Nutrient database improvement project: the influence of USDA quality and yield grade on the separable components and proximate composition of raw and cooked retail cuts from the beef chuck.

This study was designed to provide updated information on the separable components, cooking yields, and proximate composition of retail cuts from the beef chuck. Additionally, the impact the United States Department of Agriculture (USDA) Quality and Yield Grade may have on such factors was investigated. Ultimately, these data will be used in the USDA - Nutrient Data Laboratory's (NDL) National Nutrient Database for Standard Reference (SR). To represent the current United States beef supply, seventy-two carcasses were selected from six regions of the country based on USDA Yield Grade, USDA Quality Grade, gender, and genetic type. Whole beef chuck primals from selected carcasses were shipped to three university laboratories for subsequent retail cut fabrication, raw and cooked cut dissection, and proximate analyses. The incorporation of these data into the SR will improve dietary education, product labeling, and other applications both domestically and abroad, thus emphasizing the importance of accurate and relevant beef nutrient data.

Innovative retail merchandising strategies to accommodate for the growing trend of heavier carcass weights in the United States.

Three subprimals from beef carcasses, Average (mean=340.6kg) and Heavy weight (mean=461.6kg), were cut using Innovative versus Conventional cutting styles. Longer (P<0.05) processing times were required for the Heavy compared to Average and Innovative compared to Conventional. Total saleable yields were lower for the Innovative compared to Conventional for the top sirloin butt (P=0.0025) and ribeye (P<0.0001), but not for the strip loin (P=0.1416). However, yields were higher for the Heavy compared to Average for the ribeye (P=0.0054) and strip loin (P=0.0017), but not for the top sirloin butt (P=0.6797). Retail pricing increases for the Innovative compared to Conventional were 11.6% for top sirloin butt, 26.9% for ribeye, and 2.6% for strip loin. Retailers adopting innovative cutting styles to more effectively merchandise heavyweight beef must account for the decreased primary saleable yields and increased labor requirements through increased retail pricing.

Pseudomonas aeruginosa serological analysis in young children with cystic fibrosis diagnosed through newborn screening.

BACKGROUND: With newborn screening (NBS) for cystic fibrosis (CF), eradication of Pseudomonas aeruginosa (PA) is possible if PA detection occurs early. A serological response to infection likely precedes culture positivity in CF patients, so PA serological testing is very appealing in this population. However, controversies continue to exist about serology testing, titer cutoffs for enzyme-linked immunosorbent assay (ELISA) antibody tests, and their value in children with CF. METHODS: This longitudinal, prospective study collected respiratory secretions as oropharyngeal swabs or expectorated sputum for culture and also sera over 6 years in 69 patients diagnosed by NBS. Serology assessed PA antibody titers against cell lysate, exotoxin A, and elastase. A novel statistical approach with weighted receiver operating characteristic (ROC) curves was used to determine best antibody titer cutoff values to predict subsequent PA positive cultures. RESULTS: Using these weighted ROC curves, the order of sensitivity was found to be cell lysate, exotoxin A, and then elastase while age-specific cutoffs were better than fixed cutoffs previously used. Age-specific serological cutoffs both predict and detect PA respiratory infections with a higher sensitivity and specificity. Serological responses to the PA antigens determined that a response to cell lysate occurs significantly earlier than culture positivity. CONCLUSIONS: Age-specific serological cutoffs rather than fixed values against common PA antigens improve early PA identification in infants and young children diagnosed with NBS. Regular serological assessment with age-specific cutoffs in these children appears to be a worthy diagnostic tool.

Early emergence of PNH-like T cells after allogeneic stem cell transplants utilising CAMPATH-1H for T cell depletion.

