- Research progress in some important disease problems of sheep and goats: ...
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Research progress in some important disease problems of sheep and goats:
1. Advances in geeldikkop research
EM van Tonder
Regional Veterinary Laboratory
Private Bag X528
Grootfontein College of Agriculture
Middelburg C P
The rust official report on geeldikkop appeared in 1886 although it was well known to farmers in the affected areas of the Cape Province long before the time under the names of "Dikgeel" or "Geeldikkop" (Hutcheon, 1886, cited by Brown, 1959). Hutcheon believed the primary liver derangement to be of possible malarial origin.
Dixon (1894), however, was first in suspecting the dubbeltjie (Tribulus terrestris) or the larvae of a parasitic insect harbouring in the stalks of the plant to be the responsible cause of outbreaks of this disease amongst sheep and goats. His feeding experiments with dubbeltjie plants during the succeeding year nevertheless failed to produce the disease.
On account of these and also subsequent unsuccessful dosing experiments using plant material by Dixon, the belief that the dubbeltjie was involved in the aetiology of geeldikkop was abandoned for the time being. This, together with a wide range of epizootological observations again favoured an insect-transmitted infectious agent of a malarial nature as possible cause. Numerous inoculation experiments with various body fluids and organ pulp emulsions, however, failed to produce evidence to this effect (Dixon, 1899a, 1899b).
This school of thought nevertheless persisted until 1917 when large-scale outbreaks of geeldikkop for the third consecutive year through intervention by Parliament, prompted Theiler and his co-workers to indulge in comprehensive investigations into the problem. In his report, Theiler (1918) concluded that the disease could still have been of a malarial nature notwithstanding the absence of visible parasites in the blood and the negative outcome of exhaustive transmission experiments, or alternatively that its cause was of a vegetable origin. Although feeding experiments at Onderstepoort with Tribulus emanating from farms where the disease was prevalent at the time were similarly negative, Theiler however succeeded in causing geeldikkop for the first time in history in the Luckhoff area by feeding sheep with freshly harvested dubbeltjie plants. The significance of this work implicating the possible existence of a labile toxic component within the dubbeltjie, through a peculiar type of coincidence, was nevertheless overlooked in subsequent research efforts.
During the course of his investigations, Theiler not only disproved the popular theory that the larval parasite (Grammodes stolidae) present in the stems of some plants was responsible for the disease, but also observed the growth of a fungus described as a new species of Colletotrichum on Tribulus plants. Apart from hampering the growth of these plants and speculation on the possibility that it might thus modify the toxicity of the plant, no further reference was made to this particular parasite.
After Theiler's epic contribution to this field of research, ten years elapsed before further relevant information was published. In this work, three apparently similar or related conditions were either described or briefly mentioned, viz enzootic icterus (toxaemic jaundice) and "swelled head" in rams (De Kock, 1928a, 1928b, 1928c) and "dikoor" in young lambs(Steyn, 1928).
Although dikoor simulated geeldikkop very closely, its cause was attributed to Panicum maximum (blousaadgras), a grass type which predominated on the old lands on which the animals grazed. It is interesting to note that a fungus (later identified as Ustilago spp.) was again found severely to infect these plants, but it proved to have been of no significance in the aetiology of the condition when infected plant material was fed to experimental sheep. Thus it was the second time that a possible fungal cause was considered.
Nevertheless, these newly described conditions, which later proved to be unrelated entities, undoubtedly played an important part in confounding the existing concepts pertaining to the geeldikkop syndrome at the time; a situation which subsequently became more complicated and confused, and was to prevail for many years to come.
To add to the scientific tangle, Quin (1928, 1929) reported on grazing and drenching experiments performed in Victoria West and Burgersdorp where T. terrestris, immediately after collection, was again successfully employed in the production of geeldikkop; thus for the second time in history. Again, for some or other reason, these findings seem to have been ignored in subsequent research efforts into the problem.
