Fluctuations In Titers Research Articles

GMP implementation of a hybrid continuous manufacturing process for a recombinant non-mAb protein-A case study.

Advances in manufacturing technology coupled with the increased potency of new biotherapeutic modalities have created an external environment where continuous manufacturing (CM) can address a growing need. Amgen has successfully implemented a hybrid CM process for a commercial lifecycle program. In this process, the bioreactor, harvest, capture column, and viral inactivation/depth filtration unit operations were integrated together in an automated, continuous module, while the remaining downstream unit operations took place in stand-alone batch mode. CM operations are particularly suited for so-called "high mix, low volume" manufacturing plants, where a variety of molecules are manufactured in relatively low volumes. The selected molecule fit this mold and was manufactured in a low-capital micro-footprint suite attached to an existing therapeutic production facility. Use of a hybrid process within an already operating facility required less capital and minimized complexity. To enable this hybrid CM process, an established fed-batch process was converted to a perfusion process with continuous harvest. Development efforts included both process changes and the generation of a novel cell line adapted to long-term perfusion. Chromatography resins were updated, and purification processes adapted to handle variable inputs due to the fluctuations in harvest titer from the lengthy production process. A novel automated single-use (SU) viral inactivation (VI) skid was introduced, which entailed the development of a robust pH verification and alarm system, along with procedures for product isolation to allow discard of specific cycles. The CM process demonstrated consistent performance, meaning it met predefined performance criteria (including product quality attributes, or PQAs) when operated within established process parameters and manufactured according to applicable procedures. Using a 75% reduction in scale, it resulted in a five-fold reduction in process media and buffer usage, a fifteen-fold increase in mass per thaw, and an overall process productivity increase of 45-fold (as measured by grams drug substance per liter per day.) The hybrid CM process also enabled increased material demand to be met with no change in cost of goods manufactured or plant capacity, due to the repurposing of existing facility space and the flexible duration of the hybrid CM harvest. Overall, the success of the hybrid CM platform represents an exciting opportunity to reduce costs and increase process efficiency in industry.

Fluctuation of Anti-Domain 1 and Anti-β2 -Glycoprotein I Antibody Titers Over Time in Patients With Persistently Positive Antiphospholipid Antibodies.

The present study was undertaken to longitudinally evaluate titers of antibodies against β2 -glycoprotein I (anti-β2 GPI) and domain 1 (anti-D1), to identify predictors of variations in anti-β2 GPI and anti-D1 titers, and to clarify whether antibody titer fluctuations predict thrombosis in a large international cohort of patients who were persistently positive for antiphospholipid antibodies (aPL) in the APS ACTION Registry. Patients with available blood samples from at least 4 time points (at baseline [year 1] and at years 2-4 of follow-up) were included. Detection of anti-β2 GPI and anti-D1 IgG antibodies was performed using chemiluminescence (BIO-FLASH; INOVA Diagnostics). Among 230 patients in the study cohort, anti-D1 and anti-β2 GPI titers decreased significantly over time (P < 0.0001 and P=0.010, respectively). After adjustment for age, sex, and number of positive aPL tests, we found that the fluctuations in anti-D1 and anti-β2 GPI titer levels were associated with treatment with hydroxychloroquine (HCQ) at each time point. Treatment with HCQ, but not immunosuppressive agents, was associated with 1.3-fold and 1.4-fold decreases in anti-D1 and anti-β2 GPI titers, respectively. Incident vascular events were associated with 1.9-fold and 2.1-fold increases in anti-D1 and anti-β2 GPI titers, respectively. Anti-D1 and anti-β2 GPI titers at the time of thrombosis were lower compared to titers at other time points. A 1.6-fold decrease in anti-D1 titers and a 2-fold decrease in anti-β2 GPI titers conferred odds ratios for incident thrombosis of 6.0 (95% confidence interval [95% CI] 0.62-59.3) and 9.4 (95% CI 1.1-80.2), respectively. Treatment with HCQ and incident vascular events in aPL-positive patients predicted significant anti-D1 and anti-β2 GPI titer fluctuations over time. Both anti-D1 and anti-β2 GPI titers decreased around the time of thrombosis, with potential clinical relevance.

