The intestinal tract and intestinal contents were collected from 34 stunted, 5-to-14-day-old broiler chicks from eight flocks with runting and stunting syndrome (RSS) in Northern Germany to investigate intestinal lesions and the presence of enteric pathogens with a special focus on rotaviruses (RVs). Seven chicks from a healthy flock were used as controls. Severe villous atrophy was seen in chicks from six flocks with RSS but not in the control flock. Lesions were often “regionally” distributed in the middle-to-distal small intestine. Transmission electron microscopy (TEM), polyacrylamide-gel electrophoresis (PAGE), reverse-transcriptase polymerase chain reaction (RT-PCR), and seminested RT-PCR were used for detection and characterization of RVs. The PAGE allows discrimination of different RV groups, and the RT-PCR was used to verify the presence of group (gp) A RVs. RVs were detected (by all methods) in 32 of 34 chicks from the flocks with RSS. By TEM (negative staining), RV particles were observed in intestinal contents of 28 chicks from the flocks with RSS. PAGE analysis showed four RV groups: gpA, gpD, gpF, and gpG. Group A RVs were detected in four chicks from two flocks with RSS, without intestinal lesions. GpD RVs were detected in 12 chicks of five flocks with RSS, 10 of them with severe villous atrophy. GpF RVs were confirmed in four chicks from three flocks with RSS and in two birds in the control flock. GpG RVs were verified in two chicks from two flocks with RSS, one with, and one without, intestinal lesions. At present, PCR methods are only available for detection of gpA RVs. Using RT-PCR, gpA RVs were identified in samples from 22 chicks including samples of two chicks from the control flock. Statistical analysis revealed a positive correlation between presence of gpD RV and severe villous atrophy in flocks with RSS. The results suggest that gpD RV plays a major role in the pathogenesis of RSS.
During the winter of 2002–03, an unusually large number of cases of diarrhea and runting and stunting syndrome (RSS) occurred in broiler flocks of Northern Germany. RSS is a syndrome with mild clinical signs that causes severe economic problems because flocks do not grow uniformly, and the body weight at slaughter differs widely. Reovirus has received the most attention as a potential etiologic agent for RSS, but so far, it has not been possible to reproduce the disease by experimental inoculation with reovirus isolated from affected chicks (25). Researchers have suggested many other viruses, including rotavirus (RV), as the potential cause of, or as cofactors in RSS (7,9,27,28,35,40).
RVs are the major cause of diarrhea in human infants and several mammalian species. RVs have been isolated from many avian species, e.g. ducks, pheasants, chickens, turkeys, doves and wild birds, but their role as a causative agent of diarrhea varies (10,18,24,32,37,39). In pheasants and turkeys, RV has been reported as the cause of diarrhea with high mortality (12,13,17), and severe disease was induced by experimental infection (43). The clinical relevance in chickens is controversial because symptoms associated with RV infections or with the detection of RV vary between subclinical, diarrhea with growth retardation, and increased mortality (14). Experimental inoculation with RV only induced subclinical infections (23,42).
Infections with rotaviruses from different groups might explain the highly variable clinical signs. RV can be subdivided into groups (gp) A to G by serologic or—at an increasing rate—molecular methods. GpA RV, formerly denominated “typical RVs,” are mainly important as the cause of gastroenteritis in the young of many animal species and in children. RV of gpB to gpG, designated “atypical” or “non-gpA rotaviruses,” have also been found in animals and humans as the causative agents of diarrhea. GpD, gpF, and gpG RVs have been detected in several poultry species (6,14). Only limited serologic data are available from Northern Ireland and Japan. In those studies, antibodies to these viruses were found in 91% of the farms examined (16,38). The detection of gpD RV in chicken feces has been documented in Argentina, Brazil, and China (1,5,41). A detailed longitudinal study was carried out on broiler chicken in Northern Ireland where gpA, gpD, gpF, and gpG RVs were detected (20).
Our study reports the correlation between the identification of RV from different “groups,” together with the histopathologic changes in the small intestine of broiler chicks from flocks suffering from diarrhea or RSS.