CAMPATH-1H (C-1H) is widely used in vivo and / or in vitro for T cell depletion in hematopoietic SCT. This humanised monoclonal antibody is specific for CD52, a marker coexpressed on the majority of human lymphocytes with CD48 and other glycosylphosphatidyl-inositol (GPI) anchored proteins. We detected CD52 / CD48 dual expression on >99% of CD3(+) lymphocytes from normal individuals and all 15 post-SCT patients whose transplants did not utilise C-1H. By contrast, 23 / 26 patients with transplants involving C-1H (in vivo, in vitro or both) exhibited populations lacking CD52 expression that accounted for 49.7% (4.2-86.2%) of the CD3+ lymphocytes (median and range) in samples evaluated at a median of 2 months post-SCT. Most CD52- cells also lacked CD48 expression. These GPI- T cells were of either donor or mixed donor / recipient origin. They were predominant in the early months after SCT at times of profound lymphopenia and inversely correlated with the recovery of the absolute lymphocyte count (r= - 0.663, P<0.0001). The presence of CD52- cells has been correlated previously with clinical outcome after CAMPATH therapy for both malignant and nonmalignant diseases.

A DNA delivery system targeting dendritic cells for use in immunization against malaria: a rodent model.

DNA-based vaccination has emerged as a promising method of immunisation since the first demonstration of this technology. Improving the antibody responses is desirable for the protective efficacy and hence broad application of these vaccines. We examined the immunogenicity of a Plasmodium-based DNA vaccine that was targeted to antigen presenting cells by fusion to CTLA4. Fusion proteins comprising the extra-cellular domain of CTLA4, the hinge, CH2 and CH3 domains of human IgG1 and MSP-1 gene fragments were expressed in COS-7 cells. Three of the secreted proteins containing the mouse homologue of CTLA4 were shown to bind differently to the human B7-1 molecule expressed on THP-1 cells. Competition binding assays for two fusion proteins showed that binding was specific. When C57BL/6 mice were immunized with plasmids encoding the fusion proteins, antibodies against two denatured and one non-denatured MSP-1 gene fragments were successfully induced. The usefulness of this strategy in future studies of immunisaton against human malaria is discussed.

Human CD8+ CTL recognition and in vitro lysis of herpes simplex virus-infected cells by a non-MHC restricted mechanism.

Cytotoxic T lymphocytes (CTL) are important for the recognition and lysis of virally infected cells, but their effectiveness can be limited by viral immune evasion mechanisms. We investigated the immunophenotype and function of human CD8+ T cells raised in response to herpes simplex virus (HSV). The expanded population contained cells of an activated and mature phenotype, as determined by the expressions of CD25, CD45RO, CD57, CD95 and HLA-DR. Cultured cells also expressed CD45RA. These cells lysed autologous and allogeneic HSV-infected lymphoblastoid cell line (LCL) targets via a non-major histocompatibility complex (MHC) restricted recognition pathway. Inhibition assays showed the mechanism of cytotoxicity to be calcium-dependent, granule exocytosis pathway, rather than the internal disintegration pathway. Cold target competition assays indicated that a common CTL population contributed to the recognition of autologous and allogeneic-infected targets. These effectors showed recognition of infected targets which was distinct from that of K562 cells. Non-MHC restricted lysis-associated molecule 2B4 (CD244) was upregulated on culturing and made a significant contribution to lysis of FcgammaR-bearing targets in a redirected killing assay. These findings suggest that CTL can recognize virally infected cells through a combination of non-MHC restricted mechanisms and may result in more efficient lysis than classical CD8+ T cells.

Acceleration of lung disease in children with cystic fibrosis after Pseudomonas aeruginosa acquisition.

As part of the ongoing Wisconsin Cystic Fibrosis (CF) Neonatal Screening Project, we had the unique opportunity to study the longitudinal relationship between Pseudomonas aeruginosa (Pa) acquisition and infection and developing lung disease in children with CF. The primary objective was to determine whether acquisition of Pa was associated with a measurable change in the progression of lung disease. Two outcome measures were used to study 56 patients who were diagnosed through newborn screening: 1) Wisconsin additive chest radiograph score (WCXR), based on the average of scores from a pulmonologist and a radiologist, and 2) the highest forced expired volume in 1 sec (FEV(1))/forced vital capacity (FVC) ratio. We used two measures of Pa acquisition: 1) time of first positive protocol-determined oropharyngeal (with cough) culture, and 2) the magnitude of antibody titer detected by ELISA assays, using as antigen a crude cell lysate, purified exotoxin A, or an elastase toxoid prepared from three Pa strains. Other predictor variables included age, pancreatic status, height-for age, and weight-for-age-percentiles. The best regression model for predicting changes in the WCXR included time to first positive culture and antibody titer for Pa elastase. Prior to Pa acquisition, WCXR worsened by 0.45 points/year (P > 0.25); after Pa acquisition, the rate of worsening increased significantly (P < 0.001) to 1.40 points/year. Each antibody titer level (log base 2) increased the score by 0.48 points (P < 0.001). The best regression model for predicting change in the FEV(1)/FVC included only time to first positive culture. Prior to Pa acquisition, the FEV(1)/FVC ratio declined by 1.29%/year; after Pa infection, the rate of decrease significantly accelerated to 1.81%/year (P = 0.001). Our data show that Pa acquisition is associated with declining pulmonary status in children with CF, and that this effect is probably gradual rather than precipitous. Because these patients were diagnosed and treated aggressively, our estimates of the effects of Pa acquisition may be conservative. We also conclude that the WCXR appears to be more sensitive than FEV(1)/FVC in detecting early changes in lung disease associated with CF.

Laryngeal reinnervation with the hypoglossal nerve. I. Physiology, histochemistry, electromyography, and retrograde labeling in a canine model.

This study was performed to determine whether the hypoglossal nerve (cranial nerve XI [XII]) would serve as a useful donor for laryngeal reinnervation by anastomosis to the recurrent laryngeal nerve (RLN). Twenty hemilarynges in 10 dogs were studied prospectively after XII-RLN anastomosis (group A; n = 5), split XII-RLN anastomosis (group B; n = 3), XII-RLN anastomosis with a 2-cm interposition graft (group C; n = 2), no treatment (group D; n = 5), RLN section (group E; n = 2), or ansa cervicalis-RLN anastomosis (group F; n = 3). Spontaneous activity was observed monthly by infraglottic examination through permanent tracheostomies and was recorded by electromyography. Laryngeal adductory pressure and induced phonation were obtained by stimulating the RLN while passing a pressure transducer balloon or humidified air through the glottis. At sacrifice, the laryngeal muscles were stained for adenosine triphosphatase to determine the ratio of type I to type II fibers. Retrograde labeling of the brain stem was performed with horseradish peroxidase. Infraglottic examination at 6 months showed a full range of adductory motion in groups A and B during the swallow reflex, comparable with that in group D. Groups C and F showed good bulk and tone, but little spontaneous motion. Group E remained paralyzed. Stimulation of the transferred nerves caused more activity in groups A and B than in the other groups; groups C and F partially adducted at high levels. The laryngeal adductory pressure responses of groups A and B were similar to those of group D. The XII-reinnervated larynges were capable of producing normal induced phonation. Retrograde labeling of the RLN showed that the reinnervating axons originated only in the hypoglossal nucleus. Electromyography of the reinnervated adductor muscles confirmed spontaneous activity in the dogs (awake). Histochemical analysis confirmed slow-to-fast transformation of both the posterior and lateral cricoarytenoid muscles, indicating that significant reinnervation occurred. We conclude that the hypoglossal nerve functions well as a donor for adductory reinnervation of the larynx.

Laryngeal adductory pressure as a measure of post-reinnervation synkinesis.

Laryngeal adductory pressure (LAP) is the pressure induced as the vocal folds squeeze on a balloon while the recurrent laryngeal nerve (RLN) is stimulated. The LAP has been shown to vary with the frequency of stimulation, with a characteristic slope. The RLN was divided and reanastomosed 4 different ways in 12 canine hemilaryngeal preparations; the 4 subgroups represented a range of expected post-reinnervation synkinesis recovery patterns. The LAP frequency-response curve was measured before surgery and at monthly intervals for 6 months after surgery. In the "best-case" group (RLN adductor and abductor trunks each divided and reanastomosed), the slope was found to return to normal. The 2 whole RLN division-reanastomosis groups (precise realignment or 180 degrees rotation) both gave results similar to those of the "worst-case" group (RLN adductor and abductor trunks divided and transposed); these 3 subgroups were all significantly different from baseline. The slope of the LAP frequency-response curve may be a useful means of indirectly quantifying laryngeal synkinesis.

Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide.

Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm-grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI-1 (N-(3-oxododecanoyl)-L-homoserine lactone) and PAI-2 (N-butyryl-L-homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn-SOD and Fe-SOD, and the major catalase, KatA. The expression of sodA (encoding Mn-SOD) was particularly dependent on PAI-1, whereas the influence of autoinducers on Fe-SOD and KatA levels was also apparent but not to the degree observed with Mn-SOD. beta-Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI-1/2-deficient double mutant partially restored KatA activity, while the addition of PAI-1 only was sufficient for full restoration of Mn-SOD activity. In biofilm studies, catalase activity in wild-type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild-type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa.

The sigma factor RpoS (sigmaS) has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. To elucidate the role of RpoS in Pseudomonas aeruginosa physiology and pathogenesis, we constructed rpoS mutants in several strains of P. aeruginosa, including PAO1. The PAO1 rpoS mutant was subjected to various environmental stresses, and we compared the resistance phenotype of the mutant to that of the parent. The PAO1 rpoS mutant was slightly more sensitive to carbon starvation than the wild-type strain, but this phenotype was obvious only when the cells were grown in a medium supplemented with glucose as the sole carbon source. In addition, the PAO1 rpoS mutant was hypersensitive to heat shock at 50 degrees C, increased osmolarity, and prolonged exposure to high concentrations of H2O2. In accordance with the hypersensitivity to H2O2, catalase production was 60% lower in the rpoS mutant than in the parent strain. We also assessed the role of RpoS in the production of several exoproducts known to be important for virulence of P. aeruginosa. The rpoS mutant produced 50% less exotoxin A, but it produced only slightly smaller amounts of elastase and LasA protease than the parent strain. The levels of phospholipase C and casein-degrading proteases were unaffected by a mutation in rpoS in PAO1. The rpoS mutation resulted in the increased production of the phenazine antibiotic pyocyanin and the siderophore pyoverdine. This increased pyocyanin production may be responsible for the enhanced virulence of the PAO1 rpoS mutant that was observed in a rat chronic-lung-infection model. In addition, the rpoS mutant displayed an altered twitching-motility phenotype, suggesting that the colonization factors, type IV fimbriae, were affected. Finally, in an alginate-overproducing cystic fibrosis (CF) isolate, FRD1, the rpoS101::aacCI mutation almost completely abolished the production of alginate when the bacterium was grown in a liquid medium. On a solid medium, the FRD1 rpoS mutant produced approximately 70% less alginate than did the wild-type strain. Thus, our data indicate that although some of the functions of RpoS in P. aeruginosa physiology are similar to RpoS functions in other gram-negative bacteria, it also has some functions unique to this bacterium.

Psyllium improves fecal consistency and prevents enhanced secretory responses in jejunal tissues of piglets infected with ETEC.

Infection with enterotoxigenic E. coli (ETEC) induces secretory diarrhea by stimulating net secretion of fluid and electrolytes. We tested the hypothesis that ETEC potentiates jejunal ion secretion induced by other agonists and also examined whether the soluble fiber psyllium ameliorates effects of ETEC-induced pathophysiology. Noninfected or ETEC-infected piglets were given oral electrolyte solution twice daily or electrolyte solution supplemented with psyllium for 48 hr. Jejunal tissues were mounted in flux chambers and basal and stimulated ion transport responses, as reflected by short-circuit current (I(SC)) were measured. The severity of ETEC-induced diarrhea was reduced by psyllium. I(SC) responses to carbachol and 5-hydroxytryptamine were greater in tissues from infected piglets compared with noninfected controls or infected piglets given psyllium. These results suggest that psyllium ameliorates ETEC-induced diarrhea and prevents the enhanced secretory responses to calcium-mediated agonists that occur in ETEC-infected piglet jejunum.

Cleavage of influenza A virus H1 hemagglutinin by swine respiratory bacterial proteases.

