Epizootic of Clinostomum marginatum (Trematoda: Clinostomidae) in Ambystoma tigrinum from Colorado, USA: Investigation through Genomics, Histopathology, and Noninvasive Imagery.

Trematodes in the genus Clinostomum develop into large metacercariae that can sometimes achieve high intensity in their second intermediate hosts, potentially causing pathology. In 2022, there was 100% (15/15) infection prevalence of Clinostomum marginatum, with a mean of 3,125 metacercariae per salamander (range: 279-4,075) and a median of 2,949. Dissection and histopathology results indicated that C. marginatum was found in nearly all body tissues and organs of the salamanders. Parasitic infection was closely associated with chronic inflammation and fibrosis around the cysts, with heavily infected hosts exhibiting altered buoyancy and difficulty swimming. The following summer, only 22.0% (6/27) of salamanders were infected with C. marginatum; based on their size, these were probably overwintering larvae from 2022. Here, we characterize a morbidity event in tiger salamanders (Ambystoma tigrinum) from a freshwater pond in Boulder, Colorado, USA, linked to extreme clinostomid infection. After using necropsy, genomic analysis, and histopathological assessment to record the intensity and pathology associated with infections, we assessed the validity of using noninvasive, image-based methods to quantify infection. Over 2 yr, we recorded in situ video imagery of 62 larval A. tigrinum, of which a subset of larvae was collected for parasitological assessment. Infection loads of C. marginatum quantified indirectly via imagery of subcutaneous cysts correlated strongly with direct counts via necropsy and were consistent between independent reviewers, highlighting the utility of this non-invasive assessment method. All evaluated A. tigrinum were co-infected with additional parasite taxa. Prevalence of other trematodes included Ribeiroia ondatrae (80%) and Cephalogonimus americanus (77.5%), as well as the nematode Megalobatrachonema elongata (75%). To discern the impact of such high metacercarial burden of C. marginatum on tiger salamander, future work should incorporate experimental approaches to evaluate load-dependent consequences for host growth, survival, behavior, and time to metamorphosis.