Diagnosis of Enteric Disease in Small Ruminants

DIAGNOSIS OF DISEASES OF THE DIGESTIVE TRACT

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DIAGNOSIS OF ENTERIC DISEASE IN SMALL RUMINANTS David C. Van Metre, DVM, Jeff W. Tyler, DVM, MPVM, PhD, and Susan M. Stehman, MS, VMD

As with other species, accurate diagnosis of gastrointestinal disease in small ruminants requires integration of information obtained in the signalment, history, physical or necropsy examination, and ancillary laboratory tests. In some commercial flocks and herds, economic constraints may limit owner investment in ancillary testing, and the veterinarian must make a diagnosis with information obtained from the signalment, history and physical or gross necropsy examination. This article provides the practitioner with a review of the clinical signs, gross necropsy and histologic findings, and ancillary diagnostic tests of common gastrointestinal diseases of sheep and goats. Where appropriate, reviews of the pathophysiology, prevention, and control of these diseases are cited for further reading. RUMEN ACIDOSIS

Rumen acidosis (rumen lactic acidosis, grain overload, rumenitis) is a metabolic disorder that occurs after the ingestion of certain carbohydrates and their subsequent rapid ruminal fermentation. 56, 102 Although not technically an enteric disease, ruminal acidosis is a common disorder of small ruminants that can cause clinical signs similar to enteritis.

From the Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado (DCVM); the Department of Veterinary Medicine and Surgery, University of Missouri College of Veterinary Medicine, Columbia, Missouri (JWT); and the Diagnostic Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, New York (SMS)

VETERINARY CLINICS OF NORTH AMERICA: FOOD ANIMAL PRACTICE VOLUME 16 • NUMBER 1 • MARCH 2000

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Sheep and goats are prone to rumen acidosis when readily fermentable concentrates are overfed, errors in feed preparation or feedbunk management occur, or when animals are given concentrates after periods of feed deprivation. In flocks or herds fed high concentrate rations, a history of reduced or variable feed intake, diarrhea, bloat, and in dairy animals, milkfat depression may occur in instances where subclinical or mild rumen acidosis is an ongoing problem. Although considered a common sequel to rumen acidosis in cattle71 and sheep,57 laminitis is not a frequent complication of rumen acidosis in goats. 65 Rumen acidosis may also occur when groups of animals are mismatched for age or size. In such instances, timid animals are frequently denied free access to concentrates and more aggressive animals may overfeed. Clinical Signs and Necropsy Findings

In experimentally induced rumen acidosis in small ruminants, lethargy, anorexia, and reduced or absent rumen motility were observed within 12 hours after ingestion of concentrate.59, 72 Bruxism, tachycardia, and signs of mild colic, such as groaning and flank watching, are common early signs. ll, 14, 59, 72, 90 Diarrhea may be evident within 12 hours after ingestion of concentrate.72 In a recent study of rumen acidosis in 37 small ruminants, hypomotile or absent ruminal motility, soft or watery stool, and tachycardia were evident on initial examination of more than half of the animals.u Affected sheep and goats may display nasal discharge because they fail to clean the nose when depressed. ll, 59, 72, 103 Succussion of the abdomen may reveal fluid sounds on both sides of the abdomen. 83 Head pressing, ataxia, hyperpnea, recumbency, dehydration, scleral injection, and muscle twitching have also been observed.ll, 59, 72, 103 Urine pH may decrease to 5 or 6.72,103 Progression to shock and coma may occur in severe cases, with death occurring within hours.ll, 103 Differential diagnoses for signs of colic in small ruminants include rumen acidosis, enterotoxemia, intestinal obstruction, nematodiasis due to Oesophagostomum sp., and in males, urolithiasis. Subacute or chronic enterotoxemia, nematodiasis, coccidiosis, cryptosporidiosis, rotavirus, enterotoxigenic Escherichia coli (ETEC) infection (in neonates), dietary diarrhea, salmonellosis, and Johne' s disease are common diseases to consider in a small ruminant with diarrhea. At necropsy, sunken eyes may be apparent, and the rumen is distended and filled with the offending feedstuff and variable amounts of fluid. 56 Fluid contents also may be present within the small and large intestine. Diffuse or patchy hyperemia of the rumen wall may be present. The rumen papillae may be swollen, dark, and matted together. 3 Papillae may slough readily from the rumen wall, although this can occur with postmortem autolysis. Chronic cases may show patches of flat, white ruminal mucosa devoid of papillae, and multiple abscesses or foci of necrosis may be evident in the liver.3 Because ruminal fermentation continues after death of the animal, the pH of rumen contents declines

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postmortem at a rate dependent on the concentrate level of the diet and the level of feed intake prior to death. 21 Thus, rumen pH readings obtained postmortem should be interpreted in light of the prior ration, with little credence given to readings obtained several hours after death. 21 Ancillary Diagnostic Tests

Collection of rumen fluid in the live animal can be performed by ruminocentesis or by aspiration of rumen contents into a weighted stomach tube. Rumen fluid pH is influenced by the composition of the ration, the quantity ingested, and the time elapsed since ingestion. 2, 21, 23, 59, 70 A pH measurement of 5.5 or less is strongly suggestive of rumen acidosis,14, 23, 70 although healthy goats accustomed to a high grain ration may have rumen pH measurements approaching 5.0. 65 Protozoa are few or absent on microscopic examination, and gram-positive bacteria predominate.11, 103 Blood gas analysis of affected small ruminants typically reveals metabolic acidosis with various degrees of respiratory compensation. 11, 85 Elevation of the serum activity of hepatic enzymes may be present and is considered indicative of hepatocellular damage from acidosis, shock, or portal bacteremia and toxemia.59, 103 Blood electrolyte concentrations, hematocrit, and white blood cell count of affected small ruminants are variable.11, 59, 72, 90

ENTEROTOXEMIA

Enterotoxemia results from replication of and exotoxin production by Clostridium perfringens in the lumen of the gastrointestinal tract. Local and systemic disease results from the effects of potent exotoxins produced by certain types of this bacteria. The C. perfringens species are divided into five separate types (A-E) on the basis of the major lethal toxins that they produce. 9, 67, 91, 92 Although the lethal toxins are considered to be of primary importance in the pathogenesis of enterotoxemia, minor toxins such as enterotoxin may contribute to disease. 9, 67, 91 In North America, enterotoxemia of small ruminants is caused by C. perfringens types A, C, and D.6,9, 67,91,92 The disease(s) caused by each type of C. perfringens is designated as the corresponding type of enterotoxemia. Clinical Signs and Necropsy Findings Type A Enterotoxemia (Yellow Lamb Disease)

Enterotoxemia caused by C. perfringens type A manifests as an acute hemolytic disease of young lambs. 61,92 Alpha toxin is generated by

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C. perfringens type A in the gut of affected lambs. 61 Alpha toxin is an exotoxin that hydrolyzes phospholipids in cell membranes. 9, 67, 91 Once absorbed into the bloodstream, alpha toxin causes rapid, usually fatal hemolysis. 6, 61, 91, 92 Alpha toxin also causes endothelial cell damage and platelet aggregation, which may contribute to shock and organ failure. 97 This form of enterotoxemia is seen primarily in the western United States in the springtime. 61,92 Affected lambs are depressed, weak, and tachypneic. Red-tinged urine may be evident, attributable to hemoglobinuria from intravascular hemolysis. Icterus, also a result of hemolysis, is a prominent sign, hence the common name, yellow lamb disease. Diarrhea is not commonly present. Most affected lambs die within 12 hours of the onset of clinical signs. 92 Copper poisoning, leptospirosis, bacillary hemoglobinuria, onion and Brassica spp. poisoning, and hepatobiliary disease are different diagnoses to consider for icteric lambs. At necropsy, generalized icterus is evident. The liver of affected animals is pale, yellow-tinged, enlarged, and friable. The spleen may be enlarged and edematous. Dark red-brown, enlarged kidneys may be seen, with soft cortices and grossly visible infarcts evident in some cases. 61 Direct smears of the intestinal lining may reveal large numbers of gram-positive rods. Because C. perfringens type A inhabits the intestine of normal animals and proliferates rapidly in the gut postmortem, its isolation should be considered significant only from a fresh cadaver with compatible history and clinical signs (see discussion of ancillary diagnostic tests). 67 Type C Enterotoxemia

