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Streptothricosis in the Domestic Donkey (Equus Asinus Asinus). II. Bacteriological and Immunological Relationships of the strains of Dermatophilus Congolensis Isolated
Br. vet. ]. (1975), I3I, 108
STREPTOTHRICOSIS IN THE DOMESTIC DONKEY (EQUUS ASINUS ASINUS) . II. BACTERIOLOGICAL AND IMMUNOLOGICAL RELATIONSHIPS OF THE STRAINS OF DERMATOPHILUS CONGOLENSIS ISOLATED By D. H.
LLOYD* AND
M.
aLA OJO
Department of Veterinary Pathology, University of Ibadan, Nigeria SUMMARY
Fourteen Nigerian strains of Dermatophilus congolensis, including seven obtained from donkeys, were compared by cultural and biochemical tests and by means of the agar gel precipitation reaction. No differences related to host origin were found but five serologically different types were identified. The donkey isolates includes three of these types. INTRODUCTION
In 1971 an outbreak of streptothricosis occurred in a group of nine donkeys in Ibadan, Nigeria, and isolates of Dermatophilus congolensis were obtained from seven of the affected animals (Lloyd, 197 I). The studies d escribed in this report were carried out in order to determine the cultural, biochemical and immunological relationships between the seven donkey isolates and to compare them with isolates of D. congolensis obtained from other species of domestic animals in Nigeria. MATERIALS AND METHODS
Dermatophilus congolensis was isolated as described previously (Lloyd, 197 I). Cultures were preserved in the freeze-dried state and when required were seeded on to 5 per cent human blood agar and incubated at 37°C in a carbondioxide enriched atmosphere. They were maintained during the period of the tests in 5 per cent horse serum broth which had been incubated for three days at 37 °C and subsequently kept at room temperature (about 22 °C). Three- to five-day-old serum broth cultures were used as the test inocula. Media and test procedures Cultural examinations and tests were performed aerobically at 37°C. Blood agar was prepared using blood agar base (Oxoid) with the addition of 5 per
* Present address: The Hannah R esearch Institute, Ayr, Scotland,
KA65HL.
STREPTOTHRICOSIS IN THE DOMESTIC DONKEY
109
cent of human or horse blood. Five per cent serum broth was prepared from nutrient broth (Oxoid) using sterile horse serum (Oxoid). Casein decomposition was tested on casein agar prepared as described by Cowan & Steel (1965); the skim milk for this medium was made up from dried milk granules (Cadbury's Marvel) . Sugar fermentation media were prepared by the addition of 1 per cent of the test carbohydrate to Andrade peptone water (Oxoid), pH 7'2 to 7'4. Christensen's urea agar slants were prepared from urea agar base (Oxoid) with 2 per cent urea. Koser's citrate (Oxoid) was used to test for citrate utilization. For the methyl red and Voges-Proskauer reactions, 4-day, glucose-phosphate broth (Oxoid) cultures were used; two drops of 0'04 per cent methyl red solution were added to the broth for the methyl red test; the Voges-Proskauer test was carried out as described by Barritt (1936). The catalase test was p erformed by adding a drop of 3 per cent hydrogen peroxide to colonies growing on nutrient agar (Oxoid) plates. For the oxidase test, D. congolensis colonies from blood agar plates were rubbed gently against absorbent fibre-free paper previously moistened with a solution of oxidase reagent (NNN'N-tetra methyl-p-phenylene diamine dihydrochloride (BDH)) . Oxidase positive and negative control cultures were tested at the time. Motility was observed by phase-contrast examination of serum broth cultures. Immunology Antigens were prepared by the concentration of 8-day nutrient broth cultures by pervaporation through dialysis tubing (Vis king) in a forced convection at room temper ature (Oduye, 1973). The cultures were concentrated to approximately 1/20 their original volume; after centrifugation the precipitate was discarded and sodium azide added to the supernatant to a concentration of approximately 1/10,000. A n egative control antigen was prepared in the same way using sterile nutrient broth. Antigens were stored at 4°C in screw-capped bottles. The gel diffusion medium was prepared using 1'2 per cent Special Agar-Nobel (Difco), buffered at pH 8'0 with o· 10 M-phosphate buffered saline containing 1/ 10,000 sodium azide. The gel diffusion reaction was carried out on glass slides (76 mm X 38 mm) covered by 6 ml of the agar medium. Serum and antigen wells, 4 mm in diameter and 6 mm apart, were bored in three rows along the length of the slide and were filled with either serum or antigen (Fig. I ) ; slides were incubated at room temperature in a humid chamber and read at 24 h . The serum used was one capable of giving up to five precipitation lines with homologous antigen (Lloyd and Akinsete, unpublished observations) and was obtained from a Zebu animal affected with streptothricosis, at the University of Ibadan farm. 24
ANTIGENS SERUM
448 ANTIGENS
25
26
27
28
29
30
0 000 000 -~-~-~
~ O'o'()O~O 50
1
20
Fig.
