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BACKGROUND Densitometric quantification of myeloma paraproteins (M-proteins) is used to monitor secretory myeloma related disorders in humans and dogs. The previous work in dogs used agarose gel electrophoresis (AGE) but did not establish if other methods of serum protein electrophoresis, such as capillary zone electrophoresis (CZE), were comparable. OBJECTIVES We aimed to determine if the densitometric quantification of M-proteins using CZE would yield results comparable to AGE methods. METHODS Fifty-one serum samples from 22 dogs and 18 cats with confirmed monoclonal gammopathies and previously performed AGE were evaluated using CZE on a Sebia Minicap system. Samples were run in duplicate, and their M-proteins were densitometrically measured using the corrected perpendicular drop method previously described. Human-based quality control samples were used to determine the inter-run coefficient of variation (CV). Patient samples were used to calculate the intra-run CV. Method comparison was performed using simple linear regression, Passing-Bablok regression, and Bland-Altman analyses, and Medx evaluations. RESULTS Inter-run and intra-run CVs for CZE were 3.71%-7.65% and 2.89%-4.74%, respectively. Simple linear regression demonstrated an excellent correlation (r > 0.98). Passing-Bablok regression was compatible with the presence of proportional bias in the entire population, and Bland-Altman plots revealed a proportional bias in the feline cases. The Medx evaluation suggested that the two methods did not perform similarly in clinical samples with poor performance at a decision limit of 0.5 gm/dL. CONCLUSIONS Capillary zone electrophoresis is an acceptable method for M-protein densitometric quantification in canine and feline sera but cannot be used interchangeably with AGE-based evaluations.

Background Detection of free light chains (fLC) in animals relies on protein electrophoresis or the Bence-Jones protein test on urine. Objective To describe the detection of both serum fLC (sfLC) and urine fLC (ufLC) in 8 dogs and 2 cats using a commercially available human immunofixation (IF) kit. Animals Archived serum or urine samples from 27 dogs and 2 cats submitted to the Colorado State University Veterinary Diagnostic Laboratory for routine diagnostics. Methods Retrospective study evaluating the presence of fLC in dogs and cats using agarose gel electrophoresis and routine and fLC IF performed on serum and urine. The performance of the fLC IF reagents was evaluated using samples characterized by routine IF, tandem mass spectrometry, and a combination of fLC IF and western blotting. Free light chains were documented by paired electrophoresis and fLC IF. Results The fLC only myeloma case developed end-stage renal failure 5 months post initial diagnosis. All electrophoresis-defined urinary Bence-Jones proteins were labeled by the anti-free λ light chain (anti-fλ) reagent; none were labeled by the anti-free κ light chain (anti-fκ); 2 of these were identified as fκ by mass spectrometry. An electrophoretically identical protein restriction that was labeled by the anti-fλ reagent was present in the paired serum from 5/8 of cases, documenting sfLC. Conclusions and clinical importance Commercially available human IF reagents identified sfLC and ufLC in both dogs and cats. Free light chains may be nephrotoxic in dogs.

Background Equid herpesvirus type 1 (EHV-1) is ubiquitous in equine populations causing respiratory disease, and complications including late-term abortion and neurological disease. Eradication of EHV-1 from housing environments that typically contain unsealed wood and porous bedding materials can be challenging. However, consideration should be given to take advantage of the viral envelope's susceptibility to environmental conditions. Objective To determine environmental persistence of EHV-1 on materials and in environmental conditions commonly found in equine facilities. We hypothesised that environmental conditions and materials would limit environmental persistence of EHV-1 in horse housing environments. Study design Experimental study. Methods Standard inoculum of EHV-1 strain OH03 was applied to leather, polyester-cotton fabric, two bedding materials (pinewood shavings and wheat straw) and polystyrene (plastic), and placed under three different environmental conditions (4°C, indoors and outdoors). Virus titration and quantitative PCR (qPCR) were performed at six time points between 0 and 48 hours and the number of plaque-forming units (PFUs) was determined. Results Viable EHV-1 was recovered up to 48 hours from all material-environmental condition combinations, with persistence decreasing over time. In general, outdoor environment had the greatest impact, irrespective of material tested, followed by indoor environment and 4°C. On average, wood shavings had the greatest impact on persistence, followed by leather, straw, fabric and polystyrene. Main limitations The inoculum used in this study was not in a milieu consistent with nasal secretions. As such, virus particles may have been more sensitive to the materials and/or environmental conditions evaluated. Conclusions Environmental factors had variable effects on environmental persistence. Although there were significant reductions in PFUs within the first 3 hours, irrespective of environment-material evaluated, viable virus was still recovered at 48 hours likely representing a transmission risk. Barrier precautions should be used to prevent spread of EHV-1 from unrecognised environmental reservoirs.

