Zbl. Vet. Med. B, 25,668-688 (1978) @ 1978 Verlag Paul Parey, Berlin und Hamburg ISSN 05 14-7166/ASTM-Coden : ZVRBA2

University of Liverpool, Department of Veterinary Preventive Medicine, Field Station, “Leaburst”, Neston, L 647 TE, Wirral, Merseyside, England

Morphological, Cultural and Biochemical Characteristics of Dermatophilus congolensis BY MUKHTAR TAHA ABU-SAMRAI W i t h 11 figures and J tables

(Received for publication October 11, 1977)

Introduction Since the original identification of Dermatophilus congolensis by Vansaceghem in 1915 numerous investigations have been carried out (ROBERTS, 1967; STEWART, 1972; AINSWORTH and AUSTWICK,1973; BIDA and DENNIS, 1976) with a wide variety of media and atmospheric conditions. These investigations have yielded a large amount of information, which at times has been either confusing or contradictory. Until recently, when an investigation was carried out on modified techniques for the isolation on culture media of the organism from infected material (ABu-SAMRA and WALTON,1977 a) it was beIieved that carbon dioxide was essential for the isolation and growth of this organism (ROBERTS,1963 a, b; HAALSTRA, 1965). Previous work on the cultural and biochemical characteristics of Dermatophilus has shown great diversity. The micro-organisms described are almost always identical or very alike (GORDON,1964), but their comparison remained difficult since the methods used and the conditions to which the organism was subjected in different laboratories were dissimilar and thus gave inconsistent results. This led to the present study of 38 isolates (obtained from the three disease entities) under controlled laboratory conditions to find the similarities and dissimilarities between the isolates when cultured on various media under different incubation conditions and subjected to various biochemical tests. These studies may help to explain the inconsistency and diversity of findings of other workers. Present Addreu: P. 0. Box 2278, Khartoum, Sudan.

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669

Characteristics of Dermatophilus congolensis

Material and Methods Thirty-eight isolates of Dermatophilus were obtained from field cases, Nos. 1-6 were from mycotic dermatitis in sheep, 7 and 8 from strawberry foot rot in sheep and 9-38 from equine and bovine streptothricosis in Great Britain, the Sudan and Nigeria (Table 1). Table 1 Country of origin and disease from whi& Dermatophilus isolates originated Serial No.

1

Reference No. LWl

2

LWZ

3

LW3

L

LW,

5

LW,

6

Lw6

Disease

Country

Serial No.

Reference No.

Disease

.4 .-

- z .

Sudan 22

Z & ._ u:

r - c

sm

.2

E .c

Country

Great

Britain

UiVD7R

l?Fl%A 1 26

j I

UIVD23R

J

Nigeria“

UIVD30R UlVD 17R UiVD35R UlVD 27R

Sudan

I

I

’> Slope culture (Central Veterinary Laboratory, Weybridge, Surrey, England.

:>::.

Lyophilized

cultures. ’ w ’ ~material from a case of bovine streptothricosis.

Culture Lyophilized cultures of all 38 isolates were cultured aerobically, in a vacuumz and in 20 O / o carbon dioxideS at 37 OC for 2-7 days on sixteen media: Brain heart infusion agar (Oxoid, CM 375), 7 O / o horse blood-enriched agar made from blood agar base No. 2 (Oxoid, CM 271), MacConkey’s agar (Oxoid, CM 227), corn meal (Oxoid, CM 7), desoxycholate citrate agar HYNES agar (Oxoid, CM 103), nutrient agar (Oxoid, CM 3), 7 O / o horse serum-enriched agar made from nutrient agar (Oxoid), Sabouraud’s dextrose agar (Oxoid, CM 41), potato dextrose agar (Oxoid), CM 139), Edward’s medium modified (Oxoid, CM27) brilliant green agar (Oxoid, CM 263), Czapek dox agar modified (Oxoid, CM 97), 7 o / o horse blood-enriched brain heart infusion agar (Oxoid), cystine-lactose-electrolyte deficient medium (Mast Laboratories, Limited, England), chocolate blood agar prepared from 7 O / o horse blood in blood agar base No. 2 (Oxoid) and Littman ox gall (Oxoid, CM 91). -

* 3

Edwards speedivac high vacuum pump with a MCINTOSH and FILDE’S jar. Obtained by absorbing the oxygen in air in the jars, using a candle.

