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. 2022 Oct 17;9(2):625–637. doi: 10.1002/vms3.982

Study of ocular manifestations and humoral immune response in eyes of dogs with leishmaniasis

Amel F El Goulli 1,✉, Lilia Zribi 2, Rania Sanhaji 1, Ahmed Chabchoub 1, Aïda Bouratbine 2, Mohamed Gharbi 3, Hafedh Abdelmelek 4
PMCID: PMC10029893  PMID: 36253884

Abstract

Background

Ocular manifestations in dogs with leishmaniasis are frequent and complications in affected tissues can lead to blindness. Immune processes play a very important role in the pathogenesis of ocular inflammation. Therefore, the immunology of ocular manifestations in dogs with leishmaniasis remains complex and poorly understood.

Objectives

Estimation and characterisation of ocular and periocular manifestations in dogs naturally infected with Leishmania infantum and investigation of the production site of specific anti‐Leishmania infantum IgG.

Methods

The present investigation used 53 confirmed dogs infected with Leishmania infantum, presenting ocular and periocular lesions, and 10 control non‐infected dogs.

Complete macroscopic ophthalmic examination of eyelids and globes was performed. Both total and anti‐Leishmania infantum IgG antibodies were studied in sera and aqueous humour (AH) of all dogs by ELISA technique. A Goldmann–Witmer coefficient (C value) was calculated.

Results

The main ophthalmological findings were keratoconjunctivitis (71.7%; 38/53), hyperplasia of conjunctival lymphoid follicles (54.7%; 29/53), blepharitis (50.9%; 27/53) and uveitis (20.7%; 11/53). Ocular production of anti‐Leishmania infantum IgG was detected in 73.6% (39/53) of infected dogs. There was no correlation between the antibody levels in AH and sera of the same dog. The mean anti‐Leishmania infantum IgG in AH was higher in uveitis, followed by lesions affecting only the adnexa (p < 0.0001). The highest mean C values were observed for uveitis, conjunctivitis and keratitis.

Conclusions

Our findings suggest that production of anti‐Leishmania IgG in dogs infected with Leishmania infantum with ocular manifestations begin in situ and follows by a transfer of antibodies from the bloodstream to the AH.

Keywords: antibody, aqueous humour, dog, eye, Goldmann–Witmer coefficient, leishmaniasis

  • We reported here, for the first time, a significant association between follicular conjunctivitis and leishmaniasis in dogs.

  • The mean anti‐Leishmania infantum IgG in aqueous humour was higher in uveitis, followed by lesions affecting only the adnexa.

  • The highest mean C values were observed for uveitis, conjunctivitis and keratitis.

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1. INTRODUCTION

Canine leishmaniasis is a vector‐borne zoonotic disease caused by Leishmania infantum, a protozoan transmitted by sandflies. According to the World Health Organization (WHO), human leishmaniasis is considered one of the world's most neglected diseases, affecting mainly the developing countries (WHO, 2010). The annual incidence of leishmaniasis in humans due to different species of Leishmania spp. is estimated to be between 700,000 and 1 million (WHO, 2022).

The epidemiological role of both clinically and non‐clinically Leishmania infected dogs is very important as they are the main reservoirs of parasites (Bourdoiseau, 2015). Clinical signs are highly polymorphic and include general signs (weight loss, lethargy and anaemia) and specific involvements (lesions in skin, kidney and eye tissues) (Gharbi et al., 2015). Ocular manifestations in dogs with leishmaniasis are frequent with a prevalence ranging from 16% to 92% (Brito et al., 2006; Ciaramella et al., 1997; Di Pietro et al., 2016; Freitas MV de et al., 2017; Molleda et al., 1993; Peña et al., 2000). The prevalence of ocular signs as the only clinical manifestation varies between 3.72% and 16% in dogs with leishmaniasis (Brito et al., 2006; Di Pietro et al., 2016; Freitas MV de et al., 2017; Peña et al., 2000). Ocular involvement has also been reported in humans with leishmaniasis (Bouomrani et al., 2011; Ferrari et al., 1990; François et al., 1972; ModarresZadeh et al., 2007; Perrin‐Terrin et al., 2014; Satici et al., 2004).

Due to their diversity and non‐specificity, leishmaniasis is often not evoked when infection causes ocular lesions (Guyonnet et al., 2016; Peña et al., 2000). Moreover, in endemic areas, leishmaniasis is sometimes paucisymptomatic. Therefore, the management of these lesions is frequently delayed, markedly reducing the recovery rate. A plethora of direct and indirect diagnostic tools are available, such as Giemsa‐stained lymph node aspiration smear, detection of Leishmania spp. DNA in different tissue samples (skin, conjunctiva, lymph node, spleen, and bone marrow) including different PCR techniques (conventional PCR, real‐time PCR, loop‐mediated isothermal amplification), detection of specific serum antibodies using indirect enzyme‐linked immunosorbent assay (ELISA) and several rapid lateral flow devices (Gharbi et al., 2015; Lombardo et al., 2012; Solano‐Gallego et al., 2011).

The ocular manifestations are diverse, and most of the ocular tissues can be affected: blepharitis, periocular alopecia, conjunctivitis, keratoconjunctivitis, keratoconjunctivitis sicca (KCS), corneal ulcers, uveitis, orbital cellulitis and myositis of the extraocular muscles (Ciaramella et al., 1997; Molleda et al., 1993; Naranjo et al., 2010; Peña et al., 2000; Peña et al., 2008). Therefore, ocular involvement is a sentinel for leishmaniasis. Its early identification allows for more efficient therapeutic management of both leishmaniasis and ocular involvement, improving the prognosis and reducing the dog's reservoir role.

Accumulating evidence suggests that immune processes play a very important role in the pathogenesis of ocular inflammation (Garcia‐Alonso et al., 1996a; Garcia‐Alonso et al., 1996b). Therefore, the immunology of ocular manifestations in dogs with leishmaniasis remains complex and poorly understood (Garcia‐Alonso et al., 1996a).

