In the last 10 years, Clostridium difficile has been implicated as a major cause of neonatal diarrhea in pigs. C. difficile infection (CDI) affects piglets ranging in age from 1-7 days. Clinical signs of CDI include diarrhea, abdominal distention and scrotal edema with most of the pathology being attributed to toxin production by this bacteria (Toxin A and B). Currently there are no commercial vaccines against C. difficile and the use of prophylactic antibiotics has been unsatisfactory and unrewarding for swine producers. Clostridium difficile infection is currently considered one of the most common causes of nosocomial diarrhea in humans. Therefore, we hypothesize that early oral administration of probiotics to pigs will control disease after experimental infection with C. difficile. An economical, safe, and simplistic therapy to control CDI and its subclinical incidence could be very beneficial to the swine industry.
Four replicates of the experimental design were conducted involving a total of 150 new born, caesarian derived piglets. Each experiment (replicate) divided piglets into 6 different study groups. GROUP 1 negative control, GROUP 2 piglets only received non-toxigenic C. difficile strain, GROPU 3 piglets received only probiotic yogurt, GROUP 4 positive control (challenged with toxigenic C. difficile strain), GROUP 5 animals received the probiotic non-toxigenic C. difficile strain and then challenged with the toxigenic C. difficile strain, GROUP 6 received probiotic yogurt and then challenge with the toxigenic C. difficile strain. Piglets were individually housed with non-challenged piglets (Groups 1 -3) being house in separate airspace than challenged piglets (Groups 4-6). The challenge isolate originated from a field case of neonatal diarrhea in 3-6 day-old piglets with high levels of toxin detected. Piglets were fed a set amount of milk replacer via gastric intubation three times daily. All pigs were humanely euthanized 72 hours post-challenge. The experimental protocol was approved by the Iowa State University Institutional Animal Care and Use Committee.
At necropsy, gross observations at necropsy included 1) body condition, 2) dehydration status, 3) perineal fecal staining, 5) consistency of colonic contents, 6) mesocolonic edema, and the presence of 7) visible colonic luminal necrosis and were scored independently in a blinded fashion as previously described. Necropsies, clinical sign scores and gross lesion scores were completed by the same two individuals for all experiments. Fresh and formalin fixed tissues including ileum, jejunum, descending colon, cecum, and a cross section of spiral colon were collected. All tissues were submitted for histopathologic examination by a veterinary pathologist that was blinded to animal group designation. Rectal swabs collected prior to inoculation and pooled colon and cecum contents collected at necropsy were assayed for C. difficile toxins using a commercially available toxin ELISA kit providing a semi quantitatively measure of the amounts of toxin from 0 (no toxin detection) to 4+ (marked toxin detection) as indicated by the manufacturer.
For statistical analysis, three categories of scores were compared: 1) clinical signs, 2) ELISA results and mesocolonic edema, and 3) microscopic lesions. Clinical signs scores were created by summing scores for body condition, hydration status, and perineum staining. Microscopic lesion score was the sum of scores for all histopathology categories as previously described.
Overall statistical evaluation of clinical signs scores from study groups revealed no statistical difference (P>0.05). Statistical analysis revealed an overall significant difference in mesocolonic edema scores (P=0.01). Group 4 presented significant higher scores when compared to Groups 1, 2 and 5 (P= 0.04, 0.018 and 0.002 respectively). All animals were ELISA negative at the beginning of the experiment. At necropsy (72 hours post inoculation), Group 5 had significant lower levels of toxin A and B detected via ELISA in colonic content when compared to Groups 3, 4 and 6. Histopathologic examination revealed classic CDI microscopic lesions characterized by variable numbers of intestinal only observed within colon and cecum. Microscopic scores were statistically different between groups (P=0.03). Group 4 had significant higher scores when compared to Groups 2 (P=0.005) and 5 (P=0.02). No other pairwise comparisons were significantly different. Analysis of data indicated that mesocolonic edema, ELISA toxin levels and histologic lesions are highly and significant correlated (P=0.001).
This project demonstrated that the use of non-toxigenic C. difficile probiotic was effective in minimizing the histologic lesions associated with C. difficile infection. Interestingly this decrease was also seen in the non-challenged piglets that received this non-toxigenic probiotic. The Lactobacillus spp probiotic also trended to have slightly lower histologic lesions than Groups 5 and 6 although statistical significance was not reached. It could be that for the Lactobacillus spp probiotic a continuous daily dose may be necessary to provide effective protection against CDI. It was also very interesting that for the first time, to our knowledge, we were able to see a direct association between mesocolonic edema, toxin levels and histologic lesions. This supports our current understanding of CDI as a toxin mediated disease. It is very feasible that non-toxigenic C. difficile probiotic can be easily used in the field to mitigate CDI in newborn piglets.