Species-specific DNA probes for Campylobacter species isolated from pigs with proliferative enteritis

Cloned, chromosomal DNA probes from porcine isolates of Campylobacter hyointestinalis and C. mucosalis were developed for the detection and identification of these putative swine enteric pathogens. High molecular weight chromosomal DNA from each species was used to construct genomic libraries in plasmids. Recombinants were selected which hybridized strongly to the homologous organism, but not to any other species of Campylobacter. Species-specific recombinants were labeled with phosphorus-32 and tested for sensitivity by dot blot hybridization to various dilutions of DNA and bacteria from each swine species, including C. hyointestinalis, C. mucosalis, C. coli and C. jejuni. Specificity was tested by hybridizing these probes against various strains of C. hyointestinalis or C. mucosalis, and against reference strains of all other described Campylobacter species. A C. hyointestinalis-specific probe and a C. mucosalis-specific probe were identified which were capable of detecting 1 ng of DNA or 10(4) cfu by bacterial spot blotting on nylon membranes. These probes hybridized to intestinal mucosal scrapings containing C. hyointestinalis and C. mucosalis obtained from pigs with proliferative enteritis, but not to material from normal pigs. Thus, cloned, chromosomal DNA probes may be useful in the detection and identification of bacteria involved in swine proliferative enteritis.     

Plasmid profiles of six species of Campylobacter from human beings, swine, and sheep

Twenty-four isolates representing 6 species of Campylobacter were screened for plasmids. A large plasmid with an approximate molecular weight of 38 Mdal was detected in 5 C jejuni isolates originally recovered from diarrheic human beings, in one isolate of C coli recovered from diarrheic pigs, and in 1 isolate of C sputorum ssp mucosalis and 2 isolates of C hyointestinalis recovered from pigs with proliferative enteritis. One isolate of C coli and 1 isolate of C hyointestinalis contained an additional smaller plasmid with an approximate molecular weight of 1.6 Mdal; this plasmid was partially mapped by restriction endonuclease digestion. Fifteen Campylobacter isolates contained no detectable plasmids: 2 C coli, 2 C sputorum ssp mucosalis, 2 C fecalis, 1 C fetus ssp fetus, and 8 C hyointestinalis isolates. In summary, 37.5% of the Campylobacter isolates contained a 38-Mdal plasmid, with 8% having both 38 Mdal and 1.6-Mdal plasmids; 62.5% contained no detectable plasmids.     

Structural proteins of porcine enteroviruses

Analysis of the structural proteins of two strains (T80 and PE1) of cytopathogenic type 1 and two strains (V13 and T5) of cytopathogenic type II porcine enteroviruses by polyacrylamide gel electrophoresis, revealed polypeptide patterns which were generally similar to those described for other picornaviruses. However, one polypeptide (P5), of molecular weight 15 000 to 17 000, which was found in each strain of porcine enterovirus, has not been reported for other enteroviruses. A polypeptide (P4), of molecular weight 22 000, demonstrated in the type I strains, was lacking in the type II strains, and the molecular weight of the P2 polypeptide was lower in the type II strains than in the type I porcine enteroviruses.     

Cloning and characterization of a Moraxella bovis cytotoxin gene

OBJECTIVE: To identify the Moraxella bovis cytotoxin gene. PROCEDURE: Hemolytic and nonhemolytic strains of M. bovis were compared by use of western blotting to identify proteins unique to hemolytic strains. Oligonucleotide primers, designed on the basis of amino acid sequences of 2 tryptic peptides derived from 1 such protein and conserved regions of the C and B genes from members of the repeats in the structural toxin (RTX) family of bacterial toxins, were used to amplify cytotoxin-specific genes from M. bovis genomic DNA. Recombinant proteins were expressed, and antisera against these proteins were produced in rabbits. RESULTS: Several proteins ranging in molecular mass from 55 to 75 kd were unique to the hemolytic strain. An open reading frame encoding a 927-amino acid protein with a predicted molecular mass of 98.8 kd was amplified from M. bovis genomic DNA. The deduced amino acid sequence encoded by this open reading frame was homologous to RTX toxins. Antisera against the recombinant carboxy terminus encoded by this open reading frame neutralized hemolytic and cytolytic activities of native M. bovis cytotoxin. CONCLUSIONS AND CLINICAL RELEVANCE: A gene was identified in M bovis that encodes a protein with sequence homology to other RTX toxins. Results of cytotoxin neutralization assays support the hypothesis that M. bovis cytotoxin is encoded by this gene and belongs in the RTX family of bacterial exoproteins. Identification of this gene and expression of recombinant cytotoxin could facilitate the development of improved vaccines against infectious bovine keratoconjunctivitis.     

