Ophthalmic diagnostic tests, orbital anatomy, and adnexal histology of the broad-snouted caiman ( Caiman latirostris )

Ophthalmic diagnostic tests, orbital anatomy, and adnexal histologyof the broad-snouted caiman (Caiman latirostris)

Arianne P. Ori�a,* Alberto Vin�ıcius D. Oliveira,† Melissa H. Pinna,* Emanoel F. Martins Filho,*Alessandra Estrela-Lima,* Tiago C. Peixoto,* Renata Maria M. da Silva,* Fernanda O. Santana,*�Iris Daniela S. Meneses,* K�atia G. Requi~ao‡ and Ron Ofri§*School of Veterinary Medicine and Zootechny, Federal University of Bahia, UFBA, 500, Avenida Adhemar de Barros, Salvador, 40170-110, Brazil;

†Get�ulio Vargas Zoobotanic Park, Rua Alto de Ondina S/N, Ondina, Salvador, 40170-110, Brazil; ‡Private Practice, Ladeira do Acupe, 50 - Brotas, Sal-

vador, BA, 40290-160, Brazil; and §The Koret School of Veterinary Medicine, Hebrew University of Jerusalem, P.O. Box 12, Jerusalem, 76100, Israel

Address communications to:

A. P. Ori�a

Tel.: 55 71 32836749

Fax: 55 71 32836730

e-mail: [email protected]

AbstractPurpose The aim of this study was to establish normal ophthalmic parameters forselected diagnostic tests, and to describe the orbital anatomy and adnexal histology of

the broad-snouted caiman.Method A total of 35 Caiman latirostris that were free of obvious ocular diseases were

used to measure the parameters in this investigation. Ages ranged from 5 to 15 years.Ophthalmic diagnostic tests were conducted, including evaluation of tear production

with Schirmer Tear test-1 (STT1), culture of the conjunctival bacterial flora, applana-tion tonometry, conjunctival cytology, nictiating membrane incursion frequency test

(NMIFT), endodontic absorbent paper point tear test (EAPPTT), palpebral fissurelength measurement (PFL) and B-mode ultrasonography. Adnexal histology and skullsamples were studied.

Results Mean (�SD) STT1 was 3.4 � 3.6 mm/min (95% confidence interval of2.01–4.78 mm/min), intraocular pressure (IOP) was 12.9 � 6.2 mmHg, NMIFT was

6.0 � 3.5, EAPPTT was 17.1 � 2.5 mm/min, PFL was 28.9 � 3.0 mm, anteriorchamber depth was 3.1 � 0.3 mm, lens axial length was 8.4 � 0.6 mm, vitreous

chamber depth was 7.9 � 0.7 mm and axial globe length was 19.9 � 1.3 mm. For allanimals evaluated, Bacillus sp., Diphteroids and Staphylococcus sp. were predominant.

Key Words: bacterial flora, B-mode, caiman, intraocular pressure, Schirmer Tear test,ultrasound

INTRODUCTION

The Caiman latirostris, popularly known as the broad-snouted caiman, is a medium-sized crocodilian,1 with largemales measuring up to 3 m, and females up to 2 m, inbody length.2 Caimans have a wide-ranging geographicaldistribution throughout eastern and central South Amer-ica, inhabiting mangroves and other wetlands in southeast-ern Brazil, northern Argentina, Uruguay and Paraguay.3

They have a life expectancy of 50 years,4 and like all croc-odiles and alligators they are a potentially dangerousanimal when handled.5

To perform a proper diagnosis of ophthalmic disease, itis essential to know the normal morphological and physio-logical ocular parameters of the healthy eye. There are

only few published reports on the healthy crocodile eye,including intraocular pressure (IOP)6 and electroretinogra-phy7 in Alligator mississippiensis and a study of the lacrimalapparatus in crocodilians.8 Furthermore, the study of oph-thalmic diseases in crocodiles, caimans and alligators isvery limited. There are case reports of conjunctivitis,9

opacification of the nictitating membrane, cataracts andchorioretinitis,10 ophthalmia,11 eye traumas,10 and a peri-ocular abscess.12

Because of the paucity of information on the healthycrocodilian eye, we decided to establish normal parametersfor ocular diagnostic tests in broad-snouted caimans(Caiman latirostris), held in captivity at the ZoobotanicPark in Salvador-Bahia, Brazil, as well as study its orbitalanatomy and adnexal histology.

