Laboratory diagnosis of swine diseases.

The veterinary practitioner has responsibility for making final diagnoses and ultimate decisions concerning management of swine health problems and/or programs in the practice. The veterinary diagnostic laboratory can be an important supporting arm of the veterinary practice by (1) providing a consultation service, (2) providing technical assistance in performing laboratory testing and (3) collecting and disseminating information on current research. Data from cases submitted to most diagnostic laboratories are tabulated and evaluated regularly to study animal disease trends, to forewarn practitioners, and to direct concentration of research efforts.

Modern diagnostic laboratories have facilities and personnel highly trained in areas of bacteriology, chemistry, clinical pathology, immunology, mycology, parasitology, pathology, theriogenology, toxicology, and virology. Highly sophisticated equipment—such as scintillation counters, automated cell counters, autoanalyzers, atomic absorption spectrophotometers, mass spectrometers, high pressure liquid and gas chromatographic equipment, elaborate equipment for culturing bacterial anaerobes, and electron microscopes—is in routine use in many diagnostic laboratories today. Many diagnostic laboratories in the United States maintain accreditation of capabilities and competence based on standards outlined by the American Association of Veterinary Laboratory Diagnosticians.

Individual laboratories are geared toward the problems that occur in the areas in which they serve. Certain cases may be referred to specialized laboratories for more detailed analyses, if necessary. A directory of animal disease diagnostic laboratories in the United States that lists their capabilities and specialties has been prepared. The technical capabilities of the diagnostic laboratory can be used for monitoring herd health programs as well as for facilitating diagnosis in disease outbreaks.

The accuracy of laboratory results and their effectiveness in helping to resolve health problems are largely dependent on clinical evaluation of the case by the veterinary practitioner as well as selection, processing, packaging, preservation, and transport of specimens to be submitted for analysis. Recommendations concerning these topics in general and as they relate to the diagnosis of certain individual and/or groups of swine disease problems are the subject of this article.

Submission of Specimens for Laboratory Analysis

Clinical History

Telephone consultation of the veterinary practitioner with the diagnostic laboratory staff is often warranted when certain disease problems arise. The nature of the problem can be discussed, and submission of correct specimens needed to expedite the diagnosis can be assured. Provision of a clinical history, whether given by telephone or written, is an important step in the diagnostic process. The following helpful information may be included in the clinical history:

Name, address, and phone numbers of both the swine producer and the attending veterinarian

Total number of animals in the affected group

Number of animals that are currently ill

Number of animals that have died

Clinical signs observed

Lesions observed in necropsied animals

Age and weight of affected animals

Duration of the problem, date of onset

Breed of animals affected

Immunizations

Treatments administered, response

Recent additions to herd; exposure to wildlife and other domestic species

Environmental factors (housing, confinement or pasture, water source, etc.)

Feeding programs, nutrient levels, feed additives

Parasite control (internal and external): drugs and chemicals used, how often?

Information from management records (breeding dates, conception rates, farrowing interval, feed efficiency, rate of gain, etc.)

Tentative diagnosis

The following outline can be used in recording lesions observed during field necropsies:

External examination (appearance of hair coat, skin lesions-location and description, eyes, conjunctiva, ears, feet, hooves, mouth, nostrils, anus, vulva)

Respiratory system (nasal cavity, larynx, bronchi, lungs, pleura)

Circulatory system (heart, pericardium, blood vessels)

Digestive system (oral cavity, teeth, esophagus, stomach, duodenum, jejunum, ileum, colon, liver, gallbladder, pancreas)

Urinary system (kidneys, ureters, urinary bladder)

Genital system (ovaries, uterus, vagina, testicles, spermatic cord, male accessory sex glands)

Endocrine system (thyroid, parathyroid, adrenals, pituitary)

Lymphoreticular and hematopoietic systems (lymph nodes, spleen, thymus, tonsil, bone marrow)

Musculoskeletal system (muscles, bones)

Nervous system (brain, spinal cord, peripheral nerves, meninges)

A complete history is essential for performing the appropriate tests and for proper interpretation of laboratory results. Practitioners should communicate with laboratory personnel in such a manner that all requests are fulfilled and steps are taken to obtain the information needed.

Selection, Collection, and Preservation of Specimens

Specimens selected for laboratory analysis depend, of course, on the nature of the problem and the reasons for obtaining laboratory assistance. A live, untreated pig with representative clinical signs is usually the specimen of choice for laboratory analysis. However, it may be more expedient for the practitioner to collect specimens during a field necropsy, or from live animals, and send them to the laboratory via an acceptable mode of transportation. The following equipment and supplies are designed for use in performing necropsies and collecting laboratory specimens:Be certain that specimens collected are representative of the systems that appear to be involved in the disease process. Be sure that the size of samples is adequate (100 to 300 gm). Excess material can always be discarded, but it is difficult to retrieve samples if they are not collected initially.

Tissues to be submitted for histopathologic, microbiologic, and/or toxicologic examination should include gross lesions, if present, and lymph nodes draining affected areas. However, it is best to collect samples from all organs regardless of whether or not gross lesions are observed. Collect tissue with the least possible contamination. Select samples of organ tissues (lung, liver, spleen, tonsil, lymph nodes, and so forth) first, before incising the gastrointestinal tract. Individual samples should be placed in separate containers (plastic bag). Representative sections of the gastrointestinal tract (stomach, duodenum, jejunum, ileum, cecum, colon, and so forth) should also be placed in separate containers. Submission of the brain is important, especially in cases in which there are signs referable to the central nervous system. In most cases the head can be removed from the carcass, packaged, and sent intact along with other specimens. If the head is sent in this manner, the brain, ears, lymph nodes, tonsillar tissue, nasal turbinates, and other organs will be available for examination by laboratory personnel.