MATERIALS AND METHODS
Thirty-four stunted chicks were selected from eight broiler flocks in Northern Germany with a history of RSS and seven normal birds from one flock without clinical signs of RSS (control). All chicks were Ross 308 commercial broilers, which were obtained from nine broiler flocks in Northern Germany. Chicks were kept in closed (eight flocks) or in open-type houses (one flock). In most of the closed houses, the chicks were kept on one floor. Commercial starter diet and water were provided ad libitum.
The clinical signs in the affected flocks were characterized by diarrhea and/or growth depression (runting and stunting syndrome). This syndrome was observed in six flocks, and either diarrhea or growth depression occurred in two flocks. Flocks were treated with Aviapen® (Elanco Animal Health, Eli Lilly Deutschland GmbH, Gießen, Germany) (seven flocks) or Tylan® (Serumwerk Bernburg, Bernburg, Germany) (one flock) to control bacterial intestinal overgrowth and to minimize clinical signs. From each of the affected flocks, four to six chicks were selected for necropsy as soon as clinical signs were observed. Selected animals were between 5 and 14 days old. The chicks from the control flock were 10 days old at the date of necropsy.
The intestines were removed immediately after euthanatizing. The intestinal contents were collected by gentle squeezing. The intestines were ligated at the beginning of the duodenum, at the distal ileum, and at the distal part of the rectum and injected with neutral-buffered formalin until moderately distended. Additional samples were collected from the stomach, pancreas, liver, kidney, spleen, heart, lung, bursa fabricii, and metatarsal joint (in only 16 chicks) and fixed in neutral-buffered formalin.
Microbiologic examination of intestinal contents
The intestinal contents were tested for Escherichia coli and other potential bacterial pathogens using conventional methods. For a semiquantitative bacterial count of the intestine contents, a bacterial loop was plated out on Columbia agar with sheep blood (Oxoid, Wesel, Germany ), incubated at 37 C for about 24 hr, and the bacterial growth was graded on a score basis from + to +++. For E. coli detection, a bacterial loop with intestinal contents was plated out on McConkey Agar No. 3 (Oxoid), incubated for 24 hr at 37 C. The plates were evaluated for the presence of E. coli. Suspected colonies were confirmed with Enterotube (Becton Dickinson GmbH, Heidelberg, Germany). For Salmonella enterica detection, part of the small intestine content was enriched for 24 hr at 37 C in Tetrathionat brilliant-green bile-enrichment broth for microbiology (Merck, Darmstadt, Germany) and then subcultivated on Brilliant Green Agar (Oxoid) and on XLT 4 Agar (Oxoid) again for 24 hr at 37 C. After incubation, both plates were examined for presence of typical Salmonella enterica colonies. For Clostridia spp., a bacterial loop of the small intestinal content was plated out on Anaerobic-Blood-Agar with Neomycin (haipha Dr. Müller GmbH, Eppelheim, Germany) and incubated under anaerobic conditions for 24 hr.
Preparation of intestinal-content samples
From the intestinal contents, a 1:5 suspension was prepared with phosphate-buffered saline (pH 7.4). The suspensions were homogenized in an ultrasonic water bath (US$20, K. W. Meinhardt, Leipzig, Germany). Cell debris and bacteria were removed by low-speed centrifugation (3300 × g, 30 min). The supernatants were stored at −20 C until analysis.
Transmission electron microscopic (TEM) investigations
Samples of intestinal contents were investigated by conventional negative staining. The supernatants were applied to pioloform-carbon–coated, 400-mesh, copper grids; stained with 2% aqueous uranyl acetate in HEPES buffer, and examined by TEM (JEM-1010, JEOL, Tokyo, Japan) at 80 kV accelerated voltage.
Polyacrylamide gel electrophoresis (PAGE)
Intestinal content samples were analyzed for their RNA pattern using PAGE as described by Otto et al. (26). Gels were dried in a GelAir Dryer (Bio-Rad Laboratories, Munich, Germany) and scanned on a GS-700 Imaging Densitometer (Bio-Rad Laboratories). The intensity of the observed viral RNA segments was graded in four categories (+ to ++++).