Isolation of Campylobacter species from zoo animals and polymerase chain reaction-based random amplified polymorphism DNA analysis

A total of 104 fecal specimens from 30 mammals, 12 birds, and 3 reptiles at the Phoenix Zoological Gardens, Miyazaki City, Japan, were examined for the presence of Campylobacter species. All the animals examined were healthy with no fecal abnormality. Twenty-three (22.1%) thermophilic campylobacters, (9 C. jejuni, 11 C. hyointestinalis, 2 C. coli, and 1 C. lari), were isolated from 11 animals (7 mammals and 4 birds). C. jejuni and C. hyointestinalis were the predominant species isolated from these zoo animals and C. hyointestinalis was isolated frequently from simians. As selective media influence the numbers and species of campylobacters isolated, the agar medium was not supplemented with cephalothin. Campylobacters were isolated most frequently when a combination of enrichment culture and selective agar plating was performed at 42 degrees C. For the epidemiological study, a polymerase chain reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) method was used as a tool to detect the heterogeneity of amplified DNAs of Campylobacter spp. isolated from zoo animals. The two arbitrary primers used in this study enabled even closely related strains of the same Campylobacter spp. to be differentiated. RAPD analysis revealed considerable diversity among the strains, suggesting that the transmission of Campylobacter spp. among animals in a defined area occurred through different mechanisms.Examination of the genotypic diversity among the multiple clones from the same host also revealed differences between clones. These results demonstrate that campylobacter populations in zoo animals are highly divergent.     

Oocysts, IgG levels and immunoblot patterns determined for Cryptosporidium parvum in bovine examined during a visit to a farm (northeastern Spain)

Single fecal and serum samples were individually collected from 101 bovines selected at random during a visit to a farm in northeastern Spain (Group I, 26 animals aged 2-36 days; Group II, 34 animals aged 1.5-4.5 months; Group III, 41 animals aged 20-24 months). Testing for the presence of Cryptosporidium parvum oocysts in feces (Monofluo Kit Cryptosporidium, Diagnostics Pasteur, France) indicated that 26% animals were infected (81% of Group I, 15% of Group II and 0% of Group III). Serological testing (ELISA for detection of specific anti-C. parvum IgG) indicated that 59% animals were seropositive (12% of Group I, 74% of Group II and 78% of Group III). Immunoblotting results indicate that cattle sera recognize C. parvum antigens of widely varying molecular weights and that the number of antigens recognized increases with age. Immunoblots revealed that some of the sera belonging to the Group I reacted with protein fractions between 15 and 20 kDa but none recognized the 21-23 kDa antigen. Only few sera in the Group II recognized the protein fraction between 15 and 20 kDa. The recognition of 21-23 kDa fraction was observed by four sera from uninfected and seropositive animals. Sera from all the seronegative Group II animals recognized few antigens and always with molecular weight greater than 50 kDa. Serum samples from both seropositive and seronegative animals belonging to the Group III recognized antigens with molecular weight ranging 15-20 kDa. Surprisingly, the protein fractions between 21 and 28 kDa reacted with approximately 30% of the sera from seropositive animals and only one of the nine sera from seronegative animals. The recognition of 42-46 kDa antigens increased with the age and only reacted with the sera from uninfected animals.     

