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The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. Here, we identified an invariant motif in the disordered C-terminal tail of fungal Cdc14 enzymes that is required for full enzyme activity. Mutation of this motif reduced Cdc14 catalytic rate and provided a tool for studying the biological significance of high Cdc14 activity. A S. cerevisiae strain expressing the reduced-activity hypomorphic mutant allele (cdc14hm) as the sole source of Cdc14 proliferated like the wild-type parent strain but exhibited an unexpected sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. Sensitivity to echinocandins was also observed in Schizosaccharomyces pombe and Candida albicans strains lacking CDC14, suggesting this phenotype reflects a novel and conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. In C. albicans, the orthologous cdc14hm allele was sufficient to elicit echinocandin hypersensitivity and perturb cell wall integrity signaling. It also caused striking abnormalities in septum structure and the same cell separation and hyphal differentiation defects previously observed with cdc14 gene deletions. Since hyphal differentiation is important for C. albicans pathogenesis, we assessed the effect of reduced Cdc14 activity on virulence in Galleria mellonella and mouse models of invasive candidiasis. Partial reduction in Cdc14 activity via cdc14hm mutation severely impaired C. albicans virulence in both assays. Our results reveal that high Cdc14 activity is important for C. albicans cell wall integrity and pathogenesis and suggest that Cdc14 may be worth future exploration as an antifungal drug target.

The recent impact of Ebola virus disease (EVD) on public health in Africa clearly demonstrates the need for a safe and efficacious vaccine to control outbreaks and mitigate its threat to global health. ERVEBO® is an effective recombinant Vesicular Stomatitis Virus (VSV)-vectored Ebola virus vaccine (VSV-EBOV) that was approved by the FDA and EMA in late 2019 for use in prevention of EVD. Since the parental virus VSV, which was used to construct VSV-EBOV, is pathogenic for livestock and the vaccine virus may be shed at low levels by vaccinated humans, widespread deployment of the vaccine requires investigation into its infectivity and transmissibility in VSV-susceptible livestock species. We therefore performed a comprehensive clinical analysis of the VSV-EBOV vaccine virus in swine to determine its infectivity and potential for transmission. A high dose of VSV-EBOV resulted in VSV-like clinical signs in swine, with a proportion of pigs developing ulcerative vesicular lesions at the nasal injection site and feet. Uninoculated contact control pigs co-mingled with VSV-EBOV-inoculated pigs did not become infected or display any clinical signs of disease, indicating the vaccine is not readily transmissible to naïve pigs during prolonged close contact. In contrast, virulent wild-type VSV Indiana had a shorter incubation period and was transmitted to contact control pigs. These results indicate that the VSV-EBOV vaccine causes vesicular illness in swine when administered at a high dose. Moreover, the study demonstrates the VSV-EBOV vaccine is not readily transmitted to uninfected pigs, encouraging its safe use as an effective human vaccine.

Head and neck squamous cell carcinoma (HNSCC) is the 8th most common cancer in the United States predominantly affecting people over 65 years of age with an increasing rate of incidence across the world. Current therapies for HNSCC include surgical resection, chemotherapy (CT), and radiotherapy (RT). For locally advanced/unresectable HNSCC, the CT-RT combination (“chemoradiation”) has been shown to be more effective than CT or RT alone, and is currently the standard of care. Intratumoral (IT) chemotherapy-based chemoradiation has the potential to overcome the limitations of conventional systemic CT-RT that severely affects a patient’s quality of life. For realization of maximum benefits from IT CT-RT, our team has developed a radiation-controlled drug release nanoparticle formulation (paclitaxel (PTX) and CaWO4 nanoparticles (CWO NPs) co-encapsulated within a capsule formed by poly(ethylene glycol)-poly (lactic acid) (PEG-PLA), named “PEG-PLA/CWO/PTX NPs”). This unique formulation releases PTX only when it is exposed to X-ray irradiation. We have previously reported that IT-administered PEG-PLA/CWO/PTX NPs stay within the tumor for at least a month, producing significant therapeutic effects in terms of tumor suppression and survival in mouse models of HNSCC. This work demonstrates the effect of PTX stereochemistry on radiation-controlled release of the drug from a nano polymer matrix system (PEG-PLA/CWO/PTX NPs). The stereoisomertic characteristics of PTX products from two different manufacturers (“PTX-S”, and “PTX-B”) were analyzed by Raman spectroscopy, circular dichroism and 2D HMQC/NOESY NMR measurements. In their unencapsulated (free) state, PTX-S and PTX-B were comparable in their ability to kill cancer cells in vitro. However, they were found to be significantly different in water solubility; PTX-S (water solubility ≈ 4.69 μg/mL) is about 19 times more water soluble than PTX-B (water solubility ≈ 0.25 μg/mL). This difference in water solubility was found to cause a large difference in X-ray-triggered release kinetics of the PTX loaded within the PEG-PLA/CWO/PTX NPs in both in vitro and in vivo environments; PTX-S is released from PEG-PLA/CWO NPs significantly faster upon X-ray irradiation than PTX-B. This difference in release kinetics produced an interesting difference in their time-dependent therapeutic effects; at short times (< 1 month), concurrent PEG-PLA/CWO/PTX-S NPs produced a greater tumor suppression effect; on the other hand, PEG-PLA/CWO/PTX-B NPs had a longer lasting radio-sensitizing effect. In summary, the stereoisomers of PTX exhibit significantly different PK characteristics when used with controlled release carriers, even though they are pharmacologically indistinguishable in their unformulated form. Citation Format: Kaustabh Sarkar, Sandra Torresgrossa-Allen, Mark P. Langer, Gregory Durm, Sanjeev Narayanan, Bennett D. Elzey, You-Yeon Won. Effect of paclitaxel stereochemistry on x-ray-triggered release of paclitaxel from CaWO4/paclitaxel-coloaded PEG-PLA nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 304.

