Field studies of the detection, persistence and spread of the Rispens CVI988 vaccine virus and the extent of co-infection with Marek's disease virus.

OBJECTIVE: To use specific real-time qPCR to determine (1) the vaccination success of Rispens CVI988 vaccine in feathers and dust; (2) persistence of Rispens infection in vaccinated layer chickens; (3) extent of co-infection with wild-type Marek's disease virus (MDV) in vaccinated layers; and (4) presence of Rispens virus in unvaccinated broiler flocks. METHODS: Feather, dust and serum samples were collected from birds aged 3 days to 91 weeks from three layer farms. qPCR was used to detect MDV and Rispens in DNA extracted from dust and feathers. Previously tested MDV-positive dust samples from 100 broiler flocks were tested for the presence of Rispens using qPCR, while serum samples were used to detect anti-MDV antibody using ELISA. RESULTS: Overall, 66% and 93% of feather and dust samples, respectively, from Rispens-vaccinated layers were Rispens-positive. Viral load in these samples varied between farms during early life, reaching readily detectable levels at 2-3 weeks of age. Vaccinated chickens maintained a high Rispens load in feathers and dust and high MDV antibody levels until 91 weeks of age. MDV infection was detected in 6.7% of feather samples from vaccinated chickens. Rispens virus was detected in 7% of samples from unvaccinated broiler flocks. CONCLUSION: Vaccine take can be measured effectively by Rispens-specific qPCR of feathers or dust from approximately 3 weeks post vaccination. Infection with Rispens is persistent, with lifelong shedding and serological response. The detectable infection rate of vaccinated chickens with MDV is low and there is preliminary evidence of escape of Rispens virus to unvaccinated flocks.

Pathotyping of Australian isolates of Marek's disease virus in commercial broiler chickens vaccinated with herpesvirus of turkeys (HVT) or bivalent (HVT/SB1) vaccine and association with viral load in the spleen and feather dander.

OBJECTIVE: To pathotype Australian isolates of Marek's disease virus (MDV) in commercial broiler chickens using standard methods and to evaluate early markers of pathotype. METHODS: A complete 3 × 4 factorial experiment with two replicates was conducted using 648 Cobb broiler chickens in 24 isolators. The experimental factors were vaccination (unvaccinated, herpesvirus of turkeys (HVT), bivalent (HVT + SB1 strain of serotype 2 MDV)) and MDV challenge (unchallenged or 500 plaque-forming units of isolates MFP57, 02LAR or FT158). Mortality, body weight, immune-organ weights and viral load were measured to 56 days post challenge (dpc). Vaccinal protective index (PI) and virulence rank (VR) were calculated based on gross Marek's disease (MD) pathology. RESULTS: The PIs provided by the HVT and bivalent vaccines against challenge with MPF57, 02LAR, and FT158 were 84.6% 56%, 61.4% and 82.2%, 60.8%, 57.7%, respectively, leading to putative pathotypes of virulent MDV for MPF57 and very virulent MDV for 02LAR and FT158. Significantly more of the unvaccinated chickens (85.7%) had MD lesions than chickens vaccinated with either the HVT (26.8%) or bivalent vaccine (27.6%). Strong linear relationships were observed between the incidence of MD at 56 dpc and MDV load in the spleen at 7 dpc (R(2) = 0.71) and MDV load in the isolator exhaust dust at 14 dpc (R(2) = 0.57) and 21 dpc (R(2) = 0.51). Immune organ weights had a weaker association with subsequent MD incidence. CONCLUSION: Pathotyping results in broiler chickens with maternal antibody broadly agreed with those in specific-pathogen-free chickens in other studies, with some important differences. MDV load in the spleen at 7 dpc and in isolator dust at both 14 and 21 dpc was a powerful early predictor of subsequent MD incidence.

Influence of vaccine deposition site on post-vaccinal viraemia and vaccine efficacy in broiler chickens following in ovo vaccination against Marek's disease.

