Comparison of allergenic skin test with serological and bacteriological examinations of cattle for Listeria monocytogenes.

Five herds, each containing 200 to 300 cows, were surveyed for listeriosis. Since listeriosis is predominantly linked to reproductive diseases in the surveyed area, suspected cows were first selected according to the records on their previous reproductive disorders such as abortion, endometritis and sterility. By simultaneous serological, bacteriological and skin allergenic testing 40 cows suffering from listeriosis were detected. But, only seven of those cows were occasionally shedding listeriae in milk. The specific agglutinins for Listeria monocytogenes were detected in blood sera of all seven cows and the titre was from 1:20 to 1:160. The difference of skin thickness induced by allergenic test was from 1 to 2 mm. L. monocytogenes was isolated from milk of all seven cows and indirectly through the inoculation in enrichment broth, and directly only from milk of two cows. The results indicate that the positive skin allergenic and serological tests do not necessarily mean the shedding of listeriae in milk. However, the listeria-shedding in milk was always accompanied by a positive skin allergenic test and the presence of agglutinins in blood sera.

Effects of dexamethasone on shedding of Listeria monocytogenes in dairy cattle.

Ten lactating Holstein cows that had been given multiple injections of Listeria monocytogenes (serotype 4B, Scott A strain) via the intramammary route were allotted to 2 groups: group 1 (n = 5) was treated with the synthetic glucocorticoid, dexamethasone (0.04 mg/kg of body weight), for 3 consecutive days, and group 2 (n = 5) served as controls. Two days after the initial dexamethasone injection, the number of L monocytogenes in the milk had increased nearly 15-fold (1.16 log10) over pretreatment values. On day 3, Listeria numbers in the milk had increased by 1.83 log10, compared with pretreatment values. By day 4, Listeria numbers in the milk were approximately 100-fold (2.03 log10) greater than pretreatment numbers. Numbers remained high through day 7 and, by day 11, approached pretreatment numbers. Dexamethasone administration was accompanied by high total WBC and milk somatic cell counts and decreased eosinophil and lymphocyte numbers, and decreased milk production. The increase in shedding of L monocytogenes in the milk may reflect impairment of cell-mediated immune mechanisms and phagocytic cell functions that are critical for sustaining listerial immunity.

Antigenic and restriction enzyme analysis of isolates of Campylobacter fetus subsp venerealis recovered from persistently infected cattle.

Thirty-two isolates of Campylobacter fetus subsp venerealis were obtained from 1 bull and 4 heifers with experimentally induced infection. When whole-cell antigens of isolates were cross titrated with antisera to the infecting strain, isolates from 3 heifers had limited antigenic variation, whereas whole-cell antigens of isolates from 2 cattle (the bull and a heifer) differed serologically from those of the infecting strain. Changes were detected specifically in 6 heat-labile antigens. Of the 6 heat-labile factors evaluated, all were initially present on the infecting parent strain, but not on early isolates obtained from 4 of the 5 cattle. Restriction enzyme analysis revealed minor variation in the DNA fingerprints of isolates obtained from individual cattle, thus implying stability of the Campylobacter genome once persistent infection is established. Isolates with identical restriction enzyme patterns expressed different heat-labile antigens. Correlation could not be found between the DNA electrophoretic pattern and the expression of heat-labile antigens.

Vaccination of pregnant guinea pigs with Campylobacter fetus: effects of antigen dose, Campylobacter strain, and adjuvant type.

For the purpose of developing and improving vaccines to protect cattle against bovine genital campylobacteriosis, the interaction of vaccine dose, strain(s) of Campylobacter fetus, and type of adjuvant was studied in the pregnant guinea pig model. Five strains of C fetus were used in the preparation of 20 monovalent vaccines containing various doses of bacteria (0.1 mg to 20 mg, dry weight) in Freund incomplete adjuvant, Freund complete adjuvant, or aluminum hydroxide gel adjuvant. Vaccinal protection against homologous or heterologous challenge exposure with viable C fetus strains was measured in terms of infection, abortion, and serum antibody titers. Major emphasis in the study was placed on vaccines containing highly virulent C fetus subsp venerealis strains (1289 and 17761) that possess most of the heat-labile K antigens that have been identified. Vaccines incorporating strain 1289 provided greater protection against abortion than did strain 17761 vaccines (difference, 29%; P less than 0.05). A positive (P = 0.61) correlation was seen between protection from abortion and strain 1289 serum antibody titer. When the titer was greater than or equal to 4, protection was greater than 80%, whereas abortion was associated with a serum antibody titer less than or equal to 2. In an experiment comparing 2-mg vs 20-mg doses of strain 17761 prepared in aluminum hydroxide gel adjuvant, protection against challenge exposure with the heterologous strain 23 was significantly (P less than 0.05) lower than that against challenge exposure with the homologous strain at the 2-mg dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of an unclassified microaerophilic bacterium associated with gastroenteritis.