Virulence and lienotoxicity of Bordetella bronchiseptica in mice

Whole-cell suspensions (WCSs) and cell-free sonicated extracts (SEs) of seven Bordetella bronchiseptica strains were studied for lethality and lienotoxicity in mice. Lethality was assessed after intravenous and intracerebral inoculation, and lienotoxicity by splenic atrophy after intravenous inoculation. The strains represented phase I isolates with or without cytotoxin production, their phase III subcultures and a phase IV variant. The lethality and lienotoxicity of the SEs were in close positive correlation with cytotoxin production. The WCSs of all phase I strains were lethal, irrespective of their cytotoxin- and lienotoxin-producing ability. The only difference was that cytotoxic phase I strains caused splenic atrophy while the noncytotoxic phase I strain induced splenic hypertrophy in the surviving mice. The WCSs of phase III and IV variants were non-lethal and caused splenic hypertrophy even though all but one of them showed some cyto- and lienotoxic activity when their SEs were tested. The results indicate that B. bronchiseptica possesses two different mouse lethal factors: one seems to be identical with the cytotoxin, the other is associated with cell integrity and viability and, presumably, propagation in vivo. It also follows from the results that only the SEs are suitable for accurate determination of the lienotoxin-producing ability of B. bronchiseptica.     

Lectin agglutination of thermophilic Campylobacter species

Agglutination tests with lectins indicated differences in the surface composition of strains of the thermophilic (optimum temperature 42 degrees C) Campylobacter species C. coli, C. faecalis, C. hyointestinalis, C. jejuni and C. laridis. All strains examined were agglutinated by the protein-reactive agglutinins of Mangifera indica (mango) and Persea americana (avocado) and a large proportion was also agglutinated by the carbohydrate-reactive lectins of Canavalia ensiformis (Jack bean) and Triticum vulgaris (wheat germ). Reactions with other lectins varied widely between strains, even of the same species and serotype. Lectin agglutination may be useful as a supplementary procedure for characterizing individual Campylobacter isolates for epidemiological purposes.     

Porcine proliferative enteritis: serological, microbiological and pathological studies from three field epizootics.

Three outbreaks of porcine proliferative enteritis were evaluated clinically, pathologically, microbiologically and serologically. The disease was characterized by a chronic intermittent diarrhea. Pathological lesions included a thickened, turbid ileum with the microscopic appearance of proliferating ileal crypt epithelial cells. Comma shaped intracytoplasmic organisms were observed in the apical portions of the proliferating crypt epithelial cells with a Warthin-Starry silver stain. Microbiologically, both Campylobacter sputorum subspecies mucosalis and Campylobacter hyointestinalis, were cultured from ileal specimens of seven pigs with lesions of porcine proliferative enteritis. Microagglutination antibody titers were determined on sera from 12 of 14 pigs with porcine proliferative enteritis and on sera from 91 clinically normal swine. Pigs with porcine proliferative enteritis had a low antibody titer to subspecies mucosalis that ranged from 1-3 with a mean of 2.17. A varied C. hyointestinalis titer from 3-7 with mean of 4.83 was determined. Titers to either subspecies mucosalis and C. hyointestinalis were higher in non-porcine proliferative enteritis pigs. The results indicate that the presence of a positive titer to either C. hyointestinalis or subspecies mucosalis in swine is not indicative of clinical disease. The isolation of C. hyointestinalis from diseased ileal specimens (porcine proliferative enteritis) confirms previous reports implicating this agent in the disease.     

