INTEGRATED SALMONELLA MANAGEMENT

1. DINATEC DIVERSIFIED NUTRI-AGRI TECHNOLOGIES INC., PRESENTS INTEGRATED SALMONELLA MANAGEMENT

2. SALMONELLA OVERVIEW Dr. D.E. Salmon, an American bacteriologist and veterinarian, was first to isolate Salmonella in 1885. Salmonella grow best at temperatures above 10o C in environments with a pH range of 6-7.5. Salmonella are facultative anaerobes classified as genus three in the Family Enterobacteriaceae. Salmonella is a food poisoning organism that, under proper conditions, grows at extreme fast rates often doubling populations within 15 minutes. 20 million people worldwide are infected yearly by Salmonella resulting in approximately 500,000 deaths.

4. SALMONELLA OVERVIEW Non-pullorum salmonella serotypes, such as typhimurium, enteritidis and heidelberg, are the causative bacterial agents in most cases of reported food poisoning. In 1989, England and Wales reported 30,000 cases with 61 deaths. About 45, 000 cases occur annually in North America, but with a lower mortality rate (Toman, 1990). Economic losses due to salmonellosis in the USA are estimated to be in excess of US$1 billion and possibly as much as 4 billion largely as the consequence of lost wages, cost of medical treatment and decreased productivity (St. Louis et al., 1988).

5. Salmonella enteritidis Common Symptoms In most cases poultry eggs are the culprit. Illness lasts 4 to 7 days, most persons recover without antibiotic treatment. In some cases, diarrhea can be very severe enough to require hospitalization. The elderly, infants, and those with impaired immune systems may be affected severely. In these patients, the infection may spread from the intestines to the blood stream, and then to other body sites and can cause death unless the person is treated promptly with antibiotics. Symptoms are usually fever, abdominal cramps, and diarrhea beginning 12 to 72 hours after consuming a contaminated food or beverage.

6. Salmonella typhi S. typhi is the causative agent for typhoid fever in humans, the most severe type of Salmonellosis .

7. Salmonella typhimurium Less severe forms of gastrointestinal illnesses such as diarrhea are caused by many Salmonella strains and are collectively known as salmonellosis. One such strain is S. typhimurium which is able to invade the epithelial cells of the intestinal tract, survives the intracellualr defense mechanisms of the host cell, and multiplies.

8. MOVIE GOES HERE

9. SOURCES OF SALMONELLA AT THE POULTRY FARM FEED RODENTS, CATS & DOGS WATER POULTRY FLOCK DAY OLD CHIKS BROUGHT IN CONTAMINATED EQUIPMENT VERMIN/ WILD BIRDS BEDDING MATERIAL PEOPLE

10. TWO FOLD APPROACH I. Management Preventive Factors II. Chemical Treatment Considerations THEY FIT WELL TOGETHER

11. APPROACH I I. MANAGEMENT PREVENTION FACTORS

12. PURCHASING PRACTICES • Use reliable suppliers (GMP) • Use suppliers who adhere to industry guidelines published in by the U.S. Department of Agriculture for poultry animal and fish products. • Visit and observe the operations of suppliers. • Approve as suppliers only those firms who follow good practices and can routinely provide Salmonella‑negative ingredients • Add DINASAL Liquid to purchased meat meals, fish meals and soybean meals at a minimum dose level of 2 kg per metric ton.

13. INGREDIENT GUIDELEINES • Purchase ingredients only from approved suppliers. • Transport ingredients in vehicles that are clean, dry, and free from conditions conducive to Salmonella contamination. • While en route and at all other times, keep ingredients dry and protected from insects, birds, rodents and other animals. • Upon arrival Inspect incoming ingredients for evidence of water damage and for signs of fecal contamination by insects, birds, rodents, or other animals. • Reject questionable material. • Follow a program of periodic laboratory testing of ingredients to monitor for the presence of Salmonella.

14. GUIDELINES FOR PLANT PREMISES Keep premises clean. Follow good housekeeping and good sanitation practices. Continually maintain control in and around the plant premises . Prevent insects, birds, rodents, and other animals from gaining access to or remaining in the feed mill. . Keep areas within and around the mill free from refuse and trash. . Store unused equipment in a manner to discourage infestation. . Keep the grounds surrounding the mill well drained and free of unnecessary or undesirable vegetation, such as weeds and high grass. . Avoid unnecessary foot traffic from outside sources in all mill areas. Salmonella may be carried on the feet.  . Pay particular attention to foot traffic from such areas as barns, stock pens, and stock trucks to all mill areas. This includes employees who care for or raise livestock or poultry.  . Provide for sanitizing of footgear. DINATEC recommends the use of Dinaxine, our stabilized Chlorine Dioxide Solution at 10 ppm for footwear disinfecting pans.

