Comments and Recommendations:

The main finding was of enlargement of the livers, spleens and kidneys. There was some evidence of purulent air sacculitis but this was not a common finding. The gizzards contained white weed seeds, varying from 10 seeds per gizzard to forming 50% of the gizzard contents.  Varying degrees of mucoid enteritis and jaundice were also seen. A single bird was identified with a classical cholangio-hepatitis lesion. The livers were markedly enlarged, had a mosaic surface appearance and some petechiation, worst along their distal border. There was only minor evidence of respiratory lesions as seen in the birds from P1059, suggesting that a respiratory agent is unlikely to be involved.

Samples of liver, kidney, intestinal  and gizzard contents were frozen for future toxicological analysis.

Fixed samples of liver and kidney were forwarded to VLA, Lasswade for rapid histopathological interpretation.

Mortality had increased overnight. A site visit was arranged for the following morning.

Histopathology

Post Mortem Ref: P1062

Fixed liver, kidney and heart were submitted to VLA, Lasswade for histopathological examination.

The livers showed a zonal (centrilobular) pattern of fatty degeneration featuring fine vacuolation of hepatocytes and hyperplasia of the biliary epithelium in individual sections. A single section showed bile pigment within the canaliculi and larger bile ducts, indicating cholestasis. Multifocal granulopoietic hyperplasia was seen in individual sections. There was no evidence of cholangio-hepatitis.

No significant abnormalities were identified in the kidney or heart tissue submitted.

The liver appearance was suggestive of a toxic or metabolic insult. The concurrent biliary hyperplasia was more suggestive of toxin involvement. The age of the liver pathological changes was estimated at least 2-3 days old. There was reportedly no evidence of viral or bacterial involvement.

DIFFERENTIAL DIAGNOSIS

Hepatotoxicosis.

Sources include mycotoxins including aflotoxin and ochratoxin.

SITE VISIT

This took place on 22/9/99. Mortality had risen overnight.

All houses were of wooden construction, with ridge fans and side inlets.

Chopped straw litter, nipple drinkers and pan or track feeders. Each house has two feed bins.

During initial discussions, it was confirmed that two trials were occurring on the site.

1.      Chlorine Dioxide Sanitation Trial

A central header tank, fed from the mains, was being treated with 1ppm chlorine dioxide (Anthium Dioxide;Viscot) via a pump system, to supply houses 5-8 inclusive. Houses 1-4 received unsanitised mains water.

2.      Digestive Enhancer Replacement Trial

This trial replaced flavophospholipid (Flavomycin 80; Hoechst Roussel) with an alternative product (Genex; Optivite Nutrition), mixed at the feed mill and supplied to houses 1,2,5,6. Houses 3,4,7,8 received flavomycin at 20ppm.

The purpose of the visit was to observe the affected birds and identify any clinical signs of disease, examine the environment for toxic sources and observe other birds on site for clinical or post mortem evidence of the problem.

House 6 and two other houses were closely examined, which encompassed the trials occurring on the site.

Production Details

Observations

In houses 5 & 7, birds were seen panting at a recorded house temperature of 24°C, several hours after thinning, suggesting they were too hot. In general, birds appeared healthy. They were noisy, eating and drinking, and alert. The pellet quality in both houses was poor. An increase in culling was recommended, mainly due to leg problems.

In house 6, sick and dead birds in half of the house had deliberately not been picked up prior to the visit. Large, dead birds were seen lying on their sides, fronts or backs, evenly distributed across the floor. A single bird was seen, with its eyes half closed, rolling its head, which then convulsed and died on its back. A large number of birds were seen panting as in the other houses. However several birds were in more extreme respiratory distress.

In house 6, air quality measurements were taken and readings for oxygen, carbon dioxide and monoxide and ammonia were all within normal limits.

The majority of the birds were eating and drinking normally.

The pellet quality was good in house 6.

