Mechanisms of infertility associated with clinical and subclinical endometritis in high producing dairy cattle.

Clinical and subclinical endometritis are common causes of infertility and subfertility in high producing dairy cattle, delaying the onset of ovarian cyclic activity after parturition, extending luteal phases and reducing conception rates. Escherichia coli and Arcanobacterium pyogenes cause endometrial damage and inflammation. Components of microbes, such as lipopolysaccharide (LPS), are detected by Toll-like receptors on endometrial cells, leading to secretion of cytokines, chemokines and antimicrobial peptides. Long luteal phases associated with endometritis are probably caused by a switch in endometrial prostaglandin production from prostaglandin F2a (PGF) to prostaglandin E2. In addition, LPS impairs the function of the hypothalamus and pituitary, and directly perturbs ovarian granulosa cells steroidogenesis, providing mechanisms to explain the association between uterine disease and anovulatory anoestrus. Cows with uterine disease that ovulate have lower peripheral plasma progesterone concentrations that may further reduce the chance of conception associated with endometritis.

Use of bacteriophages in combination with competitive exclusion to reduce Salmonella from infected chickens.

Salmonella-spedfic bacteriophages (BP) and competitive exclusion (CE) were used to reduce Salmonella colonization in experimentally infected chickens. A "cocktail" of distinct phage (i.e., phage showing different host ranges and inducing different types of plaques on Salmonella Typhimurium [ST] cultures) was developed. The killing activity of the selected BPs on ST cultures differed significantly, as determined in in vitro killing assays. BPs were administered orally to the chickens several days prior and after ST challenge but not simultaneously. BPs were readily isolated from the feces of the BP-treated chickens approximately 48 hr after administration. A CE product consisting of a defined culture of seven different microbial species was used either alone or in combination with BP treatment. CE was administered orally at hatch. Salmonella counts in intestine, ceca, and a pool of liver/spleen were evaluated in Salmonella-challenged chickens treated with BP or with BP and CE. In both trials 1 and 2, a beneficial effect of the phage treatment on weight gain performance was evident. A reduction in Salmonella counts was detected in cecum and ileum of BP-, CE-, and BP+CE-treated chickens as compared with nontreated birds. In trial 1, BP treatment reduced ST counts to marginal levels in the ileum and reduced counts sixfold in the ceca. A reduction of Salmonella counts with BP, CE, and BP+CE treatments was evident in chickens from trial 2. Both CE and BP treatments showed differences in the reduction of Salmonella counts after challenge between spedmens obtained at days 4 and 14 postchallenge in ceca, liver/spleen, and ileum. The preliminary data presented in this report show that isolation and characterization of Salmonella-specific BP is uncomplicated and feasible on a larger scale. Results indicate a protective effect of both Salmonella-specific BPs and a defined competitive exclusion product against Salmonella colonization of experimentally infected chickens. These results are encouraging for further work on the use of BP as an effective alternative to antibiotics to reduce Salmonella infections in poultry.

Role of rpoS in acid resistance and fecal shedding of Escherichia coli O157:H7.

Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator sigma(S) and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10(1) to 10(4) CFU, we found the wild-type strain in feces of mice given lower doses (10(2) versus 10(3) CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10(4) CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P

Transcriptional analysis of the Yersinia pestis pH 6 antigen gene.

The pH 6 antigen of Yersinia pestis is a virulence protein whose gene, psaA, is positively regulated at the transcriptional level by low pH, mammalian temperature, and an upstream locus, psaE. Low pH appears to be required for initial psaA transcription, although increased temperature is necessary for full expression of the gene. In addition, psaA is monocistronic and its transcript has a relatively long 5' nontranslated region.

Molecular analysis of a cryptic plasmid isolated from avian strains of Pasteurella multocida.

Twelve small plasmids isolated from avian strains of Pasteurella multocida were examined by restriction enzyme mapping, cross-hybridization, and minicell analysis. These plasmids contained sites for several commonly used restriction enzymes and ranged in size from 3.4 to 3.8 kilobases. Restriction enzyme maps of the 12 plasmids were similar and divided the plasmids into 3 families, designated pFS1, pFS2, and pFS4. Restriction fragments of pFS1 DNA isolated from strain X-73 were used to probe AvaI/HindIII/EcoRV digests of pFS2 and pFS4 DNA. The results of these hybridization experiments demonstrated that the plasmids found in all three families shared extensive regions of homology and may have originated from a common ancestor. Escherichia coli minicells containing recombinant plasmid constructs bearing fragments of pFS1 expressed two pFS1-specific peptides, 12.5 and 28 kilodaltons in size, suggesting that some P. multocida plasmid-encoded proteins can be expressed in E. coli. These results indicate that pFS may be useful as a genetic tool for moving DNA into and out of P. multocida, since it is small, contains common restriction sites, and encodes at least two genes that are recognized and expressed in E. coli.

The Yersinia pestis V antigen is a regulatory protein necessary for Ca2(+)-dependent growth and maximal expression of low-Ca2+ response virulence genes.