670

MUKHTAR TAHAABU-SAMRA

All isolates were subcultured on slopes of LOWENSTEINJENSEN,LOFFI.EK5 serum and Dorset egg media prepared according t O CRUICKSHANK et al., (1965); and in eight liquid media: brain heart infusion (Oxoid, C M 255), peptone water (Oxoid, CM 9), nutrient broth No. 2 (Oxoid, CM 67), 7 "/o horse serum broth prepared from nutrient broth No. 2 (Oxoid), cooked meat medium (Oxoid, CM 82), Todd-Hewitt broth (Oxoid, CM 189), tryptone soya broth (Oxoid, C M 129) and selenite broth (Oxoid, CM 39). The cultures were incubated at 37 "C and examined daily until the seventh day. To study the effect of aeration on the morphology of the organism (compared to its morphology under ordinary incubation conditions) 48 hours CUItures on 7 "/u horse blood-enriched agar and brain heart infusion agar at 37 O C in carbon dioxide were subcultured in flasks containing 200 ml. nutrient broth. One set of flasks was incubated in an ordinary incubator a t 37 OC for 7 davs and the other for the same period at the same temperature in an orbital shake incubator (Gallenkamp, England) and shaken a t 100 times per minute. To study the developmental stages of cocci, isolates, 1,8 and 19 obtained from cases of mycotic dermatitis, strawberry foot rot in sheep and bovine cutaneous streptothricosis respectively, (Table 1) were randomly selected and were cultured as described for the study of the effect of aeration. The cultures were then filtered through a 0.8 pm. Sartorius membrane filter, centrifuged and the supernatant fluid discarded. 2 ml. of sterile nutrient broth was added to the sediment and mixed until a homogeneous suspension was obtained. Three series of bottles, each containing 5 ml. sterile nutrient broth were prepared, a loopful of the homogeneous suspension of each isolate was inoculated into each bottle within the respective series and these were incubated at 37 ' C . Films were made at hourly intervals for 72 hours and then every 6 hours until the seventh day. The colonial morphology of the organism on solid media was assessed according to the following criteria: size and shape of colonies (naked eye and under the light microscope), consistency, adherence to and embedding in the medium, pigment production and changes in the media. In fluid media the criteria were pellicle formation, turbidity and nature of growth. From all cultures films were stained with Giemsa. Measurements of the dimensions of the organism were made, using a stage micrometer (Graticules Limited, London) to calibrate an eyepiece micrometer (1 division = 1.2 pm).

Stains Films from brain heart infusion agar and 7 O / o horse blood-enriched agar were stained with Gram and Ziehl-Neelsen and with negative capsular, flagellar and periodic acid Schiff's stain (PAS), according to GURR(1963).

Motility The isolates were examined for motility as follows: Five colonies were picked from a 48 hour aerobic culture on 7 "/a horse blood-enriched agar and subcultured in 10 ml. of 7 O/o horse serum broth at 37 O C for forty-eight hours. A drop of the broth culture placed in the well of a Kova-Slide (ICL Scientific, California) was examined under the microscope a t hourly intervals for 12 hours and then at six-hourly intervals for 36 hours.

Characteristics of Dermatophilus congolensis

671

Haemolysis The fourth subculture of the isolates on 7 O / o horse blood-enriched agar was studied €or its ability to haemolyse goat, horse, human, ox, pig and sheep blood. Each isolate was cultured on two sets of blood agar plates with a uniform thickness of media prepared by adding 7O/o of the blood of each species to blood agar base No. 2 (Oxoid). One set of plates was incubated aerobically and in carbon dioxide at 37 O C for 48 hours and the other set for 72 hours. The zone of haemolysis was measured in the dark on an illuminated colony counter (Gallenkamp, England). Biochemical tests The isolates were tested for catalase, oxidase, methyl red, Voges-Proskauer, nitrate reduction, gelatin liquefaction, urease, indole and hydrogen sulphide production, citrate utilization and coagulation and digestion of litmus milk. The media were prepared and tested according to CRUICKSHANK et al. (1965), COWAN and STEEL(1966) and the Oxoid manual (1973). Each test medium was inoculated with five colonies icked from a 48 hours culture on brain heart infusion agar incubated at 37 C in about 2 O o / o carbon dioxide except that for catalase and oxidase the tests were carried out directly on nutrient agar plates (Oxoid) after 4 8 hours incubation in carbon dioxide a t 37 OC. For sugar metabolism, peptone water (Oxoid) was used as the basal medium to which 1 O / o Andrade’s indicator was added. Two lots of 1 O / o concentration of sugars were prepared, one with and one without 1 O / o bovine et al., 1965). The following 21 sugars were tested: serum (CRUICKSHANK adonitol, aesculin, arabinose, dextrin, dulcitol, fructose, galactose, glucose, inositol, inulin, lactose, laevulose, maltose, mannitol, raffinose, rhamnose, salicin, sorbitol, sucrose, trehalose and xylose. The inocula used for each isolate were ten colonies obtained as described above, subcultured in 10 ml. nutrient broth (Oxoid) and incubated at 37 OC for four Jays, after which 0.1 ml. was pipetted into each test sugar. Standard sugars inoculated with known strains of Bac. proteus were used for comparison. All test media were examined daily for up to 15 days before a test was judged finally as positive or negative.

8

Results The colonial morphology descriptions are based on 4 8 hour cultures except where otherwise specified. Only the dimensions of the majority of colonies per plate are given; a few larger colonies were also seen. The colonial morphology was not related to the medium or atmospheric conditions and was as follows: (I) Circular extremely fimbriated, resembling a spider’s web (Fig. 1) or a mesh of wool. The fimbriae were long, segmented and markedly branched (Fig. 2). (2) Circular, with smooth raised or flat surface or with peripherally radiating crevices resembling the segments of a peeled orange when viewed from above and slightly fimbriated. (3) Non-fimbriated colonies with a smooth or convoluted surface (Fig. 3). (4) Irregular margin and flat surface, crumb-like with many crevices (like dried mud) or with a central depression (umbilicate, Fig. 4). (5) Irregular margin and surface and cerebriform (Fig. 5), crateriform, irregularly convoluted or butterfly-like (Fig. 6). Brain heart infusion agar: Colonies 1-2 mm. in diameter were obtained under aerobic conditions and in carbon dioxide and 0.5-1 mm. diameter in a vacuum. Colonies were orange in colour, dry, compact or granular, firmly adherent and deeply embedded in the medium except for 1 4 isolates.

672

Fig.

MUKHTAR TAHAABU-SAMRA

r8 11.

