2016), can lead to hemorrhagic colitis, and in the most severe cases, a hemolytic-uremic syndrome in humans. Cattle have been concluded as the most important reservoir and carrier of STEC among ruminants (Jamshidi et al., 2015). Bovine STEC involved in economic losses may produce two types of Shigatoxins (i.e., stx1 and stx2 or both) inhibiting protein synthesis and killing the cultured Vero cells in vitro (Cobbold and Desmarchelier, 2001).

Outbreaks of STEC via food of cattle origin, especially undercooked meat and raw milk are documented indicating its importance to human health (Cobbold and Desmarchelier, 2001).

Diarrhea caused by E. coli in neonatal calves is treated via antibiotic therapy. It is logical to find alternative strategies to control NCD due to the increased incidence of antibiotic resistance and the highly limited use of antibiotics in the feed industry (Alo et al., 2018). Therefore, the determination of the target antigen is critically necessary as the first step to produce the specific therapeutic antibodies against causing agents. On the other hand, there is limited knowledge in Iran about the burden of the virulence genes of E. coli in healthy individuals, which could help evaluate the results from clinically affected individuals (Cabal et al., 2016).

Moreover, it is essential to understand the frequency of virulence factor-encoding genes in assessing the proportion of carriage. It is also required in the characterization of major unique antigens that are useful in the development of effective immunological-based preventive measures. The ranges of the prevalence of E. coli isolates containing k99, stx1, stx2, and stx1+2 were determined at 1-13%, 0.1-87%, 0.1-90%, and 0-35%, respectively (Lotfollahzadeh et al., 2004; Rezazadeh et al., 2004; Shams et al., 2010; Tahamtan et al., 2010; Dastmalchi and Ayremlou, 2012; Hashish et al., 2015; Jamshidi et al., 2015).

Since there is little information available regarding the occurrence and characteristics of ETEC and STEC isolated from calves in Sistan and Baluchistan, South East of Iran, the present study aimed to investigate the prevalence of their representative virulence genes of k99, stx1, and stx2 in E. coli isolates recovered from clinically healthy and diarrheic suckling calves.

2. Material and Methods

2.1. Sample Size and Study Design

The present study was the first comparative survey on the studied genes in Iran. The sample size, considered for the comparative research, was calculated using the formula described by Eng (2003). The numbers of subjects per group (healthy/diarrheic) were determined at 11,500 (stx1), 544 (stx2), and 437 (k99). However, each of which included 50 cases for both screening and comparing plan because it was not possible to collect and match the case-control design in the study area in line with the calculated sample size.

2.2. Specimen and Study Area

Individual fecal samples were collected during six months (September 2015 to February 2016) from the rectum of diarrheic suckling (n=50) and clinically healthy suckling calves (n=50) (age range: 1-30 days) at several farms located in Sistan and Baluchistan (30˚85' N 61˚76' E), South East of Iran. It should be noted that the sampled farms were the largest in the region. The sampled calves, which were of the cows, not the buffaloes, were traditionally maintained. Following that, the constant clinical signs observed in the examined calves were sudden onset of profuse yellow/white diarrhea leading to rapid and severe dehydration. None of the healthy individuals presented any gastrointestinal symptoms when the samples were collected. The median age of the studied calves was 13 days. The calves had eaten the colostrum at the same age and no one of which had been vaccinated against bacterial infections.

2.3. Bacteriological Examination

The samples were transported to the Laboratory of Microbiology, Faculty of Veterinary Medicine, University of Zabol, Zabol, Iran, with minimum delay in the icebox. They were then processed on the same day. One g of each fecal sample was inoculated in 5 ml of sterile Pepton water (Liofilchem, Italy) and incubated for 18-24 h at 37˚C. In the next stage, the samples were sub-cultured, and E. coli was identified biochemically. Briefly, a loop full of diluted specimens was inoculated on MacConkey agar (Liofilchem, Italy) and incubated at 37˚C for 18-24 h. Lactose fermenter (pink) colonies were streaked on Eosin Methylene Blue (EMB) agar (Liofilchem, Italy) plates and green colonies (presumptive E. coli) with metallic sheen growing on EMB confirmed as E. coli using Triple Sugar Iron Agar, IMViC test, Simmons' citrate agar, and SIM agar (Liofilchem, Italy) (Quinn et al., 2011).

