Role of environmental factors in ewes subclinical mastitis caused by Escherichia coli –shigatoxin producing

Role of environmental factors in ewes subclinical mastitis caused by Escherichia coli –shigatoxin producing:field study:

Eman A.M. El Nady¹ and Amal I. Gergis¹

¹Microbiology Department, Animal Health Research Institute (AHRI), Agriculture Research Centre (ARC), Assuit, Egyptv

Corresponding author: Eman.A.M.El Nady, Bacteriology Department, Assiut Provnicial Lab., Animal Health Research Institute, Agriculture Research Center (ARC), Egypt.

Email:  Elnadyeman@yahoo.com

Abstract

        Subclinical mastitis in ewes is primarily of bacterial origin due to contagious or environmental pathogens.The prevalence rate of subclinical mastitis is influenced  environmental factors such as feeding, management, bedding, and by housing. 104 ewes' milk samples in all were gathered from two groups: Group I ewes reared in a governmental farm under good hygienic conditions and proper feeding, and Group II, ewes reared in small private flocks that suffer from poor hygienic measures and inadequate feeding (52 samples each). The California Mastitis Test (CMT) showed that 71.5% and 80% of samples were CMT positive both in GroupI and Group II., indicating a noteworthy rise (P < 0.05) in Group II in contrast to Group I. After being isolated, E.coli was biochemically identified,  using milk samples from 22 of 104 ewes, with a total incidence a total incidence of (21.2%) testing positive for E. coli as 8 samples (15.38%) and14 samples (26.9%) in group ǀ and group ǁ respectively. The antimicrobial resistance profile of the tested E. coli strains revealed a high Multiple Antibiotic Resistance (MAR) index of 0.56. Uniplex PCR analysis of random ten E. coli strains indicated that every one of the

Ten isolated tested that every one of the ten isolated tested  100% positive for

E.coli phoA gene.

Additionally, only two E. coli isolates (20% each) had the virulence genes Stx1 and Stx2 and four out of ten isolates (40%) were found to be Shiga toxin-producing E. coli (STEC).

 Keywords: subclinical mastitis, ewes, bacteriological examination, PCR.

Introduction

     The majority of intramammary infections in dairy small ruminants are bacterial in nature and can be brought on by environmental or infectious pathogens. The most common pathogen associated with subclinical mastitis and antibiotic resistance, Escherichia coli (E. coli) is a Gram-negative opportunistic environmental infection that is correlated with inadequate hygiene practices in sheep' surroundings.(Sayed et al., 2012; Hinthong et al., 2017; Abdel-Tawab et al., 2018; Fahim et al., 2019).

Numerous pathotypes of E. coli are distinguished by the virulence factors they encode, which are linked to adhesion, hemolysin secretion, and enterotoxins. Significant variation exists in these genes' nucleotide sequences.(Manage et al., 2019).  

Numerous virulent toxins, including Shiga-like toxins (stx1 and stx2 genes), are produced by various strains of pathogenic E. coli and can cause hemolytic uremic syndrome, bloody diarrhea, and gastroenteritis in humans (Li et al., 2018).                         

The prevalence of E. Coli strains that are resistant to antibiotics varies greatly depending on the environment (Liu et al., 2020).The overuse of antibiotics, including penicillin,, tetracyclines, and sulfonamides, has contributed to the emergence of antimicrobial-resistant E. coli, particularly those sourced from animals (Navajas-Benito et al., 2018). Shiga toxin-producing E. coli (STEC) is identified by the production of Shiga toxins (Stx1 or Stx2), which interfere with the protein synthesis of host cells,resulting in the demise of cells. These toxins are known as verotoxins because of their effects on Vero cells or Shiga toxins because they resemble the toxin of Shigella dysenteriae. (Cookson et al., 2006; Wang et al., 2018).

This study aims to quantify the prevalence and extent of E. coli-induced subclinical mastitis in dairy ewes, identify the impact of environmental factors on the disease, and evaluate the pathogens' resistance to antibiotics and detection of Shiga toxins as virulence factors.

