-BOO K NUMBER Q iu a i - f y * Tfeics f^ votvi fa*) The B a lm e L i b ra ry 3 0692 1078 5918 1 University of Ghana http://ugspace.ug.edu.gh studies on the B^cTaii0L0G-y Off jfr jLL IIK FROM MIRY ffiSRDti ON THE ACCRii PIAINS A Thesis Presented to the Department o f Animal Science (Faculty o f A gricu ltu re ) U n ivers ity of Ghana, Legon. In P a r t ia l Fu lfilm ent o f the Requirements fo r the Degree o f Master o f Science (M .Sc.) (Animal Scienc e) By Derrick Amadu Ayeloo Ag. Head of Animal Science Department, U n ivers ity o f Ghana, Legon. Dr. S.N. Afoakwa, In terna l Kxaminer, Medical School, Un ivers ity o f Gljana, Legon. L r 'Ct>\ 9 • t * * • • . i t • • r r a * . . ■ Dr. K. Boakye-^Pi adorn, External Examiner, Hec.d, Department o f Pharmaceutics, U n ivers ity o f Science and TechnoJfgy, Kurnasi. June, 1973. University of Ghana http://ugspace.ug.edu.gh DECLARATION I do hereby declare that except references to other peop le 's work which have been duly acknowledged, thl3 work is the resu lt o f my own o r ig in a l research, and that th is thesis e ith er in whole, ctr in part, has not been presented fo r another degree elsewhere. University of Ghana http://ugspace.ug.edu.gh A C K N 0 W L E D & E M E IT T Thanks are due, f i r s t o f a l l , to the Department o f Animal Science fo r providing the funds and fa c i l i t i e s fo r th is viori:, and to Dr. E.IT.',7. Oppong, Ag. Head o f Department fa r his encouragement, suggestions and critic ism s . I am very h eav ily indebted to Dr. R.K.Cr. Assoku., fo r his supervision, encouragement, suggestions and co rrection o f the manuscript. I am also very g ra te fu l to Dr. R.E. Larsen fo r his suggest­ ions and encouragement, Dr. L.O. l'Igere, -who helped in the analysis o f the resu lts , and Hr. C«A. Abrahams, form erly o f th e Animal Research In s t itu te , fo r help in in i t i a l lab or at or;? techniques. Specia l thanks are a lso due to Messrs. 3.K. Qurranttey, Jumah ilahama, 3. Ewetso, and II. Iasaka (o f the U n ivers ity Hospita l) fo r th e ir help in the f i e ld and the laboratory, and to Dr. J.D. Corkish o f the Veterinary Services Laboratory, Accra, fo r his help on Tuberculin Testing. Uention must be made a lso o f Kr. Laryea and his s t a f f a t the A .R .3. Ilungua, Ur. Yeboah of the Amrahia Dairy Para, and Kallam Sidow o f Ashalley Botchway, fo r their help during the co lle c tion o f milk samples. i i i University of Ghana http://ugspace.ug.edu.gh My sincere thanks go to Kiss Gladys Lartey and Ur. H.Y. iibabio, v/ho typed the manuscript, and to a l l those who contributed in d iverse ways to making th is work a success. June , 1 973 ° Un ivers ity o f G-hana, legon. University of Ghana http://ugspace.ug.edu.gh iv TAB Tv’ 0? C0I-TT3W3 T it le o. . Declaration ■^cknowle dgement L is t o f Tables L is t o f Figures L is t o f ii.ppendice£ OifcPTEP. I : XIjUpODUCTIOS . . . CI-ItkPTUR I I : LTTSkiTtEIS RETEStf CILiPTSR I I I : LIT3SIIIB jilJD ird'HICDS (1) Dairy Farms (2 ) C ollection of l i i lk Samples (3 ) laboratory Procedures (A) Ledia . . . » . « (3 ) Standard P la te Counts fo r Bacteria . . . . . . (C) Iso la t ion and Id en tif ic a tio n o f Bacteria . . . . . . CI»K?fa IV: L j^LULTo a!ID DfiCUoiilOI'T . . . . . . (a ) Standai'd P la te (B acteria ) Counts o f Raw !,)ilk . . . » . . (B) Iso la t ion o f bacteria o f public health importance . . . (C) Iso la tion o f other important bacterid. „ . , . . . COITCnJSIOITd AITD otM.jJRY . . . iv v vi 1 5 11 11 18 20 ^0 23 25 29 CliiiPi’ER V : 41 55 University of Ghana http://ugspace.ug.edu.gh I&BLB OP COM an? S ( Cont1 d) PkGB (xi.) C 011c lu. si ons . . » «• • • « . 0 9 * 33 (B) Summary . . . . . . . . . . . . 55 References . . . . . . . . . . . . 57 Appendix . . . . . . . . . . . . 62 University of Ghana http://ugspace.ug.edu.gh VLEiJ Off FIGURES FIGURE PAG-E 1 Hand Milking at the A .R .S., Nungua . . . 13 2 Machine Milking a t Arnrahia Dairy Fi.rm . . -j 5 3 Floor plan of the ffulani Kraal . . . -\G 4- Restrainirg a ca lf during milking at the Pulani Kraals . . . . . . . . . -57 5 Milking at the Pulani Kraals . . . 19 University of Ghana http://ugspace.ug.edu.gh v i L33 T OF APPMDICjS APFEKDIX Standard Plate Counts at 37 C, the Stage o f Lactation and health Records of Cows Sampled at the &.R.S., Hun^ ua . . . 62 Standard Plate Counts at 37°C, the St^ge o f Lactation and Health Records of Cov;s Sampled at the Amrahia Dairy Farm . . . 60 Standard P!lAte Counts at 37 C, the Stage of Lactation ani. Health Records of Cows Sampled at the Fulani Kraals . . . 70 Grams Staining Method . . . . . . lb Ziehl-Neelsen litainirg Method . . . 75 Modified ^iehl-Neelsen Staining Method,,. 76 5 7 Methylene Blue Stain • • 77 University of Ghana http://ugspace.ug.edu.gh 1CHAPTER I INTRODUCTION’ Milk has been defined by the United States Public Health Services (1953) as the lactea l secretion p ractica lly free o f colostrum, obtained by the complete milking of one or more healthy cows, which contains not less than 8. 25/0 milk solid - non-fat (SNp), and not less than 3*25/- milk fa ts . Apart from the major constituents by vihich milk has been defined (Table 1 ), i t contains low core entrations (about one part per m illion ) o f iron, iodine, and copper, as well as traces of cobalt. Fhospho-lipids, stero ls, c it r ic acid, enzymes and vitamins are present in small quantities (Vanstone and Douga.ll, i 960) . Because a l l the constituents of milk are very essential for l i f e , milk has been referred to as the "most nearly perfect food elaborated by nature" (Poster, Nelson, Speck, Doetsch and Olson, 1958)• Hilk is also an excellent medium fo r the growth and the cow* s udder also provides a suitable habitat for a number o f pathogenic micro-organisms. University of Ghana http://ugspace.ug.edu.gh 2TABLE 1 MAJOR CONSTITUENTS OF MI IK CONSTITUENT H&NG-E AVERAGE % % Water 38.87-91.55 87.60 Total Solids 8.45-16.13 12.60 Eat 1.03-6.39 3.60 S o li d-Non-Fat 7.42-9.74 8.80 Prateins 2.37-4.26 3.30 lactose 4.41-5.00 4.75 Mineral Matter 0.62-0.78 0.75 Lime 0.104-0.291 O.16 Potash 0.148-0.223 0.19 Soda. .036- 0.090 0.07 Magnesia 0 .005- 0.028 0.018 Phosphoric aoid 0.146-0.310 0.23 Chloride 0.054-0.242 0.10 Source: Vanstone and Dougall (i960). University of Ghana http://ugspace.ug.edu.gh The sources of these pathogenic organisms, though usually varied, include the diseased cow, personnel, milking utensils and other equipment. Salle (1967) reported that tuberculosis, food-poisoning, in fantile diarrhea, poliomyelitis, septic to n s ilit is , and typhoid fever, are among the numerous human diseases whose organisms survive in , and are transmitted by, milk. I t is therefore absolu­ te ly necessary that good quality milk be produced for the consumer. Dairying is a developing industry in Ghana. Until recently, milk production was the sole monopoly of the Fulanis. The Fulanis keep animals from different people, and there may be as many owners as there are cattle in the kraals. The University of Ghana has a dairy herd at the Agricultural Research Station (A .R .S .), Nungua, for teaching purposes, while the •Animal Husbandry Division of the Ministry of Agriculture has a Dairy Farm at Amrahia. The herd at the A .U .S., Nungua, is mainly crossbreds (Jersey X Shorthorn; Jersey X Gudali; and Jersey X N1 dama) and a few Gudalis and N* damas. Ngere (1972), reporting on the evaluation of the present status of the dairy crossbreeding performance at A.H.3. Nungua, found that the overall average production far crossbreds was 3,225 lb s ; when the local breeds (N1 dama and Gudali) were included, the average dropped to 2,742 lbs. Though production records are lacking on Fulani farms, the production of these predominantly Sanga herds University of Ghana http://ugspace.ug.edu.gh could be put at a lib e ra l figure of 1,500 lb s , based on production records of the local breeds - N' dama and Gudali - at the A.R .S., Nungua. The friesian herd at the Amrahia dairy farm produces an average of 1 - 2 gallons per day per cow fo r a lactation period of 308 days. One of the most important problems facing the infant dairy industry in Ghana is the production of clean, healthy milk for human consumption. This problem has been successfully tackled by certain countries with well-developed dairy industries, by laying down certain regulations and standards for milk production and r ig id ly enforcing them. These regulations and standards are obviously not applicable to the Ghanaian conditions, and studies on the bacteriology of raw milk produced in th is country are therefore urgently needed. The present study, with special reference to fiecs public health importance, was therefore undertaken to investigate the bacteriology and hygiene of raw milk produced under three different farming systems in the Accra Plains of Ghana. 