2.4. Positive Control Strains

For polymerase chain reaction (PCR), a k99 serologically-positive E. coli strain and an isolate of E. coli containing stx1 and stx2 genes were used as positive controls. Double-distilled water was used as a negative control.

2.5. Polymerase Chain Reaction

The PCR was conducted for all isolates. The procedure was performed by the primers that amplify 180 bp fragment for stx1 (stx1F: 5'-ATAAATCGCCATTCGTTGACTAC-3'; stx1R: 5'-AGAACGCCCACTGAGATCATC-3') and 225 bp fragment for stx2 (stx2F: 5'-GGCACTGTCTGAAACTGCTCC-3'; stx2R: 5'-TCGCCAGTTATCTGACATTCTG-3') (Jamshidi et al., 2015). Meanwhile, in case of ETEC, oligonucleotide primers (k99F: 5'-TATTATCTTAGGTGGTATGG-3'; k99R: 5'-GGTATCCTTTAGCAGCAGTATTTC-3') that amplify a 314 bp fragment for the k99 (F5) gene was used (Franck et al., 1998).

The DNA was extracted using the boiling method (Sadeghi Bonjar et al., 2017). The PCR reaction mixture with some modification in a 25 μL volume consisted of 5.5 μl DNA, 12.5 μl Master Mix with Taq DNA Polymerase (Amplicon, Denmark), 1 μl of each primer (10 μM), and 5 μl distilled water. The amplifications were performed by a programmable thermocycler (gradient Eppendorf’s Master cycler® pro, Eppendorf, Hamburg, Germany).

Table 1 tabulates the conditions for the amplification of k99, stx1, and stx2. The amplified product was visualized by standard gel electrophoresis of 10 μl of the PCR reaction mixture on 1.5% agarose gel stained with ethidium bromide (Cinnagen, Iran). Visualized DNA fragments along with the relevant DNA marker (Fermentas, Germany) were pictured by gel documentation (Cambridge, UK).

2.6. Statistical Analysis

The data were analyzed using SPSS software (Version 16.0, USA). Moreover, the chi-square (X²) test and Fisher’s exact test were used to assess the significant differences between the diarrheic and healthy groups. Furthermore, the presence of k99, stx1, and stx2 was investigated in E. coli. A p-value less than 0.05 was considered statistically significant.

3. Results

Positive control of k99 showed the representative band (314 bp) via electrophoresis, while none of the E. coli isolates, both in clinically healthy (0%) and diarrheic (0%) suckling calves carried the k99 gene (Figure 1 A).

Figure 1. Gels represented the amplification of the selected E. coli isolates from healthy and diarrheic calves for k99 (A) Lane 1: Positive control, Lane 2: Negative control, Lane 3-5: selected studied isolates; stx1 (B) Lane 1: Negative control, Lane 2: Positive control, Lane 3 and 4: Selected investigated isolates; and stx2 (C) Lane 1: Negative control, Lane 2: Positive control, Lane 3 and 4: Selected investigated isolates. L indicates GeneRulerTM 100 bp DNA ladder marker.

In the present study, among E. coli isolated from clinically healthy suckling calves, the frequencies of the E. coli containing stx1 and stx2, or both (stx1+2) were 8%, 14%, and 4%, respectively (P<0.05). Furthermore, in E. coli isolated from diarrheic suckling calves, the corresponding frequencies were estimated at 24%, 16%, and 6%, respectively (P<0.05).

Our results demonstrated that E. coli harboring stx1, stx2, and stx1+2 in samples from diarrheic calves were more frequent than those of healthy ones. Moreover, a statistically significant difference was observed between the isolates recovered from clinically healthy and diarrheic suckling calves in terms of E. coli harboring stx1 (P<0.05).

