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1、Laboratory investigation of animal disease is critically dependent on the quality and appropriateness of the specimens collected for analysis. This chapter sets out the general standards involved in specimen collection, submission, and storage. The individual disease chapters in this Terrestrial Man
2、ual provide specific information on appropriate specimens needed in order to test for particular pathogens or toxins. Sampling may be from individual animals, from animal populations, or from the environment for a variety of purposes, such as disease diagnosis, disease surveillance, health certifica
3、tion, and monitoring of treatment and/or vaccination responses. To provide scientifically and statistically valid results the specimens collected must be appropriate for the intended purpose, and adequate in quality, volume, and number for the proposed testing. Additionally, the animals and tissues
4、sampled must be appropriately representative of the condition being investigated.Specimens must be collected using appropriate biosafety and containment measures in order to prevent contamination of the environment, animal handlers, and individuals doing the sampling as well as to prevent cross-cont
5、amination of the specimens themselves. Care should additionally be taken to avoid undue stress or injury to the animal and physical danger to those handling the animal. Biological materials should be packaged to rigorously control for leakage, and then labelled with strict adherence to the applicabl
6、e regulations guiding their transport as outlined in Chapter 1.1.2.Careful consideration must be given to the collection, containment, and storage of the specimens, including biosafety measures that must be in place to prevent contamination of the environment or exposure of other animals and humans
7、to potentially infectious materials (see Chapter 1.1.3 Standard for Managing Biorisk in Veterinary Laboratories). For information on transport of specimens see Chapter 1.1.2 Transport of Specimens of Animal Origin.The reliability of the diagnostic testing is critically dependent on the specimen(s) b
8、eing appropriate, of high quality, and representative of the disease process being investigated. Prior to sampling, consideration must be given to the type of specimen(s) needed including the purpose of the testing and the test technologies to be used. The volume or quantity of specimen must be suff
9、icient to perform initial testing, to perform any subsequent confirmatory testing and to provide sufficient residual specimen for referral or archival purposes.The purposes of testing will be aligned with the purposes for which tests are validated, as listed in Chapter 1.1.5Principles and methods of
10、 validation of diagnostic assays for infectious diseases, namely:i) Demonstration of freedom from infection in a defined population.ii) Certification of freedom from infection or presence of the agent in individual animals or their products for trade/movement purposes.iii) Eradication of disease or
11、elimination of infection from defined populations.iv) Confirmatory diagnosis of suspect or clinical cases.v) Estimation of prevalence of infection or exposure to facilitate risk analysis.vi) Determination of immune status of individual animals or populations (post-vaccination).Epidemiologically appr
12、opriate sampling plans should be developed prior to collection of specimens, as described in Section B and Appendix 1.1.1.1. These will specify the number of animals or other sampling units to be sampled.Specimens must be collected according to a sound knowledge of the epidemiology and pathogenesis
13、of the disease under investigation, or the disease syndrome to be diagnosed. This will lead to the sampling of tissues or fluids most likely to contain the infectious agent or evidence of the infection. Considerations will include the tissue predilection(s) or target organ, the duration and site of
14、infection in each tissue type and the duration and route of shedding, or the time frame in which evidence of past infection, such as an antibody response, can be detected reliably by the tests to be deployed. These considerations will also indicate the method(s) of collection to be used. In many her
15、d or flock-based disease investigations it is beneficial to collect specimens from a healthy cohort for comparative epidemiological or baseline testing (e.g. case-control and cohort approaches for diagnostic testing) and for validation purposes.Where chemical euthanasia or anaesthesia is required fo
16、r animal restraint, the impact of the chemical on the test result (e.g. toxicology testing) must be considered. Some laboratory tests are not compatible with specific blood anticoagulants and tissue preservatives, such as heparin, formalin, dry ice (exposure of the test sample to elevated levels of
17、CO2), or even freezing. While it is critical to collect specimens as aseptically as possible, equal care must be taken to avoid contamination with detergents and antiseptic treatments used to clean the collection site on the animal, as these agents may interfere with the laboratory test procedures.
