THE POSSIBLE ROLE Of AN INFECTIOUS AGENT IN THE PRODUCTION OF THE ACUTE MANIFESTATIONS OF GEELDIKKOP AND ENZOOTIC ICTERUS
- - - - I. Introductory remarks
2. Animals, infectious agents and methods
3. The haematology and chemical pathology of mild bluetongue infections:
(a) Haematology (b) Liver function
(c) Carbohydrate metabolism (d) The state of the erythrocytes (e) Kidney function
(f) Adrenal function and electrolyte balance 4. General discussion
1. Introductory remarks
Many references have been made in the preceding chapters to the facts that the acute manifestations of geeldikkop and enzootic icterus appear to be precipitated by a variety of non-specific agents, best defined for the purposes of this paper as
" stress factors ". Factors like sudden severe climatic changes, sudden changes in the nature of the natural pastures, e.g. lush growth followed by wilting and severe gastro-
intestinal stasis which could for instance be induced by these changes or by the 502
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saponins present in fast-growing invading annual plants like T. terrestris, appear to be operative in this regard in both instances (Brown, 1963). There is a considerable body of evidence, a lot of which has been presented in the preceding chapters, indi- cating that an infectious condition may be the main stressing agent which provokes the acute manifestations in the case of geeldikkop. The reader is reminded, for instance, of the severe haematological and metabolic upheavals present in the pro- dromal cases of the disease and of the hypergammaglobulinaemia which is present in typical cases from the onset of symptoms. The author has set out in an earlier paper (Brown, 1966a) a critical analysis of the symptomatology of geeldikkop and has indicated which symptoms are germane to this disease and also which symptoms belong properly to a relatively mild disease of probable viral origin.
It has been indicated in Chapter 10 that the cases of enzootic icterus which were studied fell into two categories as far as their manner of production was con- cerned, i.e. those which were precipitated by transport in confined spaces over long distances and subjected to drastic changes in diet and those which were collected during severe epizootics of the condition in which an infectious agent could well have been operative.
It was quite obvious from all our studies and those of previous workers that if an infectious agent was operative in precipitating outbreaks of either syndrome, it was not of bacterial nature. The severe epizootics of both conditions occur in the mid- and late summer months after the heavy annual rains. This is a period in which there is germination and rapid growth of a very large number of annual plants.
It is also a period in which there is a marked upsurge in insect life as was found out through bitter experience during the many summer months spent working in the field.
The epizootiology, symptomatology and haematology suggest an insect-borne virus infection. Repeated complaints from farmers in the areas concerned have been heard that the vaccine in current use against bluetongue does not give adequate protection in young animals. Some of these cases are undoubtedly due to negligence and careless use of the vaccine, but many have been shown to be geniune. The symptomatology of the disease suggests an infection which produces many symptoms reminiscent of a mild attack of bluetongue, an insect-borne disease (Brown, 1966a).
For these reasons it was therefore decided to study the effects of mild bluetongue infections on sheep and to see whether any of the chemical pathology of geeldikkop or enzootic icterus could be reproduced by viruses of this nature. Since it was postulated that the infectious agent precipitates the acute outward manifestations of a largely sub-clinical disease entity (Chapter 13) it was obviously of importance to study the effect of bluetongue viruses on sheep which were newly obtained from areas where geeldikkop and enzootic icterus normally occur as well as on sheep born and raised elsewhere.
2. Animals, infectious agents and methods
A total of thirty-nine adult Merino wethers drawn from the pool of available sheep at Onderstepoort were used for this work, together with eight clinically normal Merinos obtained during a geeldikkop-free period from a farm on which the disease has occurred regularly. The data pertaining to plasma levels of certain enzymes were supplemented by similar data obtained from six typical early cases of geeldikkop (sheep 22860 to 22865) and two early cases of enzootic icterus (sheep El and £2) obtained from affected farms during severe recent outbreaks of the two syndromes.
The clinically normal Onderstepoort and Karoo animals were infected with a strain of bluetongue virus which produced in a number of cases a clinically very mild disease entity, some seven to ten days after infection by the intravenous route.
BIOCHEMICAL STUDIES ON GEELDIKKOP AND ENZOOTIC ICTERUS
This disease entity consisted in most cases simply of a sudden fever reaction lasting two to three days, during which time body temperatures of over 104 °F were found in affected animals. In the majority of affected cases no particular clinical symptoms of note were observed, not even anorexia being shown by many of the affected animals.
In only a few instances were erosions seen on the external internasal septum and the dorsal and lateral aspects of the tongue and coronary band haemorrhages.
The virus strains used were supplied by Dr. P. G. Howell, of the Virus Research Section of this Institute, and were strains isolated in the field and designated by him "Type 1 B-Berg" and "Type 3 Cyprus".
The animals were housed, maintained and fed as described earlier in this work for the experiments performed at Onder~tepoort and the various methods employed in these studies were as used in all the work described previously. The animals were bled for the various studies before injection and thereafter every second day until the time of the expected fever reaction when daily bleedings were done until the fever had subsided. The animals were bled thereafter every second or third day for a further period of three weeks before being discharged from the experiments.
Only the data obtained from animals which showed the typical fever reaction have been used for the discussion which follows.