31 I.
38
40
16
110
BRITISH VETERINARY JOURNAL,
131, I
RESULTS
Cultural and biochemical observations Table I lists the D. congolensis cultures used in this study, the species of animals and the places from which the infected material was obtained and the date of isolation. Table II summarizes the results of the cultural and biochemical observations. The colonial morphology recorded in Table II was observed on primary cultivation from the freeze-dried state after 48 h incubation at 37°C. All cultures showed rough pinpoint to 0'5 mm colonies after 24 h, and the conversion of some of these to a smooth appearance seemed to be caused by massive zoospore production in the upper parts of the colonies. Such smooth type growth was easily removed and suspended in water, and in young cultures yielded numerous motile zoospores. Rough colonies were tough and coherent, were suspended only with difficulty, and yielded fewer motile forms. Variable numbers of rough or smooth colonies could often be found in cultures with a predominant growth of the other type, and the predominant type of growth obtained on subculture was not consistent in many cases. However, there was no obvious pattern in these changes. The strains varied in colour from a very pale grey to a grey-yellow or grey-brown; these colours became darker with age. No relationship between strain colour and host of origin was apparent. With the exception of numbers 29 and 30, all of the strains produced clearly d emarcated zones of beta haemolysis, approximately 3 mm wide, after 96 h aerobic incubation on 5 per cent horse-blood agar. Strain 29 produced a hazy zone ofhaemolysis 1'5 mm in width at this time, and strain 30 was non-haemolytic. On 5 per cent human-blood agar, a different pattern of haemolysis was apparent; strain 38 was non-haemolytic, whereas all the other strains produced narrow, hazy zones of haemolysis similar to that of strain 29 on horse-blood agar, under the same conditions of incubation. All strains decomposed casein and showed evidence of urease and catalase production. The response to the methyl red test was variable, nine of the strains gave a negative response and five gave a faintly positive reaction; these five included four donkey strains and the one horse strain. The oxidase, citrate utilization and Voges-Proskauer tests were negative for all the strains. Of the carbohydrates tested, acid was produced consistently only from glucose, and from fructose by all except strains 31 and 16. No acid was produced from lactose, sucrose, mannitol, maltose, inulin and salicin. Immunological findings The results of a comparison of the antigenic characteristics of the strains by means of the agar gel diffusion reaction, using concentrated broth culture supernatant antigens, are shown in Table III and Fig. 1. The greatest number of precipitation lines was four, and no antigen produced less than two. No lines were produced by a control antigen prepared from concentrated sterile nutrient broth. For purposes of comparison, the precipitation lines have been numbered from 1 to 4 (see Table III), 1 being closest and 4 farthest from the serum well. Five antigenic types of D. congolensis were distinguished using this
STREPTOTHRICOSIS IN THE DOMESTIC DONKEY
I I I
TABLE I SOURCES OF
Culture number I
20 38 40 31 50 16 24 25 26 27 28 29 30
D. congolensis
STRAINS USED
Date of isolation
Animal
Location in Nigeria
Bovine Bovine Bovine Bovine Bovine Sheep Horse Donkey Donkey Donkey Donkey Donkey Donkey Donkey
L.I.B.C. Kabomo, North-Central State University Farm, Ibadan, Western State University Farm, Ibadan, Western State University Farm, Ibadan, Western State Fulani herd, Forum, Benue-Plateau State University Farm, Ibadan, Western State Ibadan Polo Stables, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State
March 1969 July 197 0 April 1972 April 1972 November 1971 January 1972 August 1970 November 1970 November 1970 November 1970 November 1970 November 1970 November 1970 November 1970
