Indirect effects of heavy metals on parasites may cause shifts in snail species compositions.

We studied the direct and indirect effects of pollution on the distributions and abundances of two closely related species of pulmonate freshwater snails. Physella columbiana is more numerous at heavy metal-polluted lakes, and Lymnaea palustris is more numerous at reference lakes. Both species are present at all sites, as are predatory bluegill sunfish (Lepomis macrochirus). The direct effects examined included the snails' growth and reproduction in both the presence and absence of heavy metals and their short-term survival when exposed to large concentrations of heavy metals. The indirect effects were the species' ability to elude capture by sunfish and the diversity and abundance of parasites within the snails. We found that heavy metals had little direct effect on growth and reproduction and that both species acquired similar levels of metals in their tissues. Interestingly, P. columbiana (the more abundant species in polluted lakes) actually exhibited higher recruitment in the absence of metals than did L. palustris (reference lakes). L. palustris has life history characteristics that favor increased growth and reduced reproduction. These characteristics resulted in decreased predation of adults by gape-limited predators and a greater ability to cope with heavy parasite burdens. P. columbiana exhibited slower growth, which resulted in increased predation although higher reproduction rates may compensate.The major effect of heavy metals on species distributions was indirect on the snails' parasites. Parasites appeared to be very susceptible to metals, and this resulted in lower parasite diversity and intensities at polluted sites for both species of snails. P. columbiana may only be able to outcompete L. palustris at polluted sites due to the indirect effects of heavy metals; the negative effect of heavy metals on parasites, and a proposed negative effect of metals on the foraging ability of sunfish that favors the faster-reproducing P. columbiana.

Serum gamma glutamyl transpeptidase activity in cattle with induced fascioliasis.

Six-month-old calves were inoculated with 200 metacercariae of Fasciola hepatica over a 72 day period. Their aspartic transaminase, alkaline phosphatase, arginase and gamma glutamyl transpeptidase (GGT) values were measured at two-week intervals. Infections with a final fluke count of 19 to 87 flukes did not result in an increase of serum alkaline phosphatase or arginase compared with those in control calves. Serum aspartic transaminase values were increased significantly (P less than 0.025) above those of controls but were not increased over reference values. Seemingly, measurement of these enzymes would not be useful in assessing the degree of fluke infestation. In contrast, serum values of GGT increased 56 days after preliminary inoculation and remained high for at least 83 days after infection. The initial rise coincided with the penetration of bile ducts by migrating flukes. This relationship may be useful in further studies of fluke-induced biliary damage.

Experimentally induced Fasciola hepatica infection in young calves.

Twenty-four calves between 1 and 66 days of age (allotted to 3 age groups) were each inoculated with 100 Fasciola hepatica metacercariae to determine their susceptibility to fluke infection. Studies included measuring the immune response, as determined by enzyme-linked immunosorbent assay, fecal egg counts (to evaluate the length of the life cycle between times of metacercariae ingestion and fluke maturation), and the numbers of flukes in the liver of the calves at slaughter. Fasciola hepatica ova started appearing in the feces of calves, all ages, at 60 days after inoculations were done and reached average maximal number by 80 to 90 days. The enzyme-linked immunosorbent assay results indicated that the oldest group of calves had significantly (P less than 0.01) greater antibody concentration from 6 weeks until the 18th to 20th week after inoculation than did the 2 younger groups. At slaughter, calves in the 52- to 66-day age group had mean fluke numbers significantly (P less than 0.01) greater than did calves in the 1- to 27-day and 35- to 44-day age groups.

Scanning electron microscopy of in vitro serum-mediated destruction of juvenile Fasciola hepatica.