Tissues should be thoroughly chilled (32 to 40°F) as soon as possible after collection, prior to packing for shipment to the laboratory. Routine freezing of tissue samples is not recommended. Freezing may be necessary in special instances, and laboratory diagnosticians should be consulted.

In addition to chilling the tissue, samples should also be placed in 10 per cent buffered formalin, prepared according to the following formula: Samples from dense organs (for example, liver and kidney) should be sliced approximately 0.6 cm thick. It is not necessary to slice brain, spinal cord, eye, or lung tissue. It may be necessary to cut sections of gastrointestinal tract longitudinally to facilitate formalin fixation. To facilitate fixation and better maintenance of cellular detail, the lumen of intestine, from which sections are to be selected, may be flushed (using a syringe) with formalin solution prior to cutting and placing into the fixative. Tissues become rigid after fixation. For this reason, they should be placed in wide-mouth containers to allow removal without damage. Tissue-to-formalin ratio should be about 1:10.

Swine erythrocytes are fragile and highly susceptible to hemolysis. Techniques for obtaining satisfactory blood samples from live pigs have been described previously. Large gauge (18 to 20) needles should be used. Blood should be withdrawn slowly from the animal and discharged slowly from the syringe (with the needle removed) into the collection vial.

When blood is to be placed in tubes with an anticoagulant or other type of additive, the vial should be slowly tilted back and forth (do not shake) to make sure that there is complete mixing. The dipotassium or disodium salt of ethylenediaminetetraacetic acid (EDTA) is the most satisfactory anticoagulant to use when cell morphology is to be studied. Blood films can be prepared at the time of collection, air dried, and included with other specimens to be sent to the laboratory.Cell morphology is preserved and is more satisfactory for evaluation when the film is prepared soon after the blood sample is obtained. One to 2 ml of blood is usually adequate for hematologic evaluation.

A clean glass slide is placed on a level surface.

A very small drop (use round applicator stick) of thoroughly mixed blood is placed near one end of the slide.

Another clean slide, held at an angle of about 30°, is drawn toward the drop of blood. As it makes contact, it spreads along the end of the tilted slide.

The spreader (tilted) slide is then moved smoothly away from the original drop of blood, maintaining contact with the film slide (the greater the angle, the thicker the film).

Air dry as quickly as possible (wave slide in the air).

Blood from which serum is to be separated should be placed in sterile, chemical-free vials* and allowed to stand at room temperature (about 25°C) for one to two hours to allow adequate clotting. The sample is then centrifuged and the serum is poured or aspirated into another sterile, chemically clean vial (unless separator tubes* are used). Blood should not be submitted in syringes. Serum should then be refrigerated until arrival at the laboratory. A quantity of 5 to 10 ml of blood, from which 2 to 5 ml of serum can be obtained, is recommended for routine submissions.

Body fluids and exudates (urine; cerebrospinal fluid; pleural, pericardial, and peritoneal fluids; abscess and joint exudates, and so forth) should be collected aseptically and kept refrigerated. If cell morphology of these specimens is to be evaluated, a portion may be spread in a thin layer on a glass slide and air dried. To facilitate spreading, a drop of serum may be placed over the sample and mixed to form a slurry, which is spread evenly using another slide (similar to the technique used to prepare a blood film).

Swabs may be used to transport diagnostic samples of infectious material. It is important to prevent desiccation of bacteria, however, when specimens are submitted in this manner. Specially designed swabs that utilize a transport medium for this purpose are commercially available.† Collect as much exudate as possible on the swab. Keep refrigerated. Swabs in transport media (such as buffered tryptose broth and/or cell culture medium) may also be used for submitting specimens for virologic examination.

Fecal material and gastrointestinal contents should be placed in airtight containers and kept refrigerated.

Packing Specimens for Transport to the Laboratory

Federal regulations require that material containing toxins or viable microorganisms that may cause human disease must be packaged in a manner to withstand leakage during transport. Each specimen, whether fixed or nonfixed tissue, blood, exudate, or tissue fluid, must be placed in individual leakproof containers. Plastic bags are probably most satisfactory for tissue specimens, fecal material, and so forth. Blood tubes must be padded in some manner to prevent breakage. The individual specimens are then placed on absorbent material with good insulating quality. Coolant such as bags of crushed ice, frozen gel packs, or cans of frozen water should be placed between and on top of the specimens. If dry ice is used as coolant, it must not be placed in an airtight container and must not be in contact with the specimen. The remaining space in the shipping container, on the sides and top, are filled with insulating padding and absorbent material, such as crumpled newspaper. The shipping container should be enclosed in an outer liner constructed of cardboard, corrugated fiberboard, wood, or similar material.

The written clinical history should be put in a plastic bag (to keep it dry) and placed on top of the insulating material before the container is closed and sealed with tape. Exhibit addresses and phone numbers on the exterior of the package.

The packaged specimens should then be taken directly to the laboratory. If mail, bus, or some other commercial carrier is utilized, schedules should be determined in order that the specimens spend minimal time in transit and can be received and processed immediately upon arrival at the laboratory. Inquiries concerning mailing of specimens can be directed to the postmaster.

