NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
ETIOLOGY OF ENZOOTIC BOVINE
HAEMATURIA
347
From this it appears that the pathological process in the bladder runs almost
parallel to the recuperative mechanism of the animal's system. The disease has not
been reported in young calves, and the cattle are stated to exhibit the symptoms
after grazing for a number of years on one farm. History cards of all animals
since their purchase from the surrounding hills are maintained regularly at this
Institute. From a scrutiny of these, the time that elapses between purchase and
the apparent commencement of haematuria symptoms appears to be short, and it
appears possible therefore, that the majority of hill bulls at this Institute bring
with them a pre-existing infection, rather than contract the disease here, though
there does not appear to be any reason why such an immunity of this place should
exist. In the author's experience in 1928-29, cases had been removed from the
hills to Calcutta, but a change of locality did not produce any substantial alleviation
of the disease. It has been observed at Muktesar that a number of cases invariably
exhibit exacerbations of haematuria with snow-falls, and a gradual and continued
decrease of the bodily temperature indicates a real danger signal.
MACROSCOPIC LESIONS.
On post-mortem examination naked eye lesions of significance are not usually
discernible in any organ excepting the bladder, but occasionally the kidney or the
ureters may be abnormal. The bladder may be empty or filled with haemorrhagic
urine, which is partially or completely clotted. It has been suggested that it is
only in the basal and ventral part of the bladder that the initial lesions are formed,
but when the gravitational precipitation of the causal factor can be eliminated, such
a suggestion has no significance. On eversion of the organ, variable lesions are
encountered. The early lesions appear as small patches of congestion, and red dots
of haemorrhage up to the size of a lentil, associated with a slight thickening of the
mucous membranes are seen. The altered mucous membrane is raised above the
surrounding areas as small round yellow areas enclosed by a ring of acute congestion.
Localised gelatinous infiltration of the submucosa is seen, which is followed by the
sloughing of the overlying mucous membrane, giving rise to small ulcers, which form
the seats of continuous blood-letting until a clot forms. The ulcers are discrete,
and, when confluent, become broader or more elongated and present raised edges,
assuming a button or crater-like appearance. Since the opportunities of examining
genuine early cases are not readily obtainable, the apparent first appearance of
haematuria being possibly an intermittent attack only, the naked eye lesions are
seldom so simple as the above. On the contrary, one may find " varicosities as large
as hemp seeds, proliferations as large as nuts", rounded or cauliflower-like, rough
raised red ridges, croded bleeding patches, and submucous haemorrhages, sessile or
pediculated pyriform mucoid cysts, and considerable thickening of the bladder wall
348 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ IV, IV.
(Plate XIX, Fig. 1). Cicatrices are not very evident. Only localised areas of the
bladder may be involved or the lesions may be diffused throughout. In cases of old
standing, considerable ulceration has been detected at post mortem, although blood
was not being passed immediately prior to death. The co-existence of such a
variety and gradation of lesions, from minor changes to exhuberant growths, shows
that the lesions develop separately and continously, and not simultaneously, explain-
ing the remarkably persistent and intermittent character of haematuria.
In these studies mucoid cysts have been met with on the surface of the kidney,
and minute haemorrhages have been seen in this and other organs. Hydronephrosis
or cystic kidneys seen in haematuria cases seem to be connected with the bladder
lesions. The intestines, particularly the caecum and the proximal end of the rectum
of haematuria subjects have shown very minute sub-epithelial haemorrhages or
millet seed and pin-head sized round ulcers with raised edges and deep necrotic core,
but their significance is not known at present. Plate XIX, Fig. 2 shows polypoid
growths in the duodenum from a case of haematuria.
CLINICAL AND CHEMICAL STUDIES.
Although in the course of routine post-mortem examinations at Muktesar a few
cases of the typical bladder lesions of haematuria were undoubtedly detected earlier,
it was only after the first report by Kerr [1925] that the disease received attention.
The disease has figured in several Annual Reports of this Institute since that date.