Enteric superinfection with C. perfringens type C can result in hemorrhagic enteritis in neonatal lambs. 6, 44, 67--69, 90, 91 Type C enterotoxemia has been suspected to occur in goat kids. 45, 90 A few reports exist incriminating type C in enterotoxemia of adult small ruminants, but disease in adults appears to be quite rare. 69,92 Affected lambs are usually less than 10 days of age. 67 Dietary changes or early colonization of the neonatal intestine are considered to be potential etiologic factors in this disease. 91 Beta toxin, the principal lethal toxin of type C, causes necrosis of enterocytes in the small intestine. 9, 67,69 Loss of enterocytes allows toxin access to the deeper layers of the intestinal wall, resulting in extensive submucosal necrosis and intraluminal hemorrhage. 69 Terminally, toxin absorption into the bloodstream may cause multisystemic signs of disease. 69 Beta toxin is inactivated by exposure to proteolytic enzymes, particularly trypsin. 9, 67--69, 91 Thus, the lethal effects of beta toxin occur almost exclusively in neonates, resulting from either a low level of endogenous protease secretion or the presence of trypsin inhibitors in colostrum. 67--69, 90 Experimental reproduction of fatal type C enterotoxemia in older lambs required concurrent administration of C. perfringens type C and soybean flour, which contains an intestinal protease inhibitor.68 Affected lambs may show acute onset of listlessness and reluctance

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to nurse. Ataxia, colic, bloody diarrhea, depression, and recumbency may follow. 67-69, 91, 92 Extensor rigidity and opisthotonus are seen in some cases. 91, 92 Death may occur within hours, occasionally prior to evidence of bloody diarrhea. Less commonly, the disease may last several days.91,92 Regardless of the rate of disease progression, the fatality rate is usually very high. 92 Septicemia, meningitis, and hypoglycemia would be important differential diagnoses for cases that do not show evidence of enteritis. Other infectious agents that might cause enteritis in lambs include Salmonella spp., rotavirus, Cryptosporidium, enterotoxigenic E. coli, and in lambs 2 weeks of age or older, Eimeria spp. (coccidiosis). The predominant gross necropsy finding is severe hemorrhagic enteritis, usually most prominent in the jejunum and ileum. 6, 9, 69, 92 Occasionally the entire small intestine is involved. 69 Fibrin clots, casts of necrotic mucosa, and reddish brown blood are present within the intestinal lumen. An increased amount of fluid may be found within the peritoneal and pleural cavities and the pericardial sac; the fluid may be clear,67 straw-colored,69 or serosanguinous. 58,92 Definitive diagnosis of type C enterotoxemia requires culturing C. perfringens from the intestine of suspect cases, with subsequent typing by toxin neutralization or genotyping (see discussion of ancillary diagnostic tests). Type D Enterotoxemia (Overeating Disease, Pulpy Kidney Disease)

Clostridium perfringens type D causes enterotoxemia in small ruminants of all ages6-8, 77, 90, 92, 104-109 with the possible exception of neonatal lambs, in which type C enterotoxemia is most common. 91,92 Some reports suggest a role of C. perfringens type A in enterotoxemia of adult small ruminants, but its role in this disease remains unclear.90 Experimental intraduodenal administration of C. perfringens type A to goat kids led to transient diarrhea. 77 C. perfringens type D is not a prominent part of the normal flora of ruminants,67 although it is carried by healthy animals. 9,67 Passage of starch or plant or milk sugar into the small intestine is thought to provide the organism with substrate for rapid replication and elaboration of epsilon toxin. 8,9, 45, 90-92 Epsilon toxin is activated by intestinal and pancreatic proteases. 9, 67, 90-92 Once absorbed into the bloodstream, epsilon toxin causes loss of endothelial integrity, increased capillary permeability, and edema formation in multiple tissues. 38,67 The history in a case of type D enterotoxemia may reveal concentrate feeding, foraging on grain crops, sudden access to high quality forage, constant feeding of a high-concentrate diet, or overfeeding after a period of hunger. 39, 45, 90-92 Robust lambs or kids fed a high volume of milk or milk replacer or nursing a heavily lactating dam are at risk. Non-vaccinated animals may succumb to type D enterotoxemia even if maintained under relatively static dietary conditions. 64,90

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Type D Enterotoxemia in Sheep

In sheep, type D enterotoxemia usually presents as a peracute illness, with most affected animals simply being found dead. 64,91 If clinical signs are observed, the most prominent signs reflect central nervous system dysfunction. Affected sheep may show lethargy and ataxia early in the course of disease. Progression of the disease is rapid, and collapse, tachypnea, salivation, lateral recumbency, paddling, and opisthotonus occur within hours of the onset of clinical signs.3, 6, 8, 91, 92 Hyperesthesia may be present.3 Intact pupillary light responses, absent menace responses, variable nystagmus, and poor swallowing reflexes also have been observed in affected sheep.39 Diarrhea is not a prominent nor consistent feature of this disease in sheep.8, 92 Glucosuria is frequently present.15, 37, 57, 91 The differential diagnosis for type D enterotoxemia in sheep includes all causes of sudden death in sheep. Gastrointestinal parasitism, plant or chemical intoxication, ruminal acidosis, salmonellosis, and viral enteritis are important considerations for cases with gastrointestinal signs or lesions. Polioencephalomalacia, pregnancy toxemia, lead poisoning, hypoglycemia, salt poisoning/ water deprivation, urea poisoning, hypocalcemia, hypomagnesemia, meningitis, head trauma, and plant poisoning should be considered as differentials for the neurologic signs of type D enterotoxemia. At necropsy examination, straw or red-colored fluid containing fibrin clots is present in the peritoneal, pleural, or pericardial spaces.6-8,64 Petechial or ecchymotic hemorrhages are present on the surface of multiple organs and on the peritoneum. 3, 8, 57 Pulmonary edema and edema of the mesentery may be evident. 8 Gross lesions of the intestinal tract are frequently absent in affected sheep,3, 9, 92 although fluid intestinal contents8 and hyperemia of the abomasal, ileal, or colonic mucosa may be apparent. 9,64 Urine collected from the bladder frequently contains glucose. 3, 6, 57, 92 Softening of brain tissue with bilaterally symmetric focal areas of hemorrhage may be present in the internal capsule, thalamus, and cerebellum. 3,39 This lesion, termed focal symmetrical encephalomalacia, may be present in sheep with no other lesions suggestive of enterotoxemia. 39 Occasionally, no gross lesions are seen in sheep that die peracutely.3,6 Pulpy kidney is a term used for type D enterotoxemia; the term reflects softening of the renal cortex, which is attributed to an increased rate of renal autolysis in affected small ruminants. 6, 7, 9, 58 It is, however, a nonspecific autolytic change that is inconsistently present in affected small ruminants. 3, 90, 105, 107 The pulpy kidney lesion may not be evident if postmortem examination is carried out soon after death. 3, 8,107 Type D Enterotoxemia in Goats

The clinical signs and postmortem findings in goats affected by type D enterotoxemia differ somewhat from those seen in sheep. The clinical