4 University of Ghana http://ugspace.ug.edu.gh CHAPTER I I LITERATURE REVIEW The problem of producing clean, healthy, milk for human consumption has been realised long ago in countries of high milk production and consumption. Work has, therefore, been done to evaluate the bacteriological quality of milk, and regulations and standards have been la id down fo r production of milk far consumption (e .g . The "Milk Ordinance and Code" of the U.3. Public Health Ser- vie e s ). Sources cf bacteria in milk are varied, and since the publi­ cation of works by Ward (1900), Von-Frendenreich (1903) and Barthel (1906), i t has been established that, in many instances, the udder of the cow is a natural habitat for some of these bacteria. Darner (193°)> working on the bacteriology of aseptically drawn milk, reported bacterial counts between 53° and 4,390 per ml. on standard agar plates, which he considered 1 normal’ . But Bergey (1904) had earlie r reported that 32f* of 272 samples of milk drawn into sterile tubes contained no bacteria per m l., 48.8$i contained less than 500 bacteria per m l., and only 10.3/S contained more than 5,000 bacteria per ml. An average bacterial count o f 500 - 1,000 per m l., was reported by Foster, et a l, (1958), while Seamen (1963) found that milk leaving the udder contained between 300 and 400 bacteria per ml. Verm, Zal, R. Kotharalla and Seshacharyulu, (194k) found that mil v produced 5 University of Ghana http://ugspace.ug.edu.gh 6under different conditions showed differences in bacterial counts, though they did not say how significant these differences were. The farms with better milldng conditions showed low counts. Similar observations have also been reported by Abraham and Laryea (1968) in Ghana. They found that the University of Ghana Agricultural Farm, Nungua, had a mean bacterial count of 19j658 per m l., while the Fulani kraals had a mean of approximately 3 m illion bacteria per ml. Crowdy (1939) , however, found that milk produced in barns with beddings gave a mean of 220,000 bacteria per m l., as against 54,000 bacteria per m l., far milking in surroundings free of beddings. This author, therefore, recommended that groaning, cleaning of udders or hind quarters, trimming of ta ils to prevent their tra ilin g in d irt, milking healthy cows, healthy milkers, clean milkers’ clothes, and holding milk at low temperatures were practices to help obtain low bacterial counts. Ayers, Cook and Clement (1918) found that four simple factors were essential for the production of milk with low bacterial content, namely, (a ) sterilized utensils, (b ) clean cows with clean udders and teats, (c ) small-top pails and (d ) a holding temperature of 10°C or less. Conducting tests by varying one factor at a time, they observed that the average count of 65 samples of fresh milk was about 4,500 bacteria per m l., when only the udder and teats were not washed, but the other three conditions were satisfied . When a l l the four condi­ tions were fu lf i l le d , the mean count of the 65 samples was approximately University of Ghana http://ugspace.ug.edu.gh 2,200 bacteria per ml. Preliminary statistics from the work of Covington, Egdell and Thomas, (1952) on 171 farms, to determine the influence of various factors related to production of high- quality milk, showed that factors concerned with milking methods and sanitation were of more significance, than were factors related to building or equipment, provided the buildings and equipment are clean. De F ilippis (194-1) had argued that milk presented a chara­ cteristic total bacterial count, which was essentially independent of ttie method of production. He, however, conceded that "fau lts in milking procedure or laboratory techniques may a ffect the count temporarily". These opinions were based on the examination of bacterial counts of the outside of the udder, the milkers* hands, utensils, milk bottles, and the actual number of organisms added to milk from these sources. This view has been supported by other workers. Brew (1949) reported that he had learned from the inspection of dairy barns (for over 42 years) that there was no descernible correlation between milk quality as determined in the laboratory and the 1 score' of dairy bams. Kelly, Newman, and Hine (1917) and Hunter (1919) had agreed that, generally, bacterial content of milk was not a satisfactory index of sanitary production. McKenaie and Bowie (1946) observed that milk from certain farms, with seemingly unsatisfactory production conditions and methods, were consistently graded as satis­ factory, whereas milk from other farms with visually observable good conditions and methods, often fa iled to make the standard. They 7 University of Ghana http://ugspace.ug.edu.gh attributed, this partly to the fact that many o f the poorer farms had minimal equipment to contaminate the milk. Heeres ( 1950) and Atherton (1959) also reported finding l i t t l e relationsnip between production practices and bacteria l test results. Rrucha and fleeter (1917), working with utensils thoroughly cleaned and sanitized, but with barns ranging from very clean to unclean, found that 54/° o f the milk samples had a count of less than 10,000 bacteria per ml., and only 14 out o f 1,665 samples exceeded 50,000 bacteria per ml. I t vjas therefore concluded that the condition of the barn exerted l i t t l e measurable influence on the bacteria l content o f the milk; and in 1918, Prucba, Y/eeter and Chambers, showed that unsterilized untensils were la rge ly responsible fo r excessive bacteria l contamination of milk. Hie important micro-organisms of milk and milk products are 'tru e 1 bacteria of the sub-order Eubacteriaceae, viruses o f the order V irales, rickettsiae of the order R ickettsia les, yeasts and moulds (Poster et a l ; , 1956). These micro-organisms play a ro le either in the spoilage of milk (Walter, 1967) , in diseases outbreaks, or in the manufacture of various dairy products (Pelczar and Reid, 1965; Stainer, Dondoroff and Adelberg, 1958)* Seaman ( 1963) had reported that the typ ica l primary pathogens of millc were Corynebacterium pyogene, Streptococcus am lflctiae and Staphylococcus aureus, while Mycobac te r ium tuberculosis and Brucella abortus night be excreted in milk when the body had been extensively 8 University of Ghana http://ugspace.ug.edu.gh 9invaded,, Strep, m as titid is , B. abortus, Bacterium lipolyticum or re la ted species and some m icrococci had, however, usually been found in the udder (Domer, 1930)* Cullen and Herbert (1 967) examined b a c te r io lo g ic a lly milk samples, sJdn and teat canal swabs, throughout the la c ta tion period and found that the organisms were mainly non-pathogenic, but the fo llow in g were a lso found in r e la t iv e proportions in the two s ite s ; Coagulase negative - non-haemolyfcic staphylococci; Coagulase negative - s lig h t ly haemolytic staphylococci; Staph. aureus (producing alpha-and bcta-haemolysis), S trep, uberis , Strep, viridans ( alpha-haei.io ly t ic ) , B ac illus spp. , Actinomyces s' . , Pseudomonas spp . , Prot eus spp. , and co lifcrm organisms - mainly E. c o l i . Entero-pathogenic co liform bacteria have been recovered from milk of healthy cows, as w e ll as mill: o f cows with m astitis . TJa lter (1967) and King (19^9) had found that examination of milk from covrs with m astitis also implicated Strep, aga la c t ia e , strep . dysgalactiae. Strep, uber is and staphylococci. Abrahams and La rye a (1^£8) reported a predcsninibpc e o f Staph. albus. and micrococci in both the U n ivers ity fie sear oil Farm and the Fulani Kraals. The milk was cultured 011 Blood and I.’acConkey agar plates only. University of Ghana http://ugspace.ug.edu.gh 10 Ilorrison ai-d Ham tier ( 194 1) , Sherman, Cameron and '.Vhite (1941)» Thomas and Chandra-Sekhar (1946)j Thomas, Blodwen and Silicon (1949), Krdnan and Thornton ('1951 ) and Abdel-Ilalek and Gibson (1952) had. a l l found that the bacteria in re frigerated milk helonged to one or more o f the fo llow ing genera: Achromobacter, A lc a ligenes, Flavo bacterium, Pseudomonas, Aerobacter, Lactobacillus and Streptococcus. Steel: (1921), quoted by Dorner (1930), did not encounter any facu ltative anaerobes, and Dorner (1930), therefore, did not see any reason fo r searching fo r obligate anaerobes in n il!:, '.,'einberg, N ativelle and Prevot (1937), however, described reports of Glotridium perfringens being isolated from cheese and condensed milk. Renk (1962) also isolated th is organisn from 5 cases o f fa ta l gangrenous m istitis of cc.ttle in G-orm^ ny. The scarcity of these reports is an indication of the ra r ity with which dairy products are associated with C. perfrin.r;ens food poisoning (Yfe.lter, 1967). University of Ghana http://ugspace.ug.edu.gh CHAPTER I I I MATERIAL AND METHODS (1) DAIRY FABMS: This study was carried out on three different farra3: ( i ) The University of Ghana Agricultural Research Station (A .R .S .), Nungua; ( i i ) The Amrahia Dairy Farm cf the Animal Husbandry Division; The Fulani Kraals (Ashaley Botchway). These three farms were chosen because of the differences in management, milking procedures, and the level of hygiene on each farm. ( i ) Agricultural Research Station (ARS), Nungua; Farm A;~ Located North-east of Accra, the A .R .S., Nungua, had a dairy herd of forty-nine (49) cows this season (1972-73)j made up mostly of crossbreds. “The research efforts of the station are chiefly devoted to development of dairy cattle by crossing local breeds with Jersey and Fresians ............ 