poult enteritis complex - oie

Rev. sci. tech. Off. int. Epiz, 2000,19 (2), 565-588

Poult enteritis complex H.J. Barnes(1), J.S. Guy ( 2 ) & J.-P. Vail lancourt ( 1 )

(1) Department of Farm Animal Health and Resource Management, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606, United States of America (2] Department of Microbiology, Pathology and Parasitology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606, United States of America

Summary Poult enter i t is complex (PEC) is a general te rm tha t encompasses the infect ious in test inal d iseases of young turkeys. Some diseases, such as coronav i ra l enter i t is and stunt ing syndrome, are relat ively we l l charac te r i sed , wh i le others, such as t ransmiss ib le viral enter i t is , poul t g rowth depress ion and poult enter i t is morta l i ty syndrome, remain i l l -def ined. A l l fo rms of PEC are mul t i fac tor ia l , t ransmiss ib le and in fect ious. Sal ient c l in ical features inc lude stunt ing and poor feed ut i l isat ion tha t resul t f rom enter i t is . In the more severe fo rms, runt ing, immune dysfunct ion and mortal i ty are repor ted. Gross and mic roscop ic lesions of enter i t is are present in all fo rms but tend to be non-spec i f ic . Other lesions may be present , depending on the agents involved. The basic pathogenesis involves the fo l low ing : a) a l terat ion of the intest inal mucosa , general ly by one or more v i ruses infect ing enterocy tes ; b) in f lammat ion; c) prol i ferat ion of secondary agents, usual ly bacter ia . Non- in fec t ious fac to rs interp lay w i t h in fect ious agents to modulate the course and sever i ty of d isease. Diarrhoea is bel ieved to be pr imar i ly osmotic because of mald igest ion and malabsorp t ion , but may also have a secre tory component . Transmission is pr imar i ly faeca l -o ra l . No publ ic health s ign i f icance is recognised or suspec ted . Prevent ion is based on el iminat ing the infect ious agents f rom contaminated premises and prevent ing in t roduct ion into f locks . This is accompl ished by an ef fect ive c lean ing, d is infect ion and b iosecur i ty p rogramme. Al l - in /a l l -out product ion or separate brooding and f in ishing units are helpful . Control may require regional co-ord inat ion among all companies produc ing tu rkeys , especia l ly if t he product ion is highly concen t ra ted , and a quarant ine programme for more severe fo rms of PEC. No vacc ines or speci f ic measures for contro l l ing the organisms involved in PEC are avai lable. Treatment is suppor t ive for the viral component , wh i le ant ib iot ics, especia l ly those w i t h e f f icacy against Gram posit ive bacter ia , may help to reduce the impac t of bacter ia l in fect ions. Evidence suggests tha t PEC occurs whe reve r turkeys are raised commerc ia l ly , but th is is not w e l l documented and distr ibut ion of the var ious organisms tha t have been assoc ia ted w i t h PEC is largely unknown. The disease causes enormous economic loss, most ly f rom fa i lure of the tu rkey to reach its genet ic potent ia l .

Keywords Avian diseases - Astroviruses - Coronaviruses - Digestive diseases - Enteroviruses -Escherichia coli - Intestinal diseases - Mixed infections - Poultry diseases - Production diseases - Rotaviruses - Turkeys.

566 Rev. sci. tech. Off. int. Epiz., 19 (2)

Description of the disease Poult enteritis complex (PEC) is a general term for a group of multifactorial, transmissible, infectious diseases of young turkeys less than six weeks of age. These diseases are characterised by clinical signs of intestinal disease (enteritis), moderate to marked growth depression (stunting), retarded development (runting), impaired feed utilisation, and secondary nutritional deficiencies. Mortality is typically nil to low, but can exceed 10% in the more severe forms. Immune dysfunction generally occurs, which increases susceptibility of the flock to other infectious diseases.

Poult enteritis complex includes a number of described diseases and clinical intestinal infections of unknown cause. The characteristics evoke a production disease rather than a classical disease (Table I). The term is useful because of the limited knowledge about the causes and interactions of enteric pathogens and opportunists in young turkeys, limited availability of diagnostic procedures, lack of specific prevention methods, and the need for a generic approach to controlling all entities that comprise PEC.

Diseases encompassed by PEC include the following:

- coronaviral enteritis of turkeys ('bluecomb', infectious enteritis, transmissible enteritis) (69)

- cryptosporidiosis (41)

- maldigestion syndrome (80)

- poult malabsorption syndrome (73, 74, 75, 76)

- poult enteritis mortality syndrome (PEMS) ('spiking mortality of turkeys') (14)

- runting and stunting syndrome of turkeys (60)

- stunting syndrome ( 3 , 4 , 5 , 1 0 , 11)

- turkey viral enteritis (80) .

Table I Comparison between production and classical diseases Poult enteritis complex has more characteristics in common with a production di: and aetiology are ill-defined

Several common names, such as 'big bird - little bird syndrome' have been used in the industry. Coccidiosis could also be included, but because this is a well-recognised, specific disease, it will not be covered in this review (58). Poult enteritis complex is remarkably similar to a disease in chickens commonly referred to as runting stunting syndrome (RSS) (65). The relationship between PEC in turkeys and RSS in chickens has yet to be completely clarified, but most evidence suggests these are two distinct syndromes (23, 90).

No specific cause of PEC has been identified. The basic pathogenesis involves damage to the intestinal mucosa, usually by one or more viruses, a host inflammatory response that may intensify the mucosal damage, and subsequent proliferation and possible colonisation by intestinal bacteria and protozoa. Lesions in the intestine are best described as catarrhal enteritis (53). Impaired immunity is indicated by atrophy of lymphoid organs, fungal infections, especially crop mycosis caused by Candida albicans, and systemic bacterial infections such as colisepticaemia. Non-infectious factors such as nutrition, environment and management can have a marked influence on the course and severity of the disease.

History In the late 1940s and early 1950s, an acute diarrhoeal disease with high morbidity, and often high mortality, especially in young turkeys, occurred in flocks in several areas of the United States of America (USA). The disease was named 'mud fever' or 'bluecomb' because of the similarity to a disease in chickens. During the following two decades, bluecomb was a significant cause of economic loss in the turkey industry in Minnesota and the subject of intense research. The causative agent, a Coronavirus, was eventually isolated from affected flocks and used to reproduce the disease, which prompted the name to be changed to 'coronaviral enteritis of turkeys'.

than a classical disease, as the cause is multifactorial and the definition

Characteristic Production disease Classical disease

Cause

Clinical signs Mortality Pathology Diagnosis

Treatment Prevention/control

Experimental reproduction

Methods of study

Complex, multifactorial, interactions between non-infectious and infectious factors Not present or mild and not considered significant Typically nil to low Not present or mild and non-specific Production below genetic potential; inductive (few findings lead to generalities about flock) Rarely possible, often too late, usually of little value Requires high biosecurity, excellent management, very good nutrition Difficult or impossible to reproduce and confirm experimentally; proven by Evans' postulates (97) In flocks on farms, good records essential, emphasis on population medicine, epidemiology, etc.

Simple, usually one, or less commonly, two agents; may be infectious or non-infectious Usually present and readily recognised Variable, but usually present Typical lesions usually present Signs, lesions recognised, identification of cause; deductive (many findings lead to specific cause) Often possible, moderate to high success Vaccines and medications often useful

Generally easy to reproduce experimentally; proven by Koch's postulates Natural or experimental disease, emphasis on microbiology, pathology, toxicology, etc.

Rev. sci. tech. Off. int. Epiz., 19 (2) 567

However, while experimental infections reproduced the stunting, the mortality seen in natural infections was not typically reproduced (1). Diagnostic tests were developed and used to identify and eliminate all affected flocks in Minnesota, which was accomplished in 1976 (69). Subsequent outbreaks occurred in Quebec in the mid- to late 1980s (24), Indiana in the mid-1990s, and Virginia and North Carolina in the late 1990s (20) . Sporadic cases involving only a few flocks and with limited spread as well as occasional outbreaks in which numerous flocks are affected and the disease spreads rapidly among farms continue to occur in some turkey producing areas of the USA.

Soon after the identification of coronaviral enteritis, the existence of other intestinal diseases in young turkeys became apparent, although these tended to be less severe than coronaviral enteritis. In the late 1970s and 1980s, a number of enteric viruses were identified from poults in the USA (40, 82 , 84), United Kingdom (UK) (60 , 63 ) , France (9) and Germany (86) that were experiencing a RSS clinically similar to a disease in chickens. In addition to enteric viruses, Cryptosporidia were found to be associated with intestinal disease of poults (41) .