Gastrointestinal Diseases of Swine

Laboratory diagnosis of diseases affecting the digestive tract of swine may be difficult, and it is important to submit the correct specimen (section of gastrointestinal tract) from an untreated animal. A list of gastrointestinal diseases of swine, the specimens needed for analysis, and the laboratory tests performed for their diagnosis is presented in Table 1.

Table 1

Laboratory Diagnosis of Gastrointestinal Diseases in Swine

DISEASE	SPECIMEN	DIAGNOSTIC TEST
Colibacillosis	Duodenum Colon contents, feces	Bacterial isolation pH (alkaline)
Clostridium perfringens type C enteritis	Jejunum, ileum (fixed)	Histopathology
	Jejunum, ileum (nonfixed)	Gram stain of mucosal scraping
	Contents of jejunum and ileum, peritoneal fluid (refrigerated or frozen)	Mouse neutralization test
Salmonellosis	Jejunum, ileum, colon, liver (fixed and nonfixed), feces	Histopathology, bacterial isolation
Swine dysentery	Cecum, colon (fixed)	Histopathology
	Cecum, colon (nonfixed), feces	Dark field examination, culture, Victoria blue 4-R stain of mucosal smear, immunofluorescent antibody test
Proliferative enteritis (Campylobacter sputorum infection)	Ileum, lower jejunum (fixed)	Histopathology, Warthin-Starry stain
	Ileum, lower jejunum (nonfixed)	Electron microscopy, acid fast stain of mucosal impression smears
	Serum	Microtiter agglutination
Transmissible gastroenteritis (TGE)	Jejunum (fixed)Jejunum (nonfixed)Intestinal content (large and small), fecesSerum	HistopathologySubgross examination, fluorescent antibody test, electron microscopyElectron microscopy, pH (acid)Serum neutralization test
Other enteric viruses Rotaviruses Coronaviruses (Not TGE) Caliciviruses Adenoviruses Astroviruses	Intestinal content, feces, small intestine (nonfixed)Small intestine (fixed)	Electron microscopy, fluorescent antibody testHistopathology
Coccidiosis	Jejunum (fixed)Jejunum (nonfixed)	HistopathologyDirect microscopic examination of fresh scrapings, Wright’s stain
Cryptosporidiosis	Colon (fixed, nonfixed) Feces	Histopathology, electron microscopySmear, flotation
Stomach worms (Ascrops, Hyostrongylus, Physocephalus spp.)	Stomach	Necropsy examination
Ascaris suum	Small intestine, liver, lung Feces	Necropsy, histopathologyOva detection (flotation, smear)
Strongyloidiases	Small intestine (nonfixed)Small intestine (fixed) Feces	Microscopic exam of mucosal scrapingHistopathologyOva detection (flotation, smear)
Macracanthorhynchus hirudiaceus	Small intestine	Necropsy examination
Trichuris suis	Cecum, colonFeces	Necropsy examinationOva detection (flotation, smear)
Oesophagostomum	Colon	Necropsy examination
Gastric ulcers	Stomach (intact)Feces	Gross and histopathologyOccult blood
Intestinal hemorrhage syndrome	Small intestine, mesenteric lymph nodes	Gross necropsy examination

Respiratory Diseases of Swine

Respiratory disease in swine usually results from multiple, interrelated factors, including environmental deficiencies, as well as from infection. The infectious etiologies can often be diagnosed in the laboratory using microbiologic and histopathologic techniques and electron microscopy. Serology may be employed for the diagnosis of several of these diseases but is most applicable when used on a herd basis. It may be used for screening herd additions as an aid in preventing introduction of certain diseases. The incidence of certain respiratory diseases within a herd may best be evaluated by periodic slaughter examinations of a number of market animals. Pneumonia may also be one manifestation of a systemic disease (such as salmonellosis and pseudorabies). Therefore, other representative samples as well as tissues from the respiratory tract may be submitted ( Table 2).

Table 2

Laboratory Diagnosis of Respiratory Diseases in Swine

		PATHOLOGY		MICROBIOLOGY				SEROLOGY*
DISEASE	SPECIMENS	Gross	Histopath.	Culture	FA	EM	Virus iso.	SN	CF	HI	IFA	SF Dye	ELISA
Rhinitis
Atrophic rhinitis	Snout (head), refrig., nasal swab	X	X	X
Inclusion body rhinitis	Snout, head, refrig.	X	X
Pseudorabies	Snout, nasal swab						X	X
Pneumonia
Mycoplasma hyopneumoniae	Lung (refrig., fixed), serum	X	X	X	X				X	X			X
Bacterial	Lung (refrigerated, fixed), lymph nodes, pleural exudate (refrigerated)
Pasteurella multocida		X	X	X
Pasteurella hemolytica		X	X	X
Bordetella bronchiseptica		X	X	X
Corynebacterium pyogenes		X	X	X
Klebsiella pneumoniae		X	X	X
Streptococcus sp.		X	X	X
Hemophilus pleuropneumoniae		X	X	X					X
Viral
Influenza	Lung, lymph nodes, serum	X	X				X	X		X
Adenovirus	Lung, lymph nodes, serum		X		X	X	X	X
Transmissible gastroenteritis	Lung, lymph nodes, serum				X	X	X	X
Pseudorabies	Lung, lymph nodes, serum, tonsils, head (brain)		X		X	X	X	X
Toxoplasmosis Verminous	Same as above		X						X	X	X	X
Ascaris sp.	Fixed; nonfixed, refrig.	X	X
Metastrongylus sp.	Fixed; nonfixed, refrig.	X	X
Inhalation, environmental	Lung (fixed)		X

FA = fluorescent antibody

EM = electron microscopy

SN = serum neutralization

CF = complement fixation

HI = hemagglutination inhibition

IFA = indirect fluorescent antibody (not performed routinely in most laboratories)

SF dye = Sabin-Feldman dye test

Systemic Diseases of Swine

It is best to submit a variety of samples when a systemic disease is suspected. A list of systemic diseases of swine and specimens needed for laboratory diagnosis is presented in Table 3.