Purpose Rhabdomyosarcomas (RMS) are difficult tumors to treat with conventional therapies. Publications indicate that oncolytic virotherapy (OV) could benefit cancer patients with tumors that are refractory to conventional treatments. It is believed that the efficacy of OV can be enhanced when used in combination with other treatments. This study evaluated the response of mice with aggressive alveolar RMS (ARMS) allografts to treatment with an OV [recombinant myxoma virus (MYXVΔserp2)] in combination with a Janus kinase (JAK) inhibitor (oclacitinib). Oclacitinib is known to inhibit JAK1 and JAK2 cell signaling pathways, which should limit the antiviral Type I interferon response. However, oclacitinib does not inhibit immune pathways that promote antigen presentation, which help stimulate an anti-cancer immune response. Materials and methods To determine if MYXVΔserp2 and oclacitinib could improve outcomes in animals with ARMS, nude mice were inoculated subcutaneously with murine ARMS cells to establish tumors. Immune responses, tumor growth, and clinical signs in mice treated with combination therapy were compared to mice given placebo therapy and mice treated with OV alone. Results Combination therapy was safe; no viral DNA was detected in off-target organs, only within tumors. As predicted, viral DNA was detected in tumors of mice given oclacitinib and MYXVΔserp2 for a longer time period than mice treated with OV alone. Although tumor growth rates and median survival times were not significantly different between groups, clinical signs were less severe in mice treated with OV. Conclusion Our data indicate that MYXVΔserp2 treatment benefits mice with ARMS by reducing clinical signs of disease and improving quality of life.

Lyme disease is a tick-borne bacterial illness that occurs in areas of North America, Europe, and Asia. Early infection typically presents as generalized symptoms with an erythema migrans (EM) skin lesion. Dissemination of the pathogen Borrelia burgdorferi can result in multiple EM skin lesions or in extracutaneous manifestations such as Lyme neuroborreliosis. Metabolic biosignatures of patients with early Lyme disease can potentially provide diagnostic targets, as well as highlight metabolic pathways that contribute to pathogenesis. Sera from well-characterized patients diagnosed with either early localized Lyme disease (ELL) or early disseminated Lyme disease (EDL), plus healthy individuals (HC), from the United States were analyzed by liquid chromatography-mass spectrometry (LC-MS). Comparative analyses were performed between ELL, or EDL, or ELL combined with EDL, and the HC to develop biosignatures present in early Lyme disease. A direct comparison between ELL and EDL was also performed to develop a biosignature for stages of early Lyme disease. Metabolic pathway analysis and chemical identification of metabolites with LC-tandem mass spectrometry (LC-MS/MS) demonstrated alterations of eicosanoid, bile acid, sphingolipid, glycerophospholipid, and acylcarnitine metabolic pathways during early Lyme disease. These metabolic alterations were confirmed using a separate set of serum samples for validation. The findings demonstrated that infection of humans with B. burgdorferi alters defined metabolic pathways that are associated with inflammatory responses, liver function, lipid metabolism and mitochondrial function. Additionally, the data provide evidence that metabolic pathways can be used to mark the progression of early Lyme disease.

