Anna M. Schotthoefer

Agrochemicals increase trematode infections in a declining amphibian species

Global amphibian declines have often been attributed to disease, but ignorance of the relative importance and mode of action of potential drivers of infection has made it difficult to develop effective remediation. In a field study, here we show that the widely used herbicide, atrazine, was the best predictor (out of more than 240 plausible candidates) of the abundance of larval trematodes (parasitic flatworms) in the declining northern leopard frog Rana pipiens. The effects of atrazine were consistent across trematode taxa. The combination of atrazine and phosphate—principal agrochemicals in global corn and sorghum production—accounted for 74% of the variation in the abundance of these often debilitating larval trematodes (atrazine alone accounted for 51%). Analysis of field data supported a causal mechanism whereby both agrochemicals increase exposure and susceptibility to larval trematodes by augmenting snail intermediate hosts and suppressing amphibian immunity. A mesocosm experiment demonstrated that, relative to control tanks, atrazine tanks had immunosuppressed tadpoles, had significantly more attached algae and snails, and had tadpoles with elevated trematode loads, further supporting a causal relationship between atrazine and elevated trematode infections in amphibians. These results raise concerns about the role of atrazine and phosphate in amphibian declines, and illustrate the value of quantifying the relative importance of several possible drivers of disease risk while determining the mechanisms by which they facilitate disease emergence.

Relationship of tadpole stage to location of echinostome cercariae encystment and the consequences for tadpole survival.

The effect of echinostome infections on the survival of Rana pipiens tadpoles was examined in relation to developmental stage of tadpoles. Individual tadpoles of Gosner stages 25, 27, 32–33, and 37–39 were exposed to 1 of 4 levels of cercariae (0, 20, 50, or 100). Only tadpoles at stage 25, the earliest stage infected, died within a 5-day experimental period. This stage-specific mortality rate could be explained, in part, by the stage-specific location of encystment of cercariae, which was documented in a separate experiment. In accordance with kidney development, cercariae predominately encysted in the pronephroi during early stages of tadpole development (stages 25 through 31–32) and only in the mesonephroi and associated ducts at later stages (stages 37 through 46). As the mesonephros develops, renal capacity presumably increases. Thus, tadpoles died only when metacercariae concentrated in the functional portion of the kidney with the most limited renal capacity. As tadpoles aged, they also became less susceptible to infections. On average, 69.5% of cercariae that were exposed to stage 25–26 tadpoles successfully encysted, compared with only 8.4% of cercariae exposed to stage 37–38 tadpoles. Exposures of metamorphic frogs (poststage 46) to cercariae revealed that these individuals can become infected with echinostomes. Collectively, our data highlight the host stage–dependent dynamics of tadpole–echinostome interactions.

Rapid amplification of West Nile virus: the role of hatch-year birds.

Epizootic transmission of West Nile virus (WNV) often intensifies rapidly leading to increasing risk of human infection, but the processes underlying amplification remain poorly understood. We quantified epizootic WNV transmission in communities of mosquitoes and birds in the Chicago, Illinois (USA) region during 2005 and 2006. Using quantitative polymerase chain reaction (PCR) methods, we detected WNV in 227 of 1195 mosquito pools (19%) in 2005 and 205 of 1685 (12%) in 2006; nearly all were Culex pipiens. In both years, mosquito infection rates increased rapidly in the second half of July to a peak of 59/1000 mosquitoes in 2005 and 33/1000 in 2006, and then declined slowly. Viral RNA was detected in 11 of 998 bird sera (1.1%) in 2005 and 3 of 1285 bird sera (<1%) in 2006; 11 of the 14 virus-positive birds were hatch-year birds. Of 540 hatch-year birds, 100 (18.5%) were seropositive in 2005, but only 2.8% (14/493) tested seropositive in 2006 for WNV antibodies using inhibition enzyme-linked immunosorbent assay (ELISA). We observed significant time series cross-correlations between mosquito infection rate and proportion of virus-positive birds, proportion of hatch-year birds captured in mist nets (significant in 2006 only), seroprevalence of hatch-year birds, and number of human cases in both seasons. These associations, coupled with the predominance of WNV infection and seropositivity in hatch-year birds, indicate a key role for hatch-year birds in the amplification of epizootic transmission of WNV, and in increasing human infection risk by facilitating local viral amplification.

