An investigation of poultry litter intended for use in farm feeds showed that 0,37 %, 0,49 %, 0,25 %and
 12,3 % of the 813 samples tested were contaminated with Clostridium spp., haemolytic Escherichia coli,
 Staphylococcus aureus and 21 different species of Salmonella. The findings clearly underline the hygienically
 dangerous nature of crude poultry litter. The practical implications of the results are briefly discussed, particularly
 in view of current regulations.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.am201

Barnard, Marie-Luise

Giesecke, W.H.

Ogonowski, K.

The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. 
Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format.An investigation of poultry litter intended for use in farm feeds showed that 0,37 %, 0,49 %, 0,25 %and
 12,3 % of the 813 samples tested were contaminated with Clostridium spp., haemolytic Escherichia coli,
 Staphylococcus aureus and 21 different species of Salmonella. The findings clearly underline the hygienically
 dangerous nature of crude poultry litter. The practical implications of the results are briefly discussed, particularly
 in view of current regulations.am201

UPSpace at the University of Pretoria

Onderstepoort J. vet. Res ., 51 , 249-252 (1984) BACTERIOLOGICAL FINDINGS REGARDING THE HYGIENIC SAFETY OF POUL-TRY LITTER INTENDED AS AN INGREDIENT OF FEEDS FOR RUMINANTS K. OGONOWSKI, MARIE~LUISE BARNARD and W. H. GIESECKE, Veterinary Research Institute, P.O. Onderstepoort 01 10 ABSTRACT OGONOWSK.l , K. , BARNARD, MARIE-LUISE & GIESECKE, W. H., 1984. Bacteriological findings regarding the hygienic safety of poultry litter intended as an ingredient of feeds for ruminants . Onderstepoort Journal of Veterinary Research, 51, 249- 252 (1984). An investigation of poultry litter intended for use in farm feeds showed that 0,37 %, 0,49 %, 0,25 %and 12,3 % of the 8 13 samples tested were contaminated with Clostridium spp. , haemolytic Escherichia coli, Staphylococcus aureus and 21 different species of Salmonella . The findings clearly underline the hygienically dangerous nature of crude poultry litter. The practical implications of the results are briefly discussed, particu-larly in view of current regulations. INTRODUCTION Depending on its level of uric acid and other constitu-ents, poultry litter may be used in feeds for ruminants as a valuable source of nitrogen, provided it is also hygieni-cally safe . The significance to animal and human health of poultry litter as an ingredient of ruminant feeds has been the subject of several investigations abroad (Messer, Lovett, Murthy, Wehby, Schafer & Read, 1971; Strauch, 1983). The data indicate that the health hazards most obviously associated with feeding poultry litter to livestock are those caused by residues of chemi-cals (e.g. non-antibiotic and antibiotic remedies) from medicated feeds, pesticides, certain pathogenic micro-bial toxins (e.g. from Clostridium botulinum) and bac-teria (e.g. Salmonella spp.), all possibly present in the litter. - Owing to its importance as supplementary feed for ruminants and as a potential hazard to health (Centre of Disease Control, 1979), poultry litter sold as animal feed in South Africa is subject to the official control of the Fertilizers, Farm Feeds, Agricultural Remedies and Stock Remedies Act, 1947 (Act 36 of 1947). Regulation R. 1359 and other related regulations (Government Gazette, 1980) are explicit in their require-ments for various physical and chemical properties of poultry manure [section 12 (2)]. However, the microbio-logical requirements [section 10 (4) (b)] seem to be in-consistent and vague for the following reasons: 1. They stipulate that a registered farm feed, such as poultry manure, must be sterilized. 2. They also point out, however, that by means of such a sterilization infection or contamination with harmful micro-organisms or substances has to be reduced to levels not injurious, dangerous or detri-mental to animals fed with such a product. 3. They do not specifically mention any standard methods for the microbiological screening of re-gistered farm feeds nor any other techniques, parameters and criteria for determining their in-jurious, dangerous or detrimental nature to animal health and production. Poultry manure is a potentially hazardous ingredient in feed for ruminants and its control in terms of Act 36 of 1947 is an important factor in preventive medicine. Its importance as a source of infection and the necessity for its control have been sharply emphasized in recent months when manure has been proposed for relieving the shortage of farm feeds caused by the severe drought. As an emergency measure the Registrar of Act 36 of 194 7 was appealed to, to permit the sale of non-sterilized poultry litter as farm feed. It was therefor deemed necessary to launch an investigation on the safety of non-sterilized poultry litter from the hygienic point of view. Received 31 July 