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This article was published in 1979
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Diagnosis of Bacteroides nodosus Infections in Sheep and Cattle
D.J. Stewart, B.V.Sc., Ph.D., W.D.A. CSIRO Division of Animal Health McMaster Laboratory, Glebe. N.S.W.
The differential diagnosis of Bacteroides nodosus infections in sheep is a perplexing problem to the field veterinarian and the inability to readily differentiate between ovine benign and virulent footrot has hampered progress in footrot control particularly in areas where compulsory quarantine and treatment programmes have been introduced. The recognition of the occurrence of B. nodosus infections in cattle has added further complications in relation to the epidemiology and control of ovine virulent footrot. I propose to discuss recent research developments which may assist the veterinarian in diagnosis and differentiation of benign and virulent footrot.
CLINICAL DIAGNOSIS
Benign footrot is considered to occur on properties from which virulent footrot has been eradicated or following the introduction of sheep infected with benign B. nodosus organisms. In both benign and virulent footrot B. nodosus is the specific transmitting agent but in benign footrot the organism is less invasive due to its lower virulence. Differential diagnosis is complicated by the resemblance of early or static footrot to that of benign footrot since both produce inflammation of the interdigital skin. Virulent or progressive footrot is further characterised by extensive separation which commences at the heel, extends to the sole and toe and frequently extends down the abaxial wall with the formation of a foul-smelling, necrotic exudate. In benign or non-progressive footrot (scald) occasional separation of the horn of the heel bulbs and posterior sole region occurs but there is little accumulation of necrotic material under the separated horn (Thomas 1962, Egerton and Parsonson 1969). A further characteristic of benign footrot is that it rapidly regresses after topical treatment or after the advent of dry environmental conditions (Egerton and Parsonson 1969) whereas virulent footrot tends to persist after the onset of dry conditions unless intensive treatment is undertaken.
My own observations in pen challenge experiments indicate that there is quite a marked variation in the type of lesions produced by various benign isolates with the disease spectrum produced being distinct from that produced by virulent ones (Stewart 1979). With two ovine isolates the only lesions observed were hyperkeratosis of the posterior interdigital skin and skin-horn junction. With a further three ovine and three bovine isolates there was erythema and exudative; erosive, hyperkeratotic lesions of the interdigital skin and skin-horn junction. The horn of the axial wall of the heels possessed a corrugated appearance due to erosions in the form of shallow tissues. Separation of the soft horn of the axial wall of the heels was observed with one of the three ovine and one of the three bovine isolates but this did not extend beyond the medial plantar aspect of the heel. However, the other two ovine isolates produced more severe lesions in two out of eight feet and one out of eight feet respectively. Both isolates produced separation of the soft horn over the heel and posterior sole in one foot whereas one of the isolates also produced separation of the sole to the toe and and abaxial wall in another foot. In these under-running lesions there was a minimal amount of necrotic exudate and the lesions completely repressed within three weeks.
A further interesting observation was that when B. nodosus was re-isolated from the abaxial wall region and applied to the feet of another three sheep relatively mild lesions were produced in four feet with slight lifting of the heel in one foot only. The lesions complete regressed during the 12 week observation period.
Under pen challenge conditions thirteen virulent isolates produced severe progressive lesions with under-running of the heel and sole to the abaxial wall and toe region. The erythematous underlying tissue was covered by a layer of white necrotic exudate and the sole developed several layers of separated horn. In more chronic lesions the underlying tissue frequently possessed a moist white-yellow appearance with pockets of white necrotic exudate. The majority of feet remained infected throughout the twelve week period of observation. (Stewart 1979).
Despite the difference in character of the lesions produced by benign and virulent isolates in challenged penned sheep it is difficult and often dangerous to make a clinical diagnosis in the field following one visit. The need for repeated visits during favourable transmission periods for observation of the progression of the virulent disease is a most inefficient and tedious method of diagnosis. The added complication of separation of the horn of the heel and sole in a number of sheep in some flocks with benign footrot on lush pastures also confuses diagnosis. Under-running lesions of this nature were observed in at least one flock which I inspected with Mr. P.J. Ahrens, B.V.Sc., of the Molong district. On an adjoining property we observed severe interdigital skin lesions and under-running in lambs less than one month old but less severe lesions in their mothers. Only benign footrot isolates were cultured from both outbreaks including the lambs.
LABORATORY DIAGNOSIS
It has generally been considered that the isolation of B. nodosus by cultural methods from lesion material is too difficult for routine differential diagnosis of footrot. This belief stems from a variety of causes,
(a) The difficulty of maintaining viability of organisms during transport of lesion material from the field to the laboratory since B. nodosus is an obligate anaerobe
(b) Following culture, B. nodosus is slow growing and is normally outnumbered by other organisms. Colonies are frequently overgrown by accompanying spreading bacteria
(c) B. nodosus has rather fastidious growth requirements, not satisfied by routine Laboratory media and until recently this necessitated the incorporation of hoof powder in the basal medium. (Thomas 1958).
