Veterinary ocular microbiome: Lessons learned beyond the culture

Ocular pathogens cause many painful and vision‐threatening diseases such as infectious keratitis, uveitis, and endophthalmitis. While virulent pathogens and pathobionts play important roles in disease pathogenesis, the scientific community has long assumed disruption of the ocular surface occurs prior to microbial colonization and subsequent infection. While nonpathogenic bacteria are often detected in corneal and conjunctival cultures from healthy eyes, cultures also frequently fail to yield growth of common ocular pathogens or nonpathogenic bacteria. This prompts the following question: Is the ocular surface populated by a stable microbial population that cannot be detected using standard culture techniques? The study of the microbiome has recently become a widespread focus in physician and veterinary medicine. Research suggests a pivotal symbiotic relationship with these microbes to maintain healthy host tissues, and when altered is associated with various disease states (“dysbiosis”). The microbiota that lives within and on mammalian bodies have long been known to influence health and susceptibility to infection. However, limitations of traditional culture methods have resulted in an incomplete understanding of what many now call the “forgotten organ,” that is, the microbiome. With the introduction of high‐throughput sequencing, physician ophthalmology has recognized an ocular surface with much more diverse microbial communities than suspected based on traditional culture. This article reviews the salient features of the ocular surface microbiome and highlights important future applications following the advent of molecular techniques for microbial identification, including characterizing ocular surface microbiomes in our veterinary species and their potential role in management of infectious and inflammatory ocular diseases.     

Comparison of ear canal microbiome in rabbits with and without otitis externa using next generation DNA sequencing

BackgroundContemporary research has increasingly explored the clinical applicability of next-generation DNA sequencing (NGS) technologies in veterinary medicine, which has provided new and practical opportunities for rabbit otitis externa clinical interventions. The objectives of this study were to characterize the normal external ear canal microbiome of clinically healthy rabbits compared to otitis externa presenting rabbits, and to assess the diagnostic viability of NGS in aural veterinary medicine.MethodsSwabs from the external ear canal of 34 clinically healthy rabbits and 16 rabbits diagnosed with otitis externa were collected. Alongside bioinformatic analysis, library preparation was performed targeting the V1-V3 region of the 16S rRNA bacterial gene and the ITS-2 region for fungal DNA analysis.ResultsIn the clinically healthy group, the bacterial species with the highest relative abundances were an uncharacterized Phytoplasma from the family Acholeplasmataceae (8.74%) and Staphylococcus epidermidis (5.56%), while in the otitis group the species with the highest relative abundances were Staphylococcus aureus (12.59%), Corynebacterium lactis (9.27%), and Corynebacterium mastitidis (7.92%). Fungal species with the highest relative abundances in the healthy group were a species from the genus Cladosporium (14.46%), while in the otitis externa group the fungal species with the highest relative abundances were a species from the genus Cladosporium (9.89%) and Malassezia restricta (4.76%). Additionally, there was a significantly higher number of different bacterial and fungal species in the clinically healthy group compared to the otitis group (P = 0.00 and P = 0.00, respectively).Conclusions and clinical relevanceThis study provided evidence that the rabbit aural microbiome profile is distinctly different between a clinically healthy and an otitis state. It also highlighted new bacterial and fungal organisms of note in rabbits diagnosed with otitis externa compared to those previously thought to be the primary disease-causing organisms. Understanding the microbial population dynamics in the rabbit aural microbiome is particularly critical for helping clinicians recognize, prevent, or revert the progression of otitis.     

The intestinal microbiome of an Indo-Pacific humpback dolphin (Sousa chinensis) stranded near the Pearl River Estuary,China

The mammalian intestinal microbiome is critical for host health and disease resistance. However, the cetacean intestinal microbiota remains relatively unexplored. By using high-throughput 16S rRNA gene sequencing, we analyzed intestinal bacterial samples from an Indo-pacific humpback dolphin (Sousa chinensis) stranded near the Pearl River Estuary in China. The samples included 3 anatomical regions (foregut, midgut, and rectum) and 2 anatomical locations (content and mucus). Our analyses revealed that the dolphin intestinal bacteria contained 139 operational taxonomic units (OTUs), dominated at the phyla level by Firmicutes (47.05% in the content; 94.77% in the mucus), followed by Bacteroidetes (23.63% in the content; 1.58% in the mucus) and Gammaproteobacteria (14.82% in the content; 2.05% in the mucus). The intestinal bacteria had a small core community (15 OTUs, accounting for 99.74% of the reads), some of which could be potentially pathogenic to both human and dolphins. As an alternative to sampling the dolphin intestinal bacteria, fecal sampling could be used. Additionally, function potentials such as, xenobiotics biodegradation, beta-lactam resistance, and human disease-related pathways, were detected in the dolphin intestinal bacteria. These findings provide the first baseline knowledge of the intestinal microbiome of the Indo-Pacific humpback dolphin, which may offer new insights into cetacean conservation by using microbial surveillance.     

