Occurrence and characterization of E. coli pathotypes associated with post-weaning diarrhea in early weanling pigs in an organized farm

Introduction

Post-weaning diarrhea (PWD) represents a substantial economic challenge in global swine production and this condition affects piglets within the initial two weeks following weaning and causes severe economic losses due to slow growth, weight losses, treatment costs and early mortality (Nadeau et al., 2017; Fairbrother et al., 2005; Amezcua et al., 2002) [13,7,1] . Weaning constitutes most challenging events in a pig's life (Campbell et al., 2013) [3] . Now days, early weaning is done to enhance the pork production and weaning age shifts from 5-7 weeks to 3-4 weeks (Widowski et al., 2008) [22] . Early weaning is a significant contributor to piglet diarrhea, as it induces psychological, nutritional, environmental, and physiological stress on the piglets and disrupts the intestinal barrier function in piglets, disturbs the balance of gut microbiota, and damages the intestinal chemical, mechanical, and immunological defenses (Campbell et al., 2013) [3] . Post-weaning diarrhea is a multifactorial disease with main infectious agents are Escherichia coli, Rotavirus, Campylobacter spp., and Salmonella spp. (Rhouma et al., 2017; Luppi et al., 2016) [16,11] . Additionally, various stress factors related to weaning, including separation from the sow, changes in diet, adjustment to a new environment, mingling with pigs from different farms, and histological alterations in the small intestine, can impair the immune system's response and result in intestinal dysfunction in pigs (Lallès et al., 2004; McCracken et al., 1999) [10,12] . The prevalence of the post weaning diarrhea is around 24% in Australia (Dhungyel et al., 2017) [6] , 30% in USA (Bush, 2002), 3.57% to 14.29% in different organized farms in India (Vinodh et al., 2019) [21] and mortality reached up to 20-30% in the affected piglets (Rhouma et al., 2017) [16] . The enterotoxigenic Escherichia coli (ETEC), mainly including F4 (K88) + is main pathotype among all (Sun & Kim, 2017) [19] . Despite the known association between weaning stress and post weaning diarrhea, there is limited information on the occurrence of specific E. coli pathotypes involved in early weanling pigs on organized farms and also there is no information available of the occurrence of post weaning diarrhea in different breeds of pigs and in both sexes i.e. in male and in female piglets in an organized farm in India. Understanding the specific E. coli pathotypes associated with PWD in early weaning pigs can help to develop targeted interventions to mitigate this condition in piglets. The primary objective of the present study is: To assess the occurrence of post-weaning diarrhea in earlyweaned piglets in an organized farm and to evaluate the distribution of associated Escherichia coli pathotypes across different breeds and sex of piglets.

Materials and Methods

Selection of animals and collection of faecal samples

The study exclusively involved 238 post-weaned piglets of different breeds (141 crossbreed piglets, Large White Yorkshire breed piglets was 11, Landrace breed piglet was 69, Gurrah breed piglets was 17 in no.) in the swine production farm of ICAR-Indian Veterinary Research Institute, with diarrhea or suspected for diarrhea. Diarrhea was identified based on clinical symptoms such as frequent defecation (more than three times daily), faecal consistency (firm, semi solid, liquid), faecal colour (yellowish, greenish, brown), signs of dehydration (reduced skin pliability), dullness, and weakness. A total 54 faecal samples from the diarrheic piglets were aseptically collected using sterile faecal swabs (HiMedia laboratories Pvt Ltd. Mumbai, India) and transported in an ice box to the laboratory for E. coli diagnosis.

Screening of E. coli diarrhea E. coli pathotypes screening in faecal samples was performed by culturing in specific bacteriological media, conducting biochemical tests, and subsequently using polymerase chain reaction (PCR) with specific E. coli pathotypes primers.

Culture of bacteria

Faecal samples were immediately inoculated into sterile peptone water (HiMedia, Mumbai, India). Following a 2hour incubation at 37 °C, all broth cultures were inoculated onto MacConkey's agar (HiMedia, Mumbai, India) to promote E. coli growth. Following overnight incubation at 37 °C, at least four colonies resembling E. coli were selected and subcultured on Eosin Methylene Blue (EMB) agar (HiMedia, Mumbai, India) to observe the characteristic metallic sheen indicative of E. coli. The isolated colonies were transferred onto nutrient agar slants to obtain pure cultures, which were then subjected to standard morphological and biochemical tests.