7010 Horse blood agar: Colonies 1-2 mm. in diameter were obtained under aerobic conditions and in carbon dioxide and 0.5-1 mm. in a vacuum. In all cases the colonies were surrounded by a clear 1-2 mm. zone of haemolysis. The colonies were either greyish white, cream or light brown and were usually adherent and deeply embedded but there were exceptions.

Fig.

arbon

Characteristics of Dermatophilus congolensis

673

Nutrient agar: Colonies were 0.5-1 mm. in diameter under all incubation conditions. The colonies were white and opaque or light cream. They were either adherent and embedded in the medium, in which case they were hard and compact; or they were adherent but not embedded in the medium, in which case they were granular and soft.

Fig. 3. Non-fimbriated colony with convoluted surface on blood agar. 48 h. Culture in carbon dioxide. x 56

7 Olo Horse serum-enriched agar: Colonies were 1-1.5 mm. in diameter and light to deep cream coloured under all incubation conditions. They were hard and compact and were usually adherent and deeply embedded in the medium. 7 Olo Horse blood-enriched brain heart infusion agar: Colonies 1-2 mm. in diameter were obtained under all incubation conditions. They were light brown, hard, leathery or granular and were usually adherent and embedded in the medium. Chocolate blood agar: Colonies 0.5-1 mm. in diameter were observed under all incubation conditions. They were gray and glistening and either (a) compact, hard and adherent to the medium or (b) soft and waxy and adherent to the medium but not embedded in it.

Fig. 4. Umbilicate colonies on brain heart infusion agar. 48 h. Culture in a vacuum. x 56

674

MUKHTAK

TAHA ABU-SAMRA

Brilliant green agar: Growth was only evident by the 3rd day; the colonies reached 1.5-3 mm. by the 5th day. They were embedded in the medium and when force was used the whole colony was removed, leaving pits of varying depth. The colonies were hard, leathery, waxy or granular and were colourless or light pink.

Fig. 5. Cerebriform colony on blood agar. 48 h. Culture in a vacuum. X 56

No growth was obtained after 7 days incubation in vacuum, in carbon dioxide or under aerobic conditions on the following agar media: MCCONKEY, dextrose, corn meal, CZAPEK potato dextrose, LITTMANox gall, SABOURAUD dox, EDWARDcrystal violet and cystine lactose electrolyte-deficient medium. Dorset egg medium: Growth started within 48-72 hours and had a tendency to spread over the medium. The colonies showed at first a yellow pigment which gradually changed to orange, but 11 of the 38 isolates produced no pigment. Lofflers serum: Growth started within 2-3 days and spread laterally on the medium. Liquefaction was seen within a week in 12 isolates.

Fig. 6. Butterfly-like colony on blood enriched brain heart infusion agar. 48 h. Culture in carbon dioxide. x 56

675

Characteristics of DermatophiIus congolensis

Lowenstein- Jensen: Evidence of growth was seen within three days, when domed colonies with little or no tendency to spread laterally were observed. The organism tended to penetrate the medium so that after 7 days pits were revealed, especially at the colony margins, when the growth was lifted. Table 2 summarizes the findings obtained from culture of the 38 isolates in cooked meat medium, brain heart infusion, tryptone soya broth, TODD HEWITT broth, peptone water and horse serum broth. In selenite broth no turbidity or pellicle formation was seen. The original inoculum settled a t the bottom of the bottle and was shrunk and shrivelled.

media Cooked meat

nature of growth

pigment

homogeneous yellow

clear supernatant

floccular growth

+

-

(38)

+

-

turbidity

-

pellicle* other** formation findings

-

-

+ 13)

+

Brain heart infusion

granular or fluffy

tryptone soya broth

fluffy or yellow homogeneous

(38)

Todd Hewitt broth

yellow fluffy or homogeneous

+

+

+

+

+

1351

13)

I31

12)

13)

nutrient broth

fluffy or granular

yellow or orange

+

+

+

+

+

(33)

15)

15)

113)

116)

mucoid, f l u f f y or granular

yellow

fluffy

yellow or orange

peptone water

Serum broth

yellow or orange

(38)

+

-

-

-

IL)

+ 112)

+

+

+

+

+

(30)

18)

(8)

13)

(3)

+

-

-

138)

+

+

19)

138)

+

positive findings; - negative findings; ( ) number of isolates; 't A thin pellicle which settled on entle shaking was formed by the 3rd. day; 't'i hard fimbriated 0.5-1 mm. in diameter cofonies were seen adhering to the submerged portion of the bottle.

Nutrient broth under normal and shake incubation Similar cultural characteristics as were described earlier were seen under ordinary incubation. In shake cultures, growth was either (a) turbid, homogeneous and suspended evenly in broth (11 isolates) and settled at the bottom after standing on the bench for 24 hours, forming a thick dark yellow to orange sediment; or (b) innumerable round or ovoid beads 2-3 mm. in diameter and dark yellow to orange in colour at the bottom of the flasks, leaving a clear supernatant; the beads were slippery, turgid and quite leathery in consistency. Microscopic Structure Hyphae of varying length were seen, with the most common range 4.8-20 pm, but in fluid media the hyphae were u p to 240 pm. The following microscopic structures of the organism were seen: (1) Hyphae 1.2-2.4 Aim wide, branched or non-branched, divided closely transversely and once or twice longitudinally to form 2-4 parallel rows of 0.6 p m cocci (Fig. 7) and a few 0.6-1 p m dispersed cocci. Variants were also seen in which hyphae and cocci were in equal ratios or the cocci were more prevalent than hyphae. (2) Hyphae 1.2-1.8 pm branched (Fig. 8) or non-branched, with transverse septae close together or a t wide intervals (no longitudinal division)