© 2013 American College of Veterinary Ophthalmologists

Veterinary Ophthalmology (2013) 1–10 DOI:10.1111/vop.12115

MATERIALS AND METHODS

Thirty-five (eight male, 27 females) Caiman latirostris wereused in this investigation. The exact ages of the animalswere unknown, but they ranged between 5 and 15 years.Animal ID number, gender, body length and the testsconducted in each animal are listed in Table 1. Thecaimans were measured from tip to tail as per describedby Whittaker and colleagues.6 The study was approved bythe Authorization and Information System on Biodiversity,Brazilian Ministry of Environment, and was conducted inaccordance with the ARVO Statement for the Use ofAnimals in Ophthalmic and Vision Research. The studywas performed as part of a routine physical examinationconducted by the local veterinary staff. Therefore, a physi-cal examination was performed before the ocular examina-tion, and animals with indications of systemic disease wereexcluded from our study.

Data were collected in four stages. The first and secondstages were conducted in the animals’ enclosure, the thirdstage was performed at a private veterinary clinic, and thefourth stage conducted in the Animal Pathology Depart-ment, Federal University of Bahia. For the first and sec-ond stages of this study (except the second nictitansincursion test), a rubber band was used to tape the mouth,and the animal was restrained by 2–3 people sitting on itto prevent its movement. Data were collected in the shade,at least 30 min after the animals were captured. Prior todata collection, the eye and periocular region were exam-ined in normal light for gross abnormalities with a binoc-ular magnifying loupe 39 and a transilluminator. Anyanimals found to have gross lesions were not included inthe study. All data were collected by the same investigator(AO), except the ultrasound examination (performed byKR) and the nictitans incursion test which was conductedby four investigators.

Table 1. Animal’s identification, gender, body length and the examinations performed in each broad-snouted caiman (Caiman latirostris)

IDnumber Gender

BodyLength (m)

Conjunctivalflora STT IOP

Conjunctivalcytology EPPTT PFL NMIFT-1 NMIFT-2 Ultrasound

1 F – X2 F – X3 F – X4 F – X5 F – X6 F – X7 F – X8 M – X9 F – X10 F – X11 M 2.15 X X X X12 F 1.35 X X X X13 F 1.09 X X X X14 F 1.23 X X X X15 F 1.73 X X X X X16 F 1.56 X X X X X17 F 1.48 X X X X X18 F 1.70 X X X X X19 F 1.52 X X X X X20 F 1.45 X X X X X21 F 1.60 X X X X X22 F 1.47 X X X X X23 F 1.33 X X X X X24 F 1.64 X X X X X25 M 1.50 X26 F 1.24 X X X X27 M 1.59 X X X X28 F 1.49 X X X X29 M 1.40 X X X X30 F 1.62 X X X31 F 1.77 X X X32 M 1.69 X X X X33 M 1.76 X X X X34 F 1.51 X X X X35 M 1.64 X X X X

M, meter; STT, Schirmer Tear test; IOP, intraocular pressure; EPPTT, endodontic paper point tear test; PFL, palpebral fissure length;NMIFT, nictitating membrane incursion frequency test.The symbol ‘X’ means the animals in which the respective tests were conducted.

© 2013 American College of Veterinary Ophthalmologists, Veterinary Ophthalmology, 1–10

2 o r i�a E T A L .

First stage

Schirmer Tear test-1 (STT1) Sterile Schirmer Tear teststandardized strips (Ophthalmos�, S~ao Paulo, Brazil) wereused to measure production on the aqueous component ofthe tear film in 14 animals. No other diagnostic test orexamination was conducted prior to STT1. Testing wasconducted using sterile gloves, and without the installationof topical anesthesia, to avoid confounding the results ofthe subsequent bacterial sampling.

Sampling of conjunctival bacterial flora Sterile swabs wereused to collect samples for bacteriological culturing fromthe conjunctival fornices of both eyes of 25 animals. Swabswere immediately sent in triptose agar medium to theBacterioses Laboratory, Veterinary Hospital, Federal Uni-versity of Bahia. Culturing of the microorganisms was per-formed in Petri dishes with sheep blood agar to 6%MacConkey agar and tryptose broth, which were incubatedat 37 °C in an aerobic environment for 24–48 h. Aftergrowth, the colonies were identified based on the presenceor absence of hemolysis on blood agar and morphologicaland biochemical characteristics according to routine labo-ratory techniques.13 Yeasts were not surveyed in this study.