1984--Editor 249 During studies on the poultry litter and from investiga-tions on various commercial mixed feeds (Ogonowski, unpublished data, 1983), it became evident that, al-though extensive determinations of pathogenic bacteria (e.g. Salmonella spp.) have been performed on foods, fewer determinations have been made on animal feeds, and only very limited information is available on practi-cal techniques for collecting samples of poultry manure and screening them for pathogenic micro-organisms. The aim of this investigation was to augment the infor-mation on the bacterial content of poultry manure and to provide some relatively detailed descriptions of some of the techniques used during the investigation. MATERIALS AND METHODS Poultry litter examined The 813 samples of poultry litter examined were col-lected at 87 poultry farms distributed throughout the RSA (Fig. 1). Collection of samples Different types of fresh/old and dry/moist composite litter and almost pure manure were collected under the varying conditions of broiler, egg and breeding stock production. Where possible, samples were taken from masses of litter freshly removed during routine cleaning operations from the production units. In a few excep-tional cases, samples of litter were collected in poultry houses at sites chosen at random. Samples of approxima-tely 200-500 g each were taken at various sites and depths from each batch of litter. The samples were taken by inspectors of the Registrar Act 361194 7, who have great practical experience in the sampling of bulk consignments of different products. They are specially trained to collect samples with hy-gienic precautions, both to protect the consignments from infection and prevent them from contaminating other products and samples intended for microbiological investigations. Samples were collected in new 255 x 380 mm bags of transparent plastic. Preparatory to the sampling, an ap-proximately 100 mm-wide strip of the orifice of the bag was turned over the fingers of one hand, without touch-ing the internal surface of the bag. With the hand palm upwards, the straightened fingers with their glove-like plastic protection were then pushed in a scooping for-ward motion through the litter until an adequate amount of it was collected in the rest of the bag lying on the palm. The bag was then closed by tightly twisting its empty portion and placing it in a new 800 mf tin . This, in turn, was immediately closed and the lid completely sealed with a portable tool. Each sample container was then properly identified by means of a certificate record-ing detailed information, a corresponding sticker label BACTERIOLOGICAL FINDINGS REGARDING THE HYGIENIC SAFETY OF POULTRY LmER FIG. l Sketched map of the RSA. Nos. l , 2, 3, 4 , 5, 6 and 7 =Pretoria, Johannesburg, Durban, Bloemfontein and Kimberley , Port Elizabeth, East London and Cape Town respectively. Each dot represents one poultry farm investigated. affixed to the tin and a corresponding sample number recorded on the certificate, the label and the lid of the tin proper. The sealed samples were then transported together with their certificates to the Veterinary Research Insti-tute, Onderstepoort, where they were registered and transferred to the Section of Food Hygiene for further processing at the laboratory. Laboratory examination The samples were examined mainly for the presence of certain pathogenic bacteria. Additional examinations on several of the samples were performed for investigat-ing standard plate counts of general bacterial contami-nants. 1. I solation of Salmonella spp . Salmonella spp. were determined by means of a method, the individual steps of which are described below. ( 1) An aliquot of 20 g of each sample was placed in a 250 me sterile conical flask to which was added 200 me of sterile peptone water con-taining Tween 80 at a final concentration of 0,1 %. The suspension was mixed and incu-bated for 20-24h at± 37 oc. (2) 1 me of supernatant fluid of the incubated sus-pension (I) was used for inoculating 20 me of SBM broth* (i .e. enrichment medium con-taining selenite, brilliant green and manito!) and incubated for 20-24h at ±37 ,5 °C. (3) After incubation, 1 loopful (0 ,01 me) of SBM bouillon was spread on (a) SS agar* (i .e. Sheigella and Salmonella agar) and (b) bismuth-sulphite agar.* Both plates were then incubated for 20-24 h at ± 37,5°C. * Merck Chemicals. South Africa 250 (4) After incubation of 3a and 3b, stab cultures in triple sugar iron agar slants (TSI*) were pre-pared, using colourless, translucent single col-onies picked from SS plates (3a) and suspect Salmonella colonies. Both the TSI stab cultures were incubated again for 20-24 hat± 37,5 °C. (5) The TSI slant cultures were assessed for growth of Salmonella spp. (6) A single suspect colony was removed from either 3a and 3b or from 4 for further testing by means of the API-20E series. t (7) All cultures API positive for Salmonella spp. were serotyped by the Section of Bacteriology. (8) A sample was identified as positive for Salm-onella where culturing, API testing and sero-typing were clearly positive both in terms of growth and of the Salmonella spp. isolated. (9) If any of the cultures, API testing or serotyping, produced doubtful results, another sample of 20 g of originally registered sample was re-ex-amined. ( 1 0) A sample was Salmonella negative when cultur-ing, API testing and serotyping gave clearly negative results. 