1 (University of G-hana Calender, 1970-72). The herd ms kept in paddocks, and milked twice (3 a.m. - 6 a.m. and 1 - 3 p.m.) daily. At milking time, the cows were brought into the milking barn, and stood near the feeding troughs. A towel, dipped in Lactosan (an tiseptic ), ms used to wash the udder, which was then dried with a clean towel. A small amount of supplemental feed (usually wheat-bran) was given to each cow while being milked. 11 University of Ghana http://ugspace.ug.edu.gh The hind-legs and t a i l of each cow were restrained by a milking rope. (P ig . l ) « The hands of each milker were f ir s t washed in 1fc I&ctosan, rinsed with tap water, and then dried with a clean towel. The milkers, sitting on low stools (see Pig. 1) f i r s t stripped a small amount of milk from each func­ tional teat into a strip cup for examination. In the absence of any abnormal appearance e.g. floccules, the cows were milked by hand into clean buckets. The hands were again washed before the next cot? w a s milked. ( i i ) Amrahia Dairy Pann of the Animal Husbandry Division; Parm B: - The cows on this farm were kept in roofed, concrete pens, bedded on a mixture of saw-dust and straw. (Some of the animals were, however, le f t in the paddocks overnight, for lack of saw­ dust and straw). The animals were mostly imported Friesians, and a few Hereford X Priesian crosses. The cows were milked twice (5«15 - 6.30 a.m. and 3 - 3 p.m.) daily. At milking time, the cows were driven into the milking palour, and stood at the 'milking stand' near the feed troughs. The udder of each cow was washed with a towel clipped in 1fS Anti- G-erm 50*, and dried with a clean towel. The cows were given * Anti-G-erm 50 - Active Ingredients: Alkyl (C1^0 ^ , C1^30^, C12^ , C185^) dimethyl benzyl ammonium chlori.de........... ............. ................................ n-alkyl C1 l+30f., Cl517f', dimethyl ethyl-benzyl ammonium ch lo r id e ................................... Isopropyl alcohol ............................................................ Inert ingredients ................................................... 25T 25 30f.' 20c - 100£ University of Ghana http://ugspace.ug.edu.gh 15 Pig. 1 Hand Milking at the A.R.3. Mungua. Note: ( i ) The Clean bucket, and concrete floor, ( i i ) The Milker is sitting on a low stool. University of Ghana http://ugspace.ug.edu.gh supplemental feed (wheat-bran). A small amount of milk vas stripped fran each functional teat onto a strip cup for examination. In the absence of any abnormalities, a clean teat-cup ms attached to each teat (Fig. 2) and the cows were machine milked in sets of eight, a fter which the teat-cups were again washed in Anti-Germ 5°, rinsed in clean tap water before being used again. At the end of each milking session, the whole milking line was washed and aseptically cleaned, using both hot water and dilute formic acid. The floor of the milking palour was also washed and disinfected a fter the animals had been released for grazing, i i i ) Fulani Kraal; Farm C; - On this farm, the cattle were housed in kraals made of wooden stocks (inexpensive wood from the bush). Milking was done in these open kraals, which were heavily infested with house f l ie s (Musca daaestica) . The calves were separated fran the cows in the evening and early in the morning (about 5.30 a .m .), the cows were taken for grazing and returned at about 9 a.m. far milking. At milking time, the calves were released, one at a time (according to number of milkers available) from their separate compartment, into the main kraal to search for their dams. (F ig. 3 )» Each ca lf was allowed to suckle the dam for a short period. The Fulanis exploit the finding that the natural stimulation for milk let-down is the sucking of the teats by the ca lf (Smith, 1959). $he neck of the c a lf was then tied loosely to the foreleg of the cow (Figure 4 ). 14 University of Ghana http://ugspace.ug.edu.gh Machine Milking at Amrahia Dairy ?am. P ig. 2 University of Ghana http://ugspace.ug.edu.gh FLOOR PLAN OF THE FU LAN I KRA A L 16 X ! University of Ghana http://ugspace.ug.edu.gh 17 Pig. 4 Restraining a Calf during M ilking at the A ilan i Kraal University of Ghana http://ugspace.ug.edu.gh The hind legs and the t a i l of the cow were also restra ined by a. "milking rope”, and the cow milked by hand into calabashes, or bowls, (F ig . 5) and then bulked in kcrosine tin s fo r transportation to the market. ( 2 ) CnTXHCTTOH QF MTU? g^MPT,?S; iiilk samples were taken from cows at three different stages of lactation: (a ) Early lactation samples were those collected from cows which had not been lactating fo r more than three and a h a lf (3 ^ months ( i . e . from date of calving). (b ) Mid, lactation samples were from cows which had been lactating fo r more than three and a h a lf (3- )^ months but not more than six and a h a lf (6^) months. (c ) Cows which had been lactating for more than six and a half (&§■) months were sampled as late lactation cows. The method o f co lle c t io n o f m ilk samples depended very much on the method o f m ilking. In the case o f the A .U .S ., Nungua, and the Fulani Kraal where hand-milking was practiced , the m ilk m s mixed by gen tle s t ir r in g , using the sampling dipper, before samples were taken in to s t e r i le , la b e lle d MacCartney b o ttles with the dipper. At the Amrahia Dairy farm, however, a d irect ou tle t from the m ilk containers was used to c o lle c t the milk samples in to s t e r i le , la b e lle d MacCartney b o tt le s . Cows to be sampled were milked f i r s t , and the number o f samples taken at a time was lim ited to the number o f containers 18 University of Ghana http://ugspace.ug.edu.gh Fig. 5 I'iilk in ,at the Ij'ulani Kraals 'ote: ( i ) The milking is done in the open Kraal, into a large calabash. ( i i ) The milker ia squatting. University of Ghana http://ugspace.ug.edu.gh (e igh t) available on the farm. The udder or each cow sampled on any of the farms ms inspected fo r any abnormalities; fo r example, for non-functional te©ts and hard, indurated udders. The calving date, feeding regime and the health records of each cow, wherj available, were recorded. The milk samples in the MacCartney bottles were packed on ice in a thermos-flaak and transported to the laboratory for examination. (5 ) Li3C3-.rORY paocaptiftss: (a ) ( i ) H ood Agar - 20 gms. o f Bacto-Blood Agar Base, were -weighed in to 500 mis. of d is t i l le d water in a conical f la sk . The mixture was heated and s t ir r in g t i l l the medium was completely dissolved. The medium was then autoclaved at 15 p . s . i . (121°C) fo r 15 minutes, and allowed to cool in a water bath to 44 - 4o°C. A s t e r i le IiacCartney b o tt le o f s t e r i le defib rinated blood* (approx. 25 m is.) was added a sep tic a lly , with thorough mixing and d istribu ted with s t e r i le precautions in to s t e r i le petr i-d ish es. A ir bubbles were removed by quickly passing a bursen flame two or three times over the p la tes. * - S te r ile de fibrinated b lood :- Sheep blood obtained asep tic a lly in to s te r i le universal b o tt le s containing s t e r i le glass bead3. 20 University of Ghana http://ugspace.ug.edu.gh The p la tes were covered and allowed, to set. This medium, which was rou tin e ly used in th is study, lias been recommended, as a base which might be used in the is o la t ion % and cu ltiva tion o f many fastid ious pathogenic micro-organisms (D ifco I.anual, 1965) . ( i i ) I.iacConke.7 -igar: - 25 gms o f Bacto-MacConkey Agar, were weighed in to 500 mis o f d is t i l le d water in a con ical fla sk . The mixture was s t ir red while heating t i l l the medium was completely d issolved. I t nas then autoclaved at 15 p . s . i . (121°C) fo r 15 minutes and d istribu ted in to s t e r i le petri-d ishes as described fo r th e blood aga This medium is a d if fe r e n t ia l p la tin g medium recommended fo r use in the detection and iso la t ion ctf a l l types o f dysentery, tvphoia and para-typhoid bacteria (D ifco Mannual, 1965) . ( i i i ) 1-utriem ^gar:- This agar has a lso been recommended as a general culture medium fo r the cu lt iva tion of the m ajority of less fa stid iou s micro-organisms (D ifco 1965) . 