The two feed bins supplying house 6 were inspected. No evidence of moisture or mouldy feed was seen.

The birds were on withdrawal feed, supplemented with 17% whole wheat.

15 birds from house 6 were blood sampled. The samples were returned to the laboratory for serum separation and frozen storage.

On Site Post Mortem Examinations

H.6

11 dead birds were examined.

The birds had not eaten.

Sparse to large numbers of whole white weed seeds were seen in the gizzards of H.6 birds, contributing up to 50% of the gizzard contents

H.5 & 7

10 birds were randomly culled from each house and examined.

The livers in H.7 were paler than H.5, but no hepatomegaly or jaundice was seen.

Individual white weed seeds were seen in the gizzards, up to a maximum of 6 per bird.

Feed Deliveries

All feed from day old to processing for houses 1,2,5,6 contained Genex. This was manufactured specifically for the four houses on this site alone.

On 8/9/99, house 6 alone received a delivery of 20 tonnes of finisher and 4 tonnes of whole wheat. The rest of this 40 tonne manufactured batch was delivered on 9/9/99 to houses 1 & 2. This was the only feed delivery specific to house 6. On all other occasions, deliveries were split between houses.

Samples of all feed deliveries were stored on site. The house 6 feed delivery from 8/9/99 was returned to the veterinary laboratory for storage. Whole wheat samples were not kept.

WEED SEED ANALYSIS

The gizzard contents of a single bird were submitted to the National Institute of Agricultural Botany (NIAB) Official Seed Testing Station for analysis. The following seeds were identified;

190 whole undigested Galium aparine seeds (common name Cleavers)

19+ broken pieces of Galium seeds

1 piece of ergot

7 partly digested oat seeds

7 pieces of grit/stone

The remainder of the sample contained partially digested seed or seed coat material.

The white and black seeds were both identified as Galium aparine. The seed coat is black, and when the coat is removed, the seed is white.

INITIAL PROCESSING RESULTS

An initial thin of approximately 4000 birds from each house, caught on the morning of 22/9/99 highlighted a 9.65% reject rate in house 6 birds, 77 % of which were recorded as necrotic foci in the liver, compared to 0.77% average for the rest of the houses.

Medication

No medication was prescribed. Mortality had increased during water soluble amoxycillin medication. E.coli bacteria had been isolated from post mortem specimens, but the post mortem and histopathological findings were suggestive of a toxic insult. Supportive therapy with multivitamins, electrolytes or glucose was not instigated.

FINAL PROCESSING RESULTS

Processing rejects for house 6 aged 43 & 44 days, averaged at 4.03%, with 73% rejects due to necrotic foci in the liver.

DIAGNOSIS

Hepatotoxicity caused by an unknown agent.

DISCUSSION

The post mortem and histopathological examinations were strongly suggestive of an hepatotoxic insult. On site observations of toxicity in the single house on an eight house broiler site, suggested this was unlikely to be a farm based problem. Possible toxic sources were investigated to try to identify a factor specific to house 6.

The source flocks were common to a number of houses on the site and on other sites within the integrated operation.

Air was common to all birds on site. Air quality measurements taken in house 6 showed no abnormalities.

Mains water was treated centrally, then distributed to a number of houses.

Litter was sourced from a variety of localities, centrally treated and baled, and distributed to farm.

No wood preservatives or environmental chemicals had been used on site.

The compound feed was manufactured by a large integrated mill. Initial suspicion concerned the Genex inclusion, as little information was available regarding the ingredients of this product. Investigations revealed it was a blend of ammonium salts and formic acid on a silicate carrier, and was being included at 50% of the maximum recommended inclusion. The other concern was that this trial feed may have been incorrectly manufactured but due to the manufactured batch sizes, individual batches of feed were usually split between houses. However the whole wheat delivered on 8/9/99 was specific to house 6 only. The whole wheat is stored at the mill in a 25-30 tonne bulk bin. Deliveries occur up 3-4 times each day, direct from farm in 4-5 tonne deliveries. Delivery is by farm or grain merchant vehicle. It was possible for a delivery of whole wheat to house 6 to be sourced from a single farm delivery. This delivery most likely contained high levels of weed seeds, seen in the gizzards on post mortem examination.