Animal models are essential to cancer research, but current xenograft models are limited in their utility especially due to the lack of an immune system. Here we demonstrate that a xenograft tumor model can be developed in immunocompetent mice by tolerizing murine fetuses to human tumor cells. A375 human melanoma cells were injected into day E14 fetuses and after birth mice were challenged with A375 cells to determine their ability to develop tumors. Intravenous injections of cells resulted in metastatic-like lung tumors, which were verified to be human in origin by immunohistochemistry and PCR. These results were replicated with several other human tumor types: BxPC3 (human pancreatic adenocarcinoma), MDA-MB-231 (human breast adenocarcinoma), M21 (human melanoma), and HeLa (human cervical adenocarcinoma). Development of an immunocompetent xenograft tumor model would allow the further elucidation of the interaction of the immune system with therapy in both preclinical research and patient derived xenografts.

Green iguanas with gastrointestinal, hepatic, and reproductive disease may present to the veterinary clinic displaying non-specific clinical signs, necessitating a thorough clinical evaluation. A 15-year-old intact female green iguana (Iguana iguana) presented with a 3-week history of decreased appetite and coelomic distension. A complete blood count and plasma biochemistry analysis showed anaemia and leukopenia with an absolute lymphopenia; decreased sodium and chloride concentrations; and increased phosphorus, potassium, aspartate transaminase, and uric acid concentrations. Whole body radiographs revealed a large, round, homogenous soft tissue opacity structure in the coelom. Ultrasonography confirmed a thin-walled cystic structure located in the cranial coelomic cavity. An exploratory coeliotomy revealed a large hepatic cyst, diffusely abnormal hepatic parenchyma, and cysts on the right ovary. The right ovary and hepatic cyst were surgically excised, however the iguana died under general anesthesia. Histopathology provided a diagnosis of multifocal cholangiocarcinoma and ovarian papillary adenocarcinoma. This case report alerts the clinician to the clinical presentation, anaesthetic protocol, surgical intervention, and challenges associated with the diagnosis of a large cystic cholangiocarcinoma in a pet green iguana.

Summary The gut-brain axis is crucial to microbial-host interactions. The neurotransmitter serotonin is primarily synthesized in the gastrointestinal (GI) tract, where it is secreted into the lumen and subsequently removed by the serotonin transporter, SERT. Here, we show that serotonin decreases virulence gene expression by enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium, a murine model for EHEC. The membrane-bound histidine sensor kinase, CpxA, is a bacterial serotonin receptor. Serotonin induces dephosphorylation of CpxA, which inactivates the transcriptional factor CpxR controlling expression of virulence genes, notably those within the locus of enterocyte effacement (LEE). Increasing intestinal serotonin by genetically or pharmacologically inhibiting SERT decreases LEE expression and reduces C. rodentium loads. Conversely, inhibiting serotonin synthesis increases pathogenesis and decreases host survival. As other enteric bacteria contain CpxA, this signal exploitation may be engaged by other pathogens. Additionally, repurposing serotonin agonists to inhibit CpxA may represent a potential therapeutic intervention for enteric bacteria.

Feline infectious peritonitis and virulent, systemic calicivirus infection are caused by certain types of feline coronaviruses (FCoVs) and feline caliciviruses (FCVs), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families, the Coronaviridae and the Caliciviridae , respectively, they share a dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally and structurally conserved among these viruses and essential for viral replication, 3CLpro is considered a potential target for the design of antiviral drugs with broad-spectrum activities against these distinct and highly important viral infections. However, small-molecule inhibitors against the 3CLpro enzymes of FCoV and FCV have not been previously identified. In this study, derivatives of peptidyl compounds targeting 3CLpro were synthesized and evaluated for their activities against FCoV and FCV. The structures of compounds that showed potent dual antiviral activities with a wide margin of safety were identified and are discussed. Furthermore, the in vivo efficacy of 3CLpro inhibitors was evaluated using a mouse model of coronavirus infection. Intraperitoneal administration of two 3CLpro inhibitors in mice infected with murine hepatitis virus A59, a hepatotropic coronavirus, resulted in significant reductions in virus titers and pathological lesions in the liver compared to the findings for the controls. These results suggest that the series of 3CLpro inhibitors described here may have the potential to be further developed as therapeutic agents against these important viruses in domestic and wild cats. This study provides important insights into the structure and function relationships of 3CLpro for the design of antiviral drugs with broader antiviral activities. IMPORTANCE Feline infectious peritonitis virus (FIPV) is the leading cause of death in young cats, and virulent, systemic feline calicivirus (vs-FCV) causes a highly fatal disease in cats for which no preventive or therapeutic measure is available. The genomes of these distinct viruses, which belong to different virus families, encode a structurally and functionally conserved 3C-like protease (3CLpro) which is a potential target for broad-spectrum antiviral drug development. However, no studies have previously reported a structural platform for the design of antiviral drugs with activities against these viruses or on the efficacy of 3CLpro inhibitors against coronavirus infection in experimental animals. In this study, we explored the structure-activity relationships of the derivatives of 3CLpro inhibitors and identified inhibitors with potent dual activities against these viruses. In addition, the efficacy of the 3CLpro inhibitors was demonstrated in mice infected with a murine coronavirus. Overall, our study provides the first insight into a structural platform for anti-FIPV and anti-FCV drug development.