The extensive period of almost half a century which then followed was marked by the identification of even more suspected plants or other toxic agents, and inexhaustible efforts and intensive research into the fundamental aspects involved in the development of jaundice and photosensitization in sheep by various workers and collaborators.
At first Quin and his co-workers produced only sudden deaths as a result of methemoglobinaemia in sheep drenched with extracts of dried Tribulus (Quin, 1930), which subsequently proved to be due to the presence of inorganic nitrites in the dry plant (Rimington & Quin, 1933a, 1933b).
In subsequent work, using various fluorescent dyes by intravenous injection, Quin (1931, 1933a) produced intense photosensitization but no icterus.
This was followed by a series of drenching experiments whereby various plants were shown to cause photosensitization and/or icterus. Hypericum aethiopicum (St John's wort) produced photosensitization but no icterus Lopholaenia coriifolia (Geelbossie) caused severe liver damage and mild icterus but no photosensitization, while Lippia rehmannii (Laventelbos) and Lippia pretoriensis produced apparently typical symptoms of geeldikkop (Quin, 1933b, 1933c, 1933d).
Further work on the surgical obstruction of the normal bile flow of sheep (Quin, 1933e) eventually proved that the plant pigment phylloerythrin, a breakdown product of chlorophyll, was mainly responsible for photosensitization in experimental sheep and that it also occurred in natural cases of geeldikkop (Quin & Rimington, 1933).
However, isolation of this substance responsible for the development of photosensitization did not explain the occurrence of icterus in animals drenched with L. rehmannii but showed no photosensitivity when kept on chlorophyll-free diets, or for that matter the icterus associated with geeldikkop.
During the course of 1935, Rimington & Quin (1935) announced the isolation of an icterogenic principle from L. rehmannii which they called icterogenin and which, upon dosing to sheep, produced symptoms normally observed in geeldikkop and Lippia poisoning.
Although this active substance could be recovered from Lippia plants with ease, none of its crystalline forms, icterogenin A or B for instance, could be isolated from Panicum species presumably responsible for an outbreak of geeldikkop (Rimington, Quin & Roets, 1937), while no reference of any attempts to recover it from the dubbeltjie, could be found.
Geeldikkop, a term which developed around the particular syndrome that originally and almost exclusively occurred in certain areas of the Cape Province in close association with Tribulus infested pastures, was now commonly and conveniently used to denote virtually any form of icterus and photosensitivity in sheep and goats.
Further confusion presented itself when yet another two plants were added to the aetiological list. Van der Walt & Steyn (1939) incriminated Asaemia axillaris (vuursiektebossie) as a cause of geeldikkop but the toxic principle was never identified. In 1940 (Steyn & Van der Walt, 1941) an outbreak of icterus and photosensitization in cattle was associated with Lantana camara, an ornamental plant which, upon drenching to sheep, caused a condition essentially similar to geeldikkop and Lippia poisoning. From the latter plant Louw (1943) was able to isolate a compound capable of producing icterus and photosensitivity and named it Lantadene. In subsequent publications (Louw, 1948, 1949a, 1949b), this substance was renamed Lantadene A, while an accompanying physiologically inactive product was given the name Lantadene B.
The discovery of specific poisonous sources responsible for geeldikkop-like conditions seems to have been concluded temporarily when the toxic alga, Microcystis toxica, on account of the severe hepatotoxin it produced, was also reported as a causative agent of icterus and photosensitization.
In the period that now followed research activities on the icterus-photosensitivity syndrome was mainly concentrated on the basic chemistry of suspected plants, but for some or other reason T. terrestris was mostly excluded.
Henrici (1952), however, showed renewed interest in the chemistry of Tribulus and in her work on the starch and sugar content of the plant under various conditions reported on the appearance of a saponin-like substance in the plant on soils deficient in zinc. No evidence in support of this view was however forwarded.
After a relatively short quiescent period at the geeldikkop research front investigations were once more resumed on a large scale in 1955 by means of a joint research project between Onderstepoort and the Council for Scientific and Industrial Research. The occurrence of saponins in Tribulus as reported by Henrici (1952), was re-examined by Enslin & Wells (1956). They were able to isolate two sapogenins from Tribulus plants namely sapogenin A, which proved to be identical to Diosgenin, and sapogenin B, a new steroidal product.
