A new disease concern for pigeon fanciers is being recognized with increasing frequency. With reports of infected flocks in northern and southern California, Canada, and Australia, it now appears that Pigeon Circovirus may become a major problem among mixed-aged flocks. This is an example of a disease that has been around for at least a decade, but that went unrecognized until recently.1

Circoviruses are a newly described group that includes Pigeon Circovirus, Psittacine Beak and Feather Disease (PBFD) and Chicken Anemia Agent (CAA). Detailed laboratory studies confirm that these viruses are similar to one another (they share some portions of their DNA), but there is no known cross-over of disease agents from one group of birds to another. ³ PBFD is certainly the best known of the three, affecting more than 35 species of "hookbills" (parrots, macaws, cockatoos, and their allies). This disease results in characteristic abnormalities in feather growth, beak lesions and long-term loss of body condition. It is known that both PBFD and CAA cause disruption of the tissues comprising the lymphoid system. This system (including the spleen, thymus, Bursa of Fabricius, and both gut- and bronchial-associated lymphoid tissues) houses cells that are responsible for most immune functions of the body: making antibodies, recognizing disease organisms, processing antigens, and so forth. Because CAA and PBFD viruses harm the lymphoid organs, birds having these diseases are "immunosuppressed". That is, these viruses cause a form of acquired immunodeficiency (like human AIDS). One result is that secondary infections with other viruses, bacteria and fungi are common among birds with PBFD and CAA, even when birds are vaccinated against the secondary problems. In other words, Circoviruses compromise the birds' ability to benefit from vaccination!

Disease symptoms seen in birds with Pigeon Circovirus somewhat resemble those seen with PBFD and CAA. Pigeons initially show respiratory symptoms, weight loss, diarrhea, and stop eating, often dying after a short (2 to 5 day) period of illness. Many symptoms seen in Circovirus - infected birds are really attributable to disease caused by secondary infections that take hold because Circovirus has suppressed the bird's immune responses. Among these secondary infections, researchers have found Chlamydia (Ornithosis/Psittacosis), Herpes virus, Pasteurella (Cholera), Paramyxovirus - 1, Trichomonas (Canker), and Aspergillus (among others). ^{1, 2} Interestingly, in some Circovirus - infected flocks, there will be illness, but no mortality, while in other flocks, 100% of the squabs will die. When these pigeons are presented for post-mortem exam, the most common findings are destruction of cells in the spleen, thymus and Bursa. The latter two organs normally shrink in size after hatching and are tiny or absent in the adult bird. In the youngster, however, they are very important as sites for immune system tissues. With these primary target organs, it is not surprising that all pigeons with confirmed Circovirus infections thus far have been one year or less in age. Infections have occurred both in racing lofts (in one case a bird returned two days late from a race and other individuals were performing poorly.), and in squab operations; a variety of breeds are represented. The relatively high frequency of infections among racing pigeons suggests that inadequate attention has been paid to shipping conditions and quarantine. Dr. Leslie Woods, of U.C. Davis feels that if Pigeon Circovirus holds true to the pattern set by PBFD and CAA, then infection may be by a fecal-oral route. As yet, there are no known wild bird reservoirs for the virus, so it is uncertain how racers are encountering the disease.

Several factors conspire at present to hinder the prevention and diagnosis of Circovirus in Pigeons. So far, researchers have not been able get the virus to replicate in the lab. This step is required before rapid diagnostic tests can be developed or vaccine trials can be done. Additionally, the presence of so many secondary infections in Circovirus - positive flocks can lead to errors in treatment and diagnosis. Unfortunately, the other Circoviruses are very resistant to treatment with heat, disinfectants and detergents. 2. If this also holds for Pigeon Circovirus, it may be that the only way to salvage an infected flock is to eliminate all young birds for at least a year (ie, let the virus die - out naturally). Even this measure may fail, however, if the virus is able to exist in a "carrier state" in some adult birds. These and other details of the epidemiology (natural history and patterns of infection) of Pigeon Circovirus are completely unknown.

Our advice is to pay careful attention to hygiene and quarantine procedures when shipping birds, especially racers. Fanciers who import squabs, particularly into flocks that already have many young birds, should observe strict quarantines for 2 to 4 weeks after arrival. Until flocks can routinely be vaccinated, this newly recognized, immunosuppressive virus represents a real threat to 'open - entry' flocks and racing lofts.

References
• Woods LW. Circovirus Infections in Pigeons. Proc Annu Conf Assoc Avian Vet, 1993; 156-157.
• Gerlach H. Viruses. In Ritchie B, Harrison GJ, Harrison LR, (eds.). Avian Medicine: Principles and Application. Lake Worth, FL, Wingers Publishing, 1994; 862-948.