Probiotics, an antibiotic alternative, are widely used as feed additives for performance benefits in cattle and swine production systems. Among bacterial species contained in probiotics, Enterococcus faecium is common. Antimicrobial resistance (AMR), particularly multidrug resistance, is a common trait among enterococci because of their propensity to acquire resistance and horizontally transfer AMR genes. Also, E. faecium is an opportunistic pathogen, and in the United States, it is the second most common nosocomial pathogen. There has been no published study on AMR and virulence potential in E. faecium contained in probiotic products used in cattle and swine in the United States. Therefore, our objectives were to determine phenotypic susceptibilities or resistance to antimicrobials, virulence genes (asa1, gelE, cylA, esp, and hyl) and assess genetic diversity of E. faecium isolated from commercial products. Twenty-two commercially available E. faecium-based probiotic products used in cattle (n = 13) and swine (n = 9) were procured and E. faecium was isolated and species confirmed. Antimicrobial susceptibility testing to determine minimum inhibitory concentrations was done by micro-broth dilution method using National Antimicrobial Resistance Monitoring Systems Gram-positive Sensititre panel plate (CMV3AGPF), and categorization of strains as susceptible or resistant was as per Clinical Laboratory and Standards Institute's guidelines. E. faecium strains from 7 products (3 for swine and 4 for cattle) were pan-susceptible to the 16 antimicrobials tested. Strains from 15 products (6 for swine and 9 for cattle) exhibited resistance to at least one antimicrobial and a high proportion of strains was resistant to lincomycin (10/22), followed by tetracycline (4/22), daptomycin (4/22), ciprofloxacin (4/22), kanamycin (3/22), and penicillin (2/22). Four strains were multidrug resistant, with resistant phenotypes ranging from 3 to 6 antimicrobials or class. None of the E. faecium strains were positive for any of the virulence genes tested. The clonal relationships among the 22 E. faecium strains were determined by pulsed-field gel electrophoresis (PFGE) typing. A total of 10 PFGE patterns were observed with 22 strains and a few of the strains from different probiotic products had identical (100% Dice similarity) PFGE patterns. In conclusion, the E. faecium strains in a few commercial probiotics exhibited AMR to medically-important antimicrobials, but none contained virulence genes.

SUMMARY The enteric pathogen enterohemorrhagic Escherichia coli (EHEC) causes severe diarrhea, but the influence of the gut microbiota on EHEC infection is largely unknown. A predominant member of the microbiota, Bacteroides thetaiotaomicron (Bt), is resident at EHEC attachment sites. We show that Bt enhances EHEC virulence gene expression through the transcription factor Cra, which is functionally sensitive to sugar concentrations. This enhanced virulence accompanies increased formation of attaching and effacing (AE) lesions requisite for EHEC colonization. Infection with Citrobacter rodentium, a natural mouse pathogen homologous to EHEC, in Bt-reconstituted mice results in increased gut permeability along with exacerbated host pathology and mortality compared to mice deplete of microflora. Bt modifies the metabolite environment at infection sites, increasing metabolites involved in gluconeogenesis, with stark increases in succinate, which can be sensed by Cra. Our findings suggest that microbiota composition affects disease outcome and may explain links between microbiota composition and disease susceptibility.

Fusobacterium necrophorum, a Gram-negative, anaerobic bacterium, is an opportunistic animal and human pathogen that causes a variety of infections termed necrobacillosis. There are 2 subspecies of F. necrophorum (subsp. necrophorum and subsp. funduliforme) that differ morphologically and biochemically and in virulence. Leukotoxin, a secreted protein, is considered to be the major virulence factor. In camelids, F. necrophorum causes a variety of infections, generally involving the lips, tongue, pharynx, interdigital spaces, foot pad, larynx, mandible, or maxillary bones. The objective of the current study was to characterize the presumptive Fusobacterium isolates from a variety of necrotic infections in llama ( Lama glama) and alpaca ( Vicugna pacos) and determine whether the strains possess leukotoxin activities. A total of 7 isolates from alpaca and 2 isolates from llama were characterized. Based on growth characteristics in broth culture, and biochemical and polymerase chain reaction analyses, all 9 isolates belonged to subsp. necrophorum and possessed the putative hemagglutinin gene. Western blot analysis with antileukotoxin antibodies raised in rabbit showed the presence of leukotoxin protein in the culture supernatant of all isolates. Furthermore, flow cytometry of the culture supernatants demonstrated cytotoxicity to bovine and alpaca polymorphonuclear leukocytes (PMNs). The extent of cytotoxicity to either alpaca or bovine PMNs differed among camelid strains. The cytotoxicity of many of the camelid strains was higher ( P < 0.05) toward alpaca PMNs compared to bovine PMNs. Fusobacterium necrophorum isolates from llama and alpaca are similar to bovine isolates, and leukotoxin may be a major virulence factor.

Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, which is largely used as a surrogate EHEC model for murine infections, are exposed to several host neurotransmitters in the gut. An important chemical exchange within the gut involves the neurotransmitters epinephrine and/or norepinephrine, extensively reported to increase virulence gene expression in EHEC, acting through two bacterial adrenergic sensors: QseC and QseE. However, EHEC is unable to establish itself and cause its hallmark lesions, attaching and effacing (AE) lesions, on murine enterocytes. To address the role of these neurotransmitters during enteric infection, we employed C. rodentium. Both EHEC and C. rodentium harbor the locus of enterocyte effacement (LEE) that is necessary for AE lesion formation. Here we show that expression of the LEE, as well as that of other virulence genes in C. rodentium, is also activated by epinephrine and/or norepinephrine. Both QseC and QseE are required for LEE gene activation in C. rodentium, and the qseC and qseE mutants are attenuated for murine infection. C. rodentium has a decreased ability to colonize dopamine β-hydroxylase knockout (Dbh−/−) mice, which do not produce epinephrine and norepinephrine. Both adrenergic sensors are required for C. rodentium to sense these neurotransmitters and activate the LEE genes during infection. These data indicate that epinephrine and norepinephrine are sensed by bacterial adrenergic receptors during enteric infection to promote activation of their virulence repertoire. This is the first report of the role of these neurotransmitters during mammalian gastrointestinal (GI) infection by a noninvasive pathogen., IMPORTANCE The epinephrine and norepinephrine neurotransmitters play important roles in gut physiology and motility. Of note, epinephrine and norepinephrine play a central role in stress responses in mammals, and stress has profound effects on GI function. Bacterial enteric pathogens exploit these neurotransmitters as signals to coordinate the regulation of their virulence genes. The bacterial QseC and QseE adrenergic sensors are at the center of this regulatory cascade. C. rodentium is a noninvasive murine pathogen with a colonization mechanism similar to that of EHEC, enabling the investigation of host signals in mice. The presence of these neurotransmitters in the gut is necessary for C. rodentium to fully activate its virulence program, in a QseC/QseE-dependent manner, to successfully colonize its murine host. Our study data provide the first example of epinephrine and norepinephrine signaling within the gut to stimulate infection by a bacterial pathogen in a natural animal infection.