Few studies have examined the immunopathology of ocular manifestations in canine leishmaniasis. Intra‐cytoplasmic Leishmania spp. amastigotes in the inflammatory foci of various ocular tissues associated with immune complex deposits have been reported (Brito et al., 2010; Garcia‐Alonso et al., 1996a; Peña et al., 2008). The origin of this immunologically mediated response remains controversial. Some authors defend the hypothesis of production followed by a local deposition of immune complex after penetration of Leishmania spp. into the eye, while others favour the hypothesis that deposition of soluble immune complex from the circulation into the uveal tract plays a key role in the etiopathogenesis of the disease (Roze, 1993). To prove specific in situ or ex situ antibody production, the C value must be calculated (Jongh & Clerc, 1992). Despite its importance, C value was calculated in only two studies (Brito et al., 2006; Roze, 1990). To the best of our knowledge, the present study is the first to investigate a relatively large number (53) of dogs infected by Leishmania infantum with ocular conditions. This study aims to detail the ophthalmological changes in canine leishmaniasis and their correlation with antibody titres in sera and aqueous humour (AH).

2. MATERIAL AND METHODS

2.1. Animals

Dogs presented to the Veterinary Hospital and diagnosed with leishmaniasis by both Giemsa‐stained lymph node smears and enzyme‐linked immunosorbent assay (ELISA) over a 3‐year period (2017–2019) were examined. Only dogs with ocular lesions affecting either the eye or adnexa were included in the present study. Negative dogs for both lymph node smears and ELISA were considered as control dogs, consisting of healthy dogs (with no clinical signs) and dogs with uveitis caused by generalised lymphoma confirmed by biopsy and cytology. The control dogs with lymphoma had generalised lymph node enlargement, fever and/or weight loss. All dogs included in the study were examined clinically. To avoid enrolling dogs with uveitis caused by Ehrlichia canis, all of the dogs were confirmed to be negative for Ehrlichia canis by clinical examination, haematology and PCR. The dogs included in the study did not receive any treatment.

2.2. Clinical examination

Complete ophthalmic examination included the inspection of the eyelids and globe with a focal light source and slit‐lamp biomicroscopy. The crystalline lens, vitreous humour and fundus were assessed using direct monocular ophthalmoscopy and/or indirect binocular ophthalmoscopy. A mydriatic eye drop was used for fundoscopy. Tear production was estimated using the Schirmer tear test I. A dog was considered to have KCS when the absorbance among tear test strips was less than 10 mm at 60 s (Dodi, 2015; Giuliano, 2021; Giuliano & Moore, 2008; Williams, 2005). The presence of corneal lesions and the permeability of the nasolacrimal system were tested using the fluorescein test. Intraocular pressure was estimated using an applanation tonometer. Ocular ultrasonography was performed for all dogs presenting ocular media opacity, precluding a complete ophthalmic examination. Finally, the bulbar surface of the nictitating membrane was inspected with an atraumatic nipper after the instillation of chlorhydrate of oxybuprocaïne. Fundus photography was performed using a non‐mydriatic digital fundus camera. In order to study the symmetry of ocular signs between the two eyes of each dog, a score ranging from 0 to 3 was attributed to each ocular lesion according to its severity where, 0: absence of any lesion, 1: mild lesion, 2: moderate lesion and 3: severe lesion.

2.3. Sample collection and processing

2.3.1. Sera

Five millilitres of blood were collected in sterile dry tubes from the jugular or radial vein of each dog using a vacutainer. The blood samples were centrifuged at 3000 rpm for 15 min. Sera were collected in Eppendorf tubes and stored at ‒20°C until use.

2.3.2. Aqueous humour

Twenty minutes before the administration of both local and short general anaesthesia, one drop of the mydriatic eye drops was applied to each eye. Anaesthesia was induced by intramuscular injection of 4 μg/kg dexmedetomidine, 40 μg/kg acepromazine and 1 mg/kg ketamine. General anaesthesia was then administered via a slow intravenous injection of propofol (5 mg/kg). Local anaesthesia of the eyes was performed by the instillation of chlorhydrate of oxybuprocaïne eye drops 15 min before the above‐mentioned injections.

The AH was sampled from anaesthetised dogs after the rigorous disinfection of the eyes using a 5% solution of iodised povidone. Prior to aqueocentesis, a Barraquer eyelid speculum was placed, and limbal entry was achieved with a 29G insulin needle mounted on a 1‐ml syringe, stabilising the adjacent bulbar conjunctiva with Weiss Castroviejo fixation forceps. The volume of AH aspirated was approximately 0.3 ml per eye, which was immediately transferred to a sterile dry identified tube and placed at ‒20°C until use. Aqueous humour samples were collected from both eyes of all dogs, including those with no ocular lesions.

2.4. Serology

2.4.1. Anti‐Leishmania infantum IgG titration in sera and aqueous humour

Quantification of anti‐Leishmania infantum IgG in sera and AH was performed with an ELISA commercial kit (ID Screen® Leishmaniasis Indirect Test, Innovative diagnostics, Grabels, France) according to the manufacturer's instructions. No commercially available ELISA test is standardised for testing ocular fluid samples (Garweg et al., 2011). Thereby, the aqueous humour is considered as sera. In each ELISA plate well, 10 μl of AH was diluted at 1:20 in kit buffer, and 10 μl of sera was diluted to 1/10. The absorbance was measured using a spectrophotometer at a wavelength of 450 nm. The absorbance of each sample tested (sera and AH) was converted into a percentage based on the following formula: 100 × (sample absorbance – negative control absorbance)/(positive control absorbance − negative control absorbance). Negative and positive controls provided with the kit were added to each plate.

2.4.2. Titration of the total IgG in sera and aqueous humour

The quantification of the total IgG in sera and AH was performed using an ELISA commercial kit (Dog IgG ELISA Kit® Immunology Consultants Laboratory, Portland, USA) according to the manufacturer's recommendations. Each plate contained six standards in duplicate at concentrations ranging from 25 to 800 ng/ml. In each plate, 100 μl of each diluted standard and 100 μl of each sample (either sera or AH) at a dilution of 1:100,000 in the kit buffer were added to each well. Finally, the absorbance was measured at 450 nm using an automatic micro‐ELISA reader. Titration was performed using a four‐parameter logistic curve from standard data points that were plotted and logistically fitted with GraphPad Prism (GraphPad Prism 7.04). The concentrations of different samples were estimated using this curve. Interpolation from the generated standard curve was performed using the GraphPad Prism statistical software. Plates in which the R2 value of the standard curve was below 0.98 were run again.

