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Viral Agents Associated with Poult Enteritis and Mortality Syndrome The Role of a Small Round Virus and a Turkey Coronavirus Author s M Yu M M Ismail M A Qureshi R N Dearth H J Barnes and Y M Saif Source Avian Diseases Vol 44 No 2 Apr Jun 2000 pp 297 304 Published by American Association of Avian Pathologists Stable URL http www jstor org stable 1592543 Accessed 21 06 2014 23 44 Your use of the JSTOR archive indicates your acceptance of the Terms EID50 mean embryo infective dose GI gastrointestinal IEM immune electron microscopy PEMS poult enteritis and mortality syndrome PTA phosphotungstic acid SPF specific pathogen free SRV small round virus TCV turkey coronavirus Poult enteritis and mortality syndrome PEMS is a transmissible disease commonly af fecting young turkeys between 1 and 4 wk of age The disease is characterized by diarrhea anorexia growth depression immune dysfunc tion and high mortality 1 PEMS has caused significant losses to turkey producers in North Carolina and several other southeastern states DCorresponding author 297 This content downloaded from 195 78 109 12 on Sat 21 Jun 2014 23 44 21 PM All use subject to JSTOR Terms and Conditions 298 M Yu et al Fig 1 Turkey coronavirus detected by immune electron microscopy in intestinal samples from poults affected by the poult enteritis and mortality syndrome Bar 100 nm since its recognition in 1991 2 Although ex tensive research has been done the etiology of the disease remains controversial Coronavirus 6 and some other unidentified virus particles 4 as well as bacteria like Escherichia coli 3 have been implicated in the disease but no eti ologic relationship has been definitely estab lished In the present study we detected several viruses in intestinal samples from PEMS affect ed poults including a turkey coronavirus TCV and a small round virus SRV The pathogenicities of the TCV and SRV were stud ied in experimental infection trials to elucidate their roles in the etiology of PEMS MATERIALS AND METHODS Intestinal samples Thirty six samples of gastro intestinal GI tracts from 1 to 4 wk old poults af fected by PEMS were submitted to our laboratory over a 1 yr period The samples were collected from 36 turkey flocks in 22 turkey farms in the states of North Carolina and Indiana Samples were received frozen and contained one to six whole GI tracts Antisera Convalescent sera were collected from poults that recovered from PEMS at about 2 wk after the onset of the disease and were submitted together with intestinal samples from the same flocks The sera were inactivated at 56 C for 30 min and were then stored at 20 C until used Poults and embryos All specific pathogen free SPF turkey embryos and SPF poults originated from the SPF flock maintained by the Food Animal Health Research Program The flock is free of all rec ognized turkey pathogens including all enteric virus es Immune electron microscopy IEM The GI tracts were thawed and the contents were stripped and homogenized 1 10 w v in 0 05 M Tris HCI buffer pH 7 5 and clarified by centrifugation at 3000 x g for 30 min at 4 C The supernatants were then filtered through 0 45 pLm disposable syringe fil ters Corning Glass Work Corning NY and stored at 70 C until tested Two hundred microliters of the above supernatants were incubated overnight at 4 C with 200 Al of con valescent sera of the same flocks diluted 1 20 in 0 1 M sterile phosphate buffered saline pH 7 4 The di lutions for the supernatants and convalescent sera were chosen on the basis of preliminary trials After incubation the mixtures were ultracentrifuged for 15 min at 160 000 x g through a 50 pl cushion of 30 sucrose with a Beckman tabletop airfuge Pellets were resuspended in 400 pl of sterile distilled water and ultracentrifuged again as previously described without sucrose cushion The pellets were finally resuspended in 25 Al of sterile distilled water One drop of the resuspended solution was placed on a carbon coated 300 mesh Formvarg copper grid and stained with a drop of phosphotungstic acid PTA solution 3 PTA 0 4 sucrose pH 7 0 The grids were examined for viruses at 80 kV with a transmis sion electron microscope Philips 201 Philips No relco Eindhoven The Netherlands Virus isolation and propagation Samples con taining coronavirus only as identified by IEM were This content downloaded from 195 78 109 12 on Sat 21 Jun 2014 23 44 21 PM All use subject to JSTOR Terms and Conditions Viruses associated with PEMS 299 Fig 2 The small round virus detected by immune electron microscopy in intestinal samples from poults affected by the poult enteritis and mortality syndrome Bar 100 nm used for virus propagation About 0 2 ml of the GI tract content supernatant was inoculated into 22 day old turkey embryos via the amniotic sac After 3 4 days of incubation at 37 C the intestines of the em bryos were harvested homogenized and diluted 1 10 w v in 0 05 M Tris HCI buffer pH 7 5 The homogenates were clarified by