A retrospective study was conducted to determine the occurrence of poult enteritis syndrome (PES) in Minnesota from January 2002 to December 2007. PES is an infectious intestinal disease of young turkeys between 1 day and 7 wk of age and is characterized by diarrhea, depression, and lethargy with pale intestines and/or excessively fluid cecal contents. During the study period, samples from 1736 turkey flocks were submitted to the Minnesota Veterinary Diagnostic Laboratory for disease investigation. Of these, 151 flocks (8.7%) were PES positive. Cases of PES were seen throughout the year with higher prevalence in fall. The PES was statistically associated with age with higher occurrence in poults less than 3 wk of age. Rotavirus, small round virus (SRV), Salmonella, nonhemolytic Escherichia coli, Enterococcus, and Eimeria oocysts were detected alone or in different combinations. Reovirus and adenovirus were found in one flock each. The most commonly identified pathogens were Salmonella (85 flocks) and rotavirus (73 flocks). Of PES-affected flocks, 39 (25.8%), 66 (43.7%), and 37 (24.5%) had one, two, and three or more pathogens, respectively. Rotavirus, SRV, and reovirus occurred mostly in poults of less than 6 wk of age while Salmonella, E. coli, and Eimeria were seen in poults of all age groups. Minimum age for rotavirus detection was in 2-day-old poults. Histopathologically, moderate to severe mixed intestinal villus or lamina propria inflammatory infiltrates, necrosis of distal villus tips in intestinal specimens, and mild to severe lymphocellular depletion in thymus, bursa, and spleen were seen. Antimicrobial sensitivity patterns of bacterial isolates from PES-affected flocks revealed maximum sensitivity to trimethoprim/sulfamethoxazole and ceftiofur and a varying degree of resistance to other antimicrobials.
An acute diarrheal disease with high morbidity and mortality in young turkeys was recorded in the 1950s in the United States and was known locally as mud fever 33. Due to clinical similarities with bluecomb disease of chickens, this disease was named the “bluecomb disease.” Later, with the identification of coronavirus from these cases, the name was changed to “coronaviral enteritis of turkeys” 1,30,36. Subsequently, other intestinal diseases such as maldigestion syndrome, runting and stunting syndrome of turkeys, poult malabsorption syndrome, poult enteritis and mortality syndrome (PEMS), spiking mortality of turkeys, and turkey viral enteritis were described in young turkeys and were included in the poult enteritis complex (PEC) 4. PEC is a general term that describes infectious intestinal diseases of young turkeys and is characterized by enteritis, moderate to marked growth depression, retarded development, increased mortality (as seen in case of PEMS), and impaired feed utilization. A number of viruses (coronavirus, reovirus, astrovirus, small round virus [SRV], rotavirus, and adenovirus), bacteria (Escherichia coli, Salmonella spp., clostridia, Campylobacter, and Enterococcus), and protozoa (coccidia, cryptosporidium) have been implicated in PEC 4.
Turkey growers and poultry veterinarians in Minnesota have observed a disease syndrome in young turkeys that has been referred to as poult enteritis syndrome (PES). PES is defined as an infectious, multifactorial disease of young turkeys usually identified between 1 day and 7 wk of age. Clinical signs include diarrhea, depression, and lethargy. Gross lesions associated with PES include pale intestines and/or ceca with watery contents. Rarely, poults as old as 9 wk of age are affected. This syndrome is not usually associated with the high mortality seen in PEMS that appeared in North Carolina approximately 10 yr ago.
The presence of disease agents with enteric syndromes may vary depending on the geographical location and different management practices on farms. For example, we have not detected coronavirus in PES-affected cases in Minnesota although this virus was the primary agent recognized in PEMS in North Carolina 3,11. Thus, data on disease prevalence may better define the disease epidemiology leading to the development of meaningful approaches to disease control. In this retrospective study, we describe the occurrence of PES and its associated pathogens in Minnesota from January 2002 to December 2007.