Background: Diagnosis of paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is challenging and new tools are needed for early diagnosis as well as to understand the biochemical events that underlie the pathology in TB-IRIS. Methods: Plasma samples were obtained from participants from a randomized HIV/TB treatment strategy study (AIDS Clinical Trials Group [ACTG] A5221) with (n = 26) and without TB-IRIS (n = 22) for an untargeted metabolomics pilot study by liquid-chromatography mass spectrometry. The metabolic profile of these participants was compared at the study entry and as close to the diagnosis of TB-IRIS as possible (TB-IRIS window). Molecular features with p

Background The diagnostic performance of routine electrophoresis (agarose gel electrophoresis [AGE] and capillary zone electrophoresis [CZE]) and species-specific immunofixation (IF) for the detection of immunoglobulin paraproteins (M-proteins) and diagnosis of secretory myeloma-related disorders (sMRD) can be improved. Available canine IF targets were IgG-FC, IgA, IgM, light chain (LC), IgG4, and free LC (fLC) antibodies. Objective We aimed to review specific features associated with the presence of M-proteins in canine serum samples and the common features causing inaccurate reporting of M-proteins to improve the diagnostic performance of routine electrophoresis and IF for the detection of M-proteins. Methods Features found in AGE, CZE, routine IF, IgG4 IF, and fLC IF of 100 canine serum samples from Part 1 of this study were evaluated by simple and multivariate logistic regression to identify factors associated with the presence of M-proteins. Cases falsely called negative or positive for M-proteins were reviewed to identify the common features that could be used to increase the diagnostic performance of SPE and IF for M-protein detection. Results The presence of hypogammaglobulinemia or any peak taller than albumin was associated with an M-protein. Total protein concentrations, globulin concentrations, or peaks wider than albumin were not associated with an M-protein. Free LC sMRD cases were not diagnosed by SPE and routine IF. Cases with infectious and inflammatory etiologies had a restricted polyclonal gammopathy with multiple γ-globulin restrictions resulting in some false-positive results. SPE combined with all available IF results and the specific features identified in this study had an estimated sensitivity of 95.1% and specificity of 81.4%. Conclusions The identified criteria of this study increase the diagnostic performance of the electrophoretic evaluation for M-proteins.

The poxvirus, myxoma virus (MYXV) has shown efficacy as an oncolytic virus (OV) in some cancer models. However, MYXV replication within murine cancer models and spontaneous canine sarcomas is short-lived. In mice, successful treatment of tumors requires frequent injections with MYXV. We hypothesize that treatment of cancer with a recombinant MYXV that promotes apoptosis could improve the efficacy of MYXV. The orfC gene of walleye dermal sarcoma virus (WDSV), which induces apoptosis, was recombined into the MYXV genome (MYXVorfC). A marked increase in apoptosis was observed in cells infected with MYXVorfC. To ensure that expression of WDSV orfC by MYXV does not potentiate the pathogenesis of MYXV, we evaluated the effects of MYXVorfC inoculation in the only known host of MYXV, New Zealand white rabbits. Virus dissemination in rabbit tissues was similar for MYXVorfC and MYXV. Virus titers recovered from tissues were lower in MYXVorfC-infected rabbits as compared to MYXV-infected rabbits. Importantly, rabbits infected with MYXVorfC had a delayed onset of clinical signs and a longer median survival time than rabbits infected with MYXV. This study indicates that MYXVorfC is attenuated and suggests that MYXVorfC will be safe to use as an OV therapy in future studies.