2.4.3. Goldmann–Witmer coefficient calculation (C value)

The IgG Leishmania infantum Goldmann–Witmer coefficient (C value) was used to confirm or rule out the presence of local intraocular anti‐Leishmania infantum antibody production in the studied dogs. This was estimated as follows: (Lappin et al., 2000)

C value = (anti‐Leishmania spp. IgG % ELISA in AH /anti‐Leishmania spp. IgG % ELISA in sera) × (total IgG in sera/total IgG in AH)

According to Brito et al. (2006), Hill et al. (1995) and Pickett (2019), if the C value is higher than 1, anti‐Leishmania spp. antibodies are produced in the eyes, whereas, if the C value is below 1, this is evidence for leakage of serum antibody into the AH from the breakdown of the blood‒ocular barrier due to inflammation.

2.5. Dog groups

To study the relationship between the level of specific anti‐Leishmania infantum antibodies in AH and ocular manifestations, the dogs were divided into six groups according to the ocular structure affected:

  1. Dogs with lesions affecting only the adnexa of the eye (conjunctivitis and blepharitis)

  2. Dogs with lesions affecting the cornea (isolated keratitis, keratoconjunctivitis, KCS, ulcerative keratitis and pigmentary keratitis)

  3. Dogs with anterior uveitis

  4. Dogs with lesions affecting the ocular fundus (chorioretinitis, retinal haemorrhage and retinal atrophy)

  5. Dogs with cataracts

  6. Control dogs

2.6. Statistical analysis

To compare different distributions and mean IgG levels in AH and sera, the chi‐square test and Student's t‐test were performed with a threshold of 0.05. A Pearson's test was used to estimate the correlation between IgG concentration in sera and AH. Analysis of variance was carried out to test the relationship between clinical signs, presence of anti‐Leishmania infantum antibodies, and C value.

3. RESULTS

A total of 53 infected dogs with Leishmania infantum presenting ocular or periocular lesions were included in the present study, consisting of 38 (71.7%) males and 15 (28.3%) females (sex ratio M:F = 2.5) aged between 1 and 10 years (mean age: 4 years). The majority of dogs (47/53; 88.7%) were of large breeds weighing more than 20 kg, and only 6 (11.3%) dogs were of mixed breed. Ten dogs that were negative for both lymph node smears and ELISA were included as control dogs, consisting of 5 healthy dogs and 5 dogs with uveitis caused by generalised lymphoma.

3.1. Ophthalmological findings

There were 51/53 dogs with bilateral lesions (96.2%) (p < 0.05) and 38/53 dogs with symmetric ones (71.7%) (p < 0.05) (Table 1).

TABLE 1.

Distribution of ocular and periocular lesions, number and percentage of bilaterality and symmetry in dogs with leishmaniasis (n = 53)

Number of affected eyes Bilaterality Symmetry Number of affected dogs
Ocular sign Right eye Left eye Total (%) Number of bilateral lesions (%) p Number of symmetric lesions (%) p Total (%)
Keratoconjunctivitis 38 37 75 /106 (70.7) 37 (97.4) 2.746 25 (65.8) 0.163 38/53 (71.7)
 Chemosis 27 27 54/106 (50.9) 26 (92.8) 0.0003* 25 (89.3) 0.001* 28/53 (52.8)
 Hyperhaemia 37 35 72/106 (67.9) 34 (89.5) 0.0001* 30 (78.9) 0.008* 38/53 (71.7)
 Purulent exudate 28 26 54/106 (50.9) 26 (92.8) 0.0003* 24 (85.7) 0.004* 28/53 (52.8)
 Corneal lesion 37 32 69/106 (65.1) 31 (81.6) 0.003* 26 (68.4) 0.102 38/53 (71.7)
 Keratoconjunctivitis sicca 17 18 35/106 (33) 14 (66.7) 0.278 10 (47.6) 0.878 21/53 (39.6)
 Pigmentary keratitis 5 3 8/106 (7.5) 3 (60) NA 3 (60) NA 5/53 (9.4)
Isolated conjunctivitis 9 9 18/106 (17) 9 (100) NA 9 (100) NA 9/53 (17)
Isolated keratitis 2 2 4/106 (3.8) 2 (100) NA 2 (100) NA 2/53 (3.8)
Corneal ulcers 15 11 26/106 (24.5) 10 (62.5) 0.476 9 (56.2) 0.723 16/53 (30.2)
Hyperplasia of lymphoid follicles 29 28 57/106 (53.8) 28 (96.5) 6.605 24 (82.7) 0.008* 29/53 (54.7)
Blepharitis 27 27 54/106 (50.9) 27 (100) 2.343 25 (92.6) 0.0005* 27/53 (50.9)
 With periocular alopecia 6 6 12/106 (11.3) 6 (100) NA 6 (100) NA 6/53 (11.3)
 Without periocular alopecia 21 21 42/106 (39.6) 21 (100) NA 19 (90.5) NA 21/53 (39.6)
Anterior uveitis 11 9 20/106 (18.9) 9 (81.8) NA 3 (27.2) NA 11/53 (20.7)
 Corneal oedema 11 9 20/106 (18.9) 9 (81.8) NA 4 (36.4) NA 11/53 (20.7)
 Perilimbic neovascularisation 11 9 20/106 (18.9) 9 (81.8) NA 4 (36.4) NA 11/53 (20.7)
 Miosis 7 7 14/106 (13.2) 6 (75) NA 6 (75) NA 8/53 (15.1)
 Hypopion 7 6 13/106 (12.3) 5 (62.5) NA 3 (37.5) NA 8/53 (15.1)
 Hyphema 2 0 2/106 (1.9) 0 (0) NA 0 (0) NA 2/53 (3.8)
 Low intraocular pressure 7 5 12/106 (11.3) 5 (71.4) NA 0 (0) NA 7/53 (13.2)
Fundus lesions 5 4 9/106 (8.5) 3 (50) NA 2 (33.3) NA 6/53 (11.2)
 Chorioretinis + retinal atrophy 3 2 5/106 (4.7) 2 (66.7) NA 2 (66.7) NA 3/53 (5.7)
 Retinal haemorrhages 2 2 4/106 (3.8) 1 (33.3) NA 0 (0) NA 3/53 (5.7)
Glaucoma 2 0 2/106 (1.9) 0 (0) NA 0 (0) NA 2/53 (3.8)
Cataracts 2 2 4/106 (3.8) 2 (100) NA 2 (100) NA 2/53 (3.8)
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NA, not applicable.