Tonometry Tonometry was conducted in 14 animals,using a Tonopen� XL (TonoPen XL; Reichert Technolo-gies, New York, NY, USA) following instillation of topicalanesthesia (proxymetacaine 0.5%, Anestalcon�; Alcon, S~aoPaulo, Brazil). Measurements were taken in the centralcornea between incursions of the nictitating membrane(NM). Eyelids were not retracted manually because theanimals voluntarily kept their eyes open. Care was takento avoid applying any pressure in the neck region duringphysical restraint to prevent iatrogenic IOP alterations.

Conjunctival cytology (CC) A barren interdental brush(interdental brush conical Oral B�, Manaus, Amazonas,Brazil) was placed in the conjunctival fornix of 14 animalsand samples were collected using smooth rotational move-ments. Care was taken to avoid or minimize any trauma tothe conjunctiva. Samples were distributed onto glass slides,air-dried, and stained using the Panoptic fast method.

At the end of Stage I, corneas were stained with fluores-cein to rule out corneal ulceration.

Second stageThe tests included in the second stage were conducted ona different group of animals than those used in Stage 1(see Table 1).

Nictitating membrane incursion frequency test (NMIFT-1and NMIFT-2) The frequency of the NM incursion wasrecorded in two manners. For the first test (NMIFT-1),10 animals were captured and their mouths taped as

previously described. After several minutes, two investiga-tors sat, one on each side of the animal, and counted thenumber of incursions during 5 min. No additionalrestraint or human interference was imposed. The secondtest (NMIFT-2) was conducted several months later ineight animals. One investigator sat at a safe distance andmeasured the amount of time required for three or moreincursions. Animals were in their normal habitat, and norestraint was used. For both NMIFT-1 and NMIFT-2,time zero was the moment of the first incursion.

Endodontic absorbent paper point tear test (EAPPTT) Anendodontic absorbent paper point (Dentsply� – Color size30, Rio de Janeiro, Brazil), 28 mm in length, was insertedinto the lower conjunctival fornix of 10 animals, where itremained for 1 min (Fig. 1a). The moist portion of thepaper was measured with a digital caliper (Mitutoyo, S~aoPaulo, Brazil) immediately thereafter (Fig. 1b). The sametechnique was recently used to measure tear production inblack-tufted marmosets.14

Palpebral fissure length (PFL) Palpebral fissure lengthwas measured in 10 animals using a digital caliper (Mitu-toyo) with an accuracy of � 0.02 mm (Fig. 1c).

Third stage

B-mode ultrasonographic biometry B-mode ocular ultraso-nography was performed in 10 animals using an SA 9900(Medison, S~ao Paulo, Brazil) with a 12 MHz probe. Tenof the animals evaluated in Stage I were used for thisstudy, which was performed on a different day. Chemicalor mechanical restraint was not required, as the examina-tion was conducted in a cool, air-conditioned room. Fol-lowing application of one drop of 0.5% proxymetacaineeye drops (Anestalcon�, Alcon) and ultrasound transmis-sion gel (Carbogel UTLTM; S~ao Paulo, Brazil), the anteriorchamber (ACD), vitreous chamber (VCD), lens (LT) andglobe (AGL) and axial lengths were measured in both eyes(Fig. 2).

Fourth stage

Descriptive study of the orbit anatomy Skulls of two adultcaimans (a 20-year-old male and a 6-year-old female)belonging to the collection of the Zoobotanic Park wereused to study orbital anatomy.

Histology of the adnexa (eyelids, nictitating membrane, andHarderian and palpebral glands) The upper and lowereyelids of a female caiman, approximately 1-year old,which died due to poisoning, were studied histologically.Following the animal’s death, the cadaver was immediatelyfrozen. The frozen head was subsequently transected andplaced in a large container with a 10% formalin solution


o phth a lm i c d i agno s t i c t e s t s o f th e b r o ad - s n out e d c a im an 3

for fixation. One week later, eyelids were removed, pro-cessed and stained with hematoxylin and eosin (H & E).