Background Cyanophycin (CGP) is a sustainable polymer that can be converted to a derivative with reduced arginine content, or to completely biodegradable poly-aspartic acid, which can substitute for non-biodegradable polyacrylates. In nature, it is produced by most cyanobacteria; however, these microbes are not suitable for large-scale production due to slow growth and low polymer content. Results Cyanophycin synthetase gene (cphA) from Anabaena variabilis ATCC 29413 was PCR amplified and cloned into Escherichia coli. Different renewable media components, such as soybean meal, potato wastes and corn-derived zein hydrolysate, were evaluated for their feasibility for CGP production at shake flask level. The optimized conditions were then tested in a 7 L bioreactor; a maximum cell weight of 10.2 g L-1 was obtained. CGP comprised 23 g g-1 of cell dry matter (CDM) with molecular weight between 21.5 and 31 kDa and was composed of aspartic acid, arginine and lysine in the ratio of 1.05:1:0.2 (mass basis). Conclusion The efficient use of renewable biomass for cyanophycin production could achieve competitive price, and in return, promote the use of this platform chemical to produce innovative polymers and materials.© 2012 Society of Chemical Industry

Potential risks associated with impaired surface water quality have commonly been evaluated by indirect description of potential sources using various fecal microbial indicators and derived source-tracking methods. These approaches are valuable for assessing and monitoring the impacts of land-use changes and changes in management practices at the source of contamination. A more detailed evaluation of putative etiologically significant genetic determinants can add value to these assessments. We evaluated the utility of using a microarray that integrates virulence genes with antibiotic and heavy metal resistance genes to describe and discriminate among spatially and seasonally distinct water samples from an agricultural watershed creek in Eastern Ontario. Because microarray signals may be analyzed as binomial distributions, the significance of ambiguous signals can be easily evaluated by using available off-the-shelf software. The FAMD software was used to evaluate uncertainties in the signal data. Analysis of multilocus fingerprinting data sets containing missing data has shown that, for the tested system, any variability in microarray signals had a marginal effect on data interpretation. For the tested watershed, results suggest that in general the wet fall season increased the downstream detection of virulence and resistance genes. Thus, the tested microarray technique has the potential to rapidly describe the quality of surface waters and thus to provide a qualitative tool to augment quantitative microbial risk assessments.

The ability of pathogenic bacteria to sense and respond to periods of host stress is critical to their lifestyle. Adrenaline and norepinephrine are catecholamines that mediate acute host stress in vertebrates and invertebrates. Catecholamines are also used as environmental cues to enhance growth, motility and virulence of bacterial pathogens via specific binding receptors. Incidence of multidrug resistant and highly virulent bacterial pathogens is on the rise, and majority of the genes for antimicrobial resistance (AMR) and virulence are carried on horizontally transferable genetic elements. Conjugation machinery offers an efficient method for acquisition of AMR and virulence genes, which may be responsible for propelling the evolution of pathogenic bacteria. Here we show that norepinephrine (NE) at physiological concentrations enhances horizontal gene transfer (HGT) efficiencies of a conjugative plasmid from a clinical strain of Salmonella Typhimurium to an Escherichia coli recipient in vitro. Expressions of plasmid encoded transfer (tra) genes necessary for conjugation were also significantly upregulated in the presence of NE. Phentolamine, an α-adrenergic receptor antagonist, negated the effects of NE on conjugation more strongly than propranolol, a β-adrenergic receptor antagonist. This study for the first time provides evidence that innate mediators of acute host stress may influence evolution and adaptation of bacterial pathogens.

Objective—To evaluate the impact of oxytetracycline exposure on horizontal transfer of an antimicrobial resistance plasmid. Sample—Populations of Salmonella enterica subsp enterica serovar Typhimurium and Escherichia coli. Procedures—Mixed populations of plasmid donor (Salmonella Typhimurium) and recipient (E coli) bacteria were assigned to 1 of 2 simulated oxytetracycline dosing regimens (high peak concentration-short elimination half-life [HC-SHL] or low peak concentration—long elimination half-life [LC-LHL]) or served as untreated control replicates. Donor, recipient, and transconjugant (E coli that acquired the plasmid) bacteria populations were quantified at 12, 24, and 36 hours after oxytetracycline administration by use of culture on selective bacterial growth media. Results—The ratio of transconjugant to donor bacteria was significantly reduced in the oxytetracycline-exposed replicates, compared with the ratio for the control replicates, at 12 hours. At 24 and 36 hours, results for the HC-SHL regimens were not significantly different from results for the respective control replicates, and results for the LC-LHL regimens also were not significantly different from results for the respective control replicates. The oxytetracycline concentration at these time points (12 hours in the HC-SHL regimen and all 3 time points in the LC-LHL regimen) were in excess of the minimum inhibitory concentration of the recipient bacteria. Conclusions and Clinical Relevance—Transfer of antimicrobial resistance plasmids may be suppressed in vitro by oxytetracycline exposure at concentrations greater than the minimum inhibitory concentration of the recipient bacteria.

Summary Cattle are a primary reservoir of Escherichia coli O157:H7, a major foodborne pathogen. The organism causes haemorrhagic colitis which can lead to serious complications, including haemolytic–uraemic syndrome. Although E. coli O157:H7 is widely prevalent in cattle and cattle environments, the number of human cases remain relatively low, suggesting possible strain diversity and differences in virulence between human and bovine strains. Shiga toxins, Stx1 and Stx2, are the major virulence factors. Differences in Stx2 production between human and bovine strains have been demonstrated previously, and isolates possessing the stx2 gene, but not producing Stx2 [toxin non-producing (TNP) strains] have been identified. In this study, 150 isolates (56 human, 94 bovine) were tested by PCR for stx2 upstream regions associated with TNP and the Q933 gene, which has been previously associated with toxin production. A reverse passive latex agglutination test was used to evaluate 107 isolates (50 human, 57 bovine) for Stx1 and Stx2 production. The percentages of human and bovine isolates positive for presence of the TNP regions were similar (57.1% and 53.1% respectively), while a higher percentage of human isolates was positive for Q933 gene (89.3% versus 54.3%). Stx2 production of ≥1 : 8 was found in 86.0% of human isolates compared with 26.3% of bovine isolates. Bovine isolates with the presence of the TNP regions were associated with significantly lower Stx2 production (P 0.05) with low Stx2 production in human isolates. Therefore, Q933 was a better indicator of high Stx2 production by human and bovine isolates and may be a useful screening method to assess their potential to cause human disease.

