Rod ReeceInfectious stunting syndromes of chickens
INFECTIOUS STUNTING SYNDROMES AND RELATED DISEASES IN POULTRY
Dr Rod Reece, NSW Department of Industry & Investment, State Veterinary Diagnostic Laboratory, EMAI, Menangle NSW, Australia
INTRODUCTION
There are many causes of poor growth in broiler chickens, but in the mid 1970s a new syndrome, preferably referred to as ‘infectious stunting syndrome’ (ISS)appeared in Europe and USA, and thereafter spread rapidly throughout the world (Bracewell & Randall 1984). Mortality was not a feature, although cull rates were higher than normal. This syndrome was characterised by a 5-20% incidence (sometimes up to 50%) of stunting, with a few chickens being less than 50% the live-weight (LW) of more normal birds: severe runting (less than a third the LW of unaffected chickens), when present, usually affected only 1-5% of the flock. The affected chicks had voracious appetites and pendulous abdomens. Distension of the intestinal tract was noted on necropsy if they had recently eaten. Feathering was retarded. Affected flocks were usually detected at 4- to 14-days-old, and partial recovery of many affected chickens appeared to occur from 5-weeks-old. Oral inoculation of 1-day-old SPF chickens with intestinal homogenates prepared from affected chickens, induced a similar syndrome thus confirming this was a transmissible, ie infectious, disease. Following appearance of ISS on a farm or in a region, subsequent flock performance tended to be poorer than expected and there was intermittent clinical recurrence.Financial loss has been considerable due to poor feed conversion, excessive culling, reduced weight-for-age, greater than expected variation in weights at processing, and problems associated with processing and sale of small carcasses. Three distinct syndromes with separate target organs (namely, small intestine, pancreas, proventriculus) and thus different pathogenesis have been defined. They have been reproduced independently with bacteria-free inoculae,and it appears they have different putative aetiological agents (Reece 2001).
ISS has been described as the “disease with too many names and faces” because there has been a tendency to ascribe names to the syndrome depending upon the clinical presentation,such as pale bird syndrome, helicopter chicks, runting and stunting syndrome etc. Numerous viruses have been isolated from and/or observed in the intestinal tract of affected chickens (including enterovirus, parvovirus, reovirus, rotavirus and togavirus), but an aetiological role has been difficult to ascribe for any of them. According to Koch’s postulates (actually Henle & Koch’s), a putative aetiological agent should be present in all affected cases and be absent from non-affected cases; the presence of that agent ought to be sufficient to explain the clinical course and pathology of the disease; and isolated putative pathogens ought to be able to induce a similar disease in experimentally infected hosts, in this case chickens. As of 2010, no isolated putative aetiological agent has fulfilled these criteria and the only method of reproducing the disease remains oral inoculation of 1-day-old chickens with intestinal homogenates prepared from affected chickens, which in itself, is time-consuming, costly and sometimes problematic.Unfortunately, the criteria for diagnosis of field cases have been poorly defined at times, and therefore clinical signs and/or pathology required for confirmation of experimental transmission have varied. The literature on this group of diseases has been difficult to follow!A consideration of Evan’s Association of Causation (see Appendix 1; Evans 1976) is enlightening: these were proposed to help elucidate pathogenesis and aetiology of complex and multifactorial diseases and were appropriate for ISS even though putative aetiological agents have not yet been isolated, let alone characterised.
Funding for research into this group of diseases has tended to come from industry and the focus has been on projects that will last for only a few years but yield control programs that can be instituted by industry. This presentation is a compilation of the author’s experiences at Dept Agriculture, Veterinary Research Institute, Victoria funded by Chicken Meat Research Council; Institute of Animal Health, former Houghton Poultry Research Station, UK funded by Ministry of Agriculture, Fisheries & Food UK; and NSW DII, Elizabeth Macarthur Agricultural Institute, NSW funded by RIRDC-Chicken Meat.