Standard two-tiered testing (STTT) is the recommended algorithm for laboratory diagnosis of Lyme disease (LD). Several limitations are associated with STTT that include low sensitivity in the early stages of disease, as well as technical complexity and subjectivity associated with second-tier immunoblotting; therefore, modified two-tiered testing (MTTT) algorithms that utilize two sequential first-tier tests and eliminate immunoblotting have been evaluated. Recently, a novel MTTT that uses a VlsE chemiluminescence immunoassay followed by a C6 enzyme immunoassay has been proposed. The purpose of this study was to evaluate the performance of the VlsE/C6 MTTT using well-characterized serum samples. Serum samples from the CDC Lyme Serum Repository were tested using three MTTTs, VlsE/C6, whole-cell sonicate (WCS)/C6, and WCS/VlsE, and three STTTs (immunoblotting preceded by three different first-tier assays: VlsE, C6, and WCS). Significant differences were not observed between the results of the MTTTs assessed; however, the VlsE/C6 MTTT resulted in the highest specificity (100%) when other diseases were tested and the lowest sensitivity (75%) for LD samples. Significant differences were present between the results for various MTTTs and STTTs evaluated. Specifically, all MTTTs resulted in higher sensitivities than the STTTs for all LD groups combined and were significantly more accurate (i.e., higher proportion of correct classifications) for this group, with the exception of the WCS/ViraStripe STTT. Additionally, when other diseases were tested, only the results of the VlsE/C6 MTTT differed significantly from those of the WCS/ViraStripe STTT, with the VlsE/C6 MTTT resulting in a 6.2% higher accuracy. Overall, the VlsE/C6 MTTT offers an additional laboratory testing algorithm for LD with equivalent or enhanced performance compared to that of the other MTTTs and STTTs evaluated in this study.

Metabolites detectible in human biofluids are attractive biomarkers for the diagnosis of early Lyme disease (ELD), a vector-borne infectious disease. Urine represents an easily obtained clinical sample that can be applied for diagnostic purposes. However, few studies have explored urine for biomarkers of ELD. In this study, metabolomics approaches were applied to evaluate small molecule metabolites in urine from patients with ELD (n = 14), infectious mononucleosis (n = 14) and healthy controls (n = 14). Metabolic biosignatures for ELD versus healthy controls and ELD versus infectious mononucleosis were generated using untargeted metabolomics. Pathway analyses and metabolite identification revealed the dysregulation of several metabolic processes in ELD as compared to healthy controls or mononucleosis, including metabolism of tryptophan. Linear discriminant analyses demonstrated that individual metabolic biosignatures can correctly discriminate ELD from the other patient groups with accuracies of 71 to 100%. These data provide proof-of-concept for use of urine metabolites as biomarkers for diagnostic classification of ELD.

Lyme disease, the most commonly reported vector-borne disease in the United States, results from infection with Borrelia burgdorferi. Early clinical diagnosis of this disease is largely based on the presence of an erythematous skin lesion for individuals in high-risk regions. This, however, can be confused with other illnesses including southern tick−associated rash illness (STARI), an illness that lacks a defined etiological agent or laboratory diagnostic test, and is coprevalent with Lyme disease in portions of the eastern United States. By applying an unbiased metabolomics approach with sera retrospectively obtained from well-characterized patients, we defined biochemical and diagnostic differences between early Lyme disease and STARI. Specifically, a metabolic biosignature consisting of 261 molecular features (MFs) revealed that altered N -acyl ethanolamine and primary fatty acid amide metabolism discriminated early Lyme disease from STARI. Development of classification models with the 261-MF biosignature and testing against validation samples differentiated early Lyme disease from STARI with an accuracy of 85 to 98%. These findings revealed metabolic dissimilarity between early Lyme disease and STARI, and provide a powerful and new approach to inform patient management by objectively distinguishing early Lyme disease from an illness with nearly identical symptoms.