Detection of severe fever with thrombocytopenia syndrome virus and other viruses in cats via unbiased next-generation sequencing

We used unbiased next-generation sequencing (NGS) to detect unknown viruses in cats. Serum or plasma samples were obtained from clinically ill cats with suspected acute viral infections. Nucleic acid was extracted from serum or plasma samples to construct a complementary DNA library for NGS. Comprehensive nucleotide sequencing analyses enabled detection of the genomes of various viruses, including the severe fever with thrombocytopenia syndrome virus, feline immunodeficiency virus, feline morbillivirus, parvovirus, and Torque teno felis virus. Our findings indicate that comprehensive nucleotide analyses of serum or plasma samples can be used to detect infections with unknown viruses in cats.     

Overweight and the feline gut microbiome – a pilot study

Compared with lean humans, the gut microbiota is altered in the obese. Whether these changes are due to an obesogenic diet, and whether the microbiota contributes to adiposity is currently discussed. In the cat population, where obesity is also prevalent, gut microbiome changes associated with obesity have not been studied. Consequently, the aim of this study was to compare the gut microbiota of lean cats, with that of overweight and obese cats. Seventy‐seven rescue‐shelter cats housed for ≥3 consecutive days were included in the study. Faecal samples were obtained by rectal swab and, when available, by a paired litter box sample. Body condition was assessed using a 9‐point scoring system. DNA was extracted, and the 16S rRNA gene was amplified with a high‐throughput quantitative real‐time PCR chip. Overweight and obese cats had a significantly different gut microbiota compared to lean cats (p < 0.05), but this finding could not be linked to differences in specific bacterial groups. The rectal samples obtained higher DNA concentration than litter box samples (p < 0.0001). In conclusion, overweight and obese cats seem to have an altered gut microbiome as compared to lean cats.     

Implementation of next-generation sequencing for virus identification in veterinary diagnostic laboratories

The value of next-generation sequencing (NGS)-based applications for testing purposes in human medicine is widely recognized. Although NGS-based metagenomic screening may be of interest in veterinary medicine, in particular for intensively farmed livestock species such as pigs, there is a lack of protocols tailored to veterinary requirements, likely because of the high diversity of species and samples. Therefore, we developed an NGS-based protocol for use in veterinary virology and present here different applications in porcine medicine. To develop the protocol, each step of sample preparation was optimized using porcine samples spiked with various RNA and DNA viruses. The resulting protocol was tested with clinical samples previously confirmed to be positive for specific viruses by a diagnostic laboratory. Additionally, we validated the protocol in an NGS viral metagenomics ring trial and tested the protocol on viral multiplex reference material (NIBSC, U.K.). We applied our ViroScreen protocol successfully for 1) virus identification, 2) virus characterization, and 3) herd screening. We identified torque teno sus virus and atypical porcine pestivirus in a neurologic case, determined the full-length genome sequence of swine influenza A virus in field samples, and screened pigs using pen floor fecal samples and chewing rope liquid.     

The Gastrointestinal Microbiome: A Review

The gastrointestinal microbiome is a diverse consortium of bacteria, archaea, fungi, protozoa, and viruses that inhabit the gut of all mammals. Studies in humans and other mammals have implicated the microbiome in a range of physiologic processes that are vital to host health including energy homeostasis, metabolism, gut epithelial health, immunologic activity, and neurobehavioral development. The microbial genome confers metabolic capabilities exceeding those of the host organism alone, making the gut microbiome an active participant in host physiology. Recent advances in DNA sequencing technology and computational biology have revolutionized the field of microbiomics, permitting mechanistic evaluation of the relationships between an animal and its microbial symbionts. Changes in the gastrointestinal microbiome are associated with diseases in humans and animals including inflammatory bowel disease, asthma, obesity, metabolic syndrome, cardiovascular disease, immune‐mediated conditions, and neurodevelopmental conditions such as autism spectrum disorder. While there remains a paucity of data regarding the intestinal microbiome in small animals, recent studies have helped to characterize its role in host animal health and associated disease states. This review is intended to familiarize small animal veterinarians with recent advances in the field of microbiomics and to prime them for a future in which diagnostic tests and therapies will incorporate these developments into clinical practice.     

