The evaluation of normal ocular parameters in two breeds of hedgehogs

Abstract Objectives The purpose of this study was to evaluate conjunctival microflora and measure normal tear production and intraocular pressure (IOP) in two breeds of hedgehogs (long‐eared hedgehogs and Brandt's hedgehogs). Methods Forty‐eight hedgehogs from two different breeds were chosen for the study. Tear production was measured using the Schirmer tear test (STT) and phenol red thread test (PRTT) in both eyes. IOP was measured using a rebound tonometer. To perform microbiological sampling one drop of tetracaine was instilled in the eyes. Two sterile microswabs were used to collect samples for the microbial and fungal culture. All the microswab samples were transferred in phosphate‐buffered saline (PBS) to the laboratory for culture. Two MacConkey and two blood agar media plates were employed for each eye. Oneplate of sabouraud dextrose agar (SDA) was used for the fungal culture for each eye. Standard biochemical tests were performed to identify the isolated organisms. Results The mean STT and PRTT values were 1.6 ± 0.1 mm/min and 2.4 ± 0.3 mm/15 s in long‐eared hedgehogs and 2.2 ± 0.1 mm/min and 2.5 ± 0.3 mm/15 s in Brandt's hedgehogs, respectively. Mean (SD) Intraocular pressure of right eyes in long‐eared hedgehog and Brandt hedgehog were 19.7 ± 1.4 mmHg and 19.2 ± 2.4 mmHg, respectively. In the left eyes of long‐eared hedgehog and Brandt hedgehog mean (SD) IOP were 19.8 ± 1.5 mmHg and 19.5 ± 2.1 mmHg, respectively. In long‐eared hedgehogs, the most common bacteria were Staphylococcus epidermidis and Bacillus spp. In Brandt's hedgehogs, 24 out of 48 eyes had Staphylococcus epidermidis, which was the most commonly isolated bacterial species. Conclusions This study established reference intervals for IOP, STT and PRTT in hedgehogs and recognised and compared ocular conjunctival microflora in two breeds of hedgehogs.


INTRODUCTION
Hedgehogs are small mammals from the family of Erinaceidae. They are nocturnal animals with protective spines on the back of their body.
Seventeen species of hedgehogs are native to different parts of the world, from the Middle East and Central Asia to Australia, Africa and Europe (DK Publishing Inc, 2006). Long-eared hedgehogs (Haemiechinus auritus) are similar to European hedgehogs in appearance with larger ears and coarse fur on their limbs and faces (DK Publishing Inc, 2006). Brandt's hedgehogs (Paraechinus hypomelas) are desert-living hedgehogs with large ears whose size is similar to European hedgehogs (Yusefi et al., 2016).
In a study on 300 hedgehogs in the United Kingdom, cataract was the most common ocular finding. Fifty-seven out of 300 animals had cataracts, and among them, 54 had nuclear opacities in the lens (Williams et al., 2017). Conjunctivitis, non-ulcerative keratitis, and uveitis were the other reported common ocular findings (Williams et al., 2017). Ghaffari et al. (2012) reported normal tear production and intraocular pressure using applanation tonometer in long-eared hedgehogs.
Hedgehogs have gained popularity as exotic pets during the past decades and therefore veterinarians encounter them in the practice.
Each species and breeds of animals have their unique physiological and anatomical features. Therefore, due to the increase in the population of hedgehogs as exotic pets during past decades and the high risk of eye injuries, it seems necessary to study the natural structure of the eyes of different species of hedgehogs.
The purpose of this study was to evaluate intraocular pressure, conjunctival bacterial and fungal microflora and measure normal tear production using the Schirmer tear test (STT) and phenol red thread test (PRTT) in two breeds of hedgehogs (long-eared hedgehogs and Brandt's hedgehogs).

RESULTS
The mean weight of long-eared hedgehogs and Brandt's hedgehogs was 465.8 ± 62.5 g and 751.6 ± 37.8 g, respectively. There was a significant difference in the weights of the two breeds (p < 0.001).

Tear production
The mean ± SD of STT values in left eyes (OS) and right eyes (OD) was 2.0 ± 0.2 mm/min and 1.8 ± 0.4 m/min in the study population, respectively. No significant difference was found between OS and OD (p = 0.5). The mean ± SD of PRTT values in OS and OD was 2.5 ± 0.6 mm/15 s and 2.4 ± 0.4 mm/15 s in hedgehogs, respectively, and there was no significant difference between OS and OD (p = 0.5). The mean STT value of OD and OS and the mean PRTT value of OD and OS were computed as the mean STT OU (all eyes) and mean PRTT OU for further analysis. The mean ± SD of STT values was identical in male and female hedgehogs (both were 1.9 ± 0.3 mm/min) (p = 0.9), while the PRTT values in males (2.3 ± 0.2 mm/15 s) were slightly lower than that in females (2.6 ± 0.3 mm/15 s) (p = 0.02).
Pearson's correlation coefficient revealed that there was a positive significant linear correlation between the mean weight and the mean STT value (r 2 = 0.920, p < 0.001), but not between weight and the mean PRTT values (r 2 = 0.149, p = 0.488). There was no correlation between the STT and PRTT values (r 2 = 0.105, p = 0.6).
The intrabreed analysis revealed that there were no significant differences in the mean STT or mean PRTT values between males and females in long-eared hedgehogs (p = 0.8, p = 0.3) or Brandt's hedgehogs (p = 0.9, p = 0.06) ( Table 1).

