Higher Inoculum Doses Research Articles

Alternative Development and Processing of Fermented Beverage and Tempeh Using Green Beans from Four Genotypes of Lupinus mutabilis

In this study, the nutritional characteristics of two formulations were studied: A fermented beverage and tempeh. Green seeds from four different cultivars of tarwi (Lupinus mutabilis) were used: Andino, Guaranguito, Ecuadorian, and Peruvian. The seeds were first debittered, and then, they were fermented, using two inoculum doses and two fermentation times. For the fermented beverage, Saccaromyces cerevisae was used at doses of 1.5 and 2.5% dry weight and fermentation times of 72 and 96 h. For the tempeh, the inoculum used was the Rhizopus oligosporus strain NRRL 2710 at doses of 0.015 and 0.025% dry weight and fermentation times of 48 and 72 h. The alcoholic strength, total solid soluble, and pH in the fermented beverage were analyzed, while proximal analysis was performed for both the fermented beverage and tempeh. The results indicate that, in all cases, the Andino cultivar produced a beverage with higher alcoholic strength, regardless of the inoculum concentration and fermentation time. In both the fermented beverage and tempeh, a varietal effect was found for protein and crude fiber. The Andino cultivar had the highest protein, fat, and fiber content. The alkaloid content was similar in the beverage and tempeh, but the effect of fermentation was markedly increased with the higher inoculum dose and longer fermentation time. The alkaloid content was highest in the Peruvian cultivar, while it was similar in the remaining three cultivars. Tarwi grains have a high nutritional value, but the grain is traditionally used dry. In this study, we used green grain, which contains lower alkaloid content. Moreover, the crop cycle is shortened, and it can be used in the manufacture of fermented drinks and tempeh. In this way, we show that it is valid to use green grain instead of dried grain, constituting an improvement in the traditional use of lupine grain, and our study shows that it is possible to make both fermented drink and tempeh with green grain, showing the influence of the genotype and dosage starter.

Vinorelbine and Intermittent Cyclophosphamide Sensitize an Aggressive Myc-Driven B-Cell Lymphoma to Anti-PD-1 by an Immunological Memory Effective against Tumor Re-Challenge

We have previously shown in triple-negative breast cancer (TNBC) models that a triple therapy (TT) including intermittent cyclophosphamide (C), vinorelbine (V), and anti-PD-1 activates antigen-presenting cells (APC) and generates stem like-T cells able to control local and metastatic tumor progression. In the present manuscript, we report the generation of a highly aggressive, anti-PD-1 resistant model of a high-grade, Myc-driven B-cell non-Hodgkin's lymphoma (NHL) that can be controlled in vivo by TT but not by other chemotherapeutic agents, including cytarabine (AraC), platinum (P), and doxorubicin (D). The immunological memory elicited in tumor-bearing mice by TT (but not by other treatments) can effectively control NHL re-challenge even at very high inoculum doses. TT re-shaped the landscape of circulating innate NK cells and adaptive immune cells, including B and T cells, and significantly reduced exhausted CD4+ and CD8+ TIM3+PD-1+ T cells in the spleens of treated mice.

Sublethal infection of C3H/HeNJ against Leptospira interrogans serovar Pomona

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira spp. Leptospires can infect a variety of mammalian species. Golden Syrian hamsters are mostly used to study acute leptospirosis. However, the immunopathogenic mechanism is poorly understood because immunological reagents for hamsters are limited. This study aimed to establish C3H/HeNJ mice as an animal model for leptospirosis. Five-week-old C3H/HeNJ mice were infected with either low (103 cells) or high (106 cells) inoculum dose of Leptospira interrogans serovar Pomona. All mice were investigated for survival rate, leptospiral load and histopathology of target organs, antibody levels, and cytokine production (IFN-γ, IL-6 and IL-10) at day 28 post-infection. All infected mice survived and did not develop acute lethal infection. However, C3H/HeNJ mice infected with 106 cells of leptospires showed kidney colonization of leptospires and pathological changes in the lung and kidney including renal fibrosis. The glomerular size in PAS-D stained kidney tissues of C3H/HeNJ mice infected with 106 cells of leptospires was significantly reduced compared to that of mice infected with 103 cells of leptospires and non-infected mice. High-dose leptospires induced significantly greater levels of IFN-gamma and IL-6 than low-dose leptospires, but IL-10 level was not significantly different. Moreover, 106 leptospiral cells induced predominant IgG2a isotype suggesting Th1-like response. These results suggest that C3H/HeNJ mice may be used as a sublethal model of leptospirosis.

