Paul Mc Mullin MVB DPMP MRCVS
Poultry Health Services (formerly Hillsdown Veterinary Services)
Marsh Lane, Hemingford Grey,
Huntingdon, Cambs PE18 9EN

1  Summary
3  The Complaint
3  History
5  Initial Serological Investigations
7  Management of sero-conversion in parents
7  BroilerSubmissions
7  Post-Mortem Findings
8  Histology
8  Mycology
9  Other Submissions from progeny of the parent flock
10  Mortality Data for affected flocks
11  Epidemiology
11  Differential Diagnosis
12  Treatment
12  Prevention
13  Literature Review and Discussion
15  Conclusions
16  References

Figure 1. Broiler Parent Farm Lay-out
 

House A

...

House B

 
 

House C

...

House D

Initial Serological Examinations
The routine serological monitoring programme for the company in question calls for the broiler parent chickens to be sampled (10/air space) at no later than 14 weeks of age, with the samples being tested using a commercial Elisa test (Guildhay) for antibodies to Chick Anaemia Virus. The Ross 1 birds in the flock in question were not tested in rear so samples from all 4 houses were tested at the "point of lay" blood sampling (22 weeks of age). Follow-up tests were carried out on some of the groups at 28 weeks and 32 weeks of age.

The results of the Elisa test for each of the 4 houses at 22 weeks and of the follow-up tests, where appropriate, are shown in Table 1. Note that the normal interpretation of the test results provides 3 categories of sera, negative (Serum to Positive, S/P, ratios up to.15), suspect (S/P ratios 0.15 to 0.25), and positive (S/P ratios =>.25). Our laboratory splits the positive range into blocks of S/P ratio of 0.05 and reports the results as "titre groups". Suspects are in group 1, low positives .25-.3 in group 2, positives .3-.35 in group 3 and so on. Reporting the results in this way gives a better appreciation of the antibody response than a simple negative/suspect/positive reporting system. The results shown in Table 1 confirmed that the birds in house A had high levels of antibody and could be expected to have high levels of immunity and, hence not put progeny at risk from vertical transmission. House B (across the service area from house A) had started to sero-convert and houses C and D still had no indication of infection at 22 weeks of age. The birds in houses C and D had started to sero-convert at 28 weeks of age, and were solidly positive at 32 weeks. The trend to sero-conversion is illustrated in Figure 2.

The impression given by these results is that the infection spread slowly across the site from house A to B then C and finally D. In addition it appears that it took about 4 weeks from first infection in the house until a high proportion of birds became positive.

Management of CAV sero-conversion in parent flock.
When, at 22 weeks, it was identified that 3 of the 4 sub-flocks were either totally or partially susceptible to CAV infection, the possibility of cross-seeding birds or litter between the houses was considered. A complicating factor was the fact that a few low-grade reactions in the Elisa test for S.enteritidis carried out on the same blood samples. For this reason it was decided not to take further action at that time. In accordance with the recommendations of the test manufacturer a further 60 samples were tested from each of the three houses with low-grade positives or suspects at 24 weeks of age and none came within the positive range for the test. This led us to reconsider the possibility of encouraging the spread of CAV across the site. Since the company was integrated it was possible to elect to incubate and place the broilers even though there was a strong possibility that losses from CAV would occur. Litter was taken from the positive houses (A and B) and spread in the negative houses (C and D) when the birds were 25 weeks of age. Follow-up serological testing was carried out at weeks 28 and 32 to evaluate the response to seeding. The results were discussed above.

Broiler Submissions
Twenty one broiler flocks were submitted for investigation, one of which on 2 occasions. Table 2 shows a listing of these submissions. Each submission is given a unique accession number on arrival against which all results are recorded. The estimated hatch date shown was calculated from the date of submission minus the age in days. The estimated date on which the eggs were laid was calculated as the estimated hatch date minus 21 (days incubation) minus 5 (average egg age operated in the hatchery during this period). All of these submissions were from as-hatched placements. From these submissions a total of 236 chicks were subjected to post-mortem examination. They turned out to be evenly split between males (117 or 52%) and females (107 or 48%) suggesting that both sexes are equally susceptible to the effects of congenital CAV infection. The average age of affected progeny when presented for diagnosis was 16.6 days. The mean weight of birds submitted was only 57% of that expected for their age.

