Detecting Methemoglobinemia in Animals with a Drop of Blood.

A major concern during pesticide development and use is the impact on non-target species, such as raptors or domestic cats and dogs. Sodium nitrite and para-aminopropiophenone (PAPP) are two toxicants currently being studied for the control of invasive species, such as starlings and feral swine. When given to an animal these compounds oxidize hemoglobin, which renders it unable to carry oxygen resulting in methemoglobinemia. This study developed a method to estimate methemoglobin levels in mammals and birds by examining the efficacy of sodium nitrite to induce the conversion of hemoglobin to methemoglobin. Varying concentrations of sodium nitrite were added to aliquots of coyote, vole, feral swine, starling, and duck blood, collected from captive animals. The blood samples were analyzed spectrophotometrically to determine percent methemoglobin and digitally to determine red color values (RCV) associated with different methemoglobin levels. The avian and mammalian blood reached 100% methemoglobin levels at 200 mM and 15 mM sodium nitrite, respectively. All animals had similar RCV for a given percent methemoglobin. In conclusion, this study developed a procedure to quickly determine methemoglobin levels in mammals and birds. Furthermore, percent methemoglobin can be estimated with one standard curve from any animal species and an image of a blood spot. The technique will be useful during field studies, in agricultural areas, or in a veterinarian's office for the rapid diagnosis of methemoglobinemia in non-target animals that have eaten toxicants/baits or baited animals.

Evaluation of the Borrelia burgdorferi BBA64 protein as a protective immunogen in mice.

The Borrelia burgdorferi bba64 gene product is a surface-localized lipoprotein synthesized within mammalian and tick hosts and is involved in vector transmission of disease. These properties suggest that BBA64 may be a vaccine candidate against Lyme borreliosis. In this study, protective immunity against B. burgdorferi challenge was assessed in mice immunized with the BBA64 protein. Mice developed a high-titer antibody response following immunization with soluble recombinant BBA64 but were not protected when challenged by needle inoculation of culture-grown spirochetes. Likewise, mice passively immunized with an anti-BBA64 monoclonal antibody were not protected against needle-inoculated organisms. BBA64-immunized mice were subjected to B. burgdorferi challenge by the natural route of a tick bite, but these trials did not demonstrate significant protective immunity in either outbred or inbred strains of mice. Lipidated recombinant BBA64 produced in Escherichia coli was assessed for possible improved elicitation of a protective immune response. Although inoculation with this antigen produced a high-titer antibody response, the lipidated BBA64 also was unsuccessful in protecting mice from B. burgdorferi challenge by tick bites. Anti-BBA64 antibodies raised in rats eradicated the organisms, as evidenced by in vitro borreliacidal assays, thus demonstrating the potential for BBA64 to be effective as a protective immunogen. However, passive immunization with the same monospecific rat anti-BBA64 polyclonal serum failed to provide protection against tick bite-administered challenge. These results reveal the challenges faced in not only identifying B. burgdorferi proteins with potential protective capability but also in producing recombinant antigens conducive to preventive therapies against Lyme borreliosis.

Borrelia burgdorferi bba66 gene inactivation results in attenuated mouse infection by tick transmission.

The impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tick-mouse cycle. The BBA66-deficient mutant isolate, Bb(ΔA66), remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired Bb(ΔA66) following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of Bb(ΔA66)-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of Bb(ΔA66) with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.

Borrelia burgdorferi visualized in Ixodes scapularis tick excrement by immunofluorescence.

The enzootic cycle of Borrelia burgdorferi, the etiologic agent of Lyme disease, involves Ixodes spp. ticks and vertebrates. Resident tick Borrelia, harbored inside the midgut, are eventually expelled with the tick's saliva into the vertebrate host when a tick consumes a blood meal. During this 4- to 5-day feeding period I. scapularis will defecate onto the host's skin. Previously we detected borrelial DNA in tick feces throughout engorgement. In this study we report the microscopic examination for B. burgdorferi in nymphal excrement. Using immunofluorescence assays, we observed Borrelia in all mouse skin and capsule fecal swabs tested, although we could not culture the spirochetes. These results update our previous analysis by revealing that spirochetes can also be visualized in tick excrement. Furthermore, the results emphasize that borrelial contamination by defecation is a possibility, and that caution should be exercised by researchers investigating pathogen/host/vector interactions. The biological significance of the presence of non-culturable Borrelia in tick feces during engorgement is unclear.

