Natural and experimental infections of Arcobacter in poultry.

Arcobacter butzleri causes human enteritis and is frequently recovered from poultry carcasses. The purpose of this study was to determine 1) the natural distribution of A. butzleri in poultry and 2) its relative pathogenicity in experimentally infected poultry. Cloacal samples (n = 407) were collected on four occasions from three flocks of chickens. Overall, Arcobacter spp. were recovered from 15% of the birds; A. butzleri was identified in 1% of cloacal samples. Three experimental trials were conducted to determine the susceptibility of birds. In Trial 1, 3-d-old chicks (n = 62) were divided into three groups and infected per os with 1) A. butzleri American Type Culture Collection (ATCC) 49616, 2) a suspension of four field strains of A. butzleri isolated from retail purchased chicken, and 3) Campylobacter jejuni (positive control). Arcobacter was not detected in cloacal swabs or in cecal samples of chicks through Day 5 postinfection; C. jejuni was detected in cloacal swabs of all positive control birds. In Trial 2, 5-d-old outbred turkey poults (n = 88) were infected as described above with the addition of a group infected with a suspension of four field strains of A. butzleri from turkey meat. Arcobacter butzleri was recovered from either cloacal swabs or cecal contents of only 6.0% of birds (4 of 67); C. jejuni was recovered from 100% of the positive control birds (n = 21). In Trial 3, 3-d-old turkey poults of the highly inbred Beltsville White strain (n = 141) were experimentally inoculated. In contrast to earlier trials, A. butzleri was recovered overall from the cloacal swabs or tissues of 65% of the turkeys.

DNA sequencing and expression of the formyl coenzyme A transferase gene, frc, from Oxalobacter formigenes.

Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species utilizing an activation-decarboxylation reaction which yields formate and CO2. frc, the gene encoding formyl coenzyme A transferase, an enzyme which transfers a coenzyme A moiety to activate oxalic acid, was cloned from the bacterium Oxalobacter formigenes. DNA sequencing revealed a single open reading frame of 1,284 bp capable of encoding a 428-amino-acid protein. A presumed promoter region and a rho-independent termination sequence suggest that this gene is part of a monocistronic operon. A PCR fragment containing the open reading frame, when overexpressed in Escherichia coli, produced a product exhibiting enzymatic activity similar to the purified native enzyme. With this, the two genes necessary for bacterial catabolism of oxalate, frc and oxc, have now been cloned, sequenced, and expressed.

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.

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.

Infection of cesarean-derived colostrum-deprived 1-day-old piglets with Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii.

Neonatal piglets have been used as models to study human campylobacteriosis and helicobacteriosis. The purpose of this study was to determine the relative pathogenicities, on the basis of the duration of fecal shedding and colonization of tissues, of three Arcobacter species in 1-day-old cesarean-derived colostrum-deprived piglets. Two experiments were conducted. In experiment 1, two piglets each were infected per os with either Arcobacter butzleri ATCC 49616, Arcobacter cryaerophilus 1B ATCC 43159, Arcobacter skirrowii CCUG 10374, or the three field strains of A. butzleri (approximately 5 X 10(9) CFU per piglet). Rectal swab samples were taken prior to infection and daily thereafter for up to 7 days. Arcobacter spp. were detected at least once in rectal swab samples of all but one of the experimentally infected piglets but not in the control. At necropsy, A. butzleri was recovered from the lung, kidney, ileum, or brain tissues of the four infected piglets which had received either the field strain or the ATCC type strain of A. butzleri. A. cryaerophilus 1B was detected in rectal swab samples for up to 7 days postinfection but was not cultured from tissues at necropsy. Arcobacters were detected in the rectal swab sample of the A. skirrowii-infected piglet only on day 3 postinfection; no isolates were obtained from tissues at necropsy. No gross pathological lesions were consistently noted in the experimentally infected piglets. In experiment 2, two piglets each were infected per os with A. butzleri ATCC 49616, A. cryaerophilus 1A ATCC 43158, A. skirrowii CCUG 10374, or the single A. butzleri field strain Yard J/c (approximately 5 X 10(9) CFU per piglet). Arcobacter spp. were cultured from rectal swab samples of all but one of the experimentally infected piglets at least once. At necropsy Arcobacter spp. were cultured from the liver, kidney, ileum, or brain tissues of two of the four A. butzleri-infected piglets. However, no severe gross pathology was noted. These data suggest that Arcobacter spp., especially A. butzleri, can colonize neonatal pigs.

