ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol. 62 - No. 3-4 2007
DIAGNOSIS OF MYCOPLASMA CONJUNCTIVAE AND
CHLAMYDOPHILA SPP IN AN EPISODE OF
CONJUNCTIVITIS/KERATOCONJUNCTIVITIS IN LAMBS
Lysnyansky1, I., Levisohn1, S., Bernstein2, M., Kenigswald3, G., Blum2, S.,
Gerchman1, I., Elad2, D. and Brenner4, J.
1. Mycoplasma Laboratory,
2. Division of Bacteriology, 4Division of Virology, Kimron Veterinary Institute, 50250, Bet Dagan,
3. “Hacklait” Caesarea, Israel.
ABSTRACT
Infectious keratoconjunctivitis (IKC) is a highly contagious ocular inflammation
frequently reported in domestic sheep and goats and in wild Caprinae. Chlamydia
pecorum and Mycoplasma conjunctivae are considered as the major etiological agents
in IKC in domestic sheep. This short communication reports on the diagnosis of M.
conjunctivae and Chlamydophila spp in an episode of conjunctivitis/
keratoconjunctivitis in 3-6 weeks old lambs in a flock in the northern Negev. The
clinical signs, mainly coughing and ocular lacrimation, were first reported in January
2005 with recurrence of the disease after topical treatment with cloxacillin 16.7%
W/W as benzathine salt. The etiology of the disease in this case was supported by the
laboratory findings. M. conjunctivae was isolated from ocular swabs of all three
lambs with acute eye infection and from a lamb with severe bilateral IKC resulting in
blindness. Chlamydophila spp was demonstrated in 2 out of 3 samples that were taken
from the same lambs and from all lambs sampled after treatment, including the blind
one. It should be noted that a recent literature report implicates M. conjunctivae in an
episode of eye inflammation in young children, a previously unrecognized zoonoses
that may occur from contact in petting zoos.
INTRODUCTION
Infectious keratoconjunctivitis (IKC) is a highly contagious ocular inflammation
frequently reported in domestic sheep and goats (1) and in wild Caprinae (2). In
domestic sheep the role of Chlamydia pecorum as a major etiological agent in IKC is
well established (3). Mycoplasma conjunctivae, first identified by Barile et al (4), is
also considered a primary pathogen in establishing eye inflammation in both domestic
and wild Caprinae, without the auxiliary involvement of other microorganisms (5, 6).
Other mycoplasma species, especially respiratory pathogens or saprophytes, may also
be isolated from the corneal sac of diseased or clinically healthy animals (7,8).
Mechanic or environmental/climatic factors may act synergistically together with the
infectious agent to establish an inflammation, namely conjunctivitis, that may be
transient or progress to keratoconjunctivitis. At this stage, when the cornea is
involved, the disease may further progress toward corneal ulceration with temporary
or permanent blindness.
The importance of M. conjunctivae in the etiology of IKC is supported by
experimental infection studies (9, 10). However, under field conditions, other
microorganisms, usually Branhamella ovis or Moraxella ovis, are frequently isolated
concomitantly in addition to the possible presence of Chlamydophila spp.
In recent years, interest in M. conjunctivae infection has increased markedly due to
the impact of the disease on free-ranging ibex (Capra i. ibex) and chamois (Rupicapra
r. rupicapra) populations in the Alps and other mountain ranges in Europe (2, 11).
Diagnosis or identification of M. conjunctivae isolates may be carried out by the use
of a species-specific 16S-rRNA PCR test (6, 12, 13). Use of a gene-targeted-PCR test
detecting the lppS gene has clearly established transmission of M. conjunctivae from
asymptomatic domestic sheep to wild caprinae sharing grazing land (11). The wild
caprinae usually develop clinical IKC with resultant blindness, resulting in death
under natural conditions. While of great value in monitoring the presence of M.
conjunctivae, especially in free-ranging animals that are difficult to sample, targeted
molecular diagnosis may not detect all the etiological agents in a multifactorial
disease.
This short communication reports on the diagnosis of M. conjunctivae and
Chlamydophila spp in an episode of conjunctivitis/keratoconjunctivitis in lambs three
to six weeks of age.
CASE HISTORY
Clinical signs began in January 2005, in a sheep flock consisting of 500 ewes with 1.8
prolificacy (=900 lambs/year). During the cold season, from January through March,
cough, eye inflammation and lacrimation was noted in 3-6 weeks old lambs (30 lambs
out of approximately 300 born during this period). Three ocular swabs without
transport media were submitted for diagnosis (Sampling time I). The affected lambs
were treated with cloxacillin 16.7% W/W (as benzathine salt, Northborough, Ireland)
ointment inserted into the lower conjunctival sac for 10 days, until the clinical signs
receded. The treatment resulted in recession of the clinical signs.
Approximately two weeks after cessation of treatment clinical signs reappeared in
some of the treated animals. Six lambs, in a pen of 20, showed active conjunctivitis
(Picture A) that progressed into permanent blindness (Pictures B). Nasal and ocular
samples were taken in transport medium for diagnosis from three diseased lambs with
conjunctivitis and from two asymptomatic convalescent animals (Sample time II).
MATERIALS AND METHODS
Microbiological methods
Routine bacteriological assays for the isolation of the ocular and nasal flora are
described elsewhere (14, 15, 16). For isolation of mycoplasmas, ocular swabs
submitted for routine diagnosis (Sample time I) were inoculated into standard
Hayflick-type mycoplasma broth (17). At sampling time II, nasal and ocular swabs
were inoculated into FF (modified Friis) medium mycoplasma broth with the addition
of 67 μg/ml cefoperazone (cefobid) and 200 μg/ml bacitracin (Sigma) (17). The
cultures were propagated at 37°C in a humidified atmosphere with 5% carbon
dioxide. Identification of mycoplasma isolates was by indirect immunofluorescence of
mycoplasma colonies with species-specific polyclonal antibodies from the
Mycoplasma Reference Laboratory, Aarhus, Denmark, using standard methods (18).