Saliva, salivary gland, and hemolymph collection from Ixodes scapularis ticks.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) (1-8). To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick (3,9-13). For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle (14,15). Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva (9,16-19). As a tick feeds the host typically responds with a strong hemostatic and innate immune response (11,13,20-22). Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding (3,11,20,21,23). The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens (3,20,24-27). To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Analysis of Borrelia burgdorferi Surface Proteins as Determinants in Establishing Host Cell Interactions.

Borrelia burgdorferi infection causes Lyme borreliosis in humans, a condition which can involve a systemic spread of the organism to colonize various tissues and organs. If the infection is left untreated by antimicrobials, it can lead to manifestations including, arthritis, carditis, and/or neurological problems. Identification and characterization of B. burgdorferi outer membrane proteins that facilitate cellular attachment and invasion to establish infection continue to be investigated. In this study, we sought to further define putative cell binding properties of surface-exposed B. burgdorferi proteins by observing whether cellular adherence could be blocked by antibodies. B. burgdorferi mixed separately with monoclonal antibodies (mAbs) against outer surface protein (Osp) A, OspC, decorin-binding protein (Dbp) A, BBA64, and RevA antigens were incubated with human umbilical vein endothelial cells (HUVEC) and human neuroglial cells (H4). B. burgdorferi treated with anti-OspA, -DbpA, and -BBA64 mAbs showed a significant decrease in cellular association compared to controls, whereas B. burgdorferi treated with anti-OspC and anti-RevA showed no reduction in cellular attachment. Additionally, temporal transcriptional analyses revealed upregulated expression of bba64, ospA, and dbpA during coincubation with cells. Together, the data provide evidence that OspA, DbpA, and BBA64 function in host cell adherence and infection mechanisms.

Functional analysis of the Borrelia burgdorferi bba64 gene product in murine infection via tick infestation.

Borrelia burgdorferi, the causative agent of Lyme borreliosis, is transmitted to humans from the bite of Ixodes spp. ticks. During the borrelial tick-to-mammal life cycle, B. burgdorferi must adapt to many environmental changes by regulating several genes, including bba64. Our laboratory recently demonstrated that the bba64 gene product is necessary for mouse infectivity when B. burgdorferi is transmitted by an infected tick bite, but not via needle inoculation. In this study we investigated the phenotypic properties of a bba64 mutant strain, including 1) replication during tick engorgement, 2) migration into the nymphal salivary glands, 3) host transmission, and 4) susceptibility to the MyD88-dependent innate immune response. Results revealed that the bba64 mutant's attenuated infectivity by tick bite was not due to a growth defect inside an actively feeding nymphal tick, or failure to invade the salivary glands. These findings suggested there was either a lack of spirochete transmission to the host dermis or increased susceptibility to the host's innate immune response. Further experiments showed the bba64 mutant was not culturable from mouse skin taken at the nymphal bite site and was unable to establish infection in MyD88-deficient mice via tick infestation. Collectively, the results of this study indicate that BBA64 functions at the salivary gland-to-host delivery interface of vector transmission and is not involved in resistance to MyD88-mediated innate immunity.

Detection of Borrelia burgdorferi DNA in tick feces provides evidence for organism shedding during vector feeding.

Borrelia burgdorferi, the bacterium that causes Lyme disease, is transmitted to a susceptible host by Ixodes spp. tick bites. However, there is uncertainty whether B. burgdorferi are shed from ticks by the fecal route. In this study, B. burgdorferi-infected ticks were fed on mice while confined to a certain area of the skin by a capsule. During and after feeding, tick feces were collected and placed in Barbour-Stoenner-Kelley (BSK)-II media for cultivation and in sterile water for polymerase chain reaction (PCR) analysis. Although none of the tested samples were culture positive for B. burgdorferi, all but one of the fecal DNA samples from infected ticks were PCR positive. These results indicated that B. burgdorferi were shed from feeding ticks during defecation and suggest that the spirochetes did not remain viable once exposed to the outside environment. This finding has important ramifications for investigators interpreting B. burgdorferi-specific PCR results when conducting tick transmission experiments.

The bba64 gene of Borrelia burgdorferi, the Lyme disease agent, is critical for mammalian infection via tick bite transmission.