Post-Mortem Findings
Table 3 shows a detailed break-down of the pathological findings recorded for each of the submissions. The most common post-mortem finding recorded was anaemia of bone medulla and other tissues, affecting at least 60 % of the submitted chicks (Figure 3). The targeting of the immune system by this virus was very obvious from the severe atrophy of both the cloacal bursa and thymus (52 % of chicks). The bursal weights were recorded for 12 different submissions and the majority were found to have a bursal/bodyweight ratio below 1g/kg compared to the normal value of about 3g/kg (Table 2).

Figure 3. An affected bird showing extreme paleness of the kidney (on the right), pale tissues including the bone medulla, and virtual absence of thymus in the neck (on the left).

Among 10 of these 22 submissions severe lung lesions were a common finding (overall 25% of chicks affected). The lungs had dark red and swollen patches, mainly along their ventral margins (Figure 4.). These lesions were thought to be infarcts when first seen. A newly-available Avian Rhinotracheitis vaccine had been given to these birds at 14 days of age so it was suspected that these lesions might represent a vacinal reaction in immunosuppressed birds. However, histological examination confirmed that these lesions were caused by severe fungal growth association with a poor inflammatory response (see further comments in the sections on histology and mycology below). Kidney degeneration with the appearance of reticulated pale areas was observed in 15 % of submitted chicks.

Figure 4. An affected birds showing a dark pneumonic area in the lung. At post-mortem examination this area was much more distinctly demarcated than would appear from this photograph.

CAV syndrome in young chicks has sometimes been called "Blue-Wing Disease" due to gangrenous dermatitis affecting the wing-tips (Figure 5). Gangrenous dermatitis was, in fact, seen in a relatively small proportion of birds in these submissions (12.7%) and the lesions were observed almost anywhere on the body. They were most easily observed on the shank and foot and in these locations were associated with marked swelling. One group of birds had a submission (ref. 5959) with typical CAV syndrome at 16 days of age, and was resubmitted at 32 days (Ref. 640) at which time most of the birds submitted were affected by gangrenous dermatitis. Clostridial septicaemia characterized by haemorrhagic liquid under the skin and rapid putrefaction was most commonly observed in birds with gangrenous dermatitis.

Figure 5. An affect birds showing severe gangrenous dermatitis of the wing.

It should be noted that quite marked liver lesions due to Inclusion Body Hepatitis (see Histology) were present in 8.1% of birds submitted. Haemorrhages in various tissues (muscle, sub-cutaneous and into the caecum) also occurred, presumably as a result of a coagulation defect associated with bone medulla damage. The increased fragility of long bone recorded as "Femoral Head Necrosis" may suggest that the medullary damage can, at least in some circumstances affect the bone also. The other lesions listed were present in a small proportion of birds and are to be expected in submissions from commercial broilers.

Histology
All selected tissues were fixed in neutral buffered 10% formalin before submission to another laboratory for histological preparation and interpretation.

Table 4 summarizes the tissues selected for histological examination and the findings.  A single 32 day-old bird from a submission with gangrenous dermatitis had small nodules in the skin of the neck. The histology is strongly suggestive of Marek's Disease. No widespread occurrence of Marek's disease condemnations or transient paralysis were recognized in this company as a whole. This bird may have been immunosuppressed as a result of the CAV infection.  A variety of tissues were taken from 16-day old birds (Ref. 596) and the findings illustrate the destruction of blood-forming cells in the medulla, thymus and bursa. Trachea and lung had been collected because of the dramatic lung lesions. It was suspected that Avian Rhinotracheitis vaccination at 14 days might have induced a reaction in immunosuppressed birds. In fact the tracheas were normal and the cause of the lung lesions was a massive fungal invasion with a very poor inflammatory response.  These lesions are very different from the small circumscribed nodules which are typical of pulmonary mycosis in young chicks. Lungs from a second case (18 day old birds, Ref. 615) had similar lesions. One of the submissions exhibiting liver lesions was selected for histological examination, and this confirmed that the lesions were, in fact, due to Inclusion Body Hepatitis

