Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with “exceptional” gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these “exceptional” lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.     

Lactobacillus rhamnosus GG SpaC pilin subunit binds to the carbohydrate moieties of intestinal glycoconjugates

Lactobacillus rhamnosus GG (LGG) is a well‐established probiotic strain. The beneficial properties of this strain are partially dependent on its prolonged residence in the gastrointestinal tract, and are likely influenced by its adhesion to the intestinal mucosa. The pilin SpaC subunit, located within the Spa pili structure, is the most well studied LGG adhesion factor. However, the binding epitopes of SpaC remain largely unknown. The aim of this study was to evaluate the binding properties of SpaC to the carbohydrate moieties of intestinal glycoconjugates using a recombinant SpaC protein. In a competitive enzyme‐linked immunosorbent assay, SpaC binding was markedly reduced by addition of purified mucin and the mucin oligosaccharide fraction. Histochemical staining revealed that the binding of SpaC was drastically reduced by periodic acid treatment. Moreover, in the surface plasmon resonance‐based Biacore assay, SpaC bound strongly to the carbohydrate moieties containing β‐galactoside at the non‐reducing terminus of glycolipids. We here provide the first demonstration that SpaC binds to the oligosaccharide chains of mucins, and that the carbohydrate moieties containing β‐galactoside at the non‐reducing termini of glycoconjugates play a crucial role in this binding. Our results demonstrate the importance of carbohydrates of SpaC for mucus interactions.     

Severe nonpurulent encephalitis with mortality and feather lesions in call ducks (Anas platyrhyncha var. domestica) inoculated intravenously with H5N1 highly pathogenic avian influenza virus

One-day-old, 2-wk-old, and 4-wk-old call ducks (Anas platyrhyncha var. domestica) inoculated intravenously with the H5N1 highly pathogenic avian influenza virus A/chicken/Yamaguchi/7/2004 isolate (Ck/Yama/7/04) were examined clinically, pathologically, and virologically. Clinically, the birds exhibited mild-to-severe neurologic signs and corneal opacity. All birds in the 1-day-old group and one bird in the 4-wk-old group died within 4 days after the virus inoculation. Histologic changes were characterized by severe nonpurulent encephalitis and necrotic lesions of feather epithelium on day 3 postinoculation (PI) or later. Focal necrosis of myocardial cells, pancreatic acinar cells, skeletal myocytes, and corneal epithelial cells was observed. Viral antigens were detected in association with necrotic changes. Viruses were isolated from all examined organs including the skin with many feathers. Serum antibody against the virus was detected in all surviving birds on day 10 PI by hemagglutination-inhibition tests. These results suggest that Ck/Yama/7/04 has a pathogenicity that causes neurologic sign, nonpurulent encephalitis with mortality, and feather lesions for call ducks. Feather lesions with viral antigens and the virus isolation from the skin suggest that Ck/Yama/ 7/04 has a predilection for feathers in call ducks.     

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.     

Effects of silvopastoral areas on milk production at dual-purpose cattle farms at the semi-humid old agricultural frontier in central Nicaragua

In extensive cattle production systems, the composition of grazing areas may significantly influence productivity. In dual-purpose cattle production systems in the lowland tropics, pasture lands with trees, so-called silvopastoral areas, are considered as being important, particularly to facilitate the management of crossbred European native cattle. The aim of the study was to quantify the effects of silvopastoral areas on production at dual-purpose cattle farms in the semi-humid lowlands of central Nicaragua. The relationships between seasonal milk production and herd data, and the proportions of land use types were examined for 74 farms by stepwise regression analysis.     

Comparison of water status indicators for young peach trees

We measured a series of physiological and physical indicators and compared them to xylem sap flow, to identify the most sensitive and reliable plant water status indicator. In the growing season of 1998, 4-year-old peach trees (Prunus persica Batsch cv. 'Suncrest', grafted on 'GF 677' rootstock) were studied under two irrigation treatments, 25 l day^у and no irrigation, and during recovery. Trials were conducted near Pisa (Italy) in a peach orchard situated on a medium clay loam soil and equipped with a drip-irrigation system (four 4 l h^у drippers per tree). Measurements of leaf water potential (ƒ_W), stem water potential (ƒ_S), and leaf temperature (T_l) were taken over 5 days (from dawn to sunset) and analyzed in conjunction with climatic data, sap flow (SF), trunk diameter fluctuation (TDF) and soil water content (SWC). Physiological indicators showed substantial differences in sensitivity. The first indication of changes in water status was the decrease of stem radial growth. TDF and SF revealed significant differences between the two irrigation treatments even in the absence of differences in pre-dawn leaf water potential (pdƒ_W), up until now widely accepted as the benchmark of water status indicators. Irrigated trees showed a typical trend in SF rate during the day, while in non-irrigated plants the maximum peak of transpiration was anticipated. Measurements of water potential showed ƒ_S to be a better indicator of tree water status than ƒ_W. T_l was found to have poor sensitivity. In conclusion, we found the sensitivity of the indicators from the most to the least was: TDF >SF rate >SF cumulated = pdƒ_W=ƒ_S>mdƒ_W>T_l.     

Controlled water table management as a strategy for reducing salt loads from subsurface drainage under perennial agriculture in semi-arid Australia

Recent community based actions to ensure the sustainability of irrigation and protection of associated ecosystems in the Murrumbidgee Irrigation Area (MIA) of Australia has seen the implementation of a regional Land and Water Management Plan. This aims to improve land and water management within the irrigation area and minimise downstream impacts associated with irrigation. One of the plan objectives is to decrease current salt loads generated from subsurface drainage in perennial horticulture within the area from 20 000 tonnes/year to 17 000 tonnes/year. In order to meet such objectives Controlled Water table Management (CWM) is being investigated as a possible ‘Best Management Practice’, to reduce drainage volumes and salt loads.During 2000–2002 a trial was conducted on a 15 ha subsurface drained vineyard. This compared a traditional unmanaged subsurface drainage system with a controlled drainage system utilizing weirs to maintain water tables and changes in irrigation scheduling to maximize the potential crop use of a shallow water table. Drainage volumes, salt loads and water table elevations throughout the field were monitored to investigate the effects of controlled drainage on drain flows and salt loads.Results from the experiment showed that controlled drainage significantly reduced drainage volumes and salt loads compared to unmanaged systems. However, there were marked increases in soil salinity which will need to be carefully monitored and managed.     

Potato evapotranspiration and yield under different drip irrigation regimes

A field experiment comparing different irrigation frequencies and soil matric potential thresholds on potato evapotranspiration (ET), yield (Y) and water-use efficiency (WUE) was carried out in a loam soil. The experiment included five treatments for soil matric potential: F1 (-15 kPa), F2 (-25 kPa), F3 (-35 kPa), F4 (-45 kPa) and F5 (-55 kPa) and six treatments for irrigation frequency: N1 (once every day), N2 (once every 2 days), N3 (once every 3 days), N4 (once every 4 days), N6 (once every 6 days) and N8 (once every 8 days). Results indicate that both soil matric potential and drip irrigation frequency influenced potato ET, Y and WUE. Potato ET increased as irrigation frequency and soil matric potential increased. Comparing soil water potential, the highest ET was 63.4 mm (32.1%) more than the lowest value. Based on irrigation frequency treatments, the highest ET was 36.7 mm (19.2%) more than the lowest value. Potato Y and WUE were also found to increase as irrigation frequency increased. Potato Y increased with an increase in soil water potential then started to decrease. The highest Y and WUE values were achieved with a soil matric potential threshold of -25 kPa and an irrigation frequency of once a day.Communicated by J. Ayars     

Census and effective population sizes of white‐spotted charr (Salvelinus leucomaenis) in a fragmented landscape

Several habitat models have been proposed to predict population size for stream fishes and to guide habitat assessment and monitoring techniques. However, most models do not incorporate the potential advantage of molecular genetic markers. We conducted a field survey and microsatellite DNA analyses to quantify the relationships among genetic diversity, census/effective population size and habitat variables in fragmented populations of white‐spotted charr (Salvelinus leucomaenis). The census population size significantly increased with the stream length, the number of pools and a pool‐riffle sequence index, a proxy for channel‐unit habitat type complexity within reaches. Population density was correlated with the pool‐riffle sequence index only. Genetic diversity and effective population size were not correlated with the habitat variables or census population size. There was a lack of isolation‐by‐distance population structure in the studied populations. Our results suggest that stream length and the number of pools within reaches associated with habitat complexity are the habitat variables that explain the majority of variation in population size of white‐spotted charr. Our findings provide further evidence that census population size per se is a poor indicator of the inclusive genetic diversity within populations in a fragmented landscape.