4. Discussion

According to the literature, for many years, there have been no investigations regarding healthy calves in terms of ETEC k99 in Iran, and the most recent study was conducted in 2010. The frequencies of k99 in E. coli isolates obtained from diarrhea calves (0%) in this study are lower than those reported in Iran and abroad in the studies conducted by Lotfollahzadeh et al. (2004) in Ghaemshahr-Babol (1.07%), Rezazadeh et al. (2004) in the suburbs of Tehran (1.2%), Shams et al. (2010) in Fars Province (4.3%), and Hashish et al. (2015) in Egypt (13.3%).

These investigations were performed just in diarrheic calves, and all performed a nearly different method (conventional microbiological techniques), except for Hashish et al. (2015) who conducted a PCR method.

The various frequency of E. colik99⁺ could be related to different techniques applied for screening along with the geographical variety. Traditional microbiological methods were used by previous investigations. Many accessible diagnostic tests, hemagglutination inhibition, serum agglutination, indirect fluorescent antibody tests, and enzyme-linked immunosorbent assay have been introduced; however, PCR has been a specific and convenient method for large-scale screening of ETEC k99 for its sensitivity, simplicity, and rapidity (Younis et al., 2009; Cho and Yoon, 2014).

Many factors might contribute to the negative outcome of the PCR used in the present study. The detection limit of the PCR is related to the number of colonies forming units, and it clarifies why the target gene was not detectable (i.e., a few numbers of colony-forming units). In addition, the presence of antibiotic residues might explain the falsely negative bacteriological results because the withdrawal time may not be regarded in the studied herds by the farmers (Fricker, 1987). Since the history of prescription of antibiotics was not accessible, the farmer’s claim was considered in this connection to clarify whether or not the antibiotic was prescribed. Moreover, the lack of k99 may result from some functional changes of the receptors or the presence of immature receptors in the intestine of the suckling calves that prevented ETEC k99 to be colonized in the intestines (Quinn et al., 2011). It leads to a decline in the population of the ETEC k99 in the intestinal environment. Studies have shown that ETEC strains are more inclined to colonize in jejunum and ileum (Radostitis and Blood, 2017). Therefore, the sampling site could be suggested as a factor that alters the frequency of the isolation of E. coli harboring k99. In this connection, according to some studies (Lotfollahzadeh et al., 2004; Rezazadeh et al., 2004), the feces were collected directly from the rectum. The highest prevalence of ETEC k99 was reported in December and March (Younis et al., 2009). Although there was an attempt to perform sampling in these months, no frequency of k99 was observed for both isolates recovered from clinically healthy and diarrheic suckling calves, which indicated the need for the year-wise distribution of ETEC k99 isolation for both isolates recovered from clinically healthy and diarrheic suckling calves in the study area.

Studies showed that rotavirus infection, vaccination of the animals with combined vaccine against rotavirus, coronavirus and ETEC k99, colostrum feeding practice, animal age, Vitamin E, and selenium supplementation of pregnant cows alter the prevalence of ETEC k99 infection in healthy and/or diarrheic calves (Younis et al., 2009). In this connection, it was to match control-case samples equitably based on the history and clinical signs.

Bacterial host properties can interfere with the outcome. This lack of frequency also indicated that k99 is not the major fimbrial antigen-encoding gene and other genes are also probably involved in binding this bacterium to the target cell and should be taken into consideration for the ETEC of suckling calves of the study area for detection or prevention purpose.

Primers for stx1 and stx2 produced 180 and 255 bp PCR products, respectively, such as the control strain (Figures 1B and Figure 1C).

Recently, there have been few reports in Iran and other countries that evaluate the frequency of stx1, stx2, or stx1+2 in calves with or without diarrhea, individually.

A higher proportion of stx1 (45.2%, Tahamtan et al., 2010; 15%, Jamshidi et al., 2015; 12.5%, and Dastmalchi and Ayremlou, 2012), stx2 (88.3%, Tahamtan et al., 2010 and 19%, Jamshidi et al., 2015), and stx1+2 (34.9%, Tahamtan et al., 2010; 8%, Jamshidi et al., 2015; and 12.5%, Dastmalchi and Ayremlou, 2012) along with a lower proportion of stx2 (12.5%, Dastmalchi and Ayremlou, 2012) have been reported among E. coli from healthy calves, compared to those in the present study.