The spirochetal agent of Lyme disease, Borrelia burgdorferi, is transmitted by bites of Ixodes ticks to mammalian reservoir hosts and humans. The mechanism(s) by which the organism is trafficked from vector to host is poorly understood. In this study, we demonstrate that a B. burgdorferi mutant strain deficient in the synthesis of the bba64 gene product was incapable of infecting mice via tick bite even though the mutant was (i) infectious in mice when introduced by needle inoculation, (ii) acquired by larval ticks feeding on infected mice, and (iii) able to persist through tick molting stages. This finding of a B. burgdorferi gene required for pathogen transfer and/or survival from the tick to the susceptible host represents an important breakthrough toward understanding transmission mechanisms involved for the Lyme disease agent.

Evaluation of the control of pathogen load by an anti-Salmonella bacterium in a herd of cattle with persistent Salmonella infection.

OBJECTIVE: To determine whether an anti-Salmonella bacterium is involved in control of pathogen load in persistently infected cattle herds. ANIMALS: 24 Holstein calves experimentally infected and 39 Holstein cows naturally infected with Salmonella spp. PROCEDURES: An Escherichia coli (designated as P8E5) that possessed anti-Salmonella activity was isolated from Salmonella-negative bovine feces obtained from a herd with endemic Salmonella infection. In vitro analysis involved enumerating Salmonella enterica serovar Typhimurium coincubated with E coli P8E5. In vivo analysis involved coadministration of Salmonella spp and E coli P8E5 or an E coli control strain to neonatal Holstein calves. Fecal samples were collected on multiple days after inoculation, and quantitative PCR assay was performed by use of Salmonella-specific primers. RESULTS: E coli P8E5 reduced viability of Salmonella spp in vitro. Shedding of Salmonella organisms was diminished in calves administered E coli P8E5, whereas the control strain of E coli had no effect on shedding of Salmonella organisms. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, an E coli strain was identified that possessed bacteriocin-like activity and was able to decrease viability of Salmonella organisms in vitro and in vivo. Therefore, it is possible that this organism could be representative of native microbiota that dampen Salmonella spp in persistently infected cattle herds.

Characterization of fecal microbiota from a Salmonella endemic cattle herd as determined by oligonucleotide fingerprinting of rDNA genes.

The gastrointestinal (GI) tract microbiota is composed of complex communities. For all species examined thus far, culture and molecular analyses show that these communities are highly diverse and individuals harbor unique consortia. The objective of the current work was to examine inter-individual diversity of cattle fecal microbiota and determine whether Salmonella shedding status correlated with community richness or evenness parameters. Using a ribosomal gene array-based approach, oligonucleotide fingerprinting of ribosomal genes (OFRG), we analyzed 1440 16S genes from 19 fecal samples obtained from a cattle herd with a history of salmonellosis. Identified bacteria belonged to the phyla Firmicutes (53%), Bacteroidetes (17%), and Proteobacteria (17%). Sequence analysis of 16S rDNA gene clones revealed that Spirochaetes and Verrucomicrobia were also present in the feces. The majority of Firmicutes present in the feces belonged to the order Clostridiales, which was verified via dot blot analysis. beta-Proteobacteria represented 1.5% of the bacterial community as determined by real-time PCR. Statistical analysis of the 16S libraries from the 19 animals indicated very high levels of species richness and evenness, such that individual libraries represented unique populations. Finally, this study did not identify species that prevented Salmonella colonization or resulted from Salmonella colonization.

Comparison of the cecal microbiota of domestic and wild turkeys.