Epidemiologic evaluation of encephalitic listeriosis in goats.

OBJECTIVE: To evaluate host and environmental factors associated with the development of encephalitic listeriosis in goats. DESIGN: Retrospective analysis of diagnostic laboratory records and survey of veterinarians and goat producers. SAMPLE POPULATION: 355 goat herds accessible through laboratory records; 38 veterinarians who treated goats and 76 goat producers. PROCEDURE: Data regarding breed and use for goats affected with encephalitic listeriosis were obtained from surveys and case follow-up information. Listeria monocytogenes isolates from the brains of 7 affected goats were serotyped and subjected to DNA restriction analysis. RESULTS: Odds ratio for the development of encephalitis listeriosis in Angora (mohair-producing) goats was 22.9 by use of diagnostic laboratory records. Survey also revealed a high prevalence in herds of Angora and other breeds that subsisted on woody browse, although Angora goats feeding predominantly on hay or pasture were not affected. Listeria monocytogenes isolates from 4 Angora goats in 3 herds differed in DNA restriction patterns, although the pattern was identical in 3 other goats from another herd. CLINICAL IMPLICATIONS: Encephalitic listeriosis can be observed in all goat breeds, but a lifestyle of heavy browse consumption seems important to the development of disease in some herds.

Biphasic culture of Arcobacter spp.

Arcobacter spp. have recently been genetically differentiated as a genus distinct from Campylobacter. Physiologically, Arcobacter spp. are aerotolerant bacteria, while Campylobacter spp. are microaerophilic. However, since Arcobacter spp. have been difficult to grow to high population densities in broth media, alternative culture techniques were investigated. A biphasic culture system was developed in 25 cm2 tissue culture flasks. Biphasic culture, consisting of a solid phase of 10% bovine blood agar and a liquid phase of Brain Heart Infusion broth, was found to increase bacterial population densities by more than 2 log10 cycles for strains of A. butzleri and A. skirrowii. A strain of A. cryaerophilus, which was non-culturable in broth culture, attained population densities of 10(9) cells ml-1 in biphasic culture. Neither the addition of fetal bovine serum to the liquid nor an increase in the surface area from 25 to 75 cm2 resulted in increased cell densities.

Arcobacter-specific and Arcobacter butzleri-specific 16S rRNA-based DNA probes.

The genus Arcobacter encompasses gram-negative, aerotolerant, spiral-shaped bacteria formerly designated Campylobacter cryaerophila. Two genus-specific 16S rRNA-based oligonucleotide DNA probes (23-mer and 27-mer) were developed. The probes hybridized with strains of Arcobacter butzleri (n = 58), Arcobacter cryaerophilus (n = 19), and Arcobacter skirrowii (n = 17). The probes did not cross-react with any of the reference strains of Campylobacter, Helicobacter, including "Flexispira rappini," or Wolinella. The 27-mer hybridized with 61 Arcobacter spp. field isolates originating from late-term aborted porcine (n = 54) and equine (n = 2) fetuses and humans with enteritis (n = 5). The species of Arcobacter isolates (n = 56) recovered from aborted livestock fetuses were determined by ribotyping and were as follows: A. cryaerophilus group 1A (11 of 56; 20%), A. cryaerophilus group 1B (37 of 56; 66%), A. butzleri (5 of 56; 9%), and unknown (3 of 56; 5%). The five human field strains were identified as A. butzleri. A species-specific DNA probe (24-mer) for A. butzleri was also developed since there is evidence that this organism may be a human pathogen. This probe hybridized with previously characterized strains of A. butzleri (n = 58), with 10 field strains identified as A. butzleri by ribotyping and with 2 strains having an indeterminate ribotype. The A. butzleri-specific probe did not cross-react with strains of A. skirrowii (n = 17) and A. cryaerophilus (n = 19).