Vacuolating cytotoxin produced by avian pathogenic Escherichia coli

The purpose of this study was to determine whether avian pathogenic Escherichia coli produced cytotoxic activity. Culture supernatants of 20 E. coli strains isolated from cellulitis lesions in chickens, five E. coli strains from avian septicemia, five from swollen head syndrome, and five from the feces of healthy chickens were incubated with primary chicken embryo fibroblast (CEF) cells, primary chicken kidney (PCK) cells, a quail fibroblast cell line (QT-35), and four mammalian cell lines (human epithelioid cervical carcinoma, African green monkey kidney, Chinese hamster ovary, and human larynx epidermoid carcinoma). Cytotoxicity was observed with supernatants from the 30 avian pathogenic strains on the two primary chicken cells (CEF and PCK). The highest dilution of culture supenatant that induced cytotoxic changes in 50% of the cells was 1/64. Supernatants from the five strains from normal feces were noncytotoxic, and none of the supernatants was cytotoxic for the QT-35 or the four mammalian cell lines. The cytotoxic effect, which was observed as early as 2 hr after exposure of the cells, was maximal at 6 hr and was evident as vacuolation, morphologically indistinguishable from that previously reported for culture supernatants of Helicobacter pylori. Like the activity in H. pylori, the cytotoxicity of the avian pathogenic strains was destroyed by heating at 70 C for 30 min and by exposure to proteolytic enzymes and was retained by filtration with a 100,000 molecular weight cut-off ultrafilter. Supernatants of two vacuolating cytotoxin-positive cultures of H. pylori failed to induce vacuolation of the CEF and PCK cells but caused the characteristic vacuolation in HeLa and Vero cells. The observations suggest that avian pathogenic E. coli produce a cytotoxin that is similar to the cytotoxin of H. pylori but may be specific for avian cells.     

Cytotoxin from an ovine strain of Pasteurella haemolytica: characterisation studies and partial purification

A cell-free, water-soluble cytotoxin from an ovine strain of Pasteurella haemolytica biotype A serotype 1 killed sheep bronchoalveolar macrophages at 37 degrees C, but not at 4 degrees C or 22 degrees C. The cytotoxin was stable over the pH range 2-12, resistant to heat at 60 degrees C but inactivated at 100 degrees C or by autoclaving. Trypsin also destroyed the cytotoxin, which is therefore thought to contain a protein component essential for biological activity. A preliminary purification of the crude cytotoxin using gel-filtration column chromatography resulted in the isolation of a biologically active fraction which resolved as a single protein band and one carbohydrate band on non-dissociating polyacrylamide gels. However, this fraction resolved into approximately 16 component bands on a sodium dodecyl sulphate polyacrylamide gel.     

Detection and identification of food-borne pathogens of the genera Campylobacter, Arcobacter and Helicobacter by multiplex PCR in poultry and poultry products

The objective of this study was to develop a multiplex polymerase chain reaction (PCR) to detect and differentiate food-borne pathogens of the three genera Campylobacter, Arcobacter and Helicobacter in a single step procedure. One common reverse primer and three genus-specific forward primers were designed by hybridizing to the 16S rRNA of selected reference strains. Besides the species with significance as food-borne pathogens isolated from poultry meat--Campylobacter jejuni, Campylobacter coli, Arcobacter butzleri and Helicobacter pullorum--several other members of these genera were tested to determine the specificity of the designed multiplex PCR. In total, 20 ATCC and NCTC reference strains of Campyobacter, Arcobacter and Helicobacter were used to evaluate the PCR. Specific amplificates were obtained from all thermophilic species of Campylobacter as well as from species of Arcobacter and Helicobacter. No amplification product was obtained from the non-thermophilic Campylobacter, C. hyointestinalis and C. fetus. Furthermore, a total of 43 field strains of the three genera isolated from poultry, pigs, cattle and humans were investigated using this PCR. To confirm the classification of 10 H. pullorum strains the 16S rRNAs were sequenced. The developed PCR is a helpful diagnostic tool to detect and differentiate Campylobacter, Arcobacter and Helicobacter isolated from poultry and poultry products.     