1 2 .5 gms of Bacto-IIutrient Agar were weighed in to 500 mis o f d is t i l le d water in a con ical fla sk heated to d isso lve , and b o ttled in I.lacCartney bo ttles with s t ir r in g t i l l the medium completely d issolved in a lliqu o ts o f 10 mis. ana autoclaved a t 15 p . s . i . (121°C) fo r 15 minutes. ( i v ) ITutrient Broth: - This l ir u id medium has been recommended fo r general laboratory use fo r the cu ltiva tion of micro-organisms that are not exacting in food requirements (D ifco , 1965)• 4- gms o f jJacto-Iiutrient Agar were d issolved in 500 mis. of d is t i l le d water, 21 University of Ghana http://ugspace.ug.edu.gh 22 in conical flasks by heating and s t ir r in g , d is tribu ted in to HacCartney b o tt les and autoclaved at 1 5 p . s . i . (121°C) fa r , 15 minutes, cooled and stored in the re fr ig e ra to r fo r use. (v ) Lowenstein-Jensen Medium: 7 .5 gms o f lowenstein-Jensen medium base were weighed in to 120 mis o f d is t i l le d irater in a con ica l. The mixture was heated with constant a g ita tion t i l l i t b o iled fo r about one minute, autoc!laved a t 121°C (15 lb s . steam pressure) fo r 15 minutes, and allowed to cool to 50°C, Fresh eggs (not more than two days in storage) were washed in water, wiped with a clean towel, dipped in 75^ a lcohol wiped cleano 200 mis. o f whole egg were aseptic a l ly c o lle c ted . The te.se was then gen tly mixed with the whole egg t i l l a uniform mixture was obtained. The mixture was then d istribu ted in to s t e r i le screw-capped tubes, arranged in slanted positions in an oven, and coagulated and inspissated at 85°C - 90°C fo r 45 minutes. Lowenstein-Jensen is most popularly used fo r the iso ­ la t io n and cu lt iva tion o f Mycobacteria (Baltimore B io log ica l laboratory Manual B .B .L .; 1968). (v i ) Trypticase Spy Agar (TSA):- 40 gms T.8.A. powder were suspended in 1,000 mis. d is t i l le d mi at er in a conical f la sk , heated with frequent a g ita tion t i l l i t b o iled fo r one minute. University of Ghana http://ugspace.ug.edu.gh 23 The solution was then autoclaved at 121°C ( 15 lb s . steam pressure) fo r 15 minutes. The agar was then cooled to 45°C in a water bath and .007 ,gms. o f c rysta l v io le t added and poured in to p la tes with s t e r i le precautionary measures. 3 .3 .L . (1968) recommending that Brucella may be iso la ted from milk specimen on th is medium. S t e r i l i t y Test; A l l p la tes and IiacCartney b o ttle s o f agar media and b o ttles o f broths were incubated at 37°C fo r 1 8 - 2 4 hours (o v e r-n igh t). Contanimted p la tes showed co lon ia l growth, while contaminated broths showed tu rb id ity . These were discarded, and those p lates and broths found s t e r i le were stored in the re fr ig e ra to r and used when required. (3 ) uTjJIDARIi PLf-TS COUNTS PCR B_: CTERIA ( i ) D ilu tions:- The milk samples in the IiacCartney bo ttles were rotated tc mix thoroughly, 5 mis. of each samples p ipetted using s t e r i le p ip e tte * in to 45 mis. o f s t e r i le d is t i l le d water. 'This gave an in i t i a l d ilu tion o f 1:10, and subsequently ten -fo ld s e r ia l d ilu tions were made, with a f in a l d ilu tion o f 1:1000 fo r each sample. * A l l glass wares and d is t i l l e d water were s t e r i l is e d by autoclavint at 121°C (15 p . s . i . ) fo r 15 minutes. University of Ghana http://ugspace.ug.edu.gh 24 ( i i ) P la te s :- 1.0 ml. o f 1:100 and 1:1,000 d ilu tions o f each sample' from Farms A and B, and 0.1 ml. o f each o f 1:100 and 1:1,000 d ilu tions o f each sample from Farm C, (L im itations were determined from prelim inary studies) were p ipetted in to s t e r i le 4 -in petri-d ishes and covered up. Then 10-12 ml. o f molten Nutrient agar held at (44-46°c ) in a viater hath, were poured in to each p e tr i-d ish , taking s t e r i l e precautions. The mixture was allowed to set and was incubated at 37° C fo r 4° hours, in the inverted pos ition . A fte r incubation, the number o f co lon ies on each p la te was counted, using the Gallenkamp Colony Counter-*", The number of bacteria in 1 ml. o f the o r ig in a l m ilk sample *was ca lcu lated a s :- Q x 10° = Number o f bacteria/ml. (when 1 ml. o f each d ilu tion was used fo r p la tin g , as fo r Farms A and B ). Q x 10 x 10^ = Number o f bacteria/m l, (when 0.1 ml. o f each d ilu tion was used fo r p la tin g , as far Farm C)„ where Q = Number o f co lon ies counted in the p la te b = The d ilu tion o f sample p la ted . + Gallenkamp and Co. Ltd, Technico House, London * Source: Crowley, et a_L, ( 1969) . University of Ghana http://ugspace.ug.edu.gh 25 (c) ISOLATION Aim IMITIFICATIOI: OF B -C flB IA : (a ) S tain ing: ( i ) D irec t: The MacConkey b o tt les containing the raw milk samples were mixed by rotating and shaking several times, A loop fu l o f each sample was taken a s ep tic a lly onto 2 microscopic s lid es , and a th in film produced by spreading the drop with the edge o f another s lid e . The smear was a ir dried and fix e d by heat, and stained by (a ) Gram* s Stain and (b ) Llethylene-Blue Stain (Appendixes 4 and 7 resp ec tiv e ly ) and examined m icroscop ically. ( i i ) Cream/Sediment: S te r i le centrifuge tubes were balanced in pairs with raw milk (10-15ml.) from each sample and centrifuged at 3,000 r.p.m . fo r an hour. The cream and sediment were mixed on an agglu tination p la te with a s t e r i le loop. Three smears were made on microscopic s lid es from each sample, a ir dried and fix ed by heat. o lid e 1 : Stained by Ziehl-ITeelsen Lietaod (appendix 5) fo r a c id -fas t organisms e .g . Ilycobacterium tubercu losis. S lid e 2: Stained by the Modified Siehl-ITeelsen Method Appendix 6) fo r Brucella. S lide 3: Stained by the Grano Stained IJethod (Appendix 4 ). Each s lid e y;a:s c a re fu lly examined under the microscope. University of Ghana http://ugspace.ug.edu.gh 26 (b ) Culturing: ( i ) The mill: sample in each IiacCartney b o tt le was mixed, 'Snd loop fu l o f the sample taken a s ep tic a lly and streaked on Blood agar and IuacConkey agar. ( i i ) Each sample was a lso inoculated in to Nutrient broth. A l l the p lates and broths were incubated at 37° C fo r 2k- hours. The s ize , form, colour and the e f fe c t o f the colonies on agar (e .g . haemolysis in Blood agar) T/ere recorded. Id en tif ic a tio n o f the organisms were confirmed by sta in ing, ( i i i ) Cream/Sediment M ixture: Samples o f the cream/sediment mixture were inoculated onto lowenstein-Jensen medium, and Trypticase Soy agar. The streaking out on a l l plates vjas as described by Sirockin and Cullimore ( 1969)• The Lowenstein-Jensen agar (tubed) and Trypticase Soy agar were incubated in carbon dioxide at 37°C, and inspected every two days. ( i v ) P la tin g o f Nutrient Broth Culture on Blood and I.lacConkey Agars: A fte r incubating the Nutrient broth fo r 21+ hours, stained s lid e examination o ften showed mixed growth o f organisms. To obtain pure culture: fo r sta in ing and id en t ific a t io n , a s t e r i le loop fu l of the broth culture was sub-cultured onto Blood and hacConliey agar p lates and. incubated fo r 21+ hours. ii in g le , vjell-described colon ies we re then picked, stained and examined,, University of Ghana http://ugspace.ug.edu.gh (o ) Sero lo/ jca l (immunological) Tests: ( i ) Brucella Agglu tination Test:- Whenever Brucella organisms were suspected from stained s lid es and cu lture p la tes , s te r i le serum samples were obtained from the suspected pows and agglu tination te s t (s ) performed on these, using a 1i 10 d ilu tion o f a Brucella stained antigen*. Two-fold s e r ia l saline d ilu tions were used, and the method was as described by Oppong ( 1966) . ^ . A e S ingle Intradermal Comparative Tuberculin T es t:- Tliis test was conducted by the Veterinary Services D ivis ion . biochemical Tests:- TJhere coliform s and other members o f the Snterobacteriaceae were suspected, biochemical tests were performed to id e n t ify and d if fe re n t ia te between them. ( i ) B ile Salt (3IJIXAH TEST) : - This te s t was used to ascertain whether the coliforms detected were Escherichia, co li ; i t depended on the a b i l i t y o f E. c o l i to produce gas when growing in b i le - s a lt lactos e peptone water; a typ ica l coliforms are unable to do th is (Cruiclcshank, 1970 ) . 