Four isolates of an unclassified microaerophilic bacterium resembling Campylobacter species were characterized by growth requirements, microscopic examination, biochemical characteristics, antimicrobial susceptibility tests, and protein profile analysis. The unclassified isolates were differentiated from Campylobacter jejuni, Campylobacter coli, Campylobacter fetus subsp. fetus, Campylobacter laridis, Campylobacter pylori, and an ovine isolate. The bacterium was fusiform shaped with a corrugated surface due to the presence of periplasmic fibers and had multiple bipolar flagella. Biochemically, the bacterium was separated from the Campylobacter controls by its negative catalase reaction, negative nitrate reduction, and no growth in 1% glycine. It was also resistant to ampicillin. Protein profile analysis demonstrated nine major protein bands present in the unclassified isolates that were absent in the Campylobacter controls. The bacterium also differed from the ovine isolate by its negative catalase reaction, rapid urea hydrolysis, and susceptibility to clindamycin, erythromycin, and tetracycline. Our results showed that the unclassified bacterium was distinct from the recognized Campylobacter species.

Pathogenesis of Campylobacter fetus infections: serum resistance associated with high-molecular-weight surface proteins.

Campylobacter fetus subspecies fetus causes both systemic and diarrheal illnesses. We studied 38 strains of C. fetus isolated from 34 patients; underlying illness was present in eight (89%) of nine patients with only systemic isolates compared with three (20%) of 15 patients with only fecal isolates (P = .002). In a standardized assay of susceptibility to normal human serum, 27 (71%) strains were resistant, six (16%) had intermediate susceptibility, and five (13%) were serum sensitive. Major protein bands migrating at 100 kDa or 125 kDa on polyacrylamide gels were present in all of the 25 serum-resistant strains tested but in only four of seven serum-sensitive isolates of C. fetus from humans and animals (P = .007). The presence of these bands was associated with type A lipopolysaccharide. A low-passaged strain, 82-40, was serum resistant and contained the 100-kDa protein; however, a spontaneous mutant of this strain lacked this band and was serum sensitive. The 100-kDa and 125-kDa proteins of three strains of C. fetus were antigenically cross reactive or identical and were exposed on the surface of the C. fetus cell. Serum resistance is inherent to most C. fetus isolates from humans and is associated with the presence of cross-reactive surface proteins.

Experimental infection and abortion of pregnant guinea pigs with a unique spirillum-like bacterium isolated from aborted ovine fetuses.

Study was made of the pathogenicity of a spirillum-like, anaerobic, gram-negative bacterium, originally isolated from aborted lambs, for pregnant guinea pigs. Reproducible conditions for propagation and preservation of the bacterium were determined as requisite for the preparation of cultures for animal inoculation. A preliminary experiment was done with 10 pregnant guinea pigs to test for an infective dose of organisms that would produce abortion. High-passage cultures (n = 50) were used to inoculate these guinea pigs intraperitoneally. Six of 10 guinea pigs aborted, and the organism was cultured from fetal tissues of 5 guinea pigs. Isolates from 3 of the 6 guinea pigs were propagated through 4 passages on blood agar and used to infect 3 groups, each of 5 guinea pigs. A 4th group of 5 guinea pigs was inoculated with the original culture. Three of 5 animals in the first 3 groups, which had been given the low-passage cultures from the preliminary trial, and 2 of 5 guinea pigs in the 4th group, which had been given the original culture, aborted. Antibody against the spirillum was detected in 19 of 30 inoculated guinea pigs. The major microscopic lesions were acute suppurative placentitis and splenitis. This bacterium retained pathogenic properties sufficient to cause infection, abortion, and microscopic lesions in two-thirds of the guinea pigs, in spite of high in vitro passage. The organism has unique ultrastructures, and its genus and species are yet to be determined.

