PDF《Hong Kong》

1996 in which a total of

9578 cases were reported [5]. DNA patterns of the isolates from infected cases suggested a heterogeneous source of contamination. The epidemiology of VTEC in man and animals has been studied in Western countries and Japan [6C8] but data from South East Asia and China are sparse. In this study, faecal and carcass samples were collected from cattle and pigs in an abattoir in Hong Kong, in an attempt to study the frequency of VTEC carriage in healthy livestock in the province. Local abattoirs process over

2000000 pigs and

50000 cattle annually [9]. Since the local community consumes more pork than beef, the carriage of VTEC in pigs was of particular interest. Other virulence factors associated with VTEC strains isolated were also examined. MATERIALS AND METHODS Collection of faecal and carcass samples Rectal and carcass swabs were collected immediately after the slaughter and evisceration of animals in a Government abattoir in Hong Kong. The animals sampled were

986 adult cattle and

487 adult pigs transported to Hong Kong immediately prior to slaughter from various provinces in mainland China. The samples were collected monthly between August

1996 and December 1998. Culture for VTEC Rectal swabs were inoculated onto sorbitolC MacConkey (SMAC) agar (Oxoid Ltd, Basingstoke, UK) and incubated at

37 mC overnight. Swabs from carcasses were inoculated into VTEC enrichment broth (2 g tryptone, 1n12 g bile salt,

5 g lactose,

4 g K # HPO % , 1n5 g KH # PO % ,

5 g NaCl and 0n02 g novo- biocin per litre) and incubated overnight with gentle agitation at

37 mC. A loopful of each broth culture was streaked onto a SMAC plate. One ml was centrifuged at

13000 r.p.m. in a microcentrifuge for

5 min and the bacterial cell pellet was used for PCR. Red or pink- coloured bacterial colonies on SMAC plates were regarded as sorbitol fermenting and colourless col- onies as sorbitol non-fermenting. Detection of vt genes by PCR and DNA hybridization The bacterial pellet from each carcass broth culture and a colony sweep from the SMAC plate of each rectal swab was suspended in 0n5 ml sterile distilled water and heated at

80 mC for

15 min. The supernatant was used for PCR analysis using primers MK1 and

2 (Table 1) as described previously [10]. The

25 ?l PCR mixture consisted of

3 ?l supernatant,

50 m? KCl,

10 m? TrisCHCl (pH 8n3), 1n5 m? MgCl # ,

240 ?? dNTP,

400 ?g\ml BSA,

20 ρmole of each primer and

1 U AmpliTaq Gold DNA polymerase (PerkinCElmer Cetus, Norwalk, CT, USA). The PCR mixture was heated to

94 mC for

12 min and then subjected to

30 cycles of ampli?cation (1 min at

94 mC,

3 min at

43 mC and

1 min at

72 mC respectively), followed by a ?nal extension period of

7 min at

72 mC. Ampli?ed products were visualized by ethidium bromide staining after electrophoresis in a 2% agarose gel. PCR-ampli?ed DNA was transferred from the agarose gel to Hybond-N nylon membranes (Amersham, Arlington Heights, IL, USA) by Southern blotting [11]. The membranes were hybridized at

45 mC overnight, with probes 428-I, 428-II and vt2e (Table 1) for detection of t1, t2 and t2e (GenBank accession number M29153 [12]) respectively. These probes were labelled using the DIG DNA 3h end-labeling and detection kit (BoerhingerCMannheim, Mannheim, Germany). The overnight hybridization was followed by two strin- gency washes of the membrane at 50EC in 0n2iSSC (30 m? NaCl,

3 m? trisodium citrate, pH 7n0) with 0n1% SDS for

15 min. Labelled DNA was visualized by anti-digoxigenin enzyme-linked immunosorbent assay. Detection of VT production by vero cell assay Samples positive for t by PCR were con?rmed by a vero-cell cytotoxicity assay [13]. For each specimen,