In vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains using the PrimeFlow RNA assay.

Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses.

Serological survey for antibodies against pestiviruses in Wyoming domestic sheep.

Pestiviruses including Bovine viral diarrhea virus type 1 (BVDV-1), BVDV-2 and Border disease virus (BDV) have been reported in both sheep and cattle populations, together with the HoBi-like, an emerging group of pestiviruses. Pestivirus control programs in the United States have focused on the control of BVDV-1 and 2. The incidence of pestivirus infection in sheep in the United States and the risk of transmission between cattle and sheep populations are unknown. The aim of this study was to perform serological surveillance for pestivirus exposure in sheep from an important sheep producing state in the Unites States, Wyoming. For this, sera from 500 sheep, collected across the state of Wyoming (US) in 2015-2016, were examined by comparative virus neutralization assay against four species/proposed species of pestiviruses: BVDV-1, BVDV-2, BDV and HoBi-like virus. Rates of exposure varied between geographic regions within the state. The overall pestivirus prevalence of antibodies was 5.6%. Antibodies were most frequently detected against BVDV-1 (4%), and the highest antibody titers were also against BVDV-1. Data from this study highlights understanding of the dynamics of sheep pestivirus exposure, consideration of reference strains used for VN assays, transmission patterns, and potential vaccination history should be taken into account in implementation of control measures against pestiviruses in sheep and for successful BVDV control programs in cattle.

HoBi-Like Virus RNA Detected in Foetuses Following Challenge of Pregnant Cows that had Previously Given Birth to Calves Persistently Infected with Bovine Viral Diarrhoea Virus.

The ability of ruminant pestivirus including bovine viral diarrhoea virus (BVDV) and the related emerging pestivirus, HoBi-like virus, to establish persistent infection (PI) following foetal infection is central to keeping these viruses in circulation. Non-PI dams carrying BVDV PI calves develop high levels of immunity due constantly viral exposure. A study to determine whether the immunity developed following the generation of a BVDV PI is enough to prevent HoBi-like virus infection of a subsequent foetus was performed. This study consisted of nine pregnant cows, four had birthed BVDV-1 PI calves in a previous pregnancy, three cows had birthed BVDV-2 PIs and two had birthed pestivirus negative calves. From this, six pregnant cows were challenged with HoBi-like virus about day 85 of gestation (four BVDV-1 and two BVDV-2 cows) and three non-challenged cows (negative control). At the day of challenge, the serum neutralizing titres against the homologous BVDV strains of the first inoculation ranged from 1148 to 5793. At day 6 post-challenge, HoBi-like RNA was detected in the serum of all four BVDV-1 cows but not in the two BVDV-2 cows. The foetuses harvested from five of the exposed dams (three BVDV-1 and two BVDV-2 cows) at day 30 post-challenge were positive for HoBi-like virus RNA. The sixth cow, BVDV-1 cow #541, while pregnant at the time of exposure, had no foetus 30 days after exposure. Foetuses from HoBi-like virus exposed dams were significantly smaller and lighter than control foetuses. HoBi-like RNA was detected in samples of all challenged foetuses. The identification of viral RNA in the serum of 4 cows at day 6 post-challenge, as well viral RNA detection in all foetuses 30 days post-inoculation, indicates that the foetuses of dams with high antibodies titres against BVDV-1 or BVDV-2 would not be protected from challenge with a HoBi-like virus.

Health, social and economic consequences of dementias: a comparative national cohort study.

