Further studies on the properties of the aetiology of bovine farcy isolated from Sudanese cattle

Ix larcin du boeuf a fat l’objet d’enquêtes dans trois départements du Soudan, de janvier à avril 1978, qui ont montré que l’affection atteignait 5,19 p. 100 du cheptel bovin au Nyala (Darfur). 4.19 p. 100 au Nub Mountain (Kordofan) et 3,49 p. 100 à Omdurman (Khartoum) Un développement trés abondant de l’agent causal a été obtenu par I’incorporation dans le mdxu de cuttwe modifié de Sauton de pyruvate de sodium, le pH optimal se situant entre 7 et 9 et avec des karts de température allant de 280 k 4OT A 45 C la croissance st Iégérement ïranée, elle cesse à20 C et à 50 OC. Le germe peut suppmter I’addition de II p. 100 de chlorure de sodium dans le milieu de culture et le traxement avec 5 er 15 p 100 d’acide oxahque, pendant respectivement 20 et 5 minutes conséculwes. Les propriétés biochimiques du germe sont semblables à celles déjà décrites pour la souche du Tchad. qui est pathogène pour le cobaye. les porcs et les vea”x. Le nombre de subcultures en milieu artificiel est un facteur limitant des propriétés pathog&niques. Les souches ont montré des résistances vari& à I’INH, PAS et à la streptomycine. L’acide mycohque a été chimiquement extrait des cultures amsl que par la rechnique chromatographlque n couches minces. II a 6th idenhfié par son voint de fusion et sa tiistance à làction dlssolvanre d’un milieu à base d’eau et de “Gthanol

. Meager studies were conducted concerning the bacteriology of the disease (MOSTAFA, 1966(MOSTAFA, , 1967. Lately. SALIH, EL SANOUSI and TAG EL DIN (13) made seasonal surveys of the disease in Western and central Sudan. They also studied the predilection sites and the distribution of lesions in the affected cattle. EL SANOUSI, TAC EL DIN and ABDEL WAHAB (3), using the method of KANETSUNA and BAR-TO11 (4). were able to extract mycolic acid in quantities that permit designation of Mycobacrerium to the geenus. CHAMOISEAU (1) postulated that results and observations made on chadiense strains could be applied to sudanese strams. It is felt necessary that further investigation on the properties of the aetiology of bovine farcy in the Sudan is deemed essential. Therefore, thir work was undertaken to study the bacteriological properties of the bovine farcy organism isolated from sudanese cattle.

INTRODUCTION
Bovine farcy is an infectious disease affecting cattle in certain countries in West Africa as well as in Central and Eastern Africa. The disease is caused by Mycobacteriumfarcinogenes formerly Nocardia farcinica. The resemblance to atypical Mycobacterim was based on the presence of betahydroxy fatty acids of the mycolic type, pyrolysable with the liberatlon of tetra casanoic acid which characterizes cultues of atypical Mycobacteria. However the latter was excluded because of the pathogenicity of the farcy organism for guinea pigs and the activity on amides (1).
In the Sudan, the disease was tackled exclusively from the epidemiological and pathological (') Centra. 1   Bovine albumin and home serum werc added as filter sterilized after autoclavmg. Glucose was prepared as 20 p. 100 sterilired separately and added aseptically. A few colonies scraped from lowenstein Jensen (L. J.) medium (about 3 colonies) were used for sowing of each flask. Flasks were incubated at 37 "C in the horizontal position. Flasks were observed daily for growth.

pH
Modified sauton's medium was prepared in test tubes. pHs were selected for the study 4, 5, 6, 7, 8 and 9. IN HC1 and 0.5 N NaOH were used for adjusting pH prior autoclaving. Few tubes in each pH were sacrificed for reading of the final pH after autoclaving. The resultant pH was used for' the study. Two colonies nearly of the same size scraped from L. J. medium were used to inoculate each tube. Cultured tubes were incubated at 37 "C, and observed for four weeks.

Oxalic acid
Growth was scraped from several L. J. media and divided into two portions. One portion was resuspended in sterile 5 p. 100 oxalic acid. Samples were removed at intervals of 5, 10, 15, 20 and 25 min. washed twiçe with sterile normal saline and subcultured in fresh L. J. media. Bottles were incubated at 37 OC and observed for growth for four weeks. The other part was distributed in different concentrations of oxalic acid i. e. 5, 10, 15 and 20 p. 100 for five minutes then washed twice with sterile normal saline and subcultures were made on L. J. media, Bottles were incubated at 37 "C and observed for growth for four weeks.

Biochemical tests
The strains were tested for ability to reduce nitrate and possession of catalase, urease, arylsulphatase enzyme and hydrolysis oftween 80 by fixation of neutral red according to the procedures of VESTAL (14). Mycobacferium fortuitum was used as a positive control.

Sodium chloride tolerance
The following concentrations of sodium chloride (Anal~) were incorporated in L. J. medium 1-15 p. 100 WjV.

