S.Chitradevi*, K.Sukumar, A.Raja, P.Suresh and G.Kalaiselvi
* Corresponding Author: Assistant Professor, Department of Veterinary Microbiology, Veterinary College and Research Institute, Namakkal-637 002.
Specified pathogen free (SPF) technology is the science of developing and maintaining a species of animals free of infection by specified pathogenic micro-organisms. A specified pathogen is any micro-organism for which infection of poultry can produce a recognized disease condition. Specified pathogen free can only be applied to poultry which return negative results in accepted surveillance tests for the-presence of one or more specific pathogens. Testing must be repeated at regular, defined intervals for the SPF status of a flock to remain current. The most SPF flocks will have a limited resident gut microbial flora e.g. Streptococcus, Coliforms, Bacterioides, Clostridia spp., even after several SPF generations in isolators with wire floors. An SPF flock comprises a group of birds which share a common environment whether pen, building or isolator.
Classes of SPF flocks
Class A: Vertically transmitted agents must be shown to be absent using the most rigorous testing possible, as should be laterally transmitted agents. Nucleus breeding SPF flocks( which supply stock for production of SPF flocks) as well as SPF eggs and chickens used for testing of vaccines, production of seed viruses and monospecific antisera should be class A. Consideration should be given to maintaining Class A SPF flocks with in isolators.
Class B: Vertically transmitted agents must be excluded, but the presence of certain laterally transmitted agents is acceptable for SPF flocks which supply eggs for vaccine production. The most common example in practice would be MDV. However, vaccine manufactures should be aware of any breakdown which may occur and take measures to safe guard their product as well as performing microbiological surveillance prior to release of vaccines.
For each batch of SPF eggs supplied from a SPF flock, certification should be issued by the producer denoting the agents from which a flock has been proved free.
The SPF should have the following facilities:
Isolators for SPF poultry flocks
Stainless steel, fiber glass isolator cabinets are used to house SPF flocks for increasing the microbiological security of a flock by reducing direct contact between poultry and the operator, and spreading the risk of total loss through sub-division of the flocks. The design should also have to facilitate the waste-disposal, lighting, nest box and watering systems.
Ideally only the caretaker enters the house throughout the whole life cycle. To reduce the necessity of entering for repair and maintenance, all standard maintenance work is done during the service period when the house is empty. Important technical equipment is serviced routinely or will be replaced as a safety measure in the downtime of the house.
The caretaker enters the house through double barriers: the first barrier requires a change from street clothes and shoes into farm owned clothes and boots, the second barrier before entering the SPF-house requires a complete change to house specific clothes after a shower with hair wash. The house specific clothes remain within the house during the complete production cycle, which requires a washer and dryer for each house.
Feed and water
To minimize the risk for all SPF flocks, the feed is produced in a state of the art feed mill, using an effective heat treatment system. In this feed mill, finished feed is exclusively produced for the SPF-flocks.
Only selected raw materials from proven sources are used. Currently the feed consists of corn, wheat, soybean meal, vegetable fat plus vitamin and mineral premixes. The decontamination treatment consists of heat (85°C for 6 minutes minimum) and the addition of an acid mix after cooling.
Fat and vitamins are added after the heat treatment and need to be decontaminated separately. Fat is kept in the storage tanks at a constant temperature of 70°C and is therefore clean when added to the feed. The vitamin premix is delivered in 25 kg bags which are irradiated with 15 kgy minimum dose prior to being transported to the mill. The bags are emptied into a container in the HEPA-filtered section of the feed mill through sterile stainless steel tubes. Fat and vitamins are added to the feed in the second mixer located in the overpressure ventilated area of the mill. This mixer serves as dryer and cooler as well as final mixer for the mash feed. The process air for drying and cooling and also the air to overpressure ventilate the cooling and finished feed storage bins is HEPA-filtered.
An important part of the feed supply concept is clean delivery. A dedicated tanker type vehicle is used to deliver the feed into a storage building close to the production sites and the delivery vehicles have been thoroughly cleaned and disinfected during the weekend. Regular tests are performed internally and by an independent laboratory.
Gamma irradiation is also currently the most reliable method of decontamination of feed but very costly. Fumigation with methylbromide, ethylene oxide can also be used. Application of ethylene oxide to feed is performed with in a large pressure chamber under vacuum over treatment period of 18-24 hours. Water can also be acidified using 0.01N HCl, pH 2 is regularly practiced conferring effective sterilization and also reducing build-up of scale and slime in water lines.
Manure handling and removal
All SPF flocks are kept in family cages with manure belts. The manure is removed once or twice a week, using specially designed screw augers. These augers have a double shut-off system to ensure that there is no backflow of material into the house and that no rodents or other potential disease carriers can enter the house. This auger system can only handle wet manure which, from a management point of view, is sub-optimal.