BACKGROUND AND PURPOSE: Dementia causes morbidity, disability and mortality, and as the population ages the societal burden will grow. The direct health costs and indirect costs of lost productivity and social welfare of dementia were estimated compared with matched controls in a national register based cohort study. METHODS: Using records from the Danish National Patient Registry (1997-2009) all patients with a diagnosis of Alzheimer's disease, vascular dementia or dementia not otherwise specified and their partners were identified and compared with randomly chosen controls matched for age, gender, geographical area and civil status. Direct health costs included primary and secondary sector contacts, medical procedures and medication. Indirect costs included the effect on labor supply. All cost data were extracted from national databases. The entire cohort was followed for the entire period - before and after diagnosis. RESULTS: In all, 78 715 patients were identified and compared with 312 813 matched controls. Patients' partners were also identified and matched with a control group. Patients had lower income and higher mortality and morbidity rates and greater use of medication. Social- and health-related vulnerability was identified years prior to diagnosis. The average annual additional cost of direct healthcare costs and lost productivity in the years before diagnosis was 2082 euros per patient over and above that of matched controls, and 4544 euros per patient after the time of diagnosis. CONCLUSIONS: Dementias cause significant morbidity and mortality, consequently generating significant socioeconomic costs.

Experimental infection of calves, sheep, goats and pigs with HoBi-like viruses by direct inoculation or exposure to persistently infected calves.

HoBi-like viruses are an emerging species of pestiviruses associated with respiratory and reproductive disease in cattle and in water buffaloes. Although cattle appear to be the main natural hosts, little is know about the potential for HoBi-like viruses to be transmitted to other livestock. In this study, seronegative calves, goats and pigs, and sheep harboring pestivirus antibodies (probably due to previous exposure to BVDV) were exposed to HoBi-like viruses either by direct inoculation (GIn) or by contact with calves persistently infected with HoBi-like viruses (GEx). Both GIn and GEx groups were monitored for clinical signs, lymphocyte count, virus in buffy coats and nasal swabs up to day 18 post-inoculation (pi). Evidence of transmission of HoBi-like virus by PI calves was observed in all studied species. No difference in clinical presentation was observed between animals in the GIn or GEx groups. Evidence of infection, depending on the species included lymphocyte depletion, fever, viral RNA detection, and/or seroconversion. Depletion of lymphocytes was observed in calves and goats (35% and 50%, respectively) but not in pigs. Seroconversion was observed in at least one animal of each group and for all exposed species. The rate of seroconversion was higher in animals in the GIn experimental groups. In sheep, pre-existing moderate to high neutralizing titers against BVDV did not prevent viral replication and shed. The study demonstrated that naive cattle, goats and pigs, in addition to antibody positive sheep, can be infected by HoBi-like virus via persistently infected calf and potentially transmit the virus.

Comparison of the breadth and complexity of bovine viral diarrhea (BVDV) populations circulating in 34 persistently infected cattle generated in one outbreak.

Exposure to bovine viral diarrhea viruses (BVDV) results in acute and persistent infections. Persistent infections result from in utero exposure during the first trimester of gestation. Clinical presentation, in persistently infected cattle (PI), is highly variable. The reasons for this variation is largely unknown. The BVDV circulating in PI exist as quasispecies (swarms of individual viruses). An outbreak resulting in 34 PI cattle presented an opportunity to compare a large number of PI׳s. Methods were developed to compare the circulating viral populations within PI animals. It was found that PI animals generated in the same outbreak carry circulating viral populations that differ widely in size and diversity. Further, it was demonstrated that variation in PI viral populations could be used as a quantifiable phenotype. This observation makes it possible to test the correlation of this phenotype to other phenotypes such as growth rate, congenital defects, viral shed and cytokine expression.

Generation of calves persistently infected with HoBi-like pestivirus and comparison of methods for detection of these persistent infections.