Characterization of Campylobacter lanienae from pig feces

To isolate Campylobacter spp., the feces of healthy cattle, pigs, and broilers were examined between June 1999 and January 2000. Campylobacter lanienae strains were isolated from the feces of healthy pigs, but not from the feces of cattle or broilers. In six C. lanienae isolates, there was only 21-38% DNA-DNA homology to Campylobacter hyointestinalis subsp. lawsonii strain NCTC 12901. Thus, the primary host of C. lanienae is likely to be the pig and C. lanienae appears to be a species distinct from C. hyointestinalis subsp. lawsonii. In addition, an intervening sequence of 226 bp in the 16S rRNA gene was found in four isolates.     

The pathogenesis of turbinate atrophy in pigs caused by Bordetella bronchiseptica

The pathogenicity of 3 strains of Bordetella bronchiseptica designated B58, PV6 and B65 was compared by intranasal infection of gnotobiotic piglets. Strain B58 was a phase 1 isolate that produced haemolysin, an adhesin for calf erythrocytes, adenylate cyclase, mouse lethal factor, dermonecrotic factor and cytotoxin. B65 was a variant of B58 that produced no detectable haemolysin, adhesin or adenylate cyclase and 10-fold smaller amounts than B58 of mouse lethal factor, dermonecrotic factor and cytotoxin. Strain PV6 was a phase 1 isolate that produced only haemolysin, adhesin and adenylate cyclase. After nasal infection of gnotobiotic pigs, 10(3.2)-10(6.2) colony forming units ml-1 (cfu ml-1) of strains B58 and PV6 were cultured from nasal washings during the next 25 days. In contrast, only 10(1.0)-10(2.8) cfu ml-1 of strain B65 were recovered during the same period. Only pigs infected with strain B58 had turbinate atrophy when they were slaughtered 25 days after infection and neutralising antibody to cytotoxin was detected only in these pigs. These results suggested that the cytotoxin, which may be the same as the mouse lethal and dermonecrotic factors, was the cause of turbinate atrophy. They also support the view that the adhesin for calf erythrocytes is required for colonisation of the nasal cavity in vivo.     

Effects of challenge with a virulent genotype II strain of porcine reproductive and respiratory syndrome virus on piglets vaccinated with an attenuated genotype I strain vaccine

Porcine reproductive and respiratory syndrome virus (PRRSV) is endemic in most parts of Asia, where genotype I and II strains of diverse virulence may coexist. This study evaluated the outcome of infection with a highly virulent Asian genotype II PRRSV isolate in piglets vaccinated with a genotype I vaccine. Twenty-one 3-week-old piglets were divided in three groups: Pigs in group V (n=8) were vaccinated with an attenuated genotype I commercial PRRSV vaccine, while pigs in group U (n=8) and a control group (group C; n=5) were unvaccinated; 6 weeks later, pigs in groups V and U were challenged intranasally with a highly virulent strain of genotype II PRRSV (1×10(5) 50% tissue culture infectious doses/mL), while pigs in group C received a placebo. Over a period of 21 days after challenge, vaccinated pigs had significantly lower mortality (0/8 versus 2/8), fewer days of fever, a lower frequency of catarrhal bronchopneumonia, higher weight gains (13.4 versus 6.6 kg) and lower levels of viraemia compared to unvaccinated challenged pigs. Immunisation with a genotype I attenuated PRRSV vaccine provided partial protection against challenge with a highly virulent genotype II strain.     

Bovine diarrhea associated with Campylobacter hyointestinalis

An enteric disease affected 16 ruminating calves. The disease was characterized by a nonspecific, mild to severe diarrhea and wasting. Two calves died during the course of disease. C. hyointestinalis was isolated from 12 to 14 calves. The antibody titers of affected calves to C. hyointestinalis varied from 1:20 to 1:160. The disease was successfully treated with chloramphenicol.     

Bulk growth procedures and a button agglutination test for Campylobacter

Bulk-cell yields were obtained from 4 Campylobacter spp incubated aerobically without the use of a special atmosphere. A button agglutination test was developed for quantitation of blood serum antibodies against C fetus subsp venerealis, C fetus subsp fetus, C jejuni, and "C hyointestinalis." The test was sensitive, and different individuals reading it usually attained the same titers. Cells of C fetus subsp venerealis, C fetus subsp fetus, and "C hyointestinalis" grown aerobically in broth made satisfactory antigens for the button test, but some cell pools of C jejuni had a tendency to autoagglutinate. Cells of C jejuni grown on blood agar had less tendency to autoagglutinate.     