Mycology
In order to confirm the histological diagnosis the lung lesions were mycotic in origin 4 pieces of typically affected lung were collected (Ref.663) and placed directly on Rose Bengal Chloranfenicol Agar (Oxoid).  After incubation at 37 C. for 48 hours all four samples showed abundant growth of Aspergillus fumigatus. The probable source of infection was the straw litter. Farm staff had been complaining during this period that the straw was very variable in quality, some bales being very dusty. Hatchery contamination cannot be ruled out, however clinical Aspergillosis was not diagnosed in the progeny of other parent flocks.

Other submissions from progeny of the parent flock.
A number of other submissions were received from the progeny of the "problem" parent flock. A summary of the post-mortem findings is shown in Table 5. The first three submissions were from flocks produced during the period of the outbreak and, in fact, 2 of these (Refs. 695 and 693) had been diagnosed to have CAV syndrome in their third week of age. The findings in these cases (mainly Gumboro, respiratory infection and secondary coli-septicaemia) were fairly typical of cases seen in the progeny of other flocks. The bacterial peritonitis spreading from the area of the bursa in one of these cases was more severe than normal (Ref. 754)

The final 2 submissions listed, which refer to the same flock of chicks, are, perhaps, the most interesting.  At 10 days they were developing "field rickets" which was quite severe by the time they were 13 days old. At the second submission 6/10 chicks had acute inflammation of the bursa (including some with sub-capsular oedema) which was confirmed histologically as Infectious Bursal Disease (Gumboro). This is the earliest case of Acute Gumboro disease seen by us, though it may be that the rickets intensified the clinical signs and contributed to the mortality. The significant feature is that there were none of the lesions of CAV syndrome seen in the other flocks, nor did this flock afterwards develop CAV-like mortality, hence it delimits the end of the period of significant vertical transmission.

Mortality data for affected flocks.
All but one of the affected flocks were grown within one agricultural area of one integrated poultry company. We obtained detailed mortality and culling data for 16 house/flocks of which 8 were composed exclusively of the progeny of the affected parent flock. Of the remaining flocks some included the progeny of the unaffected house of the same parent flock (PM3 birds) and some included progeny from other parent flocks. The proportion of progeny of the affected flocks varied from 34 to 97%. The grow-out period for these birds was, on average, 40.6 days (range 38 to 42). Overall flock
percent mortality was 12.1% (range 5.1 to 20.6%).

The mean pattern of mortality for these 16 flocks is shown in Figure 6. This illustrates the dramatic increase in mortality in the 3rd week with a secondary peak in the 4th week. After 12 days of age mortality was never less than double the normal target (0.1% per day).

Table 6 attempts to place the level of mortality seen in these flocks in the context of the overall level of mortality in the same company during the period. Only the 16 CAV flocks whose mortality data are summarized above are included so the overall effect of the problem is somewhat underestimated. The data in Table 6 have been generated by calculating the total number of chicks placed, total collected dead, and total killed for all of the 16 CAV flocks. The category "Other Flocks "includes all chicks placed in other houses and/or farms during the period between the first and last CAV flock. For these the total number of birds dead and culled is estimated by subtracting the total number of broilers processed from the total number of chicks placed. The equivalent totals for the CAV flocks were then subtracted from these figures. The overall result indicates that the CAV flocks suffered 12% losses (of which 2.5% were culled) compared to only 5.8% in the remaining flocks during the same period). This single incident caused the direct loss of approximately 32.000 broilers (estimated sale value in excess of £38000). Indirect losses due to reduced weight gain and product quality and increased condemnations also seem highly likely (figures were not at this time maintained for individual house-flocks, so it is not possible to confirm this). These indirect effects could easily have doubled the financial losses.

Epidemiology
Affected flocks were hatched between September 18th and October 29th, 1993. Assuming that the eggs from the affected parent flock were being incubated 5 days after being laid then the last flock with clinical problems was from eggs produced up to October 3rd. An unaffected hatch would have been from eggs laid up to October 10th. On this basis significant vertical transmission ceased between October 3rd and 10th. If we go back to the serological data (Figure 1) and assume that sero-conversion in house D was linear between weeks 28 and 32 then we can estimate that vertical transmission in this sub-flock ceased when the birds were 50-60% positive. However, because of the variable speed of
spread of CAV infection within flocks it would be dangerous to assume that this level of sero-positive birds guarantees no CAV-related problems in the progeny in all circumstances.

Differential Diagnosis
Runting and Stunting Syndrome, field rickets and Salmonella enteritidis septicaemia were the conditions most likely to produce low weights and mortality in this age group. All of these were excluded on the absence of typical pathology. Sulphonamide toxicity and severe aflatoxicosis can induce haemorrhagic lesions and aplastic anaemia, though these would not be expected to occur in the progeny of only 1 parent flock. The combination of typical CAV pathology (marrow and lymphoid organ depletion, anaemia), including an albeit low incidence of gangrenous dermatitis, with histological findings and a knowledge of the parent flock serology led to the diagnosis of vertically-transmitted Chick Anaemia Virus infection. Viral isolation was not attempted since a positive viral isolation is not diagnostic, it was
not considered necessary.

Treatment
Medication with amoxycillin for 2-3 days was used in most flocks, sometimes on more than one occasion, in attempt to control secondary infections. Since no controlled trials were carried out it is difficult to judge the response to this treatment. Farm managers were instructed to cull all small and weak chicks.

Prevention
In the absence of an approved vaccine we rely on achieving parent flocks which are naturally exposed and uniformly immune prior to production. There is some evidence that the older a flock is when it sero-converts then the longer it will take to become immune (K.R.G. Personal Communication). A possible prevention plan based on current knowledge of the disease might be as follows:
14 Weeks. Elisa test 10 samples per house at 14 weeks of age. If all houses have some positive birds then further action should not be necessary.
16 Weeks. Any houses with no positive birds should be "seeded" by taking approximately 100 Kg of fresh litter from a known positive house and laying it in a line along the length of the house to be seeded. The birds may be fed on top of the material to encourage exposure. The "seed material" should not be allowed to heat up between collection and application. Optionally birds may also be swapped between the positive and the negative house (1-5%). Detailed records should be maintained with respect to the seeding material used, the source, and the date of application. It should be kept in mind that all seeding operations carry inherent risks of transferring pathogens over and above the intended. For this reason the source material should be selected with great care, in the following order of decreasing acceptability:

A. A positive house with birds from the same source and of the same age or younger than the negative flock.
B. Any other positive flock on the same farm.
C. A positive flock on a rearing farm in the same area.
D. The youngest available positive laying flock in the same area

22 Weeks. Elisa test 10 birds from each of the houses which were negative at 14 weeks. These samples can usually be taken from the routine 22 week sampling, though many companies will wish to check the success of seeding earlier than this. All houses should now have 50% or more birds positive. If there are still any houses negative then that house should be considered "CAV at-risk" and eggs only released for incubation when a subsequent test shows that they have converted. They should also be re-seeded as soon as possible, as described above. Re-sample and test at 2 week intervals until positive.

26 Weeks. If it is necessary to incubate eggs from "CAV at-risk" flocks it would be best to incubate the eggs from these separately, so that, if considered necessary or appropriate, antibiotic medication of the progeny may be initiated.

The above advice was written in 1994. In January 1995 a live attenuated vaccine for CAV has been licensed in the U.K. under the new abbreviated system. The use of such a vaccine will now be the preferred alternative. Unfortunately this vaccine must be applied by individual injection so flock managers may still opt to rely on field challenge rather than vaccinate routinely. It is possible that vaccination with an attenuated strains may have less adverse effect on the parent birds immune system than field challenge with a virulent strain. This remains to be determined.

