ABSTRACT. Two hundred thirty one Staphylococcus aureus isolates from bovine mastitic milk were discriminated into 60 patterns and 16 lineages by pulsed-field gel electrophoresis (PFGE). The tested isolates were also investigated using coagulase and capsule serotyping and PCR for possession of genes that encode staphylococcal enterotoxins (sea to sei), enterotoxin-like toxins (selj to selr), and toxic shock syndrome toxin (tst). One hundred seventy three of the isolates (74.9%) possessed one or more toxin genes, while no egg-yolk factor was detected in most of them. The most common combinations of toxin genes possessed by the tested isolates were sec, seg, sei, sell, and tst, or seg and sei, or sec, seg, sei, sell, seln, and tst. Two hundred and ten of the isolates (91.0%) serotyped coagulase VI, and 207 of the isolates (89.6%) expressed serotype 5 or 8 capsules. These results suggested that isolates belonging to two major lineages have spread all over Hokkaido as bovine mastitic isolates. Additionally, no remarkable difference was recognized in the identification ratio of the isolates that belonged to the two major lineages between mastitis of subclinical origin and mastitis of clinical origin. KEY WORDS: bovine mastitis, pulsed-field gel electrophoresis, staphylococcal toxin gene.

The prevalence of eae-positive Escherichia coli (eaeEC) in Japan was examined using rectal stool samples taken from 35 calves less than 1 month old, 107 calves more than 1 to 3 months old, 88 heifers more than 3 to 6 months old, 214 heifers over 6 months old, and cows from 95 farms. Screening with eae PCR revealed the prevalence to be, with increasing age, 31.4, 8.4, 26.1, and 14.5%, respectively. Of 51 selected eaeEC strains, more than 40% were serotyped as O26, O103, O111, O145, or O157, which are frequently detected as enterohemorrhagic E. coli types. Four strains were identified as recently reported intimin types , , and .Enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and attaching and effacing E. coli (AEEC) are food-borne pathogens that can cause diarrhea in humans (7,11,15). These pathogenic E. coli types often possess genes coding for Shiga toxins (stx genes) and for intimin (eae), an outer membrane protein. E. coli strains with stx genes are called Shiga toxin-producing E. coli (STEC). Cattle are considered to be the main reservoir of STEC strains, including EHEC strains (2, 3). STEC strains are classified into more than 200 O serotypes (4, 16); however, the majority of outbreaks and/or sporadic cases of hemorrhagic colitis and hemolyticuremic syndrome in humans have been caused by the members of only a few serogroups, such as O26, O111, and O157 (10,17,19). Since the strains of these limited O serogroups almost all possess eae (3, 9), this gene may be a more useful target than the stx genes for screening EHEC strains in cattle fecal samples.We used PCR to investigate the prevalence of eae-positive E. coli in cattle feces and genetically characterized the intimin types found, as well as various virulence genes seen in the isolated strains.E. coli O157:H7 strain ATCC 35150 (American Type Culture Collection, Manassas, Va.) was used as the positive control for stx 1 , stx 2 , eae, and intimin type ␥ (intimin ␥). The E. coli strains JS144, 166, VR299-2, and EPEC108, which were used as positive-control strains for intimins ␣, ␤, and ε and bundleforming pilus (bfp), respectively, were derived from the stock culture collection of the National Institute of Animal Health, Tsukuba, Japan. A total of 444 rectal stool grab samples were collected from healthy dairy cattle (35 calves under 1 month old, 107 calves more than 1 to 3 months old, 88 heifers more than 3 to 6 months old, 214 heifers more than 6 months old, and cows) on 95 farms located in the western and central parts of Japan between May and November 2001. All rectal stool samples were sampled by veterinarians at regional governmental animal hygiene centers. The samples were placed in cool boxes (4 to 8°C) and taken to the laboratory for immediate processing (usually within 24 h). Each sample of 1 g of rectal stool was enriched in 19 ml of Trypticase soy broth (Eiken, Tokyo, Japan) at 37°C for 18 h. Ten microliters of the Trypticase soy broth culture was inoculated onto MacConkey agar (MAC; Eiken). The MAC plates were incubated...

The aim of our study was to reveal the molecular basis of the serologic nontypeability of 2 Actinobacillus pleuropneumoniae field isolates. Nine field strains of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia, were isolated from pigs raised on the same farm and sent to our diagnostic laboratory for serotyping. Seven of the 9 strains were identified as serovar 15 strains by immunodiffusion tests. However, 2 strains, designated FH24-2 and FH24-5, could not be serotyped with antiserum prepared against serovars 1-15. Strain FH24-5 showed positive results in 2 serovar 15-specific PCR tests, whereas strain FH24-2 was only positive in 1 of the 2 PCR tests. The nucleotide sequence analysis of gene clusters involved in capsular polysaccharide biosynthesis of the 2 nontypeable strains revealed that both had been rendered nontypeable by the action of ISApl1, a transposable element of A. pleuropneumoniae belonging to the IS30 family. The results showed that ISApl1 of A. pleuropneumoniae can interfere with both the serologic and molecular typing methods, and that nucleotide sequence analysis across the capsular gene clusters is the best means of determining the cause of serologic nontypeability in A. pleuropneumoniae.

An indirect enzyme-linked immunosorbent assay (ELISA) using lipopolysaccharide extract as antigen was evaluated for detection of antibodies to Actinobacillus pleuropneumoniae serovar 15. The serovar 15 ELISA had a higher sensitivity and specificity than latex agglutination test for 63 and 80 sera from pigs experimentally infected and not infected with A. pleuropneumoniae, respectively. When the serovar 15 ELISA was applied to 454 field sera, high rates of seropositivity were found in pigs from farms infected with A. pleuropneumoniae serovar 15, but not in those from farms free of A. pleuropneumoniae serovar 15. The results suggest that the serovar 15 ELISA may be useful for the serological surveillance of infection with A. pleuropneumoniae serovar 15.