TABLE II CULTURAL AND BIOCHEMICAL REACTIONS
Strain
Rough/ Smooth (48 h)
20 38 40 31 50 16 24 25 26 27 28 29 30
R R R R R S S R R R R S R R
Haemolysis* (96 h) 3 3 3 3 3 3 3 3 3 3 3 3 1'5
Casein
Urease
(48 h)
(24 h)
2 2 2 2 2 1'5 2 2
+ + +2 + +2 + + + + + + + +5 +
I
2
3 I
2 2
Catalase
+ + + + + + + + + + + + + +
Methyl Red (96 h)
± ± ± ± ±
Acid production from Glucose
Fructose
+2 +2 +3 +2 +2 +2 +2 +3 +2 +2 +2 +3 +2 +2
+2 +2 +3 +3 14 +2 14 +3 +2 +2 +2 +2 +3 +2
O ther tests giving negative responses to all strains : Oxidase reaction; Citrate utilization; Vogues-Proskauer reaction; Acid production from Lactose, Sucrose, Mannitol, Maltose, Inulin and Salicin . . NO.tes: Figures for haemolysis and casein decomposition indicate width of the zone of reactIon III millimetres. Superscript figures indicate the number of days at which the observation was made. * Horse Erythrocytes.
112
BRITISH VETERINARY JOURNAL, 131,
I
TABLE III AGAR
GEL
PR E CIPITATION
LINES PRODUCED BY ANTIGENS OF THE congol e ns is STRAINS WITH BOVINE ANTISERUM
D.
DIFFERENT
Line number
2
Strain I
20 38 40 31 50 16 24 25 26 27 28 29 30
Concentrated sterile broth
+ + + + + + + + + + + +
+ + + + + + + + + + + +
3
+ + + + + + +
4
+ + + + + + + + + + + + + +
Antigen type
4 3 5 4 4 4 4 4 2 4
Note .. Line one closest, line four farthest from the serum well.
system (Table III ). Type I gives all four lines, type 2 gives lines 2, 3 and 4, type 3 gives lines I, 3 and 4, type 4 gives lines I, 2 and 4, and type 5 gives lines 3 and 4· DISCUSSION AND CONCLUS IONS
Comparison of the 14 D . congolensis strains in this study by means of growth characteristics and biochemical tests has shown that there is little variation between them. In addition, no bias in the results, with the exception of the m ethyl red test, has been shown which might be ascribable to the host origin of the isolates. In that test, five out of the eight strains of equine origin were faintly positive, whilst the remaining strains were negative. However, this is a relatively slight difference which would require confirmation using a much larger number of strains. These findings are similar to those reported by other workers, both in West Africa (Memery, 196 I; Macadam, 1964, 197 I; Anon, 1967; Vigier & Balis, 1967) and elsewhere (Gordon, 1964; EI Nageh, 1971; Meyer, 197 I). They are within the range of variation to be expected within a bacterial species (Gordon, 1964), taking into account the differences in media preparation and cultural technique which exist between laboratories (Memery, 196 I ). Comparison of the strains by means of the agar gel precipitation reaction using serum from a clinical case of bovine streptothricosis confirmed the findings of the cultural and bioch emical observations, in that no evidence of variation along host origin lines could be demonstrated. Cross reaction of all the strains in at least one line of homogeneity was apparent, but clear antigenic differences
STREPTOTHRICOSIS I N THE DOMESTIC DON KEY
11 3
between some of the strains were d emonstrated, and they could be divided into five distinct serological groups. This reaction also showed that at least three serologically different strains of D. congolensis were involved in the donkey streptothricosis outbreak (Lloyd, 1971 ). To the knowledge of the authors, the report of EI Nageh (1971 ) is the only other study dealing with the bacteriology of a donkey isolate of D. congolensis. EI Nageh (1971 ) compared strains isolated from an Abyssinian donkey, from three African cattle, and from a lamb and a horse in Britain. H e found that the six strains were identical in their biochemical reactions but was a ble to demonstrate slight immunological differences between them by m eans of direct immunofluorescent staining and agglutination tests. The results reported in this paper are in agreement with his findings.