Once again earlier inoculation experiments were repeated, using blood from affected animals during a severe outbreak (Adelaar, cited by Brown, 1959), but again without success.
In view of the dramatic advances in basic research in the fields of bile pigment and porphyrin metabolism and the genesis of icterus, a comprehensive study on the biochemistry of bile pigment excretion in sheep and the chemical pathology of various forms of jaundice was initiated (Brown, 1959). This research entailed the development of tests for hepatic function of sheep and studies on the conjugation and excretion of bile pigments and dyes, the chemical pathology of experimental bile duct ligation, icterogenin induced jaundice in comparison to natural cases of geeldikkop, haematological and histopathological changes in all conditions concerned and eventually also studies on renal function, copper metabolism and electrolyte balance. From this work it became clear that the icterus and dye retention in icterogenin poisoning and natural cases of geeldikkop were due to a biochemical lesion affecting the hepatic conjugation of bile pigments and dyes and their excretion. A close relationship between geeldikkop and enzootic icterus was also established.
Probably urged by the isolation of sapogenins from T. terrestris (Henrici, 1952; Enslin & Wells, 1956) and earlier evidence of inconsistent toxicity of the plant, appreciation for the possible role of apparently labile toxic substances present in Tribulus again materialized at this stage of the investigations. The necessity for much of the research work to be carried out under field conditions during the times that the disease occurred was fully realized, and such undertakings were then also made possible by the acquisition of a mobile laboratory unit donated by the Stock Diseases Research Fund.
During these field investigations no fewer than four sapogenins obtained from the crude saponins present in Tribulus were isolated and characterized (De Kock & Enslin, 1958). Some of these compounds were also tested for icterogenic activity, but the characteristic features of geeldikkop could not be produced (Brown & De Kock, 1959).
At the same time the possible role of a fungus in the genesis of geeldikkop, as indicated by the earlier work of Theiler (1918) and Steyn (1928), was reinvestigated.
In the course of extensive investigations conducted during 1958 and 1959, a total of 23 different fungi growing on Tribulus during outbreaks were isolated in pure culture (Brown, 1959a, 1964). These fungi which included Culvularia, Penicillium, Fusarium, Helminthosporium, Rhizopus, Alternaria, Myrothecium and Hendersonia species were dosed to sheep but at no time could the symptoms or biochemical lesions of geeldikkop be produced (Brown, 1964).
In another investigation carried out by the National Chemical Research Institute, 130 pure cultures, mostly Alternaria tenuis, were obtained from T terrestris and other plant debris collected from farms in various districts where geeldikkop occurred. Only two isolates proved to be toxic to sheep but did not produce pathological signs of geeldikkop (Gouws, 1965).
The negative outcome of these investigations, the many unanswered questions pertaining to the geeldikkop syndrome and the fact that by the methods followed no substance capable of causing geeldikkop could have been isolated from Tribulus up to that stage prompted Brown and his co-workers (Brown, 1959b, 1962, 1963, 1964, 1966; Brown & De Boom, 1966; Brown & De Kock, 1959; Brown & De Wet, 1962) to discard the idea that T terrstris had anything to do with geeldikkop, other than a secondary role, and to search for other possible explanations.
In renewed research efforts to elucidate the underlying cause of geeldikkop, the role of selenium in the aetiology of the disease was comprehensively studied (Brown & De Kock, 1959; Brown & De Wet, 1962; Brown, 1962, 1963, 1964, 1968).
During the course of these studies, the belief that geeldikkop and enzootic icterus were related, being different manifestations of the same disease only, also emerged.