Influences of melatonin and endotoxin lipopolysaccharide on goose productive performance and gut microbiota

ABSTRACT

1. This study investigated the impact of melatonin and LPS on goose growth and the intestinal microbiome.

2. Geese were injected with a control solution (C), LPS solution (L), melatonin solution (M), and both LPS and melatonin solution (LM), respectively. Faecal samples from each group were used to analyse microbial diversity and function for geese with different treatments.

3. The results showed that the M group had a little improvement in growth compared with the C group, but were much higher than the L and LM groups. A clear distinction between M and other groups was seen with regard to alpha and beta diversity in the biome. The dominant bacteria phyla were Firmicutes, Proteobacteria, and Actinobacteria spp. in all groups. Unclassified bacteria were dominant in all groups at the genus level. Significant KEGG enrichment pathways in the M group were involved in processing metabolism and genetic information, while the L group was related to processing metabolism and environmental information.

4. This study provided a foundation for future studies targeting the specific effect of important bacterial populations on goose growth performance.     

Genomic relationships computed from either next‐generation sequence or array SNP data

The use of sequence data in genomic prediction models is a topic of high interest, given the decreasing prices of current ‘next’‐generation sequencing technologies (NGS) and the theoretical possibility of directly interrogating the genomes for all causal mutations. Here, we compare by simulation how well genetic relationships (G) could be estimated using either NGS or ascertained SNP arrays. DNA sequences were simulated using the coalescence according to two scenarios: a ‘cattle’ scenario that consisted of a bottleneck followed by a split in two breeds without migration, and a ‘pig’ model where Chinese introgression into international pig breeds was simulated. We found that introgression results in a large amount of variability across the genome and between individuals, both in differentiation and in diversity. In general, NGS data allowed the most accurate estimates of G, provided enough sequencing depth was available, because shallow NGS (4×) may result in highly distorted estimates of G elements, especially if not standardized by allele frequency. However, high‐density genotyping can also result in accurate estimates of G . Given that genotyping is much less noisy than NGS data, it is suggested that specific high‐density arrays (~3M SNPs) that minimize the effects of ascertainment could be developed in the population of interest by sequencing the most influential animals and rely on those arrays for implementing genomic selection.     

Feline calicivirus: a neglected cause of feline ocular surface infections?

Objective To investigate the prevalence of feline calicivirus (FCV) infection in relation to ocular surface lesions in cats with upper respiratory tract diseases (URTD). Animals studied Ninety‐nine cats with ocular surface infection and symptoms or recent history of URTD were examined at various rescue shelters and hospitals. Procedure A complete general and ophthalmic examination was performed including Schirmer tear test, slit‐lamp biomicroscopy, fluorescein and lissamine green staining. Clinical and ocular symptoms were scored and recorded. Conjunctival samples were collected using a cytobrush, and nucleic acid extraction using RT‐PCR was carried out to analyze for the presence of various infectious agents. Results RT‐PCR detected either FCV, feline herpes virus type 1 (FHV‐1), Chlamydophila felis or Mycoplasma spp. in 63/99 samples. 30/63 samples were positive for FCV, 23/63 for C. felis, 21/63 for Mycoplasma spp., and 16/63 for FHV‐1. Out of the 30 FCV‐positive samples, 11 were positive only for FCV and in 19 samples FCV was seen in combination with other agents. FCV infection was highest in animals examined at the rescue centers and in the age group of 0–2 months. Erosive conjunctivitis was an important ocular finding. Oral ulcers were detected in all FCV‐infected cats. Conclusion Results indicate that FCV is highly prevalent in cats with URTD either as a sole infectious agent or in combination with other pathogens and therefore is a potential cause for ocular surface lesions during the URTD.     