The identification and elimination of persistently infected (PI) cattle are the most effective measures for controlling bovine pestiviruses, including bovine viral diarrhea virus (BVDV) and the emerging HoBi-like viruses. Here, colostrum-deprived calves persistently infected with HoBi-like pestivirus (HoBi-like PI calves) were generated and sampled (serum, buffy coat, and ear notches) on the day of birth (DOB) and weekly for 5 consecutive weeks. The samples were subjected to diagnostic tests for BVDV--two reverse transcriptase PCR (RT-PCR) assays, two commercial real-time RT quantitative PCR (RT-qPCR), two antigen capture enzyme-linked immunosorbent assays (ACE), and immunohistochemistry (IHC)--and to HoBi-like virus-specific RT-PCR and RT-qPCR assays. The rate of false negatives varied among the calves. The HoBi-like virus-specific RT-PCR detected HoBi-like virus in 83%, 75%, and 87% of the serum, buffy coat, and ear notch samples, respectively, while the HoBi-like RT-qPCR detected the virus in 83%, 96%, and 62%, respectively. In comparison, the BVDV RT-PCR test had a higher rate of false negatives in all tissue types, especially for the ear notch samples (missing detection in at least 68% of the samples). The commercial BVDV RT-qPCRs and IHC detected 100% of the ear notch samples as positive. While ACE based on the BVDV glycoprotein E(rns) detected infection in at least 87% of ear notches, no infections were detected using NS3-based ACE. The BVDV RT-qPCR, ACE, and IHC yielded higher levels of detection than the HoBi-like virus-specific assays, although the lack of differentiation between BVDV and HoBi-like viruses would make these tests of limited use for the control and/or surveillance of persistent HoBi-like virus infection. An improvement in HoBi-like virus tests is required before a reliable HoBi-like PI surveillance program can be designed.

Changes observed in the thymus and lymph nodes 14 days after exposure to BVDV field strains of enhanced or typical virulence in neonatal calves.

Clinical presentation following uncomplicated acute infection with bovine viral diarrhea viruses (BVDV) ranges from clinically unapparent to severe (including hemorrhagic disease and death) depending on the strain virulence. Regardless of clinical presentation, BVDV infection of cattle results in a generalized immunosuppression. BVDV immunosuppression is characterized by a reduction of circulating white blood cells (WBC) that is typically evident by day 3 post infection (PI). In infections with typical BVDV field strains WBC counts decrease until days 6-9 PI and then return to baseline values. In infections with enhanced virulence BVDV, WBC counts may continue to decline through day 14. In this study, the lymph nodes and thymus of non-infected neonatal calves and neonatal calves infected 14 days previously with either a BVDV of typical virulence or one of enhanced virulence were compared. It was found that while calves, infected with the typical virulence BVDV, had cleared BVDV, and WBC counts had returned to near baseline, the number of B-B7(+) cells in lymph nodes were reduced whereas numbers of CD4(+) cells were increased as compared to control calves. In contrast, calves infected with the high virulence strain, had not cleared the virus by day 14 and WBC counts had not returned to pre-exposure levels. Furthermore, these calves had more substantial deficits of B-B7(+) and CD4(+) cell subpopulations, compared to calves infected with a typical virulence strain. There were also an increased number of macrophages observed in both lymphoid tissues examined. The thymuses from both groups of BVDV-infected calves were significantly smaller than non-infected age matched calves. The reduction in size was accompanied by a significant depletion of the thymic cortex. These results indicate that regardless of the virulence of the infecting BVDV, infection leaves neonatal calves with deficits in specific lymphocyte subsets and lymphoid tissues that could have long-term immunosuppressive implications.

RT-PCR expression profiling of matrix metalloproteinases and their specific inhibitors in cell lines and fresh biopsies of squamous cell carcinomas of the head and neck.

The expressions of MMP2, -7, -9, -13 and TIMP1, -2, -3 were examined in biopsies and cell lines of head and neck squamous cell carcinomas (HNSCC) to determine the association between the expression profile and TNM-staging of the primary. The expressions of MMP2, -7, -9, -13 and TIMP1, -2, -3 were analyzed in 30 HNSCC biopsies, 7 HNSCC cell lines and 1 keratinocyte cell line using RT-PCR. Negative correlation was determined between N-status and MMP13-RNA expression [Kendall-tau-b -0.404 (p = 0.016), Spearman-rho -0.448 (p = 0.014)], histological grading [Kendall-tau-b -0.291 (p = 0.049), Spearman-rho -0,333 (p = 0.048)], and MMP7 and TIMP2 expression [Kendall-tau-b -0.318 (p = 0.045); Spearman-rho -0.353 (p = 0.045)]. Positive correlation was determined between M-status and MMP9-RNA expression [Kendall-tau-b 0.341 (p = 0.025), Spearman-rho 0.377 (p = 0.024)] and MMP13 and TIMP2 expression [Kendall-tau-b 0.727 (p = 0.037), Spearman-rho 0.850 (p = 0.016)]. The results point to a role of the tested MMPs and TIMPs in the metastatic spread of HNSCC.

Allelic differences in the VHOx-1 gene explain the absence of a B cell clonal dominance in the primary response of C57BL/6 mice to phthalate.

A highly conserved Id (CRIXmp-1) associated with the murine (BALB/c) humoral immune response to the hapten phthalate (Xmp) is conspicuously absent in C57BL/6 mice. The absence of this Id in C57BL/6 mice is shown here to be due to the absence of the appropriate VH gene (VHOx-1) usage in the Xmp response. To determine whether the failure to utilize this VH was due to an active suppression or to the lack of the requisite VH gene in the available repertoire, VHOx-1 gene-specific primers were used to amplify the germ-line VHOx-1 gene from genomic DNA from BALB/c and C57BL/6 mice. The germ-line coding sequence of the C57BL/6 allele of the VHOx-1 gene is 99% similar to the germ-line coding sequence of the BALB/c allele. Amplification of cDNA made from splenic RNA from C57BL/6 mice confirmed that this gene is expressed. There are four nucleotide differences that lead to three amino acid changes in the predicted protein sequence. Each change is either in or immediately adjacent to a complementarity-determining region (CDR). Two of these changes are unique to the C57BL/6 allele and are not shared with CRIXmp-1-expressing strains. These two changes are predicted to alter the Xmp binding capabilities of the C57BL/6 allelic form of this VH gene, thereby explaining the absence of the Xmp-1 clonotype, which is dominant in the primary Xmp immune response of most other strains of mice.

Transient dominance of the early primary immune response by a highly conserved B-cell clone that is distinguished by its lack of memory, high threshold of activation, and a high affinity.

We establish here that the very early primary response to the hapten phthalate (Xmp) is distinguished by a restricted heterogeneity with over 80% of the anti-Xmp antibodies expressing a single well-defined cross-reactive idiotype (CRIXmp-1) associated with a previously described highly conserved clonotype that is expressed by most inbred strains of mice and many outbred mouse populations as well. The characteristic early dominance of this one clonotype in the primary response is transient. While the CRIXmp-1 clonotype is present later in the primary and throughout the secondary response, it represents only a very small proportion of the total anti-Xmp antibody population at these times. The early dominance of the single clonotype is rapidly replaced by a heterogeneous population of antibodies. Differential activation thresholds for the primary response clonotype (CRIXmp-1) and secondary response clonotypes, and the failure of the dominant primary response clonotype to expand in the secondary response (i.e., absence of memory) suggest the presence of two distinct B-cell lineages.

Evaluation of lymphocytes' influence in luminol-dependent chemiluminescence emission of mouse spleen cells.

The focus of this study was to elucidate the influence of lymphocytes on phagocytic cell-dependent chemiluminescence (CL). CL was induced by in vitro addition of mitogens, particulate substances and Keyhole Limpet Hemocyanin (KLH)-antigen. CL was measured in spleen cells of immunologically naive mice and in mice immunized with KLH. Mice immunized with KLH and Freund's adjuvant incomplete (FA) showed a lower CL-response than mice injected with FA only. No influence on CL-activity by lymphocytes derived from immunologically naive mice could be found. Adoptive transfer of T cells from KLH-sensitized donor mice did not influence CL-activity elicited with Zymosan in spleen cells derived from KLH-unsensitized recipient mice. Preincubation of spleen cells with supernatants of T cell cultures or with T cell growth factor (TCGF) stimulated with KLH or Con A could not trigger CL-reactivity. Therefore, the lower CL-response of spleen cells derived from KLH-sensitized mice appears to be not a consequence of a suppressive cellular or lymphokine effect, but is due to direct influence of KLH on accessory cells themselves.