The underlying cause of geeldikkop was then believed to be a subclinical chronic selenosis which disrupted different enzyme systems, notably those connected with the selective permeability of cell membranes and the glycolytic cycle (Brown, 1962, 1963, 1968). The theory was based on evidence such as the presence of potentially dangerous amounts of selenium in plants from geeldikkop and enzootic icterus-prone areas (Brown & De Wet, 1962), higher levels of selenium in certain body tissues of sheep emanating from such areas (Brown, 1968) and the relationship between the selenium content of the veld and the incidence of the disease (Brown & De Wet 1962; Brown, 1968). The possibility of some or other infectious agent playing a role in precipitating either of the two manifestations of geeldikkop was also proposed (Brown, 1968).
Although this work appeared to have concluded the search for an aetiological explanation of geeldikkop, probably the most extensively and intensively researched problem in the history of this country, and general interest from the scientific establishment faded, many questions remained unanswered, the most pertinent one being the inexplicable impotence to experimentally reproduce the classical condition.
REGIONAL RESEARCH PROGRESS
Since the inception of the Regional Veterinary Laboratory, Middelburg Cape, at the end of 1%6, individual members of its staff, when requested, co-operated from time to time in the geeldikkop research programme. At this stage, activities were mostly concentrated on the possible role of selenium and the possible contribution of infectious agents in precipitating the condition (Brown, 1968).
The finalization of the research efforts of Brown and his collaborators, as well as the familiar temporary decline in the incidence of geeldikkop, caused research interest in the problem to dwindle to a level of sporadic epizootological investigations only.
Motivated by their splendid achievements in elucidating the causes of Lupinosis and facial eczema, in themselves two persistent and evasive disease conditions over many years, researchers showed renewed interest in the problem of geeldikkop. Since Lupinosis was shown to be caused by the fungus Phomopsis leptostromiformis (Van Warmelo, Marasas, Adelaar, Kellerman, Van Rensburg & Minne, 1970) and a fungus, Pithomyces chartarom, was also confirmed as the aetiological agent associated with facial eczema in South Africa (Marasas, Adelaar, Kellerman, Minne, Van Rensburg & Burroughs, 1972), the possibility that the actual cause of geeldikkop could similarly be of mycotoxicological origin, was again contemplated. Such a possibility had of course been considered in the past (Theiler, 1918; Steyn,1928; Brown, 1959a).
A new research team was thus re-established, composed of members of the Veterinary Research Institute, Onderstepoort, and the Regional Veterinary Laboratory, Middelburg Cape. Initially it was envisaged that all efforts be aimed at fungal investigations only, but on local insistence it was agreed to re-investigate the role of T terrestris in the aetiology of geeldikkop, particularly since the only successful reproduction of geeldikkop in history was obtained by feeding this plant. In addition to co-operative involvement in the fungal investigations, the re-examination of other causes of geeldikkop, and particularly the role of T. terrestris, mainly became the responsibility of the Regional Laboratory.
The first outbreak of geeldikkop in which the newly established team was to operate in a concerted effort occurred in the late summer and early autumn of 1971 at the Grootfontein College of Agriculture.
In the first experiment to exclude the possibility of a transmissible agent, blood from acute natural cases were intravenously infused into susceptible lambs, but as in previous experiments (Dixon, 1895; Paine, 1904; Theiler, 1918; Adelaar, 1959) these attempts were unsuccessful (Van Tonder, Basson & Van Rensburg, 1972).
During this outbreak and a subsequent one on the same property which occurred a month later, exhaustive observations and a thorough search were made to obtain evidence of fungal growth on T. terrestris, which was present in abundance in the wilted stage, or any other plant material that could likely have been involved. As conditions at this stage were hot and dry, no clearly discernible signs of fungal parasitism were evident and certainly not those associated with Pithomyces chartarum, the cause of facial eczema, which was also considered to be the more likely organism to be involved in the aetiology of geeldikkop-type syndromes. Several other types of fungi were however isolated, one of which was described as new species viz Pithomyces Karoo (Marasas & Schumann, 1972). Apart from having been procured from living and dead plant material, as well as from T. terrestris at the time when geeldikkop occurred, these fungi proved to be of no significance (Marasas, Adelaar, Kellerman, Minne, Van Rensburg & Burroughs, 1972; Kellerman, Van der Westhuizen, Coetzer, Roux, Marasas, Minne, Bath & Basson, 1980).