With the use of suitable anaerobic transport medium e.g. Stuart's, B. nodosus remains viable in lesion material for at least seven days at 4°C. The recent development of simplified particle free medium supporting improved growth of B. nodosus (Skerman 1975) and the incorporation of increased agar up to five per cent in the medium (Thorley 1976) has greatly facilitated the isolation of the organism.
Thomas (1962) and Egerton and Parsonson (1969) have reported that B. nodosus isolates are less proteolytic than virulent ones. Egerton and Parsonson (1969) developed a proteolytic index test utilising a casein agar plate technique which appeared to correlate with virulence. However the proteolytic index test is a variable test, subject to arbitrary interpretation and not highly specific in determining the virulence status of B. nodosus (Depiazzi and Richards, 1979; Stewart 1979).
A few years ago I started screening B. nodosus isolates with a battery of tests with the view to developing a simple, all or nothing, diagnostic test. From these studies I was able to demonstrate a definite association between virulence of B. nodosus and elastase production. A total of eighty seven isolates were examined including 54 ovine virulent, 28 ovine benign and five bovine isolates. All isolates derived from ovine virulent footrot produced elastase whereas isolates from benign footrot and bovine footrot were elastase negative. Thus there appears to be at least two distinct populations or bio-types of B. nodosus with elastase being an important virulence factor in the pathogenesis of [virulent footrot].
Recently Depiazzi and Richards (1979) have developed a degrading proteinase test which differentiated footrot and foot-scald isolates on the basis of the relative rate of decline in proteolytic activity over a period of time. They used hide powder-azure, an insoluble chromogenic substrate which can detect a number of different proteolytic enzymes. (Rinderknecht et al. 1968). It was found that proteolytic activity of foot-scald isolates declined at a much faster rate over a 12 day period than that of footrot isolates. The findings of Depiazzi and Richards (1979) were confirmed by Stewart (1979) with 22 virulent, 16 benign and two bovine isolates. The bovine isolates behaved like benign isolates in the test. We are at present investigating whether the difference in behaviour of B. nodosus in the degrading proteinase test is due to elastase activity or to another proteolytic enzyme or perhaps group of enzymes which are also associated with virulence.
B. nodosus infections in cattle feet are characterised by lameness, ulcerative and erosive inflammation of the interdigital skin and erosion and separation of the bulbar horn at the heels but the significance of cattle in the epidemiology of ovine footrot has not been fully determined. There is only one report of virulent footrot being induced in sheep by bovine lesion material (Egerton and, Parsonson 1966). However, it should be noted that pure cultures from the infected sheep induced only mild lesions in a further challenged sheep. Others have induced only mild lesions in sheep with bovine lesion material (Alexander 1962; Toussaint, Raven and Cornelisse 1971) or following natural transmission from cattle (Wilkinson et al. 2970; Laing and Egerton 1978).
The five bovine B. nodosus isolates examined were elastase negative and three of these induced only mild feet lesions in challenged sheep whereas the fourth isolate did not cause any lesions (Stewart 1979). The remaining isolate was cultured from a bovine which transmitted benign footrot to sheep under field conditions (Wilkinson et al. 1970).
In future studies it will be necessary to determine whether elastase positive isolates actually do exist in bovine foot infections as their absence would have important implications in ovine virulent footrot eradication programmes.
Further research is also aimed at improving the elastase test and investigating the existence of other proteolytic enzymes in B. nodosus.
REFERENCES
Alexander, T.M. (1962) Australian Veterinary Journal 38, 366
Depiazzi, L.J. & Richards, R.B., (1979) Australian Veterinary Journal 55, 25
Egerton, J.R. & Parsonson, I.M. (1966) Australian Veterinary Journal 42, 425
Egerton, J.R. & Parsonson, I.M. (1969) Australian Veterinary Journal 45, 345
Laing, E.A. & Egerton, J.R., (1978) Research in Veterinary Science, 24, 300
Rinderknecht, H., Geokas, M.C., Silverman, P. & Haverback, B.J. (1968). Clinica Chimica Acta 21, 197
Skerman, T.M. (1975) Journal of General Microbiology 87, 107
Stewart, D.J., (1979) Research in Veterinary Science. (in press)
Thomas, J.H. (1958) Australian Veterinary Journal 24, 413
Thomas, J.H. (1962) Australian Veterinary Journal 38, 159
Thorley, C.M. (1976) Journal of Applied Bacteriology 40, 301
Toussaint Raven, B. & Cornelisse, J.L. (1971) Veterinary Medical Review No. 2/3, 223
Wilkinson, F., Egerton, J.R. & Dixon, J. (1970) Australian [Veterinary Journal...]