Research sampling protocol

This protocol was designed for the collection of samples from bison in a uniform manner that will lead to data as free of contamination and artifact as is possible in a field setting. In addition to the complete protocol for optimum sampling situations, a short protocol is included for special situations that arise during sampling in the field. Examination of bison by biologists will involve recording of data on teeth, hair, horns and other physical characteristics. Special samples may be required for data during this phase of examination.

The WHO Laboratory Biosafety Manual places Brucella abortus in Risk Group III, indicating a high risk to persons that are involved in handling infected animals or tissues. All personnel involved in sampling should be formally advised of the risk of infection and trained in the handling of infectious tissue and the use of masks and equipment. Face masks, gloves, and protective clothing should be used in high risk situations that involve female bison that have placental lesions of brucellosis or that have aborted.

The greatest risk of infection to humans is body contact with infectious material and transmission of infection from hands to body orifices. Brucella abortus is typically present in low numbers in blood and lymphoid tissues of animals. Although most genital tissues in males and nonpregnant females have only low numbers of organisms, the infected placenta and its fluids are extremely hazardous and may contain up to 10¹³ bacteria per gram, a concentration where aerosol transmission is possible. Blood and milk are hazardous but it is unlikely that infection will occur if reasonable means are used to prevent contamination of hands and face, and spread from there to body orifices.

A. RESEARCH SAMPLING PROTOCOL

Where conditions are good, sampling of a wide range of tissues should be done to acquire the maximum amount of data. Sampling for research quality specimens may require that the tissue and fluid sample list be long (Appendix I).

For bacterial culture, the amount of body fluids or tissues will correlate directly with the detection of B. abortus. In heavy, acute infections this is rarely important. However, in chronic infections with few organisms per gram of tissue, it is important to take large samples; e.g., 100 ml blood, entire lymph nodes, large slices of organs. In lymph nodes, a small focus of B. abortus may be present in one small area of the node. Thus, to be accurate, the entire lymph node must be used as the sample for bacterial culture.

If the objective is to identify infection by B. abortus then the simple determination of the presence or absence of bacteria is sufficient. Some studies however will require knowledge of the extent of infection; i.e., the number of bacteria per gram tissue. In these cases, titration of tissue suspensions will be required.

The detection of infection in adult bison may require only a few samples; however, this is not yet known. One report from tests in adult cattle suggest that 100% detection can result from sampling of uterine caruncle, and supramammary, mandibular, and medial iliac lymph nodes (Alton et al. 1988); however, most laboratories do not achieve this detection rate. Young animals, which clear B. abortus quickly, require greater tissue sampling.

The necropsy report, where required, should follow a standard format that includes data on organ weights, size, and structure (Appendix II). To facilitate sample collection, kits for each animal to be sampled should be prepared in advance (Appendix III).

Tissues for Bacterial Culture

Organ and tissue samples should be at least 0.5 cm thick and approximately 2 to 5 cm in width and length. Tissues are collected in sterile, Whirl-Pak plastic bags. Each specimen should be labeled as to bison number, tissue, and date and time of collection. Samples should be transported on ice to the laboratory within 2 hours of collection, or quick frozen in dry ice for storage and shipment (Ewalt et al. 1989).

Tissues collected for culture (Appendix I) are those in which B. abortus is most likely to be found; the genital system and lymph nodes associated with the head and neck and with the genital system. Placenta should be carefully examined grossly noting the extent of lesions in both cotyledons and intercotyledonary placenta. Placental tissues harvested should be those with lesions. Specific lymph nodes for culture should include: bronchial, hepatic, internal iliac, mandibular, mesenteric, parotid, popliteal, prefemoral, retropharyngeal, scrotal/supramammary, and superior cervical (prescapular). If tuberculosis is suspect, medial and lateral retropharyngeal, mediastinal and tracheobronchial lymph nodes should be collected.

The following tissues, not included in the collection list for bacteriologic culturing, are recommended only for special research protocols: adrenal gland, brain, brown fat, cecum, heart, muscle (skeletal), pineal, pituitary, thymus, thyroid, and any organ with pathologic lesions.

In bison, the thymus, abomasum, liver and lungs are less likely to be infected with B. abortus (Davis et al. 1990; Davis et al. 1991). The tonsil, although it efficiently takes up pharyngeal bacteria, appears to rapidly pass B. abortus into the lymphatics and does not often yield positive cultures in cattle.