Chlamydophila identification
Conjunctival swabs were rolled onto microscope slides, air-dried, fixed with cold
acetone for 10 min and frozen at –200C till examined. These samples were tested for
Chlamydophila spp by direct immunofluorescence with a monoclonal chlamydial
group-specific fluorescein conjugate (Cellabs, Australia) according to the
manufacturers’ instructions. Samples were considered positive if 10 or more
fluorescing elements with characteristic morphology were seen per high power field.
RESULTS
Bacterial findings for ocular swabs are summarized in Table 1.
Table 1: Identification of putative pathogens in ocular swab samples
M.
Clinical status
Chlamydophila spp*
conjunctivae *
2/3
3/3
Acute infection
3/3
1/3
Symptomatic
2/2
0/2
Asymptomatic
Sampling time I
Sampling time II
(post-treatment)
*positive/teste
d
Chlamydophila spp was demonstrated in two out of two ocular samples taken from
convalescent lambs and five out of six ocular swabs from lambs with acute or recurrent
conjunctivitis (Table I). The number of fluorescent-stained elementary bodies differed
markedly among the samples. M. conjunctivae was isolated from all three ocular swabs
taken during the acute stage of the disease (Sampling time I). At sampling time II, two
animals presented with conjunctivitis and a third animal showed bilateral
keratoconjunctivitis resulting in blindness. M. conjunctivae was isolated from only one
sample after treatment (the blind animal), irrespective of the presence of clinical signs.
Each of the three ocular samples from the acute stage of disease showed a different
bacterial flora. One sample yielded mixed bacteria flora with the saprophytic Mycoplasma
arginini whereas the others yielded Moraxella ovis or Staphylococcus plasma coagulase
negative or Streptococcus α-hemolytic with no additional mycoplasma. Mycoplasma
ovipneumoniae was isolated from four out of the five nasal swabs tested.
No viral assays were attempted.
Figure Legend
A - Acute conjunctivitis in a 6 weeks old lamb. The disease was responsive to topical
treatment with antibiotics ,with disappearance of clinical signs. In some animals the lesion
reappeared after the cessation of antibiotics treatment.
B - Progression of disease to keratoconjunctivitis (bilateral blindness), and (B1)
where the necrotic center with ulceration is evident.
DISCUSSION
This study clearly demonstrates the presence of two major pathogens, M. conjunctivae and
Chlamydophila spp, in an outbreak of IKC in young lambs. This is the first identification of M.
conjunctivae in sheep in Israel. Topical treatment led to improvement of the clinical signs,
but there was resurgence of the disease after treatment was finished. Notably, isolation of
M. conjunctivae was successful in only one of the five samples from animals that had been
treated. This underscores the problems in determining the etiology of a disease in animals
after antibiotic treatment .
M .ovipneumoniae is recognized as a major pathogenic agent of the sheep respiratory tract,
together with or in the absence of Pasteurella spp. (19, 20). The organism is somewhat
fastidious and may be difficult to isolate, especially in mixed infection with nonmycoplasmal bacteria. Diagnosis of M. ovipneumoniae infection in this case is the first
report in Israel during recent years ,but the prevalence of the organism is not known since it
is not detected by mycoplasma culture methods routinely used for diagnosis. Although M.
ovipneumoniae was isolated from four out of five lambs, it is not known to what degree the
flock suffered from respiratory disease. It is not clear if there is a direct association between
respiratory infection and conjunctivitis and respiratory pathogens may be isolated from the
ocular sac in the absence of disease (7, 8). In a previous study of bovine pink-eye, we
reported on the presence in ocular secretions of Mycoplasma bovioculi in mixed infection
with Mycoplasma bovis, a major respiratory pathogen (16), suggesting potentiation or
exacerbation of the ocular disease by the respiratory disease.
In recent years there has been increasing recognition of the prevalence and importance of
disease syndromes of multifactorial etiology. It is often difficult to elucidate the role of each
of the infecting microorganisms, especially because the relative importance of each may
vary in different cases. Synergism between Mycoplasma ssp and other bacteria or viruses in
ruminants has been frequently described .)20 ,19( However, the organisms may act
sequentially rather than in concert and may not be present in the lesion or the affected
organ at the same time .
The episode of IKC described in this report is not a novel occurrence in this flock. Although
clinical signs have been reported in previous lambing seasons, no nasal or ocular swabs were
sent to the Kimron Veterinary Institute for bacterial diagnosis, and it is suggested that the
pathogens are endemic in the flock. However, this is the first time that it has been possible
to elucidate the etiology of the disease. Asymptomatic animals serve as a reservoir of
infection for the new-born lambs. The sheep may also serve as a source of infection for freeranging wild Caprinae that share the pasture, as has been reported in Europe (2, 11). Future
use of molecular diagnostic tests may simplify sampling and laboratory testing, enabling
more rapid diagnosis and appropriate treatment.
Since sheep conjunctivitis is not an emerging disease in Israel and was described in 1951 by
Komarov (21), we would suggest our practitioners be aware of the possibility that M.
conjunctivae is also considered zoonotic since this pathogen has been implicated in an
episode of eye inflammation in young children (22). Lambs, kids and small wild ruminants
are very popular in the petting zoos in Israel. Therefore, we should be alert to any clinical
signs indicative of conjunctivitis in these animals as they might potentially be hazardous for
young children.
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