At the same time two different sets of feeding experiments in sheep withT terrestris were also carried out at the Regional Veterinary Laboratory. In both experiments care was taken to use only freshly harvested plant material as this appeared to have been the only common factor in previous successful attempts to produce geeldikkop experimentally (Theiler, 1918; Quin, 1928, 1929). This, in contrast with repeated unrewarding efforts by these and subsequent workers, where before administration the collected plant material was either preserved in the dried state or subjected to some considerable delay, suggested the possibility of labile toxicity of the plant.
In the first of these experiments, the sheep were housed at the Laboratory and fed on dubbeltjie exclusively. Plant material was collected each morning from a camp in which geeldikkop occurred at the time and Tribulus was present in abundance in the wilted form. This material was made available to the sheep in the shortest possible time
In the second experiment, sheep were confined in a small movable paddock, placed on patches of wilted Tribulus in the camp where geeldikkop was experienced. The paddock was moved once or twice daily to ensure adequate feeding material, while on each occasion all other plants were meticulously removed so that only Tribulus was left to be eaten. In both these experiments, typical geeldikkop was indisputably induced with the utmost ease; thus the third time in history that this disease could be experimentally reproduced and the first time in almost half a century that elapsed since the previous successful attempt (Van Tonder, Basson & Van Rensburg, 1972). From the outcome of this work, compared with previous unsuccessful attempts where Tribulus was employed, it was postulated that a toxic substance, be it an unstable aberrant metabolite or a mycotoxin within the plant or in adhering fungal spores on its surface, even in the absence of discernable fungal involvement, could be the responsible factor.
Whereas P. chartarum, the causal organism of facial eczema, could not be isolated from Tribulus material during the 1971 outbreaks, it was, however, isolated for the first time during outbreaks in the Aberdeen area at the end of 1972 from Tribulus material devoid of conspicuous signs of fungal presence. Toxicity experiments, also employing this isolate, were however reported much later (Kellerman et al 1980).
In view of the apparent lability of the toxic factor associated with 1: terrestris, the Middelburg Laboratory, during the Aberdeen outbreak of geeldikkop, investigated various methods of extraction and preservation of the toxic substance. To confirm the toxicity of the plants, sheep were allowed to graze on wilted terrestris in the camp where the, natural outbreak occurred. Geeldikkop was again reproduced in these experimental sheep Although none of the extraction methods used proved to be successful, toxic dubbeltjie, preserved by immediate chilling in ice boxes and freezing at -150 C, reproduced classical geeldikkop when dosed to a sheep 6 weeks later in a total dose of 3 kg (Bath, Van Tonder & Basson, 1978).
In subsequent mild and also more severe outbreaks of geeldikkop in South West Africa (1973, 1974), the Central and North-western areas of the Cape Province (1976,1979) and the Orange Free State (1978), P. chartarum was isolated from T. terrestris as well as other forms of plant debris found in the toxic camps. Additionally no fewer than 29 different species of fungi, belonging to 19 genera, were also isolated from Tribulus during these investigations (Kellerman et al 1980). No specific reference to toxicity experiments with these isolates, except P. chartarum, could be found and it would therefore appear that they were regarded as non-significant.
A series of dosing experiments were however initiated, using cultures of a Karoo isolate (GA 10) of P. chartarum exclusively (Kellerman et al, 1980). In the first of these experiments, Merino sheep, bred and raised at Onderstepoort, were dosed with pure cultures at various dose rates, calculated on the equivalent of the mycotoxin, sporidesmin, contained in the culture. Apart from acute deaths at certain levels of dosing, typical signs of hepatogenous photosensitivity and icterus were shown by animals that survived long enough to do so. However the macro- and microscopical features were indeed typical of facial eczema (Mortimer,1963; Kellerman et al, 1980) and could not be confused with geeldikkop (Theiler, 1918; Brown, Le Roux & Tustin, 1960; Van Tonder et al, 1972).