Intraocular pressure
Mean ( There was no significant difference between IOP in right and left eyes (p = 0.6). Independent sample t-test revealed no significant difference between males and females in IOP (p = 0.8, p = 0.6). No significant difference was found between long-eared hedgehog and Brandt hedgehog in the IOP of right and left eyes (p = 0.5, p = 0.6).
There was no correlation between weight of animals and IOP in the study population (r 2 = −0.189, p = 0.3; r 2 = −0.117, p = 0.5)

Microbiological culture
In long-eared hedgehogs, the most common bacteria isolated from were found in 12 (24% of the total isolated bacteria), 6 (12% of the total isolated bacteria), 9 (18% of the total isolated bacteria), 3 (6% of the total isolated bacteria), 3 (6% of the total isolated bacteria), and 1 (2% of the total isolated bacteria) eyes out of 48 eyes, respectively. Only two eyes were negative for bacterial culture (Table 2).
In the fungal culture, Aspergillus spp. was found in 10 out of 48 eyes (15.6% of total isolated fungi). Penicillium spp. and Scopulariopsis spp.
were found in three (4.6% of the total isolated fungi) and three (4.6% of the total isolated fungi) eyes out of 48 eyes, respectively. All the 48 eyes had yeast genera in their fungal culture (75% of total isolated fungi). Moraxella spp. and Escherichia coli were found in two (3.5% of the total isolated bacteria), three (5.2% of the total isolated bacteria), and two (3.5% of the total isolated bacteria) eyes, respectively. All the eyes were positive for bacterial culture.

F I G U R E 1 Phoneoscopy of a normal hedgehog fundus
Fungal culture revealed 42 eyes positive for yeast genera (60.8% of the total isolated fungi). Aspergillus spp. was found in 16 eyes (23.1% of the total isolated fungi), Penicillium spp. in 8 eyes (11.5% of the total isolated fungi), and Scopulariopsis spp. only in 3 eyes (4.3% of the total isolated fungi) out of 48 eyes in Brandt's hedgehogs. Two eyes were negative for fungal cultures (Table 2).
Fundus examination revealed a holangiotic vascular pattern of vessels radiating from the optic disc on an atapetal background. It was the same in both breeds ( Figure 1).