Table 3

Laboratory Diagnosis of Systemic Infections of Swine

DISEASE	SPECIMENS REQUIRED
Bacterial diseases	Head (snout, ears, tonsils, brain, lymph nodes)
Glässer’s disease (Hemophilus parasuis)	Major organs (heart, lung, liver, spleen, kidney)
Erysipelas
Streptococcosis	Lymph nodes
Actinobacillus equuli infection	Joint (unopened), joint exudate, swab
Anthrax	Body fluids (cerebrospinal, pleural, pericardial, ascitic, urine, ocular)
Tuberculosis
Clostridial diseases*	Spinal cord, peripheral nerves
Salmonellosis
Viral diseases†
Pseudorabies
Hemagglutinating encephalomyelitis virus infection‡
Polioencephaloinyelitis
Encephaloinyocarditis
Mycoplasmal polyserositis (M. hyorhinis)
Protozoan
Eperythrozoonosis§
Toxoplasmosis‖

Also submit muscle tissue and fascia.

Refer also to Table 11.

Also submit stomach, intestine, intestinal contents, and feces.

Also submit serum and EDTA blood from adult animals; EDTA blood and blood films from baby pigs.

Also submit serum.

Table 11

Laboratory Diagnosis of Foreign Diseases of Swine

DISEASE	CLINICAL SIGNS	LESIONS	SPECIMENS REQUIRED	LABORATORY TESTS
Hog cholera	Lassitude; anorexia, fever (104–108°F); leukopenia (3000–9000/cmm); thrombocytopenia (20,000–50,000/cmm); purulent conjunctivitis; constipation followed by diarrhea; dyspnea; vomiting; piling; ataxia; posterior paresis; erythema of skin; partial alopecia (chronic form); convulsions; small litters; mummification; abortion; stillbirth; fetal malformations; weak neonatal pigs; congenital tremors; lack of response to medication	Subcutaneous hemorrhage; hemorrhagic lymph nodes; petechiae of epiglottis, kidneys, urinary bladder serosal hemorrhages; button ulcers (colon); infarcts of spleen; epiphyses of ribs widened, irregular	EDTA bloodTonsil, lymph nodes, spleen (nonfixed, refrigerated)Brain, organ tissues, lymph nodes, GI tract (fixed)	Leukocyte and thrombocyte countsFA testHistopathology
African swine fever	Fever (105°F); anorexia; depression, piling; weakness; reluctance to move; coughing; hyperpnea; dyspnea; exudative conjunctivitis; nasal discharge; diarrhea (sometimes bloody); vomiting; leukopenia; abortion	Cyanosis; hemorrhages of skin; pleural and pericardial effusion; hemorrhages of lymph nodes, serosal membranes, epiglottis, heart, gallbladder, kidneys; splenomegaly; edema of lungs, gallbladder; gastritis (hemorrhagic, sometimes ulcerative)	Heparinized bloodEDTA bloodLymph nodes, tonsil, spleen, liver, large intestine, brain (fixed and nonfixed, refrigerated)Serum	Hemabsorption testLeukocyte countHistopathology, animal inoculation, FA testCF test
Japanese encephalitis	Stillbirth; mummified fetuses; tremors, convulsions, death in neonates	Fetal mummification, malformations	SerumBrain (fetus, neonate)	SN, CF, HIVirus isolation and identification
Teschen, talfan, benign enzootic paresis	Fever (104–106°F); incoordination; ataxia; lassitude; anorexia; irritability, stiffness; falling, tripping; muscle rigidity; opisthotonus; nystagmus; convulsions; smacking lips; grinding teeth; squealing; prostration; coma (course, 3–4 days)	Nonsupportive encephalomyelitis (microscopic lesions only)	Brain, spinal cord (fixed and nonfixed, refrigerated)	Histopathology, virus isolation, and identification
Foot and mouth disease	Pyrexia; lassitude; anorexia; lameness; dehydration; weight loss; abortion, may be subclinical; mortality, from 5 (usually) to 50%	Vesicles and erosions (lips, tongue, gums, pharynx, palate, snout, coronary bands, interdigital skin, skin of metacarpus and metatarsus, teats, vulva, scrotum); myocardial necrosis	SerumVesicle fluid, skin lesions (in buffered glycerin or frozen)Heart	CF test, agar gel diffusion, SN testVirus isolation and identificationHistopathology
Swine vesicular disease	Lameness; reluctance to rise; pyrexia (104–105°F); abortion; may be subclinical; morbidity up to 65–90%; recovery in 2–3 weeks	Vesicles and erosions (buccal mucosa, tongue, snout, coronary bands, interdigital spaces, skin of metacarpus and metatarsus, tests); sloughing of sole; nonsuppurative encephalomyelitis	SerumSkin lesionsBrain, spinal cord	CF, SN testVirus isolation and identificationHistopathology
Vesicular exanthema	Pyrexia; anorexia; restlessness; drooling; lameness; diarrhea; increased abortion rate in herd; hypogalactia; recovery	Vesicles and erosions (lips, tongue, oral mucosa, snout, coronary bands, interdigital spaces, sole, skin of metacarpus and metatarsus)	SerumSkin lesions (nonfixed)	CF, SN testsVirus isolation and identification

FA = fluorescent antibody

CF = complement fixation

SN = serum neutralization

HI = hemagglutination inhibition

Arthritis and Lameness in Swine

A list of causes of arthritis and lameness in swine is presented in Table 4. Arthritis is often a manifestation of a systemic disease and may in certain stages be predominant. Vesicular diseases, which may cause lameness, are listed in Table 4, Table 11.