Materials and methods

Animals

      Study Time and Place: From December 2022 to January 2023, the study was carried out in the Assuit Governorate of Egypt. 104 dairy sheep in all were utilized to investigate different facets of subclinical mastitis., recording their hygienic measures and feeding conditions. The ewes were divided into two groups: Group I (52 dairy ewes reared in a governmental farm, receiving good feeding and maintained under good hygienic conditions) and Group II (52 dairy ewes reared in small private flocks, suffering from poor hygienic measures and inadequate feeding conditions).    

Samples:

104 milk samples in all were gathered. (52 samples in each group) according to Al-Majali Jawabreh (2003). A volume of Five milliliters of milk samples was  gathered

under sterile conditions in sterile containers and delivered right away in an ice box to the bacterial lab in the Microbiology Department at AHRI, Assuit branch, Egypt.

California Mastitis Test (CMT):

All milk samples underwent the CMT utilizing COVETO, Montaigu, France, in accordance with the Schlam et al. (1971) technique.

Bacteriological examination:

     After giving all CMT-positive samples a good shake, 1 milliliter of milk was mixed with 9 milliliters of nutritional broth, and the mixture was incubated aerobically for 18 to 24 hours at 37 °C. Lactose-fermenting colonies were inoculated onto Eosin Methylene Blue Agar (EMB) (Oxoid) and incubated aerobically at 37°C after a loopful of the inoculated broth was plated onto MacConkey Agar (Oxoid). These colonies frequently had a green metallic sheen (MacFaddin, 1985). It was believed that the mammary gland had subclinical mastitis if there were no clinical symptoms and the milk appeared normal and was both CMT and bacteriologically positive (Moawad and Osman, 2005).

Antibiotic susceptibility test:

For the recovered E. coli isolates, antimicrobial susceptibility tests were performed using the disk diffusion method (Quinn et al., 2004; CLSI, 2020). Nine antimicrobial drugs were tested against the isolated strains of E. coli: Florfenicol (FFC) 30 mcg, Enrofloxacin (EX) 5 mcg, Amoxicillin (EX) 25 mcg, Sulfamethoxazole-trimethoprim (SXT-25) 25 mcg, Streptomycin (S) 10 mcg, Ceftriaxone (CRO-30) 30 mcg, Erythromycin (E) 15 mcg, Amikacin (K) 30 mcg, and Tetracycline (TE) 30 mcg (Oxoid). The number of antibiotics to which the isolated strain was resistant and the total number of antibiotics tested are represented by (a) and (b), respectively, in the Multiple Antibiotic Resistance (MAR) index of the tested E. coli strains.

Statistical analysis:

       Software called GraphPad Prism 9.5.1 (GraphPad Software Inc., San Diego, CA, USA) was used to analyze the data. To determine whether there was a significant difference between the groups, the chi-square test was employed, with a "p" value of <0.05 considered statistically significant.

Detection of virulence genes:

       Ten phenotypic and multidrug-resistant E. Coli strains were examined for the presence of Stx1 and Stx2, and their molecular identity was verified by the detection of the phoA gene. The Animal Health Research Institute in Egypt served as the reference lab for the PCR assays, following the molecular procedures outlined below:

Extraction of DNA

      Using the QIAamp DNA Mini Kit (Qiagen, Germany), changes were made in accordance with the manufacturer's instructions to extract DNA. In short, 200 μl of the sample suspension was treated for 10 minutes at 56°C with 10 μl of proteinase K and 200 μl of lysis buffer. Following incubation, the lysate was mixed with 200 μl of 100% ethanol. After that, the sample was cleaned and centrifuged in accordance with the manufacturer's instructions.  DNA has washed with 100 μl of lysis buffer. Oligonucleotide Primers: Primers used were supplied by Metabion (Germany) and are listed in Table 1.

Amplification via PCR:

     Primers were used for uniplex PCR in a 25 µl reaction that included 6 µl of DNA template, 4.5 µl of water, 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), and 1 µl of each primer at a concentration of 20 pmol. An appliedbiosystem 2720 thermal cycler was used to carry out the reaction.