In the earlier investigations, arrangements were made to collect full clinical details
regarding haematuria, including weekly body weights, daily temperatures, etc. A
method was improvised to approximate the proportion of the admixture of blood
cells and clots to a fixed amount of urine passed. Blood smears were examined
regularly, samples of urine from each fresh case and post-mortem material were
subjected to culture examination. Experimental treatment with trypan blue, and
the benzidine test for the presence of haemolysed blood in samples of the urine,
from which the centrifuged deposits had been removed, were carried out ; and thus
the possibility of bovine piroplasmosis was first eliminated. By centrifugalising
the urine and faeces from uncomplicated haematuria cases, and by means of the
sugar floatation technique, repeated examinations were made and the possibility of
coccidia was consistently negatived. Repeated attempts to culture any protozoan
organism from urinary sediments by the use of 5 per cent. potassium dichromate
solution and of hay infusions have given negative results. As oportunities
occurred, careful post-mortem examinations were made in order to discover if the
lesions in any other organs excepting those of the urinogenital system, could be
correlated with the passage of blood in the urine. At that time the probability
ETIOLOGY OF ENZOOTIC BOVINE HAEMATURIA
349
appeared to be that " the cause of the disease was an irritant chemical substance
elaborated from the excretory products in the urine while the urine was stationary
in the bladder." Investigation of the disease on the above lines continued, but
it was increasingly felt that unless more information on some specific factors
relating to the extravasation of blood from the bladder was available, the study of
the disease was not likely to be productive of anything but negative data, and a
critical examination of all the causes suggested in literature was made. As has
already been mentioned earlier in this article, Hadwen's was the only work which
claimed to have reproduced the disease experimentally, but, even so, his views
appeared to be improbable. Therefore from theoretical considerations of available
biochemical knowledge [Datta, 1931] it was considered advisable to determine whether
there was in the system, any stagnancy of oxalic or other allied acid, such as parabanic
acid (Oxalylurea) of metabolic origin, and also whether the deficiency of calcium or
other metallic constituents of normal body tissues, or any haemophilia, existed,
which could explain the tendency to the extravasation of blood seem in the living
animal.
Since, in a few cases which had died of haematuria, the blood in the body was
found to be still in a fluid condition, and with little tendency to clot, attempts were
made to test the coagulability of blood. Various methods of determining the
coagulation time of blood were tried, and after preliminary trials with drops
of blood on glass slides, also in corked and open phials, had been made, the Loop
method of Inchley [1921] was chosen as the most suitable working method. The
serum calcium of a number of healthy and affected hill bulls was studied.
Calcium was administered in various forms to affected cases, since it was desired
to raise the calcium content of the blood in the hope of counteracting haemo-
philia, if such was present. Curiously, the same average clotting time of about
11 minutes was observed in all animals, whether in the active or quiescent stage
with regard to the passage of bloody urine. The administration, morning and
evening, of calcium lactate in the feed, and of calcium chloride solution intraven-
ously, did not produce any marked difference in the coagulability, of the blood
in affected cases. The clotting time in one case, however, rose to 18 minutes a
short time prior to death. Similarly when the serum calcium was estimated in
those haematuria subjects which were receiving calcium therapy, for purposes of
comparison with those that were not, the calcium value showed only a small
increase, but the phosphate value in the treated subject was decidedly on the
increase. Another noticeable fact in these experiments was that, even during the
period when as much as 5 per cent. of the urine passed by the animal consisted of
red corpuscles, the animal was seen to be putting on weight consistently, and this
may be ascribed to the benefits of calcium therapy. The result of chemical analy-
350 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ IV, IV.
sis on an average sample of haematuria urine, from an untreated case is given
below. The figures are per 100 c.c. of serum.
Amount of PO 4 (phosphate)
„
„
Calcium
„
„
Albumin
= 0.0148 gm.
= 0.024 gm.
= 0.060 gm.
Urea was not estimated. In order to ascertain whether there was any
hyperacidity of serum in affected cases, the PH values of some healthy and eight
haematuria subjects were determined colorimetrically, with the result that the former
gave 7.6 and the latter 7.5. There was therefore no marked discrepancy, but
whether a large proportion of neutral salts of acids were present was not deter-
mined.
With regard to the urine itself, its specific gravity, reaction and composition
were also studied. In our experience, the specific gravity of haematuria urine
varied from 1020 to 1035 in different individuals, and from the observations made
on several animals, the rise and fall in the specific gravity did not appear to bear
any relation to the percentage of blood present in the urine at different intervals.
The reaction of the urine in each case was found to be alkaline to litmus, phenol-
red, and brom-thymol blue. Samples of urine from three cases were examined
for the presence of oxalates, sugar, etc., with negative results. As was to be
expected, albumen was present.