18、Procedures requiring tissue culture of pathogens, as well as many molecular-based tests, can be negatively affected by chemicals or detergents commonly used in the manufacture or preparation of collection tools (e.g. chemicals used in manufacture of some types of swabs and detergents used in cleanin
19、g glassware).Specific information on diagnostic test methodologies and the recommended specimens, preservatives, and specimen handling procedures can be found in the individual Terrestrial Manual disease chapters or through direct consultation with the laboratory that will be performing the required
20、 testing. Procedures for collection and submission of specimens are available from most diagnostic laboratories, including national and international authorities, where the information is frequently accessible via the specific laboratorys web page. The OIE web page provides contact information for a
21、ll OIE reference laboratories (http:/oie.int/our-scientific-expertise/reference-laboratories/list-of-laboratories/).It is critical not only to collect the most diagnostically-appropriate specimens, but to also inform the laboratory of the associated disease epidemiology in order for the laboratory s
22、cientists to assign the most appropriate tests or panels of tests. Epidemiological information to be submitted with specimens is outlined in Section C of this chapter.Where investigating diseases of unknown cause multiple different specimens that represent the different stages of the disease progres
23、sion in an animal or the population of animals (e.g. the pre-clinical, early clinical, active clinical, chronically affected and convalescent phases) should be collected. Epidemiological considerations for sampling are particularly critical for diagnosing population-related diseases, as would occur
24、with beehives, flocks, and herds. Epidemiological principles used for sampling are further introduced in Section B of this chapter.Specimens can be collected ante-mortem or post-mortem. Specific considerations regarding different specimen types are as outlined below.Whole blood samples may be collec
25、ted for haematology, clinical chemistry, toxicology, direct examination for bacteria or parasites, PCR testing, immunological testing, or for culture for bacteria or viruses. Dependent on testing needs, whole blood, blood cells, and/or plasma samples can be obtained from whole blood collected into a
26、ppropriate anticoagulants. In selecting the anticoagulant to be used the collector must be aware of the laboratory tests, including PCR-based diagnostics, clinical chemistry, and toxicology, which may be negatively affected by the presence of specific anticoagulants or preservatives. Specific diseas
27、e chapters in this Terrestrial Manual provide guidance for individual tests and sample requirements. To be effective anticoagulants require that thecollected blood be thoroughly mixed with the chosen anticoagulant during or immediately following its sampling from the animal.To obtain serum, whole bl
28、ood is collected without anticoagulants and the clot is allowed to contract at ambient temperature protected from extremes of heat and cold for periods that may range from a few hours to overnight. Clear serum can be decanted or collected by pipette following physical removal of the clot, ideally fo
29、llowing gentle centrifugation to separate cell components from the serum. In the absence of a centrifuge, separation of the clot can be facilitated by tilting the freshly collected blood tube at an approximate 45 degree angle until the clot has retracted, “ringing” the clot with a sterile rod or pip
30、ette to separate the clot from the tube surface, and then removing the clot with forceps. The results of serological testing can be compromised by the quality of the sample. Bacterial contamination and red blood cell debris in serum samples can produce false positive reactions in agglutination-type
31、assays. Serological assays can be negatively impacted by haemolysis in the serum sample. Microbial contamination and haemolysis are significant concerns especially when obtaining blood and serum samples from post-mortem animals. Frequent causes of haemolysed serum and plasma include exposure to exce
32、ssive temperatures or time delays prior to separating sera from the red blood cells, blood collection using a needle of too small gauge, or failure to remove the needle when transferring the blood sample from the collection syringe.Whole blood should be collected aseptically, typically by venipunctu
33、re of the live animal. Depending on the animal and sampling situation jugular, caudal, brachial, cephalic, mammary veins or the vena cava may be used. Specific techniques for sampling small laboratory animals have been reviewed (Anon, 1993; Hem et al., 1998). Care should be taken to collect and disp