Serological assays and a two-tiered test algorithm are recommended for laboratory confirmation of Lyme disease. In the United States, the sensitivity of two-tiered testing using commercially available serology-based assays is dependent on the stage of infection and ranges from 30% in the early localized disease stage to near 100% in late-stage disease. Other variables, including subjectivity in reading Western blots, compliance with two-tiered recommendations, use of different first- and second-tier test combinations, and use of different test samples, all contribute to variation in two-tiered test performance. The availability and use of sample sets from well-characterized Lyme disease patients and controls are needed to better assess the performance of existing tests and for development of improved assays. To address this need, the Centers for Disease Control and Prevention and the National Institutes of Health prospectively collected sera from patients at all stages of Lyme disease, as well as healthy donors and patients with look-alike diseases. Patients and healthy controls were recruited using strict inclusion and exclusion criteria. Samples from all included patients were retrospectively characterized by two-tiered testing. The results from two-tiered testing corroborated the need for novel and improved diagnostics, particularly for laboratory diagnosis of earlier stages of infection. Furthermore, the two-tiered results provide a baseline with samples from well-characterized patients that can be used in comparing the sensitivity and specificity of novel diagnostics. Panels of sera and accompanying clinical and laboratory testing results are now available to Lyme disease serological test users and researchers developing novel tests.

Equine herpesvirus-1 (EHV-1) is the cause of respiratory disease, abortion and myelitis in horses worldwide. Protection following infection or vaccination is typically incomplete and this lack of protective immunity is thought to be due to the immunomodulatory properties of EHV-1. EHV-1 immune modulation is likely initiated early in the infection cycle at the respiratory epithelium, but to date, immunity to EHV-1 at the epithelial cell barrier remains poorly characterized. Thus, the purpose of this study was to use a recently established primary equine respiratory epithelial cell culture (EREC) system to characterize innate immunity to EHV-1. Differentiated ERECs were inoculated with a neuropathogenic strain of EHV-1 and cytokine responses were determined using quantitative real-time polymerase chain reaction and ELISA. Major histocompatibility complex (MHC)-I and MHC-II as well as toll-like receptor (TLR)3 and TLR9 protein expression were examined using fluorescence activated cell-sorting analysis and chemotaxis of neutrophils and monocytes were evaluated using chemotaxis assays. Infection with EHV-1 resulted in increased expression of TLR3 and 9 as well as inflammatory cytokines (IL-1, TNF-alpha, IFN-alpha, and IL-6) and chemokines (IL-8, MCP-1). In contrast, EHV-1 infection caused marked decreases of MHC-I and MHC-II expression as well as a reduction in IFN-gamma production. In summary, these results provide an initial characterization of the early immune response to EHV-1 at the epithelial cell barrier and show that, while EHV-1 maintains induction of an inflammatory response, it causes an attenuation of IFN-gamma responses and down-modulates expression of MHC-I and MHC-II, which are important molecules for antigen presentation.

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late term abortions and equine herpesvirus myeloencephalitis (EHM) and remains an important problem in horses worldwide. Despite increasing outbreaks of EHM in recent years, our understanding of EHM pathogenesis is still limited except for the knowledge that a cell-associated viremia in peripheral blood mononuclear cells (PBMCs) is a critical link between primary respiratory EHV-1 infection and secondary complications such as late-term abortion or EHM. To address this question our objective was to identify which PBMC subpopulation(s) are infected during viremia and may therefore play a role in transmitting the virus to the vascular endothelium of the spinal cord or pregnant uterus. PBMCs from 3 groups of animals were collected between days 4 and 9 following experimental infection with EHV-1 strain Findlay/OH03 or strain Ab4. PBMCs were labeled with primary antibodies selective for CD4+ or CD8+ T lymphocytes, B-lymphocytes, or monocytes and positively selected using magnetic bead separation. Cell numbers and EHV-1 genome numbers in each subpopulation were then determined using quantitative PCR for β-actin and the EHV-1 glycoprotein B, respectively. Viral genomic DNA was found in all PBMC subpopulations; the CD8+ lymphocytes were most frequently positive for viral DNA, followed by B-lymphocytes. These differences were statistically significant in horses infected with the EHV-1 strain Findlay/OH03, and ponies with Ab4. These results differ from what has been reported in in vitro studies, and indicate that different PBMC subpopulations may play different roles in EHV-1 viremia.