ACKNOWLEDGEMENTS
Thanks are due to Professor K. C. Sellers for his assistance in the preparation of this paper. D uring the period of this work Mr Lloyd was a Visiting R esearch Fellow at the University of Ibadan, supported by a grant from the Overseas Development Administration, United Kingdom Foreign and Commonwealth Office.
REFERENCES
(1967) . R apport A nnuel, 1967. Institute d'Elevage et de M ed ecine V eterinaire d es Pays Tropicaux, Laboratoire d e F a rcha. Tome II . Streptothricose B ilan D ' Activite. p. 2 I. BARRITT, M. M. (1936) . J . Path. Bact. 42,441. COWAN, S. T. & STEEL, K. T. (1965) . Manualfor the Identification of M edical Bacteria. 1st edn ., p. I 12. Cambridge : Cambridge U ni versi ty Press. EL NAGEH, M . M. (1971 ). Ann. Soc. Beige de Med. trop. 51, 239. GORDON, M. A. (1964) . J. Bact. 88, (2), 509. LLOYD, D. H. ( 1971 ). Br. vet.]. 127, 572. MACADAM, 1. ( 1964). Vet. R ec. 76,420. MACADAM, 1. ( 1971 ). Trap. Anim. Hlth . Prado 3, 225. MEMERY, G . ( 1961 ) . R ev. Elev. MM. vet. Pays trop. 14, (2), I4I. MEYER, E. (1971 ) . Beitrage zur Tropischen und Sub -tropischen L andwirtschaft und Tropen veterinarmedizin. 4, 3 I I . ODUYE, O . O. ( 1973)' Ph.D. Thesis, University of Ibadan , Nigeria. VIGIER, M. & BALIS,]. (1967) . Rev. Elev. Med. vet. Pays trap. 20, ( 1),67. ANON
(Acceptedfor publication 30 January 1974)
La Streptothricose chez l'ane domestique (Equus asinus asinus) II: Rapports bacteriologiques et inununologiques des races de Dermatophilus congolensis isolees (Lloyd et Ojo) Resume. On com para quatorze races nigeriennes de D ermatophilus congolensis, p a rmi lesquelles sept provenaient d es anes, par d es epreuves biochimiques et des epreuves de cultu re, et en utilisant la reaction de precipitation agar gel. On ne trouva a ucune difference alliee a I'origine de I'h6te mais on put identifier cinq esp eces serologiques differentes. L es isoles proven us des illles comprenaient trois de ces especes.
114
BRITISH VETERINARY JOURNAL,
13 1,
I
Streptothricose bei dO%nestizierten Eseln (Equus as;nus as;nus). D Bakteriologische und inununologische Beziehungen der isolierten Stanune von Dermatophi/us congolens;s (Lloyd und Ojo) Zusanunenfassung. Vierzehn nigerische Stamme von Dermatophilus congolensis, einschliesslich 7 von Eseln gewonnen, wurden verglichen mit Hilfe von Kulturund biochemischen Tests und der Agar-Gel-Praezipitations-Reaktion. Man fand keine den Ursprungswirten entsprechende Unterschiede; doch wurden 5 serologisch verschiedene T yp en identifiziert. Bei den von Eseln gewonnenen Erregern fanden sich 3 dieser Typen .
La estreptotricosis en el burro dODlestico (Equus as;nus as;nus) D: Relaciones bacteriol6gicas e inDlunol6gicas de las cepas aisladas de Dermatoph;lus congolens;s (Lloyd y Ojo) ResUDlen. Se compararon catorce cepas nigerianas d e Dermatophilus congolensis, incluyendo siete obtenidas de burros, m edia nte pruebas de cultivo y bioquim icas asi como por la reaccion d e la precipitacion d e la gela d e agar. No se encontraron diferencias relac ionadas al huesped d e origen pero se identificaron cinco tipos serologicamente distintos. Los aislamientos del burro incluyeron tres de estos tipos.
