Caprine abscess model of tulathromycin concentrations in interstitial fluid from tissue chambers inoculated with Corynebacterium pseudotuberculosis following subcutaneous or intrachamber administration.

Corynebacterium pseudotuberculosis causes chronic, suppurative, abscessing conditions in livestock and humans. We used an in vivo model to evaluate antimicrobial efficacy for focal abscesses caused by C. pseudotuberculosis. Tissue chambers were surgically implanted in the subcutaneous tissues of the right and left paralumbar fossa of 12 goats to serve as a model for isolated, focal abscesses. For each goat, one tissue chamber was inoculated with C. pseudotuberculosis, while the contralateral chamber served as an uninoculated control. Six goats were administered a single dose of tulathromycin at 2.5 mg/kg of body weight subcutaneously, while the other six received the same dose by injection directly into the inoculated chambers. Our objective was to compare the effects and tulathromycin concentrations in interstitial fluid (IF) samples collected from C. pseudotuberculosis-infected and control chambers following subcutaneous or intrachamber injection of tulathromycin. In addition, the effects of tulathromycin on the quantity of C. pseudotuberculosis reisolated from inoculated chambers were assessed over time. Tulathromycin IF concentrations from C. pseudotuberculosis-infected and control tissue chambers were similar to those in plasma following subcutaneous administration. Following intrachamber administration, tulathromycin IF concentrations in infected chambers were continuously above the MIC for the C. pseudotuberculosis isolate for 15 days. There were no significant differences for plasma area under the curve and elimination half-lives between subcutaneous and intrachamber administration. Six of the 12 infected chambers had no growth of C. pseudotuberculosis 15 days postadministration. Results of this study indicate that tulathromycin may be beneficial in the treatment of focal infections such as those caused by C. pseudotuberculosis.

Amplified fragment length polymorphism reveals specific epigenetic distinctions between Mycobacterium avium subspecies paratuberculosis isolates of various isolation types.

Amplified fragment length polymorphism (AFLP) was employed as a genetic analysis tool for the study of the genetic relatedness of Mycobacterium avium subsp. paratuberculosis isolates harvested from bovine fecal samples and from bovine or human tissues. This analysis revealed genetic differences between these two isolate types that were confirmed through cluster analysis. Dendrogram analysis separated these two isolate types based on the isolation scheme (tissue-associated versus fecal M. avium subsp. paratuberculosis isolates). Further sequence analysis of unique genetic regions from each isolation type revealed no genetic sequence differences. However, Clustal DNA alignments identified AFLP restriction enzyme sites that were undigested in the tissue-associated isolates. AFLP analysis also disclosed that the same AFLP restriction sites were digested in all of the fecal isolates. Sequence analysis further revealed a consensus sequence upstream of the undigested restriction sites for possible methyltransferase recognition in the tissue-associated M. avium subsp. paratuberculosis isolates.

Morphologic and cytokine profile characterization of Salmonella enterica serovar typhimurium infection in calves with bovine leukocyte adhesion deficiency.

The role of neutrophils in the pathogenesis of Salmonella enterica Typhimurium-induced ruminant and human enteritis and diarrhea has yet to be characterized with in vivo models. To address this question, the in vivo bovine ligated ileal loop model of nontyphoidal salmonellosis was used in calves with the naturally occurring bovine leukocyte adhesion deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from 4 BLAD Holstein calves homozygous for BLAD (CD18-), 1 to 5 weeks of age, were compared with 4 controls, age-matched Holstein calves negative for BLAD (CD18+). Morphologic studies revealed that infection of CD18- calves with S Typhimurium resulted in no significant tissue infiltration by neutrophils, less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion, when compared with CD18+ calves. Ultrastructurally, lesions in enterocytes induced by S Typhimurium infection in CD18- calves--including attachment and disruption of the brush border, apical membrane ruffling formation, and cellular degeneration--were similar to the ones reported in the literature for CD18- calves. Study of cytokine gene expression by quantitative real-time polymerase chain reaction revealed that early stages of acute infection (4-8 hours postinfection) were associated with increased interleukin 8 gene expression in the absence of tissue influx of neutrophils in CD18- calves, whereas later stages of infection (12 hours postinfection) were associated with increased expression of growth-related oncogene alpha in the presence of neutrophil influx in CD18+ calves. In contrast, the proinflammatory cytokines interleukin 1beta and tumor necrosis factor alpha were poorly correlated with the presence or absence of tissue neutrophils.