Two dehydroecdysone reductases act as fat body-specific 20E catalyzers in Bombyx mori.

Periodic post-embryonic changes in insects, including growth, development and metamorphosis, are strictly controlled by many compounds, including steroid hormones. The biosynthesis and clearance of 20-hydroxyecdysone (20E), the major active form of the insect steroid hormone ecdysone, result in titer fluctuations that help control insect development. The inactivation of 20E in the silkworm Bombyx mori is highly tissue-specific, with CYP18A1 and ecdysone oxidase controlling 20E inactivation specifically in the mid-silk gland and midgut, respectively. Here, we characterized silkworm 3-dehydroecdysone 3α reductase (Bm3DE3α) and 3-dehydroecdysone 3β reductase (Bm3DE3β), two enzymes involved predominantly in the C-3-mediated catalysis of 20E in fat bodies. The ubiquitous and silk gland-specific overexpression of Bm3DE3α decreased the 20E titer, resulting in larval lethality and larval-pupal transition failure, respectively. In contrast, the ubiquitous and mid-silk gland-specific overexpression of Bm3DE3β increased the 20E titer, resulting in larval growth delays and lethality at the mid-fifth larval stage, respectively. Thus, Bm3DE3α and Bm3DE3β mediate fat body-specific steroid hormone metabolism in B. mori, indicating that highly diversified 20E metabolism-related mechanisms exist in different insect species.

Fluctuations in Antibody Titers against Enterovirus D68 in Pediatric Sera Collected in a Community before, during, and after a Possible Outbreak.

We previously reported a hospital-based epidemiological study on enterovirus (EV)-D68 infection among children during the autumn of 2015, which indirectly inferred an outbreak in Sendai, Japan. In this study, stocked sera of children (aged 0-6 years; without symptoms of infectious diseases) in the Sendai community collected during 4 periods (1 year before, 6 months before, immediately after, and 1 year after the possible outbreak period) were analyzed using the neutralization antibody titer assay to determine community children's immunity levels against EV-D68 infection. The immunity levels were confirmed to have increased during the possible outbreak period and to have gradually waned over 1 year without another outbreak. These results provide background information supporting the results of our previous hospital-based surveillance study.

HSP70iQ435A-Encoding DNA Repigments Vitiligo Lesions in Sinclair Swine

Human HSP70iQ435A carries a single amino-acid modification within the dendritic cell activating region and tolerizes dendritic cells invitro. The underlying DNA was used to prevent and treat disease in vitiligo mouse models through reduced dendritic cell activation and diminished skin T-cell infiltration, suggesting the same may be useful for patients. Physiologic differences between mouse and human skin then called for studies in large animals with human-like skin. We established the efficiency of DNA jet injection into swine skin before subcloning HSP70iQ435A into clinically suitable vector pUMVC3. Vitiligo lesions in Sinclair swine were treated with plasmid DNA to measure changes in depigmentation, T-cell infiltration, expression of HSP70i in skin, serum HSP70i, and anti-HSP70i serum titers. Remarkable repigmentation following HSP70iQ435A-encoding DNA treatment persisted throughout the 6-month follow-up period. Repigmentation was accompanied by an initial influx of T cells accompanied by increased CD4/CD8 ratios, waning by week 15. Melanocytes spanned the border of repigmenting skin, suggesting that melanocyte repopulation precedes skin melanization. Serum titer fluctuations were not treatment-associated. Importantly, treatment did not interfere with melanoma immunosurveillance. These data encourage clinical testing of HSP70iQ435A.

Increasing ICA512 autoantibody titers predict development of abnormal oral glucose tolerance tests.