The survival and growth of Listeria monocytogenes and spoilage microflora during storage of fresh beef subjected to different decontamination treatments was studied. Fresh beef inoculated with a five-strain mixture of L. monocytogenes (5.18 log CFU/cm2) was left untreated (control) or was immersed (30 s) in hot water (HW; 75 degrees C), 2% lactic acid (LA; 55 degrees C), hot water followed by lactic acid (HW-LA), or lactic acid followed by hot water (LA-HW) and then stored aerobically at 4, 10, and 25 degrees C for 25, 17, and 5 days, respectively. Initial populations of L. monocytogenes were reduced by 0.82 (HW), 1.43 (LA), 2.73 (HW-LA), and 2.68 (LA-HW) log CFU/cm2. During storage, the pathogen grew at higher rates in HW than in control samples at all storage temperatures. Acid decontamination treatments (LA. HW-LA, and LA-HW) resulted in a weaker inhibition of L. monocytogenes (P0.05) at 25 degrees C than at 4 and 10 degrees C. In general, the order of effectiveness of treatments was HW-LALALA-HWHWcontrol at all storage temperatures tested. In untreated samples, the spoilage microflora was dominated by pseudomonads, while lactic acid bacteria, Enterobacteriaceae, and yeasts remained at lower concentrations during storage. Brochothrix thermosphacta was detected periodically in only a limited number of samples. Although decontamination with HW did not affect the above spoilage microbial profile, acid treatments shifted the predominant microflora in the direction of yeasts and gram-positive bacteria (lactic acid bacteria). Overall, the results of the present study indicate that decontamination with LA and combinations of LA and HW could limit growth of L. monocytogenes and inhibit pseudomonads, which are the main spoilage bacteria of fresh beef stored under aerobic conditions. However, to optimize the efficacy of such treatments, they must be applied in the appropriate sequence and followed by effective temperature control.

Lyme disease (LD), caused by Borrelia burgdorferi, is the most commonly reported tick-borne disease in the United States and Europe [1, 2]. Recent studies suggest that 300 000 cases of LD may occur in the United States each year [2]. Antibody-based diagnostics for LD are widely utilized in clinical practice [3], and the Centers for Disease Control and Prevention (CDC) recommends a 2-tier approach for serologic testing [4]. The detection of antibodies to B. burgdorferi is highly specific and sensitive in patients with late manifestations of LD; however, the sensitivity in patients with early LD is unsatisfactory (29%–40%) [3]. Direct diagnostic testing using culture or nucleic acid amplification on peripheral blood samples also has low sensitivity (≤50%) for early LD [5]. Thus, the diagnosis of early LD is usually based on recognition of the most common clinical manifestation, an erythema migrans (EM) skin lesion. Other skin lesions, however, such as tick-bite hypersensitivity reactions, STARI (southern tick associated rash illness), and certain cutaneous fungal infections, can be confused with EM [1, 6, 7]. Given the limitations of existing diagnostics for early LD, the feasibility of novel approaches that directly detect infecting spirochetes or the host's response to the pathogen should be evaluated. Modern “omic” technologies provide sensitive methods to investigate, discover, and validate individual molecules or panels of molecules as biomarkers or biosignatures of specific disease states [8, 9]. One such technology, metabolomics, allows for global analyses of low molecular mass (typically