In the 1990s, rotaviruses were associated with affected flocks in Israel (104). In the USA, a malabsorption syndrome of turkeys which was associated with skeletal abnormalities was identified in turkeys in the south-east of the country (73, 74, 75, 76), and a stunting syndrome was found to affect turkey flocks in the north-central USA (3, 4 , 5, 10, 11). Cochlosoma was another protozoan linked with intestinal disease in young turkeys in California (21) .

A particularly virulent enteric disease characterised by a sharp peak of mortality emerged in 1991 in an area of dense turkey production in western North Carolina. The disease was initially named 'spiking mortality of turkeys' because of the mortality pattern in affected flocks (Fig. 1), but was later termed 'PEMS', following the discovery of a milder clinical form of the disease in 1994 (14). In addition to high mortality, affected birds show immune dysfunction (51 , 52 , 78) and a variety of physiological abnormalities, including reduced body temperatures, reduced energy metabolism and hypothyroidism (28).

Poult enteritis mortality syndrome is readily reproduced by inoculating young poults with faeces or intestinal homogenates, or by placing susceptible birds in contact with litter from an affected flock, indicating that the most common mode of transmission is faecal-oral (17, 23) . A number of viruses, including turkey C o r o n a v i r u s (TCV) and unidentified small round viruses, along with certain strains of Escherichia coli have been associated with the disease, but no specific agent has been present in all cases or has reproduced the disease experimentally (29, 3 0 , 4 6 , 4 8 , 8 7 , 1 0 6 ) . Once a flock is affected with the ' disease, other conditions, such as colibacillosis, salmonellosis, rickets, and protozoal enterotyphlitis, occur more frequently.

The prevalence of PEMS increased from 1991 to 1996, when occurrence and severity reached a peak. Several flocks were destroyed after mortality exceeded 50%. One flock that was not destroyed experienced a total mortality of 9 6 % . Since 1996, the number and severity of outbreaks have declined and no flocks in North Carolina have been destroyed.

Fig. 1 Poult enteritis mortality syndrome Typical 'spiking' mortality pattern of a severely affected flock in western North Carolina in 1996. Day nineteen was the peak day of mortality when almost 12% of the flock died. Ordinarily, such a flock would have been killed, but this flock was kept for sampling and study until 28 days of age. Cumulative mortality at that time had reached 82.3%. Performance of the subsequent flock was normal after depopulation, cleaning and disinfection. The disease recurred the following summer, but was not sufficiently severe for the flock to be destroyed. No further flocks have been affected and production continues on the farm.


By comparing hatchmates raised at the College of Veterinary Medicine of the North Carolina State University (NCSU) with those produced on commercial farms since the mid-1980s, a type of PEC has been identified that has been named 'poult growth depression'. A formal description of the disease has yet to be published. The disease is a mild intestinal disorder that causes a reduced growth rate between two and four weeks of age in most commercial flocks. Productivity lost during this period is never fully regained, so that affected flocks are typically 10%-15% lighter at processing or require between ten and fourteen days longer to reach market weight. Analysis of flock records from integrated companies across the USA suggests that this form of PEC is so ubiquitous that the disease is generally not recognised because of its insidious nature. The disease has been experimentally reproduced by exposing seven-day-old poults to an intestinal homogenate from affected birds; the disease developed in the first two flocks placed on new farms (H.J. Barnes, J .S. Guy and G.M. Elias, unpublished data).

Production records from a flock studied at the NCSU that did not experience poult growth depression, together with data regarding ten commercial flocks marketed at the same time, are summarised in Table II. Recent average data obtained from similar flocks produced throughout the USA are included for comparison (35). On average, the unaffected flock achieved a 17.7% better growth rate, reached market weight 13.5 days earlier, and each pound of turkey cost US$0.058 less per kg of feed to produce (US$0.026/lb). Note that growth and feed efficiency of the ten commercial flocks are comparable to average production in the USA and would be considered normal.

Clinical signs Flocks are typically affected between ten and twenty-eight days of age. The first indication of PEC is often hyperactivity with excessive vocalisation. Birds are described as being noisy and 'marching' as they move continuously along feed or water lines or the walls of the house. Feed consumption drops sharply; water consumption typically increases initially and then decreases in parallel with reduced food intake. During the next twelve to twenty-four hours, the flock becomes quiet and birds huddle together near heat sources. Birds consume litter and are seen pecking at feed and sorting out larger particles. Some flocks completely refuse to eat ('feed refusal'). In most instances the feed is satisfactory; moving the feed to another flock, testing the feed under controlled conditions, or sometimes just remixing and redistributing the feed to the affected flock results in consumption. Most flocks develop diarrhoea within 24 h. Diarrhoea primarily results from the osmotic effects of undigested, unabsorbed feed. Litter rapidly deteriorates, followed by declining air quality, and birds become progressively more soiled. Insect larvae multiply in wet litter, especially when ambient temperatures are high. Many birds will have faecal staining of vent and abdominal feathers and/or pasting around the vent with faeces and urates. Size variation among birds in the flock can be observed within a few days, and lack of uniformity is clearly evident one week after onset of clinical signs. Approximately 60%-70% of the stunting can be directly attributed to decreased feed intake. Feathers, especially those of the tail, become frayed and show segmental dysplasia ('stress bars'). The feathers may break off completely at these weakened sites, leaving the bird with partial or no tail feathers, or incompletely break, which

Table II Production parameters from a flock of hens raised at the College of Veterinary Medicine of the North Carolina State University that did not experience poult growth depression, compared with ten commercial hen flocks marketed at the same time All flocks were raised on the same feeding and management programmes. Average values for hen turkeys produced in the United States of America during i are included for comparison purposes (35)

Characteristic College of Veterinary Medicine

Average (commercial flocks)

Range (commercial flocks)

(n = 10)

Average (United States of

America)

Llvablllty (%) 93.9 96.1 97.6-89.6 90.7 Average weight (lbs) 15.6 15.4 16.6-13.3 16.2 Average weight (kg) 7.08 6.99 7.53-6.03 7.35 . Age (days) 84.0 97.5 94.0-104.4 102 Average dally gain (lbs/day) 0.186 0.158 0.167-0.143 0.158 Average dally gain (g/day) 84.4 71.7 75.7-64.9 71.7 Actual FCR 1.91 2.25 2.12-2.48 _ Adjusted FCR , a l 1.84 2.19 2.09-2.42 _ NetFCR , b | 1.93 2.27 2.15-2.52 2.40 Disease condemnation (%) 0.113 0.184 0.121-0.311 _ Total condemnation (%) 1.38 0.86 0.48-1.28 1.45 Feed cost/lb of turkey (US$) 0.158 0.184 0.175-0.204 _

Feed cost/kg of turkey (US$) 0.348 0.406 0.386-0.450 -FCR: feed conversion ratio (gross weight of turkey/weight of feed consumed) a) Adjusted to a standard weight (14 lbs or 6.35 kg) to permit comparisons among flocks b) Net FCR after condemnation (net weight of turkey after processing/weight of feed consumed)

Hev.sci.tech.0ff.int.Epiz.,K[2) 569

Fig.2Feathers develop segmentai dysplasia ('stress bars'), either breakingoff completely or remaining partially attachedThe latter produces a 'helicopter' bird, as seen in this poult withexpérimental poult enteritis mortality syndrome

produces the 'helicopter' appearance (Fig. 2). Runted birdshâve an immature feather pattem and fail to grow (Fig. 3).With a few exceptions, such as the severe form of PEMS,excess mortality is generally not a feature of PEC. The typicalappearance of affected flocks is illustrated in Figures 4 and 5.