LOS~ of pathogenicity for guineapigs by subculturing
Five grams of pus scrapped from an infected lymph node were suspended in 5 ml of phosphate buffer. Five guinea pigs were inoculated intraperitoneally with one ml each of the suspension. The same lymph node was treated as previously described, and sown on L. J. medium. Growth was scraped from 5 McCartney bottles and suspended in 3 ml buffer saline. Each of three guinea pigs received one ml intraperitoneally. Guinea pigs were observed for a month, after which survivors were euthanized and autopsied.
A furtber subculture was made and three et>.. guinea pigs were inoculated and observed. The procedure was repeated till the sixth subculture.

Pathogeoicity for calves
Scrapings of growth in three MacCartne, bottles were suspended in normal saline. Three Zebu calves, 3 years of age were used. Two calves, designated A and B, were inoculated with 10 ml each intravenously. The third calf (C) was inoculated with five ml subcutaneously in the fore limb. The animais were observed for 4 months.  (7). The results were expressed as resistance ratio namely the ratio of the minimal inbibitory concentration for the farcy test strain to the minimal inhibitory concentration for the standard sensitive strain, H,, RV.

Mycolie acid deteetioo
Growth was scraped from several Lowenstein Jensen media. The method of KANETSU-NA and BARTOLI (4) was applied for extraction and characterization of mycolic acid. Further identification of mycolic acid was confirmed using thin-layer chromatography (8).

Serodiagnosis tri&
Antigen of various strains was prepared according to NICHOLLS (II) and agglutination levels were assessed using the niodified Widal test of NICHOLLS (12). The antigen was tested against sera collected from animais infected with bovine farcy. catalase positive. Normal growth occured in concentrations Urease : None of the tested strains were l-4 p. 100 within one week. In concentrations positive.
S-10 p. 100 the growth was delayed i.e. 10 days. Arylsulphatase : No enzyme detected during 3, Poor growth appeared in concentration Il p. 100 15 and 21 days incubation. Mycobacferium in 14 days, as slimy growth. 12-15 p. 100 no forruitum was found to possess arylsulphatase growth occurred up to 24-28 days. Inoculum e"7.y"E. started to disintegrate and die.
8. Loss of patbogenicity for guinea pigs Table IV EfkCL Of rubculruring 0" virulence of eliology Of h""lIw earcy "rg.luis" for g"i"ea-pigs Results are shown in table IV. Pus was formed at the site of inoculation. Peritonitis with multiple abscessation was the most prevailing lesion in a11 dead guinea pigs. White yellowish focci were spreading in the liver, kidney, spleen, mesenteric lymph nodes and pancreas. Stained smears revealed acid fat branching filaments indistinguishable from Mycobacterium farcinogenes microscopically.
The organism was recovered in L. J. medium. Survivors of the second and third subcultures showed oedema and pus at the site of inoculation, but no lesions were seen in the interna1 organs. Euthanized guinea pigs of fourth, fifth and sixth subcultures did not show any lesions.

Pathogenicity for calves
Calf (A) died after 84 days post inoculation with typical lesions of milliary form. Calf (B) was slaughtered 4 months post inoculation and no lesions were detected at post-mortem. Calf (C) no lesions were detected.

Semitivity to INH, PAS and streptomycin
Out of the eight strains tested, six strains were resistant'to the three drugs. Of the remaining two, one was sensitive to PAS and streptomycin and the other was sensitive to PAS only. 11. Mycolic acid detection 13.5 mg dry weight were isolated from each one g wet weight of bacteria. The melting point was found to be 50-52 "C.

Serodiagoosis tri&
Only traces of agglutinin were recorded.