The low-Ca2+ response is a multicomponent virulence regulon of the human-pathogenic yersiniae in which 12 known virulence genes are coordinately regulated in response to environmental cues of temperature, Ca2+, and nucleotides such as ATP. Yersinial growth also is regulated, with full growth yield being permitted at 37 degrees C only if Ca2+ or a nucleotide is present. In this study, we constructed and characterized a mutant Yersinia pestis specifically defective in the gene encoding the V antigen, one of the virulence genes of the low-Ca2+ response. An in-frame internal deletion-insertion mutation was made by removing bases 51 through 645 of lcrV and inserting 61 new bases. The altered lcrV was introduced into the low-Ca2+ response plasmid in Y. pestis by allelic exchange, and the resulting mutant was characterized for its two-dimensional protein profiles, growth, expression of an operon fusion to another low-Ca2+ response virulence operon, and virulence in mice. The mutant had lost its Ca2+ and nucleotide requirement for growth, showed diminished expression of Ca2(+)-and nucleotide-regulated virulence genes, and was avirulent in mice. The mutation could be complemented with respect to the growth property by supplying native V antigen operon sequences in trans in high copy number (on pBR322). Partial complementation of the growth defect and almost complete complementation of the virulence defect were seen with a lower-copy-number complementing replicon (a pACYC184 derivative). The data are consistent with the interpretation that V antigen is bifunctional, with a role in regulating growth and expression of low-Ca2+ response virulence genes in addition to its putative role as a secreted virulence protein.

Molecular characterization of the Clostridium difficile toxin A gene.

The gene encoding the toxin A protein of Clostridium difficile (strain VPI 10463) was cloned and sequenced. The coding region of 8,133 base pairs had a mol% G + C of 26.9 and encodes 2,710 amino acids. The deduced polypeptide has a molecular mass of ca. 308 kilodaltons. Nearly a third of the gene, at the 3' end, consists of 38 repeating sequences. The repeating units were grouped into two classes, I and II, on the basis of length and the low levels of DNA sequence similarities between them. There were seven class I repeating units, each containing 90 nucleotides, and 31 class II units, which, with two exceptions, were either 60 or 63 nucleotides in length. On the basis of DNA sequence similarities, the class II repeating units were further segregated into subclasses: 7 class IIA, 13 class IIB, 5 class IIC, and 6 class IID. The dipeptide tyrosine-phenylalanine was found in all 38 repeating units, and other amino acid sequences were unique to a specific class or subclass. This region of the protein has epitopes for the monoclonal antibody PCG-4 and includes the binding region for the Gal alpha 1-3Gal beta 1-4GlcNAc carbohydrate receptor. Located 1,350 base pairs upstream from the toxin A translation start site is the 3' end of the toxin B gene. Between the two toxin genes is a small open reading frame, which encodes a deduced polypeptide of ca. 16 or 19 kilodaltons. The role of this open reading frame is unknown.

Molecular analysis of lcrGVH, the V antigen operon of Yersinia pestis.

The lcrGVH operon of plasmid pCD1 in Yersinia pestis KIM encodes the virulence-associated V antigen, the regulatory protein LcrH, and LcrG, a protein of undefined function. In this study we sequenced lcrGVH and analyzed it for transcription initiation sites. There were three open reading frames within the sequence, 288, 981, and 507 bases in length, which could encode proteins with molecular weights and isoelectric points corresponding to those of LcrG, LcrV (V antigen), and LcrH, respectively. The predicted LcrV protein lacked an N-terminal signal sequence; however, an internal signallike sequence was present. An Escherichia coli-like promoter consensus sequence was detected upstream from lcrG. Primer extension analysis showed that (i) the transcriptional start site for lcrGVH was spaced only three bases upstream from the nearest ATG potential start site, raising the possibility that Y. pestis may use an alternate initiation codon for the V operon; (ii) there was much more primer-extended product in yersiniae grown in the absence of Ca2+ than in its presence, showing for the first time that lcrGVH is regulated at the transcriptional level by Ca2+; (iii) no separate lcrV initiation was detected, indicating that the V antigen is expressed from messages initiating at lcrG; and (iv) a non-Ca2+-regulated transcriptional start site was found upstream from lcrH, suggesting that the LcrH protein is expressed constitutively. However, two-dimensional gel analysis showed that net LcrH expression was regulated by Ca2+. We propose that lcrH lies within two differentially regulated operons, its own and lcrGVH.

lcrH, a gene necessary for virulence of Yersinia pestis and for the normal response of Y. pestis to ATP and calcium.