27 * Donated by M in istry of Pood and Agricu ltu re Laboratories, V/eybridge, ITew Ilaw. University of Ghana http://ugspace.ug.edu.gh ( i i ) Indole production t e s t : This te s t demonstrates the a b i l i t y o f certa in bacteria to decompose the amino-acid, tryptophan'e, to indole, which accumulates in the medium (Cruickshank, 1970). The method employed was as described by Cruickshank, (1970). ( i i i ) Triple Sugar Iron (T .S .I . ) Agar:- Trip le Sugar Iron agar has been recommended as a medium for use in the id en tifica tion of &ram- negative enteric pathogens, particu larly members of the Salmonella-Shigella groups (Difco Manual, 19^5). These organisms have a b il ity of fermenting lactose, saccharose, dextrose (with formation o f acid and gas) and also to produce hydrogen sulphide. T .S .I. (D ifco Manual, 19^5) tubes were inoculated with suspicious colonies from primary media, and incubated at 37°C fo r 24 hours. ( i v ) Sugar Reactions: Lactose. &lucose. Maltose. Mannitol. Sucrose and Trehalose 5 mis. o f Andrades indicator (Cruickshank 1970) were added to 5°0 mis. o f Peptone-Water base (Cruickshank 1970) and 5 mls„ o f the Peptone water base (plus indicator) were then pipetted into screw-capped tubes. (Glass Durham tubes were inserted before the caps were loosely screwed). The tubes were s te r ilised and 0.25 ml. o f s te r ile lOJi solutions o f each sugar was added. The tubes were inspected and those showing bubbles a fter storage ( in re fr igera tor) were discarded. Each tube was then inoculated a sep tic a lly , la b e lle d and incubated at 37°C fo r 24 hours. Tubes which gave negative resu lts were re-innubated fo r another 13 - 24 hours. 28 University of Ghana http://ugspace.ug.edu.gh RBSUIffS AMP DISCUSSION A total of 225 raw milk samples from the University of Ghana Agricultural Research farm, the Amrahia farm and the Pulani Kraals, a l l on the Accra Plains, were examined to provide some information on the bacteriology of raw milk on the Accra Plains. A» STANDARD PLATE (BACTERIAL) COUNTS OP RAW MIIK: Table 2 shows that the to ta l bacteria l counts of samples of raw milk from the Pulani Kraals were the highest o f the three farms. The A.R.S. (Nungua) had a bacteria l count ranging from 3,000 to 227,000 per m l., whilst the Amrahia Dairy Parm was between 3,000 and 272,000 bacteria per ml. The range fo r the Pulani Kraals was between 3>000 and 1,100,000 bacteria per ml. (Appendix 1, 2, 3 )» The Amrahia Dairy Parm, where machine-milking ms practised, had the least overa ll mean bacteria l count o f 27 , 1 6 0 bacteria per m l.; the A.R.S. (Nungua) had 61,466 bacteria per m l., and at the Pulani Kraals, the mean bacterial count o f 345*413 per ml. was the highest (Table 2 ). At the Amrahia Parm the udder of the cows were routinely washed with Anti-G-eiro 50 (an antiseptic) and dried before the animals were machine-milked; the milk was then conveyed through s te r ilis ed pipe lines into a cooling vat. This high le v e l of hygiene and milking practice obviously accounted for the low overa ll bacterial count obtained on this farm. 29 CHAPTER IV University of Ghana http://ugspace.ug.edu.gh 31 Though hand.-milld.ng ws.s p ractised at the A.R.S. (Nungua), the le v e l o f hygiene was re la .t iv e ly high. The udder o f the cows and the hands of the m ilker were washed with Lactosan (an an tisep tic ) f r ie d with a clean towel, and the cows were milked in to clean milking buckets. The degree o f b a c ter ia l contamination at the A .R .S. (Nungua) was, however, higher than that at the Anrahia Dairy Farm, probably because the hands were not clean enough, and the milking buckets not completely fre e o f contaminating bacteria . The milk could a lso have been contaminated by the often humid, dust- ridden m ilking bam . The poor b a c te r io lo g ic a l qua lity o f the milk from the Fulani Kraals might have been due to the poor m ilking hygiene, lack o f aseptic precautions end p rim itive m ilking p ractices. The ea r ly stage o f la c ta tion fo r the Amrahia Farm had a low mean o f 24,600 bacteria/m l., the A .R.S. (Nungua), 65,120 bacteria/m l., and the Fulani Kraal had a high mean of 203,760 bacteria/m l. This pattern o f low mean counts fo r Amrahia Dairy Farm and high fo r the Fulani Kraals was repeated on both farms during the mid and le.te stages o f la c ta tion (Table 2 ). This shows that the le v e l o f hygiene, and m ilking p ractices , had the s.me e f fe c t on the ba c te r ia l count on the farms irrespec­ t iv e o f the stage of la c ta tion ( i . e . farms o f good hygiene had low ba c te r ia l counts at ev :ry stage o f la c ta t io n ). . .. University of Ghana http://ugspace.ug.edu.gh 32 The mean bacterial counts for the Amrahia Dairy Farm and A.R.S. (Nungua), where the levels of hygiene and milking practices were generally high, showed very l i t t le fluctuations of differences between the stages of lactation and there was no definite pattern of increase, but the mean bacterial count for the Fulani Kraals increased with the duration of lactation (Table 2 ). On tiiis particular farm, milking was continued for well over 8 months, and weaning time was correspondingly higher. The demand, therefore, for more milk by the ca lf, the long duration of milking and the farced extraction of milk from the animals, a l l predispose the udder to infection, as evidenced by the numerous teat wounds and damaged teat canals (Appendix 3 )» According to the United States Public Health Services "Milk Ordinance and Code* (1953) Grade A raw milk far pasteurisation should not exceed 100,000 bacteria per ml., prior to mixing with other producer milk, while Grade A pasteurized milk and milk products should not exceed 20,000 bacteria per ml. Based on these requirements, Table 3» shows the grading of the raw milk from each of the three farms. 59 samples, or 78.67/* of milk, examined from the A.R.S., Nungua, was Grade A; only 16 samples, or 21.33/» of the milk, ms below the standard. 72 samples, or 96/i of the raw milk from the Amrahia Dairy Farm, was Grade A, vtfiile at the Fulani Kraals, only 13.335^ (10 samples) of the raw milk conformed with the “A" grade. University of Ghana http://ugspace.ug.edu.gh 25 sam plea were exam ined for each stage of lactation far each farm On P £ pft §■dHCDOa O S’ tu c+tr C3ffl4 t&rO0 f* 1 <-}-o o e B B 0 P0 oo &CD University of Ghana http://ugspace.ug.edu.gh The differences in grades of milk on the various farms were c lea rly a re flec tion of the d ifferen t milking practic es on eetch o f these farms. De F ilipp is (1941), who reported that milk presents a characteristic to ta l bacteria l count essentia lly independent o f method of production, nenevertheless, conceded that fau lts in milking procedure might a ffec t the count. The differences in the grades obtained in this study might, therefore, be due to the fau lts and differences in the milking procedures. This observation c learly is in contrast with the findings of Heeres (195*-*) and Atherton (1959), that there is no relationship between production practices and bacteria l test resu lts. At the Amrahia Dairy Farm, the udder of the cows was washed with 1f. Anti-G-erm 50, dried rath a clean towel before beirg machine-millced. The only possible sources o f milk contamination were probably improper cleaning o f udder, milking o f cows with sub-clinical mastitis, and using improperly s te r ilised milking-machine. Contamination from these sources on this farm was apparently very snail, as evidenced by the low mean bacteria l count (Table 2 ). At the A.R.S. Nungua, the udders of the cows were washed with %= Lactosan, dried with a clean towel, but the animals were milked by hand. Here, the contamination was only moderately; high (Tables 2 and 3 ). At the Fulani Kraals, where milking was done m the fly -in fested , open kraals, without prior washing o f the udders, the degree of bacterial contamination was extraordinary: more than 86% of the raw milk (65 camples) did not conform to the grade "A" c lassifica tion . University of Ghana http://ugspace.ug.edu.gh 35 Abrahams and laryea (1958) had reported that 100f, o f tlie , raw mxlk produced at the University Farm (A.R .S., ITungoa) was Grade A, and that only 10J5 o f the milk fran the Native Kraal (Fulani Kraals) was Grade C; 90/» o f th is milk was G-rade D» This grading was based on a United States Public Health Services Llilk Ordinance c lass ifica tion which required Grade A raw milk not to exceed 50,000 bacteria per ml. This type of c lass ifica tion cannot be found anywhere, even from the lite ra tu re . Die differences in these mean bacteria l counts from these farms were also analysed s ta t is t ica lly , using Snedecor and Cochran methods (1967). As shown in Table 5, the results of the analysis of variance do not show any sign ificant differences of the Standard Plate (Bacteria l) Counts at 37°C within either o f the three farms, or the three stages o f lactation . The results therefore agree with previous reports of Brew (1949)> K elly , et a l . (1971), Hunter (1919), and De F ilip p is (1941) that generally there was very l i t t l e relationship between production methods and bacteria l counts. University of Ghana http://ugspace.ug.edu.gh T&BLS 4- Summary of Standard Plate (Bacterial) Counts at 57°C for Analysis of Variance (Frctn Appendix 1. 