15. GUIDELINES FOR EMPLOYEE FACILITIES • Salmonella organisms can be carried on clothing, shoes, or person. • Provide adequate facilities for washing, showering, and dressing . • Instruct and train employees to maintain a high degree of cleanliness of self and work clothing.

16. GUIDELINES FOR PROCESSING AND STORAGE AREAS • Avoid accumulations of dust, spillage, and broken bags. Dispose of any material suspected of having become contaminated with Salmonella as waste unless analysis is negative for Salmonella. • Dispose any wet material as waste. • Separate ingredients and finished feeds in the storage area. Prevent air contamination by dust circulating from raw ingredient areas to processed material areas. • Keep portable equipment used for handling ingredients separate from that used for finished feeds. Segregate dirty areas from clean areas.

17. GUIDELINES FOR PROCESSING EQUIPMENT • Construct bins, blenders, mixers, conveyors, and baggers to minimize buildup or caking of material and to provide access for inspection and cleaning. • Avoid accumulations and excessive buildup of feed materials. • Clean magnets at regular intervals to prevent feed materials from building up at these points. • Inspect equipment at regular intervals • Clean equipment regularly to remove accumulations of material. • Construct and install all processing and handling equipment in a manner to minimize leakage, spillage and dust accumulation.

18. GUIDELINES FOR PACKAGING, STORAGE AND TRANSPORTATION • Do not reuse previously used bags or containers for packaging. New bags are generally considered to be free of Salmonella contamination. • Make a continuing effort to minimize storage time of ingredients and finished feeds. • Insure that equipment used to transport ingredients and finished feeds be clean, dry, and free of conditions conducive to Salmonella contamination.

19. GUIDELINES FOR DUST CONTROL • Provide adequate air filtration equipment for the pellet cooler. • Draw air from a clean area inside or outside the mill to prevent recontamination. • Periodically control and remove accumulated dust that settles in processing and storage areas. • Install a proper dust collector system to control dust and to aid in keeping the mill clean. • Periodically remove settled dust with a vacuum cleaner equipped with a highly efficient filter rather than sweeping. • Avoid using compressed air to remove dust from equipment or from clothing of personnel. • Destroy accumulation from the dust‑collector system and "sweepings“.

20. GUIDELINES FOR AIR CONTROL Avoid contamination of ingredients and finished feeds via airborne organisms • Do not draw air into the mill from areas likely to be contaminated. • Pay special attention to intake for air used for cooling of pelleted or extruded products. These products will normally be Salmonella negative as a result of the temperatures and pressures involved in these processes.

21. GUIDELINES FOR MOISTURE CONTROL Salmonella require moisture to multiply. • Keep feed materials dry at all times. • Keep roofs and ceilings leak-proof. • Construct storage‑area walls and floors in such a manner as to keep out moisture. • Avoid or correct conditions conducive to the formation of condensation in buildings and equipment. Poorly insulated pipes in the receiving area of the feed mill can result in condensation which will contribute to the spread of bacteria • Keep ingredients, finished products, containers, storage areas, and transporting vehicles as dry as possible will prevent growth of Salmonella.

22. GUIDELINES FOR PROCESS CONTROL ‑ PELLETING Temperatures reached in pelleting are critical in Salmonella control. A few seconds at 180 degrees F or twenty minutes at 165 degrees F will usually kill Salmonella organism. While these may be the target or intended temperatures, they may not be reached at the beginning of the process. For this reason, it may be necessary to recycle the first material coming through the system. Pellet mill must be carefully monitored to insure proper operation with respect to temperature.

23. GUIDELINES FOR SAMPLING AND ANALYSIS Results of laboratory examinations indicate adequacy of purchasing, handling, processing, and storage practices in procuring and producing Salmonella‑negative products. • Secure samples of ingredients upon arrival at the mill and of finished feeds at time of shipment. • Routinely subject samples to appropriate physical examination for overall quality and condition. • Employ aseptic techniques in securing and in handling samples of ingredients and finished feeds that are to be analyzed. • Use sanitized equipment and sanitize equipment between samples. Periodically submit representative samples of ingredients and finished feeds for laboratory examination. • When sampling a previously loaded vehicle or material in storage, take a tablespoon or more from each of 10 different locations.