In ovo vaccination against Marek's disease is a widely used technology in the broiler industry.A series of experiments was carried out to determine the site of vaccine deposition in the egg during automated in ovo vaccination, and the effect of vaccine deposition site and dose on vaccine responses following vaccination with cell-associated herpesvirus of turkeys in commercial broiler chickens. Vaccine deposition site following automated in ovo vaccination was principally influenced by the age of embryo, with egg size having a smaller effect. The frequency of vaccine deposition inside the embryo body increased as incubation progressed from day 17.5 to 19.5. In experiments using manual vaccine deposition intra-embryonically (IE) or extra-embryonically (EE) at day 18.5, EE vaccine deposition resulted in a significantly delayed development of post-vaccinal viraemia relative to both IE vaccination and subcutaneous vaccination at hatch. There were no effects of vaccine dose (2000, 4000 or 8000 plaque forming units) on the timing of post-vaccinal viraemia. The timing of post-vaccinal viraemia was found to be a good indicator of the level of protection provided by the vaccine against challenge with earlier viraemia associated with better protection. IE vaccine deposition induced significantly greater protection than EE deposition against challenge with a virulent strain of Marek's disease virus. IE deposition consistently produced a high level of protection (68 to 84%) irrespective of vaccine dose or challenge day, while EE vaccine deposition produced no or low levels of protection (0 to 27%) depending on the vaccine dose and day of challenge. The growth of challenged chickens was also affected by site of vaccine deposition, with significantly higher live weights at day 56 of age in IE compared with EE vaccinated groups. These data suggest that the site of vaccine deposition within the embryo is an important determinant of the success of in ovo vaccination.

Marek's disease in broiler chickens: effect of route of infection and herpesvirus of turkey-vaccination status on detection of virus from blood or spleen by polymerase chain reaction, and on weights of birds, bursa and spleen.

The polymerase chain reaction (PCR) has recently emerged as an additional tool for the monitoring and diagnosis of Marek's disease. We investigated a number of factors that may influence the interpretation of PCR results in commercial broiler chickens including the effects of route of infection and herpesvirus of turkeys (HVT)-vaccination status. We also investigated the suitability of peripheral blood lymphocytes (PBL) and spleen as tissues for Marek's disease virus (MDV) detection. HVT-vaccinated and unvaccinated commercial broiler chickens were challenged or not challenged with virulent MDV either by intraperitoneal injection or inhalation of feather dust containing the virus. Blood and spleen samples were collected at weekly intervals to day 35 post-infection for PCR of spleen or PBL. Live weight and lymphoid organ weights were also measured. Spleen and PBL were found to provide similar sensitivity of detection of MDV with a small advantage in favour of spleen. In terms of the timing of detection of MDV, intraperitoneal challenge broadly mimicked natural challenge via inhalation, although infection of birds by inhalation of infective feather dust resulted in slightly later but more complete detection of MDV in challenged birds. Vaccination with HVT delayed the detection of MDV by approximately 10 to 14 days and did not protect against the reduced growth observed in challenged chickens at day 35 post-challenge.

The intramammary inflammatory response of genetically resistant Merino ewes infected with Haemonchus contortus.

The mammary glands of 103 pasture-reared non-lactating, non-pregnant Merino ewes were infused via the teat canal with antigens prepared from the nematode Haemonchus contortus, and the inflammatory response to infusion assessed by washing the gland of its contents after 24 h and 14 days. The ewes were of two genotypes: one with proven high levels of resistance to infection with the nematode H. contortus, the other random-bred animals with relative susceptibility to infection. On day 0 of a H. contortus infection, one gland of the subgroups of both genotypes was infused with the antigen preparation. At the same time, the other gland of the random-bred ewes was infused with sterile physiological saline. A third group of infected random-bred ewes was infused with only sterile physiological saline. Similar infusions were performed on other subgroups on days 12, 21 and 35 of infection, which was then terminated with anthelmintic. A fourth group of uninfected random-bred control ewes was given both infusions 35 days after the other groups were infected. Sheep of the resistant genotype had lower worm egg counts and smaller reductions in blood packed cell volumes from day 21 of infection. Infusion of antigen had no effect on the course of infection and no effect on the response of the other gland, which had been infused with saline alone. The dominant leukocyte response from the antigen-infused gland was eosinophilia. On all days of infusion, and after both 24 h and 14 days, eosinophil counts from the resistant genotype were higher than those from their random-bred counterparts. The sheep mammary gland provides a source of eosinophils whose number is related to host genotype and stage of infection and may provide a model for the investigation of cellular responses in mucosal immunity to nematode infections.

Silicone oil tamponade to seal macular holes without position restrictions.

