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™.