The causative agent was initially isolated by passaging liver from affected chicks in SPF birds (Yuasa et alii,1979) and the same group later discovered that they could propagate the agent in MDCC-MSB1 cells, a lymphoblastoid cell line from Marek's disease lymphoma in which a cytopathic effect is induced. It causes no cytopathic effect nor alterations in chick embryo fibroblasts and chick kidney cells. The agent was resistant to ether, chloroform and pH 3.0, passed through a 25 nm membrane filter, and was inactivated partially after heating for 30 min at 80°C and completely after 15 min at 100°C (Goryo et alii, 1985). Most of the literature refers to Chick Anaemia Agent however, since it has since been shown to
be a virus (McNulty et alii, 1990, Goodwin et alii, 1991) the agent is now referred to as Chick Anaemia Virus (CAV). Specific immunofluorescent antigens are detectable in infected MDCC-MSB1 cells by an indirect immunofluorescent antibody (IFA) technique (Yuasa et alii, 1985). This makes it possible to confirm that a culture is infected and also allows the testing of field sera for antibodies. It is possible to reproduce the syndrome by inoculating day-old SPF chicks with the virus provided that such chicks are free of specific CAV maternal antibody (Yuasa et alii, 1980a). The pathogenic effect becomes progressively less if innoculation is delayed, although infections in the first week can still cause disease.
Some isolates are more effective than others in inducing disease when inoculated into 7 day old chicks. The antigenicities of various isolates were compared by cross neutralization and indirect immunofluorescent antibody methods by Yuasa and co-workers (1986). No serological distinction was recognized among the isolates. Chick inoculated at 14 days of age do not become anaemic (Yuasa et alii, 1986)

Reduced bodyweight gain and haematocrit, particularly 12-20 days after infection are characteristic of this infection (Goryo et alii, 1989). It is interesting that the incubation period in these cases was very similar to that in artificially-infected chicks in the above story. It is also, incidentally, very similar to the incubation period of acute radiation sickness in man. Many of the survivors of Hiroshima and Nagasaki died 10-15 days later from internal haemorrhage or infection as a result of the destruction of generative cells in the blood-forming organs (Weissman and Cooper, 1993). The description of the pathology of the disease usually includes anaemia with aplasia of bone marrow and atrophy of lymphoid organs, haemorrhages in muscle and enlargement and discolouration of the liver (Yuasa et alii, 1986 and 1987,
Goryo et alii, 1985 and 1989, Vielitz and Landgraf, 1988, Rosenberger and Cloud, 1989a, Lucio et alii, 1990, McIlroy et alii, 1992, Goodwin and Brown, 1992). Goryo et alii, 1989 considered that the histological lesions appear first in bone marrow and thymus and then in the bursa, spleen and liver. Swelling and intranuclear inclusion bodies in the haematopoietic precursor cells, thymocytes and reticular cells of the thymus were considered to be characteristic lesions. Hypoplasia or aplasia of bone marrow, depletion of lymphocytes in the lymphoid organs and swelling of hepatocytes occurred during the anaemic phase.

As described above, an indirect immunoflourescence test was developed to facilitate field screening of CAV antibody status of flocks. Results were shown to be the same as those obtained with the more cumbersome and expensive serum neutralization test (Yuasa et alii,1985, McNulty et alii, 1988). More recently Elisa tests have been developed which have been shown to be equally useful in detecting CAV antibody (Todd et alii, 1991, Goodwin et alii, 1992). A serological survey using IFA in Japan in 1984  found 39 of 40 commercial breeder flocks (97.5%) to be CAV sero-positive. Of the 381 individual serum samples, 357 (93.7%) were positive (Yuasa et alii. 1985). A similar survey of broiler breeder and parent and commercial layer flocks in the UK found 86 of 89 flocks (356 (72.1%) of 495 serum
samples)(McNulty et alii,1988). Yuasa and co-workers (1980) had shown that the progeny of 1 of 5 SPF flocks were not susceptible to CAV infection at one day of age. The serological surveys eventually found 2 of 19 SPF flocks in Japan and 5 of 11 such flocks in the UK to be positive for antibody to CAV. A subsequent international survey of SPF egg supply flocks (Europe 12 flocks, USA 7 and Australia 4 flock) (McNulty et alii, 1989) found that 12 of the 23 flocks were positive for CAV antibodies. Because of the pathogenicity of this virus and the possibility of egg-transmission, McNulty et alii (1989) recommended that this infection should be eliminated from SPF flocks.