Cattle are fed elevated concentrations of copper and zinc for growth promotion. The potential mechanisms of growth promotional effects of these elements are attributed to their antimicrobial activities, similar to that of antibiotics, in that gut microbial flora are altered to reduce fermentation loss of nutrients and to suppress gut pathogens. Copper and zinc fed at elevated concentrations may select for bacteria that are resistant not only to heavy metals but also to antibiotics. Our objectives were to determine the effects of feeding elevated copper and zinc on the antimicrobial susceptibilities of fecal bacteria in feedlot cattle. Twenty heifers, fed corn-based high-grain diets, were randomly assigned to treatments in a 2 x 2 factorial arrangement with 1X or 10X National Research Council recommended copper and/or zinc. Feces, collected on days 0, 14, and 32, were cultured for commensal bacteria (Escherichia coli and Enterococcus) to determine their susceptibilities to copper, zinc, and antibiotics. Fecal DNA was extracted to detect tcrB gene and quantify erm(B) and tet(M) genes. In E. coli and Enterococcus sp., minimal differences in minimum inhibitory concentrations (MICs) of copper, zinc, and antibiotics were noticed. The mean copper MIC for E. coli increased (p0.05) between days 0 and 32 and days 14 and 32. The only treatment effect detected was increased zinc MIC of E. coli isolates (p0.01). The tcrB gene was not detected in feces or in enterococcal isolates. Proportions of erm(B) and tet(M) were unaffected by copper or zinc supplementation. However, the proportion of tet(M) increased (p0.05) between days 0 and 14. Feeding elevated copper and/or zinc to feedlot cattle had marginal effects on antimicrobial susceptibilities of fecal E. coli and enterococci.

Heavy metal contamination negatively affects natural systems including plants, birds, fish and bacteria by reducing biodiversity at contaminated sites. At the Tri-State Mining District, efforts have been made to remediate sites to mitigate the detrimental effects that contamination has caused on human health. While the remediation effort has returned the site to within federal safety standards, it is unclear if this effort is sufficient to restore floral and faunal communities. Intrinsic to ecosystem and organism health is the biodiversity and composition of microbial communities. We have taken advantage of recent advances in sequencing technology and surveyed the bacterial community of remediated and reference soils as well as the intestinal microbial community of two ubiquitous rodent species to provide insight on the impacts of residual heavy metal contamination on the ecosystem. Rodents found on the remediated site had reduced body mass, smaller body size and lower body fat than animals on reference sites. Using bar-coded, massively parallel sequencing, we found that bacterial communities in both the soil and Peromyscus spp. gastrointestinal tracts had no difference in diversity between reference and remediated sites but assemblages differed in response to contamination. These results suggest that niche voids left by microbial taxa that were unable to deal with the remnant levels of heavy metals on remediated sites were replaced by taxa that could persist in this environment. Whether this replacement provided similar ecosystem services as ancestral bacterial communities is unknown.

Distillers grains, a coproduct of ethanol production from cereal grains, are composed principally of the bran, protein, and germ fractions and are commonly supplemented in ruminant diets. The objective of this study was to assess the effect of feeding wet distillers grains with solubles (WDGS) and monensin and tylosin on the prevalence and antimicrobial susceptibilities of fecal foodborne and commensal bacteria in feedlot cattle. Cattle were fed 0 or 25% WDGS in steam-flaked corn-based diets with the addition of no antimicrobials, monensin, or monensin and tylosin. Fecal samples were collected from each animal (n = 370) on d 122 and 136 of the 150-d finishing period and cultured for Escherichia coli O157. Fecal samples were also pooled by pen (n = 54) and cultured for E. coli O157, Salmonella, commensal E. coli, and Enterococcus species. Antimicrobial resistance was assessed by determining antimicrobial susceptibilities of pen bacterial isolates and quantifying antimicrobial resistance genes in fecal samples by real-time PCR. Individual animal prevalence of E. coli O157 in feces collected from cattle fed WDGS was greater (P < 0.001) compared with cattle not fed WDGS on d 122 but not on d 136. There were no treatment effects on the prevalence of E. coli O157 or Salmonella spp. in pooled fecal samples. Antimicrobial susceptibility results showed Enterococcus isolates from cattle fed monensin or monensin and tylosin had greater levels of resistance toward macrolides (P = 0.01). There was no effect of diet or antimicrobials on concentrations of 2 antimicrobial resistance genes, ermB or tetM, in fecal samples. Results from this study indicate that WDGS may have an effect on the prevalence of E. coli O157 and the concentration of selected antimicrobial resistance genes, but does not appear to affect antimicrobial susceptibility patterns in Enterococcus and generic E. coli isolates.

