2.3| BACTERIOLOGICAL AND MOLECULAR EVALUATION
Since the main pathological findings were located in the abomasum, from all necropsied animals (n = 11) were collected: (a) agar Gel Transport Swabs (Thermo Scientific™) from the abomasal mucosa surface, (b) abomasal content in microtubes (2 mL; Eppendorf™), and (c) fragments of abomasal tissue in microtubes (2 mL; Eppendorf™) for bacteriological culture and typification. For molecular diagnosis, it were also collected fragments of abomasal tissue and seven environment samples from the concentrate offered to the kid goats, feces from the sick animals, and feces from the rabbits that were kept near the kidding sector.
For C. perfringens diagnosis, samples were pre-enriched on Tarozzi medium and then streaked onto Schaedler agar with 5% sheep blood incubated under anaerobe conditions. Then, suspected colonies were submitted to multiplex PCR reactions for confirmation and typification (Meer and Songer, 1997).
For molecular diagnosis of Clostridium ventriculi , DNA extraction from the samples collected was realized by use of a commercial kit (EpicentreTM) according to the manufacturer´s instructions. Polymerase chain reaction (PCR) was used to diagnoseC. ventriculi using primers from the pyruvate decarboxylase (PDC) gene (specific to C. ventriculi ) and the 16S rRNA gene (used to detect Clostridium sp.), according to Lam-Himlin et al. (2011). A negative control containing ultra-pure water was included in the reaction.
Amplicons were visualized under UV light after electrophoresis on 1.5% agarose gels stained by GelRed™.