Irene V. Wesley

Multiplex PCR for the identification of Arcobacter and differentiation of Arcobacter butzleri from other arcobacters

A multiplex polymerase chain reaction (PCR) assay to identify Arcobacter isolates and to distinguish A. butzleri from other arcobacters is described. The test uses two primer sets. Set I targets a section of the 16S rRNA genes of Arcobacter spp. Set II amplifies a portion of the 23S rRNA genes unique to A. butzleri. Specificity of the primer sets was evaluated using ATCC reference strains of A. butzleri, A. cryaerophilus, A. skirrowii, Bacteroides spp., Campylobacter spp., Helicobacter spp. and Wolinella succinogenes. Upon PCR amplification, all of the Arcobacter isolates yielded a 1223 bp product, whereas A. butzleri ATCC 49616 exhibited both a 1223 bp and a 686 bp product. No PCR product was observed for other closely related ATCC strains (n = 37). We next analyzed by multiplex PCR field strains of Arcobacter spp. (n = 108) which had been previously characterized to the species level by either DNA-DNA hybridization, dot blot hybridization, ribotyping or by serology. The 1223 bp multiplex PCR product identified all of the isolates as Arcobacter. The presence of both the 1223 and 686 bp amplicons identified 66 strains as A. butzleri. Speciation by multiplex PCR agreed with results obtained by the other methods. The multiplex PCR assay is specific, rapid and easy to interpret and, thus, will aid in elucidating the prevalence, epidemiology and zoonotic potential of Arcobacter.

Classification of Arcobacter species isolated from aborted pig fetuses and sows with reproductive problems in Brazil

Seventeen field isolates of Arcobacter species were recovered in Brazil from aborted porcine fetal livers (n = 3), kidneys (n = 2), and thoracic fluid (n = 1). Arcobacter species were also recovered from uterine and oviductal tissues (n = 5) and a placenta from sows with reproductive problems. These isolates were initially presumed to be Arcobacter cryaerophilus on the basis of aerobic growth at 30 degree C, indoxyl acetate hydrolysis, catalase and oxidase reactions, growth on MacConkey agar, sensitivity to 3.5% sodium chloride, and susceptibility to nalidixic acid (40 mg/ml). The isolates were confirmed as Arcobacter using polymerase chain reaction, and were classified as A. cryaerophilus 1A (24%), A. cryaerophilus 1B (71%), and A. butzleri (6%) using restriction fragment length polymorphism.

Helicobacter and Arcobacter : Potential human foodborne pathogens ?

Helicobacter pylori is associated with human gastric ulcers and gastric cancer; it has been detected in water, but not in foods. Arcobacter, a newly described Campylobacter-like organism, has been associated with cases of livestock abortion and human enteritis. Arcobacter spp. have been detected in water, cattle, swine, poultry, and ground pork products. This article reviews the evidence for considering Helicobacter spp. and Arcobacter spp., especially Arcobacter butzleri, as emerging foodborne pathogens.

Detection of Arcobacter species in gastric samples from swine

Swine stomachs were surveyed for evidence of Arcobacter spp. and Helicobacter spp. infections associated with gastric ulceration. A nested PCR test targeted to the 16S rRNA was developed to detect many Arcobacter spp. and Helicobacter spp. An internal oligonucleotide probe was used for differentiation and confirmation of the PCR product. Tissue samples were obtained from the nonglandular and glandular regions of 86 swine stomachs. Evidence of infection with these microbes was detected in 51%, with 77% of the positive samples being identified as A. butzleri using a highly specific probe. Nonglandular stomach samples (44%) were more likely to be positive by PCR than samples from the glandular (23%) region. Gross lesions of any stage of gastric ulceration, ranging from parakeratosis, erosions and ulceration, were observed in 24% of stomachs examined. Of 21 samples with lesions, 52% were positive by the broadly reactive PCR assay for Arcobacter spp. and Helicobacter spp. The majority of PCR-positive samples (75%) had no gross lesions. When a single step PCR assay that was more specific for Arcobacter spp. was used on the nonglandular stomach samples, 10.4% of the 86 samples were positive. Arcobacter spp. were cultured from four of the sample stomachs. Partial sequencing of the 16S rRNA gene identified the isolates as A. butzleri (n = 2), A. cryaerophilus, (n = 1), and a mixed culture of A. butzleri and another Arcobacter spp. (n = 1). A single step PCR assay targeted to the urease gene and culturing methods were used to screen for H. pylori or other closely related urease positive bacteria, but none were found.

Specific identification of Campylobacter fetus by PCR targeting variable regions of the 16S rDNA.

Campylobacter fetus is recognized as a human and animal pathogen. The isolation and differentiation of C. fetus in diagnostic laboratories is hindered by its relatively slow growth and lack of distinguishing biochemical characteristics. We developed a fast, reliable PCR assay that specifically amplifies a 554-bp segment of the 16S rDNA from C. fetus. Fifty-two ATCC reference strains and 255 bacterial field isolates comprising the genera Campylobacter, Arcobacter, Helicobacter, Escherichia, Listeria, Salmonella, and Wolinella were evaluated using this PCR protocol. Only C. fetus strains were amplified. Sequence analysis of amplicons from ATCC and field strains of C. fetus confirmed the presence of the target DNA fragment. The detection limit of the technique was 5.9 x 10(3) CFU/ml. This PCR assay can yield reliable detection of C. fetus within 3 h after isolation of presumptive colonies on agar plates.