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signs of enteritis, manifested as diarrhea and pain, are more evident in goats, and the predominant gross and histologic lesions are found in the gastrointestinal tract. Three forms of the disease in goats are recognized: peracute, acute, and chronic. 90,lOs In peracute type D enterotoxemia, affected goats may be found dead or may display colic, abdominal distension, vocalizing, dyspnea, tachypnea, and watery diarrhea containing fibrin, mucus, or strands of blood. 90, 105, 107 Fever is common early in the course. Recumbency, dyspnea, hypersalivation, opisthotonus, and convulsions usually follow, and death usually occurs within 24 hours of the onset of clinical signs. 90, 105, 107 Glucosuria does occur, albeit inconsistently, in affected goats. 90, 105, 109 The acute form of type D enterotoxemia in goats is characterized by similar clinical signs as the peracute form, but the progression of the disease occurs over 2 to 4 days. Dehydration and acidosis may occur secondary to protracted fluid losses from diarrhea.90, 105 Recovery may occur with aggressive supportive care that includes administration of antiserum specific for the epsilon toxin. A positive response to antitoxin may aid in the diagnosis of type D enterotoxemia in a live anima1. 90 The differential diagnoses for peracute and acute type D enterotoxemia in goats includes salmonellosis, ruminal acidosis, gastrointestinal parasitism, plant or chemical intoxication, and in young kids, septicemia, type C enterotoxemia, cryptosporidiosis, and viral enteritis. Intermittent or protracted diarrhea or soft stool, depression, weakness, inappetance, weight loss, and reduced milk production are characteristic of the chronic form of enterotoxemia in goats.90, 109 The course may last several consecutive days or wax and wane over several weeks. Chronic enterotoxemia may be difficult to diagnose unless prior peracute or acute cases are known to have occurred in the herd. 90 Gastrointestinal parasitism, salmonellosis, Johne' s disease, and rumen acidosis are considered in the differential diagnoses. The most prominent gross lesion in goats with peracute or acute type D enterotoxemia is fibrinohemorrhagic colitis, usually most severe in the spiral colon. 3, 105, 107 A whitish pseudomembrane covering segments of necrotic mucosa may be evident in the colonic lumen. Luminal casts of fibrin, blood, and mucus may also be present. The colonic serosa may be hyperemic or edematous, with edema evident in the colonic mesentery and mesenteric lymph nodes. The ileum may be affected in some cases. l07 As in sheep, pulmonary edema, fluid, and fibrin in the body cavities and heart sac as well as scattered ecchymotic hemorrhages on serosal surfaces may be present. Urine in the bladder may be positive for glucose, but the absence of this finding does not rule out type D enterotoxemia. 105 As is the case for sheep, occasional cases of peracute type D enterotoxemia show no gross lesions. 90 Pulpy kidney is considered to be a less consistent finding in goats than in sheep.3 Chronic cases may show scant body fat reserves and ulcerated colonic mucosa. 3 Lesions in the central nervous system are inconsistently present in goats.7, 8, 104-109

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Ancillary Diagnostic Tests

A tentative diagnosis of each type of enterotoxemia is often based on the animal's signalment, supportive historical information, clinical signs, and gross findings at necropsy. Further laboratory testing may be pursued to confirm the diagnosis. Azotemia, increased serum osmolality, and hyperglycemia were consistent biochemical abnormalities detected late in the course of fatal, experimentally induced type D enterotoxemia in small ruminants. 8 Increases in the serum activity of hepatocellular enzymes have been observed in experimentally induced disease in sheep.37 Neutrophilic leukocytosis and hemoconcentration have been observed in affected goats. 90 Gram staining of feces from a live affected animal may show a dense population of gram-positive rods with terminal oval spores, characteristic of C. perfringens. 90 At necropsy, impression smears from the luminal surface of the intestine may be used to make a presumptive diagnosis of enterotoxemia in an animal with compatible gross findings. Impression smears can be made by making repeated blots of an exposed section of intestinal mucosa onto a glass slide. Repeated blotting of a small area of mucosa allows a gradual reduction in the amount of luminal debris transferred onto the slide and a gradual increase in the number of epithelial cells and bacteria from the enterocyte surface. A large number of uniform, gram-positive rods with terminal or subterminal spores surrounding the enterocytes is supportive of a diagnosis of enterotoxemia. 90 It is important to remember that intestinal overgrowth of C. perfringens type A occurs rapidly postmortem,67 so mucosal impression smears should be considered reliable only in the fresh cadaver. A 10- to IS-cm length of affected intestine should be tied off with string and placed in a sealed container for transport to the laboratory. Swabs can be used but are less desirable because of the possibility of drying out in transit to the laboratory. If samples are to be stored or transported, rapid cooling after collection is considered essential for reduction of bacterial overgrowth. 36 Isolation of C. perfringens from the intestinal lumen of a suspect case is supportive of, but not confirmatory for, a diagnosis of enterotoxemia.9, 67, 91, 92 Rapid postmortem proliferation of C. perfringens, type A occurs in the gut and other tissues. 9,67 Therefore, interpretation of a positive culture, particularly from autolysed tissues, is not clear-cut. The organism is relatively easily grown in vitro; in fact, C. perfringens type A grows relatively rapidly in anaerobic culture and may overgrow other potential pathogens. 67 Unlike C. perfringens type A, type C is not commonly found in normal animals and is present in very low numbers in carriers. 67 Proliferation of type C in the gut appears to occur in close temporal association with clinical disease. 88 Thus, isolation of this type in suspect cases is highly suggestive of type C enterotoxemia. C. perfringens type D is not a prominent part of the normal flora of ruminants, although it is carried by healthy animals.9, 67 In summary, isolation of C. perfringens from feces or gut contents should be considered

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significant only in cases showing compatible history and clinical signs; typing of the organism by identification of a major lethal toxin or genetic analysis is necessary to confirm the diagnosis. Intestinal contents, tissues, blood, or feces may be tested for the presence of toxin by the toxin neutralization test or immunoassays. Samples should be refrigerated or shipped on ice and processed as quickly as possible to preserve potentially labile toxins. 36 In cases of type D enterotoxemia, freezing of intestinal contents obtained soon after death helps to preserve the labile epsilon toxin. 43 Addition of preservatives such as chloroform to samples is considered unnecessary and may actually complicate interpretation of toxin neutralization testing. 36 Toxin detection in culture media containing C. perfringens may be unrewarding because of variable toxin production under in vitro conditions. 67 The toxin neutralization assay, also known as the mouse protection assay53 or mouse lethality assay,29 has traditionally been used to identify and type toxin production by C. perfringens. 96 In this test, a sample of intestinal contents, blood, or tissue extracts shown to contain C. perfringens is centrifuged and the supernatant injected into a test animal, usually a mouse or guinea pig. The toxin is detected by characteristic lethality to the test animal. The toxin is typed (alpha, beta, epsilon, or iota) by determining which toxin-specific antiserum neutralizes the lethal effect of the sample in additional test animals. The expense, labor, and ethical concerns associated with the toxin neutralization test have resulted in its limited availability and has prompted investigation of other means of demonstrating the presence of major C. perfringens toxins in animal samples. ELISA32,33 and counter immunoelectrophoresis53 have been developed for use in identifying toxin in diagnostic samples, but tests for all toxin types are not widely available.92 Polymerase chain reaction (PCR) tests that identify genes for each of the major toxins recently have been developed. 2s, 29, 62 In a recent study, PCR detection of toxin genes in clinical isolates of C. perfringens correlated highly with results of toxin neutralization testing. 62 Typing of C. perfringens isolates is available through The Clostridial Enteric Disease Center. * GASTROINTESTINAL PARASITISM Nematodes

Infestation with gastrointestinal nematodes (nematodiasis) is a common cause of ill-thrift and reduced productivity in small ruminants. The major nematode parasites of pastured sheep and goats in North America are the trichostrongylid worms of the abomasum, Hemonchus contortus, Ostertagia circumcinta, and Trichonstrongylus axei. 41, 4S, SO, 113 Other nematode *The Clostridial Enteric Disease Center, Department of Veterinary Science and Microbiology, University of Arizona, Tucson, AZ 85721.