STREPTOTHRICOSIS IN THE DOMESTIC DONKEY (EQUUS ASINUS ASINUS) . II. BACTERIOLOGICAL AND IMMUNOLOGICAL RELATIONSHIPS OF THE STRAINS OF DERMATOPHILUS CONGOLENSIS ISOLATED By D. H.
LLOYD* AND
M.
aLA OJO
Department of Veterinary Pathology, University of Ibadan, Nigeria SUMMARY
Fourteen Nigerian strains of Dermatophilus congolensis, including seven obtained from donkeys, were compared by cultural and biochemical tests and by means of the agar gel precipitation reaction. No differences related to host origin were found but five serologically different types were identified. The donkey isolates includes three of these types. INTRODUCTION
In 1971 an outbreak of streptothricosis occurred in a group of nine donkeys in Ibadan, Nigeria, and isolates of Dermatophilus congolensis were obtained from seven of the affected animals (Lloyd, 197 I). The studies d escribed in this report were carried out in order to determine the cultural, biochemical and immunological relationships between the seven donkey isolates and to compare them with isolates of D. congolensis obtained from other species of domestic animals in Nigeria. MATERIALS AND METHODS
Dermatophilus congolensis was isolated as described previously (Lloyd, 197 I). Cultures were preserved in the freeze-dried state and when required were seeded on to 5 per cent human blood agar and incubated at 37°C in a carbondioxide enriched atmosphere. They were maintained during the period of the tests in 5 per cent horse serum broth which had been incubated for three days at 37 °C and subsequently kept at room temperature (about 22 °C). Three- to five-day-old serum broth cultures were used as the test inocula. Media and test procedures Cultural examinations and tests were performed aerobically at 37°C. Blood agar was prepared using blood agar base (Oxoid) with the addition of 5 per
* Present address: The Hannah R esearch Institute, Ayr, Scotland,
KA65HL.
STREPTOTHRICOSIS IN THE DOMESTIC DONKEY
109
cent of human or horse blood. Five per cent serum broth was prepared from nutrient broth (Oxoid) using sterile horse serum (Oxoid). Casein decomposition was tested on casein agar prepared as described by Cowan & Steel (1965); the skim milk for this medium was made up from dried milk granules (Cadbury's Marvel) . Sugar fermentation media were prepared by the addition of 1 per cent of the test carbohydrate to Andrade peptone water (Oxoid), pH 7'2 to 7'4. Christensen's urea agar slants were prepared from urea agar base (Oxoid) with 2 per cent urea. Koser's citrate (Oxoid) was used to test for citrate utilization. For the methyl red and Voges-Proskauer reactions, 4-day, glucose-phosphate broth (Oxoid) cultures were used; two drops of 0'04 per cent methyl red solution were added to the broth for the methyl red test; the Voges-Proskauer test was carried out as described by Barritt (1936). The catalase test was p erformed by adding a drop of 3 per cent hydrogen peroxide to colonies growing on nutrient agar (Oxoid) plates. For the oxidase test, D. congolensis colonies from blood agar plates were rubbed gently against absorbent fibre-free paper previously moistened with a solution of oxidase reagent (NNN'N-tetra methyl-p-phenylene diamine dihydrochloride (BDH)) . Oxidase positive and negative control cultures were tested at the time. Motility was observed by phase-contrast examination of serum broth cultures. Immunology Antigens were prepared by the concentration of 8-day nutrient broth cultures by pervaporation through dialysis tubing (Vis king) in a forced convection at room temper ature (Oduye, 1973). The cultures were concentrated to approximately 1/20 their original volume; after centrifugation the precipitate was discarded and sodium azide added to the supernatant to a concentration of approximately 1/10,000. A n egative control antigen was prepared in the same way using sterile nutrient broth. Antigens were stored at 4°C in screw-capped bottles. The gel diffusion medium was prepared using 1'2 per cent Special Agar-Nobel (Difco), buffered at pH 8'0 with o· 10 M-phosphate buffered saline containing 1/ 10,000 sodium azide. The gel diffusion reaction was carried out on glass slides (76 mm X 38 mm) covered by 6 ml of the agar medium. Serum and antigen wells, 4 mm in diameter and 6 mm apart, were bored in three rows along the length of the slide and were filled with either serum or antigen (Fig. I ) ; slides were incubated at room temperature in a humid chamber and read at 24 h . The serum used was one capable of giving up to five precipitation lines with homologous antigen (Lloyd and Akinsete, unpublished observations) and was obtained from a Zebu animal affected with streptothricosis, at the University of Ibadan farm. 24
ANTIGENS SERUM
448 ANTIGENS
25
26
27
28
29
30
0 000 000 -~-~-~
~ O'o'()O~O 50
1
20
Fig.