MATERIALS AND METHODS
Collection of data
Data pertaining to PES in turkey poults were collected retrospectively from the Minnesota Veterinary Diagnostic Laboratory (MVDL), Saint Paul, Minnesota, from January 2002 to December 2007. Laboratory submissions for serology and avian influenza testing by PCR were not taken into consideration. Live or dead birds or tissues from 1736 turkey flocks were submitted to the MVDL for disease diagnosis. Gross changes in different organs were recorded at necropsy followed by collection of tissue specimens for negative stain transmission electron microscopy and microbiological, histopathological, and other examinations. The data were analyzed with respect to age group involved, monthly and seasonal occurrence, and agents identified.
Identification of enteric viruses
Intestinal contents from affected birds were collected, pooled, and examined for the presence of enteric viruses by negative contrast electron microscopy 10.
Isolation of bacteria
Tissue pools (of visceral organs excluding intestines) from affected birds at necropsy were examined for Salmonella by first enriching the samples in tetrathionate brilliant-green bile-enrichment broth for 24 hr at 37 C followed by subcultivation on brilliant green agar for 24 hr at 37 C. Salmonella-like colonies were selected and confirmed by a slide agglutination test using a polyvalent antiserum against Salmonella. For the isolation of E. coli, the tissues were plated on to MacConkey agar plates followed by incubation at 37 C for 24 hr. Suspect colonies were confirmed using biochemical tests; e.g., triple sugar iron agar, lysine, citrate, and motility-indole-ornithine. For Enterococcus isolation, tissue homogenates were plated on sheep blood agar and Columbia nalidixic acid agar. The plates were incubated at 37 C for 24 hr in a 5% CO2 atmosphere. Suspect colonies were confirmed using biochemical tests including growth in 6.5% sodium chloride and bile esculin agar 26.
Antimicrobial drug resistance
Susceptibility testing of Salmonella, E. coli, and Enterococcus isolates was carried out by the sensititre method (Trek Diagnostic System, Cleveland, OH). Briefly, isolated colonies of bacteria were inoculated in trypticase soy broth followed by 1–2 hr incubation at 37 C. The broth culture was adjusted to 0.5 McFarland standard. One hundred microliters of trypticase soy broth culture was added to Mueller Hinton broth (11 ml) and processed further for antimicrobial drug resistance. Resistance or susceptibility to antimicrobials was determined using criteria established by the National Committee for Clinical Laboratory Standards (NCCLS) 26. The bacterial isolates in intermediate category for an antimicrobial were counted as resistant to that antimicrobial.
Identification of protozoa
Pooled intestinal contents were examined for the presence of protozoa (coccidia) by the fecal flotation technique 40.
Samples of intestine, spleen, bursa, and thymus were collected in 10% neutral buffered formalin from majority of the cases. Thin sections from paraffin-embedded formalin fixed tissue blocks were cut, stained by standard hematoxylin and eosin stain 19, and then examined under a light microscope.
Occurrence of disease
During the study period, 151 (8.7%) of 1736 flocks received at MVDL were characterized as having PES. In 2002, only 2 (0.9%) of 217 flocks were PES positive. In 2007, this number increased to 52 (12%) out of a total 432 flocks (Fig. 1). Rotavirus, Salmonella, nonhemolytic E. coli, and Eimeria oocysts were detected from 2002 onwards, whereas SRV and Enterococcus were detected only after 2005. Reovirus and adenovirus were detected only in one flock each in 2003 and 2006, respectively.
PES was recorded throughout the year with higher occurrence in September and October (14%–15%) than in other months (5%–10%) (Fig. 2). During the study period, 6.4%, 8.0%, 13.0%, and 8.0% flocks were PES positive during spring, summer, fall, and winter, respectively (Fig. 3). Statistical analysis of PES versus non-PES flocks revealed significant association of PES with season (P = 0.004).
Detection of different pathogens in PES flocks
A number of enteric viruses (rotavirus, SRV, reovirus, and adenovirus) were detected alone or in combination with bacteria (Salmonella, nonhemolytic E. coli, and Enterococcus) or protozoa (Eimeria sp.) in PES flocks. Among the different pathogens detected, the proportion of Salmonella and rotavirus was significantly higher than the other pathogens (P ≤ 0.0001).