676

MUKHTAR TAHAABU-SAMRA

Fig. 7. Hyphae septated into 2-4

parallel rows of cocci. 7 days Culture in brain heart infusion. Giemsa. X 1,400

and 0.6-1 pm dispersed cocci. Another variant comprised a mixture of hyphae septatcd transversely and others septated once longitudinally, forming two parallel rows of 0.6 pm cocci (Fig. 9) and a few 0.6-1 ,um dispersed cocci. (3) Beaded hyphae 1.2-2.4 pm with oval or round segments frequently showing germinal activity (Fig. 10) together with 0.6-1 p m cocci dispersed or in clumps. (4) Cocci 0.6-1 p m dispersed (Fig. 11) or in short parallel rows forming sarcina-like cubes. A mixture of two of the above described microscopic structures of the organism was also seen.

Fig. 8. Branched hyphae with transverse septa and absence of longitudinal ones. 48 h. Culture on blood agar in carbon dioxide. Giemsa. X 1,400

Characteristics of Dermatophilus congolensis

677

Fig. 9. A mixture of hyphae, some septated transversely and others also longitudinally, giving parallel rows of cocci. 7 days Culturc i n nutrient broth. Giemsa. x 1,400

Except for nutrient broth shake cultures, where all isolates gave dispersed cocci or cocci arranged in sarcina-like cubes, there was no correlation between the morphological forms described and the various isolates, media or incubation conditions. An isolate might also show various forms in one medium under one or all three incubation conditions. Similarly the incubation condition did not influence the production of one microscopic structure in preference to others. The pattern of development of cocci in the three isolates was similar. Only 0.6-0.8 ,rtm cocci were seen within the first 8 hours of culture. These showed no evidence of germination but continued to grow, reaching 0.8-1 pm. The first evidence of germination was seen after 9 hours when a few cocci gave rise to germ tubes. Germination continued to increase steadily

Fig. 10. Beaded hyphae showing germinal activity in some segments 48 h. Culture on blood agar in carbon dioxide. Giemsa. X 1,400 Zbl. Vet. Med., Reihe B, Bd. 25, H e f t 8

47

678

MUKHTAR TAHAABU-SAMRA

until by 30-32 hours there was an equal ratio of germinating zoospores and cocci. Growth continued with increase in the length and width of the germ tubes. By 45-50 hours the morphology changed distinctly and there was a mixture of (i) hyphae 1.2-1.8 pm in diameter with transverse septations (ii) hyphae 1.2-2.4 pm with transverse and longitudinal septation giving rise to 2-4 parallel rows of cocci. (iii) 1.2-2.4 p m beaded hyphae with

Fig. 11. Dispersed cocci. 48 h. Culture on blood agar in carbon dioxide. Giemsa.

i(

1,400

swollen oval or round segments, and (iv) germinating zoospores 1-1.2 ,urn and cocci (0.6-0.8 pm). This picture persisted until 84-96 hours after which there was variation in the ratio of filaments to cocci and a marked decrease in the prevalence of beaded hyphae and germinating zoospores.

Staining Characteristics All isolates were strongly Gram positive; stained uniformly with Giemsa; non-acid fast; encapsulated and showing a marked halo with a negative capsular stain; PAS positive, the cocci staining more deeply than the hyphae. No flagellae were seen with flagellar stains. Motility Motility was first seen after two hours incubation, reached its peak by the 3rd-6th hour, started to decline by the 7th hour and was no longer demonstrable at 18 hours. Haemolysis The zone of haemolysis obtained after 48 hours incubation had increased by 72 hours. Although the zone of haemolysis under carbon dioxide was wider for some isolates than under aerobic conditions, a similar zone of haemolysis was obtained under both conditions with other isolates. Good results and wider zones of haemolysis were obtained with sheep, human, ox and goat blood. Pig and horse blood gave poor results (Table 3).

679

Characteristics of Dermatophilus congolensis

Table 3 Type and degree of haemolysis produced by 38 isolates of Dermatophilus congolensis after 48 and 72 hours incubation on blood agar prepared from blood of six species

- = NO haemolysis; a = Haemolysis; = 1-2 mm. zone of haemolysis; zone of hamolysis; ' t ; ~= 3-5 mm. zone of haemolysis.

:>:)

=

2-3

mm.

Biochemical Characteristics Table 4 summarizes the results obtained from biochemical tests. The addition of serum to sugar metabolism cultures merely increased the intensity of indicator change. Acid was produced but not gas. The positive sugar results shown in Table 4 were seen at 2-5 days. None of the isolates metabolized adonitol, aesculin, arabinose, dextrin, dulcitol, inositol, inulin, mannitol, raffinose, rhamnose, sorbitol or xylose. 47s

MUKHTAR TAHA ABU-SAMRA

680

Table 4 Summary of results of biochemical tests on 38 isolates of D. congolensis

I

I

1

I

Biochemical test Gelatin Iiqefaction.

37

1

Urease activity.’