Early phase morphological lesions and transcriptional responses of bovine ileum infected with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of chronic enteritis in ruminants (Johne's disease) and a possible etiopathologic agent in human Crohn's disease. The host-pathogen interaction in this chronic disease has largely depended on the randomly collected static lesions studied in subclinically or clinically infected animals. We have established and utilized the neonatal calf ligated ileal loop model to study the early temporal host changes during MAP infection. After inoculation of ligated ileal loop with MAP, samples were analyzed for bacterial invasion, histologic and ultrastructural morphologic changes, and gene expression at several times (0.5-12 hours) postinfection. Our results indicate that MAP invades the intestinal mucosa as early as 0.5 hour postinoculation. Distribution and migration of neutrophils, monocytes/macrophages, and goblet cells were confirmed by histopathology, scanning and transmission electron microscopy. Coincident with the morphologic analysis, we measured by real-time polymerase chain reaction gene expression of various cytokines/chemokines that are involved in the recruitment of mononuclear and polymorphonuclear leukocytes to the site of infection. We also detected expression of several other genes, including intestinal-trefoil factor, profilin, lactoferrin, and enteric ss-defensin, which may play significant roles in the early MAP infection. Thus, the calf ligated intestinal loop model may be used as a human disease model to understand the role of MAP in the pathogenesis of Crohn's disease.

Diagnostic specificity of a real-time RT-PCR in cattle for foot-and-mouth disease and swine for foot-and-mouth disease and classical swine fever based on non-invasive specimen collection.

Foot-and-mouth disease virus (FMDV) and classical swine fever virus (CSFV) are highly contagious and can cause great economic losses when introduced into disease-free regions. Accurate estimates of diagnostic specificity (Sp) are important when considering the implementation of surveillance for these agents. The purpose of this study was to estimate diagnostic Sp of a real-time reverse-transcriptase PCR assay developed for detection of FMDV in cattle and domestic swine and CSFV in domestic swine based on non-invasive specimen collection. One thousand and eighty-eight range beef cattle were sampled from thirteen geographic locations throughout Texas. One thousand and one hundred market hogs and cull sows were sampled. Results for both FMDV and CSFV were considered positive if amplification occurred at or before 40 PCR cycles, inconclusive between 40 and 45 cycles and negative otherwise. Ten cattle had nonspecific PCR amplifications for FMDV, but none were classified as positive and only one as inconclusive. Specificity (95% confidence interval) was estimated as 100% (99.7, 100). There were 19 nonspecific PCR amplifications for FMDV in sampled swine with 1 classified as positive, 6 as inconclusive, and 12 as negative. Specificity (95% confidence interval) was estimated as 99.9% (99.5, 100). There were 21 nonspecific PCR amplifications for CSFV, and 1 was classified as positive. Specificity (95% confidence interval) was estimated as 99.9% (99.5, 100). These assays have high Sp, but nonspecific PCR amplifications can occur.

ACVIM Small Animal Consensus Recommendations on the Treatment and Prevention of Uroliths in Dogs and Cats.

In an age of advancing endoscopic and lithotripsy technologies, the management of urolithiasis poses a unique opportunity to advance compassionate veterinary care, not only for patients with urolithiasis but for those with other urinary diseases as well. The following are consensus-derived, research and experience-supported, patient-centered recommendations for the treatment and prevention of uroliths in dogs and cats utilizing contemporary strategies. Ultimately, we hope that these recommendations will serve as a foundation for ongoing and future clinical research and inspiration for innovative problem solving.

Immunochemical studies on the binding properties of Brucella abortus lipopolysaccharides to bovine precipitating antibodies.

