Colin Walker’s Rota presentation in Poland

The following paper was presented by Dr Colin Walker at the International Veterinary Conference on Pigeon Pathology in Krakow , Poland on the 7th -8th June 2018.  The paper essentially covers what is known about Rota virus and the development of the vaccine up to the completion of the vaccine trials in July 2017, at which stage the manufacture and release of the vaccine became the national Boards responsibility.


It is worth noting that further papers were presented on Rota virus in Europe, in particular by Dr Dennis Rubbenstroth of Freiberg University in Germany. He has profiled all known Rota virus isolates in Europe as well as examining historical samples. He has not found the Australian Type A Rota virus G18P but has however found another G18P Rota virus that has 2 of the 8 surface proteins the same . It causes a milder disease than "our" Rota with transient diarrhoea and low mortality. To date, severe Rota disease remains an Australian problem."

Rota Virus in Pigeons. The Australian experience


Introduction

The key word that describes the entire Rota virus outbreak in Australia is “speed”. A highly infectious, high mortality disease rapidly spread throughout the country in a period of months, killing tens of thousands of pigeons and causing considerable disruption to the entire pigeon sport.


The veterinary response needed to match the severity of the problem. It was on 12thDecember 2016 that the first case was seen on the eastern seaboard of Australia and yet, by the beginning of July 2017, in only 7 months, the condition had been diagnosed, available vaccines both in Australia and overseas had been investigated for potential cross immunity, the need to make a new vaccine had been identified, a trial vaccine had, in fact, been made, vaccine trials had been completed and the vaccine was available to go to commercial production. At the same time, the nature of the disease had been investigated so that an understanding of the clinical disease caused by Rota virus had been developed. In addition, the length of time of the carrier state and the persistence of immunity had been investigated.


We quickly realised that we were dealing with a new disease in the world. All of this work had been done in Victoria, Australia.  It was a good example of the Department of the Environment and Primary Industry (DEPI), Agribio ( the diagnostic branch of DEPI ), Latrobe University, The University of Melbourne, Tredlia Biovet ( a vaccine manufacturing company ) , the Australian National Racing Pigeon Board (ANRPB) and the Victorian pigeon industry all working together in a collaborative way . Government and education organisations had combined with the pigeon industry to deliver much clinical information and a trial vaccine that could then be adapted to commercial production. It was a great result for all concerned. I think all involved learnt a lot. Many of us had made new acquaintances, some of whom became friends.


The Beginning – the first cases in Western Australia

An outbreak of disease occurred in racing pigeons in Perth, Western Australia (WA) in May 2016. Sickness and death appeared within 3 to 4 days of race birds returning from the first Pigeon Racing Federation of Western Australia (PRF) race of 2016. Affected birds developed vomiting and a green mucoid diarrhoea. Mortality rates in various lofts ranged from 10 to 50 % with an average of 25 %.The birds had been exposed in the race transporter to pigeons that had experienced similar symptoms a week or so earlier. The origin of the infection appears to be linked to illness and deaths that were not investigated in pigeons from lofts that shared a training trailer several weeks prior to the first race. Approximately 100 members competed in the race and every competitor’s loft became infected. Only the three members who did not compete remained free of the disease. At the time, the disease was diagnosed by the attending veterinarian as a combined infection of pigeon Adeno virus Type 1 and Type 2. The significance of this misdiagnosis was that the importance of quarantine was not realised. Adeno virus is not uncommon in Australia. Failure to identify the cause as a new and exotic virus meant that bird movement continued.


After several weeks the surviving WA birds regained their health and racing resumed. A normal racing season was completed in October 2016. In that same month, however, in one of the final races, the PRF transporter collected some pigeons from a country club based in Busselton, 200 km south of Perth, and transported these birds with their own to the race release point. These birds developed the disease after return.


The Department of Agriculture and Food, Western Australia (DAFWA) released a diagnostic summary 8 months later in January 2017, stating the likely cause was a Reo virus.