2. Biological test for Bacillus anthracis and Clostridium spp. ( 1) 20 g of sample was placed in a sterile specimen jar filled with sterile saline, agitated , and put into a water-bath for 60 min at 60 °C. (2) After cooling, 1 me each of supernatant fluid was administered intramuscularly to 2 adult healthy guinea-pigs. (3) The guinea-pigs were kept under close observa-tion in a well-ventilated room and regularly supplied for 12 days with food and water. * Merck Chemicals. South Africa T API Systems. France (4) If no deaths occurred during the 12 days, the corresponding guinea-pigs and sample were considered free from B . anthracis and Clostri-dium spp. (5) Dead guinea-pigs were subjected to a post-mor-tem and bacteriological investigation for B. an-thracis and Clostridium spp. For the latter, pieces of spleen, liver and heart were used to prepare in triplicate, impression smears on blood tryptose agar plates and then put into a test-tube containing cooked mincemeat broth. The plates and the tubes were incubated aero-bically, anaerobically and in a candle jar for 24 h at 37,5 °C. After incubation a loopful of an inoculated meat broth was plated on the blood tryptose agar plates and incubated again for 24 h at 37,5 °C. Haemolytic colonies from blood tryptose agar plates were picked and replated on fresh blood tryptose agar plates to obtain pure cultures. These were sent to the Section of Bac-teriology for tentative identification. (6) If dead guinea-pigs were positive for B. anthra-cis or Clostridium spp. , another 20 g of the original sample was investigated on 2 new guinea-pigs, using procedures 1-5. 3. Methods for isolating other pathogenic bacteria (I) S. aures was isolated from aerobic blood tryp-tose agar plates inoculated during the isolation procedure for B. anthracis and identified by the Section of Bacteriology. (2) E. coli: 1 g of the sample was transferred to a McCartney bottle with 15 me of MacConkey broth and incubated in a water-bath for 48 h at 44 °C. A loopful of incubated MacConkey broth was spread on blood tryptose agar and MacCon-key plates. After 24 h incubation at 37,5 °C, the suspect colonies were picked up, inoculated on blood tryptose agar plates until cultured pure and then sent for tentative identification to the Section of Bacteriology. (3) Standard plate count: 1 g of each sample was suspended in 9 me of phosphate buffer in a 20 me McCartney bottle. After agitating the sus-pension , 3 tenfold dilutions were prepared from it in phosphate buffer. One drop _{0,05 me) from each dilution was plated on TGY -agar* and Wickerhamt agar and incubated at 37 °C for 24 h and at 28 oc for 48 h respectively . Colony forming units on Wickerham plates were counted visually, and those on TGY plates with a Biomatic 16900 colony counter+. The counts per me of phosphate buffer were then calcu-lated. R ESULTS Prevalence of pathogenic bacteria other than Salmonella spp . Clostridium spp., pathogenic E. coli and S . aureus were isolated respectively from 0,37; 0,49 and 0 ,25% of the 813 samples investigated. Further identification of the 3 isolates of Clostridium spp. showed 2 C. septicum I each from 2 different farms in Transvaal and I C. perfringens from a farm in Natal. • Oxoid t Wickerham ( 195 1, c ited by G iesecke, Net & Van den Heever, 1968) :j: A/SN Foss Electronic, Denmark 251 K. OGONOWSKI, MARIE-LUISE BARNARD & W . H. GIESECKE The 4 isolates of pathogenic E. coli each originated from 3 different Transvaal units and from 1 farm in the Cape Province. The 2 isolates of S. aureus both originated from a Transvaal farm. Prevalence of Salmonella spp . Salmonella spp. were isolated 103 times from 100 (i.e. 12,3 %) of the 813 samples examined. The I 03 iso-lates of Salmonella spp. were obtained from 31 of the 87 farms investigated (20 in the Transvaal, 6 in Natal, 4 in Cape and 1 in Orange Free State). Of the 55 Transvaal, 19 Cape, 11 Natal and 2 Orange Free State farms investi-gated, those infected with Salmonella spp. were 36,4; 21,1; 54,6 and 50% respectively of the corresponding farms examined per province. Further identification of the 103 isolates of Salmonella spp. showed that the samples were contaminated with a variety of species (Table 1). TABLE I Salmonella spp. isolated from poultry litter and the corre-sponding frequency of isolation Salmonella spp. S. senftenburg ............. . S. chester .. ................ ... .. S. remo . ............. .. ................ .. S. nitra . .. ............... .... ...... .. .. . S. naware .. S. makumira ........ . .. ... ... .. ... .. S . rafo ........ . S. heidelberg . ......... .. S. ryphimurium ........ .. . .. .. ....... . S. jaja .. .. ...................... .... .. .. S. bradford . ....... .. ............. .. .. S. hessarek .. .... . ............ . .... .. S. nyeko ............................... .. S. rilburg .. .... .... .. ......... .. .. ... ... .. S. oriemalis. .. .. .. .... . .. S. selandia .. S. messina.. .. .. .... ...... . .... .. S. jubliana ... .. ........ .. ........ .. S.fulica. .. . .. . ........ .. .. ....... . Salmonella II . . . . . . . . ..... , ..... . Salmonella (rough) .. .. ..... . .. ........ . Number of isolates 29 14 10 6 5 4 4 2 2 2 2 2 2 2 I I I I I I II Additional ad hoc investigations on standard plate counts of 19 samples and the moisture content (%) of 22 samples selected at random indicated mean values ± standard deviations of 1544,26 x 106 ± 1374,26 x 106 colony forming units per gram of sample, and 18,3 ± 8,24 % moisture content. Only I of the 21 samples tested showed a moisture content (9 ,5 %) within current re-quirements. DISCUSSION The isolation from poultry litter of a variety of patho-genic bacteria and a wide range of Salmonella spp. is consistent with the results of other workers (Messer et a!., 1971 ; Strauch , 1983). From the point of view of veterinary hygiene the find-ing thus clearly confirms the potentially dangerous nature of crude poultry manure. It further implies that under conditions permitting the selling as farm feed of unsterilized poultry litter, a considerable number of poultry production units may be considered potential sources for the general spreading of especially Sal-monella spp. . From the point of view of preventive veterinary medi-cme the results clearly emphasize the importance of ap-propriate field and laboratory investigation, the necessity of follow-up measures at the farm and the urgent need for greater vigilance regarding the countrywide dissemi-nation of Salmonella not only by poultry litter as such but probably also by poultry farms and abattoirs in general . BACTERIOLOGICAL FINDINGS REGARDING THE HYGIENIC SAFETY OF POULTRY LITTER Our findings on contamination with pathogens, mois-ture content (%) and standard plate counts underline the conclusion that unless crude poultry litter is sterilized it should not be registered as farm feed , however great the shortage of feedstuffs . The necessity for sterilizing poul-try litter is nothing new (Messer et al., 1971); it has recently been re-emphasized elsewhere (Strauch, 1983), and is now confirmed by this investigation. It is surpris-ing, therefore, that the current regulations for the steril-ization of poultry litter and other farm feeds intended for registration are not as specific as the situation demands. This is further aggravated by the fact that poultry litter, though frequently heavily contaminated with pathogens, or hygienically unacceptable for other reasons, and therefore clearly unregistrable as a farm feed, may still apparently be distributed non-sterilized for other pur-poses (e.g. fertilizer), though it constitutes a danger to animal and public health. The present investigation sug-gests that amendments aimed at the elimination of such inconsistencies are necessary. ACKNOWLEDGEMENTS We wish to thank Dr Maryke Henton for tentatively identifying the bacterial isolates, and for the generous assistance and co-operation during this investigation from The Registrar Act 3611947, Department of Agricul-ture, and his field personnel. REFERENCES CENTRE OF DISEASE CONTROL, 1979. Foodborne disease out-breaks-annual summary 1978. Atlanta: U.S . Department of Health, Education and Welfare. GIESECKE W . H. , NEL, E. E. & VAN DEN HEEVER, L. W. , 1968. Blastomycotic mastitis in South Africa. Journal of the South African Veterinary Medical Association, 39, 69- 85 (S). MESSER, J. W .. LOVETT, Y., MURTHY, G. K., WEHBY, A. J. , SCHAFER, M. C & READ, R . B. Jr, 1971. An assessment of some public health problems resulting from feeding poultry litter to animals. Poultry Science, 50, 874-881. STRAUCH, D. (Ed.) , 1983. Hygienic problems of animal manures . Proceedings of a Joint Workshop of Expert Groups of the Commis-sion of the European Communities, German Veterinary Medical Society (DVG) and Food and Agricultural Organisation. Edited by D. Strauch, Institute for Animal Medicine and Animal Hygiene, University of Hohenheim, Stuttgart, Federal Republic of Germany. Printed by the Government Printer, Private Bag X85, Pretoria, 000 1 , 252 

Bacteriological findings regarding the hygienic safety of poultry litter intended as an ingredient of feeds for ruminants

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Bacteriological findings regarding the hygienic safety of poultry litter intended as an ingredient of feeds for ruminants

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