Scientists already knew healthy equine eyes can contain fungi, but only through next-generation sequencing (NGS) of DNA can we finally get an accurate idea of which fungal species are present, said Erin M. Scott, BSc, VMD, Dipl. ACVO an assistant professor in veterinary ophthalmology at Texas A&M University, in College Station, Texas.

That’s because previous work relied on growing the microorganisms in a laboratory culture, which provides useful but somewhat limited results, Scott said. Cultures mainly show the fastest-growing species, and they don’t reveal those that can’t survive in a laboratory environment.

Through advanced genomic sequencing technology, however, Scott and her fellow researchers have identified each kind of fungi found on their study horses’ eyes (as long as it represented at least 1% of the sample’s whole fungi population). And the results differ from what researchers saw previously with culture techniques.

“Our study is unique by using culture-independent molecular-based techniques, which allow for the detection of more organisms and reveal that a more diverse community exists on the ocular surface than previously thought,” said Scott.

With this new knowledge, scientists can hopefully better recognize when a horse’s eye mycobiota (fungal microbiota) is healthy and when it isn’t, Scott said. In particular, an off-balance mycobiota might red-flag those horses at greater risk of developing fungal keratitis—infected corneal ulcers.

“Horses have relatively large eyes that are prominently positioned in their head, and this anatomy combined with their outdoor living environments and exaggerated flight response makes them particularly susceptible to ocular trauma,” said Scott. “We find that horses, more so than other animals, develop corneal ulcers that become secondarily infected with opportunistic fungi.”

Mycobiota of 24 Horse Eyes Sequenced

Scott and her colleagues deep-swabbed the conjunctiva behind the lower eyelid of 12 healthy, anesthetized adult horses living at the Texas A&M research campus. The five mares lived outdoor at pasture, whereas the seven stallions lived in individual stalls within an open pavilion.

Scott’s team determined that the most common fungi in their study horses’ eyes were Leptosphaerulina (22.7%), unclassified Pleosporaceae (17.3%), Cladosporium (16.2%), and Alternaria (9.8%). Researchers had not previous identified the first two in horses’ eyes, even though sequencing revealed them to have the highest relative abundance, she said.

Previous culture-based studies had found Cladosporium and Alternaria but not the other two, Scott said. Meanwhile, cultures had shown much greater relative quantities of four other species—Aspergillus, Fusarium, Penicillium, and Scopulariopsis spp.—than the sequencing did, probably because these species grow more easily in a culture. Scott’s sequencing study didn’t identify Pencillium or Scopulariopsis spp. at all, she added.

In addition to the Leptosphaerulina and the unclassified Pleosporaceae, sequencing revealed two other fungi species never previously identified in horses’ eyes: unclassified Montagnulaceae and unclassified Trichocomaceae.

“We should not be surprised that horses have microbes naturally living on their eyes,” Scott said. If they didn’t, it would be “likely detrimental” to their eye health, she added.

Pastured Horses Have More Abundant, Varied Eye Fungi

Scott’s team also found significant differences in the mycobiota of the pastured versus stalled horses. The findings contrast with previous studies comparing the mycobiota in the eyes of stalled and pastured horses, she said.

In the new study, sequencing showed pastured horses had a much greater variety of fungal species on their eyes, and these microorganisms were richer and more abundant than in stalled horses. This might put pastured horses at greater risk of eye disease after an injury, said Scott.

In particular, the pastured mares had more Alternaria spp. and Aspergillus spp.—both of which veterinarians regularly find in infected corneal ulcers, said Scott. Although some research groups have suggested stalled horses would have more fungi in their eyes coming from the hay and bedding, Scott wonders if it’s not the large variation in plant life at pasture, combined with greater changes in temperature and humidity, that lead to the richer fungal life in pastured horses’ eyes, she said.

That doesn’t mean people shouldn’t pasture their horses, however, she added. It just suggests handlers should take care to prevent injury risks—regardless of how or where the horse lives.

“I recommend assessing stable and pasture environments to help decrease the risk of ocular trauma,” Scott said. “This includes eliminating sharp objects around the stable and low-lying tree branches and burdock plants around the pasture.

“Lastly, any signs of ocular discomfort, such as squinting, rubbing, increased tearing, redness or cloudiness to the eyes, should be evaluated by a veterinarian as soon as possible,” Scott continued. “The sooner a corneal ulcer or abrasion can be diagnosed and treated, the better the prognosis is for preventing vision-threatening microbial infections.”