24. GUIDELINES FOR SAMPLING AND ANALYSIS This sampling and analysis plan can be used as a guide or starting point for the establishment of a quality control monitoring program for Salmonella • Consider potential incidence and level of contamination, composition, form, and the various treatments to which the material has been subjected. • Total sample should weigh about half a pound. • Bulk shipments- Sample at varying intervals of time as material is unloaded or loaded • Packaged material- Take minimum 1 tablespoonful from each 20th 100‑pound bag or each 40th 50‑pound bag. • Categorize product as to whether it is homogeneous or heterogeneous in nature. • Place samples in a new or clean, secure sterile container. • Label and identify container and mail immediately to the laboratory. Salmonella‑negative ingredients and finished feeds are defined as those in which Salmonella is not detected when sampled and analyzed by the procedures outlined in these guidelines. No sampling plan will fit everyone's or every product's requirements.

25. CRITERIA FOR LABORATORY SELECTIONS Analysis for Salmonella is a procedure normally requiring use of a commercial laboratory. Selection of a competent laboratory is essential for confidence in the results. . Use FDA or AOAC recognized procedure. . Allow no variations to the recognized procedure. . Utilize positive/negative controls. . Confirm tests by using alternate tests . Specify sample size assayed (100 grams recommended). . Use a check sample program for validation. . Retain samples for a specified period of time.

26. LABORATORY METHOD Currently preferred method is that recommended by the Food and Drug Administration's (FDA) Center for Veterinary Medicine for isolating and identifying Salmonella in rendered products. Published in the Bacteriological Analytical of Official Analytical Chemists (AOAC). It is often referred to as the BAM method. The method can be obtained from FDA, AOAC, or AFIA. The respective addresses are as follows: Food and Drug Administration, 5600 Fishers Lane, Rockville, Maryland 20857  Association of Official Analytical Chemists, 1111 N. 19th Street, Arlington, Virginia 22209  American Feed Industry Association, Suite 1200, 1701 Ft. Myer Drive,Arlington, Virginia 22209

27. CHEMICAL TREATMENT APPROACH II DINATEC RECOMMENDS THE PRUDENT USE OF A CHEMICAL TREATMENT PROGRAM IN CONJUNCTION WITH A JUDICIOUS APPLICATION OF THE MANAGEMENT PREVENTION GUIDELINES

28. INTEGRATED SALMONELLA MANAGEMENT Systematic Chemical Treatment Approach DINASAL Inhibition in Raw Materials (DINASAL Liquid) and in Finished feeds (DINASAL Dry) DINAXINE Stabilized Chlorine Dioxide in drinking water and in processing plant chiller water DINAMUNE Cellular Immune/stimulation DINAFERM Aglutination

29. USAGE CHART I.S.M.BROILER DAY 42 DAY21 DAY 3 DINASAL 3.0 KG/MT DINAFERM 500 GR/MT DINAMUNE 500 GR/MT

30. SPECIFICATION BY PRODUCT • DINASAL • DINAXINE • DINAFERM • DINAMUNE

31. PASSIVE IMMUNITY

32. TYPES OF ACQUIRED IMMUNITY ACQUIRED IMMUNITY NATURALLY ACQUIRED ARTIFICIALLY ACQUIRED PASSIVE Antibodies pass from mother to fetus via placenta or to neonatal in her milk ACTIVE Antigens are introduced in vaccines; body produces antibodies and specialized lymphocytes ACTIVE Antigens enter the body naturally; body produces antibodies and specialized lymphocytes Previously produced Passive antibodies

33. Dinaxine Astabilized form of chlorine dioxide with the major advantages of being: • Odorless • Tasteless • Non corrosive Can be added to drinking water and atomized in site with no toxic effects for poultry and all livestock

34. MODE OF ACTION Dinaxines’ active ingredient, stabilized chlorine dioxide, completely disintegrates, (Oxidizes), the bacterial cell wall so that organism is never able to create selective immunity

35. Dinaxine Salmonella Reliable test results indicate that the disinfectant, Dinaxine is capable of producing a 97 to 98 percent kill of the test organism, (Salmonella typhimurium ATCC 14028) in minimal salts broth at a concentration of 10 ppm in one hour; and a 100 percent kill at a concentration of 50 ppm in 45 minutes.

36. DINASAL Highly efficacious Salmonella inhibition technology from DIVERSIFIED NUTRI_AGRI TECHNOLOGIES Inc., DINATEC

37. MODE OF ACTION DINASAL Hits the target everytime Through its’ unique blend of highly effective active ingredients and surfactants which empower it to easily penetrate the bacterial cell wall membrane into the cytoplasm. The ensuing breakdown into anions and cations results in rapid acidification of the cytoplasm interfering with DNA, RNA, Protein Synthesis and various other intercellular functions which will kill the Salmonella organism.

