To evaluate the feasibility of flexible, gas-permeable tissue culture flasks (GPF) as an alternative in vitro technique for small-scale monoclonal antibody (MAb) production, hybridomas were cultured in GPF. Cell concentration, viability, and MAb production were monitored over time, and resulting MAb were tested for functionality. Various culture conditions were evaluated, including type of culture media, seeding density, and addition of commercially available media supplements designed to enhance MAb production. Cultures grown in GPF were also compared with cultures grown in traditional rigid tissue culture flasks seeded under identical conditions. Although peak cell concentrations were highest in serum-supplemented media, MAb production was highest in serum-free media preparations. Monoclonal antibody production and cell concentrations were greatest for cultures gown in GPF at seeding densities greater than 1 x 10(6) live cells/ml, and cell growth was not observed at seeding densities of 1 x 10(3) live cells/ml. One media supplement enhanced MAb production in GPF and rigid flasks, whereas another media supplement increased production in rigid flasks, but not GPF. In 3 of the 4 hybridomas tested, cultures grown in GPF produced 1.3 to 5.1 times more MAb per ml, compared with cultures gown in rigid flasks. However, all cultures in GPF produced more total MAb than cultures in rigid flasks due to their increased volume (465 ml vs 25 ml). All MAb were functional, as determined by their respective immunoassays. The use of GPF is a reasonable alternative to the use of mice for small-scale MAb production, with additional reductions in the use of animal products through use of serum-free media.

An experimental non-lethal serodiagnostic kit was evaluated for screening candidate specific pathogen-free (SPF) Penaeus vannamei broodstock under typical field conditions. In this trial, a total of 26 adult P. vannamei was screened for the presence of infectious hypodermal and hematopoietic necrosis virus (IHHNV) using an IHHNV dot blot field kit. The non-radioactive IHHNV in situ hybridization method was used as a standard for comparison. The results showed a significant relationship between the outcome of the dot blot kit and the outcome of the in situ method. In particular, the test results using the experimental kit were consistent with the in situ hybridization method. Based on these findings, the experimental serodiagnostic kit may provide a more rapid, cost-effective diagnostic method than traditional techniques for IHHNV detection.

Mixed breed goats were infected with metacercariae of Fasciola hepatica and the resulting worm burdens were quantitated after primary and secondary exposure of the goats to the parasite. Mean length and width of the parasite recovered after all primary exposures were 1.91 ± 0.2 cm and 0.91 ± 0.2 cm, respectively. A mean of 71.8 ± 5.9% of the flukes were recovered from all of the primary infections. In the secondary infections, the mean length and width of the flukes from the physically smaller population was 0.88 ± 0.27 cm and 0.53 ± 0.19 cm, respectively. A mean of 67 ± 6.7% of the flukes were recovered from this secondary infection. It appears that the goat is susceptible to challenge infections with F. hepatica and that its response to this infection is much like that of sheep.

Fasciola hepatica metacercariae were purified in high yield, removing contaminating cyst walls and plant material by step gradients consisting of 10 ml of 60% Percoll (density = 1.08 g ml-1) and 10 ml of 50% Metrizamide (density = 1.25 g ml-1). Greater than 90% of the metacercariae applied to the density gradients were recovered. These isolated metacercariae had an in vitro excystment rate of greater than 80%, which was the same excystment rate as metacercariae not subjected to density gradient centrifugation.

The interaction between the antigens of Fasciola hepatica and the host immune response are reviewed. This paper evaluates not only more recent work, but the older literature as well. Antigens from each stage in the life cycle are considered with the idea of identifying those antigens with a potential for use in an effective vaccine. Antigens which cross-react with other parasite species are detailed as well as those that cross-react between different stages in the life cycle of F. hepatica. The objective of the review is to demonstrate for other investigators that vaccination against F. hepatica is a distinct possibility. We hope to encourage more investigators to initiate work on this aspect of an economically-important cosmopolitan parasite.

Two outbred strains of Sprague-Dawley rats were given, by intraperitoneal injection, 5, 10, 20, 30 or 50 newly excysted juvenile (NEJ) Fasciola hepatica. Ninety days post-infection all rats were killed and their livers teased apart under 10 × magnification for quantitation of the flukes present. There was no significant difference in worm numbers between the rat strains. However, several significant differences (P<0.05) between the various infection groups were observed. As the size of the infective dose were increased from 5 to 50 NEJ, the percentage of the infective dose recovered from the livers of the infected rats decreased from 36.7 to 12.1%. With the 5 and 10 NEJ infective doses, the worms recovered were large mature flukes (1.52 and 1.68 cnm respectively) and were found in the common bile ducts. In the rast receiving infections of 30 and 50 NEJ, the flukes were smaller (0.88 and 0.55 cm, respectively), immature, and were primarily located in the liver parenchyma. These findings are important in light of previous studies on the development of resistance in the rat to challenge infections in which immunity was based on both the size and the location of the flukes recovered. The results from our study indicate that in a primary infection of 20 or greater NEJ, many small immature flukes remain in the liver parnchyma, even after several months. When testing for resistance to F. hepatica in rats, these flukes may erroneously be thought to comprise a portion of the challenge infection.

The present egg isolation method is both a rapid and simple technique for recovering large numbers of Fasciola hepatica eggs (1 X 10(7) eggs/gradient) from bovine bile. Bile from infected cattle was first passed through a 45 micron screen sieve. The F. hepatica eggs were collected from the surface of the screen by backwashing with a jet of distilled water. The resultant egg suspension was layered on a 60% to 100% (v/v) linear Percoll gradient prepared in distilled water. Centrifugation at 450 g for 20 min resulted in the formation of 2 visible bands and a pellet. The top band (density of 1.075 g ml-1) contained viscous debris and crystallized bile pigments. The second visible band (density of 1.093-1.099 g ml-1) consisted of a relatively pure population of F. hepatica eggs (greater than 93%) while the pellet contained only F. hepatica egg shells.