Fusobacterium necrophorum, a Gram-negative anaerobe, causes a variety of necrotic infections in humans and animals. There are two subspecies: subsp. necrophorum and subsp. funduliforme. In cattle, subsp. necrophorum is more prevalent and production of leukotoxin is a major virulence factor. The leukotoxin operon (lktBAC) consists of three genes, lktB, lktA and lktC, of which lktA is the structural toxin gene. The subspecies identity of human F. necrophorum is less certain and it is not known whether human strains possess the leukotoxin gene or leukotoxin activity. Therefore, the objective of this study was to identify the subspecies status of four human clinical strains of F. necrophorum and determine whether they have the leukotoxin gene or leukotoxin activity. Phenotypic and genotypic characteristics suggested that the four strains belonged to subsp. funduliforme, which was confirmed by sequencing the 16S rRNA gene. Analysis of the four strains by PCR revealed the presence of the leukotoxin operon. Partial DNA sequencing identified one human strain with full-length lktA, whereas the others exhibited considerable heterogeneity in size. All strains had a leukotoxin operon promoter-containing intergenic region similar to that of bovine subsp. funduliforme strains, which was confirmed by DNA sequencing and Southern blotting. Despite variations in the lktA gene, all strains secreted leukotoxin as demonstrated by Western blotting. Flow cytometry assays revealed that the leukotoxin was toxic to human white blood cells. In conclusion, the human strains examined contained a leukotoxin gene whose gene product was biologically active. The importance of leukotoxin as a virulence factor in human fusobacterial infections needs further evaluation.

Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections.

Fusobacterium necrophorum, a gram-negative, anaerobic and rod-shaped bacterium, is generally an opportunistic pathogen and causes a wide variety of necrotic infections in animals and humans. Leukotoxin, a secreted protein, is a major virulence factor. The gene encoding the leukotoxin (lktA) in F. necrophorum has been cloned, sequenced and expressed in Escherichia coli. Because of low expression levels, problems associated with purifying full-length recombinant protein, and of the physical instability of the protein, five overlapping leukotoxin gene truncations were constructed. The recombinant polypeptides (BSBSE, SX, GAS, SH, and FINAL) were expressed in E. coli and purified by nickel-affinity chromatography. The objectives were to investigate the effectiveness of the purified truncated polypeptides to induce protective immunity in mice challenged with F. necrophorum. The polypeptides, individually or in combination, and inactivated native leukotoxin or culture supernatant of F. necrophorum were homogenized with an adjuvant and injected into mice on days 0 and 21. Blood samples were collected to measure serum anti-leukotoxin antibody titers on days 0, 21 and 42 and on day 42, mice were experimentally challenged with F. necrophorum. All polypeptides were immunogenic, with GAS polypeptide eliciting the least antibody response. Two polypeptides (BSBSE and SH) induced significant protection in mice against F. necrophorum infection. Protection was better than the full-length native leukotoxin or inactivated supernatant.The study demonstrated that the leukotoxin of F. necrophorum carries epitopes that induce protective immunity against experimental fusobacterial infection, thus providing further evidence to the importance of leukotoxin as a major virulence factor.

Leukotoxins are a group of exotoxins that produce their primary toxic effects against leukocytes, especially polymorphonuclear cells (PMNs). Leukotoxins include a variety of chemicals ranging from 9,10-epoxy 12-octadecenoate, a fatty acid derivative secreted by leukocytes themselves, to proteins such as RTX (repeats in toxin). This review focuses on leukotoxins of three species of gram-negative bacteria, Mannheimia (Pasteurella) haemolytica, Actinobacillus actinomycetemcomitans, and Fusobacterium necrophorum.

Animal models are essential to preclinical cancer research and are used clinically to determine optimal treatment regimens, but current xenograft models are limited in their utility, especially due to the lack of a competent immune system. Here we demonstrate that a xenograft tumor model can be developed in immunocompetent mice by tolerizing murine fetuses to human tumor cells. A375 human melanoma cells were injected into day E14 fetuses and, after birth, mice were challenged again with A375 cells to determine their ability to develop tumors. Intravenous injections of A375 cells after tolerization resulted in metastatic-like lung tumors, which were verified to be human in origin by immunohistochemistry and PCR. The development of lung neoplasms was dependent on fetal tolerization; non-tolerized mice did not develop tumors after injection. Interestingly, subcutaneous injected cells did not form tumors, but this was shown to be due to an innate, non-adaptive immune response and did not create lasting rejection of tumor cells. This procedure was repeated with several different tumor lines to show the universal nature of the method: BxPC3 (human pancreatic cancer), M21 (human melanoma), HeLa (human cervical cancer), and MDA231 (human breast carcinoma). Citation Format: Matthew T. Basel, Sanjeev Narayanan, Chanran Ganta, Tej B. Shrestha, Marla Pyle, Stefan H. Bossmann, Deryl L. Troyer. Developing a xenograft human tumor model in immunocompetent mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4820. doi:10.1158/1538-7445.AM2017-4820

Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA ) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the whole lktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin from F. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli . Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktA hybridizing bands between isolates of the two subspecies of F. necrophorum .

PCR amplification of the intergenic spacer region (ISR) between 16S and 23S rRNA genes among subspecies of the anaerobic bacterium Fusobacterium necrophorum gave identical patterns, with two forms of ISR identified. However, extra bands resulting from anomalous electrophoretic mobility of amplified DNA fragments with certain primer combinations were encountered. Therefore, PCR assays relying solely on banding patterns may be unreliable, and supporting sequence analysis is essential for correct culture identification.

Cefovecin is an extended-spectrum long-acting third generation cephalosporin used to treat canine infections. The study objective was to determine the effect of cefovecin on the absolute number and antimicrobial susceptibility of fecal enteric bacteria in healthy dogs. Fourteen Beagles were randomly assigned to a treated (n = 7, 8 mg/kg cefovecin subcutaneously on day 1) or untreated (n = 7) group. LC/MS was used to determine plasma cefovecin concentration on day 14. E. coli, enterococci, and Salmonella were isolated and enumerated from fecal samples collected on days 0, 3, 7, 14, and 28. Antimicrobial resistance was determined using disc diffusion, MIC, and detected using PCR for the blaCMY-2 gene on select isolates. Mean plasma concentration of cefovecin on day 14 was 9.59 μg/mL in treated dogs; untreated dogs had no measurable plasma cefovecin. The absolute number of E. coli was lower in treated dogs on day 3 (P ⩽ 0.0001), and the absolute number of cefovecin-resistant E. coli was higher in treated dogs on days 7 (P = 0.002), 14 (P = 0.004) and 28 (P ⩽ 0.0001), compared to untreated dogs. Enterococci increased and were higher in the treatment group on day 7 (P = 0.0226). Isolation of Salmonella was rare. After cefovecin treatment, beta-lactam resistance was more common in fecal E. coli from treated dogs than untreated dogs, while resistance of enterococci was not altered. On day 28, treated dogs were 3.25 times more likely to carry the blaCMY-2 gene than untreated dogs (95% CI 1.27 – 8.35). The implications of these findings in clinically ill patients require further research.