centrifugation at 3000 x g for 30 min at 4 C The supernatants were then examined by IEM as described above and used for subsequent passages in turkey embryos For the SRV isolation and propagation about 5 ml of the supernatant containing the SRV only as identified by IEM filtered through 0 45 pLm filter membrane earlier was further filtered through 0 22 pLm 0 05 pLm Millipore filter membranes The final filtrate was used for experimental infection or was inoculated into 22 day old SPF turkey embryos via the amniotic sac Intestines of turkey embryos were collected and processed by following the same steps as for TCV propagation Both SRV and TCV were titrated in turkey em bryos by a modification of a procedure described pre viously 22 Turkey embryo intestinal homogenates containing SRV or TCV were diluted serially 10 10 7 in 0 05 M Tris HCI buffer pH 7 5 Each di lution was inoculated to six SPF turkey embryos via the amniotic sac 0 2 ml each Embryos were con sidered infected when the intestines were enlarged at 3 4 days postinoculation DPI The mean embryo infective dose EID 0 was estimated by the method of Reed and Muench 13 Virus RNA extraction and electropherotyp ing Double stranded viral RNA extraction and polyacrylamide gel electrophoresis were done accord ing to the procedure described by Theil et al 20 21 In brief double stranded RNA was extracted from 1 2 g of intestinal contents and was then sub jected to electrophoresis in 7 5 polyacrylamide gel slabs with a vertical gel slab Hoefer SE 600 Phar macia Biotech Piscataway NJ The gels were stained with silver nitrate and photographed Experimental infections All SPF poults used in the different experimental groups were raised in wire cages inside high security isolation rooms pro vided with HEPA filtered intake and exhaust air All the poults were provided with the same feed and wa ter ad libitum Different experimental groups were placed in separate rooms Trial 1 Twenty one 7 day old SPF turkey poults were inoculated orally with 0 1 ml each of the 0 05 pLm filtrate described above The titer of the SRV in the filtrate was not determined The poults were wing banded and placed together with another 14 poults that served as contact exposed poults Another 21 poults were not inoculated and served as unex posed controls Poults were observed daily for dinical This content downloaded from 195 78 109 12 on Sat 21 Jun 2014 23 44 21 PM All use subject to JSTOR Terms and Conditions 300 M Yu et al signs All poults were removed from the cages and weighed individually at 3 4 5 7 11 and 21 DPI Two to six poults from each treatment were euthan atized at 3 4 5 7 11 and 21 DPI and examined for pathologic lesions Intestinal samples were col lected and examined for viruses by IEM Trial 2 Eighty three 5 day old SPF turkey poults were randomly separated into two groups One group consisted of 56 poults 27 of which were orally in oculated with 0 2 ml each of turkey embryo intestinal homogenate containing 103 EID 0 SRV The inocu lated poults were wing banded and placed together with the remaining 29 poults that served as contact exposed poults The other group of 27 poults was kept as noninoculated control Poults were observed daily for clinical signs Droppings were collected at 3 4 5 6 and 7 DPI and used for IEM examination Three to five poults from each group were necropsied at 3 4 5 7 14 and 21 DPI for examinations The poults necropsied at 7 14 and 21 DPI were weighed before euthanasia Trial 3 Thirty six SPF poults were separated into four groups Four 11 day old poults in group I were inoculated orally with 0 2 ml each of turkey embryo intestinal homogenate containing 103 EID 0 SRV Four 11 day old poults in group II were inoculated orally with 0 2 ml each of turkey embryo intestinal homogenate containing 102 EID50 TCV Four 8 day old poults in group III were inoculated orally with 0 2 ml each of turkey embryo intestinal homogenate containing 103 EID50 SRV and the same poults were given 0 2 ml turkey embryo intestinal homogenate containing 102 EID50 TCV at 11 days of age The remaining poults in each of the above groups were not inoculated and served as contact exposed poults Poults in group IV were not inoculated and served as a control group The poults were observed daily for clinical signs Three poults one inoculated and two contacts from each group were weighed and then euthanatized and examined for lesions at 2 and 4 DPI All remaining poults were weighed and eu thanatized at 7 DPI Intestines were collected for IEM examinations Statistical analysis Statistical comparisons of body weights between control poults and challenged poults were performed with the two sample t test The P value was for two tailed t tests RESULTS Virus detection The TCV Fig 1 SRV Fig 2 rotavirus and reovirus were detected either alone or incombination with other virus es Table 1 Rotaviruses were the most fre quently detected viruses followed by the SRV Coronavirus was detected in 8 of 36 samples and reovirus was detected in 5 