Detection of anti-listeriolysin O in dairy cattle experimentally infected with Listeria monocytogenes.

A dot-blot assay and an enzyme-linked immunosorbent assay (ELISA) to detect listeriosis in dairy cattle were developed that detected anti-listeriolysin O antibodies in the serum of cows experimentally infected with Listeria monocytogenes. The tests utilized purified listeriolysin O (LLO) as the detection antigen and streptolysin O (SLO) to absorb cross-reacting antibodies. The two tests were compared with an agglutination test that used formalin-killed whole L. monocytogenes cells. Blood samples were collected periodically from 17 cows after intramammary gland infection, and the development of anti-LLO antibodies was followed by an agglutination test, the dot-blot test, and the ELISA. In general, an agglutination titer of > 640 was needed for a positive dot-blot anti-LLO test for nonpregnant cows. However, 1 pregnant cow with an agglutination titer of 20 was positive in the dot-blot test. The ELISA was as sensitive as the dot-blot assay but gave a quantitative measurement to distinguish serum samples of positive reactors from cross-reactors. The specificity of the LLO-based tests was further evaluated using serum from cows that had been experimentally infected with Staphylococcus aureus, 17 of which had agglutination titers for L. monocytogenes > 640. These elevated agglutination titers were probably due to cross-reacting bacterial antigens because serum from 9 of 17 of these animals did not react to the purified LLO antigen. A positive response to the LLO-based dot-blot and ELISA assays is indicative of previous or current infection with L. monocytogenes.

Molecular cloning, DNA sequence, and gene expression of the oxalyl-coenzyme A decarboxylase gene, oxc, from the bacterium Oxalobacter formigenes.

Oxalic acid, a highly toxic by-product of metabolism, is catabolized by a limited number of bacterial species by an activation-decarboxylation reaction which yields formate and CO2. oxc, the gene encoding the oxalic acid-degrading enzyme oxalyl-coenzyme A decarboxylase, was cloned from the bacterium Oxalobacter formigenes. The DNA sequence revealed a single open reading frame of 1,704 bp capable of encoding a 568-amino-acid protein with a molecular weight of 60,691. The identification of a presumed promoter region and a rho-independent termination sequence indicates that this gene is not part of a polycistronic operon. A PCR fragment encoding the open reading frame, when overexpressed in Escherichia coli, produced a product which cross-reacted antigenically with native enzyme on Western blots (immunoblots), appeared to form homodimers spontaneously, and exhibited enzymatic activity similar to that of the purified native enzyme.

Purification and characterization of formyl-coenzyme A transferase from Oxalobacter formigenes.

Formyl-coenzyme A (formyl-CoA) transferase was purified from Oxalobacter formigenes by high-pressure liquid chromatography with hydrophobic interaction chromatography and by DEAE anion-exchange chromatography. The enzyme was a single entity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel permeation chromatography (Mr, 44,000). It had an isoelectric point of 4.7. The enzyme catalyzed the transfer of CoA from formyl-CoA to either oxalate or succinate. Apparent Km and Vmax values, respectively, were 3.0 mM and 29.6 mumols/min per mg for formyl-CoA with an excess of succinate. The maximum specific activity was 2.15 mumols of CoA transferred from formyl-CoA to oxalate per min per mg of protein.

Purification and characterization of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes.

Oxalyl-coenzyme A (oxalyl-CoA) decarboxylase was purified from Oxalobacter formigenes by high-pressure liquid chromatography with hydrophobic interaction chromatography, DEAE anion-exchange chromatography, and gel permeation chromatography. The enzyme is made up of four identical subunits (Mr, 65,000) to give the active enzyme (Mr, 260,000). The enzyme catalyzed the thiamine PPi-dependent decarboxylation of oxalyl-CoA to formate and carbon dioxide. Apparent Km and Vmax values, respectively, were 0.24 mM and 0.25 mumol/min for oxalyl-CoA and 1.1 pM and 0.14 mumol/min for thiamine pyrophosphate. The maximum specific activity was 13.5 microM oxalyl-CoA decarboxylated per min per mg of protein.

Alternative pathways for biosynthesis of leucine and other amino acids in Bacteroides ruminicola and Bacteroides fragilis.

Bacteroides ruminicola is one of several species of anaerobes that are able to reductively carboxylate isovalerate (or isovaleryl-coenzyme A) to synthesize alpha-ketoisocaproate and thus leucine. When isovalerate was not supplied to growing B. ruminicola cultures, carbon from [U-14C]glucose was used for the synthesis of leucine and other cellular amino acids. When unlabeled isovalerate was available, however, utilization of [U-14C]glucose or [2-14C]acetate for leucine synthesis was markedly and specifically reduced. Enzyme assays indicated that the key enzyme of the common isopropylmalate (IPM) pathway for leucine biosynthesis, IPM synthase, was present in B. ruminicola cell extracts. The specific activity of IPM synthase was reduced when leucine was added to the growth medium but was increased by the addition of isoleucine plus valine, whereas the addition of isovalerate had little or no effect. The activity of B. ruminicola IPM synthase was strongly inhibited by leucine, the end product of the pathway. It seems unlikely that the moderate inhibition of the enzyme by isovalerate adequately explains the regulation of carbon flow by isovalerate in growing cultures. Bacteroides fragilis apparently also uses either the isovalerate carboxylation or the IPM pathway for leucine biosynthesis. Furthermore, both of these organisms synthesize isoleucine and phenylalanine, using carbon from 2-methylbutyrate and phenylacetate, respectively, in preference to synthesis of these amino acids de novo from glucose. Thus, it appears that these organisms have the ability to regulate alternative pathways for the biosynthesis of certain amino acids and that pathways involving reductive carboxylations are likely to be favored in their natural habitats.

Fractionation of a whey growth factor for Streptococcus agalactiae into two active components containing proteins.

Previously three factors (F-1, F-2, F-3) that stimulated in vitro growth of Streptococcus agalactiae were separated from wheys of milk and colostrum by chromatographic procedures. Now F-1 was separated further into two active components, F-1a and F-1b, when rechromatographed on an anion exchange resin with distilled water as eluent. The stimulatory activity was associated with two protein peaks whereas a third protein peak that was eluted in those fractions containing lactose was not stimulatory. Most of the stimulatory activity (76 to 80%) and protein (90 to 93%) were retained by a filter with retention of greater than 1,000 MW. Two stimulatory factors were confirmed by separation of F-1 fractions by step-gradient elution on a column of octadecylsilica; one protein peak was found for each of the three solvents. Samples with protein eluted with .1 N formic acid and formic acid: methanol (1:1) had stimulatory activity, whereas the sample with protein eluted with 100% methanol was inactive. Results were similar when F-1 fractions were applied to Sep-Pak (muBondapack C18) cartridges and eluted with water, 50% methanol, and 100% methanol. Proteins in the Sep-Pak eluates with stimulatory activity (water and 50% methanol) were heterogeneous and different when separated by high-voltage paper electrophoresis. Proteose-peptone preparations with added lactose were chromatographed on columns of anion exchange resin, with distilled water as eluent, and produced two peaks of protein with elution volumes comparable to peaks 1 and 3. Lactose appeared to be a major factor in eluting the second protein peak.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum concentration of bile acids in guinea pigs as an indicator of liver damage caused by aflatoxins.