Fusobacterium necrophorum, classified into subsp. necrophorum (Fnn) and subsp. funduliforme (Fnf), is frequently associated with necrotic infections of animals and humans. The outer membrane proteins (OMP) of many Gram negative bacteria play an important role in bacterial adhesion and establishment of infection. The OMP profile of F. necrophorum has not been well characterized. We analyzed OMP of bovine strains of Fnn and Fnf and human strains of F. necrophorum. Electrophoretic separations of extracted OMP of Fnn and Fnf strains of cattle showed a total of 19 and 20 protein bands, respectively. The most prominent protein band was 40 kDa in Fnn and 37.5 kDa in Fnf. The four human clinical strains examined had more heterogeneous banding patterns and had different profiles than those of bovine Fnf strains. A total of 11 protein bands in Fnn and 13 protein bands in Fnf were recognized by sera from cattle with liver abscesses. The intensities of many of the bands in Fnn were higher than that of Fnf. We conclude that the two subspecies of F. necrophorum differ in their OMP profiles and the difference may account for differences in their virulence and involvement in the pathogenesis of necrotic infections.

Fusobacterium necrophorum, a Gram-negative anaerobe, is frequently associated with suppurative and necrotic infections of animals and humans. The organism is a major bovine pathogen, and in cattle, the common fusobacterial infections are hepatic abscesses, foot rot, and necrotic laryngitis. The species comprises two subspecies: F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme. Bacterial adhesion to the host cell surface is a critical initial step in the pathogenesis, and outer membrane proteins (OMP) play an important role in adhesion and establishment of certain Gram-negative bacterial infections. The means by which F. necrophorum attaches to epithelial or endothelial cells has not been determined. We evaluated whether OMP of F. necrophorum, isolated from a liver abscess, mediated adhesion to bovine endothelial cells (adrenal gland capillary endothelial cell line). The extent of binding of subsp. necrophorum to the endothelial cells was higher than that of F. necrophorum subsp. funduliforme. Trypsin treatment of bacterial cells decreased their binding to endothelial cells indicating the protein nature of adhesins. Preincubation of endothelial cells with OMP extracted from F. necrophorum decreased the binding of bacterial cells. In addition, binding of each subspecies to endothelial cells was inhibited by polyclonal antibodies raised against respective OMP and the antibody-mediated inhibition was subspecies specific. The western blot analysis of OMP bound to endothelial cells with anti-OMP antibodies showed four OMP of 17, 24, 40 and 74 kDa. We conclude that OMP of F. necrophorum play a role in adhesion of bacterial cells to the endothelial cells.

The Gram-positive pathogen Enterococcus faecalis is a leading agent of nosocomial infections, including urinary tract infections, surgical site infections, and bacteremia. Among the infections caused by E. faecalis , endocarditis remains a serious clinical manifestation and unique in that it is commonly acquired in a community setting. Infective endocarditis is a complex disease, with many host and microbial components contributing to the formation of bacterial biofilm-like vegetations on the aortic valve and adjacent areas within the heart. In the current study, we compared the pathogenic potential of the vancomycin-resistant E. faecalis V583 and three isogenic protease mutants (Δ gelE , Δ sprE , and Δ gelE Δ sprE mutants) in a rabbit model of enterococcal endocarditis. The bacterial burdens displayed by GelE − mutants (Δ gelE and Δ gelE Δ sprE mutants) in the heart were significantly lower than those of V583 or the SprE − mutant. Vegetations on the aortic valve infected with GelE − mutants (Δ gelE and Δ gelE Δ sprE mutants) also showed a significant increase in deposition of fibrinous matrix layer and increased chemotaxis of inflammatory cells. In support of a role for proteolytic modulation of the immune response to E. faecalis , we also demonstrate that GelE can cleave the anaphylatoxin complement C5a and that this proteolysis leads to decreased neutrophil migration in vitro . In vivo , a decreased heterophil (neutrophil-like cell) migration was observed at tissue sites infected with GelE-producing strains but not at those infected with SprE-producing strains. Taken together, these observations suggest that of the two enterococcal proteases, gelatinase is the principal mediator of pathogenesis in endocarditis.

Salmonella enterica is an important enteric pathogen consisting of many serovars that can cause severe clinical diseases in animals and humans. Rapid identification of Salmonella isolates is especially important for epidemiologic monitoring and controlling outbreaks of disease. Although immunologic and DNA-based serovar identification methods are available for rapid identification of isolates, they are time consuming or costly or both. In the current study, 2 molecular methods for identification of Salmonella serovars were developed and validated. A 70-mer oligonucleotide spotted microarray was developed that consisted of probes that detected genes responsible for genetic variation among isolates of Salmonella that can be used for serotyping. A multiplex polymerase chain reaction (PCR) assay was also developed, which is capable of identifying 42 serovars, thus providing a valuable prediction of the pathogenicity of the isolates by detecting the presence of virulence genes sseL, invA, and spvC. The gene spvC was the best predictor of pathogenicity. In a blind study, traditional serologic methods were correlated at 93.3% with the microarray-based method and 100% with the multiplex PCR-based serovar determination.