Epidemiologic study of campylobacteriosis in Iowa cattle and the possible role of unpasteurized milk as a vehicle of infection.

Bile samples were collected from 477 Iowa dairy cows and were cultured for thermophilic campylobacters. The prevalence of thermophilic campylobacters in the bile was 15.5%. Campylobacter jejuni and C coli from dairy cattle, chickens, pigs, sheep, and human beings were serotyped to develop host-species profiles. Human and cattle serologic profiles were the most similar, and human and chicken profiles shared several similarities. Epidemiologic data from 168 human cases of campylobacteriosis indicated that 23% of the cases were associated with consumption of unpasteurized milk.

Lipopolysaccharide structures of Campylobacter fetus are related to heat-stable serogroups.

To determine whether lipopolysaccharide (LPS) structures of Campylobacter fetus are related to the three known heat-stable serogroups, proteinase K-treated whole cell lysates obtained from strains of each serogroup were electrophoresed in polyacrylamide gels. All strains had smooth-type LPS with multiple high-molecular-weight repeating units. The profiles of serogroup A from C. fetus subsp. fetus and from C. fetus subsp. venerealis were identical, but they were different from those of C. fetus subsp. fetus serogroups B and AB. When we immunoblotted the LPS of these serogroups with normal or immune rabbit serum we found homologous recognition between serogroups A from C. fetus subsp. fetus and C. fetus subsp. venerealis. Similarly, serogroups AB and B from C. fetus subsp. fetus showed homologous recognition. However, antiserum against serogroup A did not recognize serogroups B and AB and vice versa. Absorption studies confirmed the identity of LPS from all serogroup A C. fetus strains and cross-reactivity of the serogroup B and AB strains with one another. Serogroup A strains were resistant to the bactericidal activity in normal human serum, whereas serogroup B and AB strains generally were susceptible; isolates from humans predominantly belonged to serogroup A. Results of these studies suggest that the LPS composition forms the basis for the heat-stable serotyping system for C. fetus and that the structural and antigenic variants are associated with differential serum susceptibility.

Plasmid content and pathogenicity of Campylobacter jejuni and Campylobacter coli strains in the pregnant guinea pig model.

Strains of Campylobacter jejuni and C coli from human and animal sources were assessed for pathogenicity by their capability to produce abortions in pregnant guinea pigs. The pathogenicity rate varied from 7% to 87% for the C jejuni strains and from 0% to 53% for the C coli strains. Plasmid carriage was not found to correlate with pathogenicity as determined by ability of the strains to produce abortions in the pregnant guinea pig model.

Campylobacter jejuni and Campylobacter coli inoculation of neonatal calves.

Three groups of neonatal calves were inoculated orally with pathogenic strains of Campylobacter jejuni or C coli. The calves developed a mild, self-limiting enteritis characterized by thick mucoid feces. Bacteremia and fecal shedding of Campylobacter were sporadic in all inoculated calves. Two groups of calves were killed 1 to 3 weeks after inoculation to study the pathogenesis of infection. Postmortem culture of tissues revealed C jejuni or C coli most frequently in the ileum, cecum, colon, and blood. Clinical or pathologic differences between C jejuni-inoculated and C coli-inoculated calves were minimal.

Pathogenicity of Campylobacter jejuni and Campylobacter coli strains in the pregnant guinea pig model.

Pathogenicity of 17 Campylobacter isolates for pregnant guinea pigs was investigated. Of 14 isolates, 12 (86%) produced rates of abortion ranging from 13% to 87%. Two isolates did not produce abortion. Reference strains of C fetus subsp venerealis produced abortion in 60% to 87% and C fetus subsp fetus produced abortion in 60% of the guinea pigs. Inoculated organisms were recovered from uterus, blood, liver, kidney, spleen, and gallbladder of the guinea pigs at rates as high as 83% for 2 ovine isolates and as low as 13% for 2 bovine and 1 human isolates. Most isolations were from the uterus. Two avian isolates were not recovered. Within the C jejuni and C coli group, the ovine and the human isolates appear to be more pathogenic. Swine, bovine, and avian isolates were less pathogenic. Seemingly, the pregnant guinea pig was a suitable and practical model for evaluating the pathogenicity of Campylobacter organisms, regardless of their host of origin.