*

Statistically significant according to the chi‐square test (p < 0.05).

Keratoconjunctival involvement (75/106 eyes; 70.7%) was present in 71.7% (38/53) of dogs with 97.4% (37/38) of bilaterality, including chemosis (54 eyes from 28 dogs), hyperaemia (72 eyes from 38 dogs), and purulent exudate (54 eyes from 28 dogs) associated with corneal lesions (69 eyes from 38 dogs). Thirty‐five eyes out of 106 (33%) from 21 dogs out of 53 (39.6%), developed KCS which was bilateral in 66.7% of dogs (14/21). Eight eyes (7.5%) from 5 dogs (9.4%) with keratoconjunctivitis presented pigmentary keratitis due to deposition of melanin on the cornea. One of these five dogs suffered from KCS, and the other four dogs had an associated hyperplasia of lymphoid follicles. Moreover, 26 eyes (24.5%) from 16 dogs (30.2%) developed corneal ulcers (Figure 1), as confirmed by the fluorescein test. We observed abnormalities only in the conjunctiva and the cornea (without intraocular signs) in 18 eyes (17%) from 9 dogs (17%) and in 4 eyes (3.8%) from 2 dogs (3.8%), respectively.

FIGURE 1.

FIGURE 1

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Hyperplasia of lymphoid follicles of the nictitating membrane characterised by numerous small ovoid lesions located in the conjunctiva of dog with leishmaniasis. A deep corneal ulcer is present

More than half (29/53; 54.7%) of the dogs showed hyperplasia of lymphoid follicles of the nictitating membrane (57/106 eyes; 53.8%) consisting of numerous small, ovoid, red, irregular structures located under the conjunctiva of the bulbar side (Figure 1). In severe cases, these follicles were apparent on the palpebral surface of the nictitating membrane. This lesion was bilateral in 28/29 dogs (96.5%).

Eyelid involvement was seen in 54 eyes (50.9%) from 27 dogs (50.9%), characterised by the presence of blepharitis with or without periocular alopecia in 11.3% (12 eyes from 6 dogs) and 39.6% (42 eyes from 21 dogs), respectively. All dogs (54 eyes from 27 dogs) presenting blepharitis had bilateral inflammation.

The vascular tunic of the eye was less commonly affected. Anterior uveitis was observed in 20 eyes (18.9%) from 11 dogs (20.7%), being bilateral in 9 of these dogs (81.8%). The uveitis was acute and fibrinous, characterised by the presence of generalised corneal oedema (20 eyes from 11 dogs) (Figure 2a), deep perilimbic neovascularisation (20 eyes from 11 dogs), iris oedema and miosis (14 eyes from 8 dogs). Exudation phenomena, such as the presence of hypopion (13 eyes from 8 dogs) (Figure 2a) or hyphema (2 eyes from 2 dogs) (Figure 2b) in the anterior chamber, and a decrease of the intraocular pressure (12 eyes from 7 dogs), were often observed.

FIGURE 2.

FIGURE 2

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Acute and fibrinous anterior uveitis in a dog naturally infected by Leishmania infantum characterised by hypopyon (a) or hyphema (b)

In the present study, 9 eyes (8.5%) from 6 dogs (11.3%) developed lesions of the fundus of which 3 were bilateral (50%), that may be indicative of sequelae of posterior uveitis. These lesions were mainly inactive chorioretinitis with black areas in tapetal fundus surrounded by small hyperreflective lesions associated with retinal atrophy at different stages in 5 eyes (4.7%) from 3 dogs (5.7%) (Figure 3) and retinal haemorrhages in 4 eyes (3.8%) from 3 dogs (5.7%).

FIGURE 3.

FIGURE 3

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Retinal atrophy in a dog naturally infected by Leishmania infantum. Note the thinning of retinal vasculature and the inactive chorioretinitis with black areas in the tapetal fundus surrounded by hyperreflective lesions

Two eyes (1.9%) from 2 dogs (3.8%) with hypertensive uveitis (Figure 4) were observed in the present study, which rapidly progressed to secondary glaucoma. Of all the observed dogs, only 4 eyes (3.8%) from 2 dogs (3.8%) showed immature cataracts.

FIGURE 4.

FIGURE 4

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Hypertensive uveitis which has progressed to secondary glaucoma in a dog with leishmaniasis. Note the increased diameter of the eyeball, the severe diffuse corneal oedema and the corneal and episcleral neovascularisation

3.2. Results of immunological study

Leishmania infantum IgG ELISA was positive in most of the AH samples (65/106 eyes; 61.3%) from infected dogs (39/53 dogs; 73.6%). Specific anti‐Leishmania infantum antibody titres in the AH of dogs with leishmaniasis were significantly (sixfold) higher than those in the AH of the two control groups (32.95 ± 36.16 vs. 5.05 ± 4.96, p = 0.012).

In the right eyes, the anti‐Leishmania infantum IgG concentration in the AH was significantly lower than that in sera (30.86 ± 32.04 vs. 158 ± 59.67; p < 0.0001). Similarly, in the left eyes, the anti‐Leishmania infantum IgG concentration in AH was significantly lower than that in sera (29.62 ± 32.11 vs. 158 ± 59.67; p < 0.0001). There was no correlation between anti‐Leishmania infantum IgG levels in AH for either right (r = 0.2069; p = 0.137) or left (r = 0.1929; p = 0.166) eyes and in sera. This means that the level of anti‐Leishmania infantum IgG in AH varies independently of the level of anti‐Leishmania infantum IgG in sera.

The mean value of the total IgG concentration in AH of the right (54.48 ± 56.52) and left (85.79 ± 103.56) eyes was significantly lower than in sera (1107 ± 432.64) (p < 0.0001). There was no correlation between the total IgG levels in AH for either right (r = 0.052; p = 0.70) or left (r = ‒0.069; p = 0.61) eyes and in sera. The levels of total IgG in AH and sera showed that their variation was independent of each other.

There was no difference between the anti‐Leishmania infantum IgG mean value in the AH of the right (30.86 ± 32.04) and left (29.62 ± 32.11) (p = 0.676) eyes but there was a significant positive correlation between the anti‐Leishmania infantum IgG levels in the AH of the right and left eyes (r = 0.87; p < 0.001). No difference was observed between the mean value of the total IgG concentration in the AH of the right (54.48 ± 56.52) and left (85.79 ± 103.56) (p = 0.162) eyes. There was a significant positive correlation (r = 0.455; p < 0.001) between the total IgG levels in the right and left eyes. This indicates that specific anti‐Leishmania infantum IgG antibodies and total IgG vary interdependently in both eyes.

There was a significant association between anti‐Leishmania infantum IgG concentration in AH and ocular lesions in either the right or left eyes (p < 0.0001). The mean value of anti‐Leishmania infantum IgG antibodies in the AH of dogs with leishmaniasis was higher in dogs with uveitis followed by dogs with other ocular and periocular lesions, generalised lymphoma, and healthy dogs (p < 0.0001). Dogs with lesions affecting only the adnexa of the eye showed the second highest (p < 0.0001) mean level of anti‐Leishmania infantum IgG concentration in AH compared to other groups (Figure 5). However, no difference was observed among dogs with cataract or fundic lesions. There was no significant association between anti‐Leishmania infantum IgG concentration in sera and ocular lesions in either the right (p = 0.376) or left (p = 0.064) eyes.

FIGURE 5.

FIGURE 5

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Variation of anti‐Leishmania infantum IgG titres in aqueous humour according to the location of the ocular lesion in dogs with leishmaniasis (n = 53) and in control dogs (n = 10)

C values higher than 1 were obtained in 26/106 eyes (24.5%) from 15/53 dogs (28.3%) with leishmaniasis, including 13 right and 13 left eyes (Table 2). The highest proportion of dogs with a Goldmann–Witmer coefficient (C value) higher than 1 was seen in the group of dogs with anterior uveitis (17/26 eyes from 10/15 dogs) (Figure 6). C values higher than 1 were detected in 17 eyes (17/22; 77.3%) from 11 dogs with anterior uveitis, including 8 right and 9 left eyes. The C value in these dogs ranged between 1.18 and 14.44. Six eyes (6/18; 33.3%) from 9 dogs with conjunctivitis as the sole manifestation had very high C values that ranged between 2.82 and 21.7. Similarly, 3 eyes (3/48; 6.2%) from 24 dogs that presented corneal lesions associated with or without conjunctivitis but without macroscopic uveal inflammation had high C values that varied between 2.94 and 9.28 (Table 2; Figure 6). C values higher than 1 were not detected in any dogs with lesions affecting the ocular fundus, with cataracts, or in the control dogs. A significant association was observed between the C value and the different types of ocular lesion in both the right (p = 0.026) and left (p = 0.020) eyes.

TABLE 2.

The Goldmann–Witmer coefficient (C value) in eyes of dogs with leishmaniasis groups (n = 53) and control dogs (n = 10)

Dog groups Ocular sign Number of dogs Number of eyes with C value >1/total number of eyes
Group 1: Adnexa lesions Conjunctivitis 9 6 /18
Blepharitis 1 0/2
Group 2: Corneal lesions Keratitis 2 3/4
Keratoconjunctivitis 22 0/44
Group 3: Anterior uveitis Anterior uveitis 11 17/22
Group 4: Fundus lesions Chorioretinitis + retinal atrophy 3 0/6
Retinal haemorrhage 3 0/6
Group 5: Cataracts Cataract 2 0/4
Group 6: Control dogs Neoplastic uveitis 5 0/10
No lesion 5 0/10
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FIGURE 6.

FIGURE 6

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Goldmann–Witmer coefficient (C value) according to ocular lesions in dogs with a C value higher than 1 (n = 15). In red threshold value of Goldmann–Witmer coefficient = 1

4. DISCUSSION

In this study, the main lesion observed in the eyes (75/106 eyes; 70.7%) of dogs infected with leishmaniasis was keratoconjunctivitis. Similar lesions have been observed in previous clinical studies, with prevalence ranging from 20% to 66.6% (Peña et al., 2000; Brito et al., 2006; Ciaramella et al., 1997; Naranjo et al., 2012). Among those dogs with keratoconjunctivitis, 35 eyes out of 106 (33%) from 21 dogs out of 53 (39.6%), developed KCS. This occurrence is higher than that reported in previous studies (Peña et al., 2000; Ciaramella et al., 1997; Freitas MV de et al., 2017; Molleda et al., 1993; Komnenou & Koutinas, 2007; Koutinas et al., 1999). The mechanism of KCS in dogs with leishmaniasis is not well understood. Some studies have shown that the decrease in tear production is due to secretory retention caused by inflammatory infiltration around the lacrimal gland ducts (Molleda et al., 1993; Baneth et al., 2008; Naranjo et al., 2005). Other authors have suggested that the deficit of lacrimal secretion is due to the direct mechanical action of parasites in the lacrimal apparatus or to damage induced by amastigotes and/or specific anti‐Leishmania spp. IgG (Ciaramella et al., 1997; Garcia‐Alonso et al., 1996a; Roze, 1986). Keratoconjunctivitis sicca can also be explained by corneal hypoesthesia due to corneal lesions (Naranjo et al., 2005; Roze, 1986; Mathers, 2000). Naranjo et al. showed that meibomian glands represent the glands most frequently affected by inflammatory infiltration in dogs with leishmaniasis. When these glands are damaged, the tear film becomes unstable, inducing the early evaporation of tears, explaining the observation of qualitative KCS with normal Schirmer test values (Naranjo et al., 2005).

The conjunctiva alone was affected in 17% of dogs (17% of eyes). Different authors have reported a prevalence of isolated conjunctivitis varying between 8% and 34.1% (Peña et al., 2000; Brito et al., 2006; Molleda et al., 1993). Peña et al. (2000) suggested that conjunctival inflammation is due to the presence of the parasite, rather than an altered immune response following infection. However, Brito et al. (2010) hypothesised that Leishmania spp. conjunctivitis is due to the presence of immune complex, which would trigger perivasculitis accompanied or not by vascular congestion.