Table 4

Laboratory Diagnosis of Lameness and Arthritis in Swine

DISEASE	SPECIMENS REQUIRED
Bacterial diseases	Joint (unopened, refrigerated), joint fluid, exudate, (refrigerated), tonsil (refrigerated), spinal column
Erysipelas
Hemophilus suis and H. parasuis infection
Streptococcosis
Corynebacterium infection
Brucella suis infection
Staphylococcus aureus infection
Mycoplasmal diseases	Joint (unopened, refrigerated), joint fluid, exudate, (refrigerated), tonsil (refrigerated)
M. hyosynoviae infection
M. hyorhinis infection
Viral diseases*—Vesicular stomatitis	Serum, skin lesions (nonfixed)
Noninfectious diseases	Joint, compact cortical bone (1-inch section from mid-shaft of femur for bone ash determination), feed
Osteochondrosis
Nutritional (calcium, phosphorus, vitamin D)
Biotin deficiency

Refer also to Table 1.

Swine reproductive Failure

Reproductive failure is one of the common reasons for requiring the assistance of a diagnostic laboratory. However, specific diagnoses are elusive and these cases often present the greatest challenge. Definitive diagnosis is made in only 25 to 35 per cent of the cases submitted to most diagnostic laboratories.8., 16. Diagnosis is often limited by the availability of samples submitted for analysis. Many causes of reproductive failure are related to management deficiencies, for which there are no laboratory tests, and their detection depends on the clinical diagnostic acuity of the veterinary practitioner. The number of available laboratory tests is limited, and most of the tests are geared toward detecting infectious organisms. Repeated sampling may be warranted if results of initial laboratory tests are negative. A few of the more obscure causes of reproductive failure are surfacing as more sophisticated analytical equipment becomes available and as new testing procedures are developed. Common causes of swine reproductive failure are listed in Table 5; specimens needed and methods of laboratory analysis are listed in Table 6.

Table 5

Causes of Reproductive Failure in Swine

VIRAL DISEASES*	FUNGI—Aspergillus sp.
Parvovirus
Picornaviruses (SMEDI; enteroviruses)	MYCOPLASMA SP.
Reoviruses
Pseudorabies	TOXINS
Coronaviruses	Mycotoxins
Influenza	Zearalenone
Infectious bovine rhinotracheitis	Ergot
	Bacterial endotoxins
BACTERIAL DISEASES	Anticoagulants
Brucella suis	Nitrate/nitrite
Leptospira sp.	Plant toxins
E. coli	Datura sp.
Streptococcus sp.	Nicotinia sp.
Staphylococcus aureus	Carbon monoxide
Staphylococcus hyicus	Penicillin
Mycobacterium tuberculosis	Diethylstilbestrol
Pasteurella multocida	Cresol
Erysipelothrix insidiosa	Vitamin D toxicity
Corynebacterium pyogenes
Pseudomonas aeruginosa	NUTRITIONAL DEFICIENCIES
Salmonella sp.	Vitamin A, B complex, E, K
Hemophilus pleuropneumoniae	Choline
Campylobacter fetus var. intestinalis	Protein
Nocardia asteroides	Iodine
Klebsiella pneumoniae	Calcium
Actinobacillus equuli	Iron
	Manganese
PROTOZOAN DISEASES
Toxoplasma gondii	ENDOCRINE FACTORS
Eperythrozoon suis
	ENVIRONMENTAL FACTORS
CHROMOSOME DEFECTS

Refer also to Table 11.

Table 6

Specimens Needed and Methods of Laboratory Analysis for Diagnosis of Reproductive Failure in Swine

SPECIMENS	LABORATORY TEST	REPRODUCTIVE DISEASE
Fetal tissue, placenta	Histopathology, culture	Bacterial, mycotic infections
	Histopathology, virus isolation, electron microscopy	Viral infections
	Histopathology, mouse inoculation	Toxoplasmosis
Fetal stomach content	Culture, virus isolation	Bacterial, viral, mycotic infections
	Dark field examination	Leptospirosis
Mummified fetus	Fluorescent antibody, electron microscopy	Parvovirus infection
Serum (dam)	Serology	Brucellosis, leptospirosis, pseudorabies, influenza, eperythrozoonosis, toxoplasmosis
Urine (dam)	CultureDark field examination, animal inoculation	Bacteriuria (infertility)Leptospirosis
Serum, plasma	Radioimmunoassay(progesterone)	Infertility, pregnancy diagnosis
EDTA blood	Hemoglobin, cytology	Eperythrozoonosis, anemia
Heparinized blood	Chromosome analysis	Reproductive failure
Feed	Chemical analysis, biologic assay	Nutritional deficiencies, toxicoses

Viral Reproductive Failure

Porcine parvovirus (PPV) is currently considered to be the most prevalent cause of infectious reproductive failure in the United States. Porcine parvovirus is stable and persists in mummified fetal tissue, where it can be detected by electron microscopy and/or immunofluorescence microscopy. Electron microscopy, immunofluorescence microscopy, and virus isolation procedures are usually performed on fetal tissues for the detection of other viruses listed. Unfortunately, infection by many of these viruses is transient and they may no longer be detectable in the tissues of fetuses and stillborn pigs submitted for examination.