Rotavirus was the most common virus identified. Of the 73 (48%) rotavirus-positive flocks (Table 1), rotavirus alone was detected in only 15 flocks (Table 2). In the remaining 58 flocks, other pathogens were also detected with rotavirus and Salmonella (14 flocks), rotavirus and Eimeria (10 flocks), and rotavirus and E. coli (seven flocks) being the predominant combinations identified (Table 2). SRV was detected in 26 (17%) flocks (Table 1). SRV alone was detected only in one flock, while in the remaining 25 flocks other pathogens were also detected. SRV and Salmonella (five flocks) and SRV, rotavirus and Salmonella (five flocks) were the predominant combinations (Table 2).
Among bacteria, Salmonella was the most prevalent; it was isolated from 85 (56%) PES-affected flocks (Table 1). Salmonella was detected alone in 17 flocks while in the remaining 68 flocks, it was detected with rotavirus (14 flocks), E. coli (eight flocks), or SRV (five flocks). In 41 flocks, Salmonella was detected in different combinations involving more than two pathogens (Table 2). Salmonella serovars belonged to B4,5; C; C1; C2; E and G serotypes and included: Agona, Anatum, Brandenburg, Bredeney, Cubana, Hadar, Heidelberg, Infantis, Kentucky, Muenster, Oranienburg, Senftenberg, Typhimurium, Uganda, and Worthington. E. coli and Enterococcus were detected in 54 (36%) and 16 (11%) flocks, respectively (Table 1). Both of these pathogens were detected either with rotavirus, SRV, or Salmonella or from flocks that involved more than two pathogens (Table 2). In 44 flocks (29%), Eimeria oocysts were also detected (Table 1). Similar to E. coli and Enterococcus, Eimeria oocysts were also observed in flocks that involved more than one pathogen.
Of PES-affected flocks, 39 (25.8%), 66 (43.7%), and 37 (24.5%) had one, two, and three or more pathogens, respectively. In one flock, as many as six pathogens (rotavirus, SRV, Salmonella, E. coli, Enterococcus, and Eimeria) were detected (Table 2). In nine flocks (6%), these pathogens were not detected and these flocks were referred to as flocks with “enteritis of unknown etiology” (Tables 1, 2).
There was a statistically significant relationship between PES and age of birds (P ≤ 0.0001). Of the 151 PES flocks, 111 (73.5%), 35 (23.2%), and 5 (3.3%) flocks had poults less than 3 wk, 3–6 wk, and 6–9 wk of age, respectively (Fig. 4). The latter two categories (3–6 wk and 6–9 wk) were combined to compare statistically the flocks less than 3 wk and more than 3 wk of age with PES. PES was significantly more in poults less than 3 wk of age as compared to poults more than 3 wk of age (P ≤ 0.001). Of 111 PES flocks with poults less than 3 wk of age, the age of the poults in 27, 67, and 17 flocks was less than 1 wk, 1–2 wk, and 2–3 wk, respectively.
As far as the distribution of various pathogens in different age groups among PES flocks is concerned, rotavirus was frequently identified in PES-affected poults up to 6 wk of age (Table 3). Only one flock older than 6 wk of age was positive for rotavirus. Occurrence of rotavirus was 48% (53 flocks) and 54% (19 flocks) in poults less than 3 wk of age and 3–6 wk of age, respectively (Table 3). Of 27 PES flocks with poults less than 1 wk of age, rotaviruses were detected in 18 (67%) flocks. Rotavirus was detected as early as in 2-day-old poults. SRV was detected in poults up to 6 wk of age but only in one flock with poults greater than 6 wk of age. SRV was detected more in poults less than 3 wk of age (20%) than in poults between 3 and 6 wk of age (9%) (Table 3). Though the rotavirus or SRVs were detected more in poults less than 3 wk of age than in poults more than 3 wk of age, but there was no statistical difference between probability of detection of these two viruses and age of PES-affected poults (Table 3). Reovirus was detected at 1–2 wk of age and adenovirus at 6–7 wk. The probability of Salmonella isolation was significantly related with the age of birds (P = 0.0015) (Table 3). Though Salmonella was isolated from poults of all age groups, isolations were more frequent up to 3 wk of age. The pattern of E. coli isolation was similar to that of Salmonella. In contrast, Enterococcus was isolated from poults of less than 3 wk of age. Analysis of data also revealed that isolation of Enterococcus was significantly higher in PES-affected poults of less than 3 wk of age than in poults older than 3 wk of age (Table 3). Detection of Eimeria sp. also had an association with age with higher occurrence at 3–6 wk (Table 3).
