A disseminated polyphasic rhabdomyositis syndrome associated with Sarcocystis neurona has been observed in both free-ranging and managed-care California sea lions (CSLs; Zalophus californianus). Histologically, affected animals show myocytes in variable stages of inflammation, degeneration, necrosis, regeneration, and scarring consistent with chronic or ongoing skeletal muscle injury. The condition can present with generalized muscle wasting, severe respiratory compromise, or megaesophagus, and may result in stranding and death from malnutrition, myoglobinuric nephropathy, or secondary infections. The underlying pathogenesis is not fully understood; an immune-mediated component has been proposed based on prior reports of lymphocytic infiltration and MHC II expression in affected tissues.
Sarcocystis neurona is an apicomplexan parasite with a heteroxenous life cycle. The Virginia opossum (Didelphis virginiana) is the recognized definitive host in North America; infective sporocysts are shed in feces and may reach marine environments by freshwater runoff. S. neurona causes severe neurologic disease in several species and has been implicated in encephalitis and mortality in multiple marine mammals. In other hosts, specific S. neurona genotypes have been linked with greater virulence or mortality.
The primary aims were to molecularly characterize S. neurona genotypes present in CSLs diagnosed with moderate to severe rhabdomyositis and to compare the genotypes identified with those previously reported from marine and terrestrial hosts along the Pacific coast of North America.
The study used archived frozen skeletal muscle samples from 25 free-ranging CSLs that live-stranded along a roughly 450-kilometer central California coastline (San Francisco to Morro Bay) and were admitted to The Marine Mammal Center (TMMC) for assessment. Cases were selected from animals that died in transport or care or were euthanized for poor prognosis due to S. neurona–associated disease (including generalized rhabdomyositis, megaesophagus, respiratory complications, myoglobinuria, protozoal cardiomyositis, and meningoencephalitis). Only animals with previously confirmed moderate or severe S. neurona–associated rhabdomyositis and archived muscle tissue were included. Two to four animals per year from 2017–2024 were randomly selected, yielding 25 cases.
DNA was extracted in triplicate from one muscle sample per animal (diaphragm, tongue, or other skeletal muscle) using a Qiagen DNeasy Blood and Tissue kit with prolonged proteinase K digestion overnight. Extracts were screened for S. neurona using a sensitive pan-apicomplexan nested PCR assay targeting the multi-copy ITS1 locus; positive amplicons were confirmed by Sanger sequencing and BLAST comparison (>99% identity reported).
For genotype characterization, one DNA replicate per animal underwent a nested or hemi-nested PCR-based multilocus sequence typing (MLST) approach targeting six polymorphic loci: three surface antigens (SnSAG1/5/6, SnSAG3, SnSAG4) and three microsatellite markers (sn3, sn7, sn9). Sequence analyses followed previously described methods.
Across the 25 CSLs, MLST identified 15 distinct S. neurona genotypes (unique combinations of antigen types and microsatellite types). Of these 15 genotypes:
A novel surface antigen type (XV) and five novel microsatellite alleles (nn, oo, pp, qq, rr) were described. The most commonly observed genotypes in this CSL series were IIg/j (6 of 25, 24%), Ib/c/d/gg (3 of 25, 12%), IIIl/m/o (2 of 25, 8%), VIIr (2 of 25, 8%), and IIi (2 of 25, 8%). Nine genotypes identified in these CSLs had not been reported previously.
Several genotypes identified in CSLs have also been documented in southern sea otters (SSOs) and other species. For example, Ia and Ib/c/d/gg were associated with fatal sarcocystosis outbreaks in SSOs in Estero Bay, CA. The IIg/j genotype has been found in SSOs from Monterey Bay and in multiple terrestrial and marine hosts historically. In contrast, genotypes strongly associated with encephalitis in pinnipeds from the Pacific Northwest (such as antigen types XIII and VI) were uncommon or absent in this CSL sample (type VI identified in only one CSL; XIII not identified).
Differences in genotype distributions between CSLs and other marine mammals may reflect variation in habitat use, prey preferences, regional opossum shedding, or hydrologic factors influencing land-to-sea transport of sporocysts.
The identification of 15 genotypes among 25 clinically affected CSLs—including nine not previously reported—demonstrates substantial S. neurona genotype diversity in this host. Because a variety of genotypes, both previously recognized and novel, were associated with the rhabdomyositis presentation, the findings suggest that parasite genotype alone may not account for the unique muscle-predominant disease phenotype observed in CSLs. The authors highlight that this pattern is consistent with a host-driven or immune-mediated mechanism contributing to the syndrome, rather than a single virulent genotype being responsible.
Key limitations reported by the authors include the relatively small sample size (n = 25) and potential difficulty detecting mixed-genotype infections using MLST methods that may preferentially amplify dominant genotypes. Samples were archival and collected as part of routine surveillance rather than prospectively for this study. The authors recommend larger studies including control groups of clinically infected and incidentally infected animals, and genotyping of opossum populations across watersheds to better define terrestrial sources, land-to-sea transmission dynamics, and environmental risk factors. Such work could clarify relationships among exposure, parasite genotype, host factors, and clinical outcomes in CSLs.
This molecular analysis of archived muscle samples from 25 CSLs with moderate to severe S. neurona–associated rhabdomyositis identified a high diversity of genotypes (15 total), including six genotypes previously reported in other hosts and nine novel genotypes. The findings support the hypothesis that host response and other non-parasite factors may play a primary role in the development of the distinctive rhabdomyositis syndrome in California sea lions. Future larger-scale and comparative investigations were recommended to delineate sources and drivers of infection and disease.