Perth is a city of 2 million people located on the west coast of Australia and has approximately 150 racing pigeon fanciers. It is 3000km from the west coast to the eastern seaboard of Australia, which is where most Australians live. The west coast is separated from the east coast by a huge desert, called the Nullabor Plain, which forms a natural barrier between the two areas. As the end of 2016 approached although the disease had not been fully diagnosed, it appeared to have been confined to WA and to have “died out”. Interest in it and diagnostic momentum were fading.


The disease spreads

Melbourne is located on the east coast of Australia, 3000km from Perth. On Monday 12 December 2016, veterinarians at the Melbourne Bird Veterinary Clinic ( MBVC ) were presented throughout the day with racing pigeons by three separate fanciers . The birds in all three lofts had vomiting and diarrhoea and about a quarter of the birds had died. All fanciers described how they had been to a pigeon sale at Kyabram ( a country town 150 km north of Melbourne ) the previous weekend on Sunday 4 December. No Western Australian pigeons were at the sale, however, it subsequently transpired that birds were offered for sale from lofts where birds were unwell with similar symptoms and mortality rates to that seen in WA. It is strongly suspected that WA birds had been introduced into these lofts in the few weeks prior to the sale. These fanciers, however, denied introducing birds from WA but did believe that their birds were affected by the same problem as that seen in WA despite not having their birds’ health problem investigated. Birds were placed from these lofts into the sale at Kyabram.


Birds from the Kyabram sale travelled to South Australia, southern New South Wales (NSW), rural Victoria and Melbourne and they took the virus with them. By the end of the year the disease had been diagnosed in Port Augusta (South Australia), Finley ( NSW ), Kyabram ( country Victoria ) and several Melbourne lofts. Throughout January 2017 an unfortunate trend developed in Melbourne, where fanciers deliberately exposed their birds to the virus, which still at this time had yet to be diagnosed. They had received veterinary advice ( from the same vet who was the attending vet in Perth ) that there was no long term way of protecting the birds through vaccination, that once birds had had the disease they could not catch it again and that it was only a matter of time until any one loft became infected. He advised that it was better to get the infection over and done with and proposed a mass infection day on 3 February 2017. A significant proportion of fanciers followed this advice, particularly those in the north-western suburbs of Melbourne.


By 11 January, 11 cases had been diagnosed in Melbourne. One of these was in Dandenong ( an outer eastern suburb ). The rest were in Melbourne’s north- west. Three were in non-racing lofts. Throughout January, the number of cases actually diagnosed in Melbourne remained low. There were, however, a significant number of lofts reporting birds with typical symptoms and mortality rates but failing to present birds for diagnosis. Social media reported that there were 100 lofts infected in Melbourne and the disease was in “plague proportions”.


The first case in Sydney was recorded on 24 January 2017.


On 16 February testing confirmed the presence of Rotavirus in pigeons in a loft in the Wide Bay region of Queensland. This was the first detection of Rotavirus in pigeons in Queensland. Throughout February, further cases were diagnosed in Melbourne. On 16 February, the disease was diagnosed in a loft in Cranbourne, just north of the Mornington Peninsula in Victoria’s south - east. On 7 March, Rota was diagnosed in a second Queensland loft, located at Gympie. The loft is a commercial squab production facility.


On 6 March, Dr Mary Lou Conway , Deputy Chief Veterinary Officer,  Animal Biosecurity and Welfare Branch, Biosecurity Tasmania, confirmed that Rota virus had been diagnosed in a pigeon loft in Devonport, Tasmania. She believed that the most likely way that the virus reached Tasmania was by a pigeon being introduced from the mainland.


By mid –April, Rota cases were being diagnosed in Adelaide. The disease became widespread relatively quickly with approximately 50 lofts becoming infected by the end of April. By this time, the disease had become well established in some areas of Sydney and in Melbourne’s north –west, primarily through the attempts by fanciers in these areas to deliberately infect their birds. One federation in the north -west of Melbourne organised two “spread the virus” training flights where fanciers were encouraged to deliberately mix their birds with infected birds.


In the last week of April, Rota was diagnosed in a racing loft in Brisbane. Over the following weeks, the disease appeared in a further 30 lofts there.


By the middle of 2017, the disease was widespread throughout all of the larger pigeon racing areas of Australia. It had taken only 6 months for the disease to become widespread and common. 90% of fanciers lofts were affected, over 100,000 birds had become unwell, and thousands had died.