38. DINASAL Will not interfere with the animals physiology or immune system in any way Will not interfere with vaccination programs

39. Dr. Richard Miles, Head of Nutrition Department, University of Florida. Phone (352) 392-1958 “I have had extensive involvement in work with probiotics and dealing with the immune system for poultry. I have never heard that any formaldehyde based product used in feed as approved by AFFCO guidelines has had any effect of immunosuppression or interference with vaccination programs. Furthermore formaldehyde solutions are used at much higher risk level with 1 day old chicks in the hatchery without observable detrimental effects. At this time there are no available studies known that confirm or deny such a statement” September 15, 2000

40. Dr. Nelson Cox, Microbiologist, Fellow, American Academy of Microbiology, USDA, Agricultural Research Services, Poultry Microbiological Research Unit, Russell Research Center, Atlanta, GA “I am involved in extensive research in the field of Salmonella control. I have not heard of any immunosuppressive effects of formaldehyde based feed additives for use in poultry as per AFFCO guidelines for the control of Salmonella. I do not know of, nor have I conducted any research that would affirm or disclaim the above. “ September 15, 2000 Phone number (706) 546-3531, E mail address ncox@saa.ars.usda.gov

41. Dr. Ruud G. Hein, Akso Nobel, Intervet Inc. Director Scientific Affairs and Services, Poultry/ Aquaculture Business Unit, “To my knowledge, I have never heard or observed of an immunosuppressive effect of any feed additive on chickens” Dated September 15, 2000

42. Frank T. Jones, Ph.D.University of ArkansasCenter of Excellence for Poultry Science “Dear Dr. Moreira: ….I was surprised to learn of that formaldehyde based feed additives are reported to have caused immune suppression and vaccine interference.  While I suppose it is not impossible, I have heard no reports of this effect in this country and I have seen no scientific studies to suggest that such an effect exists.  Indeed, since formaldehyde has been around for many years, it appears to me that such an effect would have been observed and reported at some point in time.  Formaldehyde was used for many years in hatcheries and still is used in some hatcheries here.  If such an effect exists in feed, it would seem to me that we would have seen a similar effect during the use of formaldehyde in hatcheries.

43. Frank T. Jones, Ph.D.University of ArkansasCenter of Excellence for Poultry Science It appears to me that immune competency in U. S. birds would have improved as formaldehyde usage became less.  However, we have seen no such effect.  Furthermore, there are many, many primary breeder flocks being fed formaldehyde treated feed in this country.  It seems to me that since these primary breeder birds are extremely valuable and are monitored for nearly everything (including immunity), if such an effect existed, we should have observed it here.  However, I have heard no field reports of this effect in this country…”..

44. BRIEF DISCUSSION & CONCLUSION The preceding are some of the top experts in the field and Salmonella control in the United States. It is evident that if such a condition as immune suppression existed through the use of formaldehyde based Salmonella control feed additives they would know it. It is therefore a safe assumption that since this problem has never been reported in any scientific literature, nor has there been any interest in conducting research on the matter, the purported statement has no factual basis.

45. Sources of Contamination and DINASAL action Sources of Contamination DINASALAction Fishmeal, offal, meat and bone, Soya, etc.  Immediate kill of contaminating microorganisms and prevention of recontamination.  Lorries, storage bins, hoppers, augers, etc.  Long term protection due to the non-volatile nature of the product.  Contaminated feed, litter, housing, etc.  In-bird effect helps prevent salmonella from infecting bird.  Food processing areas, cleaning and packaging. Salmonella, Listeria and Campylobacter- free feces will minimize the risk of contamination in food.

47. DINASAL

48. UsageRates Control DINASAL 4 kg/ton DINASAL Trial Data Feed: Naturally infected Layers Meal Organisms tested:Salmonella spp., Products: DINASAL, Control Survival %:  at 0 minutes 5.7  3.0 at 2 hours  n/a  3.0  at 4 hours  n/a  2.7 at 24 hours  4.3  0.7  at 48 hours 3.7  0.0

49. COMMENTS <DINASAL at 4 kg/ton kills are naturally occurring salmonella organisms in 48 hours <DINASAL kills over 99% of salmonella species in 24 hours <DINASAL continues to work effectively after 24 hours to help prevent recontamination <DINASAL eliminates salmonella from the finished feed Mix DINASAL with 3-4 tons of corn or rice hulls at the rate of 40 kg per ton and run through entire mill system in order to disinfect circuits.

50. DINAMUNE INMUNOESTIMULANT