Further antimicrobial medication was not prescribed. Mortality had risen during the course of water soluble amoxycillin. This combined with the post mortem findings, suggested clostridial bacteria were not involved. The histopathological findings were suggestive of a toxic insult, with no evidence of bacterial or viral involvement. Moderate to heavy growths of E.coli were isolated from the livers of dead birds. This may have been related to post mortem bacterial migration, reticulo-endothelial cell failure, or gastrointestinal cellular damage. These bacteria were not considered significant in the mortality. Supportive therapy was discussed, but not instigated, due to the sub-acute nature of the toxicity.

The white weed seeds have been identified as Galium aparine (Cleavers or goosegrass). These are not commonly seen to be toxic at low levels (Januszewski et al 1988, National Poisons Information Bureau verbal report), although performance can be affected (Salyi et al 1991, Mazurkiewicz et al 1991). We do not have a sample of the whole wheat delivery to allow an assessment of the quantity of weed seeds fed. NIAB commented that the quantity of seeds accumulated in the gizzards was massive. The gizzard acts as a mechanical crusher and filter, allowing suitably small particles to progress to the duodenum. The Galium seeds were very hard, and only broken with the tip of a scalpel, suggesting they would accumulate in the gizzards and be retained longer than wheat grains, preventing an accurate assessment of the levels fed (Salyi et al 1991).

The most likely source of toxicity, taking into account the house and farm circumstances, was a contaminated whole wheat delivery. The toxin may have been high levels of Galium aparine or another factor not visible in the birds at the time of post mortem. It is possible that the weed seeds were a marker of a contaminated batch of wheat, but the toxic substance may have have been digested and absorbed and thus not visible at post mortem examination. The identification of weed seeds, oat seeds and ergot in the gizzard contents, suggested that the quality of the whole wheat delivery was poor. Contamination of the wheat may have occurred at the farm store or in transit. Deliveries of whole wheat were not routinely sampled at the feed mill or farm. Thus none were available for inspection and toxicological studies.

At present, no toxicological examinations have been carried out on the stored samples, due to a potential toxin not being identified. Discussions with the VLA Weybridge and  Lasswade, Poisons Information Bureau, and Central Science Laboratory have not identified a toxin which would cause the pathological changes identified. Considered toxins included pesticides, rodenticides and molluscicides.

Toxic effects are species and dose dependent. The information available is not based on poultry exposure. The dose of exposure is unclear in this case.

Hepatotoxicity can arise from certain organochlorides (verbal communication, Dr Livesey, VLA Weybridge) but is more common with metaldehyde toxicity (verbal communication, Veterinary Poisons Information Service).

Aflotoxin and ochratoxin are hepatotoxic, although ochratoxin is primarily nephrotoxic. Aflotoxin is unlikely to occur in the UK, due to the climate, the raw materials used and mill testing procedures. A major effect is reduced performance (Leeson et al). Early performance of birds in house 6 was the best on the site. The overall processing weight for age was slightly reduced compared to the remainder of the site. This may have been the result of the extensive numbers of birds not eating in the last week.

It has been recommended that the stored samples be tested for ochratoxin and metaldehyde.

The best method of identifying the source of the toxin would be to test feed the suspect feed to a small group of chickens. This is not possible with the samples retained.

Terminal serology from house 5, 6 & 7 was taken for serum separation and refrigerated storage.

It was advised that rejects were likely to be high, as a result of chronic liver disease. It was recommended that birds were frozen and stored after processing, until the toxic risk to the food chain was established. Condemnation of all livers and cutting the carcasses to harvest the breast and leg meat was also recommended. This would potentially remove the organs containing the toxin.