Clinical signs decrease within seven to ten days after the onsetof disease but the lack of uniformity continues to increase andthe appearance of the birds remains poor for the duration ofthe life of the flock (Fig. 6). Management intervention canshorten the duration of clinical disease and the impact ofPEC on the flock. Secondary nutritional deficienciessuch as osteoporosis ('brittle bones'), rickets, and/or

Fig. 3Runting differs from stunting in thatfailure to develop is concurrentwith depressed growthRunting persists for prolonged periods, whereas stunted birds will typicallyregain a normal growth rate in a few weeks. The two birds are the sameâge. The runted bird (left) was exposed by contact to turkeys with poultenteritis mortality syndrome; the weight of this bird was 28% of theunexposed control bird (right)

Fig. 4Flock with typical signs of poult enteritis complex (central lowa, 1977)Birds are various sizes and feathering ranges from normal to poor. In theforeground is a normal sized bird standing nextto a runted bird. Thèseturkeys were raised together since placement, indicating the variability ofthe effects in individual birds, even in the same environment. The reasons forthe variability are unknown but could provide valuable insight into préventionand control

encephalomalacia may occur (57, 100). Leg problems aregreater at market-age in affected flocks (76). Susceptibility tococcidiosis in flocks with PEC may be increased if the diseaseis being controlled with a coccidiostat, due to decreased feedconsumption. The lost growth potential of the flock will notbe regained, although the rate of growth will eventually retumto normal for most birds. Flocks affected by PEC hâve lighterweights, lower average daily gains, poorer feed conversionratios, and a decreased percentage of white méat with a higherpercentage of viscera and bone when marketed. This occursbecause of the way in which nutrients are distributed tosupply and demand organs and maximal growth rates ofcommercial turkeys. During periods of decreased nutrientintake, such as those which occur in flocks affected with PEC,

Fig. 5Flock of young birds (5-6 weeks of âge) with typical signs of poultenteritis complex (western North Carolina, 1996)Note the simiiar clinical appearance of this flock with that in lowa almosttwentyyearsearlierPhoto: courtesy of MrT. Knapp

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Fig.6Flock of older birds (10-11 weeks of âge) with typical signs of poultenteritis complex (western North Carolina, 1996)Note the rough overall appearance, poorfeathering, faecal staining and lackof uniformityPhoto: courtesy of Mr T. Knapp

the limited nutrients are preferentially provided to the supplyorgans (viscera, support tissues) instead of demand organs(muscle, especially white muscle). Restoration of normalnutrition after recovery does not resuit in compensation forthe lost production of muscle mass during this period becausethere is little or no ability to exceed the maximal growth rateof the commercial turkey. The net resuit is a decreasedpercentage of méat at processing, which is directlyproportional to the severity of the épisode of PEC in the flock.Lack of uniformity in affected flocks may create problemsduring processing and in fulfilling marketing contracts.

PathologyMildly affected poults may demonstrate few external signs,apart from stunting, dirty feathers, and/or excrément aroundthe vent. Dehydration (skin folds along shanks, darkcongested tissues, dry skin), weight loss, poor feathering(notched, frayed, brittle or broken, stress marks), faecalstaining of feathers, and faeces and/or urates on feathers andaround the vent are seen in more severely affected birds.Watery, yellow-brown, flocculent liquid may drip from thevent, especially when the carcass is handled, and theabdomen often appears distended (Fig. 7).

Internai lésions are also variable according to the severity ofthe disease. However, intestinal changes consisting of pale,thin-walled intestines distended with fluid and occasional gasor mucous casts, and caeca filled with watery brown fluid andgas are observed consistently, regardless of severity (Figs 8

Fig. 7Legs of twenty-one-day-old turkey poultsOne bird (left) was exposed to poult enteritis mortality syndrome fourteendays earlier while the bird on the right is an unexposed control poult. The legof the affected bird is smaller, indicating stunting, and darker in colourbecause of dehydrationNote watery brown droppings coming from the vent

Fig. 8Intestine of a ten-day-old turkey poult affected by expérimental poultenteritis mortality syndrome, day three post exposureTail feathers are stained with pale brown liquid from the vent and intestinesare pale, translucent, thin-walled and distended with fluidNote saccular dilation of intestine. Caeca contain excess fluid

and 9). Multiple, fluid-filled, saccular distensions of the smallintestine alternating with contracted areas occur in moreseverely affected birds. The gastrointestinal tract contains littleor no ingesta, but litter is found in the ventriculus, sometimesto the point of impaction. Soft brown to orange casts areoccasionally présent in the intestinal lumen, and rarely, caecalcores. The cloaca is often distended and a firmer mass offaeces and urates may be found just intemal to the vent. Smallérosions or ulcers of the ventriculus near the entrance of theproventriculus are common in both affected and unaffectedyoung poults and should not be identified as a lésion of PEC.


Fig.9Intestine of an eleven-day-old turkey poult affected by expérimentalpoult enteritis complex, of unspecified type, day four post exposureIntestines are pale, thin-walled and distended with fluidNote saccular dilations and the poorly formed, orange-brown luminal castmixed with ingesta

In more severely affected birds, a moderate to marked muscleatrophy occurs that is most apparent around the knee jointand along the keel (Fig. 10). If the bird is dehydrated, themuscles will be dry and dark. Fat stores are depleted but donot show prominent serous atrophy. Bones may be normal,osteoporotic (thin, brittle and easily broken whenmanipulated), or rachitic (soft and flexible with widenedgrowth plates and easily eut). Kidneys are usually mildly tomoderately swollen and urates may be visible in the ureters.In severely stunted birds, adrenal glands are prominent andlymphoid organs, especially the thymus and bursa ofFabricius, are atrophie (Fig. 11). Poults with coronavirusinfection occasionally hâve caseous masses filling the lumen ofthe bursa of Fabricius. Abscesses of the yolk stalk remnant

Fig. 10Breast muscles of twenty-one-day-old turkey poultsThe poult on the left is an unexposed control poult and the poult on the rightwas exposed to poult enteritis mortality syndrome fourteen days earlier.Compared to the unexposed control poult, the affected poult has markedmuscle atrophy. The darker muscle colour in the affected bird indicatesdehydration. The gall bladder of the affected poult is distended and readilyvisible through the bodywall

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S 0.1

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0.04

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0.080.07

Bursa Thymus Spleen

• Control • Exposed

Fig.11Percentage of body weights of the bursa of Fabricius, left lobe of thethymus, and spleen from 265 control and 489 infected poults at fourteendays after contact exposure to turkeys affected with the severe form ofpoult enteritis mortality syndrome (spiking mortality of turkeys)The bursa and thymus are markedly atrophied, while the spleen is lessaffected

(vitelline or Meckel's diverticulum) occur more frequently inpoults with PEC. Crop mycosis may be identified as asecondary complication related to immune dysfunction.

Microscopic changes are often not spécifie or evencharacteristic for différent diseases within the scope of PEC.Intestinal lésions include villous changes (atrophy, bluntingand fusion), altération in the number of goblet cells, cryptepithelial hyperplasia, hyperaemia, protein loss andinfiltration of the lamina propria with mixed inflammatorycells, especially macrophages (Fig. 12). Lésions tend to be lessévident in expérimental infections with viral agents alone. Thelower small intestine and caecum are usually more severelyaffected than the duodénum. Heterophils are more numerouswhen bacterial infection is présent. Free inflammatory cells,sloughed epithelial cells and proteinaceous exudate canaccumulate in the intestinal lumen and support proliférationof intraluminal bacteria. Pancreatic acinar cells may undergovacuolar degeneration and either be depleted or engorgedwith zymogen. The latter typically are located around islets.Changes in the liver consist of Kupffer cell hyperplasia,hepatocellular atrophy, and occasionally foci of necrosisand/or mononuclear cells.

Spécifie changes in the intestine include large, basophilicinclusions within the nuclei of enterocytes produced byGroup 1 adenoviruses, adhering bacteria (Gram positivecocci, Gram positive or négative bacilli) covering villoussurfaces, and attaching effacing lésions caused byenteropathogenic E. coll. Clostridia cause focal or diffusecoagulation necrosis and lysis of the mucosal surface. Lésionscan be distinguished from autolysis by the présence ofnumerous large Gram positive bacilli and an inflammatory

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Fig. 12 Jejunum from a three-week-old poult with poult enteritis complex Lesions indicate acute catarrhal enteritis but are not specific for a causative agent. Increased goblet cells on the villous tips have been described as a lesion of turkey coronaviral enteritis, but infection with Coronavirus could not be confirmed in this flock x159; stain: haematoxylin and eosin

zone separating necrotic from viable tissue. Intravascular haemolysis is sometimes observed. Crypt necrosis with abscess formation is characteristic of Salmonella infections, especially with S. enterica subsp. arizonae and S. enterica ser. Typhimurium. Cryptosporidia can be seen within microvilli on the surface of enterocytes. Cochlosoma adheres to surface enterocytes or is free in the lumen. Spironucteus and trichomonads occur in both crypts and lumen. Spironucleus causes crypt epithelial necrosis, whereas trichomonads are not generally associated with lesions.

In other tissues of severely affected birds, lymphoid depletion may occur in the spleen, bursa of Fabricius and thymus. Infection of the bursa of Fabricius by Co ronav i r us causes degeneration, necrosis and inflammation of the epithelium, and is associated with lymphoid depletion of follicles (Fig. 13). Occasionally, fibrinoheterophilic, caseous exudate will fill the bursal lumen causing a 'bursal core' (Fig. 14) (49). Evidence of rickets may be found in bones, especially the proximal tibiotarsus. Cortical cell hyperplasia in the adrenal glands and histological indications of thyroid inactivity are evident in endocrine organs. Changes in the kidney are consistent with dehydration. Bacterial colonies are frequently numerous in yolk sac abscesses. Candida is present within the hyperplastic epithelium of the crop of birds with crop mycosis.