2 and 5 A.R.S. NUNGUA AMRAHIA DAIRY FARM FUIAKE KRAAIS GRAND TOTAL Total count far farms (per m l.) (X 10-2*-) 461.0 203.7 2,590.6 3255.3 Total count fo r Lactation stages (per m l.) (X 733.7 871.6 1,650.0 3255.3 University of Ghana http://ugspace.ug.edu.gh TABLE 5 Analysis of Variance of Standard Plate Counts Source of variations df SS MS Observed F Reau % ired F Farms 2 45,771.96 22,885.98 1.8981 6.94 18.00 Stages o f lactation 2 6, 509.01 3,254.51 0.2699 6.94 18.00 Error 4 48,229.02 12,057.26 - Total 8 100,509.99 - - University of Ghana http://ugspace.ug.edu.gh 38 B. ISOL.PlCU OF BAGTSRXi OP PUBjgG HBj' LTH fflPOiffili^E (a ) I .ycobaoterjum tubercu losi3 : Pour suspected cases of Liyco. tuaerculosls (one from the -amrahia Dairy Pam, and the other three from the Pulani Kraals) were id e n t if ie d from stained smears. Killc samples from these cows, when cultured on Lowenstein-Jensen medium at 37°C fo r four weels, and fo r S weeks on Dorset egg agar medium, however, did not show any growth. The d ingle Intradermal Comparative Tuberculin Test on the doubtful case at the -amrahia Dairy S's.im m s a lso negative (Corkish, 1973; Personal communication). The non-isolation of I.Iyco. tubercu losis, during the period o f th is research does not necessarily conclude i t s absence in the raw milk from these farms at a l l times. (b ) Brucella spp. : Attempts were a lso made to determine the incidence o f b ru ce llos is in th is study. Seven suspected cases o f b ru ce llos is were diagnosed from the Pulani Kraals, and eight cases from the Amrahia Dairy Pam ; none were detected at .a.H.rj. ITungua (Table 6) . The organism was detected in stained smears o f mil]: sediments and cultures on T,S.Jt. medium. University of Ghana http://ugspace.ug.edu.gh o wH) <+ f f lo ^ 3 H? p* H M H- p c+4 d © B 1 pHCQ Am rallia D airy Farm Standard Serum of known T itre TITRE . . _. - j crwji -F~ ^ ro -j- b +++ + . + + + + |+ + |+ + + + + | 1+ 1 1+ 1 1+ 1+ 1+ 1 + Vh + t 1+ 1+ 1+ 1+ 1+ ++ 1 1+ +++ ++ 1 I+ CT\ +++ + t ' ' ++ t n - v 4S- 1+ 1+ toCD N "ro Vji + + + + + + + + 1 + l + + + + Tryptioase Soy Agar w(D cHc+ta 83 tri 3o CD PP3 o e> l lc+H*ic+H* B H3 0) ac+ »? I c+H­a & 01o C/2O < X Vjs!VD 9 SP IO T University of Ghana http://ugspace.ug.edu.gh 1+0 Confirmatory serum agglutination test, performed on a random sample of animals and the suspects from these farms showed an infection rate of 13. 64/0 (44 animals sampled) at the inrahia Farm, and 18.45/" (38 animals sampled) at the Fulani Kraals (Table 6 ) . Though these results at Amrahia Dairy Farm, and at the Fulani Kraals were lower than the 23.47/= infection rate reported by Oppong (1966) in a survey o f bovine brucellosis in Southern Ghana, it is useful to note that the agglutination test T?as carried out on a fewer number of animals than Oppong ( 1966) and only as a confirmatory test on suspected cases. These findings suggest that a sizeable proportion of raw mill: produced by some cows on the Accra ELains is infected vdth the brucella organism, and the epidemiology of the incidence of human undulant fever in the Accra region should be examined in relation to the consumption of unpasteurized or unboiled milk by farmers and other workers. The non-isolation of brucella organism from milk samples frcm the A.JS..S., Nungua is due to the fact that since 1963 (when the disease was diagnosed on the fairo), the dairy herd and a l l heifers on this farm have been routinely -vaccinated every year against brucellosis. University of Ghana http://ugspace.ug.edu.gh C. INSOLATION OF OTTIflR IMPORTANT BACTERIA ( Other t ban ll. tubercu losis and Brucel la sp-nTTT" These other types o f b a c ter ia l are a lso o f great importance to human health, p a r ticu la r ly aembers o f the enterobacteriaceae, which have been implicated in certa in types of food poisoning -nd in fa n t ile diarrhoea. Tables 7 ?nd 8 summarise and c le a r ly show the ba c ter ia l f lo ra o f a l l t o raw m ill: samples - other than Nyco t qoercu losis and Bru ce lla spp. - iso la ted from the d iffe ren t farms. I t is evident fro:.: the resu lts that the iso la t ion of the d if fe ren t types o f bacteria are in la rg e r number in the Fulani Kraal samples than the other tyro farms. ( i ) Ba c i l lu s s~pp«: Anthrax bac illu s is the onlj' Gram-positive and variab le spore-bearing aerobic rod that is pathogenic to nan and animals; the other members o f the group are non- pathogenic, but are important from the point c f view o f b a c te r io lo g ic a l studies (Mahanta, 19^5 )• Two species o f the genus B ac illu s , were iso la ted , namely: c areas, and 3. s u b t il is . Both were indole-negative , and produced acid in glucose and sucrose. B. su b tilis produced acid in mannitol and arabinose, while B. cereus, did not. Z . sub tilis . and 3. cereus, including c lo se ly re la ted s tra in s , are genera lly encountered in raw mill: ( Post ex' -ejt a,l, 1958 ) and milk ha s been reported as being one o f the habitats 41 University of Ghana http://ugspace.ug.edu.gh Pleo - pleom orph ic oc+a Q tdl+ M x oo I oH * Bipjo ^H* M H C H t:h>*ClPca II • : « ! ro ct-|d- O OW IdCD H to CT O' o ►J B I n f ? (D <, « 1 + /1 P.O oC/Jo o . H- P O tri X + t Bacillus oereus + Bacillus 1 subtili3 Escherichia coli Staphyl0 c oc c u s SEE* 3 Wroo td W w Strentoopccus Iactobs.cillus fp j> * _______ Cri Corynebacterium '•'70 e~ Coryneba ct erium diphtheria Pseudoraon^ aeruginosa Aerobacter agrogaaeg -'.lcalif;enes viscolactis Pastcurolla S P P . c+H­Ofcf Poc+ iCDP. S» |H-DWOH£0c+Q 6 s University of Ghana http://ugspace.ug.edu.gh 25 sam ples w ere exam ined for each stage of la cta tion for each farm n (?oo k S o U i5. oo OMPo ct-C o fin £ r‘ I roV ' <­c p o t • H M CCO |o |c~.o - g B­d p­S r ) CD IS OO r-co o. t-jcf P l i t ooooo M • [ cac s ct■ pH*Cl $ p. i £ J c2 i CP ON (D £O CD £3 5 a *o o*-5*J I + <4o CO CD CD V>j oo £ >5 “nJ roo ro !+ I o 1 C O I o • It* h[o I , . » {g Vjg V-M ro ON £ O s CO roo -rCD roo cr. £ ro CD HOn O CD V^J ro -vj 8 Oj ro ro %CD ■P- CD ro roco VJlro ONo roo r: « aM fc*Q v_n r 3 O +- HCJ V-/i —v ( > roO' roCO HO n roo o € HON £ H VJl ONo ON V- fo — jT V^M | CD CD * - »-iS H fc j O r L J 1 M > c. 6 tj ; pi a E P fc»’ fi tri 3O oM £ ta 8 University of Ghana http://ugspace.ug.edu.gh of Bo cereus. and as a source of B. subtilis (Breed, Murray and Smith, 1959). The highest number cf these b a c i l l i was isolated fran the Fulani Kraals, followed by the Amrahia Dairy Farm; the A.R«30 Nungaa had the least b a c il l i count (Table 8) . The number of B. subtilis organism gradually declined with the increase in ttie length of lactation at the Fulani Kraals. B. cereus contamination of milk was also found to be quite high at the Amrahia farm during the mid-lactation period. The high incidence of these thermoduric, spcre-bearing b a c i l l i at the Fulani Kraals might be due to the general unclean­ liness of the milking equipment used, and the milking of animals with unclean udders on uncemented, open, dusty milking kraals. This conclusion is in fact in agreement with the observations of Foster and colleagues (Foster et a l . ; 1958) that a high incidence of the genus Bacillus in milk was invariably the result of the dusty coat cf cows, unclean udders and milking in dusty, open a ir . The high incidence of these aerobic b a c illi at the itarahia Farm duiing the mid lactation period, was also most probably due to accumulation of heat-resistant, sxjcore-bearing bacteria in the milking line, owing probably to fau lty cleaning after each milking session (Bryan, Bryan and Mason, 1946). 44 University of Ghana http://ugspace.ug.edu.gh The r e s t i v e l y low number o f these bacteria at the A .11 . j . Nungua, was probably because the milking buckets were washed regu la r l;7, drie d and s te r i l is e d a fte r each milking session. ( i i ) Escherichia c o li ; Gram-negative rods o f varying forms, occurring s in g ly , sometimes in pa irs and short chains, E. c o l i produces, at 44°C, gas from MacConkey b i le - s a lt broth and indole. Production o f ac id and gas at 44°C has been found to be s. sa tis fa c to ry evidence fa r E. c o l l and other fa eca l coliforms (C o llin s and Lyne, 1970). 