IV. DISCUSSION AND CONCLUSIONS
Slight increase observed in the incidence of infection in Nyala, Darfur, 5.19 p, 100 compared with the previous studies 3.120 p. 100. This could be attributed to cale merchants purchasing whole hcrds, culling apparently affccted ones for slaughtering and sending the rat for export.
However a drop in the incidences was observed in Nuba Mountains (Kordofan) 4.19 p. 100 compared with previous studies 10.2 p. 100 (13). Recently, it was noticed that cattle with apparent lesions of farcy are not attractive to merchants who only supply the slaughter houes because of condemnation of the affected carcasses without any compensation and hence the tendency towards selection. This may give an explanation to the reduction of mcidences of the disease in the slaughter houses of Nuba Mountains compared with the previous records. Most of our chemical and biochemical studies on the properties or the composition of the aetiology of bovine farcy were hindered by the poor growth obtained in various media tried before (L. J. and modified Sauton's medium). The harvested material, needed for further study, was apparently very small and it took along period to collect the required amounts. Modified Sauton's medium alone did not support a good growth, hence various additives were tried to improve the growth. A convenient medium was achieved for such a purpose by addition of sodium pyruvate (0.4 p. 100) to the modified Sauton's medium. Addition of further ingredients did not improve the growth. On the contrary a retardation in growth was observed in some combinations. We were not able to explain the mode of such retardation. As regard to the pH, neutral pH with a slight shift to alkalinity was preferred to the audity. A wide range for growth temperaturc was noted. No striking difference in growth was observed in cultures incubated at 28", 35", 37", 40 "C. However, at 45 "C there was slight delay in some strains and complete inhibition in others. At 50 "C, colonies disintegrated and died. At 20 "C, culture failed to multiply but remained alive and grew when reincubated at 37 "C after four weeks incubation at 20 OC. This was concerning strains subcultured several times. However, for primary isolation, organism failed to grow neither at 28 "C nor at 40 OC. They remained alive for four weeks at 28 "C and showed growth later when re-incubated at 37 "C. When incubated at 40 "C they failed to grow when temperature was lowered to 37 "C.
The Sudanes? strains of ~w&zcterium faucinogenes showed a remarkable resistance to sodium chloride. Though poor yet, growth occurred in concentrations up to 11 p. 100. Concentrations 5 p. 100 and 6 p. 100 delayed growth, on further incubation maximum growth was attained. Contrary to the findings of MOSTAFA (9), ou strains were able to withstand treatment with 5 p. 100 oxalic acid for up to 20 min. MOSTAFA (9) reported a maximum time of ten minutes for survival. The strains were highly pathogenic for gui& pigs. This pathogenicity is govemed by the number of subsequent subculturing in artificial medium. Partial attenuation occurred by the second subculture. Complete loss of pathogenicity was achieved by the fourth subculture. The importance of such an observation is a beneficial knowledge for vaccine production to counteract such an increasingly economical important malady. Also whenever pathogenicity of the farcy disease is to be studied, the numbers of the previous subcultures should be clearly stated. A full description to the milliary type of farcy observed in one calf is discussed elsewhere (2). Drug sensitivity tests are used not only as a guide to chemotherapy but also to support and amplify classification (6). Although the number of the strains tested were too small to draw a valid conclusion, yet, it cari be stated that most of the strains tested were primary drug resistant and hence they simulate atyp,ical mycobacteria in this respect. The studied strains showed a remarkable resistance to INH, PAS and streptomycin apart from one strain which was sensitive to PAS and streptomycin and another one which was sensitive to PAS only.
The method of KANETSUNA and BAR-TOLI (4) applied previously by EL SANOUSI, TAG EL DIN and ABDEL WAHAB (3) is a simple and reliable method for extraction of mycolic acid. At the present studies, 13.5 mg dry weight of mycolic acid were extracted from each one g wet weight of bacteria, in comparison withprevious studies 8 mgwere extracted.Though both values lie safely in the range of mycolic acid of the genus Mycobacterium yet such values could be stabilized by standardizing the ratio of ethanol to ether to be used for the extraction. The melting point was the same as in the previous studies ix. 50-52 "C.
A further evidence to the identity of mycolic acid was contimed by thin layer chromatography. When spots were washed with a 'mixture of methanol water (5 : 2 v/v), a11 spots with the exception of those corresponding to the mycolic esters, were removed. Work is in progress to use the mycolic acid extract as an intradermal injection for allergy testing in affected cattle.
The serodiagnosis of the diseases by the method of NICHOLLS (Il) though gave negative results for sera of clinically infected cattle should not be abandoned as an insensitive test unless a significant number of positive sera is tested. Also, the failure of such a test to detect agglutinin in positive sera could be attributed to the type of antibodies produced. We therefore suggest for further studies that experimental infection should be made for calves and then following the titre of the agglutinin throughout the course of the disease.
These studies showed beyond doubt that etiology of bovine farcy in the Sudan is a Mycobacterium and not a Nocardia. The strains were found to possess similar characters to those of the chadians as formerly postulated by CHAMOISEAU (1) ; perhaps the intermingling of cattle between the common borders of the two countries has facilitated dissemination of common strains.
in modilïed Sauton's medium when sodium pyruvate was incorporated with optimum pH of7-9 andawidcrange ofgrowth temperatureZZ-40 Xwith slight delay at 45 OC and no growth at 200 and 50 OC. The organism resisted 1, p. 100 sodium chloride in the medium and treatment with 5 p, 100 and 15 p. 100 oxalic acid for 20 and 5 min. respectwely. Biochemical properdes were similar to tbose of the chadian sfrain. The organisms are pathogenic for guina pigs and calves. Pathogenicity is govemed by number of subcultures in artificial medium. Strains vari& in their resistance to INH, PAS and streptomycin. Mycolic acid was extracted chemically and by thin layer chromatography and was confirmed by its melting point and by reristing dissohing mixtures of methanol and wab.
Se ha extraido de las cultivoo e, acide mic&ca quimicamente y par el técnico cromatogr&co en capas delgadas. Ha sido idcntificado par su punto de fusion y su resirtencia a ,a accidn disolvente de un medlo a base de agua y de metano1.