Multiple Levels of Triggered Factors and the Obligated Requirement of Cell-to-Cell Movement in the Mutation Repair of Cucumber Mosaic Virus with Defects in the tRNA-like Structure.

Simple SummaryBased on analysis of the tRNA-like structure (TLS) mutation in cucumber mosaic virus (CMV), mutation repair is correlated with several levels of triggered factors, including the dose of inoculation of virus mutants, the quantity effect on corresponding viral RNA, and the quality effect on corresponding viral RNA. All types of TLS mutation in different RNAs of CMV can be repaired at a low dose around the dilution end-point. At a high dose of inoculation, TLS mutations in RNA2 and RNA3, but not RNA1, can be repaired, which correlates with the relative quantity defect of RNA2 or the genome size defect of RNA3. In addition, all the above types of mutation repair necessarily require cell-to-cell movement, which presents the obligated effect of cell-to-cell movement on mutation repair.Some debilitating mutations in RNA viruses are repairable; however, the triggering factors of mutation repair remain largely unknown. In this study, multiple triggering factors of mutation repair are identified based on genetic damage to the TLS in CMV. TLS mutations in different RNAs distinctively impact viral pathogenicity and present different types of mutation repair. RNA2 relative reduction level or RNA3 sequence change resulting from TLS mutation is correlated with a high rate of mutation repair, and the TLS mutation of RNA1 fails to be repaired at the high inoculum dose. However, the TLS mutation of RNA1 can be repaired at a low dose of inoculation, particularly around the dilution end-point or in the mixed inoculation with RNA2 having a pre-termination mutation of the 2b gene, an RNAi suppressor. Taken together, TLS mutations resulting in quality or quantity defects of the viral genome or TLS mutations at low doses around the dilution end-point are likely to be repaired. Different levels of TLS mutation repair necessarily require cell-to-cell movement, therefore implying its obligated effect on the evolution of low-fitness viruses and providing a new insight into Muller’s ratchet. This study provides important information on virus evolution and the application of mild viral vaccines.

411. Significance of a Known Epidemiological Link to a COVID-19 Index Case and Severity of COVID-19 Infection

Abstract Background Several factors have been associated with severity of COVID-19 disease, but there remains a paucity of data surrounding whether the nature of exposure is impactful. Evidence demonstrating the correlation between initial viral exposure dose and disease severity exists for many viral infections. Observational studies have suggested that the exposure context, which can be considered a proxy for magnitude of viral inoculum, may influence severity of COVID-19 infection. We aimed to assess whether having a known exposure, as a proxy for higher inoculum dose to COVID-19, was associated with more severe outcomes for individuals hospitalized with COVID-19. Methods We created a retrospective cohort of community-dwelling adults hospitalized for COVID-19 in south-central Ontario from April 1, 2020 - January 14, 2021. Individuals or next of kin were contacted to ascertain exposure history. The primary outcome was death, intensive care unit (ICU) admission, or mechanical ventilation (MV) within 30 days of admission. A multivariable logistic regression model was used to determine whether a known exposure was associated with worse outcomes. Results 1097 individuals with community acquired COVID-19 required hospitalization; of these, 942 (86%) had available exposure data. In this group, the median age was 65, 44% were women, 84% lived in a private residence, 59% had a frailty score (FS) of 1 – 3 while 40% had a FS of 4 – 9, and 28% had a known exposure. Overall, the primary outcome occurred in 368/942 (39%) patients. Having a known exposure was not associated with worse outcome (OR 1.14, 95% CI 0.84–1.54, p = 0.41). Male gender (OR 1.41, 95% CI 1.06–1.89; p = 0.018), age (OR 1.01/year, 95% CI 1.00–1.03, p = 0.03), frailty (OR 1.22/point, 95% CI 1.09–1.36, p = 0.001) and living with at least one other person (OR 1.57, 95% CI 1.09–2.28, p = 0.017) were all associated with death, ICU admission, or MV within 30 days of admission. Conclusion While having a known exposure to a person with COVID-19 was not associated with worse outcome, the identified increased severity of illness associated with cohabitation suggests context of exposure may have a role in disease severity. This data and future studies can be used to guide public health recommendations to not only minimize transmission, but severity of COVID-19 infection. Disclosures All Authors: No reported disclosures

