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.

Splenic and portal venous obstruction in chronic pancreatitis. A prospective longitudinal study of a medical-surgical series of 266 patients.

The aim of this study was a prospective search for splenoportal venous obstruction (SPVO) in a medical-surgical series of 266 patients with chronic pancreatitis who were followed up a mean time of 8.2 years. SPVO was systematically searched for using ultrasonography and then confirmed by angiography or computed tomography. SPVO was found in 35 patients (13.2%) but was symptomatic in only two. Initial obstruction involved the splenic vein in 22 patients, the portal vein in 10, and the superior mesenteric vein in three. Since venous obstruction extended from the splenic to the portal vein in five patients, the prevalence of portal obstruction was 5.6% (15/266). Acute pancreatitis and pseudocysts were the probable cause of SPVO in 91.4% of our cases. Half the cases of splenic venous obstruction were related to pseudocysts of the caudal pancreas. Esophageal varices were found in two patients and gastric varices in four at the time of diagnosis and during follow-up. At the end of follow-up, 12 patients had undergone splenopancreatectomy (N = 11) or splenectomy (N = 1). Only one patient was operated on for massive esophageal variceal bleeding, and another patient died due to intractable colic variceal bleeding. In four of six patients operated on with portal vein obstruction, surgery was difficult due to venous collaterals. Ten patients were not operated on and 13 patients operated on were not treated for SPVO. The mean follow-up after diagnosis of SPVO for these final 23 patients was 28.9 months. None of these patients bled.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Danazol induced pancreatitis and hepatitis.

Danazol, a C17 alkylated anabolic steroid, has been tried as a hormonomodulator in the management of systemic lupus erythematosus. We report the case of a patient receiving 400 mg of danazol per day who developed mild pancreatitis associated with hepatitis, both induced by danazol.

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.

Mortality factors associated with chronic pancreatitis. Unidimensional and multidimensional analysis of a medical-surgical series of 240 patients.

The purpose of the study was to determine (a) the frequency and cause of mortality in patients with chronic pancreatitis; (b) the cumulative survival rates corrected by comparison of patients with a matched French population; and (c) the factors associated with mortality by a unidimensional and multidimensional analysis. The study population consisted of 240 patients (men = 208, women = 32; alcoholic = 210, nonalcoholic = 30) followed for a mean time of 8.7 yr. The status of the patients (dead or alive) was recorded in February 1987. Mean age at onset of chronic pancreatitis was 41.5 yr. Fifty-seven patients died. Mean age at time of death was 52.3 yr. "Overmortality" after 20 yr of course was 35.8% in comparison with a matched French population (p less than 0.0001). Chronic pancreatitis was the direct cause of death for only 19.3% of patients. The main causes of death have been alcoholic hepatopathy (n = 10), cancer (n = 9), postoperative mortality (n = 8). Unidimensional analysis of mortality rates showed that male sex (p less than 0.03), surgery (p less than 0.007), hepatopathy (p less than 0.01), diabetes mellitus (p less than 0.02), and absence of attack of acute pancreatitis (p less than 0.02) were associated with mortality. Multidimensional analysis showed that the following variables were linked with mortality: in a first model including the totality of the study population: surgery (p less than 0.006), hepatopathy (p less than 0.008), no attack of acute pancreatitis (p less than 0.03), male sex (p less than 0.03); in a second model excluding cirrhosis: surgery (p less than 0.001), male sex (p less than 0.06), diabetes mellitus (p less than 0.09). Nevertheless, surgery did not seem to interfere with long-term mortality. The lower mortality of patients with attacks of acute pancreatitis suggests a favorable influence for alcohol abstinence.