Body Fluids for Bacterial Culture

To limit contamination of samples of blood, skin should be disinfected with an iodine solution followed by wiping with an ethanol gauze. For culture, collect 100 ml blood using a 16 gauge needle and 50 ml syringe. Blood for serology (10 ml or more) should be harvested into small 10 ml tubes without anticoagulant. If clinical pathology studies are required, a separate collection of 5 to 10 ml of blood in anticoagulant should be taken (Appendix III).

To collect cerebrospinal fluid, incise the skin at the back of the head and separate ligaments and muscle of the neck from the skull by cutting deeply to (but not through) the surface of the meninges in the space between the occipital bone and the first cervical vertebra. Using a 10 ml syringe with 16 gauge needle, withdraw 10 ml of cerebrospinal fluid and place in sterile vacuum tube.

Allantoic fluid (10 ml) is withdrawn before the uterus is opened. Vitreous fluid from the posterior chamber of the eye is often less decomposed than fluids in other sites (Lincoln and Lane 1985). Urine (10 ml) should be collected with sterile needle (16 gauge, 2 inch) and transferred to sterile tubes.

Tissues for Histology

Histologic analysis will reveal the nature and extent of infection. Tissues listed on the check sheet (Appendix I) should be sampled for histology (Williams et al. 1993). Lymph nodes to be cultured should be sampled with one small (<O.5 cm) cross section through the middle part of the node. Tissues are fixed in standard formalin fixation and processed by standard embedment techniques with hematoxylin-eosin staining. Immunoperoxidase techniques are available for special studies (Meador et al. 1990).

Although brucellosis is the focus of these studies, other pathologic lesions in the carcass should be recorded during the necropsy, especially those lesions of diseases which may in some way promote infections by B. abortus; e.g., tuberculosis, pasteurellosis, and clostridial infections should be noted. In GYA bison, mediastinal and tracheobronchial lymph nodes should be examined for caseous lesions of tuberculosis.

Fetal Tissues

Lungs, spleen and gastric contents are the most important samples for detection of B. abortus in the bovine fetus infected in utero, and are probably most important in fetal bison (Rhyan et al. 1994). In some natural infections of pregnant cattle, meninges and choroid plexus are tropic for bacteria (Hong et al. 1991), although the incidence of brucellar meningitis is not known and the mechanisms of infection are not understood. Other organs in which lesions of brucellosis have been reported in cattle fetuses include heart (epicarditis), liver (hepatic portal inflammation), joint (fibrinopurulent arthritis), serosa (fibrinous peritonitis), and spleen (necrosis).

Table 1. Fetal tissues for sampling

B. FIELD SAMPLING PROTOCOL

When sampling is limited due to time, status of bison, or site of collection, the number of samples can be reduced to tissues which are most likely to contain bacteria (Table 2). In females, uterine secretions, spleen, mammary gland (all quarters), supramammary/scrotal lymph node, and placenta are mandatory for the maximum detection of infection. In males, scrotal lymph node, epididymis, and testicle are mandatory for sampling. In both males and females, lymph nodes, in order of preference, are: supramammary (female)/scrotal (male), parotid, prefemoral, internal iliac, superficial cervical (prescapular), medial retropharyngeal, and mandibular. Other tissues, in order of preference for sampling, are: anterior vagina, posterior uterus (endometrium), cervix, ovary, oviduct, and spleen. Where sampling of entire organs is not possible, a biopsy gun, available commercially, can be used to take small core samples of tissue.

If venipuncture is not possible, blood may be harvested by cutting a major vessel immediately after death, or by cardiac puncture. Blood collected by venipuncture or cardiac puncture should be taken using sterile equipment and techniques that include clipping of hair and disinfection of skin with iodine solution and alcohol wipes. Cardiac puncture in the dead animal provides a good source free of contamination. Where the use of sterile techniques is not feasible, samples should be collected as free of contaminating materials as possible.

Blood for bacteriologic culture should be added to an equal volume of culture medium within 2 hours of collection, or should be transported or stored at 4C.

Packaged sterile instruments should be used for each sample collection. If instruments are used multiple times, they should be sterilized after each use by being cleaned, immersed in an alcohol bath, and flamed immediately before the sample is taken.

Table 2. Tissues for short protocol

C. SAMPLE STORAGE

Tissues

Tissues to be cultured for B. abortus should be stored on ice and arrive in the laboratory within 2 hours of death of the bison. Where longer times are required, tissues in plastic bags should be placed in dry ice, or snap frozen to -70C in acetone/dry ice or in liquid nitrogen, and stored at -70C while en route to the laboratory. A safety classification system placed on tissue labels may aid in later handling of these tissue specimens in the laboratory (Appendix IV).