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worms commonly found in small ruminants include Nematodirus spp., Oesophagostomum spp., and Cooperia spp., although these are usually far less detrimental to the host than are the abomasal worms.41, 90, 113 Crossinfestation of nematodes between sheep and goats can occur. 2S, so The epidemiology, treatment, and control of nematode parasites in small ruminants has been reviewed recently.2S, 26, 4S, so Factors that influence severity of nematode infestation include host age, host immunity, host genetics, the presence of concurrent disease, stocking rate, feeding methods, nutrition, concurrent disease, geographic location, season, and weather patterns.2S, 26, 4S, so, 90 Sheep are particularly prone to parasitism because they graze close to the soil surface. Goats, who normally avoid significant parasite burdens by browsing herbage several inches above the ground, can become severely parasitized when forced to graze contaminated pastures. 2S, 80, 90 Angora goats appear to be particularly susceptible to gastrointestinal nematodiasis. 12 Adult sheep113 and possibly to a lesser extent adult goats90 develop variable degrees of immunity to nematode parasites; however, this immunity is finite and can be overwhelmed if the animal's immune function is compromised or if the animal is exposed to a high dose of infective larvae. 90, 113 Weanlings grazing contaminated pastures in the spring and summer are particularly prone to nematode infestation. 2s Although clinical disease can occur at any time of year, gastrointestinal nematodiasis is most common in warm wet times of year when larval development outside of the host occurs most rapidly.26 Death can occur with severe infestations. The history may indicate sporadic deaths of progressively weak lethargic animals occurring over a period of several days to weeks. 90 Clinical Signs and Necropsy Findings

Signs referable to anemia predominate when heavy infestation with H. contortus occurs in small ruminants. 4s, 90,113 Lethargy and exercise intolerance are common, with affected animals lagging behind others when the flock or herd is forced to move. Ataxia and collapse may occur during exercise. Severely anemic animals may die during or shortly after exercise, so caution is warranted when handling infected animals. Mucous membrane pallor, submandibular edema, tachypnea, and tachycardia are often evident on physical examination. Cardiac auscultation may reveal a hemic murmur, which is a systolic murmur caused by reduced blood viscosity.90 Diarrhea is not a predominant feature of H. contortus infestation4s, 113; in fact, scant, firm, dry fecal pellets may be seen in animals with clinical haemonchosis. 4s,90 When examination findings are suggestive of anemia, differential diagnoses include external blood loss (wounds, ectoparasites, hematuria), coagulopathies, chronic inflammatory disease, copper or cobalt deficiency, and bracken fern poisoning. Hemolytic disorders, such as eperythrozoonosis, leptospirosis, and copper, onion, or Brassica spp. toxicosis generally result in icterus as well as anemia.

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Diarrhea, dark green to black, is a more consistent feature of severe infestations with Ostertagia and Trichostrongylus infestations. 48,113 Progressive weight loss, poor weight gain in young stock, dullness, weakness, poor appetite, and poor hair or fleece quality as well as reduced milk production in dairy animals are signs of parasitic disease that can be caused by a variety of nematodes. Protein loss into the gastrointestinal tract may be severe enough to induce peripheral edema. 48, 90, 113 The intermandibular space, ventral abdomen, brisket, abdominal cavity, and the scrotum of males may show edema caused by hypoproteinemia (Fig. 1). Scrotal edema occurs secondary to ascites, with the transudate in the abdominal cavity entering the scrotal sac through the inguinal canals. Johne's disease, protein-losing nephropathies, hepatic failure, liver fluke infestation, congestive heart failure, ectoparasitism, and coccidiosis are other diseases that may produce generalized edema. For small ruminants with diarrhea, these differentials as well as salmonellosis, cryptosporidiosis, and subacute ruminal acidosis should be considered. Oesophagostomum columbianum may produce caseous nodular lesions on the surface of the small intestine. These lesions can induce intestinal adhesions, and focal or diffuse peritonitis may result if an abscessed nodule ruptures. In such cases, fever, colic, an arched back, and ileus may be evident. 90 Differential diagnoses include intestinal obstruction, rumen acidosis, type D enterotoxemia, and in males, urolithiasis. Common findings on gross examination of the carcass include emaciation, subcutaneous edema, and serous atrophy of fat on the epicar-

Figure 1. A ram with severe gastrointestinal nematodiasis. Note presence of submandibular edema, thin body condition, and pendulous scrotum.

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dium and surrounding the kidneys. Sheep and goats with severe haemonchosis may show multifocal hemorrhage and pinpoint ulceration of the abomasal mucosa. 57,90, 113 Mature H. contortus worms are large enough (2-3 cm) to be visible on close inspection of the abomasal mucosa (Fig. 2). The parasite's blood-filled gastrointestinal tract wraps around its long reproductive tract, giving it a characteristic red and white striped, barber pole color pattern. 40 The other trichostrongylids (0. circumcinta and Trichostrongylus axei) are smaller and very thin. The mature worms may be missed unless the abomasal mucosa is carefully inspected in bright light. To facilitate visualization of abomasal trichostrongylids, a necropsy knife can be scraped across the abomasal mucosa, and the scrapings placed on a slide to be examined by light microscopy. Alternatively, careful inspection with a magnifying glass may be used. Infestation of the abomasum by Ostertagia spp. may create round, nodular lesions, approximately 2 mm

Figure 2. The abomasum of a lamb with severe infestation of abomasal trichostrongylids. A, The larger Haemonchus contortus adults are visible from a distance (arrowhead). B, Magnified view of the same specimen. Note characteristic striped appearance of an adult H. contortus (arrow). (Courtesy of Robert K. Ridley, MS, DVM, PhD, Manhattan, KS.)

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in diameter, on the mucosa. These nodules are hypertrophied gastric glands containing the fourth stage larvae (L4) of O. circumcincta. 113 When distributed diffusely and in large numbers, these nodules impart a grainy, studded quality to the mucosa that resembles Moroccan leather.3, 40, 90 Histologic examination may be needed to demonstrate the L4 stage of O. circumcinta in the abomasal mucosa. Heavy infestation with intestinal nematodes may result in a nonspecific, mild catharral enteritis, with soft ingesta evident in the large intestine. 90 Intestinal nematodes usually are not visible with the naked eye, and mucosal impression smears are often needed to demonstrate the adult worms. 48,90 Intestinal and abomasal contents can be placed in 10% formalin to preserve the nematodes for microscopic examination and identification. 113 Fresh feces from live or dead animals can be placed in containers for transport to the laboratory and fecal egg counting. Exclusion of air from the container may aid in slowing larval development and hatching of the eggs. 113 If samples are to be stored for later evaluation, they should be refrigerated and not frozen. 113 Methods for quantification of nematode parasites within the abomasum and intestine have been described. 40 Ancillary Diagnostic Tests