McNulty and co-workers (1988) also carried out a longitudinal serological survey of two broiler breeder flocks and found that maternal antibody to CAV persisted until about 3 weeks of age. Both flocks began to seroconvert to CAV at 8 to 9 weeks and most birds had antibody at 18 to 24 weeks

In view of the severe lesions which are seen in 3 major organs of the immune system (bursa, thymus and bone marrow) it would be surprising if the occurrence of clinical Chick Anaemia Syndrome did not interact with a many other disease-producing organisms. The effect of CAV on lymphokine production and lymphocyte trtansformation has been examined in detail (Adair et alii, 1991). The association with bacterial gangrenous dermatitis has already been referred to. In the case reported here the most common secondary infection was pulmonary mycosis in which the histology indicated an unsatisfactory immune response to fungal invasion. Otaki and co-workers (1987) have shown that dual infection of young chicks with CAV and Marek's Disease Virus (MDV) causes much more serious mortality than either
infection on its own. There is also some evidence that HVT Marek's vaccine can actually potentiate the pathogenicity of the CAV virus, at least in SPF chicks (Otaki et alii, 1988). In chicks inoculated with CAV alone, a typical CAV infection shown by early mortality, decrease in haematocrit, severe lymphoid depletion in the lymphoid organs, and aplasia of the bone marrow occurred only when one-day-old chicks were inoculated. When both turkey herpesvirus (HVT) and CAV were inoculated, a typical CAV infection occurred even if chicks were inoculated with CAV at 8 days of age. The increased depletion of T lymphocytes in CAV-inoculated chicks may be the cause of the reduction in the protection provided by HVT in CAV challenged chicks. It has also been shown that early sub-clinical bursal disease virus infection in antibody-negative chicks extends the age to which chicks remain susceptible to anaemia induced by CAV inoculation Yuasa et alii, 1980b, Rosenberger and Cloud, 1989b). There is no doubt that clinical CAV is associated with severe immunosuppression in the affected birds. The importance of sub-clinical CAV in this regard is much less clear. Box and co-workers (1988) reported that broiler parents which had already sero-converted to CAV when hyper-immunized with an Newcastle Disease oil-based vaccine responded much more poorly than those which had not yet sero-converted. There appeared to be no effect on the response to the Infectious Bronchitis and IBD components of the oil-based vaccine. It is likely that if there is such an effect then the timing of infection may be critical. Other authors claim that there is no adverse effect of the virus in older birds (von Bulow, 1991). Goodwin and co-workers (1992b, 1993) found no significant correlation between CAV antibody status and other serological reactions except that antibody-negative (CAV) birds tended to have lower than normal IBD titres in the age range 21-29 weeks. These authors felt that this apparent association could be spurious. In this laboratory we find that the farms which sero-convert early and uniformly to CAV generally respond very well to oil-based vaccination. Finally, it should be kept in mind that broiler flocks exposed to CAV infection, especially if this occurs early in life, may suffer adverse effects as a result. McNulty and co-workers (1991) compared broiler flocks without CAV antibody at depletion with those with more than 50% of the birds positive. The economic performance of the antibody positive flocks was 13% poorer than the antibody positive flocks.  The authors proposed that this difference is the result of sub-clinical CAV infection in the absence of
vertical transmission.

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Acknowledgements
Thanks are due:
To poultry company management and farm staff for their enthusiastic help in collecting much of the information presented here.
To my colleague K.R. Gooderham for some of the post-mortem results used here and helpful suggestions.