We reported here, for the first time, a follicular conjunctivitis, which was the second main lesion observed in the present study (53.8% of the examined eyes). Conjunctiva and lymphoid tissue can react to immune‐mediated diseases, including leishmaniasis. Peña et al. (2000) reported the presence of multifocal discrete white nodules in the conjunctiva of dogs with leishmaniasis. Histological studies have shown that the conjunctiva of the nictitating membrane was the most affected tissue by the inflammatory infiltrate and also revealed the greatest degree of parasitism among different ocular and periocular structures (Molleda et al., 1993; Brito et al., 2010). A significant correlation between inflammation and the presence of the parasite in this structure has been reported previously (Brito et al., 2010). The inflammation of the nictitating membrane conjunctiva is characterised by the dominance of lymphoplasmacytic cells and enlarged lymphoid nodules corresponding to what Roze terms “leishmanioma” (Roze, 1988). In another study, granulomatous infiltrate was observed in sections of the nictitating membrane in the subepithelial region of the bulbar surface, surrounding lymphoid follicles and gland secretory ducts (Naranjo et al., 2005).

Isolated keratitis was observed in 3.8% of eyes. Although rare, these lesions have been reported by other authors (Peña et al., 2000; Brito et al., 2006; Molleda et al., 1993). In the present study, 24.5% of the eyes developed corneal ulcers at different depths. According to Molleda et al. (1993) and Brito et al. (2007), these lesions are unusual in canine leishmaniasis. These ulcers can be secondary to inflammation induced by Leishmania spp. in the cornea, tear deficiency when KCS is installed, or rubbing of the cornea when hyperplasic lymphoid follicles develop (Brito et al., 2007; Molleda et al., 1993).

Five dogs presented pigmentary keratitis. Pigmentary keratitis can be a consequence of KCS in dogs with leishmaniasis (Brito et al., 2006; Freitas MV de et al., 2017; Naranjo et al., 2012). In the present study, one of the five dogs with pigmentary keratitis had a quantitative tear deficiency. In the other four dogs, pigmented keratitis may be related to several causes including eyelid conformation, immune‐mediated keratitis, trauma, tear film disorders and mechanical abrasion (Labelle et al., 2013). Hyperplasic lymphoid follicles can cause constant irritation of the cornea that induces inflammation and keratinisation of the stratified squamous epithelium and, consequently, the deposition of pigmented cells on the surface of the cornea (Anoop et al., 2016).

In this study, over half (50.9%) of the eyes developed blepharitis associated or not with periocular alopecia and desquamation. Palpebral involvement has frequently been classified as a dermatological or non‐ophthalmological lesion (Peña et al., 2000; Ciaramella et al., 1997). These lesions can be attributed to the mechanical action of parasites that cause blood vessel inflammation and/or ischaemic and necrotising vasculitis caused by immunopathogenic mechanisms, such as the deposition of immune complex in vessels (Kaszak et al., 2015; Pumarola et al., 1991).

Anterior uveitis was observed in 18.9% of the examined eyes. This is similar to the results of several other studies (Ciaramella et al., 1997; Molleda et al., 1993; Peña et al., 2008; Naranjo et al., 2005; Amara et al., 2003; Slappendel, 1988). This lesion is due to either type III hypersensitivity or post‐therapeutic complications (Peña et al., 2000; Di Pietro et al., 2016; Garcia‐Alonso et al., 1996a). The latter cause was confirmed by Di Pietro et al. (2016), who observed that the occurrence of uveitis was significantly higher in treated dogs than in non‐treated shelter dogs (37.5% and 9.4%, respectively). This could explain the relatively low occurrence of uveitis in the examined dogs compared to other studies since none of them were treated (Peña et al., 2000; Brito et al., 2006; Freitas MV de et al., 2017). The post‐therapeutic uveal immune reaction, already observed in humans, is often associated with an allergic mechanism resulting from the destruction of Leishmania spp. in the ocular tissues of treated dogs (Amara et al., 2003; El Hassan et al., 1998). The study carried out by Garcia‐Alonso et al. suggests that the development of ophthalmic lesions is due to the direct action of intra‐cytoplasmic amastigotes in the inflammatory foci associated with type III hypersensitivity triggered by Leishmania spp. antigens and by specific and non‐specific immunoglobulin deposits (Molleda et al., 1993; Garcia‐Alonso et al., 1996a; Brito et al., 2010). Another hypothesis involves local immune production and the deposition of a specific immunoglobulin in the anterior uvea (Molleda et al., 1993). These hypotheses show that the immunology of canine leishmaniasis is complex and remains poorly understood. Two clinical forms have been previously reported for Leishmania spp. uveitis, the acute and fibrinous form, that was seen in the current study in 20 eyes, and the granulomatous form characterised by the development of multifocal nodules in the iris stroma (Peña et al., 2000; Peña et al., 2008). No granulomatous forms were observed in the current study. According to Peña et al., the nodular form often develops after the initiation of antiprotozoal therapy (Peña et al., 2000).

In this study, only six dogs showed lesions of the ocular fundus. This is in agreement with other studies (Peña et al., 2000; Freitas MV de et al., 2017; Molleda et al., 1993; Peña et al., 2008). This type of lesion could be underestimated due to an insufficient pupil response to mydriatic and deficient intraocular media transparency, making the ocular fundus ophthalmoscopically inaccessible. In addition, the absence of clinical signs, such as pain and redness, does not motivate dog owners to seek a veterinary consultation. Therefore, in isolated posterior uveitis, dogs are often diagnosed at late stages of the disease.

Posterior segment lesions remain infrequent in canine leishmaniasis and could be due to the expansion of the inflammatory process from the anterior segment, wherein chorioretinitis and retinal detachment are the most frequently observed lesions (Peña et al., 2000; Freitas MV de et al., 2017; Molleda et al., 1993; Peña et al., 2008; Brito et al., 2010; Collins & Moore, 1991). The fundus lesions observed in the present study were retinal haemorrhages or lesions of inactive chorioretinitis associated with retinal atrophy at different stages. Retinal atrophy can occur sporadically in dogs with leishmaniasis, most likely due to the deposition of immune complex (Molleda et al., 1993; Peña et al., 2008; Brito et al., 2010). Roze reported that vasodilation mostly affects the veins of the fundus as a constant finding in leishmaniasis (Molleda et al., 1993; Roze, 1986).