The NM, Harderian and palpebral glands wereobtained from a 20-year-old male that was enucleatedfollowing a fight with another animal. Immediately aftersurgical excision, these adnexa were fixed in a 10% forma-lin solution, processed routinely for histopathology andstained with H and E. Sections from the Harderian andpalpebral glands were also stained with alcian blue (AB).

Statistical analysisThe Shapiro–Wilk test was used to test data normality forSTT1, IOP, NMIFT, EAPPTT, PFL, and ultrasound

measurements. Differences between mean values of vari-ables were tested using paired Student′s t-tests. Correla-tion between quantitative variables was evaluated using thePearson correlation test.

RESULTS

Descriptive observationsNo signs of adnexal or anterior segment disease wereobserved in any of the study animals.

The lower eyelid of the broad-snouted caiman is muchmore mobile than the upper eyelid. In fact the upper eye-lid barely moves, while blinking of the lower lid causescomplete closure of the palpebral fissure. No meibomiangland openings were observed on the eyelid margin. Thepalpebral conjunctiva is pink and contains several ran-domly pigmented areas. The NM is well developed,actively mobile and not completely transparent due to thepresence of blood vessels. Its free margin has a firm dou-ble fold tissue containing yellowish pigmentation, while itsbase contains conjunctival tissue, richly vascularized withvessels of various diameters (Fig. 3a). During the ophthal-mic tests, most of the animals exhibited NM incursions,especially when approached by the examiner, though thesewere not always simultaneous in both eyes. Those incur-sions would only cease either when the test was completedor if the examiner stepped away. NM incursion was alsoobserved when the animals submerge, as soon as the eyemakes contact with the water, so that the entire eye is cov-ered by NM underwater; and when animals open theirmouths to eat, bite or attack.

The eyelids are almost completely shut when the animalis on land, especially when they are warming themselves.Eyelids open when the animal becomes excited by the pres-ence of humans, food or other alligators, and underwater.

(a)

(b)

(c)

Figure 1. Photographs of selected ocular tests performed in broad-

snouted caiman (Caiman latirostris). (a) Endodontic absorbent paper

point tear test; (b) Photograph demonstrating the absorption of the

aqueous tear portion using an endodontic absorbent paper with a

digital caliper; (c) Palpebral fissure length measurement.

Figure 2. Representative B-mode ultrasonographic image of the

broad-snouted caiman (Caiman latirostris) eye. The four dashed lines

were placed on the frozen B-scan image for measurement:

(+) Anterior chamber depth, (x) anterior/posterior axis of the lens,

(#) vitreous chamber depth, (*) axial globe length.



The broad-snouted caiman iris color varies from lightto dark green-yellow, with diffuse areas of brown to blackpigmentation in a mosaic pattern (Fig. 1a). The pupil is avertical slit and was responsive to light under voluntarycontrol. We also observed that in bright light itconstricted to a narrow slit and in scotopic conditions itassumed a circular shape.

Microbiological analysisA wide range of bacterial species, mostly Gram positive,were cultured and are summarize in Table 2.

STT1, IOP, EAPPTT, PFL, and B-mode ultrasoundDescriptive statistics results of tear production tests(STT1 and EAPPTT), IOP, PFL and ultrasoundmeasurements are presented in Table 3. There were nosignificant differences between left and right eyes for anyof these variables (Paired Student t-tests).

There was no significant correlation between IOP andbody length (Pearson’s correlation test). There was a mod-erate positive correlation between the EAPPTT and theSTT1, with borderline significance (Pearson’s correlationtest, r = 0.432, P = 0.057). There was a positive linearcorrelation between the body length and all of the

ultrasound variables measured. We found a strong, posi-tive correlation between body length and the axial lengthof the eye (r = 0.606, P = 0.005), ACD depth (r = 0.579,P = 0,007), lens thickness (r = 0.545, P = 0.013) and VCDdepth (r = 0.592, P = 0.006) which was statistically signifi-cant using Pearson’s correlation test.

Conjunctival cytologyHypercellular samples with a predominance of squamousepithelial cells and superficial keratinized cells showingmelanocytic cytoplasmic granulation in various degreeswere identified (Fig. 4). Often we saw erythrocytes, eventhough macroscopically we did not observe any conjuncti-val trauma or blood on the brush. Clusters of cellssurrounded by mucoid material, rare lymphocytes andextremely rare polymorphonuclear cells were also seen.

Nictiating membrane incursion frequency testDuring 5 min of observation (NMIFT-1), no NM incur-sions were observed.

Nictiating membrane incursion frequency test-2 wasconducted in eight animals. However, it was possible toobserve only 15 eyes, and only eight of these had three ormore incursions of NM, during the evaluation time that

(a) (b)

(c) (d)

Figure 3. Nictitating membrane (NM) of broad-snouted caiman (Caiman latirostris). (a) Macroscopic aspect of the NM covering the eye in a

living animal. Note the NM’s free margin with a firm double fold tissue with yellowish pigmentation. The NM base contains a transition area

with conjunctival tissue, richly vascularized with vessels of various calibers. Photomicrographs of the NM (b, c, d). Note: (b) insertion point of

the NM with the conjunctiva (arrow) is characterized by a change in the arrangement of collagen fibers. (c) The NM stroma is composed of

juxtaposed collagen fibers. It is, poorly vascularized, with its external aspect covered by a stratified squamous keratinized epithelium. (d) The

stroma is loose, richly vascularized and covered by a stratified squamous nonkeratinized epithelium. A linear accumulation of melanin pigment

(arrow) can be observed in the lamina propria of the NM free edge.



ranged from 20 to 60 min. Results are presented inTable 3.

Orbital anatomyThe exterior bones of the caiman skull (that come in con-tact with the overlying skin) have a pitted appearance.The orbit is completely enclosed and consists of six bones.Based on the nomenclature of the orbital bones of theAlligator mississippiensis, the frontal, lacrimal, and jugalbones form the orbital rim15 (Fig. 5). The postorbital,pterygoid, and ectopterygoid bones complete the orbit.Two holes observed in the posterior aspect of the orbitmay be the optic and round foramina (see Fig. 5a). Thenasolacrimal duct enters the skull in the medial third ofthe nasal cavity, in the olfactory chamber. Dorsal to this

opening, five holes (three larger and two smaller) wereobserved, which may be associated with unidentifiednerves and vessels (see Fig. 5b).

Histology of the adnexaThe upper eyelid is covered by conjunctiva internally andexternally by keratinized stratified squamous epithelium.In the lamina propria, aggregates of melanocytes arrangedlinearly are observed. A dermis plate delimits the presenceof dense connective tissue (tarsal plate) that correspondsmacroscopically to the central protuberance of the uppereyelid. At the insertion point of the NM into the conjunc-tiva, a change in the arrangement of collagen fibers wasobserved (Fig. 3b). In this region, the stroma is loose,richly vascularized and covered by stratified, squamous,nonkeratinized epithelium, composed of about 15 layers ofcells. The body of the NM stroma is composed of juxta-posed collagen fibers and is poorly vascularized. Its exter-nal surface is covered by stratified, squamous keratinizedepithelium with approximately half the number of layersseen in the stroma (Fig. 3c). At the free edge of the NM,a linear accumulation of melanin pigment in the laminapropria was observed (Fig. 3d).

The Harderian gland is a single structure surroundedby a capsule (Fig. 6a). Histologically, tubular secretoryunits, delimited by lobes and delicate conjunctival tissuecontaining small blood vessels and lymphoid aggregate,could be observed. Numerous cells with a slightly fibrillar(mucus) AB-positive intracytoplasmic secretory materialwere also seen (Fig. 6b).

The palpebral gland is located within the ventral eyelid,rostral to the globe and lateral to the NM. Its histologicalappearance is similar to that of the Harderian gland,except for the smaller amount of fibrillar material (notshown).

DISCUSSION

The crocodilians have good vision, hearing, smell, andsense of touch.15 However, the translucent NM, whichcovers the eye during submersion and during prehensionof food, is thought to cause defocusing16 of vision result-ing in poor resolution.15 The pupil of the broad snoutedcaimans is similar to that of many terrestrial predators.