Incidence and distribution Anecdotal evidence suggests that PEC has a world-wide distribution. However, the actual occurrence of intestinal diseases of young turkeys, and the infectious agents t h a t are associated with them, remain largely undocumented. This may be due in part to the lack of diagnostic reagents and testing, the limited turkey production in many areas of the

Fig. 13 Bursa of Fabricius from a turkey with coronaviral enteritis Degeneration, necrosis and sloughing of epithelial cells are visible together with an associated acute heterophilic inflammation. The interstitium is infiltrated by mixed inflammatory cells. Lymphoid follicles are depleted and a high level of apoptosis has occurred x62; stain: haematoxylin and eosin

Fig. 14 Bursal core in a four-week-old turkey from a flock experiencing poult enteritis complex Although this flock w a s not tested for turkey Coronavirus, the lesion is

identical to that seen in experimentally infected poults


world, the mild nature of most forms of PEC, or little awareness of the diseases that constitute PEC.

Significance Although the relationships among the various disease entities included within PEC are largely unknown, infectious intestinal disease of young turkeys is clearly very common and a significant cause of economic loss. Calculations indicate that flocks infected with TCV have increased production costs of approximately US$0.044/kg-US$0.11/kg (2 cents/lb-5 cents/lb) (55, 83) . Poult enteritis mortality syndrome devastated the turkey industry in North Carolina, causing an estimated loss of US$161 million between 1991 and 1997. Annual production of turkeys in the endemic area and State-wide substantially decreased during this period (Table III).

Although outbreaks with high mortality are dramatic and certainly costly, such episodes occur infrequently. Far greater loss results from the common, widespread occurrence of the mild forms of PEC, such as poult growth depression, which attract little attention (Table II). Based on a 10%-15% loss due to poult growth depression throughout the turkey industry of the USA, estimated potential losses to the USA turkey industry from poult growth depression would be between US$300 and US$400 million annually.

Research is continuing to identify new agents, define interactions between previously known agents, and develop diagnostic methods to determine what organisms are associated with the different forms of enteric disease. While progress is being made, the effort is small compared to the magnitude of the problem. The ability of a turkey to achieve its genetic potential with respect to growth and feed utilisation correlates directly with the health of the intestinal tract.

Table III Impact of poult enteritis mortality syndrome on turkey production in Union County, North Carolina, United States of America (USA) (endemic area), and State-wide between 1992 and 1997 (North Carolina Agricultural Statistics)

Production variable Union County (North Carolina)

State-wide (North Carolina)

Turkeys produced in 1992 (millions) 14.4 62 Turkeys produced In 1997 (millions) 5.4 53.5 Difference (millions) - 9 -8.5 Difference (%) -62.5 -13.7 Proportion of total production in 23.2 _ North Carolina In 1992 (%) Proportion of total production In 10.1 _ North Carolina in 1997 (%) Proportion of total production In the 21.5 USA in 1992 (%) Proportion of total production In the _ 17.8 USA in 1997 (%)

Description of the aetiological agents Several different viruses, bacteria and protozoa have been associated with PEC (Table IV). Those of greatest importance appear to be rotaviruses, TCV, turkey enterovirus, astrovirus, Salmonella spp., E. coli, and Cryptosporidium spp. Recently, unclassified, small, round viruses have been isolated from poults with PEMS ( 8 7 , 1 0 6 ) . Reviews have been published on viruses that cause enteritis in poultry (43) and turkeys (85).

Rotavirus Rotaviruses are classified as a separate genus within the family Reoviridae (33). Rotaviruses are non-enveloped, spherical, and have a diameter of approximately 70 nm. Intact viruses consist of two icosahedral capsid shells (approximately 50 nm and 70 nm in diameter); the particles have a distinctive 'wheel-like' appearance by negative-stain electron microscopy

Table IV Infectious agents associated with poult enteritis complex

Agent References

Viruses Aviadenovirus{s) 7,43 Alphaviruslbi 36,45 Astrovirus 61,79,95,96 Coronavirus 24,46, 49,69 Enterovirus® 44,50,66 Parvovlrus-like 98 Plcornavirus-like ld l 8 Orthoreoviws 40, 57,71 Rotaviwsiei 62 Stunting syndrome agent 3,4, 5 Unclassified small round viruses 87,106

Bacteria Campylobacter 91,101 Clostridium 37 Enterococcus 49 Escherichia coli 29,30,48 LSFOs | f) 12,68,89 Salmonella 39

Protozoa Cochlosoma 21,56 Cryptosporidium 15,22, 32, 41 Eimeria 58 Spimnucleus® 59,77,88,105 Trichomonads 72

Fungi Candida 19

a) Group 1 viruses, Group 2 viruses are uncommon In turkeys under five weeks of age b) Eastern equine encephalitis and Highlands J viruses c) At least two distinct serotypes d) Includes 'pseudoplcornavlrus' e) Group A, Group D and Rotavirus-iike viruses f) tong segmented filamentous organisms g) Previously classified as Hexamita


(EM) owing to a smooth outer rim and capsomeres of the inner capsid that radiate towards the rim. The genome is comprised of eleven linear segments of double-stranded (ds) ribonucleic acid (RNA) with a total molecular weight of approximately 12 x 1 0 6 (33).

Classification of avian rotaviruses has been based primarily on immunofluorescent antibody (FA) procedures and Polyacrylamide gel electrophoresis (PAGE) analyses of ds RNA segments. Avian rotaviruses that cross-react by FA with antisera prepared against group A mammalian rotaviruses are classified as group A avian rotaviruses (62). Rotaviruses that lack the group A antigen are referred to as 'atypical' rotaviruses. Two antigenically distinct 'atypical' avian rotaviruses have been identified in turkeys; one of these has been classified as group D, but the other one remains unclassified. Polyacrylamide gel electrophoresis analysis of ds RNA is also useful for classification of avian rotaviruses; electrophoretic migration of RNA segments is an indicator of serogroup classification and RNA profiles may be useful in epidemiological studies (62).

Rotavirus replication occurs primarily in the small intestines, in differentiated, mature epithelium lining the upper portions of intestinal villi. Based on studies with mammalian rotaviruses, diarrhoea caused by avian rotaviruses probably occurs due to destruction of mature villous epithelium and replacement by immature epithelium from crypts. Immature, undifferentiated cells which replace cells destroyed by the virus lack disaccharidases and have impaired absorptive ability. Diarrhoea is likely to be a result of the effects of both malabsorption and maldigestion (67).

In vitro propagation has been possible with turkey group A rotaviruses, but not atypical rotaviruses. Cell culture propagation of group A rotaviruses has been accomplished in a variety of cell types, including primary chicken kidney cells, primary chicken embryo liver cells and a continuous line of rhesus monkey kidney cells (MA104) (62). Serial propagation in cell culture usually requires trypsin treatment of the inoculum.

Coronavirus Turkey Coronavirus is a member of the family Coronaviridae. Members of the genus Coronavirus are RNA-containing viruses that infect a wide variety of avian and mammalian species (103). The viruses are characterised on the basis of their distinctive morphology - pleomorphic, enveloped particles with diameters of 60 nm-220 nm, with long (12 nm-24 nm), widely spaced, petal-shaped surface projections (103). A close genomic relationship between infectious bronchitis virus and TCV has been identified (16).

Turkey Coronavirus replication occurs in the epithelial lining of the bursa of Fabricius and upper portions of intestinal villi.

Diarrhoea in TCV infections, like rotavirus infections, is believed to result from malabsorption and maldigestion.

The turkey was considered to be the only natural host for TCV, but recently chickens have been infected with the virus. Experimental inoculation of young chickens resulted in intestinal infection, as determined by immunohistochemistry and virus isolation, but the chickens did not develop clinical disease (47). In addition to TCV, evidence suggests that poults may also be susceptible to infection with bovine C o r o n a v i r u s

(25, 54) , and preliminary characterisation suggests that the virus causing stunting syndrome is probably a member of the family Coronaviridae (5).

Turkey C o r o n a v i r u s may be propagated in embryonated chicken and turkey eggs by inoculation of the amniotic cavity (69). Virus replication in inoculated embryos is restricted t o intestinal epithelium and the epithelium of the bursa of Fabricius. Attempts to propagate the virus in cell culture generally have not been successful.

Enterovirus Since the discovery of enterovirus-like viral particles in the faeces of poults with diarrhoea in the UK in 1979 (66) , several viruses resembling enteroviruses, referred to as enterovirus-like viruses (ELV) or pseudopicornaviruses., have been suggested as causes of PEC (66). Enteroviruses comprise one of four genera within the family Picornaviridae (6). Picornaviridae are non-enveloped, icosahedral viruses which range from 22 nm to 30 nm in diameter. Virions lack an obvious surface structure and no surface projections are visible. These viruses possess a genome comprised of single-stranded RNA of approximately 7.5 kilobases (2.5 x 1 0 s molecular weight) (6). Genera within the family Picornaviridae art distinguished by their sensitivity to acid, buoyant density of the virion in CsCl, and clinical manifestations in the affected host (6). Enteroviruses are stable at acid pH, have a density of 1.33 g/ml in CsCl and replicate preferentially in the intestinal tract. Turkey ELVs have been classified on the basis of size, morphology, cytoplasmic replication in enterocytes and resistance to acid pH.