Serum chemistry analysis of affected animals may show hypoproteinemia caused by hypoalbuminemia. 60,90 Severe anemia (peV of 15% or less) is often present in small ruminants with severe H. contortus infestation. 9o Depending on the chronicity of infestation and the diet, secondary iron deficiency may develop, with hypochromasia and microcytosis evident on the hemogram. 9o Fecal egg counts are commonly used in diagnosis of nematode infestations in small ruminants. The fecundity of the parasite(s) involved, seasonal factors, treatment history, and the age, reproductive status, and immunity of the host influence the fecal egg count. 48, 90, 113 Furthermore, because nematodes differ in their pathogenicity, a fecal egg count does not necessarily reflect the extent of disease in an affected anima1. 48 Within a herd or flock, fecal egg counts taken from multiple individuals (10-12 animals, or 10% of the flock) provide an accurate indication of the adult worm burden present in the flock or herd and the level of ongoing egg contamination of the environment. 25, 26, 41 Fecal egg counts should be performed on individual rather than composite (mixed) fecal samples. 113 Interpretation of fecal egg counts is affected by the age of the host, the species of nematode involved, and the timing of collection relative to season and duration since anthelmintic treatment. In temperate climates in the late spring, average fecal egg counts in adult ewes of greater than 1000 H. contortus eggs per gram indicate the need for prompt anthelmintic treatment of the flock, regardless of clinical signs.25,80 At the end of the summer grazing season, average fecal egg counts of greater than 2000 eggs per gram in nonlactating adult ewes also support immediate whole-flock treatment. 25, 80 In goats, a mean

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count of more than 500 eggs per gram in adults at risk for hemonchosis indicates the need for whole herd anthelmintic treatment. 90 Because parasite burdens can change rapidly over time, repeated monitoring of fecal egg counts in a population over a year is considered the most accurate means of formulating control strategies. 25, 48, 80, 113 A single fecal egg count in the diagnosis of nematode infestation in an individual animal does not consistently reflect the level of parasitism in the individual. If clinical signs are indicative of potential parasitism and the fecal egg count is high, the diagnosis is supported. On the other hand, a low egg count or even a negative egg count in an individual does not rule out disease because of gastrointestinal nematodes. Daily variation in fecal egg shedding, the degree of infestation caused by juvenile worms, and sampling and handling factors influence the egg count on an individual sample. 48,113 A diagnosis by response to anthelmintic therapy may be necessary if no other causes for the animal's disease can be found. If the environment is heavily contaminated with infective larvae, rapid reinfestation may preclude a positive treatment response. Clinical parasitism in an individual frequently implicates a faulty control program in the population. Investigation of parasitism in the herd or flock should be considered strongly when a diagnosis of clinical parasitism in the individual animal is made. Coccidiosis

Although subclinical infection with Eimeria spp. is common in juvenile and adult small ruminants, clinical coccidiosis typically occurs in lambs and kids between the ages of 2 weeks and 6 to 8 months.24, 34, 93 Stress is considered to be an important factor in initiation of clinical disease. 27 The history may reveal a recent stressful event, such as transport, shearing, weaning, or inclement weather.35 In adults, parturition, malnutrition, or concurrent disease may precede the onset of clinical signs. Inspection of the premises may reveal overcrowding, poor sanitation, and fecal contamination of feed or water; however, coccidiosis can occur under conditions of excellent management. 27 In feeder lambs, coccidiosis commonly occurs within 1 to 3 weeks after arrival on the feedlot. 57 Infection requires ingestion of sporulated oocysts. Enteric disease results from lysis of enterocytes by certain stages of the parasite, resulting in loss of digestive and absorptive functions, absorption of bacterial toxins across the damaged intestinal wall, translocation of luminal bacteria into the gut wall, and leakage of blood, protein, and electrolyterich fluids into the gut lumen. 35 Although many species of Eimeria can infect small ruminants, only the relatively nonpathogenic species Eimeria capriovina is capable of cross-infection between sheep and goats. 90 In sheep, Eimeria crandalis, Eimeria bakuensis, Eimeria ovinoidalis, Eimeria arloingi, and Eimeria ahsata appear to be the most pathogenic. 27, 35, 78, 93 The most pathogeniC coccidia of goats include Eimeria caprina, Eimeria

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arlongi, Eimeria ninakohlyakimovae, and Eimeria christenseni. 27, 35 Clinical disease often involves concurrent infection with multiple species of Eimeria. 27 Identification of these species by oocyte characteristics has been reviewed. 27, 34, 35, 40 Clinical Signs and Necropsy Findings

Diarrhea is the most conspicuous sign of coccidiosis in small ruminants. 27 Clumping of the feces may be evident early in the course of the disease, with progression to watery, brown or yellow diarrhea occurring hours to days later. Mucus and blood may be evident in the stool in some cases,26, 27, 34, 35, 90 but this is less commonly found in small ruminants than in cattle. 35 In sheep, fecal tags on the fleece and maceration of the perineal skin may result in myiasis. 34, 35 A transient fever can develop in affected lambs. 57 Tenesmus is common in infected sheep and may result in secondary rectal prolapse. 34, 35,57 In goats, however, tenesmus is relatively uncommon. 90 Early in the course, infected kids may vocalize as if in pain and show mild bloat, depression, and restlessness. lO, 90 There is often fecal staining of the perineum and hocks. lo, 90 In lambs and kids, weight gain or weight loss and poor quality hair or fleece are common. In severe cases, dehydration, weakness, and anemia may occur, and death is not uncommon if timely treatment is not provided. Severe, nonfatal infection may result in villus blunting and scar formation in the intestinal wall, with affected animals becoming chronic poor doers. lo, 35 Differential diagnoses in lambs and kids include enterotoxemia, cryptosporidiosis, rotavirus infection, salmonellosis, nematodiasis, rumen acidosis, and diarrhea caused by a dietary change. Catarrhal or hemorrhagic enterocolitis is evident on gross examination. Close examination of the intestinal and colonic mucosa may reveal multiple raised, grayish-tan nodules, measuring 1 to 6 mm in diameter.35, 57, 90 These nodules represent areas of active coccidial replication, and large numbers of oocysts and macrogametes are visible upon microscopic examination of mucosal scrapings from these areas. Necropsy findings are considered to be more reliable means of confirming a diagnosis of coccidiosis than fecal floatation.90,93 Ancillary Diagnostic Tests

Severe blood loss into the gut lumen may result in anemia, although this may be masked by the hemoconcentration that results from dehydration. 9o In cases with extensive enterocyte loss, leukocytosis may be evident on the hemogram. Metabolic acidosis, hyponatremia, hyperkalemia, and hypocalcemia may develop in affected animals. 90 A modified McMaster floatation technique, using magnesium sulfate (specific gravity 1.18)112 or sucrose solution (specific gravity 1.101.27)35 is commonly used to detect oocysts in the feces. Because subclinical infection is common, oocysts are usually demonstrable in the feces of healthy small ruminants of a variety of ages, even those that have

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developed effective immunity.27 When performed on multiple animals in a group, quantitative fecal floatation for coccidiosis can be used to determine the level of infection in a herd or flock. 35, 112 Infection with coccidia, as evidenced by shedding of oocysts, however, is not synonymous with disease. 27, 35, 78, 90 Shedding of up to 106 oocysts per gram of feces may occur in subclinically infected lambs?8 Identification of the species of Eimeria and assessment of that species' pathogenicity for the sheep or goat may guide interpretation of oocyst counts in the feces, although this is cumbersome for the inexperienced examiner.35 In many clinical cases, counts of several hundred to several thousand oocysts per gram of feces are found 35,93 (Fig. 3). Day-to-day oocyst shedding can vary dramatically in an infected animal. 35 Thus, an antemortem diagnosis of coccidiosis in the live animal is always somewhat presumptive. A diagnosis requires demonstration of oocysts in the feces of an animal or group of animals with compatible signalment, history, and clinical signs. 90 Because enterocyte lysis precedes oocyst production, clinical signs may precede the appearance of oocysts in the feces in rare cases. The prepatent period for coccidia infections in small ruminants is 2 to 3

Figure 3. Fecal flotation from a goat with clinical coccidiosis. Note the large number of coccidial oocysts (open arrows), the single strongyle egg (small arrow), and scattered pollen grains (large arrow). (Courtesy of Robert K. Ridley, MS, DVM, PhD, Manhattan, KS.)