All material needed during a life cycle is brought into the house before final disinfection prior to the placement of day-old chicks. Other material, dead animals and eggs must pass a disinfection chamber with an effective fumigation procedure in place. All outgoing material is fumigated and the emptied chamber is fumigated again before the chamber can be opened again from the clean side.
All air delivered to a SPF-chicken house must therefore be filtered before entering the house. They currently use a system with three filter steps. The last step is a HEPA–filter which filters out 99.95 % of all particles in the air. Supply air is drawn from outside by fans and filtered in several stages, beginning with 5 µm pre-filter of approximately 90% efficiency and progressing through to a final high efficiency particulate air filter which excludes 0.3 µm diameter particles with at least 99.99% efficiency.
To insure that all air passes through the filtration system, the technical equipment must be designed to avoid false air entering the house. This is possible by using a filtered air, positive pressure (FAPP) system. The FAPP system also has active ventilation elements on the exhaust side to avoid wind pressure into the house and to keep off insects. All filters are exchanged before a new flock is placed and are checked for leaks with a particle counter.
The operating efficiency of HEPA filters should be monitored weekly through recordings of different manometers readings across each filter, and be re-challenged with DOP (dioctylpthallate) each 6-12 months by competent technical personnel.
All SPF– houses are equipped with rodent traps along the walls at a distance of only 3 m from each other. As soon as a house is depopulated, a very intensive pest control program must be carried out inside the house.
Grading, disinfection and final inspection
The final step before shipping SPF–Eggs to the customer is grading and disinfection. Although the eggs are fumigated during the transfer from the chicken house to the grading facility, the eggs may have been re-contaminated during handling and transport and are therefore disinfected again. The grading process consists of a visual inspection where soiled eggs and eggs with obvious shell defects are removed before the eggs pass through a crack detector unit and a weighing system.
Eggs with shell defects, undersized and oversized grades are directed to separate packing lines and sold to an egg breaker. Saleable SPF-eggs are sorted into three weight classes and packed into cardboard boxes. This process gives SPF eggs customers a more uniform egg size and supports automatic processes in egg handling during the vaccine production. The first disinfection is by fumigation, the final disinfection before packing the eggs by spraying. For both processes, formaldehyde free products are used.
Quality control to confirm the SPF status
Besides the visible inspection of egg quality, the SPF status of each flock has to be guaranteed at all times during production to ensure the requirements of the European Pharmacopoeia. At present, the list of agents consists of different diseases, of which absence of antibodies or antigens has to be demonstrated by validated, approved testing protocols. During the rearing period 5% of the birds have to be tested twice, during the production period 5% on a monthly basis (or 1.25% weekly).
A designated SPF flock is derived from chickens shown to be free from vertically-transmissible agents listed in below mentioned table. This is achieved by testing of 2 generations prior to the designated SPF flock.
|Agent||Test to be used||Vertical transmission||Rapid / slow spread|
|Avian adenovirus group 1||AGP, EIA||Yes||Slow|
|Avian encephalomyelitis virus||AGP, EIA||Yes||Rapid|
|Avian infectious bronchitis virus||HI, EIA||Rapid|
|Avian infectious laryngeotracheitis virus||VN, EIA||Slow|
|Avian leucosis virus||EIA for virus, VN for antibody||Yes||Slow|
|Avian nephritis virus||IS||No||Slow|
|Avian orthoreovirus||IS, EIA||Yes||Slow|
|Avian reticuloendotheliosis virus||AGP, IS, VIA||Yes||Slow|
|Chicken anaemia virus||IS, EIA, VN||Yes||Slow|
|Egg drop syndrome virus||HI, EIA||Yes||Slow|
|Infectious bursal disease virus||Serotype-1AGP, EIA, VN. Serotype -2 VN||No||Rapid|
|Influenza virus A||AGP, EIA, HI||No||Rapid|
|Marek’s disease virus||AGP||No||Rapid|
|Newcastle disease virus||HI, EIA||No||Rapid|
|Turkey rhinotrachetis virus||EIA||No||Slow|
|Mycoplasm synoviae||Agg and HI confirm positive test. EIA, HI||Yes||Slow|
|Mycoplasma Gallisepticum||Agg and HI confirm positive test. EIA, HI||Yes||Rapid|
AGP-Agar Gel Precipitation, EIA-Enzyme Immune Assays, IS – Immunostaining, Agg- Agglutination, HI – Haemagglutination Inhibition
Initial Testing Requirements for Subsequent Generations Derived from a Designated SPF Flock
Where a replacement flock is derived exclusively from a fully established SPF flock the new generation is tested prior to being designated as SPF. In addition to the tests for Salmonella and monitoring of the general health and performance of the flock, further specific testing from the age of 8 weeks is required. Tests are performed on two 5 per cent samples of the flock (minimum 10, maximum 200 birds) taken with an interval of at least 4 weeks between the ages of 12-16 weeks and 16-20 weeks.