Antimicrobial sensitivity pattern of bacterial isolates from PES-affected flocks
The maximum sensitivity of Salmonella was against trimethoprim/sulfamethoxazole (98%) followed by ceftiofur (57%) and gentamicin (51%) (Table 4). All Salmonella isolates were resistant to clindamycin, erythromycin, novobiocin, penicillin, spectinomycin, sulfathiazole, and tylosin. Resistance of Salmonella to amoxycillin, neomycin, oxytetracycline, streptomycin, sulfadimethoxine, and tetracycline ranged between 51% and 90%. Similarly, E. coli isolates were also sensitive to trimethoprim/sulfamethoxazole (98%) and ceftiofur (55%). Varying degrees of resistance were noted for the remaining antimicrobials. More than 90% of E. coli isolates were resistant to clindamycin, erythromycin, novobiocin, oxytetracycline, penicillin, spectinomycin, sulfathiazole, tetracyclines, and tylosin. In contrast, Enterococcus isolates showed sensitivity to amoxycillin (94%), penicillin (75%), and trimethoprim/sulfamethoxazole (100%). All Enterococcus isolates were resistant to clindamycin, neomycin, spectinomycin, streptomycin, sulfathiazole, and sulfadimethoxine (Table 4).
Thin walled intestines with excessively watery or fluid intestinal contents were the major necropsy findings in all PES-affected flocks. In addition, ceca were distended with loose cecal contents. Moderate to severe mixed (lymphocytic/plasmacytic/heterophilic) villus inflammatory infiltrates in the lamina propria or villi of the intestinal specimens were consistently noted. Sections with necrosis of distal villus tips with mild lymphocytic/plasmocytic villar and laminar infiltrates and clubbing of the distal villus tips were noted in many cases. In most cases, inflammatory infiltrates were diffusely distributed. Moderate numbers of protozoal (Eimeria sp.) organisms were present within intestinal villus epithelium or in the intestinal lumen in cases that were positive for coccidiosis. Changes in thymus consisted of mild to severe regional cortical lymphocellular cortiomedullary depletion and heterophilic inflammatory infiltrates surrounding the Hassel corpuscle. Mild to moderate generalized lymphocellular depletion and multifocal bursal follicular epithelial cyst changes were the major microscopic changes in bursa. Mild to moderate white pulp and/or red pulp lymphocellular depletion was observed in splenic specimens.
This study is based on data obtained from MVDL for the past 6 yr (2002–2007). During this period, 8.7% turkey flocks were found to be affected with PES. Since all PES-affected flocks are not brought to the MVDL for disease investigation, the actual number of flocks affected with PES is unknown, but may be higher. It should be realized that data in this study are from samples that were submitted to the MVDL for disease diagnosis and should not be construed as “active surveillance.” An increase in the number of PES cases from 2002 to 2007 could either be due to increased occurrence of the disease or to increased reporting and laboratory case submission. Higher occurrence of PES in fall is not surprising. For example, most cases of PEC in the southeastern United States were reported to occur between May and September 4. It could be due to more conducive environment leading to rapid multiplication of pathogens detected from PES-affected poults.