38

0

Catalase

38

0

Oxidase

0

29

9

MR

0

38

VP

0

38

lndole production

0

I

I

- ve

38

Hydrogen sulphide production

Sugar

I

+ ve

Litmus milk la, b, c l

38

I

0

I

38

Nitrate reduction

0

38

Citrate reduction

0

38

metabolism

Fructose

1

galactose

I

glucose

1

lactose

laevulose

1

maltose

1

sucrose

1

trehalose

tve

-ve

+ve

-ve

rve

-ve

tve

-ve

we

-ve

+ve

-ve

+ve

-ve

+ve

+ve

35

3

5

33

38

0

7

31

37

1

35

3

15

23

20

18

1

Discussion The environment best suited to the growth of Dermatophilus has received the attention of many workers. It was considered an obligate aerobe by THOMPSON (1954). NISBET and BANNATYNE (1955) and DEANet al. (1961); considered the organism to be aerobic and facultatively anaerobic. Better growth was claimed under anaerobic than under aerobic conditions by MACADAM (1964) and by Fox et al. (1973). Good growth was obtained in the (1958) and ROBERTS (1963 b). presence of carbon dioxide by PLOWRIGHT In this study equally good growth was obtained under aerobic conditions, in carbon dioxide and in a vacuum. The present study did not cover the growth of the organism under strictly anaerobic conditions but this was confirmed previously (ABu-SAMRA, 1971-1975). The growth of the organism under aerobic conditions and in carbon dioxide was concentrated on in the current work since those conditions are said to have a direct effect on the morphology, behaviour, germination and propagation of the organism (ROBERTS, 1961). Attempts to culture the organism in a vacuum were designed to find out whether it would propagate under those conditions and whether its morphology and life cycle are governed by the environment under which it is cultured. The maximum vacuum produced in MCINTOSHand FILDE’SJARS by an EDWARD’S speedivac high vacuum pump resulted in a colony size very similar to that in the presence of carbon dioxide and under aerobic conditions and gave the same microscopic structure. Dermatophilus will thus grow well under much reduced atmospheric pressure. Culture on solid media showed that the colonial morphology and microscopic structure of the organism were not governed by the media or by the

Characteristics of Dermatophilus congolensis

681

atmosphere of incubation. The classic filamentous and coccoid forms were observed on nearly all solid media used, whether rich or poor and whether incubated in a vacuum, in carbon dioxide or under aerobic conditions. In (1961, 1967) reported that the initial filanientous growth contrast, ROBERTS on solid media does not occur without restriction of oxygen or the presence in the medium of untreated blood or serum and that in a nutritionally poor medium either the zoospores do not germinate or the mycelia die. In this study the organism is found not to be fastidious and is extremely pleomorphic. The change from one microscopic structure to another is unpredictable and could not be related to the atmosphere of incubation, frequency of subculture, composition of the medium, pH, moisture or incubation temperature. All the isolates were subcultured from lyophilized cultures that had received the same treatment, were subcultured on the same day on one batch of media and were either incubated at 37 O C in a vacuum, in carbon dioxide or under aerobic conditions on solid media or under aerobic conditions alone in fluid and slope cultures. The various types of microscopic pattern were similar in all cases. This pleomorphism was not entirely an isolate characteristic since, if this were so, one would expect the same isolate to give uniformly either rough filamentous or smooth coccoid forms. Mkmery (1961) found it difficult to confirm or refute the three stimuli (air supply, temperature and nutrient) advocated by ROBERTS(1961) as necessary for germination of the spore. MEMERY(1961) found that the three factors were too closely linked to be studied separately and that their action overlapped. In this study the pleomorphic nature of the organism was attributed to a combination of isolate, medium, environment and temperature. ABU-SAMRA and WALTON(1977 b) found that hyphae were irregularly transformed into cocci giving rise to budding-like structures and that certain hyphae gave rise to distended segments, each segment of which functions as a zoospore, a phenomenon which was not seen among cocci formed by the budding-like structures. Fig. 10 shows beaded hyphae that stained deeply with Gram and Gienisa, with evidence of germination of some of their segments. I n addition to this type of hyphae, which was also described by HARTet al. (1967) and KHAROLEet al. (1976), all the forms described by GORDON(1964) were also seen. dextrose agar conFailure of Derrnatophilus to grow on SABOURAUD’S firmed the findings of GORDON(1964), and WEBERand SCHLIESSER (1971) and was contrary to the findings of BENTINCK-SMITH et al. (1961). Failure to grow on MACCONKEY and desoxycholate citrate agar was in (1971). The agreement with MACADAM (1964) and MACADAM and HAALSTRA dox agar and crystal unsuccessful attempt to grow the organism on CZAPEK (1971). violet confirmed the findings of MACADAM and HAALSTRA Good growth was obtained on brilliant green agar, as was seen by WEBER and SCHLIESSER (1971), but growth was delayed and less profuse than on other media and the larger colonies obtained, compared to other media, could be attributed to the longer incubation period. JENSEN the organism grew slowly with the first eviOn LOWENSTEINdence of growth at three days. WEBERand SCHLIESSER (1971) reported good growth in 2-3 days, whereas MEMERY(1961) and MACADAM and HAALSTRA (1971) reported no growth after 28 days. The medium is rich in protein but one of its components (malachite green) was reported to inhibit Dermatophilus (HUSSEL and NEUBERT,1974) and in the present study no explanation could be given as to why the isolates grew well and rapidly on this medium