Serum concentrations of glycocholic acid (GA) and glycodeoxycholic acid (GDA) and serum activity of aspartate aminotransferase (AST) were determined after guinea pigs had been fed 0, 0.005, 0.010, 0.015, 0.020, or 0.030 mg of aflatoxin B1 equivalents daily for 21 days. Mean serum concentrations for the 20 control guinea pigs were GA, 3.70 mumole/L; GDA, 0.12 mumole/L; and AST, 59.7 IU. Concentrations of GA and GDA in treated guinea pigs were significantly higher than those in controls (P less than 0.05) at doses of aflatoxin greater than or equal to 0.010 mg/day. The highest serum concentrations of bile acids were in guinea pigs given the higher doses of aflatoxin. Activities of AST in treated guinea pigs were significantly higher than those in controls for only 2 dosage levels (0.010 and 0.030 mg/day). Bile acids in the serum of guinea pigs was a more sensitive indicator of liver damage caused by aflatoxin than was AST.

Immunization of swine with heat-stable Escherichia coli enterotoxin coupled to a carrier protein does not protect suckling pigs against an Escherichia coli strain that produces heat-stable enterotoxin.

Pregnant swine were immunized parenterally with purified heat-stable Escherichia coli enterotoxin that was made antigenic by coupling it to bovine immunoglobulin G. Immunized swine had high titers of antitoxin in serum and colostrum as measured by radioimmunoassay. However, the heat-stable enterotoxin neutralizing titers of the serum and colostrum from immunized swine were comparatively low. Newborn pigs suckling their immunized dams were not protected against challenge with porcine enterotoxigenic E. coli that produce heat-stable toxin but do not produce heat-labile toxin.

Plasma alpha-fetoprotein concentrations in pregnant cows exposed to Sarcocystis cruzi, Campylobacter fetus, or Aspergillus fumigatus.

Bovine alpha-fetoprotein (AFP) was determined in maternal plasma, using radioimmunoassay in an attempt to detect and monitor fetal distress in pregnant cows. Plasma from pregnant cows in the 4th to 5th month of the gestation which had been exposed to Sarcocystis cruzi, Campylobacter fetus, or Aspergillus fumigatus was used. Plasma AFP concentrations were determined at intervals from before the cows were exposed until they had aborted or calved. The plasma AFP concentration of the exposed pregnant cattle remained at 6.5 +/- 5.0 mg/ml until 24 to 48 hours before abortion or parturition, when the value increased to 25.0 +/- 8.0 ng/ml. This pattern was similar for cattle exposed to each of the infective agents. Unlike in persons, rats, or monkeys, fetal-maternal transfer of AFP seems to be minimal in cows even with inflammation or necrosis of the placentome, Thus, changes in AFP concentrations in bovine plasma cannot be used as a diagnostic tool for fetal distress or fetal death.

Prepartum changes of plasma concentrations of prostaglandin F and 13,14-dihydro-15-ketoprostaglandin metabolites in pregnant animals exposed to Sarcocystis cruzi or Campylobacter fetus.

Pregnant cows at 4- to 5-months' of gestation were exposed to Sarcocystis cruzi or Campylobacter fetus. Plasma prostaglandin F (PGF) and 13,14-dihydro-15-ketoprostaglandin metabolite (PGM) concentrations were determined at intervals from before exposure until abortion or parturition. The plasma PGF concentration of pregnant infected cattle remained at 0.02 +/- 0.04 ng/ml until 24 to 48 hours before abortion or parturition when it increased 5-fold to 0.11 +/- 0.12 ng/ml. The plasma PGM concentration of these cattle remained at 0.10 +/- 0.07 ng/ml until 24 to 48 hours before abortion or parturition when it increased over 10-fold to 1.36 +/- 0.60 ng/ml. This change in PGF and PGM was similar to that of cattle exposed to each of the infective agents and to that of normal cows at parturition. Thus, changes in PGF and PGM concentrations in bovine plasma cannot be used as a diagnostic tool to determine fetal distress or fetal death for these infections.