Role of DNA and bacteriophage in Campylobacter auto-agglutination.

Auto-agglutinated and non-agglutinated cells of Campylobacter jejuni and C. coli were examined by transmission electronmicroscopy in phosphotungstate negative stain. Agglutination was induced by three factors (1) extracellular DNA, (2) an aggregated protein, probably a bacteriophage precursor, and (3) free phage-tail sheaths. Auto-agglutinated cells were often "leaky," with a mantle of adhering DNA. About 80% of the auto-agglutinated cells could be resuspended after treatment with DNAase. Flagella were loosely embedded in protein aggregates, especially in phage-infected cultures. They were clumped in a side-by-side arrangement by free phage-tail sheaths. These findings suggest that auto-agglutination could be minimised in suspensions of organisms intended for use in agglutination tests by harvesting early logarithmic-phase cells containing no more than a low phage population. The most common C. jejuni phage had a contractile tail, a head diameter of 60-70 nm, and an overall length of 180-210 nm. A phage isolated from C. jejuni strain 1590 was morphologically identical with C. coli phage.

Isolation of Campylobacter jejuni from an aborted caprine fetus.

Campylobacter jejuni was isolated in pure culture from the tissues and stomach contents of an aborted fetus from a mixed-breed Illinois goat herd. In the herd, 5 of 21 does aborted in late gestation. The does had diarrhea before or concurrent with the abortions. Does that did not abort were vaccinated with ovine C fetus bacterin and were given chlortetracycline orally at the rate of 75 mg/day for 2 weeks. Further abortions did not occur.

Intrauterine inoculation of Mycobacterium paratuberculosis into guinea pigs and cattle.

Mycobacterium paratuberculosis was inoculated intrauterinely into guinea pigs and cattle near the time of insemination to assess the effect and subsequent distribution of the organisms. Guinea pigs inoculated intrauterinely with 1 of 3 concentrations of M paratuberculosis or not inoculated were caged with male guinea pigs for 10 days. Guinea pigs given the largest dose had the lowest rate of pregnancy when examined. At necropsy, tissues were cultured to determine extent of infection. Abortion followed hypersensitivity reactions to johnin in some highly infected animals. Mycobacterium paratuberculosis was recovered in culture from the body and horns of the uterus of cows necropsied 1, 2, 3, and 7 days after inoculation and from 1 of 3 cows necropsied 2 weeks after inoculation. The organisms were not detected in extrauterine organs of any cattle or in uterine specimens taken at 3 or 4 weeks after inoculation. The evidence indicates that the small numbers of M paratuberculosis sometimes found in semen from paratuberculous bulls probably would be destroyed, rather than leading to systemic infection of the dam or to persistent hypersensitivity.

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.

Plasma progesterone concentration in the bovine before abortion or parturition in pregnant animals exposed to Sarcocystis cruzi, Campylobacter fetus, or Aspergillus fumigatus.

Pregnant cows in the 4th and 5th month of the gestation were exposed to Sarcocystis cruzi, Campylobacter fetus, or Aspergillus fumigatus. Plasma progesterone concentrations were determined at intervals from before the cows were exposed until they had aborted or calved. The plasma progesterone concentration of the exposed pregnant cattle remained at 3.95 +/- 2.0 ng/ml until 24 to 48 hours before abortion or parturition, when it decreased to below 1.00 ng/ml. This pattern was similar for cattle exposed to each of the infective agents. Thus, changes in progesterone concentrations in bovine plasma cannot be used as a diagnostic tool for fetal distress or fetal death.

Campylobacter fetus infection in human subjects: association with raw milk.

Within a three week period, four cases of Campylobacter fetus infection were identified in a hospital in Los Angeles County. Three of the four patients had drunk large quantities of an identical brand of commercially available certified raw milk. Campylobacter fetus subspecies jejuni was isolated from the blood of these three patients. The fourth patient, who did not drink raw milk, had Campylobacter fetus subspecies intestinalis isolated from his blood. A telephone survey comparing cases and controls for possible risk factors associated with Campylobacter fetus infection confirmed the association with raw milk (P = 0.003). In a serologic survey, evidence suggested that Campylobacter fetus infection was associated with raw milk consumption.