A similar syndrome was observed in other poultry industries especially turkeys and it was assumed these had a similar aetiology and pathogenesis: this may or may not be so. Some transmission studies utilising material from chickens were able to induce a stunting syndrome in turkeys, but on other occasions this has been unsuccessful. A somewhat similar stunting syndrome was reported in guinea fowl.
A summary of the development, structure and function of the chicken gastro-intestinal tract is attached as Appendix 2.
ISS – THE INTESTINAL OR ENTERIC FORM
EPIDEMIOLOGY
There are many reports of apparent breeder flock association of stunting syndromes: this may be a reflection of vertical transmission and/or insufficient maternal protection and/oryoung age of dams with smaller chickens at hatch and/or some other factors. It is unknown if any such association would be due to on-the-egg (ie external, a from of horizontal infection) or in-the-egg (ie true vertical transmission). There is some evidence of variation in susceptibility of broiler chicken strains to ISS based on severity and nature of the inflammatory cell response
EXPERIMENTAL TRANSMISSION STUDIES
Semi-purified (free from enveloped viruses such as avian leukosis virus, reticuloendotheliosis virus, Marek’s disease virus andinfectious bronchitis virus; as well as free from avian encephalomyelitis virus, infectious bursal disease virus, reovirus, adenovirus, chicken anaemia agent) bacteria-free intestinal homogenate (ISS-F) was able to induce typical intestinal form of ISS in chickens (with 100% of inoculated chickens having crypt lesions in mid-jejunum at 5dpi), but did not induce growth suppression nor intestinal lesions in turkeys, ducks, Japanese quail, pheasants nor guinea fowl. Intestinal lesions were similar in distribution and range of severity in several SPF chicken and commercial broiler strains. The exposure of inoculated chickens to periods of cold stress during brooding (daily at 15oC for one hour during the first week) did not accentuate intestinal lesions, but the effect of cold stress on growth rate was variable. Co-infection with either chicken anaemia agent (CAV) or avian nephritis virus (ANV) did not accentuate the intestinal lesions, but CAV inducedmusculature haemorrhages and more marked thymic and bursal atrophy than with ISS inoculae. Growth suppression of inoculated SPF Rhode Island red chickens was accentuated in males on high nutrient density feeds.
Pancreatic degeneration was not a feature in chickens inoculated with this material, nor with more than twenty other intestinal homogenates as inoculae.
Inoculation of chickens with intestinal isolates of reovirus, several avian nephritis virus strains, an astrovirus, (G-4260; AAF-7; HPRS-M), and ANV-related viruses isolated from intestinal tracts of chickens (Entero-PV2; Entero-3; EF84/700) induced mild intestinal histopathologybut no detectable crypt damage. Inoculation of chickens with ANV-F isolated from ISS-F induced minor reduction in growth rate, mild non-suppurative infiltrates in the small intestinal propria, rare apoptosis of villus enterocytes and mild interstitial nephritis, but no crypt lesions.
Chickens inoculated at 7 and 14 days-of-age developed significant intestinal lesions comparable to that observed in chickens inoculated at 1-day-old,but minor growth suppression was noted only in chickens inoculated at 7 days-of-age.
Homogenates prepared from pooled intestinal tracts or various segments (duodenum, jejunum, ileum or caecae) were similarly effective in transmitting disease, as was pancreas (have to wonder about surface contamination); but homogenates prepared from proventriculus (back flow of intestinal luminal contents does occur), kidney and liver(the putative aetiological agent was observed in macrophages of the intestinal propria, and some such cells may have entered the venous portal circulation to the liver) only induced a low incidence of mild intestinal crypt lesions. Incidence and severity of lesions declined with decreasing 10xfold dilutions of intestinal homogenates after 1/100.
Infectivity was retained after several passages through/in chickens. Others have demonstrated infectivity by oral dosing with litter from affected flocks.