Cleavage of influenza A virus hemagglutinin (HA) is required for expression of fusion activity and virus entry into cells. Extracellular proteases are responsible for the proteolytic cleavage activation of avirulent avian and mammalian influenza viruses and contribute to pathogenicity and tissue tropism. The relative contributions of host and microbial proteases to cleavage activation in natural infection remain to be established. We examined 23 respiratory bacterial pathogens and 150 aerobic bacterial isolates cultured from the nasal cavities of pigs for proteolytic activity. No evidence of secreted proteases was found for the bacterial pathogens, including Haemophilus parasuis, Pasteurella multocida, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, and Streptococcus suis. Proteolytic bacteria were isolated from 7 of 11 swine nasal samples and included Staphylococcus chromogenes, Staphylococcus hyicus, Aeromonas caviae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Enterococcus sp. Only P. aeruginosa secreted a protease, elastase, that cleaved influenza virus HA. However, compared to trypsin, the site of cleavage by elastase was shifted one amino acid in the carboxy-terminal direction and resulted in inactivation of the virus. Under the conditions of this study, we identified several bacterial isolates from the respiratory tracts of pigs that secrete proteases in vitro. However, none of these proteolytic isolates demonstrated direct cleavage activation of influenza virus HA.

Utilization of both phenotypic and molecular analyses to investigate an outbreak of multidrug-resistant Salmonella anatum in horses.

Phenotypic and molecular techniques, including antimicrobial susceptibility testing, plasmid analysis, and pulsed-field gel electrophoresis (PFGE) were used to characterize 15 isolates of multidrug-resistant (MDR) Salmonella anatum cultured during a 16 mo period from horses and a veterinary clinic environment. The isolates were resistant to multiple antimicrobial agents and could be placed into 4 groups based on their antimicrobial resistance patterns. The isolates contained multiple plasmids ranging in size from 2 to > 100 kb that could be grouped into 3 different plasmid profile patterns; these patterns did not correlate with the antimicrobial resistance groupings. Furthermore, antimicrobial resistance was conjugatively transferable. Digestion of genomic DNA from the 15 isolates with 3 different restriction endonucleases, SfiI, SpeI, and XbaI followed by PFGE revealed a highly conserved restriction endonuclease digestion pattern. In contrast, diverse banding patterns were observed with S. anatum obtained from other sources. These observations suggest that the MDR S. anatum isolates represent a common outbreak strain even though they possess different, albeit similar, antibiograms and plasmid profiles. The study showed that PFGE is a useful epidemiological tool for discriminating between unrelated and outbreak-related strains of S. anatum. In conclusion, epidemiological studies of outbreaks caused by MDR isolates of S. anatum should consist of both genotypic and phenotypic methods of analysis.

Vfr controls quorum sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa controls several genes in a cell density-dependent manner through a phenomenon termed quorum sensing. The transcriptional activator protein of the las quorum-sensing system is encoded for by the lasR gene, which is at the top of a quorum-sensing hierarchy. The activation of LasR as a transcriptional activator induces the expression of multiple genes that code for factors important for virulence, and rhlR, which encodes the transcriptional activator protein of the P. aeruginosa rhl quorum-sensing system. Elucidating the method of lasR regulation is crucial to understanding P. aeruginosa quorum sensing. In this report, we present studies on the transcriptional control of lasR. We identified two distinct transcriptional start sites for lasR that were located 201 bp (transcript T1) and 231 bp (transcript T2) upstream from the lasR start of translation. With the use of transcriptional lasRp-lacZ fusions, we showed that in P. aeruginosa, lasR expression is cell density dependent. This gene was expressed at a basal level until it was induced during the second half of log-phase growth, with expression becoming maximal during stationary-phase growth. We also showed that lasR expression was regulated through the cyclic AMP receptor protein (CRP)-binding consensus sequence in its promoter region. Our results from P. aeruginosa mutant studies and gel retardation assays indicated that this regulation was mediated by Vfr, a homolog of the Escherichia coli CRP.

A divergently transcribed open reading frame is located upstream of the Pseudomonas aeruginosa vfr gene, a homolog of Escherichia coli crp.