The effects of koruk products used as marination liquids against foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Typhimurium) inoculated on poultry meat

In this study, the effects of marination liquids (MLs) prepared using koruk juice (25% and 50%) and dried koruk pomace (1% and 2%) with or without additives [salt (1%) and thyme (0.1%)] on meat safety were determined. In the first stage, the minimum inhibition concentration (MIC) values of MLs were observed in the range of 6.50% - >50% (v/v). In the second stage, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Typhimurium were inoculated on poultry meat at high and low inoculum doses (≅ 6 log and ≅ 3 log) and marinated at 4 °C for 2, 24 and 48 h. When high inoculum doses were used, E. coli O157:H7, L. monocytogenes and S. Typhimurium counts on meats were decreased by 0.78–2.52, 0.42–1.50 and 0.58–2.00 log CFU/g, respectively. Marination with 50% koruk juice without additives (ML1) for 48 h was determined as the most effective treatment on E. coli O157:H7 and S. Typhimurium, while the highest reduction of L. monocytogenes was achieved by 50% koruk juice with additives (ML2) for 48 h. The counts of pathogens inoculated on samples at low inoculum doses were reduced to undetectable levels for all treatment times. The results of this study revealed that koruk-based MLs are effective products for providing food safety.

Co-inoculation of Bradyrhizobium stimulates the symbiosis efficiency of Rhizobium with common bean

Common bean (Phaseolus vulgaris L.) nodulates with a wide range of rhizobia. Amongst these is Bradyrhizobium, which is inefficient but able to induce profuse nodulation on this crop. Based on this observation, we tested whether co-inoculating bradyrhizobia with a more standard common bean symbiont, Rhizobium tropici, could stimulate growth and nodulation of common bean, thus contributing to a more effective symbiosis. Rhizobium tropici was co-inoculated with two Bradyrhizobium strains applied at three different doses (104, 106, and 108 CFU seed−1) under sterile conditions, and at a single dose (108 CFU seed−1) in non-sterile soil. Plant biomass, nodulation, and N accumulation in plant tissues were evaluated. Co-inoculated plants produced more nodules, and accumulated more shoot dry biomass and nitrogen than plants inoculated with R. tropici alone under gnotobiotic conditions. Significant responses were observed at the highest inoculum dose and a significant correlation between dose and shoot dry weight was observed. Co-inoculation increased biomass and N accumulation in non-sterile soil, although with a smaller magnitude. Altogether, our findings suggest that the co-inoculation with bradyrhizobia contributed to an improved symbiotic interaction between R. tropici and common beans.

A mouse mastitis model to study the effects of the intramammary infusion of a food-grade Lactococcus lactis strain.

Lactococcus lactis is one of the most important microorganisms in the dairy industry and has “generally recognized as safe” (GRAS) status. L. lactis belongs to the group of lactic acid bacteria (LAB) and is encountered in a wide range of environments. Recently, the use of the intramammary infusion of a live culture of LAB has been investigated as a new antibiotic alternative for treating mastitis in dairy ruminants. Controversial results are described in literature regarding its efficacy and safety. In this study we conducted in-depth investigation of the mammary gland immune response induced by intramammary inoculum of a live culture of L. lactis LMG 7930 using the mouse mastitis model. Overnight cultures either of L. lactis (≈ 107 CFU) or of the mastitis pathogens Staphylococcus chromogenes (≈ 105 CFU) or S. aureus (≈ 102 CFU/ml) were injected into the mouse inguinal glands. A double injection, consisting of S. chromogenes first and then L. lactis, was also investigated. Bacterial recovery from the gland and inflammatory cell infiltration were assessed. L. lactis-treated and control glands were analysed for proinflammatory cytokine production. Microbiological results showed that L. lactis was able to survive in the mammary gland 24 h post infection, as were the mastitis pathogens S. chromogenes and S. aureus. L. lactis reduced S. chromogenes survival in the glands and increased its own survival ability by coexisting with the pathogen. Histology showed that L. lactis-treated glands presented variable histological features, ranging from undamaged tissue with no inflammatory cell infiltrate to severe PMN infiltrate with focal areas of tissue damage. S. aureus-treated glands showed the most severe histological grade of inflammation despite the fact that the inoculum size was the smallest. In contrast, most S. chromogenes-treated glands showed normal structures with no infiltration or lesions. Significant increases in IL-1β and TNF-α levels were also found in L. lactis-inoculated glands. The above findings seem to suggest that food-grade L. lactis at a high-inoculum dose such as an overnight culture may elicit a suppurative inflammatory response in the mammary gland, thus becoming a potential mastitis-causing pathogen. Because of the unpredictable potential of L. lactis in acting as a potential mastitis pathogen, this organism cannot be considered a safe treatment for bovine mastitis.