OBJECTIVE: The authors performed a study to determine the effectiveness and safety of silicone oil as a substitute for gas to fill the vitreous cavity to treat macular holes. DESIGN: Multicenter, nonrandomized, interventional trial. PARTICIPANTS: Thirty-seven consecutive patients chose vitrectomy with silicone tamponade instead of gas to treat 40 eyes with stage-2 to stage-4 idiopathic age-related macular holes. Stage-2 holes constituted 40% of the holes, and stage-3 and stage-4 holes made up 60%. INTERVENTION: All eyes were treated with vitrectomy, manual detachment of the posterior vitreous face (not done for stage-4 holes), autologous serum instillation, and silicone fill of the vitreous cavity. After insertion of the oil, the patients resumed normal activity with no restriction of head or eye position except to avoid faceup position. The oil was removed after approximately 6 weeks. MAIN OUTCOME MEASURES: The authors considered the seal of the macular hole and the preoperative and postoperative logarithm of the minimum angle of resolution (logMAR) visions the most significant measures for comparison to other studies. RESULTS: Eighty percent of all holes and 86% of holes not treated previously were sealed with a single silicone tamponade of the vitreous cavity. The logMAR value of visual acuity improved an average of 0.26 (2.6 lines) to 0.61 (20/81) for all eyes and 0.34 (3.4 lines) to 0.52 (20/66) when the macular hole sealed. Completeness of fill of the vitreous cavity with silicone affected seal of the macular hole. Three of eight eyes in which open holes developed after oil removal had less than 90% fill of the vitreous cavity by silicone. Sixty-nine percent of lenses increased opacity one grade or were removed after silicone tamponade. There were no significant adverse effects arising from silicone tamponade. CONCLUSIONS: Silicone oil tamponade of macular holes is effective and safe. Silicone may be optimal for the treatment of macular holes in persons who must travel, who cannot maintain facedown positioning, or who have monocular vision. The most important factor in the successful closure of the macular hole was the completeness of fill of the vitreous cavity with silicone oil.

Air bag-related ocular trauma.

Automobile air bags have recently gained acceptance as an effective measure to reduce the morbidity and mortality associated with motor vehicle accidents. This report describes 11 cases of air bag-related ocular trauma and reviews cases previously reported in the literature, for a total of 32 patients and 39 eyes. This is the first comprehensive report on various types of ocular trauma related directly to air bag deployment. The most common type of ocular injuries seen are to the eyelids (23 patients, 28 eyes), conjunctiva (21 patients, 25 eyes), and cornea (23 patients, 28 eyes). Hyphema was frequently seen (10 patients, 11 eyes). Several serious cases of vision-threatening injuries, including retinal detachment, retinal dialysis, scleral rupture, and dislocated lens, were also reported. The following patterns were found: 55% of patients were male and 45% female; ages ranged from 2 to 81 years with a mean age of 36 years; the right eye was involved in 35% of cases, the left in 38%, and 27% were bilateral. Based on these findings, it is recommended that all patients who present with air bag-related ocular trauma undergo a complete ophthalmologic examination because the high-velocity blunt trauma causes ocular injuries that may be more serious than they initially appear. Further refinements in design and deployment need to be made to reduce the frequency and severity of air bag-related ocular injuries.

A comparison of calcium binding in Callinectes sapidus premolt and postmolt cuticle homogenates: implications for regulation of biomineralization.

Cuticle tissue homogenates (CTHs) from Callinectes sapidus premolt cuticle bound approximately 367% more Ca2+ ions than did those from the postmolt cuticle. The pH-stat assay which was used to compare in vitro CaCO3 nucleation times confirmed that the premolt CTHs had greater inhibitory activity than did the postmolt CTHs. This inhibitory activity was indicated by CaCO3 nucleation times in excess of control values. Premolt nucleation times exceeded those of postmolt samples by approximately 340%. A positive correlation was observed between Ca2+ binding and calcification inhibitory activity for both premolt and postmolt CTHs. Heat pretreatment of CTHs at 70 degrees C for a 24-hr period had no significant effect on their Ca2+ binding. However, this heat pretreatment decreased their calcification inhibitory activity. Pretreatment of CTHs with Ca2+ diminished their calcification inhibitory activity. These results are consistent with a mechanism for inhibition of biocalcification by these proteins which involves their initial reversible binding to nascent calcite nuclei growth steps and kinks, rather than their in vivo interaction with free Ca2+ ions in solution.

Thermal inactivation of butyrylcholinesterase and acetylcholinesterase.

Differences were observed in the extent of thermal inactivation of human butyrylcholinesterase (BuChE) and eel acetylcholinesterase (AChE). BuChE was more resistant to 57 degrees C inactivation than was AChE. Thermal inactivation of BuChE was reversible and followed first-order kinetics. AChE thermal inactivation was irreversible and did not follow first-order kinetics. AChE was marginally protected from thermal inactivation by the "nonspecific salts" ammonium sulfate and sodium chloride and to a greater extent by the "active site-specific salts" choline chloride, sodium acetate, and acetylcholine iodide. This protection was accompanied by a loss of absorbance at 280 nm. This data supports the hypothesis that thermal inactivation of AChE occurs by conformational scrambling and that aromatic amino acid residue(s) are involved in this process.

Contralateral occipital depression related to obstetric forceps injury to the eye.

Obstetric forceps pressure strong enough to leave a periorbital depression and corneal injury would probably be severe enough to leave an occipital depression from the opposite forceps blade. The presence of a depression at the correct occipital position would support the diagnosis of forceps injury when the birth history is unknown and the cornea has decompensated enough to make observation of the Descemet's membrane scrolls difficult. We studied six patients with known or suspected obstetric forceps injury to the cornea. Complete ocular examinations included examination for periorbital forceps depressions and posterior skull depressions 180 degrees from the affected cornea (which correlates with the opposite blade of the forceps). All of the patients with Descemet's scrolls had posterior skull depressions. This method of palpation for a contralateral skull depression may assist in the diagnosis of forceps-induced corneal decompensation.

Novel protein inhibits in vitro precipitation of calcium carbonate.

Organic molecules both coexist and interact with inorganic crystal lattices in biomineralizing tissues. Mineral precipitation and crystal morphology are tightly regulated by the actions of these molecules. Polyacrylamide gel electrophoresis studies on water soluble extracts from the cuticle of Callinectes sapidus (Atlantic blue crab) reveal the presence, in unmineralized nascent premolt cuticle, of proteins which are absent in the mineralized postmolt cuticle. In the present studies, homogenates from both premolt and postmolt C. sapidus cuticles have been tested for their effect on the in vitro precipitation of calcium carbonate. The role of protein in this process was determined by heat pretreatment and trypsin pretreatment of the cuticle homogenates prior to the precipitation assay. The results from these experiments indicate that proteins, with molecular weights of approximately 75,000 and between 10,000 and 20,000, concentrated in the C. sapidus premolt cuticle, inhibit calcium carbonate precipitation in vitro. The inhibitory activity of these proteins appears to be a result of specific interactions since trypsin, myoglobin, and ovalbumin are not inhibitory. The presence of lower amounts of these inhibitory proteins in C. sapidus postmolt cuticle may be responsible for the subsequent mineralization of this tissue.

Insulin and IGF-I stimulated RNA synthesis in primary cultures of neuronal cells: involvement of cyclic AMP and protein kinase-C.

Insulin and insulin-like growth factor I promote the growth of rat neuronal cells in primary culture. In order to investigate the mechanism of hormone signalling in this biological system, we studied the effect of cyclic AMP agonists and a protein kinase C stimulator on basal and hormone-induced RNA synthesis. Agents elevating endogenous cyclic AMP levels (forskolin, dibutyryl cyclic AMP, cholera toxin) blocked the stimulatory effects of both insulin and the growth factor; dibutyryl cyclic AMP, however, altered the binding of neither hormone. Although, unlike the aforementioned agents, phorbol ester significantly elevated basal RNA synthesis; it nevertheless inhibited the stimulation by insulin; this latter effect probably being mediated by an increase in intracellular cyclic AMP levels, as has been found in other cell types. Staurosporine, an inhibitor of protein kinase C, also blocked the effects of insulin on RNA synthesis.

Insulin and IGF-I stimulated RNA synthesis in primary cultures of neuronal cells: involvement of cyclic AMP and protein kinase-C.

