Biology Reference
In-Depth Information
as demonstrated by outbreaks that occurred in many industrialized countries
(Bonardi and Leccese
2006
). Cattle are the main reservoir of VTEC, but small
ruminants also play an important role in the transmission of these pathogens to
humans. Small ruminants represent one of the more important reservoirs of
VTEC. An increased prevalence of a wide variety of serotypes in the gut of
clinically healthy sheep has been described (Zweifel et al.
2004
;Urdahletal.
2001
; Blanco et al.
1996
).
Epidemiological studies have demonstrated that some strains of VTEC isolated
from healthy sheep have genetically distinct subtypes of Shigatoxine (
stx
1
c
and
stx
2
d
) (Pierard et al.
1998
). Shigatoxine subtypes are not commonly recovered from
the feces of patients experiencing HC or HUS, but are isolated from cases of
uncomplicated diarrhea. Furthermore, lambs, as compared to adult sheep, carry
a greater variety of atypical-VTEC serotypes that temporarily colonize intestinal
tracts. These differences may be related to the combined effect of age, physiological
differences in intestinal tracts, diets, and/or different immune response to these
pathogens (Djordjevic et al.
2004
). The aim of this study was to determine the
prevalence of VTEC in sheep slaughtered in Sardinia, where approximately three
million heads are reared, to further molecularly characterize the pathogenic profiles
of the isolated strains.
27.2 Materials and Methods
27.2.1 Sample Collection
From three slaughterhouses located in Sardinia, 380 samples from 95 animals
(50 sheep and 45 lambs) were collected that included the following matrices:
(a) scrapes of the colon and the rectum mucosa (10 g, after evisceration); (b) skin
from the abdomen and the chest (20 cm
2
next to the incision line); and (c) carcass
surfaces by sponge-swab (300 cm
2
) of regions of the chest, abdomen, and leg.
27.2.2 Rapid Screening and Strain Isolation
A 10-g portion of each sample was diluted tenfold in 90 ml of modified Tryptone
Soya Broth (m-TSB) supplemented with 20 mg/l novobiocin. Enriched broths were
incubated at 37.5
C for 18-20 h. Rapid molecular screening was used to evaluate
the presence of
vtx
genes using the PCR method of the EU Food Project with
modifications. One milliliter of each sample was subjected to (1) DNA extrac-
tion using the resin Chelex-100 (Bio-Rad, USA) and (2) amplification of
stx
1
and
stx
2
with the primer pair MK1 (5
0
-TTTACGATAGACTTCTCGAC-3
0
)and
MK2 (5
0
-CACATATAAATTATTTCGCTC-3
0
) that resulted in a PCR product of