As reported by Freitas et al., cataracts were only observed in two dogs. Although no established relation was found between the development of cataracts and leishmaniasis, the lens may be affected and opacified by changes of the AH observed in uveitis (Freitas MV de et al., 2017; Molleda et al., 1993).

One of the complications of chronic uveitis is glaucoma. The iridocorneal angle may be plugged with inflammatory cells, fibrin, blood and large molecular proteins. Anterior synechiae may also occlude the filtration angle and the ciliary cleft, leading to an increase in intraocular pressure and the subsequent appearance of hypertensive uveitis or glaucoma (Peña et al., 2000; Freitas MV de et al., 2017; Guyonnet et al., 2016; Peña et al., 2008; Plummer et al., 2021). Two dogs with hypertensive uveitis were observed in the present study, which rapidly progressed to secondary glaucoma. These results are in agreement with those of previous clinical studies, wherein only one dog with glaucoma was observed in each study (Freitas MV de et al., 2017; Peña et al., 2008).

Anti‐Leishmania infantum IgG values of the AH were significantly (sixfold) higher in dogs with Leishmania infantum than in the two groups of control dogs. Previous studies have shown that compared to healthy dogs, the total protein values in the AH of dogs with leishmaniasis are sevenfold higher (Brito et al., 2006; Novales et al., 1994). In our study, no significant correlations were observed between the concentrations of specific and total IgG in AH and sera. Their presence in AH was independent of their presence in sera, which is in agreement with previous studies (Brito et al., 2006; Novales et al., 1994; Lopez et al., 1993). Anti‐Leishmania spp. antibodies present in AH were not due, at least in their entirety, to a transfer of antibodies from the blood stream to the AH, as has been reported by Garcia‐Alonso et al. (Garcia‐Alonso et al., 1996b).

Furthermore, in the present study, no statistically significant association was found between the anti‐Leishmania infantum IgG levels in sera and ocular lesions. Whereas, a statistically significant association was observed between the level of anti‐Leishmania infantum IgG in AH and ocular manifestations, which is in agreement with the findings reported by Brito et al. (2006) and Lopez et al. (1993). These results indicate that ocular involvement is not due to specific antibodies from the sera, but rather due to specific antibodies found in the AH, providing support for the hypothesis of a local production of specific IgG.

In the present study, the highest values of anti‐Leishmania infantum IgG in AH was in dogs with anterior uveitis. Several studies conducted in dogs with leishmaniasis have reported positive correlations between uveitis and increased concentrations of total protein in AH, between uveitis and hypergammaglobulinaemia in AH, and between uveitis and titre of anti‐Leishmania infantum IgG (Garcia‐Alonso et al., 1996a; Roze, 1990; Novales et al., 1994).

The mean anti‐Leishmania infantum IgG titres in AH of dogs with leishmaniasis having ocular lesions was significantly higher than those in the healthy control group, which is in agreement with the findings reported by Brito et al. (2006). Novales et al. found that the mean protein values of the AH of healthy dogs and dogs with leishmaniasis were not significantly different (Novales et al., 1994). In addition, the statistically significant difference observed in this study between the mean anti‐Leishmania infantum IgG titres in the AH of dogs with leishmanian uveitis and dogs with non‐leishmanian uveitis (lymphoma‐associated uveitis) proved the specificity of leishmanian uveitis.

The increase of IgG concentration in AH, independently of their concentration in sera, is due to the presence of a blood–ocular barrier. In fact, the blood–aqueous barrier and blood–retinal barrier isolate AH from the peripheral blood (Katamay & Nussenblatt, 2013). In addition, AH flows continuously and unidirectionally from the posterior chamber into the anterior chamber, reducing the exchange between the blood and AH (Jongh & Clerc, 1992; Esterre et al., 1996; Hendrix et al., 2021). Furthermore, the lens capsule, Descemet's membrane and corneal stroma constitute thick anatomical barriers (Jongh & Clerc, 1992). These barriers allow for the entrance of foreign antigens into the eye but preclude their immune elimination, favouring the local immune response to the detriment of the acquisition of systemic immunological tolerance (Krawiecki, 1994; Mair & Crispin, 1989).

Until now, the eyes were considered to be an immunologically privileged site, anatomically separated from most of the immune system because of the absence of lymphatic drainage (except conjunctiva and eyelids) (Jongh & Clerc, 1992; Esterre et al., 1996; Grahn & Peiffer, 2021; Matthews, 1999). Recent studies indicate that lymphatic channels may be present in the anterior uveal tract of the dog eye (Dubin et al., 2021). Likewise, the uvea is an intensively vascularised network and, as a result, is highly susceptible to inflammatory and immune reactions. As such, both uvea and conjunctiva may play the role of an accessory lymph node for the eye (Jongh & Clerc, 1992; Romeike et al., 1998). The presence of an antigen in the eyes is followed by its passage into the bloodstream instead of its presentation to the satellite lymph node. Therefore, the primary immune response takes place at distant sites from the eye (Jongh & Clerc, 1992; Esterre et al., 1996). Subsequently, activated B and T Leishmania spp.‐sensitised lymphocytes migrate to the eye and concentrate in the limbus and uvea, forming immunocompetent sites similar to those found in a lymph node, which are responsible for the production of specific antibodies and cytotoxic cells (Jongh & Clerc, 1992; Lappin et al., 2000). Despite the elimination of the antigen, lymphoid cells that are sequestered within the uveal tract induce a relapse of inflammation following contact with the same or a similar antigen (Roze, 1993; Jongh & Clerc, 1992; Romeike et al., 1998). This may explain the local production of specific IgG in AH, as shown in this study. This observation was confirmed by the abundant presence of plasma cells with Russell bodies, found in ocular histological sections of dogs with leishmaniasis (Novales, 1991).