3. The haematology and chemical pathology of mild bluetongue infections (a) Haematology: Injection of the bluetongue virus strains produced in nine of the clinically normal Onderstepoort sheep a fair anaemia during the three weeks following the start of the reaction to the virus infection (which as has been said, lasted only two to three days itself). Haemoglobin and packed cell volume values and red cell counts fell in these sheep from 9 · 7 to 13 · 0 gm per cent, 28 to 43 per cent and 8 · 9 to 14 · 9
x
106 feu mm respectively before infection, to 6 · 6 to 8 · 6 gm per cent;18 to 27 per cent and 7 · 0 to 7 · 2
x
106 feu mm three weeks after the start of the fever reaction. Calculation of the absolute haemiltological indices in these cases showed the anaemia to be hypocythaemic normocytic and normochromic.White cell counts remained unchanged in all but two of the animals. These two cases developed mouth lesions, the appearance of which was followed by the develop- ment of a leukocytosis. Total leukocyte wunts rose in these two instances from baseline values of 5,800 to 7 · 900/cu mm to 12, I 00 to 30,000 during the development of the lesions. Differential white cell counts showed this leukocytosis to be due to a severe neutrophilia, which was accompanied by a relative lymphocytopaenia and monocytopaenia.
Three of the eight normal animals emanating from the areas where geeldikkop is prevalent showed a temperature reaction and developed subsequently a similar mild anaemia, characterized in these cases as a hypocythaemic macrocytic hypochromic anaemia. This anaemia developed during the three weeks following the start of the fever reaction. No changes in the total leukocyte counts were noted in these cases.
The decline in haemoglobin values, red cell counts and packed cell volume percentages after the start of the fever reaction in both groups of animals was not associated with a similar change in the total plasma protein levels and was thus in all likelihood not due to water retention and consequent haemodilution.
Erythrocyte sedimentation rates remained unchanged in all the animals which reacted to the infection.
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J. M. M. BROWN (b) Liver function! Most of the animals which showed a temperature reaction developed a very mild hyperbilirubinaemia; no values higher than 0·75 mg per cent of total bilirubin were recorded for plasma samples from any of the animals and the pigment present appeared to be mainly bilirubin.
The urinary excretion of coproporphyrin was studied in eight of the Onderste- poort sheep and all eight of the Karoo animals which were infected with bluetongue virus. The amount of coproporphyrin excreted daily by the individuals in the first group of animals ranged from 0 · 20 to 17 · 62 mcg/24 hours before infection. Five of these animals developed the typical fever reaction. In three of these five animals the coproporphyrin excretion slowly increased after the appearance of the fever to reach values of 30 · 40 to 40 · 36 mcg/24 hours six to seven days after the start of the fever reaction. Coproporphyrin excretion returned to the previous baseline levels after this. No change was observed in its excretion pattern in the remaining two animals which showed the typical fever of short duration. Three of the Karoo animals reacted to the infection and all three showed a similar transient increase in urinary coproporphyrin excretion as found in the three Onderstepoort sheep just mentioned.
Plasma phylloerythrin levels were not determined in any of these cases. All were however tested for photosensitivity by solar irradiation for about two hours each day for the two-week period following the start of the fever reaction, in the case of the Onderstepoort animals. The sheep emanating from the Karoo were kept in metabolism cages fully exposed to direct sunlight throughout the whole experiment. The latter animals which reacted to the infection and which showed increased urinary coproporhhyrin excretion became mildly photosensitive during the entire period over which the urinary excretion of this porphyrin was elevated.
The animals concerned showed as symptoms of this phenomenon, hyperaesthesia, pruritus, hyperaemia and mild oedema of the lips, face, eyelids and ears and exposed shorn areas of the back. Marked hyperaemia, oedema and pain in the coronary bands of all four feet were also seen in these cases. The same symptoms were discernible in the Onderstepoort sheep which reacted but to a much less obvious degree. Composite Fig. 15 shows these symptoms in one of the Karoo animals at the time when its urinary coproporphyrin excretion was maximal.
No changes of any particular significance were found in the total plasma protein levels of any of the sheep which developed a fever reaction. In come of the animals studied, the y-globulin fraction increased markedly very soon after the appearance of the fever reaction, maximum levels of this plasma protein fraction being attained about two to three weeks after the start of the fever reaction. Thereafter these y-globulin levels slowly fell once more towards the pre-infection values. Albumin levels tended to decrease as they-globulin fraction rose and vice versa towards the end of the experiments concerned. The data from one of these animals are repre- sented in Fig. 16 by tracings of cellulose acetate strip electrophoretograms made from plasma samples at the intervals indicated. The trends described above are obvious.
Plasma iron levels were studied in five of the Onderstepoort sheep and all of the animals emanating from Karoo farms. In general, no changes of any significance from the pre-infection range of 151 to 286 meg per cent found for all these cases were noticed. Total plasma copper levels remained unaltered in the same animals, the range before and after infection being II 0 to 200 meg per cent for the whole group.
BIOCHEMICAL STUDIES ON GEELDIKKOP AND ENZOOTIC ICTERUS
1 2
3 4
FIG. I 5.- Photosensitivity reaction following injection of a mild strain of bluetongue virus into a sheep emanating from an area where geeldikkop is enzootic:
1. Swelling of the face, nostrils and lips
2. Front view of the same sheep. The angle at which the ears are held is typical of what occurs as a result of oedema during a photosensitization reaction 3. Close-up view of the nasal region of the same sheep. Note the erosion on the
internasal septum
4. Swelling of the coronary band and surrounding tissues in the right forelimb of the same animal
Bromsulphalein clearance tests were performed on a number of the cases studied at various intervals throughout the fever reaction and during the two weeks which followed. In no instance was any failure to clear the dye completely from the blood within the thirty-minute test period noticed.
In three separate publications attention has been drawn to the fact that the levels of activity of certain enzymes in plasma may rise sharply some eight days after the height of the febrile reaction to bluetongue viruses and that such elevations may persist for a further week or so. The cause of the increase of these enzymes in
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