Determine if autoantibody titer magnitude and variability predict glucose abnormalities in subjects at risk for type 1 diabetes. Demographic information, longitudinal autoantibody titers, and oral glucose tolerance test (OGTT) data were obtained from the TrialNet Pathway to Prevention study. Subjects (first and second degree relatives of individuals with type 1 diabetes) with at least 2 diabetes autoantibodies were selected for analysis. Autoantibody titer means were calculated for each subject for the duration of study participation and the relationship between titer tertiles and glucose value tertiles from OGTTs (normal, impaired, and diabetes) was assessed with a proportional odds ordinal regression model. A matched pairs analysis was used to examine the relationship between changes in individual autoantibody titers and 120-minute glucose values. Titer variability was quantified using cumulative titer standard deviations. We studied 778 subjects recruited in the TrialNet Pathway to Prevention study between 2006 and 2014. Increased cumulative mean titer values for both ICA512 and GAD65 (estimated increase in proportional odds = 1.61, 95% CI = 1.39, 1.87, P < 1 × 10-9 and 1.17, 95% CI = 1.03, 1.32, P = .016, respectively) were associated with peak 120-minute glucose values. While fluctuating titer levels were observed in some subjects, no significant relationship between titer standard deviation and glucose values was observed. ICA512 autoantibody titers associate with progressive abnormalities in glucose metabolism in subjects at risk for type 1 diabetes. Fluctuations in autoantibody titers do not correlate with lower rates of progression to clinical disease.

Validation of chemiluminescent enzyme immunoassay in detection of autoantibodies in pemphigus and pemphigoid

BackgroundA novel chemiluminescent enzyme immunoassay (CLEIA) was recently developed to quantify autoantibodies specific for desmogleins (Dsgs) and BP180, the target antigens of pemphigus and pemphigoid. This assay is automated and highly accurate and efficient. ObjectiveTo validate the use of the CLEIA for detection of autoantibodies during the clinical courses of patients with pemphigus and pemphigoid. MethodsTo define cut-off values for Dsg1, Dsg3, and BP180, we evaluated 47 serum samples from patients with pemphigus foliaceus (PF), 59 from those with pemphigus vulgaris (PV), 52 from those with bullous pemphigoid (BP), and 995 from healthy individuals. We also evaluated any fluctuations in CLEIA titers according to disease activity during the clinical course of 10 cases each of PF, PV, and BP. We used clinical symptom scores, the pemphigus disease area index (PDAI) and the bullous pemphigoid disease area index (BPDAI), to evaluate disease activity. ResultsThe cut-off values for the CLEIA titers determined by the Youden index were 15.4U/mL for Dsg1, 14.9U/mL for Dsg3, and 16.8U/mL for BP180. CLEIA titers fluctuated in parallel with the PDAI/BPDAI scores in 28 of the 30 cases with PF, PV, or BP. Although the CLEIA and enzyme-linked immunosorbent assay (ELISA) values in the same samples differed substantially in some cases, the concordance rates of positive/negative results between the CLEIA and ELISA were 96% for Dsg1, 97% for Dsg3, and 96% for BP180. ConclusionThe CLEIA, a newly developed, highly effective autoantibody detection system, is as reliable as ELISA for evaluation of the clinical courses of pemphigus and pemphigoid.

Altered differential hemocyte count in 3rd instar larvae of Drosophila melanogaster as a response to chronic exposure of Acephate.