Chickens in-contact with other chickens inoculated with this material (ISS-F), grew as poorly as infected chickens and there were significant histological changes to intestines. Other workers have also demonstrated in-contact transmission.
The progeny of hyper-immunised (?) breeders were fully susceptible to infection (any passive immunity may have been masked by over-infection; or maybe maternal antibody is non-protective?).
Establishing an experimental model to exclude cross-infection, especially with ANV, was a problem that was, eventually, overcome.
CLINICAL SIGNS
The clinical signs in affected chickens were voracious appetite but poor growth; they had pendulous abdomens if they had recently fed, and there was sometimes faecal matting around the cloaca. Feathering was poor. Lameness due to osteodystrophy was noted in some affected flocks but was not a feature in experimental studies.Inexperimental studies, calculated daily growth rate was most marked in the first week (50-60% compared to controls), but was still significant in the third and fourth weeks (70-85%).
PATHOLOGY
Affected chickens were small and, although in poorer than expected bodily condition, they were not emaciated. On necropsy, if the bird had recently fed, the intestines were distended with poorly digested contents. The thymic lobes were small and often atrophic with a vestigial cortex, and the bursa of Fabricius was relatively small with small follicles. In field cases, various osteodystophieswere observed in the physis of long bones, particularly the proximal tibiotarsus. In chickens receiving high carotenoid diets, the bodywas pale.
Histologically, specific lesions were confined to the small intestine. Thesewere more prominent and more pronounced in the mid jejunum than in distal duodenum or mid small intestine,were almost absent from terminal ileum,and not present in the caecae. Initially there was oedema of the villus coria and proria with infiltration by macrophages and lymphocytes; small loose clumps of heterophils in the coria and propria were occasionally noted. Individual degenerate enterocytes were observed on the sides of villi at 1dpi. The crypt cell mitotic rate increased, the crypts were elongated and somewhat serpentine, and there was basophilia of the crypt cell cytoplasm indicating hyperplasia and immaturity; in the early stage there was a short phase of decreased villus height (a transient villus atrophy) and some enterocytes were detected in mitosis on the lower third of villi. Individual degenerate crypt enterocytes were noted, along with degeneration of surrounding macrophages. By 4-5dpimany crypts were cystic and lined by cuboidal attenuated epithelium; some of these cystic crypts contained necrotic cell debris and/or mucus. Crypt abscesses containing plugs of degenerate heterophils were rare. The cystic crypts were mostly resolved during the next few days, and by 14dpi villus height appeared normal (see below).
ISS was characterised by a massive increase in intraepithelial leukocytes, which was greater at 14dpi than at 5dpi. Monocytes and macrophages commonly infiltrated into the villus coria and propria, and were noted between enterocytes on villi and in crypts. Initially, large numbers of ChT8 (MHC-1 associated cytotoxic/suppressor) cells were associated with crypts containing degenerate enterocytes and/or were located near degenerate macrophages; whereas ChT4 (MHC-2 associated helper/inducer) cells were located predominantly within the coria and propria. By 14dpi, germinal centres (B-cells) wereforming in the coria and proria, and these were surrounded by a rim of ChT4 cells. The intraepithelial lymphocytes on villi and within crypts were ChT8 cells, and similar cells were present in the coria and propria, either loosely scattered or as clumps. The intra-epithelial ChT8+ve cells were further identified as predominantly αβ1-TCR2, whereas γδ-TCR1 cells were rare.
Structurally, there were marked alterations to the intestinal villi and crypts. Such changes were best determined in overnight fasted chickens to remove artefacts associated with luminal ingesta; and morphometrical measurements were obtained by microdissection to overcome artefacts as a result of fixation and processing. Besides transient villus atrophy at 5dpi, but a slight elongation by 14dpi, there was crypt elongation, including convoluted crypts (which were difficult to measure).The number of crypts per villus decreased. Excess goblet cells were noted on the villi due to both an absolute increase (a result of hyperplasia) and a relative increase due to loss of enterocyte chief cells.Crypt cell proliferation rate was greatly increased, possibly in part being driven by cytokines produced and released by the inflammatory cell infiltrates. The resultant migration rate of enterocytes along the villi was greatly accelerated. There was fusion of villi resulting in a haphazard arrangement of projections into the intestinal lumen. Using small inert markers, the gastrointestinal transit time of ingesta was accelerated. There was a lower density of crypts per area, probably due to separation of crypts by oedema and inflammatory cell infiltrate. Even though the chickens were smaller, the intestinal length was increased, thus giving a greater length per unit weight or metabolic body weight.