Comparative study of colonizing and noncolonizing Campylobacter jejuni

Campylobacter jejuni A74/O and A74/C are congenic strains. An oral dose of 10(5) organisms of strain A74/C colonizes chicken intestines. Strain A74/O, from which A74/C is derived, does not colonize the chicken intestines with an oral dose of 10(5) organisms. In this study, the congenic bacteria were compared to identify possible colonization mechanisms. Differences were not observed in plasmid content or by HindIII, Pst I, Acc I, HincII, Ava I, Ava II, Xba I, and BamHI restriction enzyme digestion of total DNA. Transmission electron microscopy of negatively stained samples revealed no differences between the strains. Sections of cecal tissue from nonfed day-of-hatch chicks were cultured with each strain for 2 hours and then examined by light and electron microscopy. Both strains caused necrosis of villus epithelial cells. Immunofluorescent or silver staining revealed strain A74/C located deep in numerous epithelial crypts, but strain A74/O only was present in one sample mixed with sloughed necrotic cells. Similarly, organisms were detected by transmission electron microscopy deep in crypts in tissues cultured with A74/C, but not A74/O. Cells of A74/C detected in crypts did not appear to associate with epithelial cells. The strains did not differ in chemotactic behavior to mucin or fucose.     

Transport and hindlimb exchange of peptide and serum protein amino acids in calves fed soy- or urea-based purified diets

Holstein steer calves (130 kg) were used to monitor venoarterial concentration differences of plasma peptide and serum protein amino acids across the hindlimbs. Calves were fed purified diets containing soy protein or urea as the sole source of dietary N. Twenty-four hourly feedings per day were imposed to promote "steady-state" metabolism. Negative venoarterial differences of glutamic acid (P less than .10), valine (P less than .10), lysine (P less than .05) and histidine (P less than .01) were observed in soy-fed calves and isoleucine (P less than .10) in urea-fed calves from the peptide fraction. Negative venoarterial differences of amino acids from the peptide fraction were generally similar for the two dietary treatments, except for glutamic acid (P less than .01). A large negative venoarterial difference of glutamic acid was observed when soy protein was fed and, conversely, a large positive venoarterial difference was observed when urea was fed. Two groups of serum proteins were evaluated: fraction I (primarily globulins) and fraction II (primarily albumin). Hindlimb amino acid venoarterial concentration differences for soy-fed calves were inconsistent and nonsignificant for both fractions I and II. Conversely, hindlimb negative venoarterial concentration differences in urea-fed calves for both fractions I and II were large and statistically significant for many amino acids. Fraction II venoarterial differences were much greater than differences observed in fraction I. Soy and urea treatment differences were also much more pronounced in fraction II, being statistically significant for 8 of 17 amino acids. These data indicate venoarterial concentration differences of serum protein amino acids that react differently to varied nutritional regimens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enteric campylobacter infection in gnotobiotic calves and lambs

Gnotobiotic calves and lambs were infected orally with Campylobacter jejuni, C coli or C hyointestinalis to assess pathogenicity. All animals were successfully colonised and excreted mucoid faeces but showed no other clinical signs. Campylobacters colonised the large intestine better than the small intestine, in which bacterial numbers decreased with time after infection. Campylobacters were found occasionally in the lumen of crypts in close proximity to epithelial cells and included in a mucus-like material. Lesions were mostly in the large intestine in calves whereas in lambs they were present in the ileum. In animals inoculated with C jejuni or C coli scattered crypt abscesses, focal inflammatory infiltrates in the lamina propria and goblet cell discharge were found. In lambs inoculated with C hyointestinalis only minor changes were found in the small intestine. Serum antibody response was either absent or present at a low level only from the 19th day after infection.