The effects of pure nucleotides on performance, humoral immunity, gut structure and numbers of intestinal bacteria of newly weaned pigs

Weaning is often stressful for piglets and accompanied by morphological, histological, microbial, and immunological changes along the digestive tract. Dietary nucleotides are bioactive compounds which have the potential to diminish weaning-associated challenges. The experiment was carried out with 5 litters each of 7 pigs (mixed sex), weaned at 20 d of age. One baseline pig per litter was slaughtered at d 0. The remaining 30 pigs were housed individually and randomly allocated to 2 dietary treatments: the control diet or the control diet supplemented with a mixture of nucleotides. Measurements of growth performance traits included ADFI, ADG, G:F, and BW. At d 17, fresh fecal samples were taken to determine bacterial numbers. On d 19 and 20, pigs were slaughtered and blood samples were analyzed for plasma immunoglobulins and intestinal samples were assessed for morphological traits. Digesta from the jejunum and cecum were collected for analysis of the microbiome. The ADFI was greater in the nucleotide treatment compared with the control treatment (P < 0.05), but ADG, G:F, and BW did not differ between treatments. Plasma IgA concentrations increased with age and were greater in the nucleotide (P < 0.05) compared with the control group. There were no treatment differences in plasma IgG and IgM, gut morphology, or intestinal and fecal bacterial counts. Supplemental nucleotides may increase ADFI but without having any impact on growth performance of the pigs. Greater plasma IgA concentrations indicate that adding nucleotides in the weaning diet supported humoral immunity. However, there was no effect of dietary nucleotide supplementation on the composition of the bacterial community in parts of the small and large intestine. Further research is warranted before the use of nucleotide as a feed additive in pig diet can be recommended.     

Fine mapping of a calving QTL on Bos taurus autosome 18 in Holstein cattle

Decreased calving performance not only directly impacts the economic efficiency of dairy cattle farming but also influences public concern for animal welfare. Previous studies have revealed a QTL on Bos taurus autosome (BTA) 18 that has a large effect on calving traits in Holstein cattle. In this study, fine mapping of this QTL was performed using imputed high‐density SNP chip (HD) genotypes followed by imputed next‐generation sequencing (NGS) variants. BTA18 was scanned for seven direct calving traits in 6113 bulls with imputed HD genotypes. SNP rs136283363 (BTA18: 57 548 213) was consistently the most significantly associated SNP across all seven traits [e.g. p‐value = 2.04 × 10^?59 for birth index (BI)]. To finely map the QTL region and to explore pleiotropic effects, we studied NGS variants within the targeted region (BTA18: 57 321 450–57 625 355) for associations with direct calving traits and with three conformation traits. Significant variants were prioritized, and their biological relevance to the traits was interpreted. Considering their functional relationships with direct calving traits, SIGLEC12, CD33 and CEACAM18 were proposed as candidate genes. In addition, pleiotropic effects of this QTL region on direct calving traits and conformation traits were observed. However, the extent of linkage disequilibrium combined with the lack of complete annotation and potential errors in the Bos taurus genome assembly hampered our efforts to pinpoint the causal mutation.     

Technical note: overcoming host contamination in bovine vaginal metagenomic samples with nanopore adaptive sequencing

Animal metagenomic studies, in which host-associated microbiomes are profiled, are an increasingly important contribution to our understanding of the physiological functions, health and susceptibility to diseases of livestock. One of the major challenges in these studies is host DNA contamination, which limits the sequencing capacity for metagenomic content and reduces the accuracy of metagenomic profiling. This is the first study comparing the effectiveness of different sequencing methods for profiling bovine vaginal metagenomic samples. We compared the new method of Oxford Nanopore Technologies (ONT) adaptive sequencing, which can be used to target or eliminate defined genetic sequences, to standard ONT sequencing, Illumina 16S rDNA amplicon sequencing, and Illumina shotgun sequencing. The efficiency of each method in recovering the metagenomic data and recalling the metagenomic profiles was assessed. ONT adaptive sequencing yielded a higher amount of metagenomic data than the other methods per 1 Gb of sequence data. The increased sequencing efficiency of ONT adaptive sequencing consequently reduced the amount of raw data needed to provide sufficient coverage for the metagenomic samples with high host-to-microbe DNA ratio. Additionally, the long reads generated by ONT adaptive sequencing retained the continuity of read information, which benefited the in-depth annotations for both taxonomical and functional profiles of the metagenome. The different methods resulted in the identification of different taxa. Genera Clostridium, which was identified at low abundances and categorized under Order “Unclassified Clostridiales” when using the 16S rDNA amplicon sequencing method, was identified to be the dominant genera in the sample when sequenced with the three other methods. Additionally, higher numbers of annotated genes were identified with ONT adaptive sequencing, which also produced high coverage on most of the commonly annotated genes. This study illustrates the advantages of ONT adaptive sequencing in improving the amount of metagenomic data derived from microbiome samples with high host-to-microbe DNA ratio and the advantage of long reads in preserving intact information for accurate annotations.     