On the other hand, among E. coli from diarrheic calves, a higher proportion of stx1 (86.7%, Hashish et al., 2015) and stx2 (26.7%, Hashish et al., 2015; 90%, Behzadian Nezhad et al., 2011) along with a lower proportion of stx1 (1%, Behzadian Nezhad et al., 2011; and 0.1%, Dastmalchi and Ayremlou, 2012), stx2 (0.1%, Dastmalchi and Ayremlou, 2012), and stx1+2 (0%, Dastmalchi and Ayremlou, 2012; 0%, Hashish et al., 2015; and 0%, Behzadian Nezhad et al., 2011) have been reported in the studies, compared to those in the present study.

Regardless of the health status of the calves, the fluctuation of the frequency of isolates containing stx1, stx2, and/or stx1+2 caused by many factors. These factors included varieties in geographical differences that resulted in the various distribution of bacteriophages and harboring motile genetic elements; environmental conditions, such as temperature, humidity, sunshine, and sanitary condition; and the host physiology, such as dissimilarity in the excretion of fecal and/or genetic composition that could considerably affect the prevalence of E. coli and subsequently the tested genes (Behzadian Nezhad et al., 2011).

Since the bacteriophages transfer the motile genetic element (i.e., stx-coding gene) among bacteria via transduction procedure (Quinn et al., 2011), the transduction rate and the amount of the bacteriophages may elaborate the findings of the present study. Heat stress resulted mainly from environmental conditions or environment-host physiology interaction may affect the composition of the colostrum and sensitivity of the calves of the dams for a particular pathogen. The environmental condition, in which the calf is born, affects the absorption of immunoglobulin (Ig). The Ig, particularly IgA, the dominant Ig in the colostrum and/or mucosal surface of the intestine, neutralizes the virulence factors expressed on the surface of the bacterium that may lead to a decrease in the specific pathotype of bacteria via selecting pressure.

Therefore, the correlation of the presence of the stx1, stx2, and stx1+2 positive E. coli with the content of the IgA in the colostrum of the dams and/or intestine of the calves could address the different observations among studies. In other words, the interactions between host defense, pathogens, and environment elaborate the inconsistency of the frequency of the virulence factors among the E. coli isolates studied in the different geographical regions.

In line with our findings, the dominance of stx2, compared to stx1 and stx1+2, have been reported among E. coli isolates from healthy cattle by Tahamtan et al., (2010) and Jamshidi et al. (2015). Moreover, the findings of a study conducted by Hashish et al. (2015) are consistent with our results indicating the higher frequency of stx1, compared to the stx2 and stx1+2 in diarrheic cattle. However, the results of a study performed by Behzadian Nezhad et al. (2011) are not in line with our findings in diarrheic cattle that depict the dominance of stx2, compared to the stx1 and stx1+2. Since the transcription of stx1 is iron-related, in vivo and the intestine environment is not iron-rich (Behzadian Nezhad et al., 2011), and the level of the transcription of stx1 may shift in favor of high transcription of stx2. Consequently, virulent stx1 positive isolates due to the iron-poor environment (intestine) were declined via selection pressure leading to the higher detection of stx2 positive E. coli.

Dastmalchi and Ayremlou (2012) reported in Urmia that among healthy and/or diarrheic cattle, the isolates harboring stx1 and stx2 were not dominant. This finding is not consistent with the results of the present study that can be due to various sources of information in methodology (STEC in the aforementioned study vs. E. coli in the present study).

To the best of our knowledge, this is the first report for the comparison of the frequency of k99, stx1, and stx2 genes in E. coli isolated from healthy and diarrheic calves in the South East of Iran. According to the literature, there was only one study in Iran that was approximately similar to the present study in terms of the used method; however, k99 was investigated in the present study (Dastmalchi and Ayremlou, 2012).