of 36 samples Table 1 Viruses detected by immune electron microscopy double stranded RNA genome electro pherotyping or both in the gastrointestinal tracts of poults affected by the poult enteritis and mortality syndrome No positive no sampled Positive Virus examinedA percentage SRV 16 36 44 TCV 8 36 22 RotavirusesB 26 36 72 ReovirusB 5 36 14 SRV rotaviruses 8 36 25 SRV TCV 3 36 8 SRV TCV rotaviruses 2 36 6 ASamples originated from 36 flocks on 22 turkey farms and each sample consisted of contents from one to six gastrointestinal tracts BRotaviruses were detected by either IEM or elec tropherotyping or both Reovirus was detected by electropherotyping The SRV and rotaviruses in combination were detected in 8 of 36 samples The SRV and TCV in combination were detected in 3 of 36 samples A combination of TCV SRV and ro taviruses was detected in 2 of 36 samples Avian rotavirus serogroups A D and F 17 and reo virus were detected by electropherotyping Group D rotavirus was detected more frequent ly than the other serogroups data not shown Virus isolation and propagation The TCV was isolated and passaged serially in tur key embryos via the amniotic cavity The TCV had all the typical morphologic features of co ronaviruses Embryos inoculated with TCV showed distinct intestinal lesions The whole GI tract was distended and contained greenish contents The embryos were usually stunted The SRV replicated in turkey embryos in oculated via the amniotic sac The SRV was 30 32 nm in diameter and had no distinguish ing surface features Turkey embryos inoculated with SRV had distended intestines and the giz zards were usually enlarged The titer of SRV in the embryonic intestinal homogenate reached as high as 107 EID50 ml Experimental infections Trial 1 Inocu lated and contact exposed poults showed severe diarrhea with frothy watery droppings starting at 3 DPI and lasting for about 5 days The morbidity was 100 and there was no mortal ity The ceca were severely dilated and filled This content downloaded from 195 78 109 12 on Sat 21 Jun 2014 23 44 21 PM All use subject to JSTOR Terms and Conditions Viruses associated with PEMS 301 Table 2 Mean body weights of 7 day old SPF poults in trial 1 challenged with the small round virus originated from field outbreaks Mean body weight g SD Treatment 3 DPI 4 DPI 5 DPI 7 DPI 11 DPI 21 DPI Control 126 0 4 6 147 6 10 5 165 1 10 3 187 7 12 8 285 7 30 1 530 4 30 4 Inoculated 119 8 4 6 131 1 12 3 133 9 9 8 156 5 13 7 232 6 20 4 407 7 29 8 Contact 130 6 15 4 136 4 6 6 139 8 6 7 150 9 18 2 209 6 24 1 425 7 46 7 significantly different from controls P 0 05 with yellow to brown frothy contents The poults were severely stunted and depressed be ginning as early as 4 DPI in inoculated poults and 5 DPI in contact exposed poults The body weights of these poults remained significantly depressed compared with the controls through out the 21 day experimental period Table 2 Some poults had pinpoint hemorrhages in the thymus that were not seen in the control poults The SRV was detected by IEM in in testinal contents of challenged poults at 3 4 5 and 7 DPI and no SRV was detected after 11 DPI Trial 2 Inoculated and contact exposed poults had severe watery foamy diarrhea an orexia and depression starting at 3 DPI and lasting up to 7 DPI Morbidity was 100 whereas mortality was 5 6 3 54 one inocu lated poult died at 6 DPI and two contact ex posed poults died at 4 DPI The ceca were severely dilated with yellow foamy fluids Other parts of the intestines were also filled with watery foamy contents Some spleens were enlarged Some poults had pin point hemorrhages in the thymus The inocu lated and contact exposed poults showed sig nificantly reduced weight gain at 7 DPI com pared with control poults but their body weight was similar to that of control poults by 14 and 21 DPI Table 3 The SRV was de tected by IEM in droppings between 3 and 7 DPI but not by 11 DPI Trial 3 The poults in the SRV challenged group had symptoms and lesions similar to those of the poults trial 2 The poults in the TCV challenged group had severe enteritis with yellowish loose droppings and were more de pressed and stunted than poults in the SRV challenged group The intestines from the TCV exposed poults were flaccid thin walled and filled with loose contents and the disease was acute with symptoms appearing as early as 2 DPI and lasting for about 4 days Poults in the group challenged with SRV plus TCV had more severe symptoms than poults in the groups challenged with SRV or TCV alone The thymus of some poults in all the chal lenged groups had pinpoint hemorrhages The mortality was as follows SRV group 11 TCV group 11 SRV plus TCV group 22 The body weights of poults in all the chal lenged groups were significantly depressed by 7 DPI whereas the TCV alone and SRV plus TCV challenged groups showed growth depres sion as early as 4 DPI Table 4 The mean weight gains from 2 to 7 DPI in all the three challenged groups were much lower than those in the control group The