Acephate, an organophosphate (OP) pesticide, was used to investigate the effects of its chronic exposure on hemocyte abundance in a non-target dipteran insect Drosophila melanogaster. For this purpose, six graded concentrations ranging from 1 to 6 μg/ml were selected, which are below the reported residual values (up to 14 μg/ml) of the chemical. 1st instar larvae were fed with these concentrations up to the 3rd instar stage and accordingly hemolymph smears from these larvae were prepared for differential hemocyte count. Three types of cells are found in Drosophila hemolymph, namely, plasmatocytes, lamellocytes and crystal cells. Plasmatocyte count was found to decrease with successive increase in treatment concentrations. Crystal cells showed an increasing trend in their number. Though the number of lamellocytes was very low, a bimodal response was noticed. Lamellocyte number was found to increase with the initial three concentrations, followed by a dose dependent reduction in their number. As hemocytes are directly linked to the immune system of fruit flies, fluctuations in normal titer of these cells may affect insect immunity. Hemocytes share homologies in their origin and mode of action with the immune cells of higher organisms including man. Thus the present findings suggest that immune cells of humans and other organisms may be affected adversely under chronic exposure to Acephate.

A novel therapeutic strategy for mycoplasma infectious diseases

Mycoplasma Infectious Diseases (MID) are systemic illnesses that cause vasculitis and neuritis. MID not only includes pneumonia but also diseases such as asthma, arthritis, nephritis, meningitis, encephalitis, dermatitis, pancreatitis, hepatitis, and hematologic illnesses. The broader concept of MID encompasses acute to chronic phases with diverse symptoms. Therefore, it is often confusing and difficult to identify Mycoplasma-infected patients among those with incurable diseases, such as autoimmune diseases, rheumatic diseases, nervous system disorders, and hematological disorders. Regrettably, conventional diagnosis has only been available for pneumonia, although it is critical to identify MID at early stages for effective medical treatment. A cutting-edge technology has made it possible to measure the amount of specific antibodies to species-specific mycoplasma glycolipid-antigens. This new technology provides a reliable marker to follow the state of MID by monitoring antibody titer fluctuations. A novel therapeutic strategy based on new serological diagnostics is introduced in this review.

Roles of ecdysteroids for progression of reproductive cycle in the fresh water crustacean Daphnia magna

Daphnia magna exhibits a parthenogenetic reproductive cycle linked to a moulting cycle, but regulatory mechanisms of neither moulting nor reproductive cycle are understood in daphnids. Moulting is regulated by ecdysteroids in insects. A previous study showed that a titre of ecdysteroids changed during the reproductive cycle in D. magna; however, no clear correlation among titre, moulting and reproductive cycles has been proved in daphnids. To understand endocrine mechanisms underlying the coordinated reproductive cycle, we analysed the expression of genes coding for enzymes in ecdysteroids synthesis or inactivation pathways, and the effects of 20-hydroxyecdysone (20E) on moulting and ovulation in D. magna. We cloned orthologues of neverland (nvd) and shade (shd) in the ecdysteroids synthesis pathway, and Cyp18a1 in the ecdysteroids inactivation pathway previously identified in insects. Gene expression of Cyp18a1 changed conversely with the fluctuation in ecdysteroids titre during the intermoulting period. Tissue-specific expression analysis of nvd showed a prominent expression in the gut. Furthermore, treatment of adult female D. magna with 20E inhibited moulting and/or ovulation. Our cloning and phylogenetic analyses showed that nvd and shd as well as Cyp18a1 are evolutionary conserved in D. magna, suggesting that these genes appeared in arthropods before the radiation of insects. The gene expression analysis during the reproductive cycle indicated that Cyp18a1 possibly regulates the decline of ecdysteroid titre before moulting and ovulation. Furthermore, the expression of nvd in the gut suggested that ecdysone might be synthesised in the gut. Exogenous 20E-treatment resulted in the failure of not only moulting, but also ovulation, suggesting that a low level of ecdysteroids before moulting is required for moulting and ovulation in D. magna.

Continuous Influenza Virus Production in Cell Culture Shows a Periodic Accumulation of Defective Interfering Particles

Influenza viruses are a major public health burden during seasonal epidemics and a continuous threat due to their potential to cause pandemics. Annual vaccination provides the best protection against the contagious respiratory illness caused by influenza viruses. However, the current production capacities for influenza vaccines are insufficient to meet the increasing demands. We explored the possibility to establish a continuous production process for influenza viruses using the duck-derived suspension cell line AGE1.CR. A two-stage bioreactor setup was designed in which cells were cultivated in a first stirred tank reactor where an almost constant cell concentration was maintained. Cells were then constantly fed to a second bioreactor where virus infection and replication took place. Using this two-stage reactor system, it was possible to continuously produce influenza viruses. Surprisingly, virus titers showed a periodic increase and decrease during the run-time of 17 days. These titer fluctuations were caused by the presence of defective interfering particles (DIPs), which we detected by PCR. Mathematical modeling confirmed this observation showing that constant virus titers can only emerge in the absence of DIPs. Even with very low amounts of DIPs in the seed virus and very low rates for de novo DIP generation, defective viruses rapidly accumulate and, therefore, represent a serious challenge for continuous vaccine production. Yet, the continuous replication of influenza virus using a two-stage bioreactor setup is a novel tool to study aspects of viral evolution and the impact of DIPs.

The Impact of Combined Antiretroviral Therapy on Biologic False-Positive Rapid Plasma Reagin Serologies in a Longitudinal Cohort of HIV-Infected Persons

Our objective was to determine the impact of combination antiretroviral therapy (cART) and the degree of immunosuppression on biologic false-positive (BFP) rapid plasma reagin (RPR) tests among persons infected with human immunodeficiency virus (HIV). This was a nested retrospective study of HIV-infected patients enrolled in the Johns Hopkins HIV Clinical Cohort. BFP RPR was defined as a reactive RPR and a nonreactive fluorescent treponemal antibody-absorption (FTA-ABS) test. Patients with BFP tests were compared to 2 control groups: HIV-infected patients (1) with active syphilis (reactive RPR and FTA-ABS) and (2) without current syphilis (nonreactive RPR). A persistent BFP test was defined by having at least 2 visits with consistent BFP at all visits. Of 711 patients with HIV, 96 (13.5%) had BFP tests and 342 (48.1%) had syphilis. Twenty-two of 96 (23%) had persistent BFP tests. cART use was associated with decreased odds of BFP tests compared to having syphilis (adjusted odds ratio [AOR], 0.31; 95% CI, .15-.63) and those with nonreactive RPR (AOR, 0.42; 95% CI, .22-.81). cART use was also associated with decreased odds of BFP persistence (AOR, 0.07; 95% CI, .01-.33). Neither CD4 count nor HIV RNA was significantly associated with BFP test results. Lower RPR titers, younger age, and injection drug use were associated with increased odds of BFP. The use of cART appears to decrease the odds of BFP RPR tests. This finding suggests that nontreponemal titer fluctuations in persons with HIV may reflect the influence of factors unrelated to syphilis disease activity.

Integration of stochastic simulation with multivariate analysis: Short-term facility fit prediction

This article describes a decision-support tool to help pinpoint the potential root causes of sub-optimal short-term facility fit issues in biopharmaceutical facilities. This was achieved by creating a tool that integrated stochastic simulation with advanced multivariate statistical analysis. Process fluctuations in product titers in cell culture, step yields, and chromatography eluate volumes were mimicked using Monte Carlo simulation data derived using a stochastic discrete-event simulation model. The resulting stochastic datasets, with the computed consequences on key metrics such as product mass loss and cost of goods, were examined using advanced multivariate statistical techniques. Principal component analysis combined with clustering algorithms was used to analyze the complex datasets from complete industrial batch processes for biopharmaceuticals. The challenge of visualizing the multidimensional nature of the dataset was addressed using hierarchical and k-means clustering as well as stacked parallel co-ordinate plots to help identify process fingerprints and characteristics of clusters leading to sub-optimal facility fit issues. Industrially-relevant case studies are presented that focus on technology transfer challenges for therapeutic antibodies moving from early phase to late phase clinical trials. The case study details how sub-optimal facility fit can be alleviated by allocating alternative product pool tanks. The impact of this operational change is then assessed by reviewing an updated process fingerprint.