Insulin and insulin-like growth factor I promote the growth of rat neuronal cells in primary culture. In order to investigate the mechanism of hormone signalling in this biological system, we studied the effect of cyclic AMP agonists and a protein kinase C stimulator on basal and hormone-induced RNA synthesis. Agents elevating endogenous cyclic AMP levels (forskolin, dibutyryl cyclic AMP, cholera toxin) blocked the stimulatory effects of both insulin and the growth factor; dibutyryl cyclic AMP, however, altered the binding of neither hormone. Although, unlike the aforementioned agents, phorbol ester significantly elevated basal RNA synthesis; it nevertheless inhibited the stimulation by insulin; this latter effect probably being mediated by an increase in intracellular cyclic AMP levels, as has been found in other cell types. Staurosporine, an inhibitor of protein kinase C, also blocked the effects of insulin on RNA synthesis.

Insulin and IGF-I stimulated RNA synthesis in primary cultures of neuronal cells: involvement of cyclic AMP and protein kinase-C.

Insulin and insulin-like growth factor I promote the growth of rat neuronal cells in primary culture. In order to investigate the mechanism of hormone signalling in this biological system, we studied the effect of cyclic AMP agonists and a protein kinase C stimulator on basal and hormone-induced RNA synthesis. Agents elevating endogenous cyclic AMP levels (forskolin, dibutyryl cyclic AMP, cholera toxin) blocked the stimulatory effects of both insulin and the growth factor; dibutyryl cyclic AMP, however, altered the binding of neither hormone. Although, unlike the aforementioned agents, phorbol ester significantly elevated basal RNA synthesis; it nevertheless inhibited the stimulation by insulin; this latter effect probably being mediated by an increase in intracellular cyclic AMP levels, as has been found in other cell types. Staurosporine, an inhibitor of protein kinase C, also blocked the effects of insulin on RNA synthesis.

The effect of immunosuppression with dexamethasone on Haemonchus contortus infections in genetically resistant merino sheep.

The effect of immunosuppression with the glucocorticosteroid, dexamethasone, on the susceptibility to Haemonchus contortus infection of a highly resistant Merino genotype was investigated. Higher faecal egg counts, larger worm burdens and heavier worms were recorded in immunosuppressed wethers. The characteristic globule leucocyte infiltrate in the abomasum of resistant wethers (12-month-old castrated males) was absent in immunosuppressed animals. Treatment with dexamethasone abolished differences between resistant and susceptible genotypes in faecal egg counts, worm weights, thymus weights and globule leucocyte responses to infection with H. contortus. These results suggest that an immunological basis underlies the high level of resistance to infection in the resistant genotype.

An investigation into the protective effect of various salts on the acid inactivation of human serum butyrylcholinesterase (EC 3.1.1.8).

Human serum butyrylcholinesterase (EC 3.1.1.8) loses 100% of its activity toward butyrylthiocholine in 60 min at pH 3.0. This deactivation is retarded by 1.37 M ammonium sulfate to a loss of 40% after 60 min at pH 3.0. Reneutralization experiments suggest that the mechanism for this acid inactivation does not exclusively involve hydrolysis of peptide bonds or protonation of the enzyme's active site. Studies with different anions and cations demonstrate that the order of their effectiveness as protective agents against acid inactivation closely follows the Hofmeister series. No relationship was found between catalytic activation or inhibition by salt and protection from acid inactivation. Ultraviolet difference studies at 288 nm with enzyme brought to pH 2.7 from pH 8.0 in the presence and absence of 1.37 M ammonium sulfate demonstrated no change in UV absorbance with ammonium sulfate present and approximately a 0.15 ODU rise in absorbance in the absence of ammonium sulfate. These results suggest that acidic pH conditions result in deactivating stereochemical changes in the active site of butyrylcholinesterase and that certain anions and cations, according to the Hofmeister series, are able to protect the enzyme from acid inactivation by stabilizing the active conformation of its active site.

Serial passage of Haemonchus contortus in resistant and susceptible sheep.

A recently isolated field strain of Haemonchus contortus was passaged through resistant (repeatedly reinfected) and susceptible (immunosuppressed) sheep for six and nine generations, respectively. Infectivity of the resulting serially passaged strains was not significantly different when tested in groups of 1-year-old susceptible sheep.

Differential expression of insulin-like growth factor genes in rat central nervous system.