Analyzing the PCR Results:

      The PCR products were separated by electrophoresis employing a gradient of 5V/cm on a 1.5% agarose gel (Applichem, Germany) in 1x TBE buffer at room temperature. Each gel slot had been filled with 20 µl of the uniplex PCR products for the analysis. To measure the fragment sizes, a GeneRuler 100 bp ladder (Fermentas, Germany) was employed. A gel documentation system (Alpha Innotech, Biometra) was used to take pictures of the gel, and computer software was used to analyze the results.

Table 1: Target genes, amplicon sizes and cycling conditions of PCR:

Results

The results of the study are shown in Fig. 1-4 and Tables 2-5.

Table 2: Incidence of subclinical mastitis in examinedewe’s milk samples and prevalence of E. coli strains:

* Significant statistical variations p > 0.05

% determined using the total number of samples examined (52) in every group   

Table 3: E. coli isolates' in vitro antimicrobial susceptibility test (N= 14) and MAR index.

% Determined using the total number of E.coli isolates that were examined(14).

Fig 1: Phenotype identification  using the disk diffusion method for studies of antibiotic susceptibility.

Fig 2: PCR  finding for phoA gene demonstrating amplification of 720bp.(L): ladder (100-1000 bp) plus 100 bp.Positive control (lane P), negative control (lane N), and lanes 1: 10: lanes positive for E.coli.

Fig 3: Results from PCR for the Stx1 gene indicate 614 bp. (L):100 bp plus ladder (100-1000 bp) amplification.Lanes 2 and 4 are positive for Stx1, while Lanes 1, 3, 5, 6, 7, 8, 9, and 10 are negative for Stx1. Lane P is the positive control, while Lane N is the negative control.

Fig 4: PCR findings for the Stx2 gene indicate 779 bp  amplification(L): 100 bp plus ladder (100-1000 bp).Lane P: control that is positive; Lanes 1 and 7; Lane N: negative control; Lanes 1&7  +ve for Stx2, Lanes 2,3,4,5,6,8,9, 10 negative for Stx2strains.

Table (4): Genotypic characteristics of 10 E.coli strains detected in subclinical samples of ewes' milk:

Table (5): Isolated E. coli strains confirmed by using E. coliphoA gene and Virulence genes by PCR.

Discussion

           Sheep are integral to the livelihoods and economies of rural communities in Egypt, offering essential products such as meat, milk, wool, and hair (Ramadan 2022; Aminul et al., 2021; Gaballah et al., 2022). Sub-clinical mastitis influenced by different environmental factors as feeding, management, bedding and housing (Menzies and Ramanoon, 2001). Conformation of subclinical mastitis depended on bacteriological assessments as well as the California Mastitis Test (CMT), Gebrewahid et al. 2012).

Out of 52 ewe^’s milk samples from each group I and II in the present study; 37 (71.5%) and 42 (80%) were positive by CMT, Table (3). The results of CMT test by Abd El-Tawab et al.(2018), Hawari et al. (2014), and El-Bassiony et al. (2008). were 34.01 %; 55.5% and (42.8%), respectively.The low percent of CMT test results were 6.78% and 19.8% in ewes under good and poor hygienic measurements, respectively by Sayed et al. (2012).

          Environmental pathogens such as E. coli, Pseudomonas aeruginosa, Strept. uberis, and coagulase-negative Staphylococcus sp. or infectious pathogens like Staphylococcus aureus and Streptococcus agalactiae are the primary cause of intramammary infections in dairy small ruminants(Bergonier et al., 2003).

         The primary environmental bacterial agent identified as responsible for mastitis in dairy small ruminants was Escherichia coli (Vasileiou et al., 2018). So the mammary gland  having seemingly normal milk and no clinical problems that both CMT and bacteriologically positive were consider to suffer from subclinical mastitis (Moawad and Osman, 2005). On the basis of clarifying subclinical mastitis as the presence positive results of both California Mastitis Test and E. coli isolated in the present work, 8(15.38%) and 14 (26.9%) were suffered from subclinical mastitis within groups I and II, respectively, according to  Table (2).