Reverse transcription–polymerase chain reaction (RT-PCR) for RV gpA RNA extraction
RNA was isolated from supernatants using the nucleic acid–binding system QIAamp Viral RNA Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's instructions. The extracted viral RNA was stored at −20 C.
Primers were selected on the basis of the sequence data of gene segment 6 from chicken RV 02V0002G3 (GenBank accession DQ096805) and chicken RV Ch-1 (GenBank accession X98870) using the software Primer Express (Applied Biosystems, Weiterstadt, Germany). The sequences of the primers and their localization on gene segment 6 are shown in Table 1. The product of the RT-PCR is 493 base pairs (bp) long, and after seminested amplification, a 230-bp amplicon is detectable.
The RT-PCR was conducted as previously described as a one-step RT-PCR using the Ready-to-Go (RTG) RT-PCR-Beads® (Amersham Pharmacia Biotech, Freiburg, Germany) in accordance with the manufacturer's instructions (8). Briefly, 1 μl of each of the primer solutions ARV1 and ARV2 (100 pmol/μl) was added to 10 μl of the viral, double-stranded (ds) RNA, denaturated for 5 min at 98 C, and put in liquid nitrogen. One RTG bead, dissolved with 38-μl deoxyribonuclease (DNase)-free and ribonuclease (RNase)-free water was given to the denaturated RNA–primer mix. The reaction mix was placed in a thermocycler (MiniCycler PTC 150-25H, Biozym Diagnostik, Oldendorf, Germany) and preheated to 42 C. RT-PCR was run according to the following temperature–time profile: 42 C for 30 min, 94 C for 5 min, 35 cycles of 94 C for 30 sec, 54 C for 1 min, 72 C for 1 min, and a final extension at 72 C for 10 min.
For nested PCR, 1 μl of the 1:100-diluted product of the first amplification was used as template in a 25-μl reaction mix consisting of 19 μl of DNase-free and RNase-free water, 0.25 μl of Platinum Taq–DNA polymerase (5 U/μl; Life Technologies, Karlsruhe, Germany), 2.5 μl of 10× Taq–PCR-buffer (Life Technologies), 0.75 μl of 50 mM magnesium chloride (MgCl2), 0.5 μl of 10 mM deoxynucleotide-triphosphate (dNTP-mix; Life Technologies), and 0.5 μl of each primer (ARV1 and ARV3) solution (100 pmol/μl). Amplification was carried out using the following profile: 94 C for 5 min, 35 cycles of 94 C for 30 sec, 54 C for 1 min, 72 C for 1 min, and a final extension at 72 C for 10 min.
For analysis, the products were separated on 1% agarose gels at 130 V for approximately 60 min, stained with ethidium bromide, and visualized under ultraviolet light. As size marker, a 100-bp DNA ladder (PeqLab, Erlangen, Germany) was used.
Transverse sections of intestine were cut and collected at intervals of 10 cm from the duodenum to the ileum. Four sections were placed into each embedding cassette, and cassettes were labeled in ascending order. In addition, sections of cranial and distal ileum, cecum, cranial and distal colon, and nonintestinal tissues were embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin and evaluated in a light microscope at low magnification using an ocular with a size bar. The length of villi was graded as no shortening (>600 μm long), mild shortening (400–600 μm long), and severe atrophy (<400 μm long).
The relation between the presence of viral particles and histopathologic changes was assessed by Spearman nonparametric correlation. The statistical analysis was performed with SPSS Version 10.0 (SPSS Inc., Chicago, IL). A P-value of ≤ 0.05 was considered statistically significant.
TEM investigation revealed the presence of numerous rotavirus particles in 28 out of 34 chickens from the flocks with RSS and in five birds from the control flock (Fig. 1). Reoviruses or parvovirus-like particles were only observed in one sample, respectively. Small, round, less-structured, virus-like particles (SRP), approximately 34 nm in diameter, were observed in 21 samples (Fig. 2).
