Veterinary Ophthalmology (2009) 12, 2, 97 101

Blackwell Publishing Inc

CASE REPORT

Isolated lepromatous conjunctivo-corneal granuloma in a cat

from Italy
Barbara Lamagna,* Orlando Paciello, Manuela Ragozzino,* Serenella Papparella, Serena Montagnaro
and Francesco Lamagna*
*Department of Clinical Sciences, Unit of Surgery, University of Naples Federico II, via Delpino 1, 80137 Naples, Italy; Department of Pathology and Animal
Health, Unit of Pathology, University of Naples Federico II, via Delpino 1, 80137 Naples, Italy; Unit of Infectious Diseases, Faculty of Veterinary Medicine,
University of Naples Federico II, via Delpino 1, 80137 Naples, Italy

Address communications to:

B. Lamagna
Tel.: +812536047
Fax: +812536021
e-mail: blamagna@unina.it

Abstract
Objective To describe a case of a conjunctivo-corneal mass in a cat associated with
acid-fast bacilli.
Methods A 2-year-old female black European Short-Hair cat, living outdoors in a
suburban environment in Italy, was referred for evaluation of a nodular, vascularized mass
of 2 weeks duration. The mass involved the dorsal bulbar conjunctiva at the temporal
canthus of OS and invaded the sclera and cornea. Routine ophthalmic and systemic
examination, serologic testing, cytology and histology of the mass were performed.
Mycobacterium specic polymerase chain reaction (PCR) of variable regions 1, 2
and 3 of the 16S ribosomal RNA (rRNA) gene was also performed.
Results Neutrophils, lymphocytes, macrophages and giant cells with intracytoplasmic
acid-fast bacilli were seen on cytological examination. The histological examination
conrmed the presence of a granulomatous lesion with acid-fast bacilli within
macrophages. Bacteriological culture of the material from the lesion was negative for
Mycobacterium spp. Mycobacterium 16S rRNA gene specic PCR was positive.
A diagnosis of feline leprosy was made. The owners refused any treatment, and
1 year later the lesion was still present.
Conclusions Veterinary ophthalmologists should be aware of conjunctivo-corneal leproma
as an unusual symptom of leprosy.
Key Words: acid-fast-bacilli, cat, conjunctivo-corneal granuloma, feline leprosy,
mycobacteria, toxoplasmosis

INT RO DUC T IO N

Mycobacteria may cause a variety of clinical syndromes in

cats.1 Diagnosis may be difcult primarily because of the
number of species of mycobacteria involved, many of which
are difcult to culture, and also because of the variety of
disease manifestations involved.2 Moreover, few case reports
have been described and small case series have been reported
from different geographic locations around the world.
Three manifestations of mycobacteria-associated disease
syndromes are reported in cats: classical tuberculosis, feline
atypical mycobacteriosis and feline leprosy.
Classical tuberculosis in cats, is associated with a generalized,
widespread disease, caused by infection with Mycobacterium
2009 American College of Veterinary Ophthalmologists

bovis , M. Tuberculosis and other tubercle group variants

(M. Microti).3 The alimentary tract is most commonly affected
with tubercles developing in the intestine and mesenteric
lymph nodes following ingestion of milk or meat from infected
cattle. Cutaneous lesions can develop from bite wounds with
local spread but may also develop as a consequence of dissemination from the gastrointestinal tract. Lung tubercles
may also develop.
Feline atypical mycobacteriosis is most often characterized
by panniculitis and chronic or recurrent stulous tracts and
nodules associated with a previous history of trauma and
typically affects the inguinal fat pad and ventral abdomen.
Lesions are usually associated with saprophytic mycobacteria
ubiquitous in soil and water. Potentially pathogenic species

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ET AL.

for cats include M. avium, M. fortuitum, M. phlei, M. smegmatis,

M. chelonae and M. thermoresistible.4,5
Feline leprosy refers to a Mycobacterium sp. infection in
which single or multiple granulomas form in the skin or
subcutis with acid-fast bacilli that can be difcult to culture
using routine bacteriologic methods. Gingiva, nasal plane
and internal organs may also be involved. The condition
occurs in temperate, coastal climates and has been reported
in New Zealand,6,7 Australia,810 Western Canada,11,12 the
Netherlands,13 UK14,15 USA,16 North Italy,17 and Greece.18
The causative agent is presumed to be M. lepraemurium;
however, recent studies using the polymerase chain reaction
(PCR) have recently demonstrated the presence of other
mycobacteria including novel species.9,10,19,20
A case of feline leprosy involving a 2-year-old-male cat
with a solitary lesion of the right forelimb was described in
Northern Italy in 1996.17
The precise, natural transmission of feline leprosy is
unknown, but it has been related to bites of infected rats and
cats. Mycobacterium leprae, the agent of human leprosy, has
been found in mosquitoes, eas and bed bugs. This suggests
that similar vectors may play a role in feline leprosy.
Molecular biology-based methodologies may offer a faster
and more reliable option than bacterial culture for the
diagnosis of Mycobacterial infection. PCR amplication
of the 16S rRNA gene have proven to be of value for the
diagnosis of infections caused by mycobacterial species in
veterinary medicine.21 The 16S rRNA gene is highly conserved because of structural and functional constraints on
the rRNA molecule, but regions within the gene are highly
variable and species-specic for mycobacteria.22
In this report we describe a case of a conjunctivo-corneal
mass in a cat from Southern Italy. This lesion was studied
with cytology, histopathology and PCR methods and characterized by the presence of acid-fast bacilli, resembling the
histopathologic appearance of cutaneous feline leprosy.23
CA SE REPO RT