The reptilian ocular surface is bathed in fluids secretedby the lacrimal and Harderian glands (except insnakes).8,17 The authors believe that the spreading of thetear film is accomplished by NM, rather than eyelid,movement. The nasolacrimal duct, absent in chelonians,stretches from the medial canthus to the roof of themouth, to emerge at the base or behind the vomeronasalorgan.18 However, the vomeronasal system is absent incrocodilians, marine turtles and fish.19 In Alligator missis-sippiensis, the opening of the nasolacrimal duct is locatedin the medial third of the nasal cavity,8 which is similar toits location in the broad-snouted caiman (see Fig. 5b).

Table 3. Results obtained for select ophthalmic diagnostic tests in

the Caiman latirostris eye

VariableMeanvalue

Standarddeviation

95% confidenceinterval

Schirmer Tear test (mm/min) 3.3 3.6 1.9–4.7Endodontic absorbent paperpoint (mm/min)

17.1 2.5 15.9–18.2

Intraocular pressure (mmHg) 12.9 6.1 10.5–15.3Palpebral fissure length (mm) 28.9 3.0 27.5–30.3Nictitating membrane incursionfrequency test 2

6.0 3.5 3.0–8.9

Anterior chamber depth (mm) 3.0 0.3 2.3–3.4Lens axial length (mm) 8.3 0.6 7.2–9.1Vitreous chamber depth (mm) 7.9 0.6 6.9–9.8Axial globe length (mm) 19.8 1.3 1.3–3.2Body length (m) 1.5 0.2 1.3–1.6

Table 2. Conjunctival bacterial flora isolated from healthy broad-

snouted caiman (Caiman latirostris, n = 14)

Bacterial type No. of isolations Total (%)

Gram-positiveBacillus sp. 45/96 46.8%Diphtheroids 19/96 19.7%Staphylococcus sp. 14/96 14.5%Staphylococcus epidermidis 8/96 8.3%Micrococcus sp. 7/96 7.2%Total 96/96 100%Gram-negativeEnterobacter harfnia 6/31 19.3%Alcal�ıgenes sp. 5/31 16.1%Citrobacter 4/31 12.9%Escherichia coli 4/31 12.9%Enterobacter cloacae 3/31 9.6%Pectobacterium sp. 2/31 6.4%Providencia sp. 2/31 6.4%Proteus rettigeri 1/31 3.2%Shiguella sp. 1/31 3.2%Arizona sp. 1/31 3.2%Enterobacter linquenfaciens 1/31 3.2%Enterobacter aerogenes 1/31 3.2%Total 31 100%



Tear production has been measured in several exoticand wild species.20–25 However, to the best of our knowl-edge, it has yet to be measured in crocodiles, caimans oralligators. STT1 results may be affected by physical

restraint, as well as by dirt and/or liquid coming intocontact with the eye during handling. Obviously, safetyconcerns prevented us from conducting the test withoutphysical restraint. However, every effort was made to min-imize contact between the eyes and foreign materialduring handling. Furthermore, animals were kept for

(a) (b)

Figure 4. Photomicrograph of a brush cytology sample from the conjunctiva of broad-snouted caiman (Caiman latirostris) –Panoptic fast

(10009): (a) Squamous epithelial cell with discrete melanocytic cytoplasmic pigmentation. (b) Keratinized epithelial cell and bacteria.

(a)

(b)

Figure 5. Skull from an adult male broad-snouted caiman (Caimanlatirostris). Red circles and white arrows show location of enlarged

areas. (a) Lateral view with the mandible (scale bars = 3,39 cm) and

(b) Dorsal view without the mandibule (scale bars = 2,76 cm). Note

the six bones of the orbital cavity: Fr = frontal bone; La = lacrimal

bone; Ju = jugal; Po = postorbital; Pt = pterygoid;

Ep = ectopterygoid. Note: (a) at the enlarged image the location of

the two holes in the posterior aspect of the orbit that may be the

optic and round foramina, (b) at the enlarged image the location of

the nasolacrimal duct (red arrow).

(a)

(b)

Figure 6. Harderian gland of broad-snouted caiman (Caimanlatirostris). (a) Macroscopic aspect of the gland. Note a single

structure surrounded by a capsule. (b) Photomicrograph of the gland.

Note: tubular secretory units delimited by lobes and delicate

conjunctive tissue containing small blood vessels and lymphoid

aggregate. Detail: Numerous cells with a slightly fibrillar (mucus)

alcian blue positive intracytoplasmic secretory material.



