Diagnosis of ovine fascioliasis by a dot enzyme-linked immunosorbent assay: a rapid microdiagnostic technique

A microenzyme-linked immunosorbent assay (dot-ELISA) was modified for making an immunodiagnosis of Fasciola hepatica infections in sheep. Sheep were alloted as follows: group I-3 controls and 4 principals, each inoculated with 500 metacercariae; group II-3 controls and 7 principals, each inoculated with 250 metacercariae; and group III-3 controls and 7 principals, each inoculated with 500 metacercariae. Blood and fecal samples were collected from each animal every 2 weeks for 16 weeks. Presence (or absence) of flukes was confirmed by fecal examinations and examination of dissected livers at necropsy of the sheep. The dot-ELISA incubations were done at ambient room temperature. Nitrocellulose disks dotted with 1 microliter (50 ng of protein) of F hepatica excretory/secretory products were placed in 96-well tissue culture plates. After nonspecific binding sites on the disks were bound with bovine serum albumin-triethanolamine-buffered saline solution, dilutions (1:2) of positive- and negative-control serum samples or experimental serum samples were placed in appropriate wells for a 30-minute incubation. Wells were washed (3 times), and 50 microliters of horseradish peroxidase conjugated rabbit anti-sheep immunoglobulin G was added to each well for a 30-minute incubation and then aspirated. Substrate solution (4-chloro-1-naphthol, methanol, triethanolamine-buffered saline solution, and H2O2; 50 microliters) was added for a 30-minute incubation and then aspirated. Disks were air dried for visualization: solid purple dot = positive sample, or no dot = negative sample.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prestorage heat treatment reduces pathogenicity of Penicillium expansum in apple fruit

Penicillium expansum is one of the main postharvest pathogens of apples in Israel. Heating apple fruit inoculated with P. expansum for 96 h at 38°C completely inhibited decay development. Fruit held for 24 h at 42°C or 12 h at 46°C had significantly reduced decay after an additional 14 days incubation at 20°C, compared with unheated inoculated control fruit. Mycelial growth and percentage spore germination in vitro were inversely proportional to length of time of exposure to various temperatures. The ET₅₀ for spore germination was 42, 34 and 20 h at 38, 42 and 46°C, respectively, while the ET₅₀ for mycelial growth was 48, 44 and 36 h at those temperatures. When Penicillium spores were incubated on crude extract prepared from the peel of apple fruits held 4 days at 38°C, germ tube elongation was significantly reduced, while the walls of the tubes were thicker, compared with germ tubes from spores incubated on crude extract prepared from peel of non-heated fruit. The evidence presented here supports the hypothesis that the effect of heating on the decay of apples caused by P. expansum is not only the result of direct inhibition of fungal germination and growth by high temperature, but is also partly due to the formation of an inhibitory substance in the heated peel.     

Detection of early pregnancy factor in superovulated mice

Rosette inhibition tests for the detection of early pregnancy factor (EPF) were performed on naturally ovulated and superovulated mice from day 2 of pregnancy up to 4 days after parturition. In both groups of mice, the rosette inhibition titre (RIT) increased on day 2 of pregnancy, and persisted at high levels until day 15. Thereafter, the RITs of both groups of mice decreased to the non-pregnancy range. No significant differences of the mean RITs between these two groups were observed during the high RIT period. These results showed that the superovulatory treatment did not cause any changes or interference in the detection of EPF. In order to investigate the initial time of appearance of EPF in the maternal circulation in relation to the stage of fertilization, measurement of RIT and examination of the fertilization stage were carried out on superovulated mice 1 day after mating. The mean RIT of mice with pronucleus stage ova was significantly (p less than 0.01) higher than that of mice with sperm-penetrated ova. EPF was considered to appear in the maternal peripheral blood at the pronucleus stage.     