Gene bb0318 Is Critical for the Oxidative Stress Response and Infectivity of Borrelia burgdorferi.

A greater understanding of the molecular mechanisms that Borrelia burgdorferi uses to survive during mammalian infection is critical for the development of novel diagnostic and therapeutic tools to improve the clinical management of Lyme disease. By use of an in vivo expression technology (IVET)-based approach to identify B. burgdorferi genes expressed in vivo, we discovered the bb0318 gene, which is thought to encode the ATPase component of a putative riboflavin ABC transport system. Riboflavin is a critical metabolite enabling all organisms to maintain redox homeostasis. B. burgdorferi appears to lack the metabolic capacity for de novo synthesis of riboflavin and so likely relies on scavenging riboflavin from the host environment. In this study, we sought to investigate the role of bb0318 in B. burgdorferi pathogenesis. No in vitro growth defect was observed for the Δbb0318 clone. However, the mutant spirochetes displayed reduced levels of survival when exposed to exogenous hydrogen peroxide or murine macrophages. Spirochetes lacking bb0318 were found to have a 100-fold-higher 50% infectious dose than spirochetes containing bb0318 In addition, at a high inoculum dose, bb0318 was found to be important for effective spirochete dissemination to deep tissues for as long as 3 weeks postinoculation and to be critical for B. burgdorferi infection of mouse hearts. Together, these data implicate bb0318 in the oxidative stress response of B. burgdorferi and indicate the contribution of bb0318 to B. burgdorferi mammalian infectivity.

Survival of foodborne pathogens in unripe grape products

Antibacterial effects of unripe grape products (verjuice and sour grape sauce) which are particularly rich in antioxidants and organic acids were evaluated. Survival of Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium and Staphylococcus aureus which were inoculated in unripe grape products at different inoculum doses (2 and 6 log CFU/mL) was determined. All the samples were kept at room temperature (approximately 25 °C) for 0, 5, 15 and 30 min after inoculation with pathogens, separately. The unripe grape products which were inoculated at low inoculum dose inhibited all tested pathogens in 5 min, while it took 15 min for high inoculum dose. The pH, titratable acidity (tartaric acid %) and total sugar content values of the samples were between 2.14–2.74, 2.29–7.10% and 1.19–17.65 g/L, respectively. It was concluded that unripe grape products are microbiologically safe products and they have self-protection systems even when they were contaminated with pathogens at high levels.

Assessment of Aspergillus fumigatus burden in lungs of intratracheally-challenged turkeys (Meleagris gallopavo) by quantitative PCR, galactomannan enzyme immunoassay, and quantitative culture.

Aspergillus fumigatus remains a major respiratory pathogen in birds and treatment is still difficult. We challenged different groups of few-day-old turkeys via intratracheal aerosolisation with increasing concentrations (10(5) up to 10(8)) of conidia using a MicroSprayer(®) device. The fungal burden was assessed by real-time PCR, galactomannan dosage, CFU counting and histopathological evaluation in order to provide a comparison of these results within each inoculum groups. Significant mortality, occurring in the first 96h after inoculation, was only observed at the highest inoculum dose. Culture counts, GM index and qPCR results on the one hand and inoculum size on the other hand appeared to be clearly correlated. The mean fungal burden detected by qPCR was 1.3log10 units higher than the mean values obtained by CFU measurement. The new model and the markers will be used to evaluate the efficacy of antifungal treatments that could be used in poultry farms.