The binding properties of Brucella abortus smooth lipopolysaccharides (LPS) to the precipitating antibodies in the serum of a B. abortus S2308 infected cow were studied by quantitative precipitin and precipitin-inhibition assays. Crude B. abortus lipopolysaccharides (cLPS) and chemical modification products of strains 1119.3, 19 and 2308 (virulent) were used for this study. In the quantitative precipitin assay, 13 micrograms of cLPS precipitated about 4.0-5.2 micrograms nitrogen of precipitating antibodies in 10 microliter of serum. After alkaline treatment (0.125 N NaOH at 56 degrees C for 1 hr), the alkali-treated lipopolysaccharides (aLPS) from S19 and S1119.3 maintained the maximum precipitability, but the precipitation profile was changed from a sigmoidal to a bell-shaped curve, and aLPS from S2308 was reduced to about 40% of its reactivity. These results indicate that some of the antigenic determinants blocked by other molecules were reshielded by alkaline treatment, and possibly that structural differences exist between virulent (S2308) and nonvirulent (S19 and S1119.3) strains. Among the monosaccharides and glycosides tested for inhibition of precipitation, methyl alpha-D-mannose was most active and about 3.3 times as active as D-mannopyranose (DMan) and only 1/200 as active as acid hapten or the carbohydrate moiety of cLPS prepared from S1119.3 (expressed as a monosaccharide of mol. wt 200) implying that the carbohydrate determinants of this antigen are probably related to a DMan alpha 1----linked compound. The precipitability of the antibodies in this serum was greatly reduced after absorption with cLPS and the precipitin reaction was completely inhibited by the carbohydrate moiety of LPS (O-antigen) S1119.3 demonstrating that the common antigenic determinants (hapten) of B. abortus LPS reacting with these precipitating antibodies are in the region of the carbohydrate moiety of the LPS.

Animal models of Salmonella infections: enteritis versus typhoid fever.

The most common disease syndromes caused by Salmonella serotypes in humans, typhoid fever and enteritis, can be modeled using Salmonella enterica serotype Typhimurium infections in mice and calves, respectively. This article reviews murine typhoid and bovine enteritis and discusses strengths, limitations and distinctive features of these animal models.

An imposed oscillating electrical field improves the recovery of function in neurologically complete paraplegic dogs.

We show that an applied electric field in which the polarity is reversed every 15 minutes can improve the outcome from severe, acute spinal cord injury in dogs. This study utilized naturally injured, neurologically complete paraplegic dogs as a model for human spinal cord injury. The recovery of paraplegic dogs treated with oscillating electric field stimulation (OFS) (approximately 500 to 600 microV/mm; n = 20) was compared with that of sham-treated animals (n = 14). Active and sham stimulators were fabricated in West Lafayette, Indiana. They were coded, randomized, sterilized, and packaged in Warsaw, Indiana, and returned to Purdue University for blinded surgical implantation. The stimulators were of a previously unpublished design and meet the requirements for phase I human clinical testing. All dogs were treated within 18 days of the onset of paraplegia. During the experimental applications, all received the highest standard of conventional management, including surgical decompression, spinal stabilization (if required), and acute administration of methylprednisolone sodium succinate. A radiologic and neurologic examination was performed on every dog entering the study, the latter consisting of standard reflex testing, urologic tests, urodynamic testing, tests for deep and superficial pain appreciation, proprioceptive placing of the hind limbs, ambulation, and evoked potential testing. Dogs were evaluated before and after surgery and at 6 weeks and 6 months after surgery. A greater proportion of experimentally treated dogs than of sham-treated animals showed improvement in every category of functional evaluation at both the 6-week and 6-month recheck, with no reverse trend. Statistical significance was not reached in comparisons of some individual categories of functional evaluation between sham-treated and OFS-treated dogs (ambulation, proprioceptive placing); an early trend towards significance was shown in others (deep pain), and significance was reached in evaluations of superficial pain appreciation. An average of all individual scores for all categories of blinded behavioral evaluation (combined neurologic score) was used to compare group outcomes. At the 6-month recheck period, the combined neurologic score of OFS-treated dogs was significantly better than that of control dogs (p = 0.047; Mann-Whitney, two-tailed).

Cisplatin versus cisplatin combined with piroxicam in a canine model of human invasive urinary bladder cancer.

PURPOSE: More than 12,000 people are expected to die from invasive transitional cell carcinoma (TCC) of the urinary bladder each year in the United States, indicating that more effective therapy is needed. Drugs inhibiting cyclooxygenase (cox) have recently been found to have chemopreventive and antitumor activity and may potentiate the effects of chemotherapy. The purpose of this study was to determine whether cisplatin combined with the cox-inhibitor piroxicam would induce remission more frequently than cisplatin alone in a relevant animal model of human invasive TCC. METHODS: Pet dogs with naturally occurring, histopathologically confirmed, measurable TCC of the urinary bladder were randomized to receive cisplatin (60 mg/m2 i.v. every 21 days) or cisplatin (same dosage) combined with piroxicam (0.3 mg/kg orally every 24 h). Complete staging was performed prior to and at 6-week intervals during therapy. RESULTS: After eight dogs had been evaluated in each treatment group, a significant difference in remission rate was noted (Fisher's Exact test, P < 0.004). Tumor responses in the cisplatin/piroxicam group included two complete remissions (CR), four partial remissions (PR), two stable disease (SD), and no progressive disease (PD). Tumor responses to cisplatin alone in eight dogs were no CR, no PR, four SD, and four PD. Six additional dogs were treated with cisplatin/piroxicam, and in total 10 of 14 dogs had remission (two CR, eight PR). Renal toxicity of cisplatin/ piroxicam was frequent and dose limiting. CONCLUSIONS: Cisplatin/piroxicam induced remission more frequently than cisplatin alone in a canine model of human invasive TCC. Strategies to reduce renal toxicity need to be developed prior to evaluation of cisplatin/piroxicam in humans or general use of this treatment in pet dogs.