Ribeiroia ondatrae Cercariae Are Consumed by Aquatic Invertebrate Predators

Trematodes amplify asexually in their snail intermediate hosts, resulting in the potential release of hundreds to thousands of free-living cercariae per day for the life of the snail. The high number of cercariae released into the environment undoubtedly increases the probability of transmission. Although many individual cercariae successfully infect another host in their life cycle, most fail. Factors that prevent successful transmission of cercariae are poorly understood. Microcrustaceans and fish have been observed to eat cercariae of some species, although the possibility that predation represents a significant source of mortality for cercariae has been largely unexplored. We tested the cercariophagic activity of several freshwater invertebrates on Ribeiroia ondatrae, a trematode that causes limb deformities in amphibians. Individuals of potential predators were placed into wells of multiwell plates with 10–15 cercariae, and numbers of cercariae remaining over time were recorded and compared with numbers in control wells that contained no predators. Of the species tested, Hydra sp., damselfly (Odonata, Coenagrionidae) larvae, dragonfly (Odonata, Libellulidae), larvae, and copepods (Cyclopoida) consumed cercariae. In some cases, 80– 90% of the cercariae offered to damselfly and dragonfly larvae were consumed within 10 min. In most cases, predators continued to consume cercariae at the same average rates when offered cercariae together with individuals of an alternate prey item. Hydra sp. ate fewer cercariae in these trials. Our findings suggest the need for field and laboratory studies to further explore the effects of predators on transmission of R. ondatrae to amphibian larvae. In addition, the results suggest that conservation of the biodiversity and numbers of aquatic predators may limit adverse impacts of trematode infections in vertebrate hosts.

Effects of wetland vs. landscape variables on parasite communities of Rana pipiens: links to anthropogenic factors

The emergence of several diseases affecting amphibian populations worldwide has prompted investigations into determinants of the occurrence and abundance of parasites in frogs. To understand the spatial scales and identify specific environmental factors that determine risks of parasitism in frogs, helminth communities in metamorphic frogs of the northern leopard frog (Rana pipiens) were examined in relation to wetland and landscape factors at local (1 km) and regional (10 km) spatial extents in an agricultural region of Minnesota (USA) using regression analyses, ordination, and variance partitioning techniques. Greater amounts of forested and woody wetland habitats, shorter distances between woody wetlands, and smaller-sized open water patches in surrounding landscapes were the most consistently positive correlates with the abundances, richness, and diversity of helminths found in the frogs. Wetland and local landscape variables were suggested as most important for larval trematode abundances, whereas local and regional landscape variables appeared most important for adult helminths. As previously reported, the sum concentration of atrazine and its metabolite desethylatrazine, was the strongest predictor of larval trematode communities. In this report, we highlight the additional influences of landscape factors. In particular, our data suggest that anthropogenic activities that have resulted in the loss of the availability and connectivity of suitable habitats in the surrounding landscapes of wetlands are associated with declines in helminth richness and abundance, but that alteration of wetland water quality through eutrophication or pesticide contamination may facilitate the transmission of certain parasite taxa when they are present at wetlands. Although additional research is needed to quantify the negative effects of parasitism on frog populations, efforts to reduce inputs of agrochemicals into wetlands to limit larval trematode infections may be warranted, given the current high rates of amphibian declines and extinction events.

Predator diversity, intraguild predation, and indirect effects drive parasite transmission

Significance Humans are altering biodiversity globally and infectious diseases are on the rise; thus, there is considerable interest in understanding how changes to biodiversity affect disease risk. We show that the diversity of predators that consume parasites was the best negative predictor of infections in frogs, suggesting that predation on parasites can be an important mechanism of disease reduction. Follow-up experiments, field data, and mathematical models revealed that intraguild predators, predators that consume both hosts and parasites, decrease macroparasite infections per host less than predators that only consume parasites, representing a general trait of predators that predicts their ability to reduce disease. Consequently, managing assemblages of non-intraguild and intraguild predators is an underutilized tool to minimize human and wildlife diseases. Humans are altering biodiversity globally and infectious diseases are on the rise; thus, there is interest in understanding how changes to biodiversity affect disease. Here, we explore how predator diversity shapes parasite transmission. In a mesocosm experiment that manipulated predator (larval dragonflies and damselflies) density and diversity, non-intraguild (non-IG) predators that only consume free-living cercariae (parasitic trematodes) reduced metacercarial infections in tadpoles, whereas intraguild (IG) predators that consume both parasites and tadpole hosts did not. This likely occurred because IG predators reduced tadpole densities and anticercarial behaviors, increasing per capita exposure rates of the surviving tadpoles (i.e., via density- and trait-mediated effects) despite the consumption of parasites. A mathematical model demonstrated that non-IG predators reduce macroparasite infections, but IG predation weakens this “dilution effect” and can even amplify parasite burdens. Consistent with the experiment and model, a wetland survey revealed that the diversity of IG predators was unrelated to metacercarial burdens in amphibians, but the diversity of non-IG predators was negatively correlated with infections. These results are strikingly similar to generalities that have emerged from the predator diversity–pest biocontrol literature, suggesting that there may be general mechanisms for pest control and that biocontrol research might inform disease management and vice versa. In summary, we identified a general trait of predators—where they fall on an IG predation continuum—that predicts their ability to reduce infections and possibly pests in general. Consequently, managing assemblages of predators represents an underused tool for the management of human and wildlife diseases and pest populations.