Interim Results about examinations regarding various viruses:
The presence of various viruses in pigeons suffering from Young Bird Sickness was determined at the Universities of Leipzig and Gießen.
• Adenovirus and Polyomavirus PCR (polymerase chain reaction) tests of the livers were negative as were subsequent attempts at culturing.
• PCR tests regarding Herpesvirus was positive in 15% which could also be confirmed through cultures.
• The presence of Circovirus could be confirmed in 90% with the PCR test and 100% of all blood tests were positive as were all tests of the oil gland.

The trigger of young bird sickness is the Circovirus in connection with other disease-causing agents. At the FCI-level a related study is running that concentrates itself on how Circovirus is spread. From this comes the question of how one can most effectively fight the virus.

British Homing World, January 23/04

Investigations into circovirus infection in pigeons 2003

a) Introduction

Young bird sickness continues to cause problems in racing pigeons. Work funded by the British Homing World Show of the Year 2002, reported in British Homing World in January 2003, demonstrated that around two-thirds of sick young pigeons had evidence of infection with a virus called pigeon circovirus. This virus damages an organ called the bursa of Fabricius, interfering with the normal functioning of the immune system of the bird and predisposing to a range of different secondary infections. Affected birds show signs such as diarrhoea, weight loss, vomiting, respiratory signs, and may die. Until recently the diagnosis of this disease involved microscopic examination of preserved slices of bursa of Fabricius, a technique that requires access to post mortem material and that sometimes gives false negatives.

It was recognised that this figure of two-thirds of young pigeons being infected with circovirus could be an underestimation, and it was agreed that there was a need for more sensitive tests and tests that could be carried out in live birds. Such tests would not only help in the diagnosis of this disease, they could also prove useful in finding out more about this disease, for example where it comes from, how it spreads, and whether certain management practices are more likely or less likely to precipitate circovirus infection.

As reported in British Homing World in January 2003, preliminary work with a laboratory in Edinburgh in 2002 had suggested that a test called the polymerase chain reaction test or PCR test was showing promising results and was worthy of further investigation. Additional funding was therefore provided by The British Homing World Show of the Year 2003 to allow further development of the PCR test for pigeon circovirus by SAC Veterinary Services Auchincruive and Biobest Laboratories, Edinburgh.

b) PCR test development in 2003 - liver samples

Numerous samples of tissues from pigeons with and without microscopic evidence of pigeon circovirus infection had been retained in the deep freeze from the 2002 investigations. These samples, additional material collected from pigeons in 2003, and samples of tissues from birds other than pigeons, were then tested by the PCR under strictly controlled laboratory conditions. Initial tests showed that samples of liver were suitable for testing because liver appeared to be a good source of virus and because the liver is an organ easily sampled at post mortem examination. By varying the testing procedures the PCR was repeatedly refined until it appeared to be capable of detecting circovirus in the liver of infected birds but without giving false positive reactions in uninfected birds.

For any new test such as a PCR to be acceptable in the UK or overseas, it must first be rigorously and independently validated. The laboratory methods must be carefully described, covering all important steps in the process such as extraction of she DNA from the liver, the times and temperatures of different thermal cycles, running the gels, and interpretation of the final results. Using these standard procedures, a laboratory must then test a certain number of samples in a 'blind' fashion, without knowing the origin of the samples and what results would be expected. The final document, including testing protocols and the results, is then subjected to an independent quality assurance audit, and the test will only be validated if the quality assurance is satisfactory.

As part of this validation procedure, the laboratory was sent 60 samples of liver, individually labelled but whose identity was unknown to the laboratory. Fifteen samples were from young pigeons with microscopic evidence of circovirus infection in their bursa of Fabricius; nine samples were from young pigeons in which no microscopic evidence of circovirus infection had been found but in which the history was consistent with circovirus infection; six samples were from young pigeons in which no microscopic evidence of circovirus infection had been found and in which circovirus was not suspected; fifteen samples were from adult pigeons; and fifteen samples were from various species of wild bird other than pigeons. The results of the PCR on samples of liver for pigeon circovirus are given in Table 1.

The PCR test detected pigeon circovirus in the liver of all fifteen young pigeons in which microscopic evidence of this disease had previously been found, and was negative in the 15 'non-pigeon' livers, showing the accuracy of he test. The PCR test was also negative in the six young pigeons in which circovirus was not suspected. Six young pigeons with signs consistent circovirus infection but in which microscopic examination of the burst previously been found to be negative were in fact positive for circovirus the PCR - this probably reflects the limited sensitivity of microscopy, a finding also reported by other research workers, and reinforces the suspicion that circovirus infection is even more widespread than realised. Pigeon circo is primarily an infection of young pigeons, and this was reflected in the finding that 14 of the 15 samples from adults were negative. One adult sample however was postfive, suggesting that some adult pigeons may can virus. Overall, the results indicate that a positive PCR on liver is a strong indicator of circovirus infection, and that a negative PCR on liver is an indication that the sample is free from circovirus.