Turkey ELVs have been demonstrated to replicate preferentially in the jejunum and ileum (50). Virus replication occurs principally in villous epithelium located halfway between the tip and base of the villus.

Turkey ELVs may be propagated in embryonated turkey eggs (44). Laboratory propagation is accomplished by amniotic inoculation of embryonated turkey eggs; virus replication occurs in embryo intestines.

Astrovirus Members of the genus Astrovirus are small, roughly spherical viruses, 28 nm to 31 nm in diameter (79). The viruses possess a characteristic morphological feature, namely: a five- or

Rev. sa. tech. Off. int. Epiz., 19 (2) 575

six-pointed star which covers the surface of approximately10% of virus particles. The biochemical structure of avianastroviruses is largely unknown, as thèse viruses hâve notbeen propagated in vitro.

SalmonellaSalmonella art Gram négative, non-spore-forming bacteriawhich are members of the family Enterobacteriaceae. Motile,non-host-adapted serotypes of Salmonella are referred to asparatyphoid Salmonella. Of the serotypes of Salmonellaidentified in turkeys with PEC, paratyphoid serotypes aremost common. Serotypes are identified using theKaufmann-White method, based on antigenic différencesidentified in both somatic and flagellar antigens (39).

Paratyphoid salmonella hâve an extensive host range. Thebacteria can be isolated from a wide variety of speciesincluding humans and other mammals, avian species andreptiles.

Salmonella art readily cultivated in the laboratory asnutritional and growth requirements are simple (102). Thebacteria are facultatively anaerobic and grow well under bothaérobic and anaerobic conditions. Salmonella can be culturedon a variety of simple culture média. Optimal growth is at37°C and pH 7, but can occur over a wide range oftempératures (5°C-45°C) and pH (pH 4-9).

Escherichia coliEscherichia coli are Gram négative, non-spore-formingbacteria which also are members of the familyEnterobacteriaceae. Identification of E. coli is based onbiochemical characteristics, serotyping and virulenceproperties. Serotyping is determined by différences in O(somatic), H (flagellar), and K (capsular) antigens. Virulenceproperties which allow catégorisation of strains of E. coliinclude élaboration of enterotoxins and shiga toxins, and theprésence of certain gènes, such as the E. coli attaching andeffacing (eae) gène (70).

Escherichia coli hâve not generally been considered to beintestinal pathogens of poultry; however, enteropathogenicstrains of E. coli hâve recently been associated with PEC (48).Enteropathogenic strains of E. coli art identified bycharacteristic attaching and effacing lésions in intestinaltissues (Fig. 15), absence of enterotoxin and shiga toxinproduction, lack of cell invasiveness, and présence of the eaegène (70).

Escherichia coli hâve a wide host range including mammalian,avian, reptilian and amphibian species. EnteropathogenicE. coli hâve been identified in a variety of mammalian andavian species, including turkeys.

Escherichia coli art readily cultivated in the laboratory usingordinary culture média. Escherichia coli, like Salmonella spp.,

Fig. 15Jéjunum from a poult experimentally infected with enteropathogenicEscherichia coli, strain R985Near the tip and along the side of this villus are bacterial colonies embeddedin the brush border of the enterocytes, causing attaching and effacinglésions. Note the micro-erosion and vacuolation of enterocytes associatedwith one of the colonies and infiltration of the lamina propria withheterophilsx255; stain: GiemsaReprinted from Guy ef a/. (48)

are facultatively anaerobic and grow well under both aérobicand anaerobic conditions. Eschenchia coli hâve simplenutritional and growth requirements.

CryptosporidiumCryptosporidia are small coccidian parasites that replicatewithin the microvilli of the intestinal tracts of vertebrates. Twospecies hâve been identified as causes of intestinal infection inturkeys, namely: Cryptosporidium meleagndis and C. baileyi.However, C. meleagndis is probably the prédominant speciesassociated with PEC, as C. baileyi infection is restricted inturkeys to the cloaca, bursa of Fabricius and respiratorySystem (22).

Replication of C. meleagndis in vivo occurs primarily in themiddle and lower small intestine of turkeys. Parasites arefound within the brush border of intestinal epithelium(Fig. 16). Attempts to propagate cryptosporidia in vivo hâvenot been successful.

EpidemiologyThe occurrence of PEC is influenced by environmentalconditions and farm location. In gênerai, PEC tends to bemore évident during the warmer seasons of the year. This is


Fig. 17 Relative differences in average body weights between a small commercial flock raised at the College of Veterinary Medicine (CVM) of the North Carolina State University (represented by the zero baseline) and hatchmates raised on a commercial farm Flocks received the same poults and feed and were subject to the same management programme. After two weeks, the average weights of birds in the commercial flock exceeded those of the CVM flock by nearly 10%. However, between the second and third weeks, the commercial flock experienced mild enteritis without appreciable mortality, which was named 'poult growth depression'. This led to a marked decrease in growth rate, which was most apparent at six weeks of age when average weight of the commercial birds was over 20% lower than the CVM flock. Growth rate returned to normal and some modest compensatory growth occurred during the seventh and eighth weeks which narrowed the gap between the two flocks. However, the percentage difference in the average weights remained relatively unchanged from the ninth week until processing

Clinicopathological findings In monitoring flocks for clinical evidence of enteric disease, indicators other than mortality need to be established as this is rarely elevated in most cases of PEC. When a flock with clinical signs is identified, a visit to the farm is necessary to assess the environment, feed and management, and to collect samples. The most accurate information will be obtained early in the course of the disease. Six to ten representative affected birds should be selected for necropsy. If mortality is occurring, dead birds should be examined in addition to the live ones selected for necropsy. Six to ten serum samples, a composite sample of droppings, a water sample, and a feed sample from the feeders need to be collected. Samples of serum and droppings can be taken from any birds in the flock or from those selected for necropsy. The flock should be revisited approximately three weeks later to collect a set of convalescent serum samples. If evaluation of the birds is not possible when clinical signs are present, or if the onset of the disease is uncertain, normal two- to three-week-old turkeys can be placed into the flock as sentinels and sampled six to eight days later.

The following is a necropsy and sampling protocol which has been developed by the authors for investigating outbreaks of PEC. Birds must be killed individually and necroscopy performed immediately. Critical microscopic evaluation of the intestinal mucosal surface is only possible when autolysis has been minimised. Similarly, artifact resulting from exposure of the tissues to water, scraping, or rough handling must be

avoided. After the bird has been killed, the investigator should open the abdomen, isolate the duodenum, and remove a cross-section of descending and ascending duodenum including pancreas. The duodenal segments should be opened and placed into fixative (10% buffered neutral formalin is adequate for most evaluations).

Any ingesta present in a 2-cm segment of jejunum at Meckel's diverticulum should be gently expressed, the segment should then be removed, and placed into fixative. Fixation will be adequate without the intestine being opened if the intestine is empty or contains fluid only. An adjacent area of jejunum is then opened, the mucosa blotted on absorbent towelling to remove any adhering ingesta, and touch impressions are made on one half of two microscopic slides. The slides can be briefly air-dried, and blotted on towelling to remove any thick areas, and then air drying of the slides completed. An area of the mucosa should be scraped and placed into the centre of a few drops of warm physiological saline. A coverslip is placed on the mucosal scraping and gently pressed to spread the sample. The saline and the sample should not be mixed together before placing the coverslip.

For immunofluorescent procedures and virus isolation, two 2-cm segments of jejunum should be removed from the opposite, unsampled, cut end and snap frozen in liquid nitrogen or on dry ice. Alternatively, a 0.5-cm ring can be frozen directly in cryostatic mounting medium for immunofluorescence.


A 2-cm segment of ileum and adjacent proximal caeca should be placed into fixative. Adjacent pieces of ileum can be frozen for virus isolation and immunofluorescence. Another segment of the ileum is opened and mucosal touch impressions are made on the other half of the microscopic slides. A wet smear from the ileum or caecum is not necessary. The remaining intestinal segments should be opened to examine for lesions, and then those from Meckel's diverticulum to the cloaca pooled, frozen and stored at - 7 0 ° C in case experimental studies are required. Samples collected in the field should be kept refrigerated, but not frozen, and submitted to a diagnostic laboratory as rapidly as possible.