PREVALENCE OF SKELETAL AND EYE MALFORMATIONS IN FROGS FROM NORTH-CENTRAL UNITED STATES: ESTIMATIONS BASED ON COLLECTIONS FROM RANDOMLY SELECTED SITES

Skeletal malformation rates for several frog species were determined in a set of randomly selected wetlands in the north-central USA over three consecutive years. In 1998, 62 sites yielded 389 metamorphic frogs, nine (2.3%) of which had skeletal or eye malformations. A subset of the original sites was surveyed in the following 2 yr. In 1999, 1,085 metamorphic frogs were collected from 36 sites and 17 (1.6%) had skeletal or eye malformations, while in 2000, examination of 1,131 metamorphs yielded 16 (1.4%) with skeletal or eye malformations. Hindlimb malformations predominated in all three years, but other abnormalities, involving forelimb, eye, and pelvis were also found. Northern leopard frogs (Rana pipiens) constituted the majority of collected metamorphs as well as most of the malformed specimens. However, malformations were also noted in mink frogs (R. septentrionalis), wood frogs (R. sylvatica), and gray tree frogs (Hyla spp.). The malformed specimens were found in clustered sites in all three years but the cluster locations were not the same in any year. The malformation rates reported here are higher than the 0.3% rate determined for metamorphic frogs collected from similar sites in Minnesota in the 1960s, and thus, appear to represent an elevation of an earlier baseline malformation rate.

Intestinal infections in humans in the Rocky Mountain region, United States.

Abstract To evaluate the seasonal prevalence of human intestinal parasites in the western states of Colorado, Utah, New Mexico, and Montana, fecal samples were examined as part of routine diagnostic testing from patients experiencing gastrointestinal discomfort in August (summer) 2006, January (winter), and April (spring) 2007. Parasite identification in positive samples was confirmed using light microscopy after wet mount and trichrome staining techniques. Seventy-eight of the 1,083 patients surveyed (7.2%) in August tested positive for at least 1 species of intestinal parasite. Forty-eight of 726 (6.6%) patients and 51 of 795 (6.4%) patients tested positive for at least 1 species in January and April, respectively. Blastocystis sp. was the most prevalent, followed by Giardia lamblia. Approximately 25% of the parasite occurrences were multiple infections involving fecal-oral transmitted species. Co-infections with Entamoeba spp. and Blastocystis sp. were common, suggesting a possible fecal-oral transmission for the latter parasite. Entamoeba spp. were more likely to co-occur than independently. Other species detected included Endolimax nana, Diphyllobothrium latum, Hymenolepis nana, Dientamoeba fragilis, and Iodamoeba butschlii.

Effects of low-temperature flea maintenance on the transmission of Yersinia pestis by Oropsylla montana.

Yersinia pestis, the causative agent of plague, is primarily a rodent-associated, flea-borne zoonosis maintained in sylvatic foci throughout western North America. Transmission to humans is mediated most commonly by the flea vector Oropsylla montana and occurs predominantly in the southwestern United States. With few exceptions, previous studies showed O. montana to be an inefficient vector at transmitting Y. pestis at ambient temperatures, particularly when such fleas were fed on susceptible hosts more than a few days after ingesting an infectious blood meal. We examined whether holding fleas at subambient temperatures affected the transmissibility of Y. pestis by this vector. An infectious blood meal containing a virulent Y. pestis strain (CO96-3188) was given to colony-reared O. montana fleas. Potentially infected fleas were maintained at different temperatures (6°C, 10°C, 15°C, or 23°C). Transmission efficiencies were tested by allowing up to 15 infectious fleas to feed on each of 7 naïve CD-1 mice on days 1-4, 7, 10, 14, 17, and 21 postinfection (p.i.). Mice were monitored for signs of infection for 21 days after exposure to infectious fleas. Fleas held at 6°C, 10°C, and 15°C were able to effectively transmit at every time point p.i. The percentage of transmission to naïve mice by fleas maintained at low temperatures (46.0% at 6°C, 71.4% at 10°C, 66.7% at 15°C) was higher than for fleas maintained at 23°C (25.4%) and indicates that O. montana fleas efficiently transmit Y. pestis at low temperatures. Moreover, pooled percent per flea transmission efficiencies for flea cohorts maintained at temperatures of 10°C and 15°C (8.67% and 7.87%, respectively) showed a statistically significant difference in the pooled percent per flea transmission efficiency from fleas maintained at 23°C (1.94%). This is the first comprehensive study to demonstrate efficient transmission of Y. pestis by O. montana fleas maintained at temperatures as low as 6°C. Our findings further contribute to the understanding of plague ecology in temperate climates by providing support for the hypothesis that Y. pestis is able to overwinter within the flea gut and potentially cause infection during the following transmission season. The findings also might hold implications for explaining the focality of plague in tropical regions.