TCV plus SRV chal lenged group was the lowest followed by the TCV only group and the SRV only group The TCV was detected by IEM at 2 and 4 DPI DISCUSSION We have detected and isolated TCV and SRV from field samples of PEMS affected poults We have also demonstrated that both TCV and SRV were pathogenic and contagious and they initiated diseases similar to PEMS in SPF poults Poults challenged with SRV showed severe diarrhea growth depression and varied mor tality in trials 1 2 and 3 The mortality rates in trials 1 2 and 3 were 0 5 6 and 11 respectively The growth depression in trials 1 2 and 3 was inconsistent In trial 1 significant growth depression occurred from 4 DPI through 21 DPI In trial 2 the growth depres sion was significant only at 7 DPI and the poults had body weight comparable to controls at 14 and 21 DPI In trial 3 the growth de pression was significant in all experimental groups at 7 DPI The variations might be be cause of the difference in virus origin and dos This content downloaded from 195 78 109 12 on Sat 21 Jun 2014 23 44 21 PM All use subject to JSTOR Terms and Conditions 302 M Yu et al Table 3 Mean body weights of 7 day old SPF poults from trial 2 challenged with turkey embryo prop agated small round virus Mean body weights g SD Treatment 7 DPI 14 DPI 21 DPI Control 164 8 36 0 263 0 22 1 422 8 27 9 Contact 119 2 27 1 272 8 24 2 443 0 18 9 Inoculated 142 3 10 7 282 4 32 7 483 5 45 1 significantly different from controls P 0 05 age The SRV induced a disease similar to the mild form of PEMS in turkey poults Conceiv ably under field conditions the SRV infection in a flock of poults could cause significant con sequences The TCV alone was able to cause severe en teritis significant growth depression and mor tality in turkey poults Although the prevalence of TCV as detected by IEM in this study was low this might be because of the low sensitivity of IEM Moreover because the disease caused by TCV was very acute the TCV could be de tected by IEM only during a limited period af ter infection Poults challenged with SRV plus TCV showed the most severe clinical responses and the mortality was the highest Because the mor tality in PEMS is usually high it is highly pos sible that most PEMS outbreaks are caused by concomitant or sequential infections of two or more viruses such as SRV and TCV SRV and TCV may be important agents in the etiology of PEMS Barnes and Guy 1 speculated that TCV or some other viruses may be primarily responsible for initiating the en teritis growth depression and increased suscep tibility to bacterial infections which might ac count for the mortality Our experiments indi cate that SRV and TCV not only initiate en teritis and growth depression but also cause mortality We conclude that a combined infec tion of SRV and TCV could be responsible for the outbreaks of the severe forms of PEMS whereas an infection by SRV or TCV alone can initiate different milder forms of PEMS Rotaviruses especially serogroup D were the most frequently detected viruses This finding was consistent with a previous report 18 In experimentally infected turkeys turkey rotavi ruses did not cause mortality Under field con ditions clinical signs caused by rotavirus infec tion varied in severity with diarrhea and wet litter as the predominant signs 9 In our study rotaviruses were frequently found in combina tion with other viruses such as SRV and TCV Rotaviruses had been detected in combination with other viruses in diarrheic turkey poults in earlier studies 15 18 Their pathogenicity in combination with SRV TCV or both in the etiology of PEMS needs to be evaluated Reovirus was the least detected virus in this study Reovirus has been found in feces of healthy turkey poults in other studies in our laboratory unpubl data Because reovirus can be commonly found in the digestive and respi ratory tracts of clinically normal chickens and turkeys 16 it is unlikely to play an important role in the etiology of PEMS Table 4 Mean body weights mean body weight gains and mortality in SPF poults from trial 3 challenged with the SRV the TCV and the SRV plus the TCV Mean body Mean body weights g SD weight gain g from 2 Treatment 2 DPI 4 DPI 7 DPI to 7 DPI Mortality Control 143 3 33 1 188 7 27 3 283 3 9 61 140 0 0 0 9 TCV 97 3 33 1 108 7 36 2 161 5 19 1 64 2 11 1 9 SRV 146 3 21 9 194 0 52 3 253 2 21 8 106 9 11 1 9 SRV TCV 117 7 41 0 137 3 14 0 136 3 38 0 18 6 22 2 9 significantly different from controls P 0 05 This content downloaded from 195 78 109 12 on Sat 21 Jun 2014 23 44 21 PM All use subject to JSTOR Terms and Conditions Viruses associated with PEMS 303 The SRV or TCV infection caused hemor rhagic lesions in the thymus which raised the question of a possible effect on the turkey im mune system In another study 12 SRV and TCV were thought to predispose the poults to infection by other opportunistic agents such as E coli This may happen via either a per manent or transitory blockage or dysfunction of the immune system The PEMS poults hav