Pathogenic Consequences of Vaginal Infection with CCR5-Tropic Simian-Human Immunodeficiency Virus SHIV SF162P3N

We previously reported efficient transmission of the pathogenic R5 simian-human immunodeficiency virus SHIV(SF162P3N) isolate in Indian rhesus macaques by intravenous and intrarectal inoculations, with a switch to CXCR4 coreceptor usage in ∼50% of infected animals that progressed rapidly to disease. Since women continue to be disproportionately affected by HIV, we developed an animal model based on the intravaginal challenge of female rhesus monkeys with SHIV(SF162P3N) and sought to validate the utility of this model to study relevant aspects of HIV transmission and pathogenesis. The effect of viral dose on infection outcome was evaluated to determine the optimal conditions for the evaluation of HIV-1 preventive and therapeutic strategies. We found that the virus can successfully cross the vaginal mucosal surface to establish infection and induce disease with coreceptor switch, but with lower efficiencies compared to intravenous and rectal transmissions. In contrast to intrarectal infection, peak and cumulative viral load over a 1 year-infection period were significantly greater in macaques exposed intravaginally to lower rather than higher inoculum doses. Moreover, low and transient viremia was observed only in macaques that were challenged intravaginally twice within the same day with a high dose of virus, which can be seen as doubling the dose. Taken together, these results show that SHIV(SF162P3N) can successfully transmit across the genital mucosa, undergo coreceptor switch, and induce disease. However, the administered dose appears to impact SHIV(SF162P3N) vaginal infection outcome in an unexpected manner.

Highly Pathogenic Influenza A(H5N1) Virus Survival in Complex Artificial Aquatic Biotopes

BackgroundVery little is known regarding the persistence of Highly Pathogenic Avian Influenza (HPAI) H5N1 viruses in aquatic environments in tropical countries, although environmental materials have been suggested to play a role as reservoirs and sources of transmission for H5N1 viruses.Methodology/Principal FindingsThe survival of HPAI H5N1 viruses in experimental aquatic biotopes (water, mud, aquatic flora and fauna) relevant to field conditions in Cambodia was investigated. Artificial aquatic biotopes, including simple ones containing only mud and water, and complex biotopes involving the presence of aquatic flora and fauna, were set up. They were experimentally contaminated with H5N1 virus. The persistence of HPAI H5N1 virus (local avian and human isolates) was determined by virus isolation in embryonated chicken eggs and by real-time reverse-polymerase chain reaction. Persistence of infectious virus did not exceed 4 days, and was only identified in rain water. No infectious virus particles were detected in pond and lake water or mud even when high inoculum doses were used. However, viral RNA persisted up to 20 days in rain water and 7 days in pond or lake water. Viral RNA was also detected in mud samples, up to 14 days post-contamination in several cases. Infectious virus and viral RNA was detected in few cases in the aquatic fauna and flora, especially in bivalves and labyrinth fish, although these organisms seemed to be mostly passive carriers of the virus rather than host allowing virus replication.Conclusions/SignificanceAlthough several factors for the survival and persistence of HPAI viruses in the environment are still to be elucidated, and are particularly hard to control in laboratory conditions, our results, along with previous data, support the idea that environmental surveillance is of major relevance for avian influenza control programs.

Nipah Virus Transmission in a Hamster Model

Based on epidemiological data, it is believed that human-to-human transmission plays an important role in Nipah virus outbreaks. No experimental data are currently available on the potential routes of human-to-human transmission of Nipah virus. In a first dose-finding experiment in Syrian hamsters, it was shown that Nipah virus was predominantly shed via the respiratory tract within nasal and oropharyngeal secretions. Although Nipah viral RNA was detected in urogenital and rectal swabs, no infectious virus was recovered from these samples, suggesting no viable virus was shed via these routes. In addition, hamsters inoculated with high doses shed significantly higher amounts of viable Nipah virus particles in comparison with hamsters infected with lower inoculum doses. Using the highest inoculum dose, three potential routes of Nipah virus transmission were investigated in the hamster model: transmission via fomites, transmission via direct contact and transmission via aerosols. It was demonstrated that Nipah virus is transmitted efficiently via direct contact and inefficiently via fomites, but not via aerosols. These findings are in line with epidemiological data which suggest that direct contact with nasal and oropharyngeal secretions of Nipah virus infected individuals resulted in greater risk of Nipah virus infection. The data provide new and much-needed insights into the modes and efficiency of Nipah virus transmission and have important public health implications with regards to the risk assessment and management of future Nipah virus outbreaks.

Phenotypic and Genotypic Evidence for l -Fucose Utilization by Campylobacter jejuni

Campylobacter jejuni remains among the leading causes of bacterial food-borne illness. The current understanding of Campylobacter physiology suggests that it is asaccharolytic and is unable to catabolize exogenous carbohydrates. Contrary to this paradigm, we provide evidence for l-fucose utilization by C. jejuni. The fucose phenotype, shown in chemically defined medium, is strain specific and linked to an 11-open reading frame (ORF) plasticity region of the bacterial chromosome. By constructing a mutation in fucP (encoding a putative fucose permease), one of the genes in the plasticity region, we found that this locus is required for fucose utilization. Consistent with their function in fucose utilization, transcription of the genes in the locus is highly inducible by fucose. PCR screening revealed a broad distribution of this genetic locus in strains derived from various host species, and the presence of this locus was consistently associated with fucose utilization. Birds inoculated with the fucP mutant strain alone were colonized at a level comparable to that by the wild-type strain; however, in cocolonization experiments, the mutant was significantly outcompeted by the wild-type strain when birds were inoculated with a low dose (10⁵ CFU per bird). This advantage was not observed when birds were inoculated at a higher inoculum dose (10⁸ CFU per bird). These results demonstrated a previously undescribed substrate that supports growth of C. jejuni and identified the genetic locus associated with the utilization of this substrate. These findings substantially enhance our understanding of the metabolic repertoire of C. jejuni and the role of metabolic diversity in Campylobacter pathobiology.

Bioluminescence Imaging ofClavibacter michiganensissubsp.michiganensisInfection of Tomato Seeds and Plants

Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of C. michiganensis subsp. michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the C. michiganensis subsp. michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent C. michiganensis subsp. michiganensis strain using the modified Tn1409 containing a promoterless lux reporter. A total of 19 bioluminescent C. michiganensis subsp. michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent C. michiganensis subsp. michiganensis to help better elucidate the C. michiganensis subsp. michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of C. michiganensis subsp. nebraskensis with Tn1409::lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the agriculturally important C. michiganensis.

Experimental infection of humans with A2 respiratory syncytial virus

Development of a readily available challenge model of human respiratory syncytial virus (RSV) infection would be a useful tool for evaluation of antiviral agents and vaccine candidates. Accordingly, we evaluated a new challenge pool of RSV A2 virus, provided by the National Institutes of Allergy and Infectious Diseases. Healthy adults subjects were inoculated intranasally with various dilutions of the pool, and virus shedding, clinical symptoms, and immune response were studied. In a preliminary study of 36 randomly selected volunteers, stratified by serum neutralization titer both higher inoculum dose (4.7 log 10 TCID 50 virus) and lower antibody titer (≤10.36 log 2), were associated with infection. In a second confirmatory study 12 of 13 (92%) subjects selected for low serum neutralizing activity shed virus after challenge with the high inoculum of virus. Mean peak virus shedding was 2.2 log 10 TCID 50/mL nasal wash with a mean duration of 4 days. None of the subjects developed signs or symptoms of lower airway disease, although respiratory symptom scores and nasal mucus weight were temporally correlated with virus shedding. Prescreening for low levels of neutralizing antibody may allow selection of subjects with relatively higher susceptibility to experimental infections. This model provides a safe and efficient mechanism for proof-of-concept studies of anti-viral agents and RSV vaccines, as well as for investigation of immune responses to infection.

Wide variations in herpes simplex virus type 1 inoculum dose and latency-associated transcript expression phenotype do not alter the establishment of latency in the rabbit eye model.