The Pseudomonas aeruginosa homolog of the Escherichia coli global transcriptional regulator CRP (or CAP) was recently identified and designated Vfr (S. E. H. West, A. K. Sample, and L. J. Runyen-Janecky, J. Bacteriol. 176:7532-7542, 1994). Nucleotide sequence analysis of the region 5' to vfr identified a 423-bp open reading frame (ORF), which was designated orfX. The deduced amino acid sequence of ORFX was 53% identical and 87% similar to a divergent ORF of unknown function located 5' to the E. coli crp gene. When orfX was expressed from a phage T7 promoter in E. coli, a protein with an apparent molecular mass of approximately 18 kDa was produced. We constructed a chromosomal deletion of the region containing the 5' end of orfX (orfX'), vfr, and the 3' end of trpC (trpC') in P. aeruginosa strains PAO1 and PA103. The cloned vfr gene restored Vfr-dependent production of exotoxin A and protease in the PA103 orfX'-vfr-trpC' deletion mutant, suggesting that ORFX is not required for Vfr production or activity. To determine whether transcription of orfX and vfr are controlled by the same mechanisms that control transcription of the region of the divergent ORF (dorf) and of crp, we compared the vfr-orfX and crp-dorf intergenic regions. Using S1 nuclease analysis, we determined that the distance between the orfX and vfr transcriptional start sites was 105 bp. Thus, the P. aeruginosa orfX and vfr promoters are arranged in a back-to-back orientation rather than the face-to-face orientation of the dorf and crp promoters. A CRP recognition site is associated with each promoter in the crp-dorf intergenic region; binding of the CRP-cyclic AMP complex to the stronger dorf CRP recognition site activates transcription from the dorf promoter and represses transcription from the crp promoter. The vfr-orfX intergenic region does not contain an obvious CRP recognition site. In addition, vfr was not required for transcription of orfX. Unlike the dorf and crp mRNAs, the 5' ends of the orfX and vfr mRNAs were not complementary. Thus, the orfX mRNA cannot hybridize to the 5' end of the vfr mRNA to inhibit vfr transcription, a mechanism that has been postulated to control crp transcription in E. coli.

Control of an outbreak of salmonellosis caused by drug-resistant Salmonella anatum in horses at a veterinary hospital and measures to prevent future infections.

Salmonella anatum was isolated from horses treated at a private veterinary clinic or at a university veterinary medical teaching hospital. All isolates were resistant to most commonly used antibiotics. Because of the severity of disease resulting from outbreaks of infections with drug-resistant strains of S anatum, an epidemiologic investigation was conducted. Enteric bacteria, including S anatum, that were resistant to most antibiotics were isolated from the private veterinary clinic environment. Salmonella anatum was not isolated from the university teaching hospital environment. To prevent transmission, disinfection and isolation protocols were reviewed, and changes were implemented, including discontinuing use of power sprayers for cleaning, improving a two-step disinfection process, restricting movement of horses, and enhancing awareness of Salmonella spp transmission. Communication and prompt action are pivotal in preventing dissemination of resistant strains of Salmonella spp in a clinic or hospital environment.

Construction of an Actinobacillus pleuropneumoniae-Escherichia coli cosmid cloning vector [Gene 160 (1995) 81-86].

We constructed a cosmid vector, pSW206, which should be useful for the construction of genomic libraries of Actinobacillus pleuropneumoniae (Apl) DNA. pSW206 is based on the broad-host-range plasmid RK2 and can be introduced into Apl by conjugation.

Construction of Actinobacillus pleuropneumoniae-Escherichia coli shuttle vectors: expression of antibiotic-resistance genes.

We constructed several cloning vectors, designated pGZRS-18/19 and pGZRS-38/39, which were based on an endogenous Actinobacillus pleuropneumoniae (Apl) 4.3-kb plasmid. They carry the lacZ alpha-complementation fragment and MCS from pUC18/19, and either the bla gene under the control of a putative Apl promoter or the KmR gene from Tn903. These vectors replicate in representative strains of Apl serotypes 1 and 7, Escherichia coli, Pasteurella haemolytica (Ph) and Haemophilus (Actinobacillus) actinomycetemcomitans. We also found that Apl and Ph did not express genes under the control of the lacZ or bla promoters, suggesting that their RNA polymerases may not utilize these promoters.