Four piglets, 3-4-days old, that had died during an outbreak of high morbidity, low mortality, neonatal diarrhoea in a 1000 sow commercial pig herd were submitted for necropsy. Details of treatment prior to death were not available.
At gross necropsy all four carcasses were well preserved with adequate body fat reserves. Stomachs were filled with milk. There was mild mesocolonic oedema, and small intestinal and colonic contents were soft and yellow in all four. No gross changes were noted in intestinal, caecal or colonic mucosa. Other body systems were unremarkable.
At least 6 sections from representative areas along the length of the small intestine, a section of caecum and at least six sections of spiral colon were sampled for histopathology. They were fixed in buffered formalin, processed routinely and stained with haematoxylin and eosin.
On histopathological examination three of the piglets had mild (n = 1) to severe (n = 2), multifocal, superficial fibrinosuppurative and erosive colitis with neutrophils and fibrin spilling from lamina propria through the eroded epithelium and into the lumen ('volcano lesions') (Fig. 1). In the other piglet there was a mild, multifocal, suppurative mesocolitis. In addition, in two of the piglets there was acute, mild, superficial, suppurative enteritis, with superficial necrosis and microthrombosis in one of the piglets. All four piglets also had mild atrophic enteritis. 
Colonic contents were positive for C. difficile toxins A/B using using Premier Elisa Kit Toxins A&B (Meridian Bioscience Inc.)
Fifteen muL colonic contents were treated with 50 muL (96 %) ethanol. Fifteen muL of each mixture was transferred individually to plates containing Brazier's cefoxitin cycloserine egg-yolk (CCEY) medium (Lister, 2014). Plates were incubated anaerobically (10%H2, 10%CO2 and 80%N2) at 34  C for 48 h. 'Broken glass' colonies, typical of C. difficile, were transferred to blood agar plates and incubated for a further 48 h. Chelex-100 chelating resin was used for whole-cell DNA extraction of the resulting colonies. PCR-amplification of the DNA was performed with BioMix Red mastermix (Bioline) and CD-16s primers, 5'-CTG GGG TGA AGT CGT AAC AAG G-3', 6'-GCG CCC TTT GTA GCT TGA CC-3' (Eurofins MWG). PCR products were transferred to a heating block, set to 75  C, for 45 min to concentrate products to c.20 muL, before electrophoresis on a 3 % agarose gel with GelGreen nucleic acid stain (Biotium). The gel was placed under ultraviolet light in a closed chamber of Bio-Rad Universal Hood II. Results were visualised with Bio-Rad Quantity One (4-6-1) 1-dimensional analysis software. PCR ribotypes were successfully obtained for three piglets; two were ribotype 078 and one was ribotype 110. The fourth piglet's sample failed to yield a pure culture after treatment with 96 % ethanol and anaerobic incubation with CCEY medium.
 Clostridium perfringens (C. perfringens) was isolated by direct anaerobic culture of intestinal contents using pre-reduced 5 % Columbia sheep blood agar (SBA) and fastidious anaerobe blood agar with nheomycin (E&O Laboratories, Scotland). C. perfringens alpha toxin was detected in a pooled sample of small intestinal contents using a sandwich ELISA, testing for alpha (alpha), beta (beta), epsilon (e) toxins and C. perfringens Antigen (BioX Diagnostics, Belgium).
Rotavirus group B was detected in small intestinal contents in two of the piglets using modified versions of previously described PCR methods. PCRs for porcine coronaviruses and porcine reproductive and respiratory virus were negative using modified versions of previously described methods. No antigen was detected using anti-Cryptosporidium parvum monoclonal antibody labelled with fluorescein isothiocyanate (Bio-X Diagnostics, Belgium, Catalogue Number BIO 073).