Rotavirus and Salmonella were the agents present in the greatest proportion in PES cases. Significantly higher detection of rotavirus and Salmonella from PES cases suggests that the causality path should be further explored. Rotaviruses are the major cause of diarrhea in human infants and several mammalian species. Rotaviruses have also been detected in many avian species like pheasants, turkeys, ducks, chickens, and wild birds, but their role as a causative agent of diarrhea varies 9,18,23,43,44. In turkeys and pheasants, rotavirus has been reported as a cause of diarrhea 18,22,37. Rotavirus has also been associated with PEC 4 and runting and stunting syndrome in broiler chickens 28. Analysis of 10-yr data (1993–2003) of poult enteritis in California turkeys revealed that rotavirus-like viruses (RVLVs; all viruses that have rotavirus-like appearance on electron microscopy) were identified in 46% of enteric virus positive poult enteritis cases 48. In the present study, rotavirus was detected in poults less than 6 wk of age. These results are similar to those of Reynolds et al.35 who monitored four turkey flocks for the presence of enteric viruses from placement until 7 wk of age. During the first 4 wk of life, astrovirus was the most frequently detected virus followed by RVLVs and rotavirus. In one flock, rotavirus and astrovirus were detected in samples collected at 3 days of age. Theil and Saif 46 detected rotavirus in commercial turkeys between the ages of 3 and 35 days. Woolcock and Shivaprasad 48 also detected a majority of RVLVs by 36 days of age. In a longitudinal survey of enteric viruses in eight commercial turkey operations, rotavirus was the only virus detected prior to placement 31. In the present study, the minimum age at which rotavirus was detected was in 2-day-old poults. Further, rotaviruses were detected in 18 flocks that had poults up to 1 wk of age. Detection of rotavirus at such an early age may indicate either fecal contamination of hatching eggs and/or vertical transmission of rotaviruses 31,35. However, further studies are needed to confirm or refute these observations and their relevance to Minnesota PES cases.
SRVs were detected in young poults either alone or in combination with other pathogens. Enterovirus, astrovirus, enterovirus-like particles, and picornavirus have all been referred to as SRVs and their sizes vary from 15 to 30 nm. Though SRVs have distinct morphologies visible by electron microscopy 5, the characteristic physical properties may become less prominent due to the emergence of new subgroups and strains within these viruses 17. Due to this, there are chances of misidentification or misclassification of a virus on electron microscopy 47. Thus, it is often difficult to determine if SRVs detected on electron microscopy are actually enterovirus or astrovirus or other SRVs. We can only confirm the viral genus by using other methods including molecular methods. The chance of these SRVs being astroviruses is greater because we have detected astroviruses from intestinal contents of some of the PES-affected flocks (unpublished data) by RT-PCR while these same samples were negative when examined by electron microscopy. Astroviruses have also been detected from turkey poults suffering from enteritis 12,25,34. Enterovirus-like viral particles were identified for the first time in the feces of poults with diarrhea in the United Kingdom 24. Subsequently, the detection of enterovirus in the feces of young turkeys has been reported by different workers 35,37,38.
Viruses other than rotavirus and SRV associated with PEC are turkey coronavirus 11,16,45, reovirus 15,41, and adenovirus 39,42. Adenovirus in the present study was detected only from one flock. This finding is in agreement with that of Woolcock and Shivaprasad 48 who also reported low positivity of adenovirus in poult enteritis cases by electron microscopy. Reovirus was also detected only from one flock. No coronavirus was detected in the present study indicating that either it is not associated with PES or their concentration was too low to be detected by electron microscopy.
Amongst bacteria, Salmonella and E. coli are important pathogens associated with PEC 4,7,29. Salmonella was isolated from PES-affected poults of all age groups (with more probability of occurrence in younger birds). E. coli was isolated from poults of all age groups but Enterococcus was more prevalent in poults less than 3 wk of age. The role of these bacteria as primary pathogen or as co-pathogen/s with enteric viruses cannot be determined. Perhaps these bacteria prolong the illness after the birds are infected with enteric viruses or they may increase the severity of enteric disease in concert with enteric viruses. Salmonella serovars isolated in the present study are referred to as paratyphoid salmonellae. These serovars can cause disease in young turkeys under stressful conditions and generally colonize the intestinal tract. Salmonella and enteric viruses particularly the rotavirus (if transmitted vertically) can occupy a niche in the intestines of day-old poults and may lead to PES at a very young age.
In the present study, E. coli isolates were of nonhemolytic type and were isolated along with other pathogen(s). E. coli is an opportunistic pathogen and has the ability to cause disease under stress conditions. Enteroinvasive strains and enteropathogenic strains of E. coli (EPEC) have been reported to play a significant role in PEMS 3. Guy et al.13 reported that inoculation of turkey poults with EPEC failed to produce any effect on the birds. However, birds coinfected with turkey coronavirus and EPEC demonstrated diarrhea, stunting, mortality, lymphoid organ atrophy, and marked colonization of EPEC. This finding supports the results of the present study that E. coli was isolated along with other pathogens from PES-affected flocks (52 of 54 flocks). Species of the genus Enterococcus comprise a large proportion of the autochthonous microflora associated with gastrointestinal tracts of animals and are frequently responsible for significant morbidity in predisposed humans 8. We do not know at present the significance of Enterococcus in PES cases. Similar to E. coli, Enterococcus was also isolated along with other pathogens from PES cases.