Samples of lymphoid organs, especially the bursa of Fabricius, liver, and any gross lesions, should also be collected and placed into fixative. If turkey Coronavirus is suspected, one half of the bursa should be frozen for immunofluorescence. Weighing of the birds and lymphoid organs may be desirable to determine their relative weights. Location and removal of all of the thymus is not necessary. The authors have found that the left lobes proximal to the thyroid gland are readily accessible and constitute approximately 5 8 % of total thymic tissue.

Jejunal wet smears should be kept warm and evaluated as soon as possible. As the saline cools, motility of protozoa will diminish or cease, rendering detection more difficult. The interface between the mucosal scraping and saline should be examined for motile protozoa under the low power lens of a microscope. By not mixing the saline and mucosal scraping together, the organisms can be easily observed as they swim from the sample into the surrounding fluid. If motile protozoa are seen, their identity can be confirmed by using the high power lens. Cochlosoma moves at a moderate pace with seemingly random spinning, twirling, rotational movements. The large ventral sucker is clearly evident giving the organism the appearance of a snail shell. Spironucleus has a rapid, darting, directed movement. The appearance is suggestive of a mouse with a pointed nose and trailing tail. Trichomonads display a jerking motion because of their undulating membranes, but show little movement, often appearing to have the distal ends fixed on pieces of debris. The undulating membrane suggests fingers waving within a sock or thin mitten. Detection of protozoa in the mid-jejunum, especially if numerous, is considered to be more significant than detection in the lower gut or in very low numbers. Some protozoa are normally found in the caecum. In addition, small, rapidly darting spiral bacteria, typical o f Campylobacter, may be identified. In a well-prepared wet smear, the mucosal sample will consist of a confluent layer of the sheared tips of villi. Identification of coccidia, Cryptosporidia and long-segmented filamentous bacteria in the mucosal sample may be possible.

One of the mucosal impression smears should be stained with Gram's stain and the other with a cellular stain such as Wright's, Giemsa, or a combination of the two. High numbers of large Gram positive bacilli are indicative of clostridial proliferation and possible necrotic enteritis. Protozoa may be seen in the Gram-stained smear but the resolution is not as good as in the cytological smear. Any motile protozoa observed on wet smears, coccidia and Cryptosporidia, together with the types and numbers of bacteria can be determined by examining the cytological smears. The number of inflammatory cells, especially heterophils, will provide an indication of the degree of enteritis. Haemolysis of erythrocytes is usually seen when high numbers of clostridia-like bacteria are present. Additional ileal smears can be stained with auramine-0 or a modified acid-fast stain to demonstrate Cryptosporidia, which are most numerous in the ileum. If indicated, caecal smears can be prepared in the same way as jejunal and ileal smears to examine for coccidia.

Feed should be analysed for ingredients that may cause diarrhoea, such as sodium and mycotoxms. Similarly, water should be checked for levels of sodium, sulphates, nitrates and magnesium. The composite sample of droppings is examined for viruses and bacteria as described below, and for protozoan cysts using a faecal flotation. Faecal samples can be archived for possible future use by addition of an equal amount of 2 0 % dimethyl sulphoxide in physiological saline, mixing thoroughly, dispensing in 1 ml amounts, snap freezing, and storing at -70°C . Levels of digestive enzymes, especially the disaccharidases found in the enterocyte brush border, can be determined using frozen intestinal segments. The levels of these enzymes are decreased in birds affected by PEC (4). The ability to absorb D-xylose is another physiological parameter that can be measured to determine the status of intestinal function (40) . Paired serums are examined for antibodies against known enteric pathogens. For discovery of new pathogens, convalescent serum can be used against tissues collected during the acute stage of the disease in an immunofluorescent assay (64).

Isolation and identification of infectious agents Methods used for detecting enteric viruses involved in PEC vary considerably depending on the agent of interest (81).

Diagnosis of infection with Rotavirus is generally based on detection of viruses in faeces using EM, detection of viral antigens using FA procedures, or demonstration of RNA of Rotavirus in faeces using PAGE (62). Detection of Rotavirus in faeces by direct EM is a sensitive diagnostic approach and this method will detect rotaviruses of all serogroups (94) . Immune EM and FA require specific antisera; however, these procedures may be used to identify specific serogroups. Detection of RNA of Rotavirus in faeces using PAGE has been demonstrated to be almost as sensitive as EM (94).


Diagnosis of TCV infection requires detection of virus, viral antigen, or virus-specific antibodies using virus isolation, FA, EM, or serology (69). Virus isolation may be accomplished by amniotic inoculation of embryonated turkey eggs, with subsequent identification of viral antigen in embryo intestines using FA procedures. Electron microscopy may be used to detect TCV in intestinal tissues and bursa of Fabricius of infected turkeys; however, identification can be confused by the presence of cell membrane fragments ('fringed particles') that may resemble Coronavirus particles (42) . Immune EM is a preferable procedure in that TCV may be specifically identified with TCV-specific antiserum. Serological detection of infection may be accomplished by an indirect FA procedure using frozen sections of infected turkey embryo intestines as a source of antigen. In areas where serology for TCV infection is unavailable, the commercial enzyme-linked immunosorbent assay (ELISA) for infectious bronchitis of chickens may be useful. A low level of cross-reaction occurs in the test, thereby detecting high titres to TCV.

Diagnosis of turkey enterovirus infections is most commonly accomplished by EM examination of droppings or intestinal contents (66). Both direct and immune EM procedures for detection of turkey enteroviruses have been described. In addition, diagnosis may be accomplished by detection of viral antigens in tissues or faeces using FA or antigen-capture ELISA.

Infections with Astrovirus may be diagnosed either by direct EM or immune EM of droppings or intestinal contents (79). Using direct EM, Astrovirus spp. may be confused with other small round viruses, such as Enterovirus; thus, diagnosis depends on identification of particles with characteristic size and surface structure. Immune EM is therefore the preferred diagnostic procedure.

Diagnosis of infection with Salmonella is dependent on laboratory culture and identification. Clinical samples that are most commonly recommended for laboratory culture are the caudal ileum, caeca, caecal tonsils, and caecal contents. However, samples of droppings or intestinal contents and extra-intestinal tissues (liver, spleen, heart or kidney) may also be cultured; many paratyphoid Salmonella species may become systemically disseminated in infected turkeys (39) .

Standard methods for isolation and identification of Salmonella generally follow the sequence shown below:

a) enrichment to encourage growth of small numbers of Salmonella in clinical samples

b) plating on selective agar media to obtain isolated colonies

c) identification based on biochemical and serological tests.

Several enrichment media are commonly used including tetrathionate broth and selenite-cysteine broth. Selective media used include brilliant green agar, xylose-lysine-desoxycholate (XLD) agar, bismuth sulphite agar and

Hektoen enteric agar. Culture media are generally incubated for 2 4 h at 37°C, but incubation of enrichment media at elevated temperatures (42°C) is often used to suppress growth of other bacteria in samples containing faecal material (intestinal tissues and droppings/intestinal contents).

Biochemical and serological tests are used to confirm the genus identity and serotype of bacteria isolated on selective agar plates. Biochemical identification is based on the fermentation pattern of the isolate using a variety of carbohydrates. Serological identification is usually determined using slide and/or tube agglutination tests (102) .

Diagnosis of infection with E. coli is dependent on laboratory culture and identification coupled with identification of virulence factors (70) . Cultivation of E. coli is usually performed using selective media such as eosin-methylene blue agar, MacConkey agar, or tergitol-7 agar. Presumptive diagnosis may be made from the character of colonies produced on these plates; definitive diagnosis is based on biochemical reactions. Enteropathogenic strains may be identified by occurrence of characteristic attaching and effacing lesions in intestines (Fig. 15), detection of the eae gene of E. cóli using polymerase chain reaction procedures, and absence of enterotoxin and shiga toxin production (70). Serotyping is rarely sufficient for identifying enteropathogenic E. coli. In addition, this method is expensive and only a small number of reference laboratories can perform the test reliably.

Cryptosporidium can be diagnosed by identifying oocysts in intestinal tissues, intestinal contents, or samples of droppings (22). In histological sections stained with haematoxylin and eosin, parasites appear as basophilic bodies of 2 pm-6 µm, along the surface of intestinal epithelial cells (Fig. 16). Diagnosis may also be accomplished by identification of oocysts in intestinal contents and droppings using concentration procedures followed by microscopic detection. Various microscopic procedures are available for identifying oocysts of Cryptosporidium. These include bright-field or phase contrast microscopy, acid-fast staining, negative staining, or staining with auramine-0 and examination with a fluorescence microscope.