Effects of color stimulation and information on the copying performance of attention-problem adolescents.

The optimal stimulation theory proposes that hyperactive children are less tolerant of lower levels of arousal than nonhyperactive children and should thus derive greater gains from stimulation added to repetitive copying tasks than do comparisons. To test this hypothesis, 16 adolescents, rating high on attention and behavior problems, were matched on the basis of age and poor handwriting performance to 16 controls. Matched pairs were randomly assigned to treatment order (high-stimulation colored letters followed in 2 weeks by low-stimulation black letters or the reverse order) and to level of information (color added to difficult letter parts or added to randomly selected letters), counterbalanced for treatment order and level of information within each order. Errors and activity were subjected to a mixed-design analysis of covariance, with IQ the covariate. The major findings indicated that attention-problem adolescents performed better with high-stimulation task stimuli than with low, relative to the opposite performance pattern of controls. Different responding was significant for experimental but not for control children.

Efficacy of enzyme-linked immunosorbent assay in serodiagnosis of aspergillosis.

Sera from 43 cases of aspergillosis, 73 cases of other bronchopulmonary diseases, and 50 healthy persons were examined for the presence of antibodies to Aspergillus fumigatus. Enzyme-linked immunosorbent assay proved to be more efficacious than the immunodiffusion test and counterimmunoelectrophoresis in the cases of allergic bronchopulmonary and invasive aspergillosis.

Bacteria and zymosan opsonized with histone, dextran sulfate, and polyanetholesulfonate trigger intense chemiluminescence in human blood leukocytes and platelets and in mouse macrophages: modulation by metabolic inhibitors in relation to leukocyte-bacteria interactions in inflammatory sites.

Human blood leukocytes and platelets and mouse peritoneal macrophages emit very rapid and very intense Luminol-dependent chemiluminescence (CL) signals when treated with streptococci, staphylococci, or with zymosan, which have been preopsonized with arginine-rich histone, dextran sulfate or polyanetholesulfonate (liquoid). Liquoid alone at 10-30 micrograms/2 X 10(5) leukocytes also triggers intense CL responses in the absence of a carrier. Strong CL can also be triggered, and at the same levels, when the various polyelectrolytes are simply mixed with the bacteria or zymosan and added to the leukocyte suspensions. The CL responses induced by the polyelectrolyte-bacteria complexes greatly exceed those triggered in leukocytes by antibody-complement-coated particles. Liquoid also shows a unique property of markedly augmenting CL signals which have already been induced by other ligand-coated bacteria or zymosan particles. Streptococci and staphylococci were found to be much superior to zymosan, Gram-positive bacilli, or E. coli as carriers for the various polyelectrolytes in the CL reaction. Neither protamine sulfate, lysozyme, myeloperoxidase, crystalline ribonuclease (all cationic in nature), chondroitin sulfate, heparin, nor alginate sulfate acted as ligands for triggering CL, when used to opsonize bacteria or zymosan. The induction of CL in blood leukocytes by the various ligand-coated bacteria is markedly inhibited by azide, KCN catalase, aminotriazole, and EDTA, agents known to inhibit the production of oxygen radicals following stimulation of leukocytes by opsonized bacteria. Two children diagnosed for chronic granulomatous diseases (CGD) of childhood and an apparently healthy sister of one of the male patients completely failed to respond with CL either to the polyelectrolyte-bacteria complexes, liquoid or antibody-coated bacteria and zymosan. It is proposed that liquoid be employed for the rapid screening of defects in certain oxygen-dependent metabolic processes in both PMNs and macrophages. It is also suggested that polyelectrolytes like the ones described in this study may markedly enhance the bactericidal properties of leukocytes and macrophages towards both extracellular and intracellular microorganisms and may perhaps also augment the tumoricidal effects of activated macrophages.