All samples are collected and tested individually. Blood samples for antibody tests and suitable samples for testing for leucosis antigen are collected. Only when all tests have confirmed the absence of infection may the new generation be designated as SPF.
Routine Testing of Designated SPF Flocks
General examination and necropsy.
Clinical examination is carried out at least once per week throughout the life of the flock in order to verify that the birds are free from fowl-pox virus and signs of any other infection. In the event of mortality exceeding 0.1 per cent per week, necropsy is performed on all available carcasses to verify that there is no sign of infection. Where appropriate histopathological and /or microbiological / virological studies are performed to confirm diagnosis.
Specific examination for tuberculosis lesions is carried out and histological samples from any suspected lesions are specifically stained to verify freedom from Mycobacterium avium. Caecal contents of all available carcasses are examined microbiologically for the presence of Salmonella spp. using the techniques described below. Where appropriate, caecal samples from up to 5 birds may be pooled.
Tests to be Conducted at the End of the Laying Period
Following the last egg collection, final testing to confirm the absence of vertically-transmissible agents is performed. After the last egg collection, a minimum of 5 per cent of the flock (minimum 10, maximum 200) is retained for at least 4 weeks. Blood samples are collected from every bird in the group during the 4-week period with at least 1.25 per cent of the birds (25 per cent of the sample) being bled not earlier than 4 weeks after the final egg collection. Serum samples are tested for vertically-transmissible agents using the methods indicated. Where sampling is performed cent of the sample) are tested each week during this period. Alternatively, within 4 weeks of the final egg collection blood and/or other suitable sample materials are collected from at least 5 per cent of the flock and tested for the presence of vertically-transmissible agents using validated nucleic acid amplification techniques
Action to be Taken in the Event of Detection of a Specified Agent
If evidence is found of contamination of the flock by an agent listed as slowly spreading, all materials derived from the flock during the 4-week period immediately preceding the date on which the positive sample was collected are considered unsatisfactory. Similarly, if evidence is found of contamination of the flock by an agent listed as rapidly spreading, all materials derived from the flock during the 2-week period immediately preceding the date on which the positive sample was collected are considered unsatisfactory. Any product manufactured with such materials, and for which the use of SPF materials is required, is considered unsatisfactory and must be discarded; any quality control tests conducted using the materials are invalid.
Producers must notify users of all eggs of the evidence of contamination as soon as possible following the outbreak. Any flock in which an outbreak of any specified agent is confirmed may not be redesignated as an SPF flock. Any progeny derived from that flock during or after the 4-week period prior to the last negative sample being collected may not be designated as SPF.
Advantages of SPF Eggs
- Usage of SPF Eggs in the production of vaccine assures abscence of extraneous agents in poultry and human vaccines.
- SPF eggs are predominantly preferred over an ordinary fertile egg for virus growth because of absence of specific antibodies in SPF egg yolks. This characteristic of SPF eggs, in-turn allows to attain considerably higher titre levels of virus than that of ordinary eggs.
- The international standards of quality demand usage of only SPF eggs for the production of poultry and human vaccines. WHO, EGG and USDA standards for production of vaccine call for the use of SPF eggs.
- SPF eggs totally avoid the hazardous risk of embryo – transmitted and laterally transmitted viral agents as contaminants in virological and immunological research work.
- SPF eggs ensure reproducibility of research results in the field of virology and immunology
- Use of SPF eggs provides international acceptance to the research work of Virologists, Biotechnologists, Immunologists, Cellbiologists and Oncologists.
- Kenji Furuta, Hitoshi Ohashi, Jitsuo Odana & Shizuo SA TO· Performance of 3 successive generations of specified-pathogenfree chickens maintained as a closed flock,Laboratory Animals (1980) 14, 107-112.
- EUROPEAN PHARMACOPOEIA 5.1 2.2. SPF chicken flocks for vaccines, 04/2005:50202
- M, Seemann G. and Cuxhaven, Fertile eggs – a valuable product for vaccine production, Lohman information Vol. 43 (2), Oct. 2008, Page 37.
- Luginbuhl, R.E, The commercial production of specific pathogen free eggs and chickens: The evolution of an industry. Avian Diseases 44: 632 – 637, 2000.