Decisional tool to assess current and future process robustness in an antibody purification facility

Increases in cell culture titers in existing facilities have prompted efforts to identify strategies that alleviate purification bottlenecks while controlling costs. This article describes the application of a database-driven dynamic simulation tool to identify optimal purification sizing strategies and visualize their robustness to future titer increases. The tool harnessed the benefits of MySQL to capture the process, business, and risk features of multiple purification options and better manage the large datasets required for uncertainty analysis and optimization. The database was linked to a discrete-event simulation engine so as to model the dynamic features of biopharmaceutical manufacture and impact of resource constraints. For a given titer, the tool performed brute force optimization so as to identify optimal purification sizing strategies that minimized the batch material cost while maintaining the schedule. The tool was applied to industrial case studies based on a platform monoclonal antibody purification process in a multisuite clinical scale manufacturing facility. The case studies assessed the robustness of optimal strategies to batch-to-batch titer variability and extended this to assess the long-term fit of the platform process as titers increase from 1 to 10 g/L, given a range of equipment sizes available to enable scale intensification efforts. Novel visualization plots consisting of multiple Pareto frontiers with tie-lines connecting the position of optimal configurations over a given titer range were constructed. These enabled rapid identification of robust purification configurations given titer fluctuations and the facility limit that the purification suites could handle in terms of the maximum titer and hence harvest load.

Global Wolbachia prevalence, titer fluctuations and their potential of causing cytoplasmic incompatibilities in tsetse flies and hybrids of Glossina morsitans subgroup species

We demonstrate the high applicability of a novel VNTR-based (Variable-Number-Tandem-Repeat) molecular screening tool for fingerprinting Wolbachia-infections in tsetse flies. The VNTR-141 locus provides reliable and concise differentiation between Wolbachia strains deriving from Glossina morsitans morsitans, Glossina morsitans centralis, and Glossina brevipalpis. Moreover, we show that certain Wolbachia-infections in Glossina spp. are capable of escaping standard PCR screening methods by ‘hiding’ as low-titer infections below the detection threshold. By applying a highly sensitive PCR-blot technique to our Glossina specimen, we were able to enhance the symbiont detection limit substantially and, consequently, trace unequivocally Wolbachia-infections at high prevalence in laboratory-reared G. swynnertoni individuals. To our knowledge, Wolbachia-persistence was reported exclusively for field-collected samples, and at low prevalence only. Finally, we highlight the substantially higher Wolbachia titer levels found in hybrid Glossina compared to non-hybrid hosts and the possible impact of these titers on hybrid host fitness that potentially trigger incipient speciation in tsetse flies.

Recent topics on the regulatory mechanism of ecdysteroidogenesis by the prothoracic glands in insects.

Molting and metamorphosis are strictly regulated by steroid hormones known as ecdysteroids. It is now widely recognized that ecdysteroid biosynthesis (ecdysteroidogenesis) in the prothoracic gland (PG) is regulated by the tropic factor prothoracicotropic hormone (PTTH). However, the importance of PTTH in the induction of molting and metamorphosis remains unclear, and other mechanisms are thought to be involved in the regulation of ecdysteroidogenesis by the PG. Recently, new regulatory mechanisms, prothoracicostatic factors, and neural regulation have been explored using the silkworm, Bombyx mori, and two circulating prothoracicostatic factors, prothoracicostatic peptide (PTSP) and Bommo-myosuppressin (BMS), have been identified. Whereas PTTH and BMS are secreted from the brain, PTSP is secreted from the peripheral neurosecretory system – the epiproctodeal gland – during the molting stage. The molecular basis of neural regulation of ecdysteroidogenesis has been revealed for the first time in B. mori. The innervating neurons supply both Bommo-FMRF related peptide (BRFa) and orcokinin to maintain low levels of ecdysteroids during the feeding stage. These complex regulatory mechanisms – involving tropic and static factors, peripheral neurosecretory cells as well as the central neuroendocrine system, and neural regulation in addition to circulating factors collaborate to regulate ecdysteroidogenesis. Thus, together they create the finely tuned fluctuations in ecdysteroid titers needed in the hemolymph during insect development.