The tegumental surface of immature Fasciola hepatica was damaged when incubated in vitro with serum collected from an experimentally infected calf. Degeneration of the tegumental surface was observed by scanning electron microscopy (SEM) at 4 hr. after incubation. Decomposition was observed 8 to 12 hr after incubation and complete destruction of the tegument occurred by 16 hr. The flukes became inactive after 8 to 12 hr of incubation. None of the above findings were observed for the tegument of flukes incubated in tissue culture media or in media containing normal calf serum and the trematodes remained motile throughout the incubation period. Latex particles were used as an immunological marker for SEM studies to determine if gamma globulin could be responsible for the observed changes and, if so, the site of antibody attachment. The coated latex particles covered the entire surface of flukes recovered from mice 5 days after infection with metacercariae. In contrast, latex particles coated with either normal gamma globulin or gamma globulin from serum of the experimentally infected calf that had been adsorbed with disrupted adult flukes were not attached to the surface of the flukes. Absorption of the serum with disrupted, adult flukes decreased the concentrations of immunoglobulins (Ig)G1 and G2 whereas IgA and IgM were apparently not affected.

An enzyme-linked immunosorbent assay for diagnosis of Fasciola hepatica infection in cattle.

An enzyme-linked immunosorbent assay (ELISA) was investigated for the diagnosis of Fasciola hepatica infection in cattle. Studies included examination of 4 antigen preparations: freshly collected fluke antigen (FFA), dead fluke antigen (DFA), lyophilized fluke antigen (LFA), and partially purified antigen (PPA) for activity and use of an ELISA with FFA and LFA for diagnosis of experimentally and naturally occurring fascioliasis in cattle. The ELISA, using FFA, detected F hepatica antibody in calves as early as 4 weeks after exposure to this parasite. The DFA exhibited slightly less activity, and LFA did not have diagnostic value until 9 to 10 weeks after exposure. The PPA produced high background readings with noninfected control sera and was not considered sufficiently specific for further use. The long incubation and short incubation ELISA procedures can be used for diagnostic work. Both were equally sensitive, with optical density readings of known positive sera routinely 2.5 times those of sera from normal controls. The repeatability of these tests was also excellent, and only slight variation in optical density was observed in ELISA was performed on representative known positive and negative sera with similar reagents in replicate tests.

The pathogenesis of primary and secondary infection with Fasciola hepatica in mice.

Primary and secondary infections of F. hepatica in mice were compared to determine how prior exposure to the parasite affected host response. Mice with primary parenchymal Fasciola infections initially had hemorrhagic tunnels filled with inflammatory cells and connective tissue. These lesions were progressive and became most severe 30 days after exposure as the parasites entered the bile ducts. At this time there was much hyperplasia and thickening of all layers of the duct system near the parasites and occasionally severe periportal fibrosis. By 2 months after exposure regeneration of the damaged liver cells was complete although hyperplasia persisted in bile ducts containing flukes. In mice with secondary infections (mice exposed 40 to 50 days after first infection), the inflammatory response was faster and shorter. Most lesions were resolved by 30 days after the second exposure. There was little difference in histology of primary and secondary infections during the chronic phase of the disease.

Host-parasite relationships of Fasciola hepatica in the white mouse. VIII. Successful vaccination with culture incubate antigens and antigens from sonic disruption of immature worms.

Mice were successfully vaccinated using culture incubate and sonicate antigens from 16-day-old flukes. Various injection schedules using the culture incubate antigen decreased challenge worm counts by 54 to 86%. The best results were achieved when the culture incubate was injected at 12 and 24 hr of incubation. Host mortality in the natural immunity controls ranged from 33 to 42%, and in the vaccinated animals from 12.5 to 25%. Functional antigens were present by 12 hr of incubation. A single immunizing injection with the sonicate antigen decreased challenge worm counts by 86%. Two immunizing injections with this antigen decreased challenge worm counts by 82%. However, the pathologic process in the liver was more severe than in animals that received a single injection.

Host-parasite relationships of Fasciola hepatica in the white mouse. VII. effects of anti-worm incubate sera on transferred worms and successful vaccination with a crude incubate antigen.

Mouse antisera against the 16-day-old worm incubate and sera from 25-day infections in mice debilitated migrating flukes in recipient animals as measured by worm recovery and host mortality. Mouse antisomatic and 100-day infection sera produced no such effects. Host mortality was significantly lower after challenge in mice given one ip immunizing injection of the worm incubate; however, worm recovery was not significantly reduced. Injections at 2, 7, 12, and 24 hr with the worm incubate elaborated over a 24-hr period protected 75% of the mice from infection after challenge, and reduced the worm burden by 83.3%.