GENESIS OF THE DEFINITE CLUE.
The etiological researches upon haematuria took a definitely hopeful turn on
the 7th of March, 1932, when the author discovered some very interesting ' bodies '
in the course of histological studies upon specimens of preserved bladder and
kidney tissues, collected from local haematuria cases (Plate XXIV, Fig. 2). When,
some peculiar spherical bodies were detected in the urine of all the six clinical
cases of the disease then at Muktesar, the securing of ' data of considerable
value and promise' was mentioned in the Annual Report of this Institute
for 1931-32 (p. 15). They appeared to differ from body cells. They were globu-
lar bodies whose nuclei could be discerned with difficulty in unstained wet smears ;
these bodies appeared to be absent from the urine from healthy cases, but were
present in the quiescent stages of haematuria. To ascertain if these bodies were
present in samples of haematuria urine from other provinces in India, enquiries
were made from the Heads of Veterinary Departments in the provinces, where
the disease occurs—viz., Bengal, Madras and the Punjab. On the arrival of samples
ETIOLOGY OF ENZOOTIC BOVINE HAEMATURIA
351
from the provinces the same obscure structures were detected, and the possibility
of a Schistosome origin was suggested. In the Annual Report for 1932-1933 (p. 15)
the failure to obtain confirmation of the above suggestion has been mentioned, but
the observation of ' a definite host reaction ' against the ' bodies ' left no doubt
that they were parasitic and foreign (Plate XXIX, Fig. 1). In size they were larger
than the usual body cells, and they definitely had a greater depth of focus. Since
an occasional' body ' appeared to have engulfed rare red corpuscles, or to possess
a vacuole they were described in the above Report as ' peculiar macrophage-like
cells,' and the possibility that these were Entamoeba was pursued by means of the
cultural technique of Boeck and Drbohlav [1925]. The first results have already
been mentioned in the Annual Report of 1932-33. Before giving a preliminary
description of the causative parasite, which appears to be responsible for this
disease, it is essential that the minute histology of the condition be first described
in the light of fresh knowledge, since the current views are faulty in some respects.
PATHOLOGICAL HISTROLOGY.
The Urinary Bladder. One would not expect to detect a case of
bovine haematuria at its very commencement with a view to studying
the pathological process in its earliest stage. Young and old lesions are, however,
present in most cases and these may be utilized for histopathological
studies. The disease is primarily an affection of the submucous layer, inflam-
atory exudation into which leads to necrosis and consequent ulceration
of the mucous membrane. The bladder is intensely congested, and the blood vessels
supplying the mucous and submucous layers are greatly dilated (Plate XX, Fig. 2).
The capillary vessels are often ruptured, and extravasation of blood around them is
to be seen. In some cases however the whole of the bladder wall is extensively in-
filtrated with blood and its pigments. The vascular endothelium is damaged to a
certain extent, and the exudation of serum to the perivascular regions leads to a
pushing apart of the surrounding connective tissue. Sometimes oedema is very
pronounced, and is easily discernible immediately below the muscularis mucosa and
in the strands of tissue, which intervene between muscle fibres.
The diversity of the pathological processes and the extent of their severity seen
in each case, depend upon the degree of the parasitic infestation, rate of division of
the parasites, and upon whether the lethal effects are repeated over a length of time.
The susceptibility of the affected bovine or its bodily resistance must also be a
factor of importance in the pathogenesis.
As a result of the inflammatory exudation, the epithelial lining of the bladder
is swollen and elevated, the inflammatory products separating the mucosa
from the thickened submucous layer. The extravasated blood and exuda-
354 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ IV, IV.
& 2). So-called "bird's nest bodies," of epithelioma are however never to be seen,
and real metastasis into other organs has not been found.
Another abnormality present in the sections consists of scattered minute
collections of cellular infiltration, immediately beneath the epithelium and also
around the ulcerated or haemorrhagic lesions. The cells are generally of the
mononuclear type, consisting of young fibroblasts, lymphocytes, and endothelial
cells with an admixture of so-called plasma cells. The most interesting picture is
however to be seen below the clots and ulcers. The central core of an ulcer consists
of nectrotic material with cellular debris and fibrin. Around the degenerative core
is a cellular reaction zone, the margins of which contain the invading parasites.
