The gut microbiota is essential for the development and maintenance of the hosts’ immune system. Disturbances to the gut microbiota in early life stages can result in long-lasting impacts on host health. This study aimed to determine if topical ivermectin treatment for endemic hookworm (Uncinaria sanguinis) infection in endangered Australian sea lion (Neophoca cinerea) pups resulted in gut microbial changes. The gut microbiota was characterised for untreated (control) (n = 23) and treated (n = 23) Australian sea lion pups sampled during the 2019 and 2020/21 breeding seasons at Seal Bay, Kangaroo Island. Samples were collected pre- and post-treatment on up to four occasions over a four-to-five-month period. The gut microbiota of untreated (control) and treated pups in both seasons were dominated by five bacterial phyla, Fusobacteria, Firmicutes, Proteobacteria, Actinobacteria, and Bacteroides. A significant difference in alpha diversity between treatment groups was seen in pups sampled during the 2020/21 breeding season (p = 0.008), with higher richness and diversity in treated pups. Modeling the impact of individual pup identification (ID), capture, pup weight (kg), standard length (cm), age, and sex on beta diversity revealed that pup ID accounted for most of the variation (35% in 2019 and 42% in 2020/21), with pup ID, capture, and age being the only significant contributors to microbial variation (p < 0.05). There were no statistically significant differences in the composition of the microbiota between treatment groups in both the 2019 and 2020/21 breeding seasons, indicating that topical ivermectin treatment did not alter the composition of the gut microbiota. To our knowledge, this is the first study to characterise the gut microbiota of free-ranging Australian pinniped pups, compare the composition across multiple time points, and consider the impact of parasitic treatment on the overall diversity and microbial composition of the gut microbiota. Importantly, the lack of compositional changes in the gut microbiota with topical ivermectin treatment supports the utility of topical ivermectin as a safe and minimally invasive management strategy to enhance pup survival in this endangered species. 

Faecal samples were collected from neonatal Australian sea lion pups at Seal Bay Conservation Park on Kangaroo Island, South Australia (35.99°S, 137.32°E). Pups were sampled during the 2019 winter (n = 160) and 2020/21 summer (n = 184) breeding seasons as part of an ivermectin treatment trial (Lindsay et al. unpublished). Faecal DNA was extracted from FecalSwabTM media (200µL) using the ISOLATE II Fecal DNA Kit (Bioline, Sydney, Australia) following the manufacturer’s protocol. Faecal DNA was tested for PCR competency by a 16S PCR using methods described by Fulham et al. (2020) using forward primer 27F and reverse primer 1492R (Lane, 1991). DNA was submitted to the Ramaciotti Centre for Genomics (University of New South Wales, Sydney, Australia) for 16SV1-3 amplicon sequencing with an Illumina Miseq v3 2x300 bp sequencing kit using primers 27F and 519R, producing a ~530bp fragment (Lane, 1991). Demultiplexed paired-end sequences were analysed using Quantitative Insights Into Microbial Ecology 2 (QIIME 2) version 2022.2 software (Bolyen et al., 2019).

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