The extent to which production methods alter intestinal microbial communities of livestock is currently unknown. As the intestinal microbiota may affect animal health, nutrition, and food safety, a baseline comparison of the cecal communities of domestic and wild turkeys was performed. Oligonucleotide fingerprinting of ribosomal RNA (rRNA) genes (OFRG) of 2,990 16S rRNA clones and dot blot quantification of dominant populations were used to identify the dominant bacterial taxa. Seventy-three percent of all the clones belonged to as yet uncultured genera. However, at a higher phylogenetic level, the OFRG library was composed of 54% Bacteroidetes clones (52% of the domestic library clones, 56% of the wild library clones), 30% Firmicutes clones (33% of the domestic library clones, 32% of the wild library clones), 3% Proteobacteria clones (5% domestic, 2% wild), and 3% Deferribacteres clones (4% domestic, 1% wild). Seven percent of the clones were unidentifiable (6% domestic, 9% wild). Bacteroidetes clones included the genera Alistipes, Prevotella, Megamonas, and Bacteroides. Of the Clostridiales clones, groups IV, IX, and XIV including genera Faecalibacterium, Megasphaera, Phascolarctobacterium, and Papillibacter were predominant. Lactobacillus, Enterococcus, and Streptococcus bacilli were also identified. beta- delta- and gamma-proteobacterial genera included Acinetobacter, Sutterella, and Escherichia. Deferribacteres clones showed high similarity to Mucispirillum schaedleri. Statistical comparison of the domestic and wild turkey clone libraries indicated similar levels of community richness and evenness despite the fact that the two libraries shared only 30% of the total clone operational taxonomic units. Together these results indicate that although high level taxonomic community structure is similar, high-density turkey production causes considerable divergence of the genera found in the ceca of commercial birds from those of their wild counterparts.

The role of proton motive force in expression of the Staphylococcus aureus cid and lrg operons.

The Staphylococcus aureus cidABC and lrgAB operons have been shown to play a key role in the regulation of murein hydrolase activity and cell death in a manner thought to be analogous to bacteriophage-encoded holins and anti-holins respectively. Because of these functions, it has been proposed that the regulation of these operons is tightly controlled and responsive to key metabolic signals. The current study revealed the presence of two overlapping regulatory pathways controlling cidABC and lrgAB expression, one dependent on acetic acid and the other dependent on proton motive force (PMF). The latter pathway was analysed using agents that affect various aspects of the PMF. Gramicidin and carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimicrobial agents that dissipate the DeltapH and membrane potential (DeltaPsi), both enhanced lrgAB expression. Restoration of the PMF by incubation of the bacteria in the presence of glucose restored lrgAB expression back to the uninduced state. In addition, valinomycin, which specifically collapses the DeltaPsi, also induced lrgAB expression. In contrast, nigericin, which dissipates the DeltapH component of the PMF, was found to have a minimal effect on DeltaPsi and lrgAB transcription. Finally, the DeltaPsi-inducible expression of lrgAB was shown to be dependent on the previously characterized LytSR two-component regulatory system that is involved in the regulation of autolysis. The results of this study support a model in which the LytSR regulatory system responds to a collapse in DeltaPsi by inducing the transcription of the lrgAB operon.

A LysR-type regulator, CidR, is required for induction of the Staphylococcus aureus cidABC operon.

The Staphylococcus aureus cidABC and lrgAB operons have been shown to regulate murein hydrolase activity and affect antibiotic tolerance. The cid operon enhances murein hydrolase activity and antibiotic sensitivity, whereas the lrg operon inhibits these processes. Based on these findings and the structural similarities of the cidA and lrgA gene products to the bacteriophage holin family of proteins, we have proposed that the cid and lrg operons encode holin- and antiholin-like proteins, respectively, that function to control the murein hydrolase activity produced by the bacteria. Analysis of cid operon transcription revealed the presence of two transcripts, one spanning all three cid genes and whose expression is induced by growth in the presence of acetic acid and the other spanning cidB and cidC only that is produced in a sigma B-dependent manner. The cidABC operon lies immediately downstream from the cidR gene, encoding a potential LysR-type transcriptional regulator. In this study, we demonstrate that cidR is involved in the regulation of cidABC expression. Northern blot analyses revealed that the cidR gene product positively regulates cidABC expression by increasing transcription in the presence of acetic acid produced as a result of the metabolism of glucose. As expected for an operon that encodes a positive effector of murein hydrolase activity, the upregulation of cidABC expression resulted in increased murein hydrolase activity produced by these cells. Furthermore, it was demonstrated that antibiotic tolerance and stationary-phase survival of S. aureus are affected by the cidR gene. Taken together, these results demonstrate that the cidR gene product functions as a transcriptional activator of cidABC transcription in response to acetic acid accumulation in the growth medium.

The Staphylococcus aureus cidC gene encodes a pyruvate oxidase that affects acetate metabolism and cell death in stationary phase.