Few studies have investigated the immunopathology of ocular manifestations in canine leishmaniasis. They have suggested a production and a local deposition of immune complex after the penetration of Leishmania spp. into the eye, followed by a deposition of soluble immune complex from the circulation in the uveal tract (Peña et al., 2008; Garcia‐Alonso et al., 1996a; Brito et al., 2010; Roze, 1993). Garcia‐Alonso et al. (1996b) indicated that anti‐Leishmania infantum IgG originates from the general circulation after the breakdown of the blood‒aqueous barrier, without excluding the possibility of local immunoglobulin synthesis. Lopez et al. reported on the local production of IgG, independent of plasma production, particularly during uveitis (Lopez et al., 1993). However, to prove specific in situ antibody production, the C value must be calculated (Jongh & Clerc, 1992). A C value higher than 1 suggests that the specific antibody detected in AH is present at a concentration too high to be explained by serum leakage into the eye (Lappin et al., 2000). Only two studies have previously calculated this value, namely Roze, who found a C value higher than 1 in only 4.76% of dogs (1/21), which could not highlight local antibody production, and Brito et al., who found a C value higher than 1 in 56% of dogs (14/25) and subsequently demonstrated the local synthesis of specific IgG in dogs with leishmaniasis with ocular manifestations (Brito et al., 2006; Roze, 1990).

In the present study, the C value was higher than 1 in 28.3% (15/53) of the dogs. A significant increase in the C values was observed in dogs with leishmaniasis having uveitis, conjunctivitis and keratitis. However, no C values greater than 1 were estimated in dogs with cataracts or involving the ocular fundus. In their study, Brito et al. (2006) reported that the mean total protein concentration of the AH in dogs infected with Leishmania chagasi and presenting uveitis was higher than that in dogs presenting other ophthalmic manifestations, such as conjunctivitis, KCS, keratitis and ulcerative keratitis. Thereby, the high C value in uveitis, found in the current study, was expected as reported by Brito et al. (2006), but the high C value observed in dogs with only conjunctivitis and/or keratitis was unexpected. This could be explained by the fact that intraocular involvement may not be detected during ophthalmologic examination, especially during early stages of ocular leishmaniasis (Peña et al., 2000; Molleda et al., 1993). In general, in the early stages of uveal inflammation, uvea, which represents the primary centre of lymphoid activity in the eye, produces locally specific antibodies in large quantities, which will result in a very high concentration in AH (Roze, 1993). Subsequently, these antibodies combine with the antigens found in situ to form immune complexes. These immune complexes are responsible for the onset and maintenance of uveal inflammation, and consequently for the tissue disturbance of the various ocular structures which became clinically visible. Uveal inflammation can breakdown the blood‒ocular barriers, allowing immunoglobulins to diffuse passively from sera, masking the local production in ocular compartments (Roze, 1993; Jongh & Clerc, 1992; Garweg et al., 2011; Villard et al., 2016). Furthermore, inflammation induced a significant degradation of macromolecules, which masks the local production of antibodies (Roze, 1990). This may explain the lower C value observed during uveitis compared to that found in conjunctivitis. The presence of immune complex can also decrease the antibody concentration in AH, modifying the C value (Lopez et al., 1993).

In the current study, ocular lesions were bilateral in 96.2% of dogs, which is in agreement with previous descriptions and can be explained by the systemic nature of leishmaniasis and the migration of parasitised macrophages (Peña et al., 2000; Brito et al., 2006; Freitas MV de et al., 2017; Molleda et al., 1993). This observation was supported by a significant positive correlation between the levels of anti‐Leishmania infantum antibodies in the AH of the right and left eyes reported in this study. Nevertheless, it has been reported that during the early stages of the disease, unilateral eye damage can be observed (Brito et al., 2006). However, the latter often evolves with chronicity in bilateral and symmetrical lesions (Brito et al., 2006; Freitas MV de et al., 2017).

5. CONCLUSION

Ocular and periocular involvement in dogs naturally infected by L. infantum are common. Keratoconjunctivitis, follicular hyperplasia and blepharitis were found to be the most frequent lesions reported herein followed by uveitis. Serological analyses revealed the local production of anti‐Leishmania antibodies and the presence of a significant association between the production level of these antibodies and ocular clinical signs. Consequently, it suggest that the humoral immune response is of importance in the pathogenesis of ocular leishmaniasis. This in situ production of antibodies was observed in dogs with uveitis, but also with conjunctivitis, and keratitis. Developed in paucisymptomatic dogs infected by Leishmania infantum, these ocular lesions can be confused with other diseases by veterinarians. Dogs from endemic areas of leishmaniasis with even minor ocular conditions should be deeply examined and screened for Leishmnia infantum infection.

AUTHOR CONTRIBUTIONS

El Goulli Amel Founa: Conceptualization, data curation, formal analysis, investigation, methodology, writing original draft, writing review and editing. Lilia Zribi: investigation; methodology; resources; validation; visualisation; writing – review & editing. Rania Sanhaji: investigation. Ahmed Chabchoub: writing – review & editing. Bouratbine Aida: methodology, resources, validation & visialization. Mohamed Gharbi: formal analysis; funding acquisition; supervision; validation; writing – review & editing. Hafedh Abdelmelek: supervision; writing – review & editing.

CONFLICT OF INTEREST

The authors declare that there are no conflict of interest.

ETHICAL STATEMENT

The whole study was performed in accordance to the Tunisian code of practice for the care and use of animals for scientific purposes. The Tunisian Association of Laboratory Animals Science (ATSAL) approved the study's protocol (No. 0117 ATSAL).

PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.982.

ACKNOWLEDGEMENTS

The authors wish to thank Pr Médiha Khamassi Khbou (National School of Veterinary Medicine, Tunisia) for her invaluable help in the statistical study and Pr Sabine Chahory (National Veterinary School of Alfort, France) for her useful and constructive recommendations. We also pay tribute to the memory of Professor Abderrazek Ghorbel who initiated this work without being able to finish it.

El Goulli, A. F. , Zribi, L. , Sanhaji, R. , Chabchoub, A. , Bouratbine, A. , Gharbi, M. , & Abdelmelek, H. (2023). Study of ocular manifestations and humoral immune response in eyes of dogs with leishmaniasis. Veterinary Medicine and Science, 9, 625–637. 10.1002/vms3.982

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request. cd_value_code=text

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. cd_value_code=text

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