Infection with equine herpesvirus-1 (EHV-1) causes respiratory disease, late-term abortions and equine herpesvirus myeloencephalitis (EHM). Our understanding of EHM pathogenesis is limited except for the knowledge that EHV-1 infected, circulating peripheral blood mononuclear cells (PBMC) transport virus to the central nervous system vasculature causing endothelial cell infection leading to development of EHM. Our objective was to develop a model of CNS endothelial cell infection using EHV-1 infected, autologous PBMC. PBMCs, carotid artery and brain endothelial cells (EC) from 14 horses were harvested and grown to confluency. PBMC or ConA-stimulated PBMCs (ConA-PBMCs) were infected with EHV-1, and sedimented directly onto EC monolayers ('contact'), or placed in inserts on a porous membrane above the EC monolayer ('no contact'). Cells were cultured in medium with or without EHV-1 virus neutralizing antibody. Viral infection of ECs was detected by cytopathic effect. Both brain and carotid artery ECs became infected when cultured with EHV-1 infected PBMCs or ConA-PBMCs, either in direct contact or no contact: infection was higher in carotid artery than in brain ECs, and when using ConA-PBMCs compared to PBMCs. Virus neutralizing antibody eliminated infection of ECs in the no contact model only. This was consistent with cell-to-cell spread of EHV-1 infection from leucocytes to ECs, demonstrating the importance of this mode of infection in the presence of antibody, and the utility of this model for study of cellular interactions in EHV-1 infection of ECs.

Infection of dogs with canine influenza virus (CIV) is considered widespread throughout the United States following the first isolation of CIV in 2004. While vaccination against influenza A infection is a common and important practice for disease control, antiviral therapy can serve as a valuable adjunct in controlling the impact of the disease. In this study, we examined the antiviral activity of nitazoxanide (NTZ) and tizoxanide (TIZ) against three CIV isolatesin vitro. NTZ and TIZ inhibited virus replication of all CIVs with 50% and 90% inhibitory concentrations ranging from 0.17 to 0.21 μMand from 0.60 to 0.76 μM, respectively. These results suggest that NTZ and TIZ are effective against CIV and may be useful for treatment of canine influenza in dogs but further investigation of thein vivoefficacy against CIV as well as the drug's potential for toxicity in dogs is needed.

Microorganisms persisting in slaughter plant environments may develop acid resistance and be translocated to other environmental surfaces or products. The objective of this study was to evaluate the potential of Escherichia coli O157:H7 to form biofilms and maintain acid resistance, under different culture habituation scenarios, on stainless steel coupons (2 x 5 x 0.08 cm), in the presence of beef carcass decontamination runoff fluids (washings). Coupons were stored in test tubes with unsterilized water washings (WW; pH 6.94) or lactic acid washings (LAW; pH 4.98), which were inoculated with E. coli O157:H7 (10(3)-10(4)CFU/ml) and incubated at 15 (24 or 48 h) or 35 degrees C (7 or 24 h), simulating different habituation scenarios on sites of a slaughter plant, including sanitation and overnight drying, during consecutive operational shifts. Acid resistance (AR) of planktonic and detached E. coli O157:H7 cells was assessed in tryptic soy broth adjusted to pH 3.5 with lactic acid. The highest pre-drying attachment and AR of E. coli O157:H7 were observed after 24h at 35 degrees C and 48 h at 15 degrees C. Drying reduced (P