A second trial was then conducted, where sheep raised in the Karoo were dosed with cultures of P. chartarom immediately on arrival at Onderstepoort, while a similar group of sheep, in addition to the administration of the fungal material, were allowed to feed on Tribulus pastures. Whilst the animals included in the first part of the experiment developed photosensitivity accompanied by pathological changes consistent with facial eczema, those kept on Tribulus grazing after being dosed with P. chartarom cultures, not only developed photosensitivity but also showed pathological and particularly microscopic changes characteristic of both facial eczema and geeldikkop (Theiler, 1918; Brown et al, 1960; Mortimer, 1963; Van Tonder et al, 1972). Two control sheep running on T. terrestris pastures at the same time, without being dosed with P. chartarom, although they did not become photosensitive, nevertheless showed subclinical signs of geeldikkop as was evidenced by localized macroscopical lesions in the liver and the microscopical demonstration of deposits of crystalloid material in the liver cells which are characteristically found in geeldikkop and not in facial eczema (Theiler, 1918; Brown et al, 1960; Mortimer, 1963; Van Tonder et al, 1972).
In the final experiment, Karoo sheep, one group on predominantly T terrestris pastures and another group on veld with almost no T terrestris present, were dosed at realistically reduced dosage rates so as to simulate natural outbreaks of sporidesmin photosensitivity, while a new supposedly subclinical dosing level was also included. Control animals were included in both groups.
A notably larger proportion of the dosed sheep on predominantly T terrestris pastures, as well as one control animal, became photosensitive, and had typical histopathological lesions of geeldikkop in their livers. Furthermore, similar characteristic lesions were also encountered in cases classified as subclinically affected, while focal lesions and crystalloid material in the bile ducts were also seen in the other apparently unaffected control animals.
Only two animals from the group dosed on the veld with almost no T terrestris developed photosensitivity and showed typical facial eczema lesions in their livers, while some of them, which were dosed at the higher levels but failed to show signs of photosensitivity, had similar mild to more severe liver lesions. None of the control sheep in this group showed lesions of facial eczema or geeldikkop (Kellerman et al, 1980).
In all, these experiments failed to elucidate the true cause of geeldikkop, although the important role of T. terrestris in the aetiology of the condition was again emphasized and the dissimilarity of geeldikkop and facial eczema was also clearly demonstrated. Evidence to the effect that P. chartarum could be of some aetiological significance in the occurrence of geeldikkop, even in a more remote capacity, also seems to be inconclusive.
In pursuance of the success attained by preserving toxic T1'ibulus material through freezing (Bath et al, 1978), further efforts were made during subsequent major or minor outbreaks of geeldikkop, not only to determine the optimal temperature for preservation and maintenance of toxicity of the preserved material, but also to examine methods for the extraction of the toxic substance.
Investigations were carried out in the following districts during each particular year that geeldikkop occurred: 1976 (Aberdeen, De Aar, Middelburg, Richmond), 1978 (Carnarvon), 1979 (Jansenville, Middelburg, Murraysburg, Richmond), 1981 (Middelburg, Graaff-Reinet, Jansenville, Beaufort West), 1983 (Steytlerville, Middelburg, Murraysburg) and 1986 (Richmond, Carnarvon, Murraysburg).
In each case it was endeavoured to visit farms where outbreaks of geeldikkop had just commenced and to collect and process T1'ibulus plants from the same camp where the sheep were running at the time. On each occasion experimental fistulated sheep were either given freshly harvested material at the onset of activities or freshly preserved dubbeltjie on arrival at the Laboratory in order to confirm its toxicity.
In all these investigations, it was found that classical geeldikkop could be caused at will whenever toxic T terrestris was available (Bath & Smith, 1979). Failures to do so were almost always accompanied by unforeseen circumstances rendering the dubbeltjie non-toxic, such as delays in the collection of material in relation to the onset of the outbreak and sudden spells of rain. In most of these cases, the visual appearance of the T1'ibulus plants also deviated from the recognised toxic form.