682

MUKHTARTAHAA B U - S A M R A

No growth was obtained on potato dextrose agar, as reported by DEAN et al. (1961) whereas CHODNIK (1956) reported scanty growth on this medium. The organism did not grow on corn meal agar, LITTMANox gall agar, or on cystine lactose electrolyte-deficient medium. It was inhibited and destroyed i1i selenite broth. The reasons for failure to grow in these media may be lack of nutrients (corn meal agar and cystine lactose electrolyte-deficient medium) and the presence of bile (LITTMAN ox gall agar) and sodium biselenite (selenite broth). Apart from M ~ ~ M E (1961), RY who reported no growth on Dorset egg medium, the finding that the organism grows well on this medium, producing more than one shade of pigment, substantiates similar results reported by other workers. O n nutrient agar, except for the scarcity of aerial hyphae, an observa(1971), all the isolates gave tion also recorded by MACADAM and HAALSTRA good growth similar to that on blood agar. This was contrary to WEBERand SCHLIESSER (1971) who reported poor growth on nutrient agar. Attempts to culture Devmatophilus on horse blood-enriched brain heart infusion agar gave very good results, comparable to those on blood agar and serum enriched nutrient agar. In this study growth patterns similar to those reported by other workers were observed i n nutrient broth, peptone water and serum broth. In the current work Devmatophilus grew equally well in brain heart broth, tryptone soya broth and cooked meat medium, infusion, TODD-HEWITT all those media being relatively rich and free from inhibitors. The effect of aeration on the organism was to convert all the isolates into either a fine powdery sediment or a mass of spheres. Smears revealed a complete change to 0.6-1 ,urn. diameter cocci. Mechanical agitation may have accelerated the release of the cocci formed by budding-like structures (ABu-SAMRA1977) or the effect may have been the result of lack of nutrients as recorded by ROBERTS(1961) since the nutrients in the medium were not replenished. The culture of pure cocci resulted in the production of germ tubes that developed and ended in the formation of beaded hyphae in addition to segmented ones. Growth continued until a stage was reached when germination of spores ceased and there was also a marked reduction in the prevalence of beaded hyphae. These changes may have been due to deficiency of nutrients in the medium (ROBERTS, 1961). There is general agreement in the literature, which is confirmed in this study, that Devmatophilus is Gram positive, non-acid fast and encapsulated. The organism was PAS positive and GORDON(1964) reported that in tissues the organism showed up well with this stain. No flagellae were seen under the light microscope; they were probably too small. Workers who studied the motility of the organism in semi-solid agar and by the hanging-drop technique agree that it is motile. The present work confirmed this but only after 2 hours, with a peak at 3-6 hours. The first 2 hours were probably needed for motile cocci to be set free. The ability of the organism to haemolyse blood of various species was studied because the organism was observed to have lost its haemolytic ability on horse blood agar after the fourth subculture on this medium. HART et al. (1967) observed that continued subculture in serum broth led to loss of haemolytic ability which was regained by passage through the chick embryo chorioallantois. Table 3 shows that the organism gave very poor results on horse blood and good results on blood from other species except for the pig.

Characteristics of Dermatophilus congolensis

683

Although biochemical characteristics have received attention from many (1973) found them ill-defined and mostly workers AINSWORTH and AUSTWICK negative. In the present study all the isolates are urease and catalase positive; liquefy gelatin (except for one isolate); produce a clot, peptonize or digest litmus milk, liquefy, digest or clear LOFFLERSserum and metabolise glucose. These findings substantiate what has been reported elsewhere. In this study none of the isolates reduced nitrate or utilized citrate, produced indole or hydrogen sulphide; all gave a negative V. P. test. All this is in accordance with what has been reported elsewhere. The extent to which the findings obtained in this study with other biochemical tests agree or disagree with the findings of other workers is summarized in Table 5. In conclusion, only very slight differences were observed between the 38 isolates of Devmatophilus in their cultural, morphological, haemolytic and biochemical characteristics. Such differences as were found could not be correTable Extent to which results of biochemical tests agree or disagree with those of other workers This study

Biochernicat test

+ve

-ve

Oxidase

29

9

MR test

0

Arabinose Dulcitol lnositot Dextrin Fructose

I

Galactose

Negative ( - 4

NIL

WEBER and SCHLIESSER,(1971); Fox et a l , (1973); LLOVDand OJO. (1975).

38

EL-NAGEH, (1971IjLLOYDand and OJO 11975)*.

Fox et al, (1973);MACADAMand HAALSTRA, (1971); KRUZE et al, 11975).

0

38

BEN~INCK-SMITH et al, (1961). HART e t at, (1967); MACADAMand HAALSTRA, (1971); FISCHMAN et a1 11971).

0

38

KELLEYet al, (1964); FISCHMAN et al, 11971).

NIL

35

3

GORDON,(1964); LLOVOand OJO, (1975)?

NIL

I 1 1

I

5

33

MACADAMand HAALSTRA, (1971); DEAN et al, (1961); KELLEVet al, KRUZEet at. (1975). I(1964):HARTet al. (1967).

lnulin

0

38

KELLEVet al, (1964); Fox et al, (1973).

Lactose

7

31

HART et al, (1967)*; MACADAMEL-NAGEH,(1971); KING et al, (1971); and H A A L S T R A(1971)*. . KRUZE et al. (1975).

Laevutose

37

1

MACADAM and HAALSTRA, (1971); CHODNIK, (1956); FISCHMAN et al, 11971). KRUZEet al, 11975).

Maltose

35

3

KING et al, (19711; MACADAM WEBER and SCHLIESSER, (19711; Fox et and HAALSTRA, 11971)* at, (1973);LLDVDand OJO, (1975).

Mannitol

0

38

THOMPSON, (1954); MACADAM and HAALSTRA,(1971)*.

