P. N. NANDA AND M. ABDUSSALAM 229
of the disease were available, one in an Hissar Dale
ram and five in Bikaneris. Two animals of the
latter breed were in an advanced stage and well on
the way to recovery. The other four were more or
less early cases. Conjunctival scrapings were
obtained by means of slides with well ground edges
from the lining of lids and nictitating membrane
and spread in the form of thin smears on clean dry
slides. These were air dried, fixed in methyl alco-
hol, and stained by Giemsa's method. Under the
oil immersion objective of the microscope, Ricket-
tsia conjunctivae Coles could be detected in a large
number of epithelial cells in all the four early cases.
Besides the usually heavily infected conjunctival
cells, one could also see clean epithelial cells, neutro-
philes and a few red blood corpuscles. In the two
advanced cases, no Rickettsia were found and in
place of neutrophiles, lymphocytes predominated.
This is in conformity with the observations of
Coles [1931, 1].
The casual protista, Rickettsia conjunctivae, is
markedly pleomorphic and in Giemsa stained pre-
parations we could see spherical or sub-spherical
solid forms, ring forms, imperfect rings or 'horse-
shoe' forms, triangles with rounded angles and
bipolar forms. Their dimensions varied from
0.4 µ to 1.8 µ at the maximum diameter. In
smears prepared in acute stages of the disease,
most of the cells were found to be infected and a
majority of them were packed with the parasite;
in some cells it was diffusely scattered or lay in
clumps which had no tendency to hug the nucleus.
With Giemsa's stain the protista takes a pale blue
or lilac colour, the solid forms and the 'poles' of
the bipolar and triangular forms taking a reddish
hue. No degeneration of the invaded cell was
found.
Donatien and Lestoquard [1938] have studied
the developmental cycle of this protista in experi-
mentally infected animals.
TRANSMISSION
The natural mode of transmission of the disease
is unknown but the comparative frequency of out-
breaks in those parts of the year when flies (Musca
spp.) are abundant at Hissar, suggests that these
insects may be responsible for its natural dissemi-
nation. Another possible mode of (mechanical)
transmission is the long grass on which the sheep
graze in certain seasons. Edgar [1931] has also
noticed that contact infection in Australian sheep
occurred only when sheep were grazed onlong grass;
it did not occur when they were kept on short pas-
ture.
Artificially, the disease is readily transmissible
from cases in the early stages, to healthy animals.
Conjunctival scrapings of an early case were dilut-
ed with sterile normal saline solution and inoculat-
ed sub-conjunctivally in two healthy lambs and
intra-ocularly in a third one. These healthy lambs
were six months old and were obtained from a
flock in which the disease was not known to have
occurred previously. Two days after these in-
jections all the three lambs developed lacryma-
tion and conjunctivitis followed by other typical
symptoms of the disease. Later the experiment
was repeated in a simpler manner. The conjunc-
tival surface of an affected eye was touched with
a finger which was immediately rubbed in the
healthy eyes of another lamb. On the third day
this lamb also developed typical symptoms of the
disease. It is evident from these experiments
that the disease is readily transmissible mechanical-
ly with an incubation period of two to three days.
Edgar [1931] was also able to transmit the
disease by instillation of infected lacrymal secretion
into healthy eyes but he failed to affect transmission
with filtrates of infected tears obtained by passage
through Seitz, Berkfeld, Chamberland F. and
Plaster of Paris Candles. By centrifuging dilute
lacrymal secretion at 3,000 r. p. m. for two hours
he obtained a non-infective supernatant portion
and an infective deposit.
DIAGNOSIS
Clinical diagnosis of this disease is simple and
is based on the occurrence of typical symptoms in a
large number of sheep in the same flock. It can,
however, be easily confirmed by microscopic exami-
nation of Giemsa stained conjunctival scrapings
obtained from an early case, when a large number
of conjunctival cells will be found to contain
Rickettsia conjunctivae. It must, however, be
distinguished from another, apparently harmless,
intra-cellular organism of the conjunctival epi-
thelium of sheep encountered by Coles [1931, 2]
in South Africa and by Donatien et Lestoquard
[1939] in Algeria. According to Coles [1931, 2]
this organism is distinguished by its less marked
pleomorphism, somewhat larger size and that it
does not occur in clumps; a deeply staining mate-
rial in the body of the organism is arranged more
or less in the form of a quarter to half moon, the
rest of the organism staining very faintly. Unlike
R. conjunctivae it shows no red with Giemsa, but
the half moon part stains deep blue and the rest,
a very faint blue. The present writers have not
encountered this organism at Hissar.
The presence of melarim granules in the epi-
thelial cells of the conjunctiva should also be kept
in mind, since they may be mistaken for Rickettsia.
They can, however, be readily distinguished by
their brown colour and by being visible in
unstained preparations.