Differentiation between Transmissible Gastroenteritis Virus and Porcine Respiratory Coronavirus Using a cDNA Probe

A plasmid, pG3BS, containing a cDNA clone from the 5′ coding region of the peplomer glycoprotein gene appears to be specific for enteric transmissible gastroenteritis virus (TGEV) strains and for live-attenuated TGEV vaccines. This cDNA probe is used to differentiate porcine respiratory coronavirus (PRCV) isolates from TGEV field and vaccine strains by a slot blot hybridization assay. Probe pG3BS also hybridizes to canine coronavirus (CCV) RNA but does not hybridize to antigenically related feline infectious peritonitis virus (FIPV) RNA. The RNAs of 13 enteric TGEV isolates from the United States, Japan, and England, 4 US-licensed live-attenuated TGEV vaccines, and antigenically closely related CCV were detected by pG3BS. The RNAs of FIPV and 3 US isolates of PRCV did not react with pG3BS but were detected by a TGEV-derived plasmid, pRP3. Pigs infected with either PRCV or TGEV test serologically positive for TGEV antibody by the serum neutralization test. Characterization of the virus circulating in a swine herd by the pG3BS probe will differentiate between an enteric TGEV and a respiratory PRCV infection.

Characterization of Listeria monocytogenes isolates by Southern blot hybridization

A synthetic deoxyribonucleotide probe for virulent Listeria monocytogenes, designated ADO7, was evaluated for its ability to identify restriction fragments of L. monocytogenes with nucleic acid sequences homologous with the beta-hemolysin gene by Southern blot hybridization of clinical and food isolates. The synthetic probe hybridized with three restriction fragments (approximately 1.1, 0.86, and 0.76 kb) of the serotype 1/2A isolates. Southern blot hybridization of the serogroup 4B isolates indicated that the nucleic acid sequences homologous with the beta-hemolysin gene probe were limited to a single restriction fragment of approximately 1 kb.

The use of listeriolysin O in an ELISA, a skin test and a lymphocyte blastogenesis assay on sheep experimentally infected with Listeria monocytogenes, Listeria ivanovii or Listeria innocua☆

Purified listeriolysin O (LLO) was evaluated as a specific antigen to detect both humoral and cell mediated immune responses of sheep infected with Listeria monocytogenes. Six sheep (two in each group) were orally inoculated with 10(10) organisms of L. monocytogenes, L. ivanovii, or L. innocua. Only the L. monocytogenes inoculated sheep had an elevated temperature (> 42 degrees C) and after 15 days had anti-LLO antibodies as assessed by an ELISA. In a blastogenesis assay, only peripheral blood mononuclear cells (PBMC) from L. monocytogenes-infected sheep responded to LLO, while PBMC from all the sheep responded somewhat to heat-killed L. monocytogenes bacteria. In a skin test, only L. monocytogenes-infected sheep exhibited a positive reaction to injected LLO, while all the Listeria-infected sheep reacted to heat-killed bacteria. On day 120 postinfection, all of the sheep were orally inoculated with L. monocytogenes. Only the four that had not been previously given L. monocytogenes exhibited an elevated temperature (> 42 degrees C). 80 days later, sera from all of the animals were positive for anti-LLO antibodies. Thus, prior exposure to L. ivanovii or L. innocua does not protect against a L. monocytogenes challenge. These results suggest LLO is an excellent antigen for use in detecting Listeria infection in sheep. However, whether LLO will be useful in differentiating chronically infected animals from animals that have recovered, has yet to be investigated.

Restriction Enzyme Analysis in the Epidemiology of Listeria Monocytogenes

Listeria monocytogenes is a foodborne bacterial pathogen which is transmitted to humans via consumption of dairy products and vegetables. The purpose of this study was to examine the genetic diversity of L. monocytogenes by restriction enzyme analysis and to localize the hemolysin gene by hybridization with a synthetic oligomer specific for the β-hemolysin (β- listeriolysin) toxin gene. Reference strains of serotypes 1A (1/2A), IB (1/2B), 1C (1/2C), 3A, 3B, 3C, 4A, 4B, 4AB, 4C, 4D, and 4E and 39 isolates obtained from food and clinical specimens were serotyped and genomic DNA analyzed for restriction enzyme patterns. Three groups of isolates were examined: two groups were derived from environmental samples and food products, and unrelated sporadic clinical cases. The third set consisted of isolates recovered during the recent listeriosis outbreak in Los Angeles County, California. Isolates were identified as serogroup 1A (factor 1) or as serotype 4B (factor 6). Reference strains exhibited a distinct electro- phoretic pattern after Hha 1 digestion. Isolates assigned to the same serotype exhibited different restriction patterns after Hha 1 digestion. However, when restriction fragments were blotted using the Southern method and probed with a synthetic oligonucleotide specific for the β-hemolysin gene, hybridization occurred with a limited number of restriction fragments. Ten strains recovered from Mexican-style soft cheese incriminated in the California listeriosis outbreak were antigenically identified as serogroup 4B (factor 6). All 10 strains exhibited identical restriction patterns after cleavage with Hha I. Only a single restriction fragment (0.9 kb) hybridized with the β-hemolysin gene probe. This suggests a common contaminating source of Listeria for the cheese samples examined.