31 I.
38
40
16
110
BRITISH VETERINARY JOURNAL,
131, I
RESULTS
Cultural and biochemical observations Table I lists the D. congolensis cultures used in this study, the species of animals and the places from which the infected material was obtained and the date of isolation. Table II summarizes the results of the cultural and biochemical observations. The colonial morphology recorded in Table II was observed on primary cultivation from the freeze-dried state after 48 h incubation at 37°C. All cultures showed rough pinpoint to 0'5 mm colonies after 24 h, and the conversion of some of these to a smooth appearance seemed to be caused by massive zoospore production in the upper parts of the colonies. Such smooth type growth was easily removed and suspended in water, and in young cultures yielded numerous motile zoospores. Rough colonies were tough and coherent, were suspended only with difficulty, and yielded fewer motile forms. Variable numbers of rough or smooth colonies could often be found in cultures with a predominant growth of the other type, and the predominant type of growth obtained on subculture was not consistent in many cases. However, there was no obvious pattern in these changes. The strains varied in colour from a very pale grey to a grey-yellow or grey-brown; these colours became darker with age. No relationship between strain colour and host of origin was apparent. With the exception of numbers 29 and 30, all of the strains produced clearly d emarcated zones of beta haemolysis, approximately 3 mm wide, after 96 h aerobic incubation on 5 per cent horse-blood agar. Strain 29 produced a hazy zone ofhaemolysis 1'5 mm in width at this time, and strain 30 was non-haemolytic. On 5 per cent human-blood agar, a different pattern of haemolysis was apparent; strain 38 was non-haemolytic, whereas all the other strains produced narrow, hazy zones of haemolysis similar to that of strain 29 on horse-blood agar, under the same conditions of incubation. All strains decomposed casein and showed evidence of urease and catalase production. The response to the methyl red test was variable, nine of the strains gave a negative response and five gave a faintly positive reaction; these five included four donkey strains and the one horse strain. The oxidase, citrate utilization and Voges-Proskauer tests were negative for all the strains. Of the carbohydrates tested, acid was produced consistently only from glucose, and from fructose by all except strains 31 and 16. No acid was produced from lactose, sucrose, mannitol, maltose, inulin and salicin. Immunological findings The results of a comparison of the antigenic characteristics of the strains by means of the agar gel diffusion reaction, using concentrated broth culture supernatant antigens, are shown in Table III and Fig. 1. The greatest number of precipitation lines was four, and no antigen produced less than two. No lines were produced by a control antigen prepared from concentrated sterile nutrient broth. For purposes of comparison, the precipitation lines have been numbered from 1 to 4 (see Table III), 1 being closest and 4 farthest from the serum well. Five antigenic types of D. congolensis were distinguished using this
STREPTOTHRICOSIS IN THE DOMESTIC DONKEY
I I I
TABLE I SOURCES OF
Culture number I
20 38 40 31 50 16 24 25 26 27 28 29 30
D. congolensis
STRAINS USED
Date of isolation
Animal
Location in Nigeria
Bovine Bovine Bovine Bovine Bovine Sheep Horse Donkey Donkey Donkey Donkey Donkey Donkey Donkey
L.I.B.C. Kabomo, North-Central State University Farm, Ibadan, Western State University Farm, Ibadan, Western State University Farm, Ibadan, Western State Fulani herd, Forum, Benue-Plateau State University Farm, Ibadan, Western State Ibadan Polo Stables, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State Moor Plantation, Ibadan, Western State
March 1969 July 197 0 April 1972 April 1972 November 1971 January 1972 August 1970 November 1970 November 1970 November 1970 November 1970 November 1970 November 1970 November 1970