The diagnostic value of viral serology, especially for PPV and picornaviruses (SMEDI), is limited to testing paired samples from sows to demonstrate a rise in titer. It would be necessary to collect serum from sows and gilts at breeding time and again following clinical evidence of reproductive failure. These procedures are probably impractical in most swine operations. Results of serologic tests on pericardial fluid of fetal piglets may be of diagnostic value. Serologic tests for detection of picornaviruses are not performed routinely in most diagnostic laboratories because they are quite costly owing to the large number of serotypes involved.

Bacterial Reproductive Failure

Various bacteria (see Table 5) have been implicated in reproductive diseases of swine. Correlation of bacterial isolations with histologic lesions in fetal tissues, placenta, and so forth adds credence to the diagnosis. Bacteriologic examination of vaginal exudates, and swabs thereof, is not considered worthwhile because of the presence of contaminants and normal flora from the lower reproductive tract.

Bacteriuria in sows has been correlated with reduced litter size, prolonged interval between farrowings, and lower fertility rate. Midstream urine samples can be collected in a sterile plastic bag fixed in a container fitted with a long handle. The bag should have a cover that is kept closed, except when collecting the sample. Slides with cystine lactose electrolyte deficient (CLED) culture medium may also be dipped in the urine aseptically. The number of colonies on these slides after 24 hours’ incubation at 37°C gives an impression of the real numbers of bacteria in the urine.

Bacterial endotoxins may play a major role in swine reproductive failure. Abortion has been induced experimentally in pregnant swine injected with crude E. coli endotoxin. Diagnosis or confirmation of spontaneous endotoxic abortion, however, is difficult because reliable tests for the detection of endotoxins in blood or tissues are not currently available for use in swine.

Miscellaneous Infectious Causes of Swine Reproductive Failure

Species of Toxoplasma, Mycoplasma, Aspergillus, and Eperythrozoon have been listed as infectious causes of porcine reproductive disease, but their incidence seems to be low. Perhaps more effort should be directed toward their detection.

Mycotoxin-Induced Reproductive Failure in Swine

Reproductive disorders including infertility, constant estrus, pseudopregnancy, reduced litter size, small offspring, malformation, juvenile hyperestrogenism, and probably fetal resorption as well as “splayleg” syndrome in newborn pigs have been attributed to ingestion by pregnant sows of feed containing grain contaminated with fusarium (F2) toxin (zearalenone). Lesions (squamous metaplasia in the uterus, uterine ducts, cervix, vagina, and mammary glands) attributed to activity of fusarium toxins were observed grossly and histologically. Hypogalactia, resulting in neonatal death, and abortion have been attributed to ergot toxicity.

Diagnosis of mycotoxicosis by means other than signs and lesions is often difficult. Contaminated feedstuffs that contained the mycotoxin may have been completely consumed prior to manifestations of signs of toxicity and would no longer be available for analysis. Methods for detecting mycotoxins in tissues and body fluids are not in routine use. (Refer to the section on laboratory diagnosis of toxicosis in swine for information on sampling feedstuffs for mycotoxin analysis.)

Nutritional and Toxic Causes of Swine Reproductive Failure

Deficiencies of various nutrients (see Table 5) have been associated with subestrus, anestrus, abortion, fetal malformations and stillbirths, neonatal death, splayleg syndrome and poor survivability in newborn pigs. Hemoglobinemia (less than 9 gm per 100 ml) has been associated with stillbirth.

Certain toxins (such as cresol, dicumerol compounds, and nitrate) have caused abortion in sows. Plant toxins such as tobacco and jimsonweed have caused teratogenic effects in porcine fetuses. Laboratory confirmation may be difficult in these cases, but submission of feed, water, fetuses, placenta and EDTA blood for analysis may be helpful.

Progesterone Determination in Pregnancy and Fertility Diagnosis

Serum or plasma progesterone radioimmunoassay may be used in determining pregnancy status in sows. When serum samples are collected 17 to 24 days after mating, progesterone levels greater than 9 ng per ml indicate pregnancy; levels less than 9 ng per ml indicate nonpregnancy in samples collected during this period. There is also evidence suggesting that fertility status can be evaluated when progesterone levels are determined in serum collected from sows 10 to 15 days after mating.

Chromosome Analysis

An increasing number of reports of reproductive failure in swine—including embryonic death, developmental abnormalities in fetuses, stillbirths, neonatal death, and reduced numbers of pigs born per litter—are being correlated with translocation-type chromosomal defects.1., 9., 13., 20. Chromosomal changes may be found in the sire and dam as well as in the offspring.

Consultation with laboratory personnel where the analyses are to be performed is recommended prior to collecting samples for submission. Ten ml of blood must be collected aseptically with 10 IU of heparin. Samples should be kept at room temperature, about 25° C; they should be protected from freezing as well as overheating during transit. The samples should be processed within 24 hours but may be usable for up to 48 hours after collection if handled properly.

Diagnosis of Boar Problems in Swine Reproductive Failure

Many boar problems relating to herd infertility must be evaluated clinically. Semen evaluation for motility, malformations of sperm, and concentration should probably be done as soon as possible after collection of the sample. The diagnostic laboratory may be of assistance only if it is in close proximity and the samples can be processed within a short time.