TABLE 1. PCR RESULTS FOR PIGEON CIRCOVIRUS - LIVER

c) PCR test development in 2003 - samples of intestine

Having shown that the PCR can be successfully applied as a diagnostic tool using samples of liver, the same procedure is currently being evaluated on intestinal samples, with additional comparisons with liver samples tested previously from the same birds. As before, the laboratory was sent individually labelled samples, this time of intestine, but whose identity was unknown to the laboratory. Twelve samples were from young pigeons with micros evidence of circovirus infection in their bursa of Fabricius; six samples were from young pigeons in which no microscopic evidence of circovirus had been found but in which the history was consistent with circovirus infection; four samples were from young pigeons in which no microscopic evidence of circovirus infection had been found and in which circovirus was not suspected; six samples were from adult pigeons; and five samples were from birds other than pigeons (greenfinches). The results of the PCR on samples of intestine for pigeon circovirus are given in Table 2.

TABLE 2. PCR RESULTS FOR PIGEON CIRCOVIRUS - INTESTINE

The PCR test detected pigeon circovirus in the intestine of nine out of twelve young pigeons with circovirus lesions in the bursa of Fabricius. Earlier, the PCR had detected circovirus in the liver of all fifteen pigeons with circovirus lesions (Table 1), suggesting that liver is a better source of circovirus than intestine. Similarly, the PCR detected circovirus in the intestine of two out of six young pigeons without circovirus lesions but with a history consistent with circovirus infection, whereas the PCR had earlier detected circovirus in the liver of six of nine young pigeons in similar circumstances. Again, this suggests that liver may be a better source of virus than intestine. The PCR did not detect pigeon circovirus in the intestine of six adult pigeons, four young pigeons in which circovirus infection was not suspected, nor five non-pigeons. These results indicate that a positive PCR on intestine is a strong indication of the presence of circovirus in the intestine, but that a negative PCR on intestine does not rule out the possibility that the bird is carrying circovirus elsewhere.

d) Conclusions

Funding from the British Homing World Show of the Year 2002 established the importance of pigeon circovirus as a significant disease in young pigeons. Subsequent funding by the British Homing World Show of the Year 2003 has enabled the development of a PCR test capable of detecting circovirus in samples of liver, and to a lesser extent intestine. This test can be used for diagnostic purposes, ideally on samples of liver, and is more sensitive than the previously used test that involved microscopic examination of preserved sections of bursa of Fabricius. The ability of the PCR to detect circovirus in samples of intestine suggests that the next logical development would be to test samples such as faeces, swabs, eggs etc that can be collected from live birds. Using the PCR on such samples would then allow the virus to be 'tracked' in a loft situation, opening up the possibility of assessing the impact of different management procedures on the course of this infection.

Tom Pennycott, December 2003
Avian Health Unit,
SAC Veterinary Science Division
Auchincruive
Ayr KA6 5AE

British Homing World, January 3/03

Investigation into Circovirus Infection in Pigeons 2002

Background

Earlier work funded by the Scottish Homing Union Trust Fund showed that in the year 2000 infection with circovirus was very common in young pigeons in Scotland, showinq signs such as vomiting, diarrhoea, weight loss and death. The work also showed that there could be a link between circovirus infection and paramyxovirus infection, because circovirus damages the immune system of the bird, predisposing to secondary diseases (including paramyxovirus) and also reducing the ability of the bird to respond to vaccines against paramyxovirus. Infection with paramyxovirus can, therefore, follow infection with circovirus, even in pigeons that have been vaccinated against paramyxovirus.

Additional funding was received from the British Homing World Show of the Year 2002 to allow further investigation of pigeon diseases, especially circovirus infection. Until very recently, the diagnosis of circovirus infection relied on the microscopic examination of preserved portions of a particular organ, the bursa of Fabricius, after removal from the dead bird at post mortem examination. Improved methods of detecting circovirus by culture or by other virus demonstration techniques are essential if we are to find out more about this increasingly important disease or if a vaccine is to be developed, but clearly it would be unfair and unrealistic to expect individual pigeon fanciers to pay for this work. The British Homing World Show of the Year 2002 therefore agreed to pay for the collection and examination of material from a limited number of suspected cases of circovirus and for the storage and subsequent delivery of positive samples to another laboratory working on this virus. It was hoped that this approach would result in three main benefits: It would tell individual fanciers if circovirus was present in their birds. It would allow an estimation to be made as to how common the virus was in young pigeons in general. It would provide a source of positive material for other laboratories that may, in the long. term, be able to produce a vaccine against pigeon circovirus or develop better diagnostic tests.

How common was circovirus infection in pigeons in 2002?

Microscopic examination of preserved sections of bursa of Fabricius was carried out on 37 sick young pigeons submitted between May 2002 and October 2002, of which 24 (65%) showed microscopic evidence of infection with circovirus. This is very similar to the figure of 75% found in Scotland in 2002, and also to the figure of 66% found in pigeons in Northern Ireland between 1995 and 2000. Clearly infection with circovirus is still very common in sick young pigeons and the recent development of more sensitive tests suggests that these figures may be an underestimation. Positive birds showed a range of clinical signs such as weight loss, diarrhoea and death, and less often vomiting, poor performance, nervous signs or respiratory signs.

Secondary Infections association with circovirus infection

Infection with circovirus damages the immune system of the bird and allows other infectious agents to cause problems. The funding from the British Homing World Show of the Year allowed a more detailed examination of the pigeons submitted for post mortem examination. The bacterium E. coli was isolated from many of the birds with circovirus infection, but the significance of this is uncertain because E. coli is found in the intestines of healthy pigeons. Salmonella was isolated from two birds with circovirus, and the internal protozoan parasite Hexamita was found in five birds. Surprisingly, only one bird had damage to the liver caused by an adenovirus or herpesvirus infection. Paramyxovirus was isolated from one and suspected in another two birds. Other organisms included the fungus Aspergillus and the protozoan Trichomonas, the cause of canker. However, the organism recovered most frequently with the exception of E. coli was Chlamydia (the cause of ornithosis) which was found in 41% of birds with circovirus.

Ornithosis in Pigeons

The disease ornithosis of pigeons is caused by a micro-organism called Chlamydia, recently re-named Chlamydophila, an organism with some of the properties of conventional bacteria and some of the properties of viruses. The same organism can cause disease in humans and birds of the parrot family, in which the disease may be called psittacosis, or alternatively the disease may be called chlamydiosis. So this disease complex has several different names.

Ornithosis in pigeons may be one of several causes of respiratory disease or may cause diarrhoea. In both situations infection can result in the death of severely affected birds. Adult birds may be apparently healthy but carry this organism, passing it to other adults or young birds. It can spread via the droppings or the respiratory tract and so could spread while the birds are in the transporters. Not only is ornithosis important in pigeons - disease can sometimes spread to humans also, causing flu-like signs, headaches and a high temperature, and sometimes spreading to the lungs to cause coughing and pneumonia. Very rarely meningitis and death can occur in humans.

Between May and October 2002, 40.9% of sick young pigeons with circovirus inclusions in the bursa of Fabricius were also positive for Chlamydophila (Chlamydia) psittaci by a test called the polymerase chain reaction (PCR) test, as were 46.2% of sick young pigeons without circovirus inclusions. This overall chlamydia-positive rate of 15 out of 35 young pigeons (42.9%) compares with 4 of 55 sick young birds (7.3%) tested positive for chlamydia by PCR between April and November 1992. It is unclear what role pigeon circovirus has played in this increase in the number of chlamydia-positive birds.

Development of an improved test for the detection of circovirus infection

Until very recently, the diagnosis of circovirus infection relied on the microscopic examination of preserved portions of the bursa of Fabricius after its removal from the dead bird at post mortem examination. However, detecting virus in live birds by culture or by other virus demonstration techniques is essential if we are to find out more about this increasingly important disease. Workers at Stormont, Northern Ireland, recently succeeded in sequencing the genetic DNA of pigeon circovirus and based on that information a laboratory in Scotland is hoping to develop a very sensitive PCR lest to detect pigeon circovirus DNA. This preliminary work has been funded by the British Homing World Show of the Year and is showing promising results. Twenty-five samples of a range of tissues from five pigeons with circovirus inclusions in the bursa were first tested by an existing PCR test that detects a related circovirus of parrots called Psittacine Beak and Feather Disease virus. Although this virus is related to the pigeon circovirus, all 25 samples were negative. However, the new PCR test being developed in Scotland specifically for pigeon circovirus gave strong positive results for some of the samples from each of the five birds known to be infected with circovirus. Much more work needs to be done on the development of this test, but the results so far indicate that it is worth taking further.

Conclusions

- The results from 2002 indicate that pigeon circovirus is still widespread in sick young pigeons.

- The results from 2002 have also revealed that ornithosis, a disease that can spread to humans, appears to be more common in young pigeons than it was 10 years ago.

- The development of a more sensitive test for pigeon circovirus is showing promising results.

British Homing World Show of the Year 2003

It has just been announced that the British Homing World Show of the Year is to fund further work in 2003. I will be present at the Show in Blackpool on January 18th and 19th 2003 and will be happy to discuss the results so far.

TOM PENNYCOTT