High concentration of copper, fed as copper sulfate, is often used to increase growth rates in swine. Bacteria exposed to copper may acquire resistance, and in Enterococcus faecium and Enterococcus faecalis, a plasmid-borne transferable copper resistance (tcrB) gene that confers copper resistance has been reported. Our objectives were to determine the occurrence of tcrB in fecal enterococci from weaned piglets fed diets with a normal supplemental level (16.5 ppm) or an elevated supplemental level (125 ppm) of copper and to determine the association of tcrB with copper, erythromycin, and vancomycin resistance. A total of 323 enterococcal isolates were examined and 15 (4.6%) isolates (14 E. faecium and 1 E. faecalis) were positive for tcrB. Fifteen tcrB-positive and 15 randomly chosen tcrB-negative isolates from piglets fed the normal supplemental level of copper were tested for erm(B), tet(M), vanA, and vanB genes and susceptibilities to copper, erythromycin, tetracyclines, and vancomycin. All tcrB-positive and -negative isolates contained erm(B) and tet(M), but not vanA and vanB. The mean minimum inhibitory concentration of copper for tcrB-positive (21.1 mM) was higher (p0.001) compared with tcrB-negative isolates (6.1 mM). All isolates were resistant to erythromycin and tetracyclines and susceptible to vancomycin. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains raises the possibility that copper supplementation may exert selection pressure for antibiotic-resistant enterococci. This study is the first report on the occurrence of the tcrB gene in enterococci isolated from swine in the United States.

Influenza virus infects the respiratory system of human and animals causing mild to severe illness which could lead to death. Although vaccines are available, there is still a great need for influenza antiviral drugs to reduce disease progression and virus transmission. Currently two classes (M2 channel blockers and neuraminidase inhibitors) of FDA-approved influenza antiviral drugs are available, but there are great concerns of emergence of viral resistance. Therefore, timely development of new antiviral drugs against influenza viruses is crucial. Plant-derived polyphenols have been studied for antioxidant activity, anti-carcinogenic, and cardio- and neuroprotective actions. Recently, some polyphenols, such as resveratrol and epigallocatechin gallate, showed significant anti-influenza activity in vitro and/or in vivo. Therefore we investigated selected polyphenols for their antiviral activity against influenza A and B viruses. Among the polyphenols we tested, isoquercetin inhibited the replication of both influenza A and B viruses at the lowest effective concentration. In a double treatment of isoquercetin and amantadine, synergistic effects were observed on the reduction of viral replication in vitro. The serial passages of virus in the presence of isoquercetin did not lead to the emergence of resistant virus, and the addition of isoquercetin to amantadine or oseltamivir treatment suppressed the emergence of amantadine- or oseltamivir-resistant virus. In a mouse model of influenza virus infection, isoquercetin administered intraperitoneally to mice inoculated with human influenza A virus significantly decreased the virus titers and pathological changes in the lung. Our results suggest that isoquercetin may have the potential to be developed as a therapeutic agent for the treatment of influenza virus infection and for the suppression of resistance in combination therapy with existing drugs.

Rapid diagnosis and treatment of disease is often based on the identification and characterization of causative agents derived from phenotypic characteristics. Current methods can be laborious and time-consuming, often requiring many skilled personnel and a large amount of lab space. The objective of our study was to develop a spotted microarray for rapid identification and characterization of bacterial pathogens and their antimicrobial resistance genes. Our spotted microarray consists of 489 70mer probes that detect 40 bacterial pathogens of medical, veterinary and zoonotic importance (including 15 NIAID Category A, B and C pathogens); associated genes that encode resistance for antimicrobial and metal resistance; and DNA elements that are important for horizontal gene transfer among bacteria. High specificity and reliability of the microarray was achieved for bacterial pathogens of animal and human importance by validating MDR pathogenic bacteria as pure cultures or by following their inoculation in complex and highly organic sample matrices, such as soil and manure.

Successful printing and hybridization is essential for efficient and reliable data acquisition in a spotted microarray experiments. In this study we demonstrated that printing a 25 mer (printed 25 mer) with a standard 70 mer probe in each spot followed by the use of a fluorescently labeled 25 mer complement in the hybridization mixture ensures monitoring overall printing quality of the chip. This system can also be used as a control to evaluate adequate hybridization, washing, and alignment of spots to position the tracking grids during scanning. A print correction value incorporated in data analysis enhances consistency and reliability of results.

DNA finger printing (restriction fragment lining than in cattle with normal rumens. The length polymorphism analysis) was employed positive correlation between incidence of liver to genetically compare Fusobacterium abscesses and ruminal lesions was the basis for necrophorum isolates of liver abscesses, the hypothesis that F. necrophorum in liver ruminal wall, and ruminal contents from the abscesses is of ruminal origin. Our approach same animal. Fusobacterium necrophorum to documenting that F. necrophorum of liver isolates from liver abscesses were genetically abscesses originates in the rumen was to show identical to the corresponding isolates from the that the rumen wall and liver abscess isolates ruminal wall in eight of nine animals tested. are genetically identical and, thus, are progeny This genetic similarity between the isolates of a single cell. supports the hypothesis that F. necrophorum in liver abscesses originates from the ruminal wall.

The most commonly recognized tick-borne coinfection in most of the eastern USA is Lyme borreliosis and babesiosis, caused by the spirochete Borrelia burgdorferi and the erythrocytic parasite Babesia microti respectively, accounting for ~80% of coinfections. Studies of clinical outcomes indicate that coinfected patients have more severe symptoms and longer duration of illness than patients with Lyme borreliosis alone. Little is known about the immune related mechanisms during tick-borne coinfections. This study used a murine model to examine whether defined disease conditions differed in severity in mice infected solely with B. burgdorferi and in mice coinfected with B. microti and B. burgdorferi. C3H.HeN and BALB/c mice cohorts were coinfected or singly infected and then monitored experimentally for 1, 7, 10, 14, 21 and 60 days after inoculation. Carditis and arthritis was determined by blinded histopathological evaluation of myocardium and tibiotarsal joints. Cytokine measurements were made on lymph node and spleen supernatants for IFN-γ, interleukin (IL-4), IL-10 and TNF-α. Coinfected C3H and BALB/c mice had a significant increase in carditis severity at day 7. In addition, a significant increase in arthritis severity was observed at day 21 and 60 for BALB/c mice. This clinical observation was correlated with a significant increase in the expression of IFN-γ and TNF-α and with a significant reduction of IL-4 and IL-10. NIH P20RR016443-04