Acremonium and trichosporon fungal keratoconjunctivitis in a Leopard Gecko (Eublepharis macularius)

A 6‐year‐old male leopard gecko (Eublepharis macularius) was presented with a 2‐year history of recurrent dysecdysis involving the ocular surface of both eyes. Ophthalmic examination revealed ocular surface desiccation and multifocal superficial ulcerative keratitis with patchy remnants of retained shed. Other abnormalities included stomatitis and mandibular and maxillary osteomyelitis. Topical and systemic antibiotic therapy, oral vitamin A, and improved husbandry conditions resolved the stomatitis and osteomyelitis but did not improve the ocular surface. Corneal cytology collected with a cytobrush revealed branching hyphae and budding yeast consistent with fungal keratitis. Fungal culture grew Acremonium sp. and Trichosporon sp. The addition of topical antifungal therapy improved the ocular surface health, but the patient was euthanized 7 weeks after initial presentation for persistent vomiting and dyspnea. Necropsy was declined. This case describes the first case of fungal keratitis caused by Acremonium sp. and Trichosporon sp. in a reptile.     

A genome‐wide association study for fat‐related traits computed by image analysis in Japanese Black cattle

The objective of this study was to identify genomic regions associated with fat‐related traits using a Japanese Black cattle population in Hyogo. From 1836 animals, those with high or low values were selected on the basis of corrected phenotype and then pooled into high and low groups (n = 100 each), respectively. DNA pool‐based genome‐wide association study (GWAS) was performed using Illumina BovineSNP50 BeadChip v2 with three replicate assays for each pooled sample. GWAS detected that two single nucleotide polymorphisms (SNPs) on BTA7 (ARS‐BFGL‐NGS‐35463 and Hapmap23838‐BTA‐163815) and one SNP on BTA12 (ARS‐BFGL‐NGS‐2915) significantly affected fat percentage (FAR). The significance of ARS‐BFGL‐NGS‐35463 on BTA7 was confirmed by individual genotyping in all pooled samples. Moreover, association analysis between SNP and FAR in 803 Japanese Black cattle revealed a significant effect of SNP on FAR. Thus, further investigation of these regions is required to identify FAR‐associated genes and mutations, which can lead to the development of DNA markers for marker‐assisted selection for the genetic improvement of beef quality.     

Viral infections shared between water buffaloes and small ruminants in Switzerland

Importation of exotic animals that may harbor infectious agents poses risks for native species with potentially severe impacts on animal health and animal production. Although the Asian water buffalo (Bubalus bubalis) population in Europe is steadily increasing, its susceptibility to viral infections and its role for interspecies transmission is largely unknown. To identify viral infections that are shared between exotic water buffaloes and native small ruminants, we collected blood samples from 3 Swiss farms on which water buffaloes were kept either without, or together with, sheep or goats. These samples were analyzed by next-generation sequencing (NGS) as well as by selected conventional tests, including PCR, ELISA, and in some cases a virus neutralization test. By NGS, a novel virus of the genus Gemykrogvirus (GyKV; Genomoviridae) was first detected in the buffaloes on one farm, and subsequently confirmed by PCR, and was also detected in the co-housed sheep. In contrast, this virus was not detected in buffaloes on the farms without sheep. Moreover, conventional methods identified a number of viral infections that were not shared between the exotic and the native animals, and provided evidence for potential roles of water buffaloes in the epidemiology of ruminant pestiviruses, especially bovine viral diarrhea virus, bluetongue virus, and possibly bovine alphaherpesvirus 2. Our results clearly indicate that water buffaloes are susceptible to interspecies viral transmission and may act as intermediate hosts, or even as reservoirs, for these viruses.     