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weeks, depending on the species of Eimeria involved. 27,35 Thus, if a naive animal is acutely exposed to a heavy load of infective oocysts, clinical coccidiosis may develop approximately 2 weeks later, at which time the fecal flotation may be negative for oocysts. In such cases, oocyst shedding in the feces usually follows within a few days of the onset of clinical signs, and fecal floatation may have to be repeated several days later to demonstrate the oocystS. 27 NEONATAL DIARRHEA

Enteritis caused by Cryptosporidium parvum,35, 90 rotavirus,66, 82, 98, 99 and ETEC47, 52, 73, 90, 110 occurs in lambs and kids. The clinical and pathologic features of enteritis due to these organisms in lambs and kids are similar to that seen in calves. This material is discussed in greater detail in "The Differentiation of Gastrointestinal Diseases of Calves" in this issue. In lambs, enteritis caused by ETEC typically occurs in the first 2 to 3 days of life. 52, 110 As in calves, diarrhea results from ETEC adhering to the enterocytes of the small intestine and inducing hypersecretion via release of the heat-stable enterotoxin, STa. Loss of bicarbonate-rich small intestinal fluid in scours rapidly leads to dehydration and acidosis. Surface fimbrial antigens on ovine ETEC isolates include K99 and F41, expressed singly or together. 52 Neonatal goat kids may also develop enteritis from K99-positive strains of ETEC.73, 90 The diagnosis is confirmed through Gram staining of impression smears of the ileum and demonstration of the fimbiral antigen in E. coli isolates from feces or intestinal contents. 52, 73, 90, 110 Certain media promote expression of fimbriae by E. coli, whereas other media may suppress expression, so selection of media is critical in identification of ETEC.50 Diarrhea caused by C. parvum infection typically affects lambs and kids younger than 3 weeks of age, with the peak number of cases occurring in animals between 5 and 10 days of age. 35, 73 Enteritis caused by C. parvum is considered to be the most common cause of diarrhea in neonatal goat kids. 73 As a sole agent, C. parvum usually induces mild enteritis, but it can complicate enteritis from other agents to produce potentially fatal enteritis. 35 Group B rotavirus has been identified as a cause of diarrhea in lambs in Ohio98 and Wyoming. 99 Affected lambs, 2 to 14 days of age, voided yellow, semiliquid stool for 2 to 3 days. Lethargy, excessive salivation, weight loss, and occasional death were also seen. Because commercial kits detect only group A rotavirus, veterinary diagnostic laboratories that rely only on these kits to diagnose rotavirus infections are not able to detect group B rotavirus in samples. 99 Demonstration of group B rotavirus in feces or intestinal contents requires immune electron microscopy or cultivation of the virus in cell culture. Fecal or intestinal contents submitted for these tests should be obtained from lambs early in the course of clinical disease. 99 Samples should be chilled but not frozen. 99

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Group A rotavirus has been demonstrated concomitant with C. parvum in samples of diarrheic feces from 1- to 2-week-old kids in Canada. 82 Demonstration of caprine group A rotavirus in feces or gut contents can be performed using commercially available latex agglutination tests, enzyme-linked immunosorbent assays, and gel electrophoresis.66, 82 SALMONELLOSIS

Salmonella typhimurium is the most common serotype associated with gastroenteritis and enterocolitis in sheep.31, 57, 81 Reports of caprine salmonellosis are few, with Salmonella dublin and S. typhimurium involved in most reported cases. 31,90 S. typhimurium DT104, isolated with increasing frequency from humans and animals in recent years, has been isolated from sheep in the northwestern United States5 and from a goat in the Eastern United States. 4 Salmonella arizonae is considered hostadapted to sheep and has been isolated from cases of proliferative rhinitis, abortion, and enteritis as well as from clinically healthy sheep.13, 47, 63, 79 Several other serotypes have been isolated on a less frequent basis from sporadic cases of enterocolitis in small ruminants. 81, 90 Infection with pathogenic serotypes of Salmonella can result in acute or chronic enteritis, septicemia, abortion, and chronic shedding of the organism in the feces in healthy or recovered animals. 13, 31, 47, 51, 63, 79, 90 Infection may occur through contact with animals suffering from clinical infection, subclinically infected carriers, contaminated feed or water, and contact with Salmonella in the environment. As in other species, oral ingestion is the most common means of infection. Physiologic stress is a critical factor in initiation of salmonellosis. Comingling of animals from multiple sources, weaning, crowding, feed deprivation, transport, concurrent disease, malnutrition, fecal contamination of feed or water, and poor hygiene may be evident in the history or on inspection of the premises. 57, 81, 90 Because feeder lambs may encounter several of these stressors during transition from the flock to the feedlot, salmonellosis tends to occur in these animals within days of arrival on the feedlot. 31,57 Clinical Signs and Necropsy Findings

Small ruminants with enteric salmonellosis initially may show lethargy, drooped ears, an arched back, separation from the group, reduced appetite or anorexia, and fever. Diarrhea usually develops within hours of the onset of illness. 31, 90 Although blood, fibrin, and mucus may be evident in diarrheic feces, a lack of these findings does not preclude a diagnosis of salmonellosis. Affected feeder lambs and goats may show yellowish to green-brown diarrhea. 31,90 Progressive weakness and dehydration develop if fluid losses in diarrhea become severe. In cases that develop septic or endotoxic shock, tachycardia, tachypnea, and a subnor-

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mal rectal temperature are frequently present. Death may occur after a disease course of 1 to 5 days. Case fatality rates vary with the serotype involved and the resistance of the host. Chronic enterocolitis, with recurrent bouts of diarrhea lasting for days or weeks, may occur in sheep and goats. 81,90 Differential diagnoses in adult small ruminants include coccidiosis, gastrointestinal nematodiasis, Johne' s disease, enterotoxemia, and ruminal acidosis. In neonates, viral or cryptosporidial enteritis, coccidiosis, and ETEC are important differential diagnoses; for septicemia caused by Salmonella, colisepticemia, type D enterotoxemia, and in kids, mycoplasma septicemia should be considered. In sheep, the location of enteric lesions at gross necropsy examination varies with the stage of disease. In acute cases, lesions are predominantly found in the abomasum and proximal small intestine, whereas subacute and chronic cases show lesions centered in the ileum, cecum, and proximal colon. 81 Acute cases tend to show severe mucosal reddening of affected segments of gut, with focal areas of intraluminal hemorrhage occasionally evident. Serosal lesions are rare in acute cases, but mild darkening of the serosa overlying reddened mucosa may be seen. Cases with concurrent septicemia may show petechiae and ecchymoses on multiple organs, hemorrhagic and edematous lymph nodes, and catharral cholecystitis in addition to variable gastroenteric lesions. 81 Subacute and chronic cases consistently show edema of affected segments of bowel and mesenteric lymph nodes. 81 Petechial or ecchymotic hemorrhages may be evident on the serosa of the ileum and colon. The mucosa of affected segments may be ulcerated or necrotic, with mucoid or catarrhal luminal exudate commonly seen. In some chronic cases, pale, cauliflowerlike areas of mucosa may be present in the distal ileum and proximal colon. These lesions are created by foci of mucosal hyperplasia located adjacent to areas of mucosal cell depletion. 81 Common gross necropsy findings in goats90 are similar to those of sheep, although the association of gross lesions with disease progression described in sheep has not been described in goats. The organism may be readily isolated from the affected segments of bowel in enteritis cases. The wall of the gallbladder, liver, mesenteric lymph nodes, heart, blood, spleen, and bone marrow may be submitted for culture in cases of suspected septicemia. 3, 81, 90

Ancillary Diagnostic Tests

In acute salmonellosis, leukopenia, followed by a rebound leukocytosis with a left shift, may be seen in the hemogram of affected animals. 90 Toxic changes (hypersegmentation of nuclei, Dohle bodies, azurophilic cytoplasm) may be evident on cytologic examination of the neutrophils. Metabolic acidosis, hemoconcentration, hyponatremia, hypochloremia, and variable serum potassium concentration frequently develop in animals with profound fluid losses in diarrhea. Chronic cases may develop