The mucosal brush border enzyme activities in the jejunum were significantly reduced in ISS-inoculated chickens compared to controls, especially disaccharidases andaminidases (thus reducing ability to break down and absorb carbohydrates and amino acids, leading to malabsorption and possibly initiating an osmotic-type diarrhoea), and glucosidase (reflecting a reduction of enterocyte protein synthesis) and maltate dehydrogenase (reflecting mitochondrial and cytoplasmic damage within enterocytes): these were more marked at 5dpi than at 14dpi. There was a slight increase in intestinal brush border alkaline phosphatase activity of inoculated chickens at 14dpi compared to controls but the significance of this was uncertain (elevated iAP may be protective against luminal acidity?). Taken together, these results indicated biochemical immaturity of villus enterocytes,probably as a consequence of accelerated migration rate along the villi (there was probably insufficient time in transit along the villi for effective biochemical maturation of the enterocyte brush border), thus impairing nutrient absorption, as well as damage to enterocytes per se (Dr Ed Hall, University of Liverpool UK).
Circulating concentrations of growth hormone were greater in 5dpi inoculated chickens than in the controls, but similar in both groups at 14dpi, whereas insulin-like growth factor (produced by liver in response to growth hormone)was not significantly altered either in absolute or relative terms (Dr C.Goddard, Roslin Lab, UK).
A sequential electron-microscopic examination was undertaken by Dr Judith Frazier (Houghton, UK) and summarised here. Small dense cytoplasmic inclusions containing crystalline arrays of picornavirus-like particles (approximately 20nm diameter) (PVLP) were noted within enterocytes on the sides of villi at 18 hours-pi, and in crypt enterocytes at 36 hours-pi. They occurred in the cytoplasm of macrophages between enterocytes and within villus coria and in propria around crypts from 2dpi. These PVLP inclusions were not observed after 5dpi. The small cystic crypts noted more prevalently in the early phase, were lined by attenuated squamous epithelial cells (which had few brush border microvilli) and intestinal myofibroblasts, joined together by occasional desmosomes, but these cysts lacked a distinct basement membrane. The larger cystic crypts were lined by cuboidal enterocytes on a basement membrane. Many of the dilated and cystic crypts contained cellular debris and occasional granulocytes. By 7dpi, there were fewer dilated and/or cystic crypts, and these declined in prevalence over the remaining period of the study (to 28dpi). Intraepithelial leukocytes between enterocytes in crypts and upon the sides of villi were noted at 3dpi and became extremely prominent from 11-28dpi: these were both lymphocytes and macrophages, From 7dpi, the fibroblasts and myofibroblasts in the propria around crypts, and especially within villus coria, were arranged in a haphazard manner, that is, not aligned parallel with the long axis of the villi.
PATHOGENESIS
The virus (PVLP) invaded and damaged villus and crypt enterocytes, rather than inducing confluent enterocyte necrosis with ulceration. This invoked a marked inflammatory response with lymphocytes and macrophages. Repair of crypts and attempted restoration of intestinal structure and function resulted in enterocytes on the villi that were biochemically immature and therefore inefficient at nutrient absorption.
During the first week of life post-hatch, the gastro-intestinal tract of chickens adapts to ingested food, and infection of small intestines at this crucial stage damages structure and function and thereby has a significant impact on growth, feed conversion efficiency, and nutrient absorption and utilisation.