TABLE II CULTURAL AND BIOCHEMICAL REACTIONS
Strain
Rough/ Smooth (48 h)
20 38 40 31 50 16 24 25 26 27 28 29 30
R R R R R S S R R R R S R R
Haemolysis* (96 h) 3 3 3 3 3 3 3 3 3 3 3 3 1'5
Casein
Urease
(48 h)
(24 h)
2 2 2 2 2 1'5 2 2
+ + +2 + +2 + + + + + + + +5 +
I
2
3 I
2 2
Catalase
+ + + + + + + + + + + + + +
Methyl Red (96 h)
± ± ± ± ±
Acid production from Glucose
Fructose
+2 +2 +3 +2 +2 +2 +2 +3 +2 +2 +2 +3 +2 +2
+2 +2 +3 +3 14 +2 14 +3 +2 +2 +2 +2 +3 +2
O ther tests giving negative responses to all strains : Oxidase reaction; Citrate utilization; Vogues-Proskauer reaction; Acid production from Lactose, Sucrose, Mannitol, Maltose, Inulin and Salicin . . NO.tes: Figures for haemolysis and casein decomposition indicate width of the zone of reactIon III millimetres. Superscript figures indicate the number of days at which the observation was made. * Horse Erythrocytes.
112
BRITISH VETERINARY JOURNAL, 131,
I
TABLE III AGAR
GEL
PR E CIPITATION
LINES PRODUCED BY ANTIGENS OF THE congol e ns is STRAINS WITH BOVINE ANTISERUM
D.
DIFFERENT
Line number
2
Strain I
20 38 40 31 50 16 24 25 26 27 28 29 30
Concentrated sterile broth
+ + + + + + + + + + + +
+ + + + + + + + + + + +
3
+ + + + + + +
4
+ + + + + + + + + + + + + +
Antigen type
4 3 5 4 4 4 4 4 2 4
Note .. Line one closest, line four farthest from the serum well.
system (Table III ). Type I gives all four lines, type 2 gives lines 2, 3 and 4, type 3 gives lines I, 3 and 4, type 4 gives lines I, 2 and 4, and type 5 gives lines 3 and 4· DISCUSSION AND CONCLUS IONS
Comparison of the 14 D . congolensis strains in this study by means of growth characteristics and biochemical tests has shown that there is little variation between them. In addition, no bias in the results, with the exception of the m ethyl red test, has been shown which might be ascribable to the host origin of the isolates. In that test, five out of the eight strains of equine origin were faintly positive, whilst the remaining strains were negative. However, this is a relatively slight difference which would require confirmation using a much larger number of strains. These findings are similar to those reported by other workers, both in West Africa (Memery, 196 I; Macadam, 1964, 197 I; Anon, 1967; Vigier & Balis, 1967) and elsewhere (Gordon, 1964; EI Nageh, 1971; Meyer, 197 I). They are within the range of variation to be expected within a bacterial species (Gordon, 1964), taking into account the differences in media preparation and cultural technique which exist between laboratories (Memery, 196 I ). Comparison of the strains by means of the agar gel precipitation reaction using serum from a clinical case of bovine streptothricosis confirmed the findings of the cultural and bioch emical observations, in that no evidence of variation along host origin lines could be demonstrated. Cross reaction of all the strains in at least one line of homogeneity was apparent, but clear antigenic differences
STREPTOTHRICOSIS I N THE DOMESTIC DON KEY
11 3
between some of the strains were d emonstrated, and they could be divided into five distinct serological groups. This reaction also showed that at least three serologically different strains of D. congolensis were involved in the donkey streptothricosis outbreak (Lloyd, 1971 ). To the knowledge of the authors, the report of EI Nageh (1971 ) is the only other study dealing with the bacteriology of a donkey isolate of D. congolensis. EI Nageh (1971 ) compared strains isolated from an Abyssinian donkey, from three African cattle, and from a lamb and a horse in Britain. H e found that the six strains were identical in their biochemical reactions but was a ble to demonstrate slight immunological differences between them by m eans of direct immunofluorescent staining and agglutination tests. The results reported in this paper are in agreement with his findings.