34、ense blood samples as gently as possible to prevent damage to red blood cells, which causes haemolysis. Blood and sera are typically shipped and stored cool (or frozen in the case of sera) in non-breakable vials, tubes, or bottles; however for some laboratory tests that require viable peripheral blo
35、od mononuclear cells, the blood must be packaged, transported and stored so as to prevent exposure to temperature extremes. For some tests, aliquots of specimens can be dried onto a piece of untreated, or specifically-treated commercial filter paper designed for stabilised sample transport and stora
36、ge.Faeces can be collected freshly voided or preferably directly from the rectum/cloaca for tests such as culture for microorganisms, parasite examination, or faecal occult blood determination; or can be collected for culture and molecular-based diagnostics from the rectum/cloaca using cotton, dacro
37、n, or gauze-tipped swabs, dependent on the volume of sample required by the specific test methodology. Samples collected on swabs should be kept moist by placing them in the transport media recommended for use with the specific test to be performed, which may range from sterile saline to culture med
38、ia containing antimicrobials or stabilisers. Faecal specimens should be kept chilled (e.g. refrigerated at 4C or on ice) and tested as soon after collection as possible to minimise the negative impacts on test results caused by death of the targeted microorganism, bacterial overgrowth or hatching of
39、 parasite eggs. Double-packaging of faecal samples in screw cap or sealable containers that are subsequently contained within sealed plastic bags will help prevent cross-contamination of samples and associated packaging materials. Faeces contained only in rectal exam gloves, plastic bags, or rubber-
40、stoppered tubes are unsuitable as they are very frequently comprised by bacterial growth with gas production that can rupture plastic bags, displace stoppers, and allow leakage of the specimen.Epithelial tissue in the form of biopsies or skin-scrapings; swabs of oral, nasal, pharyngeal, and gastroin
41、testinal surfaces, as well as plucked hair or wool can be used variously for direct examinations or laboratory tests to identify surface parasites such as mites and lice, fungal, bacterial or viral infections, allergic reactions, and neoplasia. The specimens should be collected aseptically and prese
42、rved as specified for the intended test(s). Deep skin-scrapings obtained using the edge of a scalpel blade are useful for burrowing mites. Feather tips have been validated for use in the detection of viral antigen for Mareks disease, and used as a sample for molecular detection of additional avian d
43、iseases. Epithelial tissues, particularly those associated with vesicular lesions and collected into viral transport media, can be critical in the laboratory diagnosis of specific viral infections such as foot and mouth disease.The surface of the eye can be sampled by swabbing or ocular scraping, en
44、suring that cells rather than mucopurulent discharge or lacrimal fluids are collected for testing. Specimens from the conjunctiva are typically collected by holding the palpebra apart and gently swabbing the surface of the eye with a cotton, dacron, or gauze swab that has been pre-moistened with ste
45、rile saline or equivalent media. Such swabs should be kept moist in saline or transport media specifically recommended for use with the testing to be performed. Biopsies from the third eyelid of sheep have been used as a lymphoid-rich tissue for prion detection.Preputial and vaginal wash fluids and
46、swabs of the cervix and urethra can be used as specimens for investigation of reproductive disease. The swabs should be kept moist following collection by placing in the recommended volume of transport media required by the laboratory test, typically sterile saline or specified culture media. Semen
47、specimens are typically obtained using an artificial vagina or by extrusion of the penis and artificial stimulation. Avoid contamination of the specimen with antiseptic or detergent solutions used to prepare the animal/site for sampling.Secretions can be collected directly into a vial or tube, or ca
48、n be collected using swabs. Vesicular fluids provide a highly concentrated source of pathogen for diagnostic testing, and can be collected from unruptured vesicles using a sterile needle and syringe, and immediately transferred to a securely sealed vial or tube. Specifically developed sampling tools
49、, such as probang cups, can be used for collecting cellular material and mucus from the pharynx of livestock. Cotton ropes that animals are allowed to mouth and chew have been validated for use in collecting saliva specimens from domestic swine.Milk can be collected from individual animals or from bulk milk in tanks pooled from multiple animals in a herd. The teat(s) used for s