We are investigating the functions of the three proteins encoded by the V operon (lcrGVH) of the low-calcium response virulence plasmid pCD1 of Yersinia pestis KIM5. The purpose of this study was to define the role of the 18-kilodalton protein encoded by lcrH, the third gene of the V operon. Using marker exchange mutagenesis, we constructed a Y. pestis mutant that failed to express the LcrH protein. This LcrH- mutant was "ATP blind" in that it failed to show altered growth and V-antigen expression at 37 degrees C when 18 mM ATP was present. It also showed only a partial response to 2.5 mM Ca2+. The parental Y. pestis strain showed full growth yield at 37 degrees C and depressed expression of V antigen and of yop (yersinial pCD1-encoded outer membrane protein) genes in response to ATP or Ca2+. In contrast, the LcrH- mutant failed to grow at 37 degrees C in the presence of ATP and showed only limited growth when Ca2+ was present. V-antigen expression in the mutant was not depressed by ATP and only partially depressed by Ca2+. These findings show that LcrH is necessary for the normal response of Y. pestis to ATP and that LcrH contributes to Ca2+ responsiveness. The mutant also showed abnormal yopJ expression, indicating that LcrH also is necessary for normal yop regulation. The LcrH- mutant was avirulent in mice, probably because of its compromised growth at 37 degrees C. These findings indicate that the responses of Y. pestis to ATP and Ca2+ are distinct and that lcrH encodes a protein that is an important mediator of Ca2+ and ATP regulation of pCD1-encoded virulence determinant(s) in Y. pestis.

Using treatment plans for quality assurance monitoring in a residential center.

Treatment plans for children in residential centers are required for accreditation and reimbursement, and they can provide important insights to the quality of care. One residential center has developed a system for reviewing treatment plans as part of its quality assurance (QA) monitoring. QA staff use checklists to review plans to assure that they are complete and technically correct and to evaluate the quality and appropriateness of care as revealed in the treatment strategy.

Studies on bacterial synergism in mice infected with Bacteroides intermedius and Fusobacterium necrophorum.

This study was undertaken to characterize the kinetics of possible bacterial synergy using a mouse model of mixed intraabdominal infection with Bacteroides intermedius and Fusobacterium necrophorum. Female CD-1 mice were injected intraperitoneally with B. intermedius, F. necrophorum or mixtures of both organisms. Generalized septic peritonitis developed within 24 hr, with abscess formation occurring after one to two wk in survivors with the mixed infection. Involvement of the reticuloendothelial system was evidenced by dose-dependent hepatosplenomegaly, which appeared during the first wk postinfection and progressed throughout the course of the experiment. Indirect immunofluorescence confirmed the presence of both species of bacteria in frozen sections of liver tissue. The median lethal dose (LD50) was 2.11 x 10(9) for the mixture, 3.03 X 10(9) for B. intermedius alone, and 1.07 X 10(9) for F. necrophorum alone. The median abscess-producing dose (AD50), the dose required to produce abscesses in fifty percent of the surviving mice at two wk, was approx. 1/100 of the LD50 dose. The AD50 for intrahepatic abscesses was 2.8 x 10(8) for the mixture, whereas the AD50 for intraabdominal abscesses occurring in any site was 5.14 X 10(7). Both Bacteroides and Fusobacterium persisted in tissue for at least 22 wk following mixed infection. The persistence of the Bacteroides in tissue represents a synergistic result of mixed infection with Fusobacterium and contributed to the chronicity of intraabdominal abscess formation. Bacteroides, injected alone, did not produce abscesses at any of the doses tested. However, when passaged (isolated from mixed infection hepatic abscesses) B. intermedius was used, the bacteria did induce abscesses.

Enhancement of Bacteroides intermedius growth by Fusobacterium necrophorum.

Previous work from this laboratory has demonstrated the persistence of Bacteroides intermedius in the livers of mice receiving an intraperitoneal inoculum of B. intermedius and Fusobacterium necrophorum. This study was undertaken to determine whether F. necrophorum enhanced the in vitro growth of B. intermedius. Tryptose phosphate broth did not support the growth of B. intermedius alone, but the bacterium did survive in a tryptose phosphate broth culture of F. necrophorum. B. intermedius cultured in F. necrophorum-conditioned tryptose phosphate broth grew impressively, reaching maximal absorbance at 24 h after inoculation. The growth of B. intermedius in F. necrophorum-conditioned tryptose phosphate broth was proportional to the amount of conditioned medium present. The B. intermedius growth-stimulating factor was detectable in conditioned medium 8 h after inoculation with F. necrophorum and could be detected throughout the 96-h incubation period. Growth-factor-active fractions eluted from a Sephadex G-100 column did not absorb at 280 nm and were retained on the column until 4 column volumes were eluted. The growth factor was nondialyzable and stable to boiling, lyophilization, extraction with hot aqueous phenol, and trypsin digestion. The factor was inactivated by exposure to pH 2.0 in the pepsin digestion protocol. Significant amounts of hexose, methyl pentose, and 2-keto-3-deoxyoctonate were detected in pooled growth-factor-active fractions eluted from the Sephadex column. This pool was also active in the Limulus lysate endotoxin assay. These results suggest that the B. intermedius growth-stimulating factor produced by F. necrophorum is a lipopolysaccharide.

Comparison of antimicrobial susceptibility patterns among coagulase-negative staphylococci.

Coagulase-negative staphylococci, identified as S. epidermidis, S. saprophyticus, S. haemolyticus, S. hominis, and S. warneri, were tested for susceptibility to a number of antimicrobial agents by disk agar diffusion and broth microdilution methods, S. warneri and S. saprophyticus were found to be the most susceptible, and S. haemolyticus and S. epidermidis were found to be the least susceptible. Differences in antimicrobial susceptibility patterns are discussed, along with their epidemiological and therapeutic implications.