DEAN et al, (1961); EL-NAGEH, (1971); LLOVDand OJO, (1975).

Raffinose

0

EL-NAGEH(1971).

0

38 38

MEMERY, 11961).

Rhamnose

15

23

__ 20

HART et al, 11967)*; MACADAMGORDON,(1964); KING et at, (1971); Fox and HAALSTRA, 11971)*; KRUZE et at, 11973) e l at, 11975).

18

MEMERV,(1961); MACADAM and HAALSTRA,(1971)*.

38

E~-Nageh, 11971).

Sucrose

Xylose 't

Other workers' findings Positive (+ve)

I

0

= Some strains

I

only.

HARTe t al, (19671; MACADAM and HAALSTRA, (1971)~KRUZE et at, 11975).

WEBER and SCHLIESSER,(1971). BENTINCK-SMITH et at, (1961);KELLEYet al. (1964); KRUZEet at, 11975).

MACADAM,11964 b).

1 KING e t

al, (1971); FOXet at, (1973).

1

684

MUKHTAR TAHAABU-SAMRA

lated with the animal species from which the isolates were obtained and they were not sufficiently constant to allow Derrnatophilus to be divided into precise strains. The term “isolates” is therefore preferred when discussing the findings. Thus the proposed three species Dermatophilus congolensis, D. derrnatonornus and D. pedis (AUSTWICK, 1958) could all be accommodated within a single genus and species D. congolensis, as was suggested by GORDON (1964).

Summary The cultural and biochemical characteristics of 38 isolates of Dermatophilus from cases of bovine and equine streptothricosis and from ovine mycotic dermatitis and strawberry foot rot, in Great Britain, the Sudan and Nigeria, were studied. All isolates grew well under aerobic conditions, in carbon dioxide and in a vacuum on various solid media, except that no growth was obtained on dox agar, corn meal agar or cystine lactose LITTMANox gall agar, CZAPEK electrolyte-deficient medium. All isolates grew well under aerobic conditions in various liquid media including brain heart infusion, cooked meat medium, broth. tryptone soya broth and TODD-HEWITT The colonial morphology of the isolates on various media under the previously mentioned incubation conditions was examined, together with the microscopic structure of the organism. In addition to its well recognized forms, a beaded type of hypha is described and is considered an important stage in the life cycle. The effect of aeration on the morphology and development of the organism was studied in nutrient broth shake cultures, and a description is given of the stages of development of pure cocci from three isolates which were followed over a period of a week. A study of the haemolytic activity of the organism on the red cells of cattle, goats, horses, man, pigs and sheep showed that after the 4th subculture on horse blood its ability to haemolyse horse red cells was lost, whereas its ability to haemolyse red cells from other species persisted. The zones of haemolysis obtained under aerobic conditions did not differ appreciably from those in the presence of carbon dioxide. Pig red cells were poorly haemolysed. Acid but no gas was produced from fructose, galactose, glucose, lactose, laevulose, maltose, sucrose and trehalose. No isolate metabolized adonitol, aesculin, dextrin, dulcitol, inositol, inulin, mannitol, raffinose, rhamnose, sorbitol or xylose. The organism is oxidase positive. The minor differences found among the 38 isolates in their cultural, morphological and biochemical characteristics could not be related to the host species from which the isolates were obtained, and were not sufficiently constant to allow for the division of the genus into three species, or even to justify the isolates being considered to be distinct strains. These findings confirm the view that Derrnatophilus congolensis, D. dermatonornus and D. pedis should form a single genus and species Derrnatophilus congolensis. Acknowledgements The author wishes to thank the British Council for their generous financial assistance which made this work possible, Emeritus Professor E. G. WHITE, Mr. G. S. WALTON,Department of Veterinary Preventive Medicine, University of Liverpool, for their valuable help and continued encouragement. Thanks are also extended to Dr. LIBEROAJELLO,Centre for Disease Control,

Characteristics of Dermatophilus congolensis

685

Atlanta, Georgia, and Dr. G. A. PEPIN,Central Veterinary Laboratory, Ministry of Agriculture, Fisheries and Food England, for confirming the Department identification of some of the isolates; and to Dr. C . 0. DAWSON, of Veterinary Pathology, University of Glasgow Veterinary School, Mr. D. H . LLOYD, the Hannah Research Institute, Ayr, Scotland and veterinary practitioners for supplying infected material or lyophilized isolates.