Reduced conception rate and litter size have been correlated with certain infections of the male genital tract. Semen may be submitted for microbiologic and electron microscopic examinations. Several infectious agents (Brucella sp., Staphylococcus aureus, E. coli, Streptococcus sp., Corynebacterium suis, picornaviruses, and parvoviruses) have been isolated from semen and the genital tracts of boars.

Urinary Diseases of Swine

Primary disease of the urinary system in swine is not common. Corynebacterium suis causes ascending infection in sows and gilts, resulting in cystitis, ureteritis, nephritis, and subsequent toxemia and death a few weeks after breeding. Asymptomatic boars harboring the organism are thought to be instrumental in mechanical transmission. Diagnosis is based on the presence of gross and microscopic lesions in the urinary bladder, ureters, and kidneys and isolation of the bacterium from these tissues and urine. Urine or swabs from the sheath of suspected boars may also be submittted for culture.

Kidney worm disease (Stephanurus dentatus) may be diagnosed by gross examination of adult worms in the renal pelvices and perirenal fat or of damage by larval migration in the liver and peritoneal cavity and the detection of ova in the urine.

Skin Diseases in Swine

Skin biopsies (both fixed and nonfixed) and skin scrapings for histopathologic, electron microscopic, and microbiologic examination may be submitted routinely for the diagnosis of dermatologic problems ( Table 7, Table 11). Biopsies that include the full thickness of the affected skin are the most desirable specimens. They should be taken at the margin of circumscribed lesions and should include a portion of the normal tissue. Serum and feed may be submitted when nutritional deficiencies are suspected. Serologic tests and virologic examination of vesicle fluid should always be performed to differentiate the vesicular diseases.

Table 7

Laboratory Diagnosis of Skin Diseases of Swine

DISEASE	SPECIMENS REQUIRED
External parasitism	Skin biopsy (fixed, nonfixed), Skin scrapings (include exudate from external ear)
Lice (Hematopinus suis)
Sarcoptic mange
Demodectic mange
Bacterial diseases	Skin biopsies (fixed and nonfixed, refrigerated); swabs of exudate
Subcutaneous abscesses
Erysipelas
Exudative epidermitis (greasy pig disease)
Dermatophilus congolensis
Viral diseases*
Swine pox	Skin biopsy (fixed and nonfixed, refrigerated)
Vesicular stomatitis	Serum, skin lesion (nonfixed)
Noninfectious dermatoses	Skin biopsy (fixed)
Parakeratosis	Serum
Photosensitization	Feed
Sunburn
Pityriasis rosea
Biotin deficiency
Neoplasms	Skin biopsy (fixed)

Refer also to Table 11.

Nutritional and Metabolic Diseases of Swine

Most swine rations are composed of grain and protein supplements fortified with vitamins and minerals supplying all the nutritional requirements. The occurrence of clinical disease due to a single nutrient deficiency is rare. Problems often arise from mistakes in mixing feed, when multiple deficiencies develop simultaneously. Nutritional disease may result from the interaction of certain nutrients. Excess calcium in the ration interferes with utilization of zinc. Gossypol toxicity is less likely if ferrous sulfate is added to the ration. Salt toxicity is prevented if there is adequate drinking water. The protein avidin in egg albumin inactivates biotin, causing dermatitis and lameness. Vitamin E may be utilized rapidly and rendered unavailable if there is excessive oxidized fat in the ration. The specimens needed for laboratory investigation of certain nutritional and metabolic diseases are presented in Table 8.

Table 8

Laboratory Diagnosis of Nutritional and Metabolic Diseases of Swine

DISEASE	SPECIMENS REQUIRED
Vitamin A deficiency	Serum, liver (frozen)
Vitamin B6, B12, riboflavin, pantothenic acid, thiamine, niacin	Feed
Vitamin D deficiency, calcium-phosphorous imbalance	Cortical bone (1-inch section from mid-shaft of femur); feed; serum
Vitamin E/selenium-responsive disease	Heart, liver, skeletal muscle (fixed); feed
Postpartum hypocalcemia	Serum (prior to calcium therapy)
Iron-deficiency anemia	EDTA blood, serum
Parakeratosis	Serum, feed, skin biopsy (fixed)
Protein, amino acid deficiency	Serum, feed
Copper deficiency	Liver
Choline, biotin, magnesium, potassium, sodium, iodine, manganese deficiencies	Feed

Toxicosis in Swine

A list of toxins that may affect swine, along with specimens needed for laboratory diagnosis, is presented in Table 9. Mycotoxins causing disease in swine are listed in Table 10. The diagnosis of toxicity in swine is usually based upon the demonstration of chemical toxins in tissues of affected animals as well as ingested feedstuff’s, water, and so forth. Contaminated feed may be completely consumed and unavailable for analysis when an outbreak of poisoning occurs. This may also occur in cases of chronic toxicosis (such as ingestion of mycotoxins and anticoagulant compounds for long periods) and nutrition-related problems resulting from mistakes in mixing and the like. To insure the availability of proper specimens, routine periodic collection and storage of feed samples and/or individual ingredients of mixed feeds for possible future analysis are warranted. The following procedure for obtaining feed samples for mycotoxin analysis has been suggested. Sampling is most effective if small samples are taken at intervals from a moving stream of grain. If possible, the recommended quantity for a composite sample from which such sampling is to be performed should be 10 lb (4.54 kg). Probe sampling is most effective when performed soon after grain has been blended. Samples should be dry for transport and storage (dried at 80 to 90°C for about 30 hours as soon as possible after collection). If fungal culture is to be performed, feed samples should be dried at 60°C for 6 to 12 hours. Dried samples should be placed in cloth or paper bags. Plastic bags may be used for high moisture samples only if they are to be preserved by refrigeration or freezing.