Mosaic Symptoms Induced by Cucumber mosaic virus in Polygala myrtifolia in France and New Zealand.

Myrtle-leaf milkwort or sweet pea shrub (Polygala myrtifolia L.), family Polygalaceae, is a shrub from South Africa and is well adapted to Mediterranean-type conditions and used as an ornamental plant in gardens and pots or as cut flowers. During 2002 and 2003, mosaic symptoms and leaf distortion were observed in P. myrtifolia in Menton, Roquebrune-Cap Martin, Golfe Juan, and Antibes (Alpes Maritimes Department, France) in public gardens and potted plants. Occasionally, white streaks were observed in flowers. Cucumber mosaic virus (CMV) was identified in samples collected from the four locations on the basis of transmission to and symptoms exhibited by a range of diagnostic host plants (1), observation of isometric particles (≅30 nm) in crude sap preparations from the infected plants by electron microscopy, and positive reaction using double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with polyclonal antibodies raised against CMV (2). Each isolate was shown to be a group II CMV strain (3) using double-immunodiffusion analysis. During 2004, CMV was also detected using DAS-ELISA in P. myrtifolia samples collected in New Zealand (Christchurch, Akaroa, and Roturoa). To confirm that CMV was responsible for pathogenicity, the Menton isolate was isolated from local lesions on Vigna unguiculata, amplified in Nicotiana tabacum cv. Xanthi-nc, and then mechanically inoculated into 1-year-old P. myrtifolia, P. myrtifolia cv. Grandiflora, and P. myrtifolia cv. Compacta (synonymous to cv. Nana) plants. The D strain of CMV, a reference tomato strain from subgroup I (2), was used for comparison. All experimental plants were propagated from cuttings, grown hydroponically and all tested negative for CMV using DAS-ELISA prior to inoculation. At 12 weeks postinoculation, systemic symptoms were observed on leaves from all inoculated plants (10 plants per genotype for the Menton isolate and 5 plants per genotype for the D strain), except for two P. myrtifolia plants inoculated with the Menton isolate. CMV was detected in apical, noninoculated leaves using DAS-ELISA in all symptomatic plants. A total recovery from symptoms was observed in P. myrtifolia and P. myrtifolia cv. Grandiflora but not in P. myrtifolia cv. Compacta at 6 months postinoculation (mpi) in 7 of 15, 10 of 15, and 15 of 15 DAS-ELISA positive plants, respectively. At 7 mpi, the plants were pruned and planted in soil and at 8 mpi, CMV was detected using DAS-ELISA in most of the plants, and symptoms developed in a few stems of some of the plants. Tessitori et al. (4) described similar symptoms and have detected CMV in P. myrtifolia from Italy, but they did not reproduce the disease in healthy plants. Our results show that CMV is responsible for the symptoms observed and that both CMV subgroups are infectious in P. myrtifolia. Since P. myrtifolia is generally vegetatively propagated by cuttings, frequent CMV tests on the mother stock plants are recommended because of fluctuations in virus titer and symptom expression in some genotypes. To our knowledge, this is the first report of this CMV host in France and New Zealand. A voucher specimen will be deposited at the Station de Pathologie Végétale at INRA, Montfavet.

Recognition patterns of Neospora caninum tachyzoite antigens by bovine IgG at different IFAT titres.