Public health significance Other than the relationship between bacteria, such as Campylobacter, Salmonella, and certain strains of E. coli, which are associated with PEC and can cause food-borne illnesses in people, and concerns regarding the transfer of antibiotic resistance between those micro-organisms infecting animals and those infecting humans, no public health risk is known to arise from PEC. No significant illness has been reported in farm workers, growers, service personnel, researchers, or others who have been in contact with affected flocks or birds, and no evidence exists to suggest that pathogens associated with the various forms of PEC can infect

580 Rev. sci. tech. Off. int. Epiz, 19 (2)

humans. However, this is an area of limited knowledge and the lack of information should not be interpreted as evidence that infections do not occur. For example, cryptosporidiosis caused by C. baileyi, a pathogen that can occur in turkeys, although more frequently found in chickens, was identified in an immune compromised individual (27) . Any potential pathogen, especially those that occur in the digestive tract of food-producing animals, should be considered as a possible public health risk and the necessary precautions should be taken during processing to prevent carcass and product contamination.

Prevention and control methods Management of PEC is primarily supportive. An integrated approach is required because no single factor can control this enteric problem. Therefore, current intervention strategies incorporate both drug and management components, and actions are required before and after an outbreak has occurred. Clinical evidence shows that eggs from new breeder hens (less than seven weeks in production) should not be used for at-risk farms because smaller poults are more susceptible to PEC.

Given the viral nature of PEC, no simple solution exists. Supportive care is needed at the onset of clinical signs. This includes water-soluble multiple vitamin preparations with vitamin E at twice the recommended level (because of the antioxidant properties of vitamin E, which help stabilise enterocytes), and water-soluble antibiotic treatment. Response to antibiotics can be variable. Generally those that possess activity against Gram positive bacteria, such as tylosin, lincomycin, or penicillin, are most effective. Once the disease is present, antibiotic treatment may help speed improvement (2, 99) and limit mortality, but typically has little impact on stunting. Broad-spectrum antibacterials are not recommended during the first ten days of age because of a possible negative impact on normal intestinal microflora. Probiotics have been used without much success. If coccidia are present, the on-going anticoccidial programme must be evaluated.

Palliative care is not complete without sustained efforts to optimise the environment. If birds show huddling, a slight increase of ambient temperature (1°C-2°C) is often required. For example, higher mortality due to PEMS has been observed at lower temperatures during brooding, even under dry conditions. When litter moisture increased, mortality abo increased (31). Every effort should be made to keep the litter as dry as possible (using ventilation, tilling, or top dressing with fresh litter). Various lighting programmes did not affect the mortality or stunting that occurs in PEMS (93).

Anorexia accounts for much of the stunting and growth depression, and any action that will increase feed intake should have a positive effect on PEC. Remixing feed,

frequently walking through the flock to encourage movement, manually turning feed lines on and off, top dressing feed with rolled oats, whole grain, confectionery sprinkles, calf milk replacer, etc., and even using flashing strobe lights have all been used to encourage sick poults to eat. Alimentation through water is often more successful, as the birds continue to drink even after ceasing to eat. Electrolytes such as the World Health Organization rehydration solution (3.5 g sodium chloride, 2.5 g sodium bicarbonate, 1.5 g potassium chloride and 20 g dextrose per litre) in the drinking water may reduce the effect of PEC on a flock. However, efforts to reduce the impact of PEMS by adding sucrose and potassium phosphate to the drinking water only delayed mortality (28). Care is needed when adding carbohydrates to drinking water because of bacterial multiplication. Milk replacer has been used to treat coronaviral enteritis but can cause problems in automatic water lines (69). Supplementation with betaine, an osmoregulator, may help to control diarrhoea (34).

The feed used must be of top quality. A starter feed with a low percentage of protein (24%-26%) is recommended. This protein level contributes to maintaining the pH of the upper small intestine, which may help to preserve intestinal integrity. The response of the poult to PEC is influenced by nutrition. To be effective, diet modifications should be instigated early in the outbreak. Poults fed complex diets containing several protein sources seem to perform better. Highly digestible, nutritious ingredients (e.g. fish meal [if well stabilised with antioxidants] or dried whole egg powder) can alleviate some of the impact of the disease (13, 34) . However, this may not be economically or technically practical. Such diets are costly and most feed mills are not able to produce small special batches of feed for isolated cases. However, changes in pellet size and texture of crumbles may be possible and beneficial. Special rations with concentrated essential nutrients may be useful to partially counteract the lowered feed intake of affected poults. Another approach is to segregate the birds from an affected flock into groups and feed each group according to size and stage of development rather than age. Runted birds (body weights of less than 50% of average body weight is a useful measure) should be rigorously culled. This will improve flock uniformity and may even permit the remaining birds to perform better.

A good relationship between 'service people' (personnel employed by poultry production companies who supervise production and move between farms), veterinarians and nutritionists is paramount to shorten delays in improving the environment, the management and the nutrition or feed presentation for birds affected by PEC.

Prevention Although research is being conducted on potential vaccines, and maternally-acquired antibodies have been shown to aid in the protection of poults against infections with Rotavirus during the first week of life (92), the problem of PEC is not likely to be solved in the near future. Therefore, the best


method to control PEC is to prevent transmission. Given the infectious nature of the disease, significant efforts have been targeted at improving cleaning, disinfection and biosecurity (in particular, limiting movement of people from farm to farm) (38).

The resistance of the viruses associated with PEC is incompletely known. Of six different treatments, only formaldehyde inactivated agents that cause PEMS (18). McLoughlin et al. were able to eliminate runting and stunting syndrome from contaminated premises by depopulation followed by thorough cleaning and disinfection (60). Performance is generally better when only one age group of turkeys is kept on a farm, such as all-in/all-out operations or separate brooding and finishing farms. In addition, better flocks tend to be produced in farms which allow at least two weeks before restocking, including at least a full week after cleaning and disinfection. However, immediate removal of litter from a contaminated house is not recommended. The relatively high number of organisms present in the litter immediately after the flock is removed increases the chance of dispersal and spread to other nearby flocks during litter removal. Litter should not be taken out of the house for at least one week after the flock has been removed, to allow a reduction in numbers of pathogens through natural mortality. When used litter from a quarantined farm is removed, great care needs to be taken to spread this litter away from at-risk farms (those with young birds would be at highest risk). Vehicles used to move the litter (which should be covered) must be thoroughly washed and disinfected afterwards. Serious attention needs to be devoted to developing cost-effective technology for rearing poults on raised slats or wire floors. In limited trials, birds raised in this way have superior performance compared to hatchmates reared on litter (T. Knapp, personal communication).

Water sanitation is also important. Chlorination and filtration post treatment should be considered. Superchlorination of the tank medicator, lines and drinkers should also be performed before restocking.

A strict rodent control programme must be implemented. Each barn should also be bird-proof to prevent direct contact between wild birds and turkeys. The vicinity of the farm should also be evaluated and nesting areas for wild birds eliminated. Circumstantial evidence suggests that flies and darkling beetles can serve as mechanical carriers and transmit organisms from one flock to another. Control programmes against these insects are essential, and should be co-ordinated regionally to be successful. Keeping curtains raised and using power ventilation has been found to be effective in reducing flies in bird houses.

Dead turkeys are a reservoir for disease. The carcasses need to be removed from the flock promptly and buried, incinerated, composted, or disposed of in an approved manner.

Severely affected farms are normally placed under quarantine. Depopulation should also be considered, primarily if co-ordination with farms in the same area can be achieved. A key element of quarantine is a posted sign that prominently declares that the farm is under quarantine. Visits by non-farm personnel must be minimised. All such visitors should take extra precautions if they must enter the flock. A stringent biosecurity protocol must be followed when entering and exiting. Traffic patterns must also be modified in order to limit any traffic carrying or travelling to susceptible birds. Every individual likely to visit the farm ('service people', veterinarians, truck drivers, employees of utilities companies, etc.) must be informed as soon as a farm is placed under quarantine. All equipment or material should remain on the farm. When birds are sent to the processing plant, the route from the farm to the processing plant should be designed, as much as possible, to avoid driving past at-risk farms. Processing of these birds at the end of the week is also preferable, as this allows time for cleaned and disinfected equipment and trucks to dry completely before being reused.

Regional level management Efforts to control disease in regions of high farm, concentration cannot rely solely on single farm level prevention methods. A regional perspective is essential to co-ordinate the movement of service personnel (e.g. contractors involved in live haul or disposal of dead birds). In high-density areas, all-in/all-out production or off-site brooding will probably become the only viable option. An added key ingredient will be stringent, non-compromising, biosecurity. Finally, the frequent emergence or re-emergence of infectious diseases in poultry in the context of global trading will require enhanced communications among poultry companies in the same region. Regional depopulation/repopulation programmes may be required to effectively control infectious diseases, particularly enteric disorders. Zone raising (a regional all-in/all-out system) may eventually be required.