A 2-year-old female European Short-Hair cat, living outdoors

in a suburban environment, was presented for evaluation of
a 2 weeks duration nodular vascularized mass arising in the
dorsal bulbar conjunctiva at the temporal canthus of OS and
invading the sclera and cornea (Fig. 1).
No other ocular abnormalities were seen on biomicroscopic
and indirect ophthalmoscopic examination. Physical examination was within normal limits. Chest radiographs and
abdominal ultrasonography did not show any important
alteration. Blood cell count showed a mild lymphocytosis
(8.000 per L) while the biochemical prole was normal.
Serologically, FeLV and FIV virus were negative, and
toxoplasmosis IgG titer was 1 : 640.
Cytobrush samples and a biopsy of the mass were
obtained. The tissue specimen was divided in two parts. One
part was xed in 10% neutral buffered formalin and parafn
embedded. The other was used for bacterial culture.

Cytology revealed the presence of numerous neutrophils

and macrophages with abundant foamy cytoplasm often
packed with negatively stained, rod like structures arranged
in the cytoplasm like needles thrown on the ground. Occasionally, multinucleated Langerhans type cells containing
numerous rod-like structures were seen (Fig. 2).
Histopathologically the lesions consisted of multifocal
to coalescing pyogranulomas with central necrosis. Multinucleated giant cells were present associated with vacuolated
macrophages containing unstained rod-like structures. Ziehl
Neelsen staining revealed many acid-fast bacilli within macrophages and giant cells. Similar lesions have been described in
the feline multi-bacillary lepromatous leprosy syndrome2,18
(Fig. 3).
For mycobacterial culture, fresh refrigerated tissue was
sliced with a scalpel blade and homogenized by mechanical
squashing in the presence of 4% sulfuric acid as decontaminating solution.24 After neutralization with 6% sodium hydroxide,
samples were inoculated with Lowenstein-Jensen and Coletsos
egg based solid media as well as Bactec MGIT 960 liquid
medium. Cultures were negative at 3 months.18
PCR of variable regions 1, 2 and 3 of the Mycobacterium
16S ribosomal RNA (rRNA) gene was performed on parafn
embedded tissue.
From each parafn block, ten 30 m tissue sections
were obtained using a microtome mounted with a sterile
disposable blade that was discarded after each case. Sections
were deparafnized using two 1-mL xylene and 80% ethanol
washes, dried in a rotary evaporator and nally processed
with the High Pure PCR Template (Roche) for DNA
extraction.
To ensure that sufcient DNA was extracted from parafn
xed tissue, 3 g of the extracted DNA was subjected to
electrophoresis in a 1.5% agarose gel.
PCR was carried out with 1 g of DNA template. In the
second round of nested PCR, 10 L of the rst-round PCR
was used as template. The reaction mix (50 L) contained
1 m of each primer, 8 L of dNTPs mix (2.5 mm each),
2.5 U Takara LA Taq (Takara BIO Inc., Japan), and 10 LA
PCR buffer II plus MgCl2 (Takara BIO Inc.).
The primer combination, Mycobacterium 16S rRNA
gene2,25 17F (sense 5-CATGCAAGTCGAACGGAAAG-3)
and 525R (antisense 5-TTTCACGAACAACGCGACAA-3),
selected from published reports, were used in the rst-round
PCR (approximately 500 bp). The primer combination that
amplies a smaller portion of the same region, Mycobacterium
16S rRNA gene 53F (sense 5-GAGTGGCGAACGGGT
GAGTAA-3) and 485R (antisense 5-TTACGCCCAGT
AATTCCGGACAA-3), selected from published reports,
was used second round of nested PCR.
In the rst and second round of PCR, the reaction mixtures were kept at 94 C for 2 min, and then cycled 35 times
with denaturation at 94 C for 1 min, 2 min annealing at
60 C and 2 min strand extension at 72 C, followed by a
nal extension step at 72 C for 10 min. In the second-round
PCR, conditions were identical to the rst-round PCR.2

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Figure 1. Two-year-old female European cat with a conjunctivocorneal mass of the OS at presentation. A nodular vascularized mass
arising in the dorsal bulbar conjunctiva at the temporal canthus of OS
and invading the sclera and cornea is apparent.