Identification of Neofabraea species causing bull's eye rot of apple in Poland and their direct detection in apple fruit using multiplex PCR

Based on partial sequence analysis of the β‐tubulin gene, 19 isolates of fungi causing bull's eye rot on apple in Poland were classified into species: Neofabraea alba, N. perennans and N. kienholzii. To the authors’ knowledge, the detection of N. kienholzii is the second in Europe and the first in Poland. Species affiliation of these fungi was confirmed by a new species‐specific multiplex PCR assay developed on the basis of previously published methods. The new protocol allowed for the specific identification of bull's eye rot‐causing species, both from pure cultures and directly from the skin of diseased or apparently healthy apples. In 550 samples of diseased fruits collected from nine cold storage rooms located in three regions of Poland, in 2011 and 2012, N. alba was detected as the predominant species causing bull's eye rot, occurring on average in 94% of the tested samples. Neofabraea perennans was found in a minority of apple samples, N. kienholzii was found only in two apple samples, while N. malicorticis was not detected in any sample tested. In tests on 120 apparently healthy fruits, only N. perennans was detected in a single sample. The results of genetic diversity analyses of bull's eye rot‐causing fungi based on the β‐tubulin gene sequence and an ISSR (inter‐simple sequence repeat) PCR assay with two primers were consistent, showing the expected segregation of tested isolates with respect to their species boundaries. However, the genetic distance between N. perennans and N. malicorticis was very low, as reported previously.     

Genetic parameters for body weight, reproduction, and parasite resistance traits in the Creole goat

We estimated the genetic parameters for BW, reproduction, and parasite resistance traits to implement a breeding program for the Creole goat. The traits were preweaning BW at 70 d of age (BW70d), BW at 11 mo of age (BW11), fecal egg count at 11 mo of age (FEC11) for all animals, packed cell volumes of lactating does (PCV), and their fertility (FER) and litter size (LS). We analyzed about 30 yr of data, which included 18,450 records on 11,970 animals from the INRA experimental flock in Guadeloupe (French West Indies). Heritability estimates were low for reproduction traits (0.11 ± 0.02 for LS and FER) to moderate for production traits (0.32 ± 0.03 for BW11; 0.20 ± 0.03 and 0.08 ± 0.02 for the direct and maternal heritability estimates of BW70d, respectively). Heritability estimates for gastrointestinal nematode resistance traits were situated in an intermediate range (0.13 ± 0.05 for PCV and 0.18 ± 0.04 for FEC11). Genetic correlations between FER, PCV, BW11, and the maternal effect of BW70d were altogether positive, whereas LS and FEC11 were almost uncorrelated phenotypically and genetically. These correlations are very favorable for setting up a breeding program, making it possible to improve BW, reproduction, and parasite resistance traits simultaneously.     

In vivo lipopolysaccharide injection alters CD4+CD25+ cell properties in chickens

In chickens, thymic CD4(+)CD25(+) cells are characterized as regulatory T cells. The objectives of this experiment were to study the effects of an in vivo lipopolysaccharide (LPS) injection on the percentage of CD4(+)CD25(+) cells in peripheral organs and the suppressive properties of splenic CD4(+)CD25(+) cells in chickens. Chickens were injected with LPS and CD4(+)CD25(+) cells were analyzed at 1, 2, 3, 5, and 10 d post LPS injection. The LPS injection increased CD4(+)CD25(+) cell percentage approximately 5-fold in the blood at 1 d post LPS injection (P < 0.001), 3-fold in the thymus at 3 d post LPS injection (P = 0.001), and 2.5-fold in the spleen at 2 d post LPS injection (P = 0.001) compared with the no-LPS-injected group. The LPS injection did not alter the CD4(+)CD25(+) cell percentage in the cecal tonsil (P = 0.162), lung (P = 0.098), or bone marrow (P = 0.071) at any time point measured. At 2 d post LPS injection, splenic CD4(+)CD25(+) cells lost their suppressive ability (P < 0.001). At 5 d post LPS injection, splenic CD4(+)CD25(+) cells not only regained their suppressive ability, but also became supersuppressive (P < 0.001). Splenic CD4(+)CD25(+) cells at 5 d post LPS injection produced 5.5-fold more (P = 0.005) IL-10 mRNA than splenic CD4(+)CD25(+) cells at 0 and 2 d post LPS injection. In conclusion, chicken regulatory T cells are differentially activated to facilitate immune response during the early stage of inflammation and to facilitate immune suppression at a later stage of inflammation.