        In present work, the ewes in group II significant increase (p > 0.05) in the frequency of subclinical mastitis than ewes in group I, Table (2), that Ewes in Group I received a good management and good feeding, while ewes in group II suffered from poor management, poor hygienic conditions and bad feeding as they were in nomadic rearing system but grazing on the rubbish and roughages in streets. Differences in management, food, flock size, breed, and dam parity, as well as lactation time, season, case definition, and diagnostic criteria, are the primary factors influencing the prevalence of subclinical mastitis in ewes. (Batavani et al. 2003).

     The frequency  of subclinical mastitis in ewes caused by E. coli through many studies varied widely as Awad and Awad (2021), Gebrewahid et al.(2012),Mork et al. (2007), Abdallah et al. (2018), and Majeed (2020) were 8.67%,17%, 7.3%, 44.4% and 37.2%; correspondingly, in ewe's milk samples E. coli were isolated in a percentage 0.51% by El- Bassiony et al.  (2008).

The primary environmental bacterial agent identified as responsible for mastitis was Escherichia coli (Vasileiou et al., 2018).The discrepancies in infection rates may be attributed to variations in sample sizes, the populations studied, and the distinct epidemiological and ecological contexts.

In the present work, California Mastitis test  revealed a greater frequency of  subclinical mastitis than bacteriological examination (Table 3), because California Mastitis Test has been standardized for cow's milk and it is most accurate in bovine (Batavani et al., 2003). CMT test is useful as screening test in ovine species but false positive results can be obtained due to the presence of non-infectious factors as ewe's normal milk typically contains more cells, nuclear fragments, cytoplasmic particles, and lipids (Al-Majali & Jawabreh, 2003; Moawad &Osman, 2005; Lafi, 2006 and Donovan et al., 1992).

The phenotypic analysis of the milk samples highlighted significant concerns regarding the maintenance of hygienic practices at the collection sites. Escherichia coli strains isolated were linked to environmental pathogens that contribute to subclinical mastitis, marking them as the predominant members of coliform species. The primary sources of coliform bacteria that lead to subclinical mastitis include water supplies, bedding materials, sewage, and other environmental contaminants. The incidence of coliform-related subclinical mastitis tends to rise during climatic conditions that favor the proliferation of these microbes. Typically, the entry point for these environmental pathogens into the udder tissue is through the teat orifice (Emon et al., 2024). The increased susceptibility of sheep to E. coli infections can be linked to several factors, including their immune system responses, heightened vulnerability to stress during transportation, abrupt environmental changes, and the presence of viral infections, all of which contribute to their increased risk of bacterial infections, as noted by Pavan et al. (2022).

Identification of the causative organism and antimicrobial susceptibility testing besides treatment or culling of untreatable animals are very important for control of sub-clinical mastitis, as the infected animals with subclinical mastitis act as primary source of infection to another healthy contact ones . In vitro the present antimicrobial susceptibility testing showed that E. coli isolated strains were highly sensitive to Enerofloxacin (100%), Florfenicol (92.8 %) and Ceftriaxone (92.8%). Conversely, resistance was noted at 85.7% for Amoxicillin, Sulfamethoxazole – trimethoprim 71.4%, Erythromycin 64.28%, Tetracycline 42.85% and Streptomycin 35.7%; Table (4) and Fig. (1).In the current investigation, the Escherichia coli isolates demonstrated MDR characteristics, as noted by Jamali et al. (2018). Hassen et al. (2020) and Bedada and Hiko (2011) found that E. coli was fully susceptible to streptomycin, yet completely resistant to erythromycin.While the moderate resistance levels of E. coli strains for sulphamethoxazole - trimethoprim and erythromycin reported by Awad and Awad (2021); Kindu et al. (2019);42.85% and Streptomycin 35.7%; Table (4) and Fig. (1).In the current investigation, the Escherichia coli isolates demonstrated MDR characteristics, as noted by Jamali et al. (2018). Hassen et al. (2020) and Bedada and Hiko (2011) found that E. coli was fully susceptible to streptomycin, yet completely resistant to erythromycin.While the moderate resistance levels of E. coli strains for sulphamethoxazole - trimethoprim and erythromycin reported by Awad and Awad (2021); Kindu et al. (2019),Briscoe et al. (2005) and  Bharathi et al. (2008).                                                                                     