Because electron microscopy is unable to distinguish among gpA RV and other RV groups, all samples were subjected to testing by PAGE, which is able to discern the RV of all groups. For detection at higher sensitivity, RT-PCR was performed only for gpA RV. The frequency of RV detection in each flock is shown in Table 2. GpA RVs were most commonly detected. They were detected by RT-PCR in 20 chicks and by seminested PCR in 26 out of the 34 birds from the flocks with RSS and in two chicks from the control flock. Only four cases could be confirmed by PAGE (Fig. 3). GpD RV was identified in 12 samples from six flocks with RSS by PAGE, in four cases, together with gpA RV (Fig. 3). Atypical, non-gpD RVs, which are most likely electropherotypes gpF and gpG, were found in six chicks from flocks with RSS and in two chicks from the control flock, respectively (Fig. 3).
As expected, bacterial growth could be detected in all intestinal samples. We found bacterial growth between + and +++, which consisted of different colonies. The most predominant bacteria were gram-positive cocci. The rate of bacterial growth was dependent on whether an antibiotic treatment (Aviapen® or Tylosin®) took place before we euthanatized the birds for examination.
Escherichia coli could be detected in small amounts in nearly all samples of the distal part of the small intestine. The E. coli isolates were agglutinated with anti-avian E. coli O:1, O:2, and O:78 hyperimmunsera from BioVac (Beaucouze Cedex, France), to find out whether E. coli serovars that are specifically pathogenic for chickens might be involved. Escherichia coli O:1, O:2, and O:78 could not be detected in the intestine of the chickens investigated.
Clostridia and salmonellae could not be detected. We concluded, from our bacteriologic findings, that a specific bacterial infection of the intestine as a primary effect could be excluded.
All chicks from the control flock had long slender villi and short crypts (Fig. 4). The length of the villi decreased from duodenum and cranial jejunum to the distal small intestine. The villus/crypt ratio was between 6:1 and 8:1 in the duodenum and cranial to the middle small intestine and around 4:1 in the distal small intestine and ileum.
Animals from flocks with RSS
Lesions were found only in the intestinal tract. They were characterized by moderate-to-severe villous atrophy, fusion of villous tips, and attenuation of surface epithelium (Fig. 5). In severe villous atrophy, villus/crypt ratios were reduced to 1:1 or 1:2 (Fig. 5). Crypt hyperplasia was frequently associated with villous atrophy (Fig. 5) but was also seen independently. An increased number of dilated crypts with flattened epithelium and crypts containing apoptotic cells within the crypt epithelium were seen in chicks with villous atrophy and crypt hyperplasia (Fig. 6A,B).
Flocks could be divided into two groups based on the severity and extent of the lesions: severe villous atrophy was found in flocks A, B, C, E, F, and G; and mild or no villous shortening and crypt hyperplasia were observed in intestines of chicks from flocks D and H. The number of chicks from flocks A, B, C, E, F, and G with severe villous atrophy varied within these flocks from two of four to four of four affected. Lesions were predominantly found in the middle-to-distal small intestine (in 16 chicks), but in 11 chicks, extended into the cranial jejunum and in six chicks into the ileum. Findings are summarized in Table 3. No lesions were seen in the cecum and the colon.
Correlation between intestinal lesions and detection of RV groups
Morphologic lesions showing severe villous atrophy and detection of RVs are summarized in Table 4. Morphologic lesions were not seen in the intestines of the control chicks. In the intestinal contents, no gpD RVs were detected, but gpA RVs (very low titers) and gpF RVs were identified. Mild intestinal lesions were seen in flocks D and H, and gpA RV (low titers and high titers) were detected in all chicks; low titers of gpD RV were found in one chick of flock D only. Severe villous atrophy occurred in flocks A, B, C, E, F, and G. Only gpD RV was detected in flock B (in two out of four chicks), and both gpA RV (low titers) and gpD RV were detected in flocks A, C, E, and G. GpA, gpD, and gpF RVs were found in flocks A and G, and gpA, gpF, and gpG RVs were detected in flock F. The correlation between presence of virus and severe villous atrophy was significant for gpD RV (P ≤ 0.015) but not for gpA RV or SRP.