ACKNOWLEDGEMENTS
Thanks are due to Professor K. C. Sellers for his assistance in the preparation of this paper. D uring the period of this work Mr Lloyd was a Visiting R esearch Fellow at the University of Ibadan, supported by a grant from the Overseas Development Administration, United Kingdom Foreign and Commonwealth Office.
REFERENCES
(1967) . R apport A nnuel, 1967. Institute d'Elevage et de M ed ecine V eterinaire d es Pays Tropicaux, Laboratoire d e F a rcha. Tome II . Streptothricose B ilan D ' Activite. p. 2 I. BARRITT, M. M. (1936) . J . Path. Bact. 42,441. COWAN, S. T. & STEEL, K. T. (1965) . Manualfor the Identification of M edical Bacteria. 1st edn ., p. I 12. Cambridge : Cambridge U ni versi ty Press. EL NAGEH, M . M. (1971 ). Ann. Soc. Beige de Med. trop. 51, 239. GORDON, M. A. (1964) . J. Bact. 88, (2), 509. LLOYD, D. H. ( 1971 ). Br. vet.]. 127, 572. MACADAM, 1. ( 1964). Vet. R ec. 76,420. MACADAM, 1. ( 1971 ). Trap. Anim. Hlth . Prado 3, 225. MEMERY, G . ( 1961 ) . R ev. Elev. MM. vet. Pays trop. 14, (2), I4I. MEYER, E. (1971 ) . Beitrage zur Tropischen und Sub -tropischen L andwirtschaft und Tropen veterinarmedizin. 4, 3 I I . ODUYE, O . O. ( 1973)' Ph.D. Thesis, University of Ibadan , Nigeria. VIGIER, M. & BALIS,]. (1967) . Rev. Elev. Med. vet. Pays trap. 20, ( 1),67. ANON
(Acceptedfor publication 30 January 1974)
La Streptothricose chez l'ane domestique (Equus asinus asinus) II: Rapports bacteriologiques et inununologiques des races de Dermatophilus congolensis isolees (Lloyd et Ojo) Resume. On com para quatorze races nigeriennes de D ermatophilus congolensis, p a rmi lesquelles sept provenaient d es anes, par d es epreuves biochimiques et des epreuves de cultu re, et en utilisant la reaction de precipitation agar gel. On ne trouva a ucune difference alliee a I'origine de I'h6te mais on put identifier cinq esp eces serologiques differentes. L es isoles proven us des illles comprenaient trois de ces especes.
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Streptothricose bei dO%nestizierten Eseln (Equus as;nus as;nus). D Bakteriologische und inununologische Beziehungen der isolierten Stanune von Dermatophi/us congolens;s (Lloyd und Ojo) Zusanunenfassung. Vierzehn nigerische Stamme von Dermatophilus congolensis, einschliesslich 7 von Eseln gewonnen, wurden verglichen mit Hilfe von Kulturund biochemischen Tests und der Agar-Gel-Praezipitations-Reaktion. Man fand keine den Ursprungswirten entsprechende Unterschiede; doch wurden 5 serologisch verschiedene T yp en identifiziert. Bei den von Eseln gewonnenen Erregern fanden sich 3 dieser Typen .
La estreptotricosis en el burro dODlestico (Equus as;nus as;nus) D: Relaciones bacteriol6gicas e inDlunol6gicas de las cepas aisladas de Dermatoph;lus congolens;s (Lloyd y Ojo) ResUDlen. Se compararon catorce cepas nigerianas d e Dermatophilus congolensis, incluyendo siete obtenidas de burros, m edia nte pruebas de cultivo y bioquim icas asi como por la reaccion d e la precipitacion d e la gela d e agar. No se encontraron diferencias relac ionadas al huesped d e origen pero se identificaron cinco tipos serologicamente distintos. Los aislamientos del burro incluyeron tres de estos tipos.