Plate XXI, Fig. 1 represents an active haemorrhagic ulcer involving the mucosa and
submucosal layer. Just below the haemorrhagic ulceration a colony of a number
of parasites, enclosed in a capsule and lying in a pool of acute extravasation and
with little attachment to the supporting bladder tissue around it, is noticed (Plate
XXI, Fig. 2). This picture leaves little doubt about the parasitic etiology.
In regard to the parasites, it has been stated that they are found in the epi-
thelial nodes on the lining membrane of the bladder, and in the lumen of blood
vessels which are more or less altered. The occurrence of the parasites in the reac-
tion zone around the degenerated areas of the submucosa has also been mentioned.
Further the parasites may be found considerable distance beyond the initial reaction
zone, and may even be seen making their way along the fibrous septa between the
muscle bundles. The parasites may be numerous in the active lesions, but occa-
sionally it may be difficult to find them. In the chronic cases, the muscular layer
and the subserosa may become involved. In the more acute cases however, the
parasites are situated exclusively in the submucosa. In acute attacks and early
lesions, the parasites are easily recognised but in chronic cases this may need some
perseverance.
Kidney. The parenchyma of this organ shows haemorrhagic areas, and cloudy
swelling in places. The glomeruli show varying degrees of dilation and degeneration
and the epithelial cells of the tubules are unrecognisable. The parasites have been
detected in this organ also. (Plate XXVI, Fig. 1.)
THE PARASITE.
The parasite varies in shape and size, and can be seen both in a free and an
encysted stage. Its cytoplasm appears to be sharply defined, having an alveolar
or reticulated appearance. Inclusion bodies and ingested red corpuscles have been
seen, but bacteria seem to be singularly absent amongst the ingested material. The
parasite possesses definite histolytic powers, and appears to grow by the absorption
PLATE XXIX (a).
Free hand drawing illustrating the characteristic features of the parasite, as seen in
Sections.
Figures 1, 2, 3 and 4. Parasites showing cytoplasm, nucleus, karyosome and linin
net-work.
Figure 3. Shows nuclear division into four, and Figure 4 shows a parasite divided
into two.
Figure 5. Shows rod-shaped chromatoid, body and glycogen vacuoles.
Figure 6. Shows a fragmented nucleus.
Figure 7. Shows a nucleus and glycogen vacuole.
Figure 8. Dividing nucleus and irregular shaped parasite.
Figure 9. Divided nucleus with an irregular mass of glycogen.
Figure 10. Two ingested red cells and glycogen vacuole.
Figure 11. Dividing nuclei.
Figure 12. Massing of chromatin into three irregular clumps.
Figure 13. Inclusion bodies in parasites. Section stained with iron hæmatoxylin.
ETIOLOGY OF ENZOOTIC BOVINE HAEMATURIA
355
of food material through its body surface. Granules of blood pigment have been seen
in its cytoplasm, and the ingested erythrocytes appear to have undergone some shrin-
kage. In the smallest sizes, the cytoplasm is clear and shows no ingested bodies.
Contractile vacuoles do not appear to be present, but so-called chromatoid bodies
and glycogen vacuoles are occasionally found. Microphotographs of collections of
the parasite as seen in histological sections are appended hereto. A diagrammatic
representation of the characteristic features of the individual parasites is given
(Plate XXIX (a)). In shape the parasites are usually spheroidal (Plate XXVII,
Fig. 2), but irregular shapes are also seen. Occasional prolongations of the cytoplasm
(Plate XXV, Fig. 2), are met with, and these are probably pseudopodia. Some para-
sites are elongate, as seen in (Plate XXI, Fig. 2). No suggestion of the presence of
flagella or cilia have been detected. Perhaps the spheroidal shape indicates a state
of rest, and the elongate shape that of activity. Differentiation into endo-and
ectoplasm is not discernible in sections, but the sharp outline of the parasite
obviously indicates such a hyaline ectoplasmic structure. The nucleus of the para-
site is the most outstanding feature. It is vesicular and round, and possesses a
centrally situated dot, the karyosome. The nuclear membrane is very definite,
and presents on its inner surface a series of bead-like small granules of chromatin
arranged uniformly. The clumping of the peripheral chromatin beads into three
irregular masses on the internal surface of the nuclear membrane has been seen in
one or two exceptional parasites (Plate XXIX (a), Fig. 12). From the karyosome a
network, presumably of linin, is seen to radiate to the nuclear membrane. On
careful examination, the nuclear membrane is found to be an even circle of highly
refractive material of some depth of focus. The encysted and free forms of the
parasite, encountered in sections from half a dozen cases of haematuria have been
measured in microns as follows :—
Free forms.