In this field study, 34 broiler chicks from eight flocks with runting and stunting syndrome (RSS) were examined to determine whether rotaviruses are major contributors to the pathogenesis of RSS. Rotavirus infection in avian species is frequently associated with outbreaks of diarrhea (14), but the association of rotavirus infection and RSS in broilers has not been established (34). The study was designed to investigate morphologic lesions of the intestinal mucosa and the presence of pathogens in the intestinal contents in the same chicks, allowing an evaluation of whether there is a direct association between occurrence of lesions and the presence of infectious agents.
In RSS, transient diarrhea is followed by growth depression (30). Intestinal lesions, characterized by villous atrophy, were frequent in the chicks examined from flocks with RSS but not in the controls. The observed villous atrophy may account for the clinical sign of diarrhea described in RSS. Villous atrophy is commonly induced by enteropathic viruses (e.g., rotavirus, coronavirus, torovirus) in different animal species and humans (2,21,29). These infectious agents have a tropism for the mature enteroabsorptive cells lining the intestinal villi and destroy those cells during their replication. The lack of mature enterocytes decreases the absorption of nutrients and electrolytes as well as the digestion of nutrients by enzymes localized in the microvillous region of mature enterocytes thus causing malabsorption, maldigestion, and osmotic retention of water (4). The severity of clinical signs may vary depending upon the extent of enterocyte loss (i.e., the severity of villous atrophy and the extension of lesions along the small intestine). Mild lesions in the small intestine may even be clinically compensated by an increased absorption of fluids in the large intestine, resulting in subclinical infection (4).
Lesions were observed in about 50% of the chicks from RSS-affected flocks. The lack of lesions in the other chicks might be because not all chicks were susceptible to infection or because they were in different phases of the disease and recovery. Frequently, the lesions were not found throughout the small intestine but had, instead, a regional distribution in the middle-to-caudal small intestine, and extensive sampling of the intestine was necessary to identify them. A regional distribution of lesions has also been described in bovine rotavirus infection with lesions predominating in the upper small intestine and in porcine rotavirus infection with most severe lesions in the middle small intestine (11,31). In poultry, different target areas were observed for gpA and gpD RVs. GpA RV grew optimally in the duodenum of experimentally infected chickens, and gpD RV favored the jejunum and ileum (15). Therefore, lesions in the chicks examined are most likely associated with gpD RV.
Villous atrophy is a transient condition if there is no damage to the immature crypt epithelial cells inhibiting their proliferation (3). Increased proliferation within crypts (crypt hyperplasia) was frequently observed in chicks with lesions, indicating recovery. The recovery time frame could not be determined under field conditions. The delayed weight gain in chicks with RSS might be attributed to either a delayed or incomplete recovery of the intestinal mucosa or to the failure to compensate the transiently reduced weight gain during the short time span until slaughter.
RVs were the most common microbiologic pathogens identified in intestinal samples of 32 broiler chicks from the eight flocks with RSS. PAGE was used to distinguish different groups of RV. With this method, the 11 segments of dsRNA (ranging from 0.2 to 2.1 ×106 in molecular weight) of the avian RV genome produce an electrophoretic migration pattern in polyacrylamide gel characteristic for each RV group (36). In four chicks, a migration pattern characteristic for gpA RV was observed: segments 7, 8, and 9 migrated triplet-like in mammalian gpA RV, and segment 5 migrated very close to segment 4, unlike their mammalian counterparts (36). Furthermore, the migration of segments 10 and 11 were difficult to resolve. Typically, the pattern of the RNA segments of avian gpA RV was, in all four cases, 5–1–3–2 (36).
The genome profiles of 20 RV strains were different from each other and different from those of avian gpA RV. The RNA profiles were 5–2–2–2, 4–1–2–2–2, or 4–2–2–3, respectively. These RNA migration patterns have been described for gpD, gpF, or gpG RVs, respectively (22). The prototypes of these groups of RV are strains 132 (gpD RV), A4 (gpF RV), and 555 (gpG RV), which were detected in gut contents of chicken in Northern Ireland (20).