3. c o l i , a common micro-organism of the in te s tin a l tra c t o f various domestic animals, including cow (ijih&nta, 1965; Poster et a l . , 1958; Cruickshank, 1970), is commonly found in cow dung, and therefore an easy contaminant o f m ilk. The Fulani Kraals, with poor m ilking hygiene, and -.vhere hand m ilking was rou tinely practised in open kraals, showed the highest (2(4 - 60Jj) S. c o l i iso la tion s , fo llow ed by the A .ft.S . Nungua (12 - 32/s)> where hand m ilking was a lso p ractised but the standard o f m ilking hygiene was considerably h i ghe r . The jimrahia Dairy Farm, where machine-milking was practised , showed the lowest percentage (4 - 20) o f E. co_li isolh .tions, This was probably due to the high le v e l o f hygiene, and the r e la t iv e ly , low E. c o l l . , contamination could 45 University of Ghana http://ugspace.ug.edu.gh 46 on ly be due to fa u lty cleaning o f udder, improperly s t e r i l is e d teat cups or animals with sub -c lin ica l 3. c o l i m astit is . 3. c o li is o rd in arily non-pathogenic, when con fineu to the in te s tin a l tra ct o f young calves (hahanta, 1 965) but has been associated with m astitis in cows (S a lle , 1967), and abortion in ewes (Cruickshank, 1970)« The presence o f E. c o l i in some o f the raw milk could a lso be viewed with some in te res t, 13. c o l i has also been accepted as a normal and probably b en e fic ia l inhabitant o f the human howel, but i t has been implicated in some cases o f append icitis , gas troen ter itis and urinary in fections (Tay lor, 1966) . ( i i i ) 5taphvlococcus spp. : I t is evident fran Table 8 that the number o f iso la tion s o f the species o f th is genus was the la rgest of a l l the types of bacteria iso la ted from the various farms. 'The Fulani Kraals showed a high incidence (76 f) o f staphylococcal contamination, the degree o f contamination, increasing on each farm with the prolongation of m illin g (Table 8 ) . This high le v e l o f contamination is l ik e ly to be dangerous. This high staphylococcal contamination at the Fulani Kraals could be explained by the observation that the m ajority o f cases of bovine m astitis on th is University of Ghana http://ugspace.ug.edu.gh farm was caused by staphylococcus species (Apjsendi:: 3 ) anl ranged from chronic to per acute. Some specics o f staphylococcus ( i . e . Staph, aureusj have keen found on the udders o f cows and on human skin and nay e a s il;- gain access in to the raw m ilk; the organism may even be present in milk obtained under the most hygienic conditions (Poster, _et a l„ '1958)- Staphylococci have been incrim inated in abortion in liv e s to ck (S tab le forth and Galloway, 1959) m astitis in most domestic animals (l.iahanta, 19&5; S a lle , 1.967; Seamn, 19^3) and osteom ye litis , broncho­ pneumonia septicaemia and lo c a lis e d abscesses in humans (Croikshank, 1970). The enterotoxin produced by th is organism during grovjth in milk and milk products has been responsible fo r certa in human cases o f food poisoning (a a lle , 1967). ( i v ) streptococcus spp .: The highest number o f strep tococci was iso la ted from the Pulani Kraals (Table 8 ), and about 80;. o f the m ilking cows in the Pulani Kraals had some from o f defect or the other, o f the small udders (Appendix 3)0 Lost of the animals on this farm had either two or three functional tea ts , and sometimes had open wounds 011 the tea ts . The loss o f one or more quarters o f the udders o f these cows was in variab ly due to m astitis , and th is genera lly accounted 47 University of Ghana http://ugspace.ug.edu.gh fo r the higher incidence of streptococci, staphylococci and Bscherichia coli on this particular farm. Streptococci have been isolated from the mouth, nose, threat, genital tract, and faeces of healthy animals and man, and also from milk and milk products (Mahanta, 1965) «• Several species ( Strep, pyogenes. Strep, agalactiae. Strep. dysgalactiae. Strep, uberis and Strep, zooepidemicus) are known to cause mastitis, which nay lead to the loss at one or more quarters of the udder, and some species (e «g . Strepo pyogenes) are infectious to man, causing scarlet-fever and septic sore-throat (Mahanta, 1965; Sa lle , 1967; Foster, et a^, 1958). (v ) Corynebacterium spp,,: Two species of this genus, C. diphtheria and C. pyogenes, were isolated from the three farms. Corynebacterium spp, . like the other genera isolated, showed a tendency to increase in number with increasing stage of lactation, on a l l the farms (Table 8 ). I t is interesting to note that C. diphtheria was practically absent from A.R.LL Nungua, Tihereas the Fulani Kraals, as usual, shewed a high incidence. G. d iphtheria, has been implicated in epidemics o f diphtheria among drinkers o f raw milk (Poster, et a l . , 1958), vrhile C. pyogenes is the cause of the so -ca lled bovine 48 University of Ghana http://ugspace.ug.edu.gh 1 summer-mastitis1 — a fulm inating, per-acute mastis o f e ith er dr}' cows, m ilkers or h e ife rs (Seaman, 1963)* ( v i ) Lactobacillus spp. : The highest percentage of L a c tob a c illi (2S) v:as iso la ted from mill: obtained a t the Fulani Kraals. These organisms were also regu la r ly is o la ted from the j-mrahia Barm. Very few numbers o f these organisms were, however, is o la ted from A .R .S ., Nungua, and th is i s d i f f i c u l t to explain. Iactobacilli are invariably found in milk and. milk products, and Lactobacillus acidophilus has been used for the preparation of sour milk drinks used in hospitals (Mahanta 1965; Briggs and Briggs, 1954> Orla-Jensen, 1919; Seaman, 1963). This group of micro-organisms, stric tly speaking, cannot be called pathogenic, but their intimate association with dental-caries makes them important as a public health hazard. (Mahanta, 1965)* These isolations of Lactobacilli cannot be directly related to the health of the animals, but it is important to note that since they occur in the intestine of mammalian animals, and in faeces and saliva of certain species (Cruicksbank, 1970), any management and milking systems which easily expose the milk to faeces, dust and saliva as occurs regularly in the Fulani Kraals, are liab le to be easily contaminated. 49 University of Ghana http://ugspace.ug.edu.gh ( v i i ) Pasteurella sun. : This organism was iso la ted only from the Pulani Kraals (Table 8 ) . Since these organisms are usually found in the upper resp ira tory tract o f normal ca lves, and the udder o f the cow was not cleaned before m illin g a t the Pulani Kraals, i t was probable that the udder became contaminated by the suckling c a l f ; the organisms eventually fin d in g th e ir way in to the m ilk. Kilk-borne Pasteurella in fe c t ion of human beings has not been reported, probably because pasteurization ea s ily destroys these bacteria (ifa lte r , 1967)1 Organisms o f the Pasteurella group are liovrever, w idely d istributed in nature, and are frequen tly found in the upper a ir passages o f normal ca ttle (Mahanta, 19&5)* Bovine m astitis has been reported by Barum (1954), Packer (194-6), Rude, Johnson, and O'Conner, (1961), Schlotthauer (1944) and Tucker ( 1953) as having been caused by th is organism. ( v i i i ) Pseudomonas aeruginosa: Small numbers o f Pseudomon; s aeruginosa were iso la ted from milk during the ea r ly and la te stages o f la c ta tion at K .3 .S ., ITungua and a t the Amrahia Parm; the organism was, however, iso la ted from a l l the stages o f la c ta tion at the Pulani Kraals (Table 8) . The organism which re s is ts drying fo r several weelss when contained in pus o r exudates (Mahanta, 1965;, e a s ily contaminates milk when cows with teat and udder wounds are milked ind iscrim inate ly, a 50 University of Ghana http://ugspace.ug.edu.gh 51 practices prevalent a t the Fulani Kraals. Ps. aeruginosa is a lso frequently present, although in small numbers, in normal in te s tin a l f lo ra o f man and animals, and has been implicated in certa in human urinary tra ct in fec tion s (Cruickshank, 1970). Ihe presence o f Pseudomonas in milk is a lso very undesirable, fo r they are v e rs a t ile spoilage agents with pronounced biochemical a c t iv ity , espec ia lly on protein and fa ts (Poster et a l . , 195o). jierobacter aerogenes: Again, there was a gradual increase in A. aerogenes iso la tion s r e la t iv e to stage o f la c ta tion at the Fulani Kraals, but at both the A .R .3 ., Nungua and the Amrahia Dairy Farms, on ly a few iso la tion s were recorded during the early part o f the la cta tion period (Table 8 ). A. aero genes has been found in m il]: and milk products and is one o f the main causes o f ropiness o f m ilk, which is o f great economic importance to the milk processor (Foster j e t , * ! . , 195— * -eaman, 1963) . ..lca ligenes v is c o la c t is : The Fulani Kraal had the highast percentage iso la tion s o f —lca lig en e s v is c o la c t is , and a few o f these organisms were iso la ted from the *jsrahi». Farm. No iso la tion s were recorded at the Braigua farra. University of Ghana http://ugspace.ug.edu.gh Poster and colleagues (Poster, _et. a l . , 1958) had. reported that ii. v isco lactis. usually found in so ils , manure and dairy utensils, had been implicated in outbreaks of ropiness of milk and cream. - Pungus; Ss.coharom.yces 3pp.; Only two milk samples from AoH.S. ITungua (Table 8) showed these large, ovoid ce lls in clusters, and they are re latively non-pathogenic. It is interesting, f in a lly , to note that despite the presence of coliforms of a l l kinds in samples from a l l the farms, no salmonellae vjas isolated, however, 3^ enter i t idi s , 3. new port. 3. thompson and typhi-murium have a l l been implicated in milk-borne outbreaks of human salmonellosis (Barry, 1966) . 