The latency-associated transcript (LAT) is required for efficient reactivation of herpes simplex virus type 1 from latent infection in the rabbit eye model, but LAT's mechanism of action is unknown. In addition to reactivation, the LAT region seems to correspond to multiple functions, with some LAT deletion mutants exhibiting increased virulence, increased neuronal death, and restricted establishment of latency. While a LAT promoter deletion mutant (17DeltaPst) seems to be primarily restricted in reactivation in the rabbit, subtle effects on virulence or the establishment of latency cannot be precluded at the normal high levels of virus inoculum used in the rabbit model. Since such additional LAT phenotypes may be more evident with lower doses of virus, we evaluated the influence of initial viral inoculum and LAT expression on the progression of acute infection and the establishment of latency. We have assayed both virus recovery rates and viral genome loads in rabbit corneas and trigeminal ganglia. Our results show that (i) in the corneas and trigeminal ganglia, the maximum amount of virus present during acute infection is independent of the LAT genotype and inoculum dose, although greater viral yields are obtained earlier with higher inoculum doses, and (ii) the range in numbers of latent genomes detected in the ganglia is independent of the inoculum dose and the LAT genotype and therefore no difference in establishment of latency is observed.

Moderate Inflammatory Reaction During Experimental Escherichia coli Mastitis in Primiparous Cows

Nineteen primiparous cows were experimentally infected in 2 quarters with 1 x 10(4) (group A) or 1 x 10(6) (group B) cfu of Escherichia coli P4:O32 per quarter within 2 to 4 wk after parturition. Blood and milk samples were collected from all primiparous cows at regular time intervals from d -4 to d +3 relative to inoculation. Milk production rapidly decreased in both groups during E. coli mastitis, but recovery appeared to be faster in group B at d + 1 postinfusion (p.i.). The milk production losses in the noninfected quarters were substantial on the day of inoculation, which is probably due to pronounced systemic effects. However, on d + 2 p.i. milk production in the noninfected quarters nearly reached preinfection levels, indicating a moderate clinical severity following intramammary inoculation. None of the other severity criteria evolved towards a severe response pattern. Reticulorumen motility was inhibited in both groups during E. coli mastitis. The clinical episode was short lasting in both groups. Rectal temperature, heart rate, blood leukocyte count, number of colony-forming units, milk somatic cell count and several indicators for the disintegration of the blood-milk barrier returned to normal values within 24 to 72 h p.i. Primiparous cows reacted with a moderate inflammatory response following intramammary infusion with a relatively high dose of E. coli. Despite the use of a high inoculum dose, primiparous cows in both groups showed pronounced resistance against severe intramammary E. coli infection. A possible effect of the inoculum dose could be present, however, further research into the effect of the inoculum dose on the inflammatory response should be performed.

Inhibition of pneumococcal adherence to human nasopharyngeal epithelial cells by anti-PsaA antibodies.

The role of pneumococcal (Pnc) surface adhesin A (PsaA) in the adherence of Streptococcus pneumoniae (pneumococcus) to host cells is not well defined. We examined the effect of anti-PsaA antibodies in an inhibition of adherence assay using Detroit 562 nasopharyngeal human epithelial cells. Rabbit polyclonal (Pab) anti-recombinant PsaA (rPsaA) sera, a purified mouse monoclonal antibody (MAb) (MAb 6F62G8E12), and 22 healthy adult sera with known anti-PsaA IgG levels (obtained by enzyme-linked immunosorbent assay) were evaluated for their abilities to inhibit Pnc adherence to confluent monolayers (measured as percent reduction in CFU counts compared to those of uninhibited controls). Pnc adherence was dependent on capsular phenotype (no or low adherence for opaque strains). With an inoculum of 10(4) to 10(5) bacteria/well, the mean +/- standard deviation count in controls was 163 +/- 32 CFU/well for transparent strains. Low adherence was observed for a PsaA-minus mutant even at higher inoculum doses. Mean percent inhibitions of adherence with Pab and MAb were 54 and 50%, respectively. Adult sera showed inhibition in a dose-response fashion with a range of 98 to 8%, depending on the serum anti-PsaA antibody concentration. Absorption of Pab with rPsaA restored Pnc adherence to control levels. Absorption of sera with a PsaA-minus mutant did not result in a significant decrease (P >0.05) of inhibition of adherence activity. Additionally, nearly 100% of Pnc adherence was inhibited by lipidated rPsaA at 2.5 micro g/ml. Our data support the argument that PsaA is an adhesin that mediates Pnc adherence to human nasopharyngeal cells. This functional assay may be useful in evaluating antibodies elicited in response to PsaA vaccination.