If preliminary processing is to be done in the field, tissue suspensions can be prepared and frozen. Lymph node biopsy specimens <3 mm in diameter, taken by biopsy gun, should be rinsed in sterile saline and ground to suspension in a Ten Broeck tissue grinder. Samples from solid organs (liver, lung, etc.) should be cut into small segments, ground in a Ten Broeck tissue grinder using 10 ml of medium. Whole lymph nodes should be cleaned of fat, connective tissue and blood vessels, and rinsed in sterile saline. The lymph node is cut into 1 cm cubes and each cube is processed by grinding in a tissue grinder to suspension.

Fluids

Fluids can be directly streaked onto solid media or inoculated into tubes of fluid media. Whole blood is stored at 37C for 2 hr to facilitate clotting and serum collection. Tubes are centrifuged and serum collected. Milk is centrifuged at 7000g for 15 minutes, the skim milk is discarded, and the cream and sediment are inoculated separately onto solid media.

REFERENCES

• Alton GG, Jones LM, Angus RD, et al. Techniques for the brucellosis laboratory. Paris: Institut National de la Recerche Agronomique, 1988.

• Davis, DS, Templeton JW, Ficht TA, Williams JD, Kopec JD and Adams LG. Brucella abortus in captive bison. I. Serology, bacteriology, pathogenesis, and transmission to cattle. J. Wildlife Dis 26:360-371, 1990.

• Davis DS, Templeton JW, Ficht TA, Huber JD, Angus RD and Adams LG. Brucella abortus in bison. 2. Evaluation of strain 19 vaccination of pregnant cows. J Wildlife Dis 27:258-264, 1991.

• Ewalt D, et al. The effect of freezing on the isolation of Brucella abortus from bovine milk.

• Project Report, Nat Vet Services Lab, 1989.

• Hong CB, Donahue JM, Giles, RC Jr, Poonacha KB, Tuttle PA and Cheville NF. Brucella abortus-associated meningitis in aborted bovine fetuses. Vet Pathol 28:492-496, 1991.

• Lincoln SD and Lane VM. Postmortem magnesium concentration in bovine vitreous humor. Am J Vet Res 46:160, 1985.

• Meador VP, Hagemoser WA and Deyoe BL. Histopathologic findings in Brucella abortus-infected, pregnant goats. Am J Vet Res 49:274-280, 1988.

• Meyer ME and Meagher M. Brucella abortus infection in the free-ranging bison (Bison bison) herd in Yellowstone National Park. National Symposium on Brucellosis in the Greater Yellowstone Area. (in press)

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• Olsen SC, Cheville NF, Kunkle RA, Palmer MV and Jensen AE. Bacterial survival, lymph node changes, and serological response of bison (Bison bison) vaccinated with Brucella abortus strain RB51 or strain 19. Am J Vet Res (in press)

• Rhyan JC, Quinn WJ, Stackhouse LS, Henderson JJ, Ewalt DR, Payeur JB, Johnson M and Meagher M. Abortion in free-ranging, Yellowstone National Park bison (Bison bison) due to Brucella abortus biovar 1. J Wildlife Dis 30:445-446, 1994.

• Williams ES, Thorne ET, Anderson SL and Herriges JD Jr. Brucellosis in free-ranging bison

• (Bison bison) from Teton County, Wyoming. J Wildlife Dis 29:118-122, 1993.

APPENDIX I. TISSUE/FLUIDS SAMPLE LIST SUMMARY

* Tissue in sterile plastic bag unless other is specified.

** Lymph nodes for bacteriologic culture should be sampled from one side, lymph nodes for histology should be taken from the opposite side of the animal. In chronic infection, only one small area of the lymph node is likely to have culturable bacteria; if bacteriologic data is to be precise, suspensions of the entire lymph node must be cultured.

APPENDIX II. RESEARCH NECROPSY PROTOCOL

The carcass is that of a (development, fat/muscle, sex) bison (Bison bison) measuring ________cm in length. Temperature, rigor, lividity. Skin and hair coat in general. Eye: color, pupils, corneae, lens, sclerae. Ears: lesions, discharge. Nose: turbinates. Oral cavity: teeth, tongue, tonsils, salivary duct orifices. Neck: enlargments. Lymph nodes: general or local enlargement. Thorax: size, symmetry. Mammary glands: size, teats, masses. Abdomen: shape, scars (location, age). Genitalia: lesions, scars, discharge. Legs: asymmetries, deformities, edema. Horns. Hooves: lesions. Subcutaneous fat: amount, color. Peritoneal cavity: fluid, serosal surfaces, abnormal mesenteric relations. Pleural cavities: serosal surfaces, mediastinum.