This study evaluated the behavior of Escherichia coli O157:H7 during aerobic storage, after storage in vacuum packages, on beef inoculated with cultures prepared (35 degrees C, 24 h) in tryptic soy broth without dextrose (TSB), nonacid hot water carcass decontamination runoff fluids (washings; pH 6.0; WASH), cells from biofilms formed on stainless steel coupons in WASH (WETB), or WETB dried (25 degrees C, 12 h) before harvesting of cells (DRYB). These inocula were applied to fresh beef pieces (40 cm2), which were then left untreated or treated by immersion in hot water (75 degrees C) followed by 2% lactic acid (55 degrees C; hot water/lactic acid [HW/LA]), for 30 s each. Inoculated samples were vacuum packaged and stored at 0 (30, 60, or 90 days), 4 (7 or 14 days), or 12 degrees C (4 or 8 days) and subsequently transferred to retail packages for aerobic storage at 7 degrees C for 5 days. Populations of E. coli O157:H117, regardless of inoculum type, remained generally unchanged (P > 0.05) after aerobic storage (7 degrees C, 5 days) of untreated or HW/LA-treated beef samples previously stored in vacuum packages at 0 or 4 degrees C. However, reductions in E. coli O157:H7 levels were generally obtained when vacuum packaged, untreated beef samples previously stored at 12 degrees C were transitioned to aerobic conditions. Additionally, despite similar (P > 0.05) levels of E. coli O157:H7 cells of TSB, WASH, WETB, and DRYB origin on vacuum-packaged, untreated samples after 8 days of storage at 12 degrees C, subsequent aerobic storage resulted in larger (P < 0.05) reductions of cells of WETB and DRYB origin than for cells of TSB and WASH origin. For HW/LA-treated beef previously stored at 12 degrees C in vacuum packages, populations of E. coli O157:H7 remained largely unchanged after aerobic storage in retail packages. Results thus indicated that aerobic storage of beef (7 degees C, 5 days) previously stored in vacuum packages at 0 or 4 degrees C did not lead to E. coli O157:H7 population changes, whereas transition from vacuum packages stored under mildly abusive temperature (12 degrees C) to aerobic storage may have caused injury and death to the pathogen.

This study evaluated the impact of inoculum preparation and storage conditions on the response of Escherichia coli O157:H7 exposed to consumer-induced stresses simulating undercooking and digestion. Lean beef tissue samples were inoculated with E. coli O157:H7 cultures prepared in tryptic soy broth or meat decontamination runoff fluids (WASH) or detached from moist biofilms or dried biofilms formed on stainless steel coupons immersed in inoculated WASH. After inoculation, the samples were left untreated or dipped for 30 s each in hot (75°C) water followed by lactic acid (2%, 55°C), vacuum packaged, stored at 4 (28 days) or 12°C (16 days), and periodically transferred to aerobic storage (7°C for 5 days). During storage, samples were exposed to sequential heat (55°C; 20 min) and simulated gastric fluid (adjusted to pH 1.0 with HCl; 90 min) stresses simulating consumption of undercooked beef. Under the conditions of this study, cells originating from inocula of planktonic cells were, in general, more resistant to heat and acid than cells from cultures grown as biofilms and detached prior to meat inoculation. Heat and acid tolerance of cells on meat stored at 4°C was lower than that of cells on nondecontaminated meat stored at 12°C, where growth occurred during storage. Decontamination of fresh beef resulted in injury that inhibited subsequent growth of surviving cells at 12°C, as well as in decreases in resistance to subsequent heat and acid stresses. The shift of pathogen cells on beef stored under vacuum at 4°C to aerobic storage did not affect cell populations or subsequent survival after sequential exposure to heat and simulated gastric fluid. However, the transfer of meat stored under vacuum at 12°C to aerobic storage resulted in reduction in pathogen counts during aerobic storage and sensitization of survivors to the effects of sequential heat and acid exposure.

Recently, the product of equine herpesvirus type 1 (EHV-1) ORF1, a homolog to HSV-1 pUL56, was shown to modulate MHC-I expression and innate immunity. Here, we investigated modulation of respiratory epithelial immunity by EHV-1 pUL56 and compared responses to those of PBMCs, which are important target cells that allow cell-associated EHV-1 viremia.The salient observations are as follows: (i) EHV-1 significantly down-modulated MHC-I and MHC-II expression in equine respiratory epithelial cells (ERECs). MHC-I expression remained unaffected in PBMCs and MHC-II expression was increased. (ii) Infection with an EHV-1 ORF1 deletion mutant partially restored MHC-I and MHC-II expression and altered IFN-alpha and IL-10 mRNA expression. (iii) Deletion of EHV-1 ORF1 also significantly increased chemokine expression and chemotaxis of monocytes and neutrophils in ERECs. Collectively, these results suggest a role for EHV-1 pUL56 in modulation of antigen presentation, cytokine expression and chemotaxis at the respiratory epithelium, but not in PBMC.