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hypoproteinemia secondary to prolonged exudation into the gut lumen. Anemia of chronic inflammatory disease may be evident in such cases. 90 Confirmation of a diagnosis of salmonellosis requires isolation of the organism from the feces, gut contents, blood, or tissues of an affected animal. Swabs from the lumen of affected segments of gut, luminal contents, or feces can be chilled and submitted for culture and sensitivity testing. Because large numbers of other enteric bacteria are usually present in these samples, several grams of feces, intestinal contents, or intestine should be submitted. JOHNE'S DISEASE

Johne's disease (JD), or paratuberculosis, is a chronic, granulomatous enterocolitis of adult domestic and wild ruminants caused by Mycobacterium avium subspecies paratuberculosis. lOl Although transmission in utero is considered possible, lambs and kids are thought to be infected primarily through oral contact with feces from clinically or subclinically infected adults. 18, 86, 89, 90, 94, 95 Transmission through infected colostrum or milk is also possible.1 8, 94, 95 Once ingested, the organism is engulfed by macrophages in the intestinal wall, but effective intracellular killing of the organism often fails to occur. Spread of M. avium subsp. paratuberculosis within the intestinal wall and to extraintestinal sites occurs through migration of infected macrophages. A slowly progressive, granulomatous inflammatory enterocolitis develops, eventually resulting in a protein-losing enteropathy and progressive wasting. Weight loss is a consequence of malabsorption and protein loss caused by the inflammatory response in the bowel wall. 18 Clinical signs of JD do not develop until several months to years after infection is initiated. 18, 86, 89, 90, 94, 95 Infected animals may shed M. avium subsp. paratuberculosis in their feces, however, long before they show signs of clinical disease, potentially leading to spread of the infection to other animals. Clinical Signs and Necropsy Findings

Owing to the chronic, insidious nature of JD, clinical signs of the disease may be present in only one or a few adults in a heavily infected population. 89,90 The most consistent clinical sign of JD in small ruminants is weight IOSS.16-l9, 42, 86, 87, 89, 90, 94, 95, 100 In dairy goats, reduced milk production may precede weight loss.90 The age of onset of weight loss typically ranges from 2 to 7 years of age89 but tends to be younger in small ruminants than in cattle. 94,95 Clinical JD may be seen in yearling sheep and goats. 86, 89 Occasionally, the history reveals that a stressful event, such as parturition or transport, preceded the onset of weight loss.86, 90 Diarrhea is a far less common clinical sign in small ruminants than in cattle, occurring in only 100/0 to 20% of affected small ruminants. 16, 20,87, 89, 90, 94, 95 When present, diarrhea tends to develop late in the disease

j

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course. In those rare instances, the feces may be watery or may merely be clumped and softer than normal89 (Fig. 4). Affected animals usually maintain a good appetite and are afebrile, although poor appetite, weakness, dehydration, and lethargy may be evident in the terminal stages of the disease. 42,94 Peripheral edema, wool break, and poor fleece quality may occasionally be seen in ovine cases. 94,9S Flaky skin and a poor haircoat may be seen in affected goats. 90 Acid-fast stains of fecal smears may show the characteristic acidfast bacilli in clumps, although this finding is only suggestive for JD.22 Advanced JD may be complicated by concurrent, severe gastrointestinal parasite infestation in both sheep 16 and goats. 89 Thus, a history of a poor or incomplete response to anthelmintic therapy and adequate parasite control measures might be indicative of JD in a sheep or goat with wasting disease. Johne's disease should be suspected in small ruminants suffering from weight loss in the face of good nutrition, parasite control, and dental health. Differential diagnosis in small ruminants includes malnutrition (macronutrients or micronutrients), dental disease, gastrointestinal parasitism, liver flukes, the internal form of caseous lymphadenitis, ovine progressive pneumonia (sheep), caprine arthritis encephalitis virus infection (goats), scrapie, chronic bacterial pneumonia, and chronic renal or hepatic disease. Impaired access to feed caused by inadequate feeder space or social hierarchy should also be ruled OUt. 89 In Suffolk sheep, abomasal emptying defect should be considered also as a differential diagnosis for weight loss and anorexia.

Figure 4. Clumped feces from a ewe with advanced Johne's disease.

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At necropsy, a lack of fat stores and reduced muscle mass are evident. Gross lesions of the intestinal tract may be subtle or nonexistent,16, 86 although lesions of gastrointestinal parasitism are often found. 16, 89 When present, gross lesions of JD are most commonly found in the ileum and ileocecal junction. 16, 18-20, 87 Thickening of the bowel wall, cording of the serosal and mesenteric lymphatics, and enlargement of the mesenteric and ileocecal lymph nodes may be apparent. 16, 75, 76, 89 Corrugation of the intestinal mucosa, a relatively common finding in clinically affected cattle, is not consistently present in affected small ruminants. Carrigan and Seaman16 observed corrugation of the ileal mucosa in 48% of sheep with clinical JD, whereas only 380/0 of affected goats showed this lesion in a separate study.42 Edema, ascites, and hydropericardium may be evident in severely hypoproteinemic animals. 18-20 Goats may show nodular foci of caseation, occasionally with mineralization evident, in the intestinal wall and mesenteric lymph nodes. 18, 100 In light of the frequently subtle and variable nature of gross lesions with JD in small ruminants and the possible presence of coexistent lesions of gastrointestinal parasitism, the veterinarian performing necropsy examination of an adult small ruminant with wasting disease should strongly consider histologic examination for JD. Sections of the ileum, ileocecal junction, and the associated mesenteric lymph nodes should be submitted for microscopic examination and acid-fast or immunohistochemical staining. Smears of the mucosa of the distal ileum and the cut surface of the ileocecal lymph node can be submitted for acidfast staining; detection of clumped acid-fast organisms is highly suggestive of JD.87 Owing to the difficulty in culturing ovine strains of M. avium susbsp. paratuberculosis, histopathology has been used as the defining test for diagnosis of JD in sheep.2o, 49,75 Ancillary Diagnostic Tests

Hypoproteinemia, characterized by hypoalbuminemia and normal serum concentrations of globulin, may be evident on serum biochemistry analysis of affected small ruminants. 54, 84, 89, 90 The serum total calcium concentration may be low as a result of hypoalbuminemia. 54 Mild anemia also may be present; centrifugation of blood for 10 minutes may be necessary to allow for adequate packing of the smaller red blood cell in sheep and goats. 89 Because weight loss in small ruminants is caused by a variety of diseases and management problems, a definitive diagnosis of JD in sheep and goats cannot be made by clinical signs alone. Definitive diagnosis of JD in ruminants is complicated by the lack of a single, antemortem, diagnostic test with absolute sensitivity and specificity.89,90, 94,95 Methods used to obtain a definitive diagnosis include culture or polymerase chain reaction (PCR) to identify the organism in feces or tissues, histologic examination of biopsy or necropsy specimens, and serologic tests that indicate the animal's immune response to the organ-

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ism. Application and interpretation of these tests for diagnosis and control of JD in small ruminants has been reviewed. 94,95 Culture