Dastmalchi and Ayremlou (2012) determined the dissemination of the STEC in feces of 2-6-month-old healthy and diarrheic calves in Urmia, Iran. The isolates of fecal samples were tested for stx1, stx2, and other virulence marker genes. It is worth mentioning that the isolates containing stx1, stx2, and stx1+2 were more prevalent in healthy calves (12.5%, 12.5%, and 12.5%), compared to the diarrheic ones (0.1%, 0.1%, and 0%), which was not consistent with the results of this study. The variety of the abundance between healthy and diarrheic ones reported by Dastmalchi and Ayremlou (2012), compared to the present study, irrespective of the variety in the sources of information in methodology (STEC in their study vs. E. coli in the present study), may result from the variety in the animal age, farm management/conditions, and/or geographical location (Behzadian Nezhad et al., 2011), which require more investigation and examination.

Some studies have revealed that strains possessing only stx2 are potentially more virulent than those harboring stx1 or even strains carrying both stx1 and stx2 (Tahamtan et al., 2010). Furthermore, stx2 harboring bacteria has more ability to survive in the intestine (Behzadian Nezhad et al., 2011) which consequently increases the possibility of lateral gene transfer leading to incorporating virulence genes or genetic recombination to emerge new virulent strain causing diarrhea (Cabal et al., 2016). These explanations may elaborate the reason for the function of the stx2 positive isolates in diarrheic calves despite their fewer frequency, compared to the healthy calves in a study conducted by Dastmalchi and Ayremlou (2012).

Notably, the iron content of the intestine between the two groups (healthy and diarrheic) should be taken into account to elaborate the compared data (Behzadian Nezhad et al., 2011). Further investigation to test this hypothesis is suggested locally, regionally, and nationally.

The findings in this study are important for public health and preventive veterinary medicine since the isolates recovered from both healthy and diarrheic suckling calves in the study area contained stx1, stx2, or stx1+2, and the main virulence factors/markers for zoonotic bacteria (i.e., STEC). Moreover, despite the low frequency of the isolates containing stx1+2, compared to the isolates harboring stx1 and stx2 in this study, clinically healthy cattle could be considered the primary reservoir of the STEC strains which may help transfer the pathotype to human or other animals in the study area due to the statistically equal distribution of isolates containing stx1+2 between clinically healthy and diarrheic suckling calves of the study area.

Moreover, the stx1 can act as a virulence factor among E. coli isolates of diarrheic suckling calves of the study area indicating that it could be regarded as the main factors for the pathogenicity of E. coli in the study area and could be used for the diagnosis of a virulent strain of E. coli isolated from calves.

Our analysis may be biased towards two E. coli pathotypes; therefore, it is recommended to consider all pathotypes when comparing E. coli from stools. The present case-control study aimed to investigate and analyze three genes represented in two E. coli pathotypes, isolated from suckling calves with and without diarrhea and accompanied with fairly matched case-control samples. The critical decision to evaluate only STEC and ETEC was not the best since there was no information in the area and selected representative genes. Our study, as a preliminary survey, compared the frequency of three genes of E. coli among the isolates from suckling calves with and without diarrhea to propose a probable candidate gene to control the diarrhea measures.

5. Conclusion

The isolates recovered from both clinically healthy and diarrheic suckling calves in the study area contained the remarkable virulence factors of E. coli, stx1 (dominant in diarrheic calves), stx2, or stx1+2, alarming for public health. Furthermore, k99 was not regarded as the major fimbrial antigen-encoding gene, and other genes (i.e., encoding fimbriae) should also be taken into consideration for ETEC when formulating a strategy for health purposes in the study area. Future epidemiological studies on the stx1 gene, other than k99 and stx2, are suggested for calf diarrhea in the study area.

Authors' Contribution

All authors have approved the final article. S. S. designed the study. N. K. and S. S. performed the experiment and data analysis, respectively. A. R. designed the study, interpreted the data, and drafted the article. S. S. and A. R. revised the manuscript critically for the important scientific content.

Ethics

This study was carried out according to the legal requirements of the relevant local authority.

Conflict of Interest

The authors declare that they have no conflict of interest.

Grant Support

This study was funded by the University of Zabol, Zabol, Iran (grant No. UOZ/GR/9618/32).

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