HEART

• Congenital abnormalities. Weight ______g. Epicardium: surface, fat (amount, atrophy), chambers (size, content). Endocardium. Valves: descriptions. Myocardium: thickness, color, consistency, lesions. Coronary blood vessels: caliber, lining, walls.

LUNGS

• Lungs: pleural surfaces, hilar blood vessels, bronchi, hilar lymph nodes, cut surface (color, wetness, appearance of bronchi, blood vessels, general consistency). Weight: right ______g; left ______g.

SPLEEN

• Size. Surface. Consistency. Hilar vessels. Cut surface: color, texture, trabeculae, white pulp. Weight ______g.

GASTROINTESTINAL TRACT

• Esophagus: dilatation. Abomasum: mucos (thickening of rugae, edema), color. Small intestine: note Peyer's patches and other intestinal lymphoid tissues. Cecum. Colon.

LIVER

• Surface and cut surface: lobule architecture, color, consistency. Focal lesions. Weight: ______g. Gallbladder: Bile amount, color, consistency, mucosa, wall thickness. Extrahepatic bile ducts: caliber, wall thickness, abnormalities.

PANCREAS

• Size, color, consistency. Lobulations, islet, ducts.

ADRENALS

• Size, consistency. Cut section: color, width of zones. Lesions.

URINARY TRACT

• Kidney: Surfaces and cut surfaces: color, cortical thickness, striation, glomeruli visible. Weight: left ______g: right ______g. Pelvis: size, architecture, arcuate arteries. Ureter size, color, thickness. Lesions. Bladder: contents (volume, color, clarity), mucosa, wall and ureteral orifices.

GENITAL SYSTEM

• Testicles: size, diameter, lesions. Epididymis: size, lesions. Penis. Urethra. Prostate. Seminal vesicles: size, uniformity, location, appearance and consistency of abnormal areas. Vagina. Ovary. Oviduct. Uterus. Size, mucosa, lesions.

PREGNANT UTERUS

• Placentomes: size, color, lesions, exudate. Intercotyledonary placenta: color, opaque, flecks, exudate. Arteries and veins. Allantoic fluid: color, amount, exudates. Amnion. Fetus: crown-rump measurement.

VASCULAR SYSTEM

• Aorta: caliber, elasticity, intima, renal artery orifices, other branches. Abdominal veins.

LYMPHATIC SYSTEM

• Focal or systemic enlargement. Edema. Hemorrhages. Dilatation of lymphatics.

NECK ORGANS

• Thyroid: size, color, consistency, nodularity. Parathyroids. Thymus: weight______g. Color consistency. Larynx. Salivary glands.

MUSCLE AND SKELETAL SYSTEMS

• Local or general abnormalities not covered under carcass description. Skeletal muscle. Pallor, atrophy, parasites. Joints: swollen, synovial fluid increased, synovial membranes, proliferative lesions.

BONE MARROW

• Color, consistency (mushy, cellular, firm, fibrous, gelatinous).

BRAIN

• Skull. Meninges: opacity, texture, fluid. Cerebral cortex: convolutions, sulci, color, consistency, symmetry. Blood vessels. Brain weight ______g. Ventricles: choroid plexus.

Pituitary. Pineal.

APPENDIX III. CHECK LIST FOR SAMPLING KIT

Postmortem equipment should include: bone cutter, rib cutter, forceps (12+), cleaver, hatchet, clipper (rechargeable), knives (2), metric ruler, scissors, scalpel (12+), saw. Disinfectants and gauze should be included in each kit.

APPENDIX IV. SAFETY CLASSIFICATION OF ANIMAL TISSUE

Tissues and body fluids of bison that may be infected with Brucella spp. should be collected, processed, and transported within the confines of appropriate containment. There is great variability in the risks at different ages, stages of infection, and stage of pregnancy in animals with brucellosis. The following classification may be helpful in the laboratory processing and transport of noncultured tissues and body fluids from animals in which selected tissues have been cultured.

Class 0 = No risk. Animal not infected.

Class 1 = Low risk. Tissues (genital tract, lymph node, blood) are culture negative and not pregnant, but animal was known to have been infected previously.

Class 2 = Moderate risk. Animal infected, not pregnant, blood and genital secretions are culture negative. Lymph nodes or other tissues are culture positive.

Class 3 = High risk. Any tissue that is known to contain B. abortus. Other tissues and fluids of unknown culture status that are obtained from an animal that is infected, pregnant, blood culture positive, lymph node positive >1 x 10³/g. Example: placenta, fetus, vaginal exudate, lymph node, blood.