DISCUSSION
The present study reported commensal bacterial and fungal microflora and mean STT, PRTT and IOP in two breeds of hedgehogs.
The tear film is the first refractive surface of the eye. It is vital to health of the eye by protecting and nourishing the cornea (Gelatt, 2020). STT is the conventional method for measurement of tear production. Mean STT has been reported in sedated long-eared hedgehogs as 1.7 ± 1.2 mm/min (Ghaffari et al., 2012). The mean STT value of long-eared hedgehogs in our study was 1.6 ± 0.1 mm/min. Brandt's hedgehogs had higher STT values (2.2 ± 0.1 mm/min) than long-eared hedgehogs in this study.
An alternative method for assessing tear secretion in humans and animals is PRTT. It is more suitable to use in animals with small palpe-bral fissure lengths like hedgehogs (Rajaei et al., 2013). The mean PRTT value in hedgehogs in this study was about 2.5 mm/15 s, which was lower than mean PRTT values of Syrian hamsters (6.8 mm/15 s) (Rajaei et al., 2013) and rabbits (20.8 mm/15 s) (Biricik et al., 2005).
Results of this study revealed that weight and tear secretion had positive linear correlation in hedgehogs. In contrast, Rajaei et al. (2013) found no correlation between tear production and the weight of the animals in the Syrian hamster. The female hamster has been reported to have higher weight compared to male hamsters (Rajaei et al., 2013).
Intraocular pressure is the balance between secretion and drainage of aqueous humor (AH). AH is secreted by the ciliary body and drained via both conventional and unconventional pathways. An increase and decrease in IOP is usually associated with glaucoma and uveitis respectively (Gelatt, 2020).
Intraocular pressure has been reported in long-eared hedgehogs using an applanation tonometer under general anaesthesia as 20.1 ± 4 mmHg ranging from 11.5 to 26.5 mmHg (Ghaffari et al., 2012). This study was conducted without general or topical anaesthesia during the time of IOP measurement.
In another study, the mean IOP in 10 healthy adult hedgehogs have been reported as 12.6±1.8 mmHg using rebound tonometer (Williams et al., 2017). The Mean IOP in the present study was 19 mmHg and the maximum difference in mean IOP between females and males was 0.4 mmHg, which was not statistically or clinically significant.
The population of commensal bacteria and fungi that interact with each other and immune system are important for regulation of the ocular surface health and prevention of opportunistic pathogens overgrowth (Gelatt, 2020).
In nearly all small mammal and lagomorph species, Gram-positive bacteria have been reported as the dominant normal conjunctival bacterial flora in health and disease as these bacterial species are found to be normal skin bacteria (Ansari Cooper et al., 2001;Faghihi et al., 2018;Faghihi & Rajaei, 2019). In rabbits, Staphylococcus species were found in nearly 57% of 70 healthy subjects. Micrococcus and Bacillus spp. were the second and third isolated populations of bacteria in healthy rabbits (Cooper et al., 2001). In Persian squirrels, 83% of the bacterial population was Staphylococcus spp. followed by Corynebacterium spp. (56%), and Streptococcus spp. (53%) as the second and third most common cultivable bacteria (Faghihi et al., 2018). The results of the present study revealed the dominance of Staphylococcus epidermidis as a Gram-positive bacterium.
Gram-negative bacteria were found in a very small population in the eyes of both long-eared and Brandt's hedgehogs. Corynebacterium spp. was isolated from two hedgehogs in this study. Bacillus spp. was common among animals in this study while Moraxella spp.
and Escherichia coli have been isolated in small numbers. Bacillus spp.
Many factors have been reported to influence conjunctival microflora, from sampling and culture methods, climates, and habitats to the gender and age of animal species (Gelatt, 2020).
Contact with faeces has been reported to increase Gram-negative bacteria in the conjunctiva of rodents, especially in captivity (Spinelli et al., 2010). In this study, a few numbers of Gram-negative bacteria were observed in the bacterial cultures and just one colony of Escherichia coli was cultured in an eye. The animals in this study were kept in a captive colony under sanitary conditions, which might be the reason for the low number of faecal Gram-negative bacteria.
In cats, the number of Gram-negative bacteria was reported to be increased during cold seasons (Aftab et al., 2019). In clinically normal dogs, the number of positive bacterial cultures was different during cold and warm seasons (Wang et al., 2008). In the present study the sampling was performed in the spring and the animals was housed indoor with constant temperature and humidity.
The effects of gender on bacterial flora in humans have been investigated, and women have been reported to have more cultured bacterial flora and less goblet cell density in alteration of sex hormones. In Persian squirrels (Faghihi et al., 2018), horses (Johns et al., 2011), and pigs (Davidson et al., 1994), the result was the same as humans. In the present study, no significant differences were observed between the breeds and genders.
Age can influence the immune system and the commensal bacterial population in the body. In humans, a strong correlation has been found between age and conjunctival bacterial flora (Wen et al., 2017). In the present study, the exact age of the animals was unknown.
Aspergillus spp., Penicillium spp., Scopulariopsis spp. and yeast genera were found as fungal conjunctival flora of hedgehogs in our study.
Aspergillus nidulans, Cladosporium spp. and Aspergillus fumigatus were isolated from horses with normal eyes (Gemensky-Metzler et al., 2005). All of the isolated fungi from hedgehogs in this study have been isolated from the conjunctiva of humans with normal eyes (Williamson et al., 1968). Aspergillus spp., Penicillium spp., Cladosporium spp. and Acremonium spp. were reported as fungal flora of clinically normal donkeys (Nardoni et al., 2007). The fungal flora of dogs in the south part of the France was same as the flora of hedgehogs in our study (Verneuil et al., 2014). Yeast genera have been isolated from the eyes, ears, and skin of dogs in health and disease (Prado et al., 2008).
There are some limitations in the current study. The first limitation was the environment of the animals. All studied animals were from a colony, and they were not in their natural habitat at the time of this study.
In conclusion, this study established reference intervals for IOP, STT and PRTT in hedgehogs and recognised and compared ocular conjunc-tival microflora in two breeds of hedgehogs. Future studies on the conjunctival microflora of hedgehogs in health and disease conditions are recommended.

ACKNOWLEDGMENTS
We would like to thank the department of clinical science, Science and Research Branch, Islamic Azad University (IAU), Tehran, Iran, for their approval and supports for conducting an investigation.

CONFLICT OF INTEREST
All authors declare that they have no conflict of interest.

FUNDING
No specific grants have been received from any financial institutions in the public, commercial or non-profit sectors.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
All applicable international, national and/or institutional guidelines for the care and use of animals were followed

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1002/vms3.1055