Humoral and cell-mediated immune responses in chickens with infectious bursal disease.

Primary and secondary immune responses to Newcastle disease virus (NDV) was evaluated in chickens infected with infectious bursal disease virus (IBDV) at one and 28 days of age. The geometric mean primary hemagglutination-inhibition antibody titers (GMT) of chickens infected with IBDV at one day of age was significantly lower (P less than or equal to 0.01) than those infected at 28 days of age. Infection with IBDV had no influence on secondary immune response to NDV. The effect of IBDV infection at one day of age on the cell-mediated immunity of chickens was evaluated by skin allograft acceptance or survival time. There was no significant difference between the percentage of grafts accepted in IBDV infected and noninfected control chickens. However, the mean graft survival time in the IBDV infected chickens was significantly longer (P less than or equal to 0.05) than those in the control group. This suggested a suppression of cell-mediated immunity due to IBDV infection.

SDS-soluble and peptidoglycan-bound proteins in the outer membrane-peptidoglycan complex of Brucella abortus.

Outer membrane-peptidoglycan complex from Brucella abortus was separated from cytoplasmic membrane and cytosol by either sucrose density gradient fractionation or differential (rate) centrifugation of surface labeled cells disrupted by sonication without the use of detergents. The outer membrane-peptidoglycan complex had a buoyant density of 1.22 gm/ml and contained 67 labeled SDS-soluble proteins when examined by SDS-PAGE. Included were four major bands exhibiting molecular masses of 88k, 40k, 35.7k and 26k daltons corresponding to previously described group 1, 2 and 3 outer membrane proteins. Lysozyme treatment of outer membrane-peptidoglycan complex increased its buoyant density to 1.25 gm/ml and released eight additional peptidoglycan-linked proteins.

The myth of Brucella L-forms and possible involvement of Brucella penicillin binding proteins (PBPs) in pathogenicity.

Brucella spp. L-forms have been proposed to be stationary phase organisms in the evolution of new variants and enduring entities in the host in complicated cases of brucellosis and during latent brucellosis. In vitro formation of Brucella L-forms has been achieved by treating the cells with sub-lethal doses of penicillin. Interestingly, Brucella spp. have classified during the evolution into two groups, penicillin susceptible or penicillin resistant, yet both types grow on 20 microg/ml of methicillin. Strains proven susceptible to penicillin grew in the presence of methicillin as L-forms as demonstrated by light and electron microscopy. In addition, the B. melitensis vaccine strain Rev.1, a penicillin susceptible organism, responded to sheep serum by development of L-form-like structures unlike wild type, strain 16M. The two strains grew normally in sheep macrophages. We propose, for the first time, a model that associates Brucella pathogenicity with the structure and activity of two of their penicillin binding proteins (PBPs). According to the model, PBP1 has evolved as the major cell wall synthesizing enzyme of the genus, capable of responding to host serum growth factor(s) necessary for Brucella survival in the host. This property is associated with high avidity to beta-lactam antibiotics. PBP2 complements the activity of PBP1. New beta-lactam antibiotics and improved vaccines might be developed based on this property.

Molecular fingerprinting confirms extensive cow-to-cow intra-herd transmission of a single Mycobacterium bovis strain.