Strains of M. avium subsp. paratuberculosis obtained from sheep are extremely difficult to grow using conventional culture techniques. Positive cultures from feces or tissues have been obtained from only 8% to 15% of sheep with confirmed, clinical JD.16, 22, 87 Owing to its poor sensitivity, conventional culture has not proved to be a useful antemortem or postmortem diagnostic test for clinical or subclinical JD in sheep.22, 87, 89,94,95 Promising advances in experimental culture techniques may lead to improved sensitivity for culture of ovine isolates. l , 55, 111 Goats, on the other hand, are usually infected with strains of M. avium susbsp. paratuberculosis that are more readily grown in vitro, and feces, ileum, or mesenteric lymph nodes can be submitted for culture. Fecal culture has been shown to detect 76% to 86% of goats with clinical JD.42,100 As in cattle, the sensitivity of fecal culture in detecting subclinical infection in goats is limited by the tendency for the organism to be shed intermittently and in low numbers in the feces during the early stages of infection. Periodic fecal culture of the entire adult goat herd can be used as part of a test and cull program or to eventually establish proof of freedom from infection. 94 Rarely, goats may be infected with the more fastidious ovine strains, particularly if comingled with infected sheep. In such instances, fecal culture would be a poor choice for diagnosis of clinical or subclinical JD in goats. 94 Polymerase Chain Reaction

Detection of DNA sequences unique to M. avium subsp. paratuberculosis has been used to detect infection in sheep with histologically confirmed JD.22,46 When applied to samples of ileum from 12 diseased sheep, peR detected the organism's DNA in all cases. 46 The specificity of peR was also 100% when it was used to test samples from 10 clinically normal, histologically negative sheep from an unaffected flock. Sensitivity of detection of infection was lower in diseased sheep when the peR was applied to blood, liver, or the ileocecal lymph node. 46 Fecal testing by peR was positive for 16 of 25 diseased sheep (64%) in another study.22 The sensitivity of peR in detecting early or subclinical infection is unknown. To reduce the potential for contamination of samples, aseptic technique is necessary when acquiring tissues for peR testing. Individual containers are required for collection of fecal samples, and gloves used to collect feces should be changed between animals. The relative high cost and limited availability of peR currently limit its use for diagnosis of JD in small ruminants.

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Histologic Examination

Shulaw and colleagues87 detected acid-fast bacteria in the ileocecal lymph node of only 25% of sheep. Although sinus histiocytosis was present on histologic examination of all of the ileocecal lymph nodes examined in that study, this lesion is present in other inflammatory conditions and is not specific for JD. 87 Gezon and colleagues42 found histopathologic evidence of JD in 32% of mesenteric and ileocecal lymph nodes examined from goats with clinical disease. Thus, ileocecal lymph node biopsy may not consistently provide definitive proof of JD in sheep or goats. Biopsy of the distal ileum and the ileocecal lymph node may result in more sensitive detection of lesions. Immunohistochemical staining may increase the detection rate of mycobacteria in tissues.?5 Serologic Testing

Serologic tests are attractive as antemortem diagnostic tests because of their low cost and ease of collection. Serologic tests used to diagnose JD in small ruminants include the agar gel immunodiffusion (AGID) test, the enzyme-linked immunosorbent assay (ELISA), and the complement fixation (CF) test. Because the humoral immune response and clinical signs develop late in the course of JD, these tests all lack sensitivity in detection of subclinically infected animals. 18, 49, 89, 94, 95 Thus, the use of serologic tests to detect latent JD in apparently healthy animals has limited diagnostic value. The CF test, ELISA, and AGID test possess good sensitivity (87100%) in detecting JD in sheep and goats showing clinical signs of the disease. 94 The ELISA and CF tests, however, have been shown to detect cross-reacting antibodies to Corynebacterium pseudotuberculosis in sheep, resulting in poor test specificity in flocks infected with this bacteria. 30, 49, 74 The AGID test does not appear to be influenced by antibodies to C. psuedotuberculosis. 30, 49, 87 No fully validated and licensed ELISA tests for JD in small ruminants are presently available in the United States. Thus, although the AGID is an insensitive assay for detection of subclinical infection in small ruminants, it appears to be the most specific serologic test available and has good sensitivity for detection of animals in the later stages of clinical JD.94 Because animals with advanced, clinical JD are more likely to be seropositive and are likely to shed more organisms in their feces, AGID testing of the population detects the most heavily infected animals in the population. 18, 19,76,89,94 By testing the thinnest animals (body condition score of 2 or less) in a flock or herd, the veterinarian can apply the AGID to a subset of animals most likely to test positive if their poor condition is truly caused by JD. 94 This testing method may be chosen as an initial step in determining if JD is present in a population. In goats, combination of AGID testing and fecal culturing has been recommended to identify infected animals within a herd. 42, 89

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The prevalence of JD in a flock or herd influences the interpretation of a positive result on a serologic test for JD in an individual anima1. 94 The predictive value, or reliability, of a positive test is higher in populations with a high prevalence of disease than in those with low prevalence. In other words, in a population with a large proportion of diseased animals, there is greater probability that a positive test on an animal with wasting disease is a true positive result. On the other hand, if wasting disease is rare in the flock or herd, the predictive value of a positive test is lower; that is, there is a better chance that any given positive serologic test result is actually a false (spurious) positive result. Thus, if the flock or herd history, culture or histopathology results, and examination findings support or confirm the presence of JD in the population, a positive AGID test is reliable as an indicator of JD in a small ruminant with wasting disease.

References 1. Aduriz H, Juste RA, Cortabarria N: Lack of mycobactin dependence of mycobacteria isolated on Middlebrook 7H11 from clinical cases of ovine paratuberculosis. Vet Microbiol 45:211-217, 1995 2. Angelov G, Nikolov Y, Angelov A: Changes in acid-base parameters, blood sugar, and blood lactate in experimental acute rumen acidosis in sheep. Indian Veterinary Journal 73:309-324, 1996 3. Barker IK, Van Dreumel AA, Palmer N: The alimentary system. In Jubb KVF, Kennedy PC, Palmer N: Pathology of the Domestic Animals, ed 4, vol 2. San Diego, Academic Press, 1993, pp 1-318 4. Benson CE, Munro DS: Salmonella typhimurium DT104 in the Northeastern USA [letter]. Vet Rec 141:503-504, 1997 5. Besser TE, Gay CC, Gay JM, et al: Salmonellosis associated with S. typhimurium DT104 in the USA [letter]. Vet Rec 140:75, 1997 6. Blackwell TE: Enteritis and diarrhea. Vet Clin North Am Food Anim Pract 5:557570, 1983 7. Blackwell TE, Butler DG: Clinical signs, treatment, and postmortem lesions in dairy goats with enterotoxemia: 13 cases (1979-1982). J Am Vet Med Assoc 200:214-217, 1992 8. Blackwell TE, Butler DG, Prescott JF, et al: Differences in signs and lesions in sheep and goats associated with enterotoxemia induced by intraduodenal infusion of Clostridium perfringens type D. Am J Vet Res 52:1147-1152, 1991 9. Borriello Sp, Carman RJ: Clostridial diseases of the gastrointestinal tract in animals. In Borriello SP (ed): Clostridia in Gastrointestinal Disease. Boca Raton, Florida, CRC, 1992, pp 195-221 10. Bowen JS: Coccidiosis in goats. In Annual Compilation of Papers on the Health and Disease of Small Ruminants. Nashville, American Association of Small Ruminant Practitioners, 1997, pp 184-186 11. Braun U, Rihs T, Schefer U: Ruminal lactic acidosis in sheep and goats. Vet Rec 130:343-349, 1992 12. Bretzlaff K: Special problems of hair goats. Vet Clin North Am Food Anim Pract 6:721-735, 1990 13. Brogden KA, Meehan JT, Lehmkuhl HD: Salmonella arizonae infection and colonisation of the upper respiratory tract of sheep. Vet Rec 135:410-411, 1994 14. Bulgin MS: Toxicoses most frequently seen in sheep. In Proceedings of Small Ruminants for the Mixed Practitioner, 70th Western Veterinary Conference, Las Vegas, NV, 1998, pp 22-28 15. Bullen H, Battey I: Enterotoxaemia of sheep. Vet Rec 69:1268-1276, 1957

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