In six of the eight flocks with RSS, 12 birds (one to four per flock) excreted a large number of gpD RV particles. Ten out of the 12 chicks shedding gpD RV had severe villous atrophy. The other RV groups were found less frequently by PAGE. GpA RV was found in four chicks from two flocks, and none of the chicks had intestinal lesions. GpF RV was identified in four birds from two flocks with RSS and villous atrophy but was also identified in two chicks of the control flock. GpG RV was present in two animals, respectively, in one flock with, and one without, intestinal lesions. This suggests that there are differences in pathogenicity of the different RV groups, with gpD RV inducing the most severe lesions. This was confirmed by statistical analysis.
Negative staining is a routine method to diagnose intestinal virus infections by electron microscopy. In the intestinal contents of chicks from flocks with RSS, and particularly in chicks with severe villous atrophy, RVs were detected frequently by TEM in 28 of 34 chicks from flocks with RSS and in five of seven chicks from control flocks (Fig. 1). GpA and non-gpA RV particles can not be discriminated by TEM because of their identical morphology (Fig. 1). Therefore, that method cannot be used to differentiate the pathogenic RV from other RV groups that are not associated with clinical signs.
TEM showed that non-gpA RV particles have a high stability because intact particles were found even after fecal preparations had been stored at 20 C for 24–48 hr. Their stability was similar to that of gpA RV particles and not mammalian gpB or gpC RVs, which seem to be less stable (6).
Negative staining also revealed the presence of SRPs in fecal preparations of 21 chicks. Based on size, these SRPs might represent astrovirus or entero/entero-like viruses, which have been shown to cause enteritis in different avian and mammalian species (19,33). Astrovirus was also isolated from cases of RSS (44). Because SRPs were found in chicks from flocks with RSS (18 of 34 chicks) and in controls (three of seven chicks) and there was no correlation between the presence of SRPs alone or in combination with RVs and villous atrophy, there is no indication that the SRPs detected in the present study contributed to the disease.
Because PAGE is a method with comparatively low sensitivity, RT-PCR and nested RT-PCR specifically for the detection of gpA RVs were used to increase detection sensitivity. Unfortunately, PCR methods were only possible for the detection of avian gpA RV because sequence data of avian non-gpA RVs are not yet published in GenBank. The comparison of the results from PCR and PAGE revealed that 26 chicks from flocks with RSS and two of the seven chicks from the control flock excreted gpA RV at a low titer only (<105 virus particles per ml). Mixed infections with gpA and gpD RVs were frequent, but there were also flocks with either gpA or gpD RV infections only. The remarkably increased number of chicks positive for gpA RV identified by PCR testing suggests that there should be even more chicks positive for gpD RV. This may also be the explanation as to why no gpD RVs were identified in one of the flocks in which chicks had severe villous atrophy.
Based on the findings from this field study, it was shown for the first time that gpD RV appears to be a major contributor to the development of RSS, whereas gpA RV is shed frequently at low titers without clinical signs. Conflicting results from the literature concerning the role of RV in diarrhea/RSS are most likely caused by the inability of methods (TEM) to distinguish RV groups. For further investigations, sensitive diagnostic assays have to be established on the basis of genomic characterization of avian non-gpA RVs. Furthermore, experimental infections with strains of different groups of avian RV will be necessary to confirm these findings.
We wish to thank Prof. Ian N. Clarke, University of Southampton, Department of Molecular Microbiology, for reviewing the English language in the manuscript. We acknowledge the outstanding work of Jutta Prudlo, Petra Eppler, Renate Danner, Monika Godat, Sabine Lied, and Monika Schwebs.
Nucleotide sequence of the primers used and their position on gene segment 6 of chicken gpA RV
Occurrence of gpA, gpD, gpF and gpG RVs in intestinal samples of 41 chicks from 9 flocks of Northern Germany
Distribution of severe villous atrophy in chicks from flocks with RSS and a control flock
Correlation between severe villous atrophy and detection of RV group in the intestinal contents.A