In this study we have characterized M. bovis isolates from a herd of cattle in Uvalde, Texas in which 52 of the 193 animals selected at random in 1994 from a herd of 331 were caudal fold skin-test positive. Thirty-two of 52 skin-test positive cattle had gross lesions at slaughter, and isolations of M. bovis were made from 29 animals. The herd was comprised of Red Devon cattle purchased between 1978 and 1980 (n = 26) and breeding bulls (n = 3) introduced at later times, and all were tuberculosis test negative at the time of purchase. Other animals were natural additions (offspring) of these cattle. One additional animal, a Holstein present on the ranch at the time of purchase in 1976, was retained to nurse orphaned and weak calves. Using several molecular fingerprinting techniques we have verified a clonal relationship among the M. bovis isolates consistent with infection originating with a single strain. The molecular fingerprint patterns demonstrate the stability of the profiles despite persistence and spread of the organism within the herd for two decades and confirms their use in epidemiological tracing.

Immune responsiveness and lymphoreticular morphology in cattle fed hypo- and hyperalimentative diets.

Immune responses and lymphoreticular morphology were studied in 3 groups of yearling Hereford steers fed hypoalimentative, maintenance and hyperalimentative diets for 142 days. Significant decreases in plasma protein levels and circulating lymphocyte levels occurred in low energy intake steers. Percent circulating lymphocytes bearing surface immunoglobulins and serum levels of IgG and IgM did not vary significantly within or between groups. Antibody responses to Brucella abortus bacterin inoculated on day 63 were similar in all 3 groups. Antibody responses to chicken erythrocytes inoculated on day 88 were significantly lower in hypoalimentated steers. Differences between groups in lymphocyte blastogenic responses to phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) were not significant. Hyperalimentated steers had significantly depressed PWM responses compared to a baseline value established for that group. In addition, hypoalimentated steers tended to have elevated responses to PHA, although differences were not significant. There were no significant differences between groups in dermal hypersensitivity responses to tuberculin following sensitization with viable Bacillus Calmette-Guerin (BCG). The thymuses of hypoalimentated steers were markedly atrophied but lymph nodes and splenic white pulp were normal. Thymus, lymph nodes and spleen were normal in hyperalimentated steers.

The long-term culture of bovine monocyte-derived macrophages and their use in the study of intracellular proliferation of Brucella abortus.

Although the immune response to Brucella abortus is multifaceted, the key event in contending with this pathogen appears to be the interaction of the organism with cells of the mononuclear phagocyte system. A cell culture system was developed which allowed the long-term maintenance of blood monocyte-derived macrophages in Teflon culture vessels in a relatively unstimulated state. The assay system was optimized for timing of bacteria-macrophage interaction and numbers of bacteria and macrophages used in each assay. Interaction of B. abortus strain 2308 with bovine mononuclear phagocytes from animals phenotypically resistant and susceptible to infection with B. abortus was investigated. This cell culture and assay system should provide a useful model for the investigation of intracellular parasitism in cattle.

Intracellular survival of Brucella abortus, Mycobacterium bovis BCG, Salmonella dublin, and Salmonella typhimurium in macrophages from cattle genetically resistant to Brucella abortus.

Peripheral blood monocyte-derived macrophages were obtained from a herd of cows selected, bred, and confirmed as resistant or susceptible by in vivo challenge of Brucella abortus Strain 2308. The ability to control in vitro intracellular bacterial replication of B. abortus Strain 2308, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Montreal Strain 9003, Salmonella dublin Strain 5631, and Salmonella typhimurium Strain 14028 was evaluated in a bactericidal assay. The macrophages from resistant cattle were significantly superior (P < 0.05) in controlling intracellular growth of B. abortus, M. bovis BCG, S. dublin but not of S, typhimurium than macrophages from susceptible animals. Controls of all four pathogens correlated strongly with each other in resistant or susceptible macrophages. Data from resistant cattle had a tighter grouping than that of susceptible cattle, while data from susceptible cattle overlapped considerably with data from resistant animals. Therefore, this assay was considered a phenotypic marker of the resistant trait. For each bacterial species a percent bacterial survival value was used as a cut-off point to designate animals as resistant or susceptible. These data were compared with the in vivo challenged resistant or susceptible classification by using the Chi-square analyses. A cut-off point of 70 percent bacterial survival for B. abortus designated 14 cattle as susceptible and seven as resistant and this correlated 100 percent with the number of animals designated as to the relevant category by in vivo challenge. A value of 65 percent bacterial survival for M. bovis BCG, and 100 percent bacterial survival for S. dublin correlated highly with actual numbers of animals designated as susceptible or resistant.

Induction of lymphocyte responsiveness by the outer membrane-peptidoglycan complex of rough strains of Brucella abortus.

The outer membrane-peptidoglycan complex (OM-PG) from rough strains of Brucella abortus was tested for its ability to induce lymphocyte responsiveness in cattle. Six groups of heifers were immunized with varying doses and administration schedules of rough OM-PG and assayed for responsiveness of their lymphocytes in proliferation assays in vitro. All OM-PG preparations were emulsified in a commercial adjuvant for administration. Two other groups of heifers were immunized with strain 19 vaccine or adjuvant alone. Three groups of heifers received two inoculations of OM-PG antigens from a naturally-occurring rough strain at a 57-day interval. The doses of OM-PG given in these three groups were 400 micrograms, 1200 micrograms, and 4000 micrograms at each inoculation. The frequency of cows that responded in lymphocyte proliferation assays increased with the dose of OM-PG given. Two groups received single inoculations of OM-PG, either 2400 micrograms or 8000 micrograms. Although there were responsive cows in these immunization groups, their frequency was lower than in the groups receiving the same total dose in two inoculations. A sixth group of cows was inoculated with OM-PG from a rough transposon mutant of B. abortus, and the frequency of responsive cows in this immunization group was comparable to that of responsive cows immunized with the same dose of OM-PG from the spontaneous rough mutant. In comparisons of cows inoculated with strain 19 to those inoculated with OM-PG preparations, differences were observed in the relative responsiveness of their lymphocytes to whole cells and OM-PG in the in vitro lymphocyte proliferation assays. These differences suggested that lymphocytes stimulated by strain 19 vaccination have different specificities than those stimulated by immunization with OM-PG of rough mutant strains of B. abortus.

Mechanisms of binding of Brucella abortus to mononuclear phagocytes from cows naturally resistant or susceptible to brucellosis.

During the course of bovine brucellosis, Brucella abortus adheres to and infects cells of the mononuclear phagocyte system. Potential mechanisms of binding, as measured by numbers of phagocytosed bacteria, were studied in two populations of cattle genetically resistant (R) or susceptible (S) to infection with B. abortus. Live B. abortus gained entry into cultured bovine macrophages without organism-specific opsonization. Bacterial entry into macrophages from R was inhibited by the peptide RGDS, outer membrane-peptidoglycan complex from B. abortus strain RB51, anti-LFA-1 monoclonal antibody, anti-C3 antiserum, fibronectin, purified O-antigen from B. abortus lipopolysaccharide, mannan and heat-aggregated IgG. Bacterial entry into macrophages from S was inhibited by outer membrane-peptidoglycan complex, anti-LFA-1 monoclonal antibody, O-antigen and heat-aggregated IgG. The peptide RGES did not inhibit entry into macrophages from R or S. These data support the existence of organism-related receptors on monocyte-derived macrophages for B. abortus which mediate binding in the absence of serum. Secondly, there are demonstrable differences in mechanisms of binding of B. abortus to cells from cattle genetically resistant or susceptible to infection by this organism. These findings further substantiate the importance of phagocytosis and clearance functions of the mononuclear phagocyte system in resistance to bovine brucellosis. Perpetuation of infection in susceptible cattle may occur by establishing an intracellular reservoir of viable organisms. Further studies are necessary to investigate receptor affinities, and the potential for an alternate receptor for this organism in S cattle.

Survival of smooth, rough and transposon mutant strains of Brucella abortus in bovine mammary macrophages.

Transposon mutants offer a unique way to evaluate the role of lipopolysaccharide (LPS) by producing a theoretical single-gene difference between the original strain and the transposon mutant strain. Comparative survival of Brucella abortus smooth strain 2308, rough RB51, smooth strain 19, and two transposon mutant strains (rough strain 2308::Tn5 Lac Z [m106] and rough strain 19::Tn5 Lac Z [m3], was tested in restrictive bovine mammary macrophages that were able to effectively reduce the percentage of intracellular bacterial survival and permissive bovine mammary macrophages that were unable to control the intracellular replication of B. abortus. The theoretical single-gene difference between strain 19 and strain 19::Tn5 lac Z [m3] and between smooth virulent strain 2308 and rough transposon mutant 2308::Tn5 lacZ [m106] is likely related to differences in LPS content or structure. Significant (P less than 0.05) reduction in the survival of rough strain 19::Tn5 Lac Z [m3] with no significant reduction in the rough transposon mutant strain 2308::Tn5 lacZ [m106] indicated that at least one factor other than LPS contributes to the intracellular survival of B. abortus in bovine macrophages.