Table 9

Laboratory Diagnosis of Toxicoses of Swine

TOXIN	SPECIMENS REQUIRED	AMOUNT	COMMENTS
Sodium	Brain (cerebrum)SerumFeedSpinal fluid	5 gm2 ml25 gm1 ml	Fixed; nonfixed, refrig.
Iron	Kidney, liverWhole bloodSerumFeces	10 ml5 ml100 gm	Fixed
Copper	Kidney, liver	50 gm (ea.)	Nonfixed, refrig.
Anticoagulants (e.g., Warfarin)	FeedLiver	100 gm100 gm	Fixed; nonfixed, refrig.
Vitamin D	Necropsy tissuesFeedFeed	Complete set100 gm5 kg	FixedFor chemical analysis Rat feeding tests
Hygromycin B	Eyes (lens)		Fixed
Rumensin	HeartLiverStomach contentFeed		FixedNonfixed, refrigerated
Phenylarsonic compounds (arsanilic acid, 3-nitro, 4-nitro, p-ureidobenzine-arsonic acid)	Peripheral and cranial nervesLiver, kidney	100 gm (ea.)	FixedNonfixed, refrigerated (tissue levels may not be diagnostic)
Strychnine	Stomach content Liver, kidney Urine	500 gm 50 gm (ea.) 10 ml	Nonfixed, refrigerated
Mercury	Liver, kidney	50 gm (ea.)	Nonfixed, refrigerated
Ammonia	Whole blood, serumUrine	5 ml5 ml	Frozen
Sulfates (water quality)	Drinking water	500 ml	Chemical-free container
Chlorinated hydrocarbons, Pentachlorophenol, Polychlorinated biphenyls	Brain		Nonfixed, refrigerated
	Stomach contentsLiver, kidneyBloodFeed	100 gm500 gm10 ml250 gm	Nonfixed, refrigerated
Organophosphates; carbamates	FeedStomach contentsLiverBloodBrain	100 gm50 gm500 gm10 ml
	Peripheral nerves		Nonfixed, refrigerated Fixed (haloxon)
Coal tar/phenols	Liver		Fixed
Sodium fluoroacetate	Stomach contents	500 gm	All available; frozen
Amaranthus retroflexus (pigweed)	Kidney		Fixed
Nicotinia sp. (tobacco)	Fetal teratology		Gross examination
Crotolaria sp.	LiverFeed		FixedGross and microscopic exam for seeds
Gossypol	Heart, liverFeed		Fixed
Datura sp. (jimsonweed)	Stomach content; urine		Alkaloid assay + gross examination for plant material
Cocklebur	Stomach contents		Gross examination for plant material
Arsenic	Liver	50 gm	Refrigerated
	Kidney	50 gm	Refrigerated
Lead	Liver	50 gm	Refrigerated
	Kidney	50 gm	Refrigerated
	Blood	20 ml	Clotted or heparinized (consult with laboratory personnel)
	Feces	50 gm	Refrigerated
Nitrite	FeedWaterFertilizerBody fluids	500 gm1000 ml500 gm50 ml

Table 10

Mycotoxins Causing Swine Disease

MYCOTOXIN	FUNGUS	SUBSTRATE	DISEASE CONDITION
Aflatoxins	Aspergillus flavus	Cottonseed, corn, peanuts, sorghum	Hepatic damage, hemorrhages, retarded growth, neoplasia
Citrinin	Penicilliumviridicatum	Barley, corn, feed	Nephrosis, perirenal edema, hepatic damage
Emetic factor	Fusarium sp.	Corn, wheat	Emesis
Ergot	Claviceps purpurea	Cereal grains	Hypogalactia, gangrene, CNS signs, reproductive failure
Ochratoxin A	Aspergillusochraceus	Corn, barley, legumes	Nephrosis
Oxalic acid	Aspergillus niger	Hay, cereal grain	Nephritis, clotting defect
Rubratoxin	Penicillium rubrum	Corn	Hepatic damage, hemorrhages
Trichothecene (T-2 Toxin)	Fusarium tricinctum	Corn, hay	Gastroenteritis, hemorrhages, hematopoietic depression
Tremorigen Penitrem A	Penicillium cyclopium and P. palitans	Corn, feed	Tremors, convulsions, ataxia
Zearalenone (F-2 Toxin)	Fusarium graminearum	Corn	Vulvar swelling, prolonged estrus, mammary enlargement, preputial swelling, abortion

Foreign Diseases of Swine

Several diseases do not currently affect swine in the United States (Table 11). These diseases are difficult to differentiate clinically and pathologically and require laboratory assistance for their diagnosis.

Equipment	Supplies
Knife	Swabs (aerobic and anaerobic)
Steel	Blood vials (no additives; EDTA)
Stone	Plastic bags
Forceps	Sterile syringes
Scissors	Sterile needles
Saw	Wide-mouth containers with 10% formalin
Cleaver	pH paper
Gloves	Shipping container
Coveralls	Refrigerant
Boots	Marking pen or pencil; note pad/submission forms
Pail; brush	Disinfectant

37 to 40% formaldehyde solution	100.0 ml
Distilled water	900.0 ml
Sodium phosphate, monobasic (NaHPO4)	4.0 gm
Sodium phosphate, dibasic (Na2HPO4)	6.5 gm