The Staphylococcus aureus cid and lrg operons have previously been shown to affect murein hydrolase activity and antibiotic tolerance. Based on their similarities to the holin family of proteins it was proposed that the functions of the cidA and lrgA gene products are analogous to bacteriophage-encoded holin and antiholin proteins respectively. The cid operon expresses two overlapping transcripts, one that spans the cidA, cidB and cidC genes and whose expression is induced by the acetic acid generated by aerobic growth in the presence of excess glucose, and the other that spans the cidB and cidC genes only and is expressed in a sigma B-dependent manner. In the study presented here, we have focused primarily on the third gene of this operon, cidC. A sequence analysis of the cidC gene product suggested that it encodes a pyruvate oxidase that catalyses the oxidative decarboxylation of pyruvate yielding acetate and CO(2). Indeed, a ferricyanide-based spectrophotometric assay revealed that the cidC mutant produced decreased pyruvate oxidase activity relative to the parental and complemented strains. In the presence of excess glucose the cidC mutant accumulated normal levels of acetic acid in the growth medium, likely because of the activity of the pyruvate dehydrogenase complex. However, in contrast to the wild type and complemented strains, the pH of the cidC mutant culture began to increase gradually until it was able to utilize the acetate for a secondary round of growth. Finally, a mutation in cidA caused reduced cell lysis in stationary phase but only minimally affected cell death. These results indicate that the cidC gene product is involved in the generation of acetic acid that contributes to the cell death and lysis that occurs in high-glucose stationary phase cultures, while the cidA gene product, a putative holin, controls lysis of the dying cells.

Acetic acid induces expression of the Staphylococcus aureus cidABC and lrgAB murein hydrolase regulator operons.

The Staphylococcus aureus lrg and cid operons encode homologous proteins that regulate extracellular murein hydrolase activity and penicillin tolerance in a diametrically opposing manner. Although their specific regulatory functions remain unknown, it has been postulated that the functions of CidA and LrgA are analogous to those of bacteriophage holins and antiholins, respectively, and that these proteins serve as molecular control elements of bacterial programmed cell death. Although these studies demonstrated that cidBC transcription is abundant in sigmaB-proficient strains, cidABC transcription was only minimally expressed under standard growth conditions. In this study, we demonstrate that cidABC and lrgAB transcription in the clinical isolate UAMS-1 is induced by growth in the presence of 35 mM glucose and that this enhances murein hydrolase activity and decreases tolerance to vancomycin and rifampin. The effect of glucose on murein hydrolase activity was not observed in the cidA mutant, indicating that the induction of this activity was dependent on enhanced cidABC expression. Furthermore, we demonstrate that the effects of glucose on cidABC and lrgAB transcription are mediated by the generation of acetic acid produced by the metabolism of this and other carbon sources. These results shed new light on the control of the S. aureus cidABC and lrgAB genes and demonstrate that these operons, as well as murein hydrolase activity and antibiotic tolerance, are responsive to carbohydrate metabolism.

The Staphylococcus aureus cidAB operon: evaluation of its role in regulation of murein hydrolase activity and penicillin tolerance.

Recent studies have shown that expression of the Staphylococcus aureus lrgAB operon inhibits murein hydrolase activity and decreases sensitivity to penicillin-induced killing. It was proposed that the lrgAB gene products function in a manner analogous to an antiholin, inhibiting a putative holin from transporting murein hydrolases out of the cell. In the present study the cidAB operon was identified and characterized based on the similarity of the cidA and cidB gene products to the products of the lrgAB operon. Zymographic and quantitative analyses of murein hydrolase activity revealed that mutation of the cidA gene results in decreased extracellular murein hydrolase activity compared to that of S. aureus RN6390, the parental strain. Complementation of cidA expression restored the wild-type phenotype, indicating that expression of the cidAB operon has a positive influence on extracellular murein hydrolase activity. The cidA mutant also displayed a significant decrease in sensitivity to the killing effects of penicillin. However, complementation of the cidA defect did not restore penicillin sensitivity to wild-type levels. Reverse transcriptase PCR also revealed that cidAB is maximally expressed during early exponential growth, opposite of what was previously observed for lrgAB expression. Based on these results, we propose that the cidAB operon encodes the holin-like counterpart of the lrgAB operon and acts in a manner opposite from that of lrgAB by increasing extracellular murein hydrolase activity and increasing sensitivity to penicillin-induced killing.