52 University of Ghana http://ugspace.ug.edu.gh CHAPTER V CONCDJJIQI-B AilD STOJHARY (A ) COIIC HJSIONS Very elaborate equipment is not a p re -requ is ite fo r producing m ilk o f lor; b a c ter ia l count, or o f the best grade; simple m ilking hygiene and decent storage methods are very essen tia l and necessary. The Fulani farmers should be encouraged to adopt simple sanitary precautions, l ik e mshing the udders o f the cons and hands o f milkers n ith soap before m ilking, and a lso keeping the calabashes, bowls and kerosine containers clean and dry. The absence o f p o s it iv e cases o f tdycobacte r ium tubercu losis , in the present study, should not lead to s. wrong assumption that milk produced 011 these farms is tubercu losis-fr e e , fo r except at A .H .3 ., Hungua, none o f the herds are tuberculin tested yearly* Even a ttested herds should be tested yea rly (S tab le forth and Galloway, 1959)* Regular b a c te r io lo g ica l examination and se ro log ica l testing o f a l l m illin g herds w i l l be necessary to ensure good qua lity m ilk. k ilk produced on a l l these farms is sold unpasteurised to consumers and th is , th erefore, ca lls fo r e f fe c t iv e measures to protect the health o f the public from the hazards o f milk-borne diseases. I t is th erefore recommended that: (a ) A cen tra l milk depot be set up to buy a l l raw milk from the producing sources, pasteurise and s e l l to the public. 53 University of Ghana http://ugspace.ug.edu.gh 54 (b ) Public Health inspectors should periodically v is it these farms to advise on, and to enforce the maintenance of proper hygienic milking conditions. - (c ) A leg islation should lie passed to regulate the conditions under which a l l cows should "be milked and this should be vigorously enforced. (d) The present bacteriological data could be 'the basis by which the National Standards Board might use to advise the Government on a ’Milk Ordinance Code*. University of Ghana http://ugspace.ug.edu.gh (B) summary The bacteriological quality of raw milk, and the distribution of the various types of bacteria, from three farms (A.R.S. Nungua, Amrahia Dairy Parm and the Pulani Kraals) of different management, hygiene and milking practices, were determined at three different stages of lactation — Early-lactations 1 - 3*5 months; Mid-lacta­ tion: 3.5 — 6.5 months, and let e-lactation: over 6.5 months. The overall results indicate that raw milk produced by cows at the Ministry of Agriculture Parm (Amrahia) was by fa r hygienic a l ly superior to milk produced at A.R.S. (Nungua) and the PUlani Kraals. The standard of hygiene at the two former farms compared highly favourably with that of w ell established dairy farm elsewhere. The Amrahia Dairy Ite-rm had the least overall mean bacterial count of 27,000 per ml.; the A.R .S,, Nungia, had 61,000 per m l., and the Pulani Kraals gave a mean total of 3 Per mlo There were, however, no sta tistica lly significant differences between stages of lactation and mean bacterial count. The Amrahia Dairy Parm produced 36o00% Grade A raw milk, whilst only 13.33/° of the raw milk produced at the Pulani Kraals came within Grade A. 78.67/° of "the to tal number of raw milk samples from A.R .S ., Nungua conformed with the G-rade A. Jg. cereus. B. su b tilis , E. c o li , Staph, spp. . Strep, spp.. C. pyogenes, C, diphtheria, Lactobacillus spp. . Pasteurella spp. . 55 University of Ghana http://ugspace.ug.edu.gh 56 Pseudomonas agruHnosa. Aerobacte r aeroKenes. and A loa ligenes v is co la c t i s , were iso la ted from milk produced at the three f s x n s . There wc.s predominance o f Staphlococci on a l l three f arris . Si:: cases o f b ru ce llos is from the Amrahia Dairy Fam, and seven from the Fulani Ejraals were iso la ted . None was i fo l . 't e d from the A .S .3. ITungua. Pour cases o f suspected tuberculosis (one from the Amrahia Dairy yarn and three from the Fulani Kraals) were investigated , but a i l proved negative a fte r culturing, and tuberculin tes tin g . I t is in teres tin g to note that despite the high percentage o f co lifc ru s iso la ted from milk samples from the Fulani E rasls , and indeed from the other farms, no salmonellae -were isols.ted in any o f the samples. The public health importance o f the bacteria is o la ted was discussed, and a few suggestions as to how to protect the public from milk-borne diseases were o ffe red . University of Ghana http://ugspace.ug.edu.gh 57 r e f e r e n c e s 1. Abdel-Llalek, Y. and Gibson, T. (1952). Studies in the Bacteriology of Milk. IV. The G-ram Negative Rods in Milk. J. Dairy Re s. 1_2, 294-301. ' 2 . Abrahams, C.A. and Laryea, A.M. ( 1968) . A preliminary investigation of the bacteriology of raw milk produced by cows on the Accra Plains. Ghana Med. J. 2> 100-103. 3. Andrey, J. J r., and Frazier, W.C. (l959)» Psychrophiles in milk held two days in faim bulk cooling tanks. J. Dairy Sci. 42, 1781-1784. 4. Atherton, H.V. (1959). A f ie ld study of the sanitary care of milking equipment on dairy farms. J. Milk Food Technol. 22, 273-278. 5. Ayers, SJi., Cook, L.B. and Clemmer, P.W. (1918). The four essential factors in the production of milk of low bacterial content. U.S. Dept. Agric. Bu ll. 642.. 63. 6. Baltimore B iological Laboratory (BBL) Manual (1968) . Products and Laboratory Procedures. Cockeysville, Maryland. 21030. U.S.A. 7. Barthel, C. (1906). K. Landbr. Akad. Handl. Och Tidskr. 45. 48-56 (Abstract in Espt. Sta. Rec, 1907) 18, 75. 8. Banm, D.A, (1954). A herd outbreak of mastitis caused by Pasteurella multocida. Can. J. Comp. Med. Vet. Sc. 18, 113-119. " 9. Bergey, D.H. (1904). Source and nature of bacteria in milk. Penn. Dept. Agr. Bull. 125. 1-40. 10. Breed, 3 .R ., Murray, 3.G.D. and Smith, F .R. (1959). Bergey* s Manual of Determinative Bacteriology 7th Edition. V/illiams and Wilkins Co., Baltimore. 11. Brew, J.B. (1949). Sanitary milk control in retrospect. J. Milk Food Technol. J_2, 193-195. 12. Briggs, M. and Briggs, C.A.E. (1954). The Lactobacilli: a review of the literature with special reference to taxonomy. Dairy Sci, Abstract. l£ } 252- 268. 13. Bryan, C.S. Bryan, H.S. and Mason, K. (1946) . Heat-resistant bacteria from an unclean milking machine invade the udder of cow. Milk Plant Monthly 23.» 30-32. University of Ghana http://ugspace.ug.edu.gh 58 14. Collins, C.H. and Lyne, P.M. (1970). Microbiologioal Methods Butterworth Sc. Publ. London. 15. Covington, J .L., Egdell, J.47. and Thomas, S.B0 (1953). Recent investigations on thermoduric organisms in milk, 13th Int. Dairy Congress (The Hague) Proc. 2, 139-147. 16. • Crowdjr, S.H. (1939). Notes on the contamination of milk by bacteria in the tropics. Trop. Agr. Trin. 16, 124-129. " 17. Crowley, FT., Bradley, J.M. and Darrell, J.H. (1969). Practical Bacteriology. Butterworths, London. 18. - Cruicksharik, R. (1970). Medical Microbiology. McG-raw-Hill, London. 19. -Cullen, G.A. and Hebert, C.N. ( 1967) . Some ecological obser­ vations on micro-organisms inhabiting bovine, skin, teat canals and milk. Br. Vet. J. 123, 14. 20. ' De F ilipp is , V. (1941). The bacterial count of raw milk. (Some irreducible factors in the bacterial count and their seasonal incidence). Dairy Sci. Abstr. 193-194. 21. - Difco Manual (1965) . Dehydrated culture media for Microbio­ log ical and Clinical Laboratory Procedures. 9th Edition. Difco Laboratories Inc ., Detroit, Michigan. 22. -Dorner, W. (1930). The bacterial flo ra of aseptically drawn milk. N.Y. (G-eneva) Agri. Expt. Sta. Tech. Bull. Vo l.165,, 23. Economic Survey of Ghana (1969). Domeatic Livestock Popula­ tion. Central Bureau of Statistics, Accra. 24. Erdman, I .E . and Thornton, R.H. (1951 )• Psychrophilic Bacteria in Edmonton milk and cream. I I . Kinds. Ca. Jour, of Technol. 22, 238-242. 25. -Poster, E.M., Nelson, F.E., Speck, M.L. Doetsch, I-t.N. and Olson, C.J. (1958). Dairy Microbiology. MacMillan and Co. Ltd., London. 26. G-ibson, T. and Abdel-Malek, Y. (1957)• The development of bacterial populations in milk. Can. J. Microbiol. 2, 203-213. 27. Harrigan, 1/.?. and MeCane, E.M. (1966). Laboratory Methods in Microbiology. Academic Press, London. 28. Heeres, H. (1950). The connection between the qua lity of milk and the judging of farm conditions (English Sumraa r y ) . Neth. Milk Dairy J. 10-21. 29. Hunter, 0..!. ( 1 91 9) • The colon-aerogenes group o f mill:. J. Dairy Sci. g, 108-129. University of Ghana http://ugspace.ug.edu.gh