Origin, where did this disease come from?

Where did this virus come from? It probably did not come from anywhere. The virus, or at least its parts, has probably been in Australia for a long time but in a slightly different form.


The sequenced virus has significant similarities to Rota viruses isolated previously from the Red Fox (Vulpes vulpes) and also from the Spotted neck dove ( Streptopelia sp ).  Red foxes are a common and widespread feral pest in Australia. Active extermination programs are in place due to the damage they inflict on native Australian mammals and birds but their populations appear remarkably resilient. Foxes are found in all Australian environments and are surprisingly common not only in rural areas but also residential suburbs and indeed large city centres. Three species of Streptopelia dove were introduced into Australia towards the end of the 19th century from south-east Asia. All have established large feral populations. Streptopelia chinensis and Streptopelia tigrina are found on the eastern seaboard while Streptopelia senegalis is located on the west coast. The eastern and western populations are discreet, being geographically separated not only by distance but by the huge central desert areas of Australia.


So where did this disease come from? The suggestion is that an antigenic shift in a  Streptopelia Rota virus has occurred  or alternatively a viral recombination has occurred that has altered the virus sufficiently that both the pattern of disease it causes  and the species it affects have changed.


Reaching a diagnosis


Throughout December 2016 and January 2017, extensive diagnostic work involving autopsy, bacteriology, histopathology, electron microscopy, virus culture and genetic sequencing was done in Victoria. When the initial unwell birds were presented to veterinarians at the MBVC on 12 December a fairly standard diagnostic path was followed. Following clinical examination and standard screening tests including microscopic examination of the faeces and crop flushes several birds were selected for autopsy. Full sets of tissue samples were collected for histopathology, swabs for microscopic examination, bacterial culture and antibiotic sensitivity (M C and S) were taken from the liver and bowel and swabs were collected for  PPMV (Pigeon Paramyxovirus) and Circo virus PCR ( Polymerase Chain Reaction) tests. The PCRs returned negative results while the bacterial cultures grew normal bacteria.


Gross autopsy was unremarkable, however, on histology the lesion looked viral, according to the pathologists at AgriBio, so electron microscopy was performed (at the Australian Animal Health  Laboratory (AAHL) ) and abundant viral particles were seen. Back at AgriBio, viral cultures in embryonated eggs were commenced. Isolation proved tricky, but identification of a Rotavirus on next generation sequencing allowed some modifications, which led to isolation, but not amplification. (Staff at AgriBio, including Dr Christina McCowan, plan to do more work on this.)


Next generation sequencing and Sanger PCR confirmed the virus to be an A group rotavirus, sub type G18P,of  previously undescribed genotype.


Further testing in Victoria on samples collected in Western Australia in May 2016, showed the disease in Western Australia to be caused by the same virus.


Diagnostic Features


Autopsy and histology changes


As mentioned above gross autopsy is unremarkable with no reliable gross abnormalities. Some birds have enlarged spleens, many do not. Sometimes the liver is slightly pale or perhaps a bit friable. Essentially however autopsy is not diagnostic.


Histologically the principle lesion is hepatic necrosis seen as dissociation, rounding up and hypereosinophlia of hepatocytes with shrinking and distortion of nuclei, often with little or no inflammation. A moderate to marked macrophage response, some with prominent cytoplasmic vacuolation and intravacuolar cellular debris and yellow/brown granular pigment is seen. Inflammatory infiltrates (lymphocytic or lymphohistiocytic) were often, but not always seen in the spleen. Histiocytic infiltrates (associated with resolving the necrosis) and sometimes a lymphocytic infiltrate are seen in the liver but are not reliable features. Gut lesions are inconsistent with most birds not having them at all.  Other organs are not reliably involved.


Professor Amir Noormohammadi at the University of Melbourne accidentally found Rota virus in the kidneys of some birds on electron microscopy (EM). However there is no significant renal lesion, apart from urate nephrosis in some birds which is thought to be probably secondary to dehydration (which is not surprising with vomiting and diarrhoea). The indication is that the virus is not causing any pathology in the kