Indirect enzyme-linked immunosorbent assay for detection of Brucella melitensis-specific antibodies in goat milk.

Brucella melitensis is the cause of brucellosis in sheep and goats, which often results in abortion. Few cases of B. melitensis infection in goats have occurred in the United States over the last 25 years. However, vigilance must be maintained, as it is for the bovine milk industry, to ensure that brucellosis is not introduced into the U.S. goat population. The objective of this study was to develop a sensitive and specific indirect enzyme-linked immunosorbent assay (iELISA) for the detection of B. melitensis-specific antibodies in goat milk. Brucella salt-extractable protein extract was employed as an antigen, and a horseradish peroxidase-labeled polyclonal anti-goat antibody was used as an anti-species conjugate. Thirteen of 13 (100%) individual infected goat milk samples tested positive and 134 of 134 (100%) uninfected bulk milk samples tested negative by the developed iELISA. Three positive milk samples with high, medium, and low absorbance values were used to simulate one positive animal in an otherwise negative herd. By this estimation, one high-titer animal could be detected in a herd of >1,600 animals. Detection estimates for medium- and low-titer animals were one positive animal per herd of <200 and 50 animals, respectively. Based on this estimation, it is recommended that herds be sampled in groups of 50 animals or less for bulk milk testing. The iELISA developed for this study was found to be sensitive and specific and shows potential for use as a bulk milk test for the detection of B. melitensis-specific antibodies in goat milk.

Erysipelothrix rhusiopathiae: genetic characterization of midwest US isolates and live commercial vaccines using pulsed-field gel electrophoresis.

This is the first report of molecular characterization of US erysipelas field isolates and vaccine strains of Erysipelothrix rhusiopathiae by pulsed-field gel electrophoresis (PFGE). Erysipelas in pigs is mainly caused by E. rhusiopathiae serotypes 1a, 1b, and 2. In 2001, erysipelas reemerged as a clinical problem in pigs in the midwestern United States. In this work 90 erysipelas isolates (58 recent and 28 archived field isolates as well as 4 live-vaccine strains) were genetically characterized. Because of the limited availability of antiserum, 74/90 isolates (44/58 recent isolates) were serotyped. The serotype of the majority (79.6%) of the 44 recent isolates tested was determined to be 1a, 13.6% were serotype 1b, and 6.8% of recent isolates were serologically untypeable. Among all 90 isolates, 23 different PFGE patterns were identified. There were 43 isolates identified as serotype 1a with 4 genetic patterns: 38/43, 1A(I); 3/43, 1A(III); 1/43, 1B(V); and 1/43, 3B. Sixteen serotype 1b isolates had 11 unique genetic patterns: 4/16 were genotype 1B(III), 2/16 were genotype 3A(I), and 1/16 was in genotype groups 1A(V), 1A(VI), 1A(VII), 1B(I), 1B(IV), 1B(VII), 2, 4, and 5. Six genetic patterns were distinguished among the 10 serotype 2 isolates: 1A(IV) (1/10), 1A(V) (1/10), 1B(VI) (1/10), 2 (4/10), 7 (1/10), and 8 (2/8). Erysipelas vaccine strains (modified live) were similar to each other but different from current field strains, sharing 78.6% identity with the most prevalent genotype 1A(I) based on the PFGE-SmaI pattern. Compared with serotyping, PFGE genotyping is a more distinguishing technique, easy to perform and not dependent on the limited availability of antiserum.

Porcine circovirus type 2 (PCV-2) coinfections in US field cases of postweaning multisystemic wasting syndrome (PMWS).

The prevalence of different pathogens detected in combination with porcine circovirus type 2 (PCV-2) was studied retrospectively in field cases of postweaning multisystemic wasting syndrome (PMWS) diagnosed at the Iowa State University Veterinary Diagnostic Laboratory, Ames, Iowa, between January 2000, and September 2001. The presence of PCV-2 antigen in lymphoid tissues and/or lung, demonstrated by immunohistochemistry, together with moderate to severe lymphoid depletion and/or granulomatous lymphadenitis, was used as the criteria for the diagnosis of PMWS. A total of 484 cases fulfilled these criteria. Most of the cases (294/369) of PMWS occurred in pigs between the ages of 8 and 18 weeks, with a peak at 10 weeks of age. Porcine reproductive and respiratory syndrome virus was detected in 51.9% of the cases, Mycoplasma hyopneumoniae in 35.5%, bacterial septicemia in 14.0%, bacterial pneumonia in 7.6%, swine influenza virus in 5.4%, and PCV-2 alone in 1.9%. In cases with bacterial septicemia the most frequently isolated pathogen was Streptococcus suis. In cases with bacterial pneumonia, Pasteurella multocida was the most prevalent.

Evidence for transfer of CMY-2 AmpC beta-lactamase plasmids between Escherichia coli and Salmonella isolates from food animals and humans.

Escherichia coli is an important pathogen that shows increasing antimicrobial resistance in isolates from both animals and humans. Our laboratory recently described Salmonella isolates from food animals and humans that expressed an identical plasmid-mediated, AmpC-like beta-lactamase, CMY-2. In the present study, 59 of 377 E. coli isolates from cattle and swine (15.6%) and 6 of 1,017 (0.6%) isolates of human E. coli from the same geographic region were resistant to both cephamycins and extended-spectrum cephalosporins. An ampC gene could be amplified with CMY-2 primers in 94.8% of animal and 33% of human isolates. Molecular epidemiological studies of chromosomal DNA revealed little clonal relatedness among the animal and human E. coli isolates harboring the CMY-2 gene. The ampC genes from 10 animal and human E. coli isolates were sequenced, and all carried an identical CMY-2 gene. Additionally, all were able to transfer a plasmid containing the CMY-2 gene to a laboratory strain of E. coli. CMY-2 plasmids demonstrated two different plasmid patterns that each showed strong similarities to previously described Salmonella CMY-2 plasmids. Additionally, Southern blot analyses using a CMY-2 probe demonstrated conserved fragments among many of the CMY-2 plasmids identified in Salmonella and E. coli isolates from food animals and humans. These data demonstrate that common plasmids have been transferred between animal-associated Salmonella and E. coli, and identical CMY-2 genes carried by similar plasmids have been identified in humans, suggesting that the CMY-2 plasmid has undergone transfer between different bacterial species and may have been transmitted between food animals and humans.

Effects of heat stress on the antimicrobial drug resistance of Escherichia coli of the intestinal flora of swine.

The effects of heat stress on the antimicrobial drug resistance of Escherichia coli of the intestinal tract of swine were studied in animals from a farm that had not been supplementing antimicrobials in feed for the past 10 years. In one study, 10 finisher hogs were heat stressed (34 degrees C) for 24 h. Antimicrobial resistance levels after stress were significantly higher (P < 0.05) when compared with pre-stress levels for amikacin, ampicillin, cephalothin, neomycin and tetracycline from faecal samples. This high level of resistance persisted to slaughter that occurred at 10 days post-stress for most of the antimicrobials mentioned. In a second study, samples of different sections of the gastrointestinal tract were collected after heat stress and compared with control, non-stressed animals. Results indicated that E. coli which colonized the ileum and caecum had a higher level of resistance to ampicillin and tetracycline than the E. coli which colonized the colon and rectum. When animals were exposed to heat stress, resistance to ampicillin and tetracycline of E. coli in the lower digestive tract increased (P < 0.05) to a level similar to that observed in the ileum and caecum. Based on these findings, an investigation was made to test the hypothesis that (a) an increase in intestinal motility increases shedding of resistant E. coli and (b) heat stress induces a reduction in intestinal transit time in swine. For each study, two groups of three, randomly selected finisher hogs each were formed (treated and control groups). In study (a), induction of increased motility and peristalsis was obtained using an intramuscular injection of the cholinergic drug neostigmine methylsulphate. Escherichia coli isolates were obtained from the ileum, caecum, colon and rectum after animals were slaughtered. A higher level of ampicillin-resistant E. coli was found in the caecum (40%) than in other segments of the intestinal tract. In treated animals, level of resistance increased for organisms from the colon and rectum. Similar results were obtained for tetracycline resistance. In study (b), intestinal transit time was measured using chromium-EDTA as a marker. Swine were euthanized and samples were collected throughout the intestinal tract (duodenum to rectum) 8 h after administration of the marker to control and heat-stressed animals. Results indicated a reduced transit time for the stressed group. These findings corroborate the initial hypothesis that an outflow of resistant organisms moves from the upper tract (ileum and caecum) to the lower tract (colon and rectum).

Detection of Lawsonia intracellularis in swine using polymerase chain reaction methodology.

The polymerase chain reaction (PCR) was evaluated for its usefulness as a diagnostic tool to detect Lawsonia (ileal symbiont) intracellularis. Porcine ilea were collected from swine cases submitted to the Iowa State University Veterinary Diagnostic Laboratory between December 1, 1994, and June 30, 1995. Sampling was random, with no regard to health status. There were 621 ileum scrapings evaluated using the PCR technique. Thirty-five of the samples were positive, either by PCR or conventional diagnostic methods such as histology and Warthin-Starry silver stain. These 35 samples were further evaluated by Warthin-Starry silver stain and indirect immunofluorescent antibody test (IFAT) to confirm the presence of L. intracellularis in the tissue sections. Of the 26 samples positive by PCR, 22 were positive by IFAT. Sixteen of the 22 were also positive when stained with Warthin-Starry and evaluated microscopically for typical bacteria. Nine of the original samples were negative by all 3 techniques. PCR appears more sensitive and specific for L. intracellularis detection than Warthin-Starry stain and IFAT. This study provides evidence that PCR may be useful as a reference standard for the detection of L. intracellularis. PCR may be an appropriate monitoring tool for swine herds because it is a rapid procedure that could be applied to batch testing. Although the test is currently too laborious and expensive for routine diagnostic use, there may be situations in which it is justified because of the advantages of greater sensitivity and specificity.

Effects of cold stress on the antimicrobial drug resistance of Escherichia coli of the intestinal flora of swine.

Faecal samples were initially collected from pigs of different age groups, over periods considered to be seasonally normal and stable (baseline), and during times in which drastic drops in environmental temperature (cold stress) occurred. Baseline bacterial resistance to ampicillin and tetracycline were significantly higher (P < 0.05) in younger than in older pigs. Also, when animals were exposed to excessively cold conditions, there was a significant (P < 0.05) increase in ampicillin and tetracycline resistance in Escherichia coli for animals of all age groups. These results may indicate that factors other than feeding or use of antibiotics may play a role in establishing or maintaining the antibiotic resistance microflora of pigs, especially in those operations where animals are maintained outdoors, with minimal protection against extreme weather conditions.

Porcine reproductive and respiratory syndrome virus: routes of excretion.

This study was conducted to delineate potential sites of exit and duration of shedding of porcine reproductive and respiratory syndrome virus (PRRSV). Two experiments of 6 pigs each were conducted. Pigs were farrowed in isolation, weaned at 7 days of age, and housed in individual HEPA filtered isolation chambers. In each experiment, 3 pigs served as controls and 3 were inoculated intranasally with PRRSV (ATCC VR-2402) at 3 weeks of age. In a first experiment, on days 7, 14, 21, 28, 35, and 42 post-inoculation (p.i.), pigs were anesthetized and intubated. The following samples were collected: serum, saliva, conjunctival swabs, urine by cystocentesis, and feces. Upon recovery from anesthesia, the endotracheal tube was removed, rinsed, and the rinse retained. In the second experiment, the sampling schedule was expanded and serum, saliva, and oropharyngeal samples were collected from day 55 to day 124 p.i. at 14 day intervals. Virus was isolated in porcine alveolar macrophages up to day 14 from urine, day 21 from serum, day 35 from endotracheal tube rinse, day 42 from saliva, and day 84 from oropharyngeal samples. No virus was recovered from conjunctival swabs, fecal samples, or negative control samples. This is the first report of isolation of PRRSV from saliva. Virus-contaminated saliva, especially when considered in the context of social dominance behavior among pigs, may plan an important role in PRRSV transmission. These results support previous reports of persistent infection with PRRSV with prolonged recovery of virus from tonsils of swine.

Comparison of MICs of ceftiofur and other antimicrobial agents against bacterial pathogens of swine from the United States, Canada, and Denmark.

The MICs of ceftiofur and other antimicrobial agents, tested for comparison, for 515 bacterial isolates of pigs from the United States, Canada, and Denmark with various diseases were compared. The organisms tested included Actinobacillus pleuropneumoniae, Escherichia coli, Pasteurella multocida, Salmonella choleraesuis, Salmonella typhimurium, Streptococcus suis, Streptococcus dysgalactiae subsp. equisimilis, Streptococcus equi subsp. equi, and Streptococcus equi subsp. zooepidemicus. In addition to ceftiofur, the following antimicrobial agents or combinations were tested: enrofloxacin, ampicillin, sulfamethazine, trimethoprim-sulfadiazine (1:19), erythromycin, lincomycin, spectinomycin, lincomycin-spectinomycin (1:8), tilmicosin, and tetracycline. Tilmicosin was only tested against the U.S. isolates. Overall, ceftiofur and enrofloxacin were the most active antimicrobial agents tested against all isolates, with MICs inhibiting 90% of isolates tested (MIC90s) of < or = 2.0 and < or = 1.0 microgram/ml, respectively. Erythromycin, sulfamethazine, spectinomycin, and lincomycin demonstrated limited activity against all of the organisms tested, with MIC90s of > or = 8.0, > or = 256.0, > or = 32.0, and > or = 16.0 micrograms/ml, respectively. Trimethoprim-sulfadiazine was active against isolates of A. pleuropneumoniae, S. choleraesuis, S. typhimurium, P. multocida, S. equi, and S. suis (MIC90s, < or = 0.5 microgram/ml) but was less active against the E. coli strains tested (MIC90, > 16.0 micrograms/ml). Ampicillin was active against the P. multocida, S. suis, and S. equi isolates tested (MIC90s, 0.5, 0.06, and 0.06 micrograms/ml, respectively) and was moderately active against S. typhimurium (MIC90s, 2.0 micrograms/ml). However, this antimicrobial agent was much less active when it was tested against A. pleuropneumoniae, S. cholerae-suis, and E. coli (MIC90s, 16.0, > 32.0, and 32.0 micrograms/ml, respectively). Against the U.S. isolates of A. pleuropneumoniae and P. multocida, tilmicosin was moderately active (MIC90s, 4.0 and 8.0 micrograms/ml, respectively). However, this compound was not active against the remaining U.S. isolates (MIC90s, > 64.0 micrograms/ml). Differences in the MICs from one country to another were not detected with enrofloxacin, ceftiofur, or lincomycin for the strains tested, but variations in the MICs of the remaining antimicrobial agents were observed.

Effect of environmental variables in turkey confinement houses on airborne Aspergillus and mycoflora composition.

Environmental conditions and airborne mycoflora were measured concurrently in 10 turkey confinement houses during warm and cold weather. The following variables in the environment were measured: numbers of feed- and litter-associated yeast and mold fungi, temperature, relative humidity, airspeed, carbon dioxide and ammonia concentration, airborne bacteria, and airborne particulate mass, particle number, and particle size distribution. Winter air in turkey confinement houses contained significantly higher concentrations of Aspergillus, Scopulariopsis, and Mucor sp. and significantly lower concentrations of Cladosporium, Fusarium, and Alternaria sp. when compared with summer air. Significantly greater numbers of Mucor sp. were recovered per cubic meter of air where the current turkey flock was present less than 100 d when compared to houses where the current flock resided 100 d or more. Management decisions regarding control of the internal environment of turkey confinement houses apparently influence airborne mycoflora composition.

Cecal spirochetosis in commercial laying hens.

Cecal spirochetosis in chickens has been associated with enteric disease and reduced egg production in the United States and Europe. This report describes spirochete overgrowth of cecal mucosa in chickens from a flock of 100,000 commercial layers experiencing diarrhea and a 5% drop in egg production. Spirochetes were demonstrated in the ceca by darkfield and light microscopy. Apical surfaces of cecal enterocytes were covered by a dense layer of spirochetes aligned parallel to each other and perpendicular to the mucosal surface. Weakly beta-hemolytic, indole-negative spirochetes were isolated from the ceca on BJ media under anaerobic conditions at 42 C. Chicken cecal spirochetosis may represent an economically significant enteric disease of laying hens which has heretofore been infrequently recognized.

Evaluation of rotavirus infection and diarrhea in Iowa commercial pigs based on an epidemiologic study of a population represented by diagnostic laboratory cases.

Group A, B, and C rotaviruses were identified in 9% (96/1,048) of pig fecal specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory during 1987 and 1988. Six of the rotaviruses were group B, 5 were group C, and the remaining 89% were group A. Of the rotavirus cases with more than 1 serotype, 5 were multiple group A serotypes, 1 involved a group A and B serotype, and 1 included 2 group C serotypes. A retrospective epidemiologic evaluation of pig diarrhea in herds of origin was done using data obtained from the accession records of the rotavirus and 88 matched nonrotavirus pig diarrhea control cases. Herds from which rotavirus cases were derived experienced lower morbidity, mortality, and case fatality rates than matched control herds. The incidence of diarrhea decreased rapidly among all pigs from birth to 3 weeks of age. The peak incidence for piglet diarrhea occurred in February, and a moderate rise occurred in August-September. Definitive evidence for transmissible gastroenteritis virus was found in 12% of nonrotavirus cases but none of the rotavirus cases in which it was sought. Other pathogenic microorganisms were identified less frequently and inconsistently.

Effect of building ventilation design on environment and performance of turkeys.

Environmental variables in 10 commercial turkey confinement buildings, representing 2 natural ventilation designs, were measured during summer and the following winter. Sliding doors spaced at intervals along the walls of 5 of the buildings provided about 35% opening, and continuous wall curtains provided 60 to 80% opening in the other 5 buildings. Environmental variables assessed included airspeed; temperature; relative humidity; gases; particle number, size, and mass per cubic meter of air; and colonies of bacteria, yeasts, and other fungi per cubic meter of air. Colonies of yeasts and other fungi were quantitated in feed and litter. For most of the variables evaluated, significant differences were not attributable to building ventilation design; however, in winter, the total mass of particulate matter per cubic meter of air was higher in the curtain-type houses, compared with sliding door-type houses. Ammonia concentration in the air of sliding door-type houses progressively increased during summer and winter sampling periods. A significant effect of building ventilation design on turkey performance was not detected when using mortality, average daily gain, feed conversion, condemnations at slaughter, or average individual bird weight as measures of production.

Experimental transmission of an apparent viral pneumonia in conventional and gnotobiotic pigs.

Endemic pneumonia in five- to eight-week-old pigs induced microscopic lesions of proliferative interstitial pneumonia which were compatible with a viral aetiology. The disease was transmitted experimentally to conventional and gnotobiotic pigs by means of a lung homogenate filtered through a 0.22 micron filter. No common viral respiratory pathogens of pigs were isolated. Two types of virus particles were observed in cell culture by electron microscopy; one was about 70 nm in diameter and had an envelope and short surface spicules, the other also had an envelope, was elongated, pleomorphic, measured 80 x 320 nm and was coated by antibodies.

Comparison of DNA fingerprints and somatic serotypes of serogroup B and E Pasteurella multocida isolates.

The DNA fingerprint profiles and somatic serotypes of 71 Pasteurella multocida capsule serogroup B isolates, 13 capsule serogroup E isolates, and 16 somatic reference serotype strains were compared. Each of the 16 reference somatic serotypes had a unique DNA fingerprint profile with the HhaI restriction endonuclease. Fifty-four serogroup B isolates (isolated from classical cases of hemorrhagic septicemia) reacted with somatic serotype 2 or 5 antiserum and had DNA fingerprint profiles which resembled that of the serotype 2 reference strain. Seven DNA fingerprint profiles were found among 16 serogroup B strains representing other somatic serotypes. The DNA fingerprints of these isolates were different from the fingerprints of the 16 somatic reference serotype strains. All 13 serogroup E isolates had identical somatic serotypes and identical DNA fingerprint profiles when the HhaI endonuclease was used. The HhaI fingerprint profile of the serogroup E isolates did not match any fingerprint profile of the reference somatic serotype strains. Following DNA profiling with the HhaI endonuclease, the 13 serogroup E isolates were differentiated sequentially with HpaII restriction endonuclease. A descriptive identification epithet for P. multocida isolates was constructed. The descriptive epithet consists of serologic identification and sequential DNA profiles with restriction endonucleases HhaI and HpaII, respectively. DNA fingerprinting of P. multocida is a precise characterization method. In conjunction with serologic typing, it can further classify P. multocida isolates for epidemiologic studies.