Current status and future perspectives for sequencing livestock genomes

Only in recent years, the draft sequences for several agricultural animals have been assembled. Assembling an individual animal’s entire genome sequence or specific region(s) of interest is increasingly important for agricultural researchers to perform genetic comparisons between animals with different performance. We review the current status for several sequenced agricultural species and suggest that next generation sequencing (NGS) technology with decreased sequencing cost and increased speed of sequencing can benefit agricultural researchers. By taking advantage of advanced NGS technologies, genes and chromosomal regions that are more labile to the influence of environmental factors could be pinpointed. A more long term goal would be addressing the question of how animals respond at the molecular and cellular levels to different environmental models (e.g. nutrition). Upon revealing important genes and gene-environment interactions, the rate of genetic improvement can also be accelerated. It is clear that NGS technologies will be able to assist animal scientists to efficiently raise animals and to better prevent infectious diseases so that overall costs of animal production can be decreased.     

Microbiota characterization of sheep milk and its association with somatic cell count using 16s rRNA gene sequencing

This work aimed to use 16S ribosomal RNA sequencing with the Illumina MiSeq platform to describe the milk microbiota from 50 healthy Assaf ewes. The global observed microbial community for clinically healthy milk samples analysed was complex and showed a vast diversity. The core microbiota of the sheep milk includes five genera: Staphylococcus, Lactobacillus, Corynebacterium, Streptococcus and Escherichia/Shigella. Although there are some differences, some of these genera are common with the microbiota core pattern of milk from other species, especially with dairy cows. The microbial composition of the studied samples, based on the definition of amplicon sequence variants, was analysed through a correlation network. A preliminary analysis by grouping the milk samples based on their somatic cell count (SCC), which is considered an indicator of subclinical mastitis (SM), showed certain differences for the core of the samples identified as SM. The differences in the microbiota diversity pattern among samples might also suggest that subclinical mastitis would be associated with the significant increase in some genera that are inhabitants of the mammary gland and a remarkable concomitant reduction in the microbial diversity. Additionally, we have also presented here a preliminary analysis to assess the impact of the sheep milk microbiome on SCC, as an indicator of subclinical mastitis. The results here reported provide a first characterization of the sheep milk microbiota and settle the basis for future studies in this field.     

Next-generation sequencing capacity and capabilities within the National Animal Health Laboratory Network

With the cost of next-generation sequencing (NGS) decreasing, this technology is rapidly being integrated into the workflows of veterinary clinical and diagnostic laboratories nationwide. The mission of the U.S. Department of Agriculture–National Animal Health Laboratory Network (NAHLN) is in part to evaluate new technologies and develop standardized processes for deploying these technologies to network laboratories for improving detection and response to emerging and foreign animal diseases. Thus, in 2018, the NAHLN identified the integration of NGS into the network as a top priority. In order to assess the current state of preparedness across NAHLN laboratories and to identify which have the capability for performing NGS, a questionnaire was developed by the NAHLN Methods Technical Working Group and submitted to all NAHLN laboratories in December 2018. Thirty of 59 laboratories completed the questionnaire, of which 18 (60%) reported having some sequencing capability. Multiple sequencing platforms and reagents were identified, and limited standardized quality control parameters were reported. Our results confirm that NGS capacity is available within the NAHLN, but several gaps remain. Gaps include not having sufficient personnel trained in bioinformatics and data interpretation, lack of standardized methods and equipment, and maintenance of sufficient computing capacity to meet the growing demand for this technology.     

Characterization of ocular gland morphology and tear composition of pinnipeds

Objective The importance of tear film integrity to ocular health in terrestrial mammals is well established, however, in marine mammals, the role of the tear film in protection of the ocular surface is not known. In an effort to better understand the function of tears in maintaining health of the marine mammal eye surface, we examined ocular glands of the California sea lion and began to characterize the biochemical nature of the tear film of pinnipeds. Procedures Glands dissected from California sea lion eyelids and adnexa were examined for gross morphology, sectioned for microscopic analysis, and stained with hematoxylin and eosin. The tear film was examined using interferometry. Tears were collected from humans and pinnipeds for the analysis of protein and carbohydrate content. Results The sea lion has sebaceous glands in the lid, but these glands are different in size and orientation compared with typical meibomian glands of terrestrial mammals. Two other accessory ocular glands located dorsotemporally and medially appeared to be identical in morphology, with tubulo‐acinar morphology. An outer lipid layer on the ocular surface of the sea lion was not detected using interferometry, consistent with the absence of typical meibomian glands. Similar to human tears, the tears of pinnipeds contain several proteins but the ratio of carbohydrate to protein was greater than that in human tears. Conclusions Our findings indicate that the ocular gland architecture and biochemical nature of the tear film of pinnipeds have evolved to adapt to the challenges of an aquatic environment.