A sensitive solution-hybridization assay was used to investigate the expression of genes encoding insulin-like growth factors I and II (IGF-I and -II) in the rat central nervous system (CNS). mRNAs for both IGFs are synthesized throughout the CNS of adult rats but exhibit distinct regional differences for each growth factor. IGF-I mRNA is 8-10 times more abundant in the cervical-thoracic spinal cord and in the olfactory bulb than in whole brain and is enriched 3-fold in the midbrain and cerebellum. IGF-II mRNA is minimally enriched in the medulla-pons and cerebellum but is 3-5 times less abundant in the midbrain and corpus striatum than in total brain. During CNS development the content of IGF-I and IGF-II mRNAs is highest at embryonic day 14 and declines by a factor of 3-4 at birth, to values found in adult brain. Embryonic neurons and glia synthesize IGF-I mRNA during short-term cell culture; only glia produce IGF-II mRNA. These observations show that IGF-I and IGF-II are differentially expressed in the developing and adult CNS and suggest that each growth factor may play a unique role in the mammalian nervous system.

Characterization of a neuronal subtype of insulin-like growth factor I receptor.

Primary neuronal cultures from fetal rat brain were utilized to investigate the possible role of insulin-like growth factor I (IGF-I) in neuronal growth and differentiation. 125I-IGF-I binding to intact cultured neurons was specific and saturable with an apparent Kd of 7.0 +/- 1.2 nM and a Bmax of 1.8 +/- 0.3 pmol/mg protein. Binding of 125I-IGF-I to neurons was inhibited by IGF-I, followed by IGF-II and insulin. 7 S nerve growth factor, but not beta-nerve growth factor, also inhibited 125I-IGF-I binding. A similar binding site was detected on brain membranes. Affinity cross-linking of 125I-IGF-I to intact cultured neurons revealed, under reducing conditions, a major binding moiety with an Mr of 115,000 and a minor component at Mr 260,000. The former represents a neuronal type of the IGF-I receptor alpha subunit, whereas the latter probably represents an alpha dimer. The Mr = 115,000 binding component for 125I-IGF-I was also present in membranes prepared from postnatal whole brain. In contrast, the binding moiety in cultured glial cells was of Mr = 135,000, which was identical to the IGF-I receptor alpha subunit of placenta. Thus mature brain, despite its cellular heterogeneity, expresses a structural subtype of IGF-I receptor which appears to be unique to differentiated neurons. Moreover, glial and neuronal cultures secreted a polypeptide which specifically bound IGF-I; the apparent Mr of this binding protein was determined by affinity cross-linking to be approximately 35,000. The presence of neuronal IGF-I receptors and binding proteins suggested that IGF-I may exert neurotrophic effects on developing neurons. This possibility was supported by the observation that IGF-I markedly stimulated neuronal RNA synthesis.

Phorbol ester receptors and protein kinase C in primary neuronal cultures: development and stimulation of endogenous phosphorylation.

Embryonic rat neurons cultured in defined medium, essentially in the absence of glia, were highly enriched in phorbol ester receptors. The neurons displayed a single class of phorbol 12,13-dibutyrate binding sites with a maximum binding capacity, after 10 d in culture, of 18.6 pmol/mg protein and an apparent dissociation constant of 7.1 nM. Phorbol ester binding sites were associated with protein kinase C, which represented a major protein kinase activity in primary neuronal cultures. Ca2+-phosphatidylserine-sensitive phosphorylation of endogenous substrates was more marked than that observed in the presence of cyclic AMP or Ca2+ and calmodulin. Phorbol ester receptors and protein kinase C levels were critically dependent on the culture age. Thus, about a 20-fold increase in binding sites occurred during the first week in culture and was accompanied by a corresponding increase in Ca2+-phosphatidylserine-sensitive protein phosphorylation in soluble neuronal extracts. These changes largely paralleled a similar rise in phorbol ester binding during fetal development in vivo. The apparent induction of phorbol ester receptors was specific relative to other cellular proteins and could be inhibited by cycloheximide or Actinomycin D. Phosphorylation of endogenous substrates in intact cultured neurons paralleled the age-dependent increase in protein kinase C. Furthermore, 32P incorporation into several major phosphoproteins was markedly augmented by treating the neuronal cultures with phorbol esters. Such phosphorylation events may provide a clue to the significance of protein kinase C in developing neurons.