Actinobacillus (Haemophilus) pleuropneumoniae: use of coagglutination and complement fixation to determine the relationship between presence of organism and antibody titer in slaughterhouse pigs.

The conventional culture method was compared to coagglutination for detection of Actinobacillus (Haemophilus) pleuropneumoniae in 425 sets of pig lungs. Sera from the same animals were evaluated for antibodies to A. pleuropneumoniae by the complement fixation (CF) test. All samples were collected at 2 packing plants in Iowa. In 2 nonvaccinated herds with no history of respiratory disease, the difference between standard culture results and coagglutination was highly significant (P less than 0.001). None of the 57 pigs in this group were positive for A. pleuropneumoniae by conventional culture, but 7 were positive by the coagglutination test. There were 15 animals with CF titers between 1:8 and 1:32. Animals from 6 herds vaccinated for A. pleuropneumoniae and without recent respiratory problems were evaluated. One out of 118 animals tested was positive for A. pleuropneumoniae by standard culture as compared to 9 positive by coagglutination. The difference in positive results between culture and coagglutination was highly significant (P less than 0.001). Twenty-eight animals had CF titers to A. pleuropneumoniae (1:4 to greater than or equal to 1:128). Two hundred fifty lungs and sera samples were collected from 7 herds which had recently experienced varying degrees of respiratory disease. Thirty-nine lungs were positive for A. pleuropneumoniae by culture and 182 were positive by coagglutination. The number of positives detected by coagglutination was significantly different (P less than 0.001) from the number positive by culture. There were 172 animals with antibody titers ranging from suspect to greater than or equal to 1:128. There were significantly fewer positive animals detected by standard culture than with the CF test (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Infusion of adrenergic receptor agonists and antagonists into the locus coeruleus and ventricular system of the brain. Effects on swim-motivated and spontaneous motor activity.

These studies examined how pharmacological stimulation and blockade of alpha receptors would affect active motor behavior in rats. In experiment I, alpha-2 receptor antagonists (piperoxane, yohimbine) and agonists [clonidine, norepinephrine (NE)] were infused into various locations in the ventricular system of the brain, including the locus coeruleus region, and motor activity was measured. Activity was measured principally in a swim test but spontaneous (ambulatory) activity was also recorded while drugs were being infused. When infused into the locus coeruleus region, small doses of the antagonists piperoxane and yohimbine depressed activity in the swim test while infusion of the agonists clonidine and NE had the opposite effect of stimulating activity. These effects were highly specific to the region of the locus coeruleus, since infusions of these drugs into other nearby locations in the ventricular system or use of larger doses had different, often opposite effects. This was especially true of clonidine and NE which profoundly depressed activity when infused posterior to the locus coeruleus, particularly over the dorsal vagal complex. Infusion of small doses of these drugs into the lateral ventricle had effects similar to infusion into the locus coeruleus region, though less pronounced. Changes in spontaneous motor activity were also observed, but this measure differentiated the groups less well than did the swim test. In experiment II, the predominantly postsynaptic receptor agonists isoproterenol (beta agonist) and phenylephrine (alpha-1 agonist) were infused into the ventricular system. Since infusions of piperoxane and yohimbine into the locus coeruleus that decreased activity in experiment I increase the release of NE by blocking alpha-2 inhibitory receptors on cell bodies and dendrites of the locus coeruleus, experiment II tested whether ventricular infusion of predominantly postsynaptic receptor agonists would also decrease activity in the swim test. Both isoproterenol and phenylephrine produced this effect, but did so selectively with respect to dose and location of infusion in the ventricular system. These findings are consistent with recent results relating to the mechanism that underlies stress-induced depression of active behavior.