41.3x12.0
33/3 x 13.3
34.7 x 14.7
29.3 x 17.3
34.7 x 14.7
44.0 x 18.7
39.3 x 14.7
48.0x12.0
53.3 x 10.7
41.3x17.3
Cysts.
14.4 x 9.4
28.3 x 28.3
24.3 x 17.5
31.7x22.9
356 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [ IV, IV.
Free forms.
29.3 × 13.3
24.0 × 10.7
16.7 × 9.3
21.3 × 10.7
13.3 × 9.3
22.7 × 16.0
33.3 × 29.3
44.0×25.3
24.3 × 21.4
30.0 × 16.4
17.5 × 17.0
28.5×30.0
15.7 × 12.8
34.7 × 14.7
33.3 × 13.3
37.3 × 9.3
32.0×20.0
60.0 × 14.7
36.0 × 12.0
41.3×21.3
36.0 × 17.3
40.0 × 13.3
40.0 × 10.7
36.7×32.0
48.0 × 32.0
46.7×20.0
44.0 × 16.0
40.0 × 16.0
56.0 × 40.0
30.7×25.3
25.3 × 22.7
34.7 × 30.7
30.7×25.3
18.6 × 16.4
20.0×20.0
Cysts.
28.3×16.9
The cysts are rare in sections and are found lying somewhat separated from the
host's tissues. A thick double.contoured capsule with the evidence of one, two or
ETIOLOGY OF ENZOOTIC BOVINE HAEMATURIA
357
three nuclei has been seen (Plate XXIII, Fig. 2 and Plate XXV, Fig. 1). The size
of the parasites is important in order to distinguish them from body cells, such as,
epithelial cells, pus cells, macrophages or other bloated cells. The size of the
double contoured cyst is helpful in the identification of the parasite. It is in the
smallest stages of the parasite that one has to be particularly careful in
distinguishing them from bloated or wandering cells.
In the nondividing stage, the nucleus of the parasite varies from 5.7 to 7.3
microns. Nuclear division takes place by a process of binary fission, and the
maximum of up to four nuclei has been seen in dividing individuals.
The parasite is best studied under the 1-6th objective with frequent use of
the 1-12th oil immersion lens. It has also been studied in the fresh condition in
wet smears, prepared from the clear and bloody urine obtained from hæmaturia
cases. Since urinary sediments contain a variety of cells one has to examine the
former from a large number of healthy cattle, before forming conclusions regarding
the structures encountered in the urine of haematuria subjects. Parasites of
spherical shape, which may show an ectoplasm, and several times larger than body
cells, with inclusion bodies and a hazy nucleus in unstained preparations, are to
be seen in haematuria urine. Highly vacuolated and granular bodies probably
represent degenerated parasites. Spherical double-contoured encysted parasites,
the largest measuring about 35.4×30.8 microns, are to be found. Rarely a small
pore-like structure has been discerned in the cyst wall. On the warm stage parasites
with digit-like or blunt dome-shaped pseudopodium have been seen. Under the
microscope the parasites have not shown any attempt to cross the field nor have
any simultaneous retraction and protrusion of processes been seen. They would
thus appear to be in a somewhat moribund state in the urine.
From the above description, it will be appreciated that the parasite has no
features of the phylla ciliophora, mastigophora and sporozoa, but would appear to
belong to phylum Rhizopoda. The parasite is characterised by a nucleus with
a small spherical karyosome and a peripheral layer of fine chromatin beads,
justifying for it a place in the genus Entamoeba. The size of the free forms and
the cyst is much larger than that of all the known species of Entamoebæ, and
the host is a bovine. Definite histolytic powers are possessed. In view of these
considerations, and the fact he has received considerable help from his wife
Mrs. Kamala Datta the author suggests the designation E. Kamala, n. sp. for the
large protozoan parasite causing bovine haematuria.
DISCUSSION.
It will be of interest now to consider whether the course and nature of bovine
haematuria, as also its general morbid anatomy and histology, are in conformity
with what is known about natural and experimental amoebiases.
F