Figure 3. Histological section (6 m). There is a central area of

necrosis surrounded by numerous lymphocytes and neutrophils that
overlay macrophages. H&E, original magnication 400.

On the basis of these ndings the diagnosis of feline

leprosy was supposed and a treatment with clindamicin
(12.5 mg/kg PO SID) in association with doxycycline
(10 mg/kg PO SID) and topical tetracycline/sulfamethyltiazole
ointment was prescribed.
The owners were not able to give any drugs to the cat and
refused any surgical treatment. One year later, on telephone
follow-up the owners indicated that the mass was still
present but it was impossible to approach the cat, which
lived outdoors.
D I S C U S S IO N

Figure 2. Smear made from cytobrush samples. Neutrophils and

a multinucleated giant cell containing numerous rod like structures
red-stained. Ziehl-Neelsen original magnication 1000.

PCR products were visualized on 1.5% agarose gels with

ethidium bromide staining,2 and the sizes of the respective
amplied products were estimated by comparing the mobilities
with a 100-bp ladder (Takara BIO Inc. Japan).
DNA extract from Mycobacterium bovis, Mycobacterium
avium and DNA extract of a negative control were tested
simultaneously with the selected DNA extracts to ensure the
consistency of the amplication results.
PCR amplication of 16S rRNA gene in the V2 and
V3 region identied Actinomycetales organisms in the
conjunctivo-corneal lesion of the cat.

To the authors knowledge, this is the rst report of a granulomatous conjunctivo-corneal mass caused by acid-fast
bacilli, as a feature of feline leprosy. Recently, mycobacterial
keratitis has been identied in cats residing in semi-rural
Australia.10
Interestingly, the examination of the data concerning
feline leprosy cases from Italy shows a uniform age distribution,
with both (the conjunctivo-corneal mass described in this
report and the case of Roccabianca17) occurring in cats about
2 years of age. Malik et al.9 have suggested that feline leprosy
in Australia comprises two different clinical syndromes, one
tending to occur in young cats (< 4 years) and caused typically
by M. lepraemurium, characterized by an aggressive clinical
course, and another in old cats (more than 9 years old) caused
by a single novel mycobacterial species, characterized by a
protracted clinical progression.
The causative agent of both feline leprosy cases from Italy
has not yet been well identied. Ocular manifestations of
human leprosy (Hansens Disease), caused by infection with
M. leprae, include madarosis, subconjunctival brosis, punctate
epithelial keratopathy, posterior subcapsular cataract, corneal

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hyperesthesia, lagophtalmos, corneal pannus, entropion,

prominent corneal nerves, iridocyclitis, focal avascular
keratitis, scleritis, interstitial keratitis, iris pearls and ocular
clofazimine crystals.26,27 Fibrous histiocytoid leproma in the
cornea has also been reported.28 Recently, a corneal leproma
as an initial feature of lepromatous leprosy has been
described in a 72-year-old woman. It was characterized by a
pinkish nodular mass involving more than half of the cornea,
The mass was composed of foamy macrophages with numerous
acid-fast bacilli.29
Ocular manifestations of feline leprosy are very rare in
cats. One report described keratopathy in two cats in the
northwestern United States that was also characterized
histopathologically by acid-fast bacilli.30 They reported that
the corneal lesions were progressive and eshy in appearance.
Histopathologically, the lesions contained neutrophils,
mononuclear inammatory cells and bacteria within macrophages. The histopathologic appearance was considered to
be similar to that seen in the skin of cats with feline leprosy.
It has also been hypothesized that Florida Spots, nonprogressive greyish supercial stromal opacities that are seen
in cats and dogs most commonly in the southeastern states of
the USA,31 are caused by mycobacterial organisms,32 but the
causal organism is not yet well dened.
To speculate on the causes of the infection in our case we
can consider that the cat lived outdoors and most likely she
had contact with infected rats or other cats. It has also been
postulated that mycobacteria may normally reside in soil or
stagnant watery environments that favor the proliferation of
saprophytic mycobacteria, and subsequently become inoculated into the conjunctival mucosa through contamination of
traumatic injuries (from other cats or another mammalian
vector) or via a biting insect vector.
It is unlikely that, a presumptive saprophytic mycobacterium,
the causative agent of the corneoconjunctival granuloma
described here, may be a public health concern. Saprophytic
mycobacteria are not considered contagious and highly
pathogenetic in humans.33
However, reports of mycobacterial diseases in humans due
to species generally considered saprophytic have become
more numerous.34
Therefore, contact precautions with Mycobacterium-infected
pets are advised, especially for immunosuppressed people.35
CON C LUSIO N S

Veterinary ophthalmologists should be aware of conjunctivocorneal leproma as an unusual symptom of leprosy, as

discussed here.
Other investigations are necessary to further dene the
clinical and pathological features of this disease in cats.
A CKN O WLEDGMENT

Authors thank Joanne Stellato for the proofreading of the

manuscript.

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