Biochemical test

Identification of E. coli pathotypes using polymerase chain reaction (PCR) Extraction of bacterial DNA

Bacterial DNA was extracted following the procedure outlined by Dashti et al. (2009) [5] . Briefly 1ml broth culture was pelleted by centrifugation for 10 min. The bacterial pellet was resuspended in 50 µL of nuclease free water (NFW), then heated for 5 minutes and immediately chilled for 10 minutes to lyse the bacteria. The lysate was centrifuged again, and the supernatant was directly used as the template for PCR.

Identification of E. coli pathotype by PCR

All PCR assays in this study were conducted in a 15 µL reaction mixture, comprising 7.5 µL Thermo Scientific Taq Green PCR Mastermix (GeneDirex, Uttar Pradesh, India), 0.5 µL forward primer, 0.5 µL reverse primer, 5 µL nuclease-free water, and 1.5 µL bacterial DNA extract. [18] TCC TTC ATC CTT TCA ATG GCT TT The PCR conditions for all genes (eae, stx1, lt) included 35 cycles, with each cycle consisting of an initial denaturation at 95 °C for 5 minutes, followed by denaturation at 95 °C for 30 seconds, annealing at 56 °C for 30 seconds, elongation at 72 °C for 30 seconds, and a final extension at 72 °C for 7 minutes. Following PCR amplification, the amplified PCR products were analyzed by gel electrophoresis in 2% agarose gel containing ethidium bromide (0.5 µg/mL) (Sambrook and Russel, 2001) [17] . Oligonucleotides primers and other PCR reagents were procured from Thermo-fisher scientific, USA.

Statistical analysis

The current study was carried out for a period of 3 months from April, 2024 to June, 2024.The occurrence of E coli infection in post weaned diarrhoic piglets were determined as below.

Occurrence rate = Total number of positive cases of Diarrhoic piglets associated with E. coli infection/Total number of cases reported/screened (during specified period) X 100 During the study, total strength of male piglets was 130 and female piglets was 108. No of diarrheic samples collected were 54 in no. (22 from male and 32 from female piglets), out of them, total no. of positive samples was 27 (14 from male and 13 from female piglets). Therefore, the sex-wise occurrence of diarrhea-causing E. coli was approximately ≈10.8% in male, ≈12.0% in female.

Results

Occurrence of Escherichia coli diarrhea in Piglets

Sex of Piglet

Discussion

The study aimed to evaluate the occurrence of Escherichia coli pathotypes associated with post-weaning diarrhea (PWD) in early weanling pigs from an organized farm, and the findings reveal several key insights into the prevalence and distribution of E. coli infections among different breeds and sexes of piglets.

The overall occurrence of diarrhea-causing E. coli was approximately 11.34%, a significant finding that underscores the high burden of this pathogen in post-weaned piglets. This high prevalence aligns with previous studies that report E. coli as a major contributor to PWD, with varying occurrence rates depending on geographical location and management practices (Hong, 2006) [9] . The high rate of E. coli infections highlights the importance of effective management strategies and preventative measures to mitigate the impact of this pathogen. Breed-wise analysis revealed varying occurrences of E. coli infection. Crossbred piglets had an occurrence rate of approximately 14.2%, which is consistent with reports that crossbreeds are often more susceptible to E. coli infections due to their diverse genetic backgrounds and the Large White Yorkshire piglets exhibited the highest occurrence rate of 27.3%, which may be attributed to breed-specific susceptibility or management factors specific to this breed (Parkunan et al., 2015) [15] . Conversely, Landrace and Gurrah breeds had lower occurrence rates of 4.3% and 5.9%, respectively, suggesting that these breeds may have better natural resistance or differing management practices that reduce their susceptibility to E. coli infections (Pal & Chakravarty, 2019) [14] . Sex-wise analysis of the positive samples showed a higher occurrence in female piglets (12.0%) compared to male piglets (10.8%). This slight difference in occurrence rates by sex is consistent with some studies suggesting that female piglets may be more susceptible to gastrointestinal infections due to differences in immune responses or social interactions (Gao et al., 2022) [8] . However, the difference observed in this study is relatively modest, indicating that while sex may play a role in susceptibility, other factors such as management practices and environmental conditions likely have a more substantial impact. The diagnostic methods used, including culture on specific media, biochemical tests, and PCR, provided a comprehensive approach to identifying E. coli infections.

The presence of the eae gene in 24 out of 27 positive samples suggests that the majority of the infections around 10.08% were caused by enteropathogenic E. coli (EPEC), which is known to play a significant role in PWD (Vidal et al., 2019) [20] . The use of PCR for detecting specific toxic genes like eae, stx 1 , and lt is crucial for accurate diagnosis and understanding the pathogenic mechanisms of the infections.

Acknowledgement

AcknowledgmentThe authors are thankful to Director of ICAR-IVRI, Head of Division of Medicine of Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India for providing necessary facilities and support.

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