Zusammenfassung Morphologische, kulturelle und biochemische Eigenschaften von Dermatophilus congolensis Es wurden die kulturellen und biochemischen Eigenschaften von 38 Dermatophilus-Isolaten aus Fallen boviner und equiner Streptotrichose sowie der ,,Mycotic dermatitis" und ,,Strawberry foot rot'' der Schafe in GroBbritannien, dem Sudan und Nigeria untersucht. Alle Isolate wuchsen unter aerobeii Bedingungen gut, und zwar sowohl in CO.) als auch im Vakuum auf verschiedenen Festnahrboden. Auf LITTMAN dox Agar, Maismehlagar und Cystin-Laktose Medium ox gall Agar, CZAPEK ohne Elektrolyte wurde kein Wachstum beobachtet. Alle Isolate wuchsen unter aeroben Bedingungen gut in verschiedenen Flussignahrboden wie TryptonBouillon, Fleischbouillon und Hirn-Herz-BodSoja-Bouillon, TODD-HEWITT, lon. Es wurde die Morphologie der Koloiiien dieser Isolate in verschiedenen Nahrmedien unter den oben genannten Wachstumsbedingungen untersucht, und daruber hinaus die mikroskopische Struktur der Keime. AuBer den gut sichtbaren Formen wird eine ,,geldrollenahnliche" Hyphenart beschrieben; diese wird als wichtiges Stadium im Lebenszyklus gewertet. Es wurde weiterhin die Auswirkuiig der Beluftuiig auf Morphologie und Eiitwicklung der Keime in Nahrbouillon-Schuttelkulturen untersucht. Die Entwicklungsschritte von reinen Rundformen aus drei Isolaten, verfolgt uber einen Zeitraum von einer Woche, werdeii beschrieben. Untersuchungen uber die hamolytische Aktivitat der Keime auf Erythrozyten von Mensch, Rind, Pferd, Ziege, Schwein und Schaf zeigten, dai3 nach der 4. Subkultur in Pferdeblut die Fahigkeit, Pferdeerythrozyten zu hamolysieren, verloren ging, wahrend die hamolysierende Eigenschaft fur Eryhrozyten anderer Spezies erhalten blieb. Die unter aeroben Bedingungen erhaltenen Hamolysezonen uiiterschieden sich nicht wesentlich voii deneii in CO,. Schweineerythrozyten wurden nur schlecht hamolysiert. Saureprodukte, jedoch keine Gase wurden n i t folgenden Substanzen gebildet : Fruktose, Galaktose, Glukose, Laktose, Laevulose, Maltose, Sukrose und Trehalose. Keiiies der Isolate lronnte Adonitol, Askuliii, Dextrin, Dulcitol, Inositol, Inulin, Mannitol, Raffinose, Rhamnose, Sorbitol oder Xylose metabolisieren. Die Keime sind Oxidase-positiv. Es konnte kein Zusammenhang zwischen der Art des Wirtstieres und den geringfiigigen Unterschieden in den kulturellen, morphologischen und biochemischen Eigenschaften der 38 Isolate gefundeii werden. Diese geringen Unterschiede reichen nicht aus, um eine Einteilung der Gattuiig in drei Arten zu erlauben oder um eine eindeutige Unterscheidung in verschiedene Stamme zu rechtfertigen. Diese Ergebnisse bestatigen die Meinung, dai3 Dermatophilus congolensis, D. derniatonomus und D. pedis in einer einzigen Gattung und Arc Dermatophilus congolensis zusammengefaBt werden sollten.

686

MUKHTAR TAHAABU-SAMRA

RCsum6 Proprichis inorphologiques, en culture et biochimiques de Dermatophilus congolensis O n a examink les propriktks en culture et biochimiques de 38 isolements de Dermatophilus B partir de cas de streptotrichose bovine et kquine, de des moutons en Angleterre, au Soudan et au Nigeria. Toutes les souches ont bien poussk sur diffkrents milieux solides en akrobiose, en CO, et sous vide. I1 n’y a pas eu de croissance sur LITTMANox gall Agar, sur CZAPEK dox Agar, sur agar B la farine de mais et sur un milieu cystine-lactose sans klectrolytes. Toutes les souches ont bien poussk dans des conditions akribies dans diffkrents milieux liquides comme bouillon tryptonbouillon de viande et bouillon cerveau-coeur. soja, bouillon de TODD-HEWITT, O n a examink la morphologie des colonies de ces souches dans diffkrents milieux et dans les conditions de croissances citkes et la strucutre microscopique des germes. Une sorte d’hyphe semblable B une p i k e de monnaie est dkcrite en plus des formes bien visibles; cette forme a ktk considkrke comme un stade important du cycle vital. O n a recherche en plus l’action de l’akration sur la morphologie et le dkveloppement des germes dans des bouillons nutritifs agitks. La marche de dkveloppement de formes rondes pures B partir de trois isolements est dkcrite durant un laps de temps d’une semaine. Des recherches sur l’activitk hkmolytique des germes sur des krythrocytes humains, de bovin, de cheval, de chhre, de porc et de mouton ont montrk qu’aprb la quatireme sous-culture, le pouvoir d’hkmolyse des krythrocytes de cheval se perd alors que cette propriktk demeure pour les autres espices. Les zones d’hkmolyse en condition akrobie ne se diffkrencient pas de celles en CO,. Les krythrocytes de porc ne furent que ma1 hkmolysks. Une production acide sans gaz a ktk observke avec le fructose, galactose, glucose, lactose, lkvulose, maltose, sucrose et trkhalose. Aucune souche n’a modifik l’adonitol, esculine, dextrine, dulcitol, inositol, inuline, mannitols, raffinose, rhamnose, sorbitol ou xylose. Les germes sont oxydase positive. On n’a pas trouvk de rapport entre l’espkce h8te et les diffkrences infimes dans le propriktks de culture, morphologiques et biochimiques des 38 souches. Ces petites diffkrences ne suffisent pas B classer le germe en trois especes ou B justifier une nette difference entre les diffkrentes souches. Ces rksultats confirment I’impression que Dermatophilus conoglensis, D. dermatonomus et 11. pedis doivent 4tre rangks dans un seul genre et doivent &re regroup& dans l’espkce Dermatophilus congolensis. Resumen Propiedades morfol6gicas, culturales y bioquimicas de Dermatophilus congolensis Se examinaron las propiedades culturales y bioquimicas de 38 aislamientos de Dermatophilus en casos de estreptotricosis bovina y equina, asi como de la