International trade considerations Day-old turkey poults At least some of the infectious agents associated with PEC occur wherever turkeys are produced, and the complex has a world-wide distribution. However, information is limited and additional study is needed on the specific organisms, including distribution, and role in intestinal diseases of young turkeys. Transmission is predominantly faecal-oral. At present, Salmonella is the only organism associated with PEC that is known to be vertically transmitted. Breeder flocks, hatching eggs and poults should be tested to ensure freedom from Salmonella, particularly S. enterica subsp. arizonae, and the serovars S. Typhimurium and S. Enteriditis. None of the viruses involved in the complex have been shown to be vertically transmitted, but several have only recently been


identified and their biology is unknown. No epidemiological

evidence exists to suggest that PEC is associated with specific

breeder flocks.

Finished product Turkey meat or other products destined for consumption are not a source of the infectious agents that cause PEC in young turkeys. However, these products should be wholesome and not contaminated with any bacteria associated with PEC, which could potentially cause food-borne illness in humans.

Acknowledgements Drs D.P. Wages and E. Gonder provided invaluable insights on control, prevention and treatment of PEC. Dr D. Carver shared her findings on the comparative epidemiology of

PEMS and turkey Coronavirus. Dr M. Stringham contributed

information on pests as vectors of PEC and their control. The

authors express their appreciation to Dr S. Clark and Roche

Animal Nutrition and Health for supporting a conference on

coronaviral enteritis and related disorders, the proceedings of

which provide the poultry industry with the most

comprehensive, up-to-date source of information on enteric

diseases of turkeys (20).

Complexe entéritique du dindonneau H.J. Barnes, J.S. Guy & J.-P. Vaillancourt

Résumé Le « complexe entér i t ique du d indonneau » est un terme génér ique qui englobe diverses maladies intest inales infect ieuses des jeunes d indons. Certaines maladies, te l les que l 'entéri te à Coronavirus et le syndrome de malabsorbt ion sont re lat ivement bien décr i tes tandis que d'autres, comme l 'entéri te virale t ransmiss ib le , le retard de cro issance du d indonneau et le syndrome de mortalité par entéri te des dindonneaux, restent mal connues. Toutes les formes du complexe entér i t ique du dindonneau sont mul t i factor ie l les, t ransmissib les et infect ieuses. L'arrêt de cro issance et les t roubles de l 'assimi lat ion dus à l'entérite sont les signes cl in iques les plus marquants. Dans les formes plus graves, des cas de malabsorpt ion, une inhibi t ion de l ' immunité et des cas de mortal i té sont s ignalés. On observe des lésions macroscop iques et mic roscop iques dans toutes les formes d 'entér i te, mais elles ne semblent guère spéc i f iques. D'autres lésions peuvent être observées, selon l 'agent responsable. La pathogénie implique essent ie l lement : a) une al térat ion de la muqueuse intest inale, généra lement par un ou plusieurs v i rus in fectant les entérocytes ; b) une inf lammation ; c) une prol i férat ion d'agents secondai res , habi tue l lement des bactér ies. Des fac teurs non infect ieux interagissent avec les agents infect ieux, modulant ainsi l 'évolution et la gravité de la maladie. La diarrhée résulte essentiel lement des t roubles osmot iques liés à une mauvaise digest ion et à une malabsorpt ion, mais elle peut également avoir une composante sécréto i re . La t ransmiss ion se fa i t essent ie l lement par voie féca le-ora le . Aucune inc idence grave sur la santé publ ique n'a été reconnue ou suspectée. La prophylaxie consis te à él iminer les agents infect ieux des locaux contaminés et à empêcher leur in t roduct ion dans les élevages. Pour ce fa i re , il faut mettre en place des programmes ef f icaces de net toyage, de désinfect ion et de bio-sécur i té . La condui te en bande unique et la séparat ion des unités de couvaison et de f in issage sont également uti les. La lutte contre la maladie peut nécessi ter une coordinat ion régionale entre tous les éleveurs de dindons, surtout lorsque la product ion est t rès concen t rée , ainsi qu'un programme de quarantaine pour les formes les plus graves du complexe


entér i t ique du d indonneau. Il n'existe aucun vacc in ou mesure spéc i f iques pour lutter contre les agents responsables de ce complexe. Il existe, en revanche, un t ra i tement pour la composante v i ra le , tandis que les ant ib iot iques, no tamment ceux qui sont e f f i caces cont re les bactér ies qui prennent la co lorat ion de Gram, peuvent cont r ibuer à réduire l ' impact des in fect ions bactér iennes. Il semble que le complexe entér i t ique du d indonneau soit présent dans tous les élevages commerc iaux de d indes, mais les in format ions à ce sujet sont incomplè tes et la répart i t ion des divers agents associés à ce complexe reste en grande part ie méconnue. La maladie occas ionne de lourdes pertes économiques , essent ie l lement dues au fa i t que les dindes ne peuvent expr imer tou t leur potent iel génét ique.

Mots-clés Astrovirus - Coronavirus - Dindes - Entérovirus - Escherichia coli - Infections mixtes -Maladies aviaires - Maladies digestives - Maladies entéritiques - Maladies des volailles - Maladies d'élevage intensif - Rotavirus.

Complejo de enteritis del pavipollo H.J. Barnes, J.S. Guy & J.-P. Vaillancourt

Resumen "Comple jo de enter i t is del pav ipo l lo" (CEP) es un té rmino general que designa las enfermedades intest inales in fecc iosas de los pavos de corta edad. A lgunas de estas enfermedades están bien caracter izadas (enter i t is coronav i ra l o síndrome de mala absorc ión , por e jemplo) , mientras que otras conservan aún su parte de mister io (como la enter i t is v iral t ransmis ib le , la inh ib ic ión del c rec imiento del pol luelo o el síndrome de morta l idad de pavipol los por enteri t is). Todas las fo rmas del CEP son mul t i factor ia les, t ransmis ib les e in fecc iosas. Entre sus mani fes tac iones cl ín icas destacan el ret raso del c rec imiento y el escaso aprovechamiento de los al imentos provocados por la enter i t is. En los casos más graves se han descr i to enanismo, d is func iones del s istema inmuni tar io y mor ta l idad. Aunque se observen todo t ipo de lesiones macro y m ic roscóp icas propias de la enter i t is , éstas t ienden a ser inespecí f icas, y en func ión del agente in fecc ioso habrá también otras lesiones. La patogénesis más común t rae aparejada la s iguiente s intomatología: a) a l terac iones de la mucosa intest inal , debidas en general a la in fecc ión de enteroc i tos por uno o más v i rus; b) in f lamac ión; c) pro l i ferac ión de agentes in fecc iosos secundar ios , genera lmente bacter ias. Hay fac to res de t i po no in fecc ioso que in teracc ionan con los agentes in fecc iosos, modulando así el curso y la gravedad de la en fermedad. La d iarrea, a la que se atr ibuye un carác ter pr inc ipalmente osmót ico asoc iado a la mala digest ión y la mala absorc ión , puede tener además un componente secretor. La en fermedad, que de momento parece i r re levante en términos de salud públ ica, se t ransmi te bás icamente por vía feca l /o ra l . Las medidas prof i láct icas se basan en el pr incipio de el iminar el agente in fecc ioso de las insta lac iones contaminadas y prevenir su in t roducc ión en las bandadas. Para ser e f icaces, esas medidas deben t raduc i rse en un programa adecuado de l impieza, des in fecc ión y segur idad b io lógica. También resul tan de ayuda la ap l icac ión de un sistema de ro tac ión comple ta de incubadora (all-in/all-out) o la separac ión de las unidades de incubac ión y de engorde. Pero luchar ef icazmente contra la enfermedad exige la coord inac ión a escala regional de todas las granjas productoras de pavos (espec ia lmente


cuando la producción esté muy concentrada), así como un programa de cuarentena para las formas más graves del CEP, considerando sobre todo que no existen vacunas ni medidas específicas para combatir a los microorganismos involucrados. En caso de infección vírica, el tratamiento constituirá una simple medida de apoyo, mientras que el uso de antibióticos, sobre todo cuando son eficaces contra bacterias grampositivas, puede ser útil para reducir la incidencia de infecciones bacterianas. Aunque falten pruebas concluyentes al respecto, y además se ignore en buena medida la distribución de los diversos microorganismos relacionados con el CEP, de la información existente puede deducirse que esta patología está presente en todas las instalaciones industriales de cría de pavos. El CEP provoca pérdidas económicas muy cuantiosas, debidas principalmente a la incapacidad de los pavos para realizar plenamente su potencial genético.

Palabras clave Astrovirus - Coronavirus - Enfermedades asociadas a la producción intensiva -Enfermedades aviares - Enfermedades de las aves de corral - Enfermedades digestivas -Enfermedades intestinales - Enterovirus - Escherichia coli - Infecciones mixtas - Pavos -Rotavirus.

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