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Introduction
In literature several reports of bovine cerebral babesiosis caused by Babesia bovis, Babesia bigemina and Babesia argentina causing neurological signs like nervousness,
circling, paddling of limbs, ataxia, convulsions, paraplegia, coma and death have been described. (Tehernmoretz, 1943; Zlotnick, 1953 cited by Riek, 1963; Lavine, 1961; Callow and MaGavin, 1963; Jubb and Kennedy, 1963; Soulsby, 1968; Rogers, 1971; Ristic, 1981 and Hadani et al., 1982). Most authors describe clogging of brain capillaries by parasitised erythrocytes and some have also reported such clogging in other organs like kidney, lung, liver, spleen heart and skin.
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In the laboratory, it has been the experience of the authors of this report that when positive blood samples were kept at room temperature for a few hours the smears made from such samples seldom revealed the presence of Babesia organisms. This particular fact of disappearance of the parasites in smears made from aged samples of blood was initially attributed to the severe hemolytic behaviour of the parasites. The wet-mount method of diagnosing haemoprotozoan infections (Setty, 1975) provided the necessary breakthrough, in that the parasitised erythrocyte clumps were still evident in old samples of blood suggesting that a mechanism other than hemolysis was responsible for not finding the parasites in smears made from aged samples of blood. It was inferred that in blood samples that are a few hours old, the erythrocyte clumps either settled to the bottom of the tube or did not appear in the smears made or if in the smear it got washed out during the staining process rendering the smears negative. Several hypotheses have been postulated to explain the mechanism of clumping of parasitised erythrocytes. Losos et al. (l980) and Ristic and Krier (l981) suggested an immune mechanism for such preponderance of parasitised erythrocytes (clumps) in brain capillaries. Jan Klein (l982) described that human patients with malaia have extra-ordinarily high levels of on immunoglobulin M (IgM) causing spontaneous agglutination of erythrocytes.
Therefore in the present investigation an attempt was made to find out the possible
role of IgM in the formation of erythrocyte clumps be splitting the IgM molecule, if any, into its monomers by mild reduction with 0.01 M-2-Mercaptoethanol as suggested by Jan Klein (l982).
Materials and Methods
Fresh blood samples from cattle with acute babesiosis were collected in vials using either sodium citrate or heparin as an anticoagulant. Eleven samples from cattle with parasitaemia ranging from 2 to 10 % were initially examined by stained blood smears as well as the wet-mount method (Setty, 1975). From each case, two 2 ml samples of blood were collected in vials with anticoagulant, one sample as control and the other sample for treatment with 2 to 3 drops of 0.01 M solution of 2-Mercaptoethanol. Both samples were allowed to
stand at room temperature for a few hours or left overnight taking care to mix the samples by gentle shaking 2 or 3 times. When ready to examine, both the control and the 2-Mercaptoethanol treated samples were subjected to wet-mount screening method of Setty (l975). Five minutes before examination, 1 to 2 drops of 0.1 % new Methylene Blue stain was added to each sample to stain the erythrocytes and the parasites. A drop of this sample was placed on slide, covered with a cover slip and examined under the oil immersion lens.
Results and discussion
Of the eleven samples examined erythrocyte clumps were detected in all the untreated controls (Fig: 1, 2 and 3) while in nine of the Mercaptoethanol (MCE) treated samples no such clumps were detected indicating
that MCE has prevented the formation of such clumps. Remaining two of the MCE treated samples had clotted preventing microscopy. The new Methylene Blue stain had given a blue tint to the erythrocytes and the parasites had a basophilic stain. The size of the clumps in untreated samples varied from very small comprising of a few erythrocytes to very large comprising of hundreds of erythrocytes.
No satisfactory explanation has been given in literature for the agglutination of parasitised erythrocytes. Callow and McGavin (l963) described intravascular clogging in brain as a heavy brain infection. Rogers (l971) attributes the cause to shock syndrome and multiplication of the parasites and an antigen-antibody reaction coating the erythrocyte surface making them sticky and adhere together. Ristic (l981) considers the phenomenon as due to erythrocyte
charge neutralized by the parasite antigen favouring agglutination Ristic and Krier (l981) consider brain as a site of predilection providing biochemical requirements of the parasite besides suggesting that an immune mechanism resulting in such aggregation of erythrocytes. Losos et al. (l980) point towards a structural change on the erythrocyte surface with antigen-antibody complexes on the surface leading to agglutination. As described, at least some of the authors have pointed towards an immune mechanism resulting in formation of such intravascular clumps blocking the capillaries. Jan Klein (l982) describes that human patients with malaria have high levels of IgM causing spontaneous agglutination of erythrocytes and that IgM could be split into its monomers by mild reduction with 0.01 M 2-Mercaptoethanol at a neutral pH.
As in human malaria, a similar mechanism probably occurs in bovine babesiosis with the involvement of IgM. The IgM molecule being pentavalent having ten antigen-combining sites might have resulted in the formation of large thrombi involving several infected erythrocytes. It is a known fact that parasitised erythrocytes get a coating of parasite antigen on their surface making them susceptible to the action of IgM leading to the formation of
aggregates of parasitised cells. In cattle with babesiosis intravascular aggregates of erythrocytes have been described in other organs besides brain (Callow and McGavin, 1963 and Rogers, 1971). This suggests that the agglutination of erythrocytes occurs in general circulation and when such clumps are carried to vital organs like brain, capillary blockage occurs leading to ischemia causing neurological clinical signs and death.
In the present investigation the finding that Mercaptoethanol treated blood samples did not show any erythrocytes aggregates strongly suggests the involvement of IgM. A quantitative estimation of immunoglobulins in bovine babesiosis could throw more light on the subject and appears to be the most necessary step in understanding the pathogenesis of in
vivo erythrocyte agglutination in bovine
babesiosis.
Bibliography
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Callow,
L.L. and McGavin, M.D. (1963). Cerebral Babesiosis due to B. argentina. Australian Vet. J. 39: 15-21.
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Jan Klein
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Jubb,
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Rogers, R.J.
(1971). Observations on the pathology of B. argentina infections in cattle. Australian Vet. J., 47: 242-247.
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Setty,
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Authors
Corresponding address:
Dr.D.R.L.
Setty,
Former Professor and Head, Dept of Vet Medicine, Veterinary College, Hebbal, Bangalore-560 024
Residence:
114/2. C.N. Palya, II stage, Nagasandra post, Bangalore-560 073, India.
Phone:0091-80-28391792
E-mail: drlsetty@rediffmail.com
Dr. R.N.Ramachandra,
Associate Principal Scientist, Schering-Plough Animal Health Corporation, 21401 West Centre Road,
Elkhorn, NE 68022, USA.
E- mail: rangappa.ramachandra@spcorp.com
Dr.Daniel Rajasekharan,
Staff pathologist, MPI Research, 54943, North Main Street, Mattawan, MI 49071-9399, USA.
E-mail:
danraj@aol.com |