Through lack of sufficient freezing space, potentially harmful T1'ibulus was also stored at ordinary refrigeration temperatures (0 - 4 0 C). Such material was still found to be toxic when tested up to 4 months later. Frozen T terrestris on the other hand was found to have retained its toxicity when administered to experimental sheep approximately 8 months later. In both instances, these periods cannot be regarded as the endpoint of preserved toxicity, since supplies of material thus preserved became exhausted (Bath & Smith, 1981).
Repeated attempts to extract the toxic substance from freshly collected or preserved confirmed poisonous plants by various methods proved unsuccessful. This was regarded as being due to the toxic substance either being insoluble in the extraction fluids (distilled water, saline and vinegar solutions) used, or that it was ensconced within the plant in such a way that it could not be regained at all, or in sufficient concentrations, to render the extracts toxic. The employment of mechanical devices in order to effect various degrees of contusion and maceration of the plant material prior to extraction proved to be equally unsuccessful, thus making the efficiency of the solvents used to become suspect (Bath, Mollett & Smith, 1980).
Preservation and/or extraction using volatile substances, such as ether and acetone, proved unsuccessful in all respects.
Progress became evident however, when alcohol, either pure or in various dilutions, was employed as extraction medium. Eventually treatment of macerated freshly harvested or frozen plant material, the whole process completed under cold room conditions (0 - 4 0 C), proved to be the most rewarding procedure. Clinical geeldikkop could often be caused by administration of these extracts, while the residual plant material failed to do so (Bath & Smith, 1981). Inconsistent results often obtained with the bulky alcoholic extracts of confirmed toxic T terrestris prompted investigations into the possibility of increasing the concentration of the toxic substance in the prepared extracts.
In order to increase the concentration of the toxic factor in the extracted product, bulk freeze-drying was resorted to, whereby the initial volume could be reduced to a variable extent on reconstitution of the freeze-dried material. This proved to be most successful in the sense that geeldikkop could be produced more readily with extracted material, which in any case could be stored and handled more conveniently. Apart from obtaining more potent extracts, dosing experiments to date have also confirmed the potency of the freeze-dried extracts to last for at least 2 years (Bath, Joubert & Smith, 1986). The success of the entire procedure of preservation and extraction of the toxic principle in T terrestris is substantiated by the fact that geeldikkop can be successfully induced b)f the administration of reconstituted freeze-dried extracts, whereas it is not possible to do so with the residual plant material. More-over it was found that geeldikkop could be reproduced with the extracts derived from about 4 kg of preserved toxic T terrestris, whereas :t 3 kg of the same material given intact, was required to produce similar results (Bath & Smith, 1981; Bath, Joubert & Smith, 1986).
Since doubt existed whether all possible fluids were regained from the soaked plant material by the use of a locally devised compressor (Bath, Mollett & Smith, 1980), a centrifugal system was developed, and through comparative operations has already proved to afford a more efficient method for the collection of extracts (Joubert, Mollett & Smith, 1986). Comparative tests are underway to determine whether, apart from diminished losses in volume, higher concentrations of the toxic substance could also be effected by this method.
From the afore-said, it is evident that the Regional Veterinary Laboratory featured prominently in advances made in geeldikkop research. Activities aimed to incriminate or exclude a fungal cause, were mostly co-operative but extensive, and were also fundamental in proving fungal involvement to be of negligible importance.
The Regional Laboratory and staff were however mainly involved in re-investigations of the role played by T terrestris. In this field, highly significant advances have been made and great strides taken towards elucidation of the cause of geeldikkop. Not only has T terrestris in its toxic stages repeatedly and indisputably been shown to cause geeldikkop, and has toxic material been successfully preserved for periods of up to two years, but the toxic principle has also been successfully extracted.
Presently, all efforts are directed towards the characterization and identification of the toxic substance or substances.
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Karoo Agric, Vol 3, No 7, 1986