Multiple Antimicrobial Resistance index (MAR) was 0.56, Table (3).The tested E. coli isolated   strains' MAR index value was high due to MAR index value just ≥ 0.2 was regarded high, Subramani &Vignesh (2012).The findings align with previous studies indicating a high level resistance to multiple drugs (MDR) among Escherichia coli isolates, particularly against antimicrobial classes deemed critical by the World Health Organization (WHO) for human health.Strains were classed as multiple resistant when they displayed resistance to three or more types of antimicrobial drugs (Schwarz et al., 2010).These results of the antimicrobial susceptibility test of Escherichia coli from mastitic sheep have indicated varying prevalence and resistance patterns, which may be influenced by regional differences in antimicrobial usage, the accessibility of over-the-counter antibiotics, and the quality of veterinary care available.

The inappropriate use of antibiotics, characterized by low concentrations, short treatment durations, and improper intervals not only leads to suboptimal clinical outcomes but also results in substantial economic repercussions (Zufferey et al., 2021). Furthermore, the spread of infected microorganisms, coupled with the transfer of resistance genes to other microbes, contributes to the emergence of multidrug-resistant strains (Kindu et al., 2019).

E.coli have several virulence factors. Shiga toxins Stx1 and Stx2 are produced by E. coli (STEC) strains that produce Shiga-like toxins.are considered the significant food-borne pathogen groupings. STEC strain is a significant  cause of gastroenteritis,acute renal failure in children  hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC)(Beutin et al., 2004 and Petro et al., 2019). Bovine and ovine are being the primary STEC strain- reservoirs (Zschock et al. 2000).

     In the current study uniplex PCR examination of ten strains of  E. coli were examined for phoA E.coli gene identification  and the existance of two virulence genes for stx1 and stx2 . All ten isolates bacteria E. coli tested were positive for the E. col iphoA gene (100%), Only two E.coli isolates had the virulence genes Stx1 and Stx2 identified  Table (5&6) and Fig. (2).Four out of ten E.coli isolates(40%) were found to be shiga toxigenic E.coli (STEC) based on uniplex PCR results.  ,Only two E. coli isolates (20%) had the virulence genes Stx1 and Stx2 identified, as shown in Table( 5 & 6 ) and Fig.,( 3 & 4).Out nine E. coli isolates three were include both Stx1 and Stx2; two isolates only had Stx1 detected, and one isolate only had Stx2 (El-Khabaz et al., 2022). Stx1, Stx2 were present in E. coli strains that were recovered from samples of mastitic milk (Osman et al, 2012,Ismail &Abutarbush, 2020 ).

The percentages of E. coli field isolates with Stx1 and Stx2 genes were 27.3 and 6.7%, respectively. These genes for virulence were discovered alone or in various combinations

 Awad et al. (2020) detected that eight (88.9%) and four (44.4%) of the nine E. coli strains that were analyzed have Stx1 and Stx2, respectively. However, 3 strains (33.3%) have both the stx1 and stx2 genes,Sayed (2014). The differences indistribution of the E. coli virulence genes found in E.coli isolates may be due to differences in study geographical areas Moussa et al. (2010).  

Conclusion

The Environmental conditions as hygienic measurement, management, bedding, housing and feeding  have a significant part in prevalence of ovine' subclinical mastitis, so significant  increase (p > 0.05) in prevalence of subclinical mastitis in ewes in group II than ewes in groupI. The primary environmental pathogen responsible for subclinical mastitis in ewes was Escherichia coli. The isolated Escherichia coli strains had high Multiple Antimicrobial resistance index. E. coli isolates harbor virulence Shiga toxin genes (Stx1 &Stx2) and all tested strains have E. coliphoA gene.

Recommendations

       The implications of these results underscore the critical requirement  for improved oversight  and management practices in dairy farming to mitigate the risks associated with MDR E. coli. The relationship between environmental conditions and the frequency of drug-resistant strains highlights the importance of adopting best practices in animal husbandry. By addressing the factors contributing to udder inflammation and improving overall farm hygiene, it may be possible to minimize the incidence of mastitis and the consequence  spread of resistant pathogens. This approach is essential for safeguarding both animal health and public safety in the context of food production.