Most of the bacterial isolates from PES flocks were sensitive to trimethoprim/sulfamethoxazole and ceftiofur. These results are similar to those of Pedersen et al.32 who reported that Salmonella serovars from Danish turkeys were sensitive to colistin, ceftiofur, and amoxycillin with clavulanic acid. Olah et al.27 reported varying degree of resistance to tetracycline, sulfamethoxazole, gentamicin, and streptomycin in Salmonella isolated from turkeys in Midwest region of the United States. Our results are also similar to those of Malik et al.20 who reported that isolates of Salmonella and E. coli from chickens in Minnesota were maximally sensitive to trimethoprim/sulfamethoxazole. Hayes et al.14 reported that Enterococcus sp. isolated from turkey meat in the United States were resistant to tetracyclines and erythromycin. In the present study, Enterococcus isolates were resistant to tetracyclines, streptomycin, clindamycin, neomycin, sulfathiazole, and sulfadimethoxine.
Most cases of coccidiosis occurred in PES-affected poults greater than 3 wk of age. The life cycle of Eimeria, the causative agent of coccidiosis, is typically completed within 4–6 days 21. However, occurrence of disease in a flock also depends upon parasitic load in the farm, presence or absence of coccidiosis in previous flocks, cleanliness at farm, disposal of Eimeria-contaminated litter from a previous flock, rodent activity, environmental conditions, etc. Presence of these factors on a farm or vaccination against coccidiosis may lead to identification of Eimeria sp. in younger poults.
Nine flocks in the present study were categorized as flocks with enteritis of unknown etiology. In these flocks, enteric pathogens identified in other PES-affected flocks could not be detected. This may be due to the presence of a low quantity of enteric viruses in the intestinal contents that could not be detected by electron microscopy. Another factor could possibly be the noninfectious etiology; the managemental or nutritional factors might have led to enteritis in poults.
Statistical relationship between PES and age of birds suggests that young poults are more susceptible to PES and that the syndrome would affect the growth potential of affected birds. Experimental inoculation of 14-day-old turkey poults with intestinal contents (positive for rotavirus, astrovirus, and Salmonella) from PES-affected birds resulted in significantly lower body weights than controls. Overall growth depression due to PES treatment was 31.8% (N. Jindal, unpubl. data). Considering such a growth depression and its occurrence, it would appear that PES causes considerable economic losses for turkey producers. In the present study, 24.5% PES-affected flocks had three or more pathogens. Surprisingly, one of the flock had six pathogens. In such situations, increasingly additive adverse effects (particularly on growth) can be expected. Though we do not know at present up to which age the PES-affected birds will harbor these pathogens, future studies will shed light on the pathogenesis of PES and the ill effects produced at a later stage. Pathological changes in gastrointestinal tract, similar to those observed in the present study, have earlier been reported in poults affected with rotavirus 49, stunting syndrome 2, and spiking mortality syndrome 6. The depletion of lymphocytes in lymphoid organs observed in the present study has the support of Teixeira et al.45 who also observed depletion of lymphoid organs in turkey poults affected with PEC. Such a depletion may predispose the affected birds to secondary infections and may further deteriorate poult health by hampering vaccinal immunity. In conclusion, this retrospective study reveals the occurrence of PES in Minnesota turkeys less than 6 wk of age. Rotavirus, SRV, Salmonella, E. coli, Enterococcus, and Eimeria were the primary pathogens associated with this syndrome.
This work was supported in part by a grant from the Rapid Agricultural Response Fund, University of Minnesota.
Occurrence of various pathogens in PES-affected flocks during 2002–2007
Detection of pathogens alone or in combination: PES-affected flocks during 2002–2007
Occurrence of pathogens in PES-affected flocks of different age groups during 2002–2007
Antimicrobial resistance pattern of bacteria isolated from PES-affected flocks during 2002–2007