This study describes qualitative and quantitative antibody response in cows naturally infected with Neospora caninum. The study was carried out with 269 serum samples obtained from 24 cows over a period of 15 weeks. Prior to sample collection, the cows were tested with ELISA. The 269 samples were screened with IFAT and categorized into seven IFAT titre groups (< 1 : 80, 1 : 80, 1 : 200, 1 : 600, 1 : 1000, 1 : 2000, > 1 : 2000). The samples were finally analysed by Western blotting. Seven immunodominant antigens (approximately 18-, approximately 25-, approximately 33-, approximately 35-36-, approximately 45-46-, approximately 47-, approximately 60-62 kDa) and five minor antigens (approximately 25, approximately 51, approximately 64, approximately 77, approximately 116 kDa) were recognized by cow sera. The recognition of approximately 46 kDa antigen by cow sera was common to samples with IFAT titre 1 : 80 and above. Another common antigen was the approximately 18 kDa antigen, which was recognized by samples with IFAT titre 1 : 200 and above. The most remarkable observation was the presence of the 45-46 kDa, the 77 kDa, and absence of the 18 kDa antigenic bands in samples with IFAT titre 1 : 80. This observation was consistent even in the face of fluctuating antibody titre where serum antibody titres from an animal exceeded then failed to reach 1 : 80. Antibody fluctuation was observed across all cows (pregnant and aborted) with no discernible fluctuation pattern. However, the fluctuation in antibody titre observed appeared to be most remarkable in initially ELISA-negative pregnant cows, and to a lesser extent in ELISA-positive pregnant cows, and ELISA-positive aborted cows. Although there was fluctuation in antibody titre, the banding patterns of N. caninum tachyzoite antigens by cows within the same IFAT titre group remained similar.

Host plant pubescence: Effect on silverleaf whitefly, Bemisia argentifolii, fourth instar and pharate adult dimensions and ecdysteroid titer fluctuations

Host plant pubescence: Effect on silverleaf whitefly, Bemisia argentifolii, fourth instar and pharate adult dimensions and ecdysteroid titer fluctuations

Host plant pubescence: Effect on silverleaf whitefly, Bemisia argentifolii, fourth instar and pharate adult dimensions and ecdysteroid titer fluctuations

The ability to generate physiologically synchronous groups of insects is vital to the performance of investigations designed to test insect responses to intrinsic and extrinsic stimuli. During a given instar, the silverleaf whitefly, Bemisia argentifolii, increase in depth but not in length or width. A staging system to identify physiologically synchronous 4th instar and pharate adult silverleaf whiteflies based on increasing body depth and the development of the adult eye has been described previously. This study determined the effect of host plant identity on ecdysteroid fluctuations during the 4th instar and pharate adult stages, and on the depth, length and width dimensions of 4th instar/pharate adult whiteflies. When grown on the pubescent-leafed green bean, tomato and poinsettia plants, these stages were significantly shorter and narrower, but attained greater depth than when grown on the glabrous-leafed cotton, collard and sweet potato plants. Thus, leaf pubescence is associated with reduced length and width dimensions, but increased depth dimensions in 4(th) instars and pharate adults. For all host plants, nymphal ecdysteroid titers peaked just prior to the initiation of adult development. However, when reared on pubescent-leafed plants, the initiation of adult development typically occurred in nymphs that had attained a depth of 0.2 to 0.25 mm (Stage 3 - 4). When reared on glabrous-leafed plants, the initiation of adult development typically occurred earlier, in nymphs that had attained a depth of only 0.15-0.18 mm (Stage 2 Old - early 3). Therefore, based on ecdysteroid concentration, it appears that Stage-2, -3 and -4/5 nymphs reared on pubescent-leafed plants are physiologically equivalent to Stage-1, -2 Young and -2 Old/3, respectively, nymphs reared on glabrous-leafed plants. The host plant affected the width but not the height of the nymphal-adult premolt ecdysteroid peak. However, leaf pubescence was not the determining factor. Thus, host plant identity affects physiological events as well as structural characteristics during whitefly nymphal and adult development.