30 min in the shade before performing ophthalmic tests tominimize the potential effect of any confounding factor.Even though it has been recommended that eyes of ani-mals be covered with cloth to minimize handling stress,we chose to leave the eyes uncovered in the present studyto not interfere with the results.

Endodontic absorbent paper point tear test is widelyused in dentistry during endodontic procedures, due to itshigh absorption properties. However, its use in measuringthe aqueous component of the tear film is not yet wide-spread and requires additional studies in other species aswell as standardization of size, raw material, make and dyeapplication.14 Comparing results of the two tear produc-tion tests employed in this study, the mean value of tearproduction was higher using EAPPTT (see Table 3). Thistrend was also observed in black-tufted marmosets.14

There are several possible explanations for this difference,including differences in strip width and absorbance prop-erties. Furthermore, the EAPPTT strip sits deeper in thefornix than the STT strip, resulting in greater contactwith the tear lake present in the large conjunctival sac ofthese animals.

Endodontic absorbent paper point tear test was used inblack-tufted marmosets because these animals have smalleyes, making the use of conventional Schirmer strips verydifficult.14 While this is not a factor in the large caimaneye, we believe that the low values measured by STT makeEAPPTT a more reliable test in this species. It was easy touse, and the strip was readily inserted by one person. Whileboth strips were moved by NM incursions, the dislodgingof the STT strip was greater than that of the EAPPTT.

Both tests measure only the aqueous component of thetear film. Rehorek et al.8 report on the presence of mucinand lipid products in the Alligator mississippiensis tear film.Based on alcian blue staining in the Harderian gland (seeFig. 6) we suspect similar mucin production in the caiman.However, neither study included a quantitative evaluationof the mucin and lipid components of the tear film. Thelow STT1 values and NM incursion frequency of the cai-man lead us to suspect that these layers play a very impor-tant role in maintaining tear film integrity in this species.

Guineas pig and chinchillas also have low blink frequen-cies, with a mean value of 3.4 � 1.1 in 20 min in theformer26 and 2.7 � 1.0 for males and 2.5 � 0.6 for femalesin 10 min of testing in the latter.27 The mean value foundfor NMIFT in the broad-snouted caiman was very low, withan interval of approximately 6 min between incursions. Onthe other hand, dogs, cats, horses, cattle, and pigs have highblink frequencies (between 1 and 25 times/min),28 as domarmosets with a mean frequency of 20.3 � 5.9 in 5 min.14

Again, we suspect that mucin and lipid components of thecaiman tear film are produced in sufficient quantities tomaintain film stability for such long periods without theneed for meibomian glands and their secretion.

The histological description of the caiman Harderiangland is quite similar to that of the Alligator mississippien-

sis.8 In the latter, it has been reported that there are morelipid droplets scattered in a diffuse and uniform manner inthe Harderian gland compared to the palpebral gland.8

The Harderian gland duct orifices are located in the ven-tral part of the orbit.8 Substantial amounts of lacrimalgland secretions including fat, mucous and serous productshave also been described in alligators.8

According to several authors, crocodilians have a bony ora very tough cartilaginous tarsus in the upper eyelid that iscapable of powerful closure.10,29 This structure was not evi-dent in the specimen evaluated in this study. Additionalstudies are needed to determine whether or not a tarsus withsuch characteristics is a common finding in this species.

The arrangement of collagen fibers in NM is similar tothat of the corneal stroma, resulting in a transparentmembrane (see Fig. 3).30 We did not find the cartilagi-nous plate which has been reported in the alligator NM.8

The pitted appearance of the dorsal surface of the cai-man‘s skull is in marked contrast to the smooth skull ofmost domestic species and humans. Though the physio-logical mechanism for this appearance is unknown, it hasbeen speculated that it is a result of direct fusion of theskin to the skull.31 Other researchers suggest that it mayresult from the local action of osteoclasts.32

Intraocular pressure was measured in several exotic andwildlife species,25,33–37 but in reptiles, it has only beenreported for red-38 and yellow-footed tortoises,39 logger-head sea turtles40 and alligators.6 Our results are similarto those reported in most reptiles, but were higher thanthose reported for sea turtles.40 We did not find theinverse relationship between IOP and body length whichhas been reported in Alligator mississippiensis.6 We believethat this is because Whittaker et al.6 studied animals ofdifferent age groups, including both juveniles (bodylength < 50 cm with mean IOP: 23.7 � 2.1 mmHg) andadults (body length > 50 cm with mean IOP:11.6 � 0.5 mmHg), while our animals were all adults ofsimilar size (see Table 1). It should be emphasized that inthis study it was not possible to investigate the effect ofphysical restraint and body position on caiman IOP. It ispossible that our physical restraint methods may haveaffected both the STT1 and IOP results, but due to theaggressiveness of the animals it was not possible toperform any kind of examination without restraint.

Since 1982 when A-mode ultrasound was first used forevaluating canine eyes,41 ultrasonographic studies havebeen performed in exotic and wildlife including rabbits,ferrets, rhesus monkeys, capybaras, guinea pigs, camels,and elephants.35,41–47 However, only one study, of foursnake species, has been conducted in reptiles.48

The ultrasound image of the broad-snouted caiman eye issimilar to that of mammalian species and differs from thatof snakes, as the latter possess a spectacle andsubspectacular space.48 The measurements obtained for theaxial length of the broad-snouted caiman eye are similar tothose of the human eye, which ranges from 21.0 to



27.5 mm.49,50 The anterior/posterior axis of the broad-snouted caiman lens (8.3 � 0.6 mm) is longer than thatreported in dogs (6.7 � 1.0 mm),51 and cats(7.7 � 0.23 mm)52 and much longer than that reported inhumans (4.2 � 0.06 mm).53 This probably allows signifi-cant lenticular refraction to occur underwater, thus com-pensating for loss of corneal refraction. The ratio betweenthe length of the lenticular anterior/posterior axis and theglobe axial length in the broad-snouted caiman is 1:2.4, sim-ilar to the range of values (1:1.9 to 1:2) reported in snakes.48

The constricted caiman pupil is a vertical slit, somewhatsimilar in shape to that of the domestic cat (see Fig. 1a).As in other nocturnal species, this shape probably protectsthe retina from excessive amounts of daytime light whileincreasing the depth of focus.5 The iridal muscle is stri-ated, and the sphincter is under voluntary control, thusmaking it difficult to examine the posterior segment of theeye.10 Mydriasis may be achieved during general anesthe-sia, following relaxation of the sphincter muscle, orthrough the use of curare.10

In this study, we isolated bacteria in 100% of samplescollected from 50 eyes. Most (75.6%) of the isolates weregram-positive (see Table 2), which is consistent withresults in other exotic and wildlife species.35,54–56 How-ever, it is important to remember that the normal bacterialflora may vary with season,18 geographic location, nutri-tion, differences in culture techniques,57 population den-sity55 and contact with other animals.22

Similar to observations in other studies,58,59 brush cytol-ogy was performed with minimal irritation and discomfortand allowed the collection of representative material inquality and quantity sufficient for effective assessment (seeFig. 4). As we observed no macroscopic conjunctival traumaor blood on the brush following sampling, microconjuncti-val lesions may explain the presence of erythrocytes inslides. We observed that the predominant cell pattern wassimilar to that reported in mammalian species. The pres-ence of pigmentation in the cytoplasm of the conjunctivalcells has been reported in horses, sheep, and cattle.60 Indogs and cats, it suggests the presence of pigmentary hyper-plasia, which usually occurs in cases of keratoconjunctivitissicca, vitamin A deficiency, chronic ocular surface diseaseand injury by mechanical irritants.58 While we couldobserve conjunctival pigmentation both microscopicallyand macroscopically, the animals we studied showed nosigns of adnexal or anterior segment disease.

The determination of values for different ophthalmictests in healthy broad-snouted caiman (Caiman latirostris)represents an important aid in the diagnosis, treatmentand prevention of eye diseases in this species.

ACKNOWLEDGMENTS

The authors acknowledge Gerson de Oliveira Norberto(Coordinator of the Getulio Vargas Zoobotanic Park,Salvador, BA, Brazil), Aline M. Pontes Ori�a and all veteri-

naries, technicians and veterinary students who contrib-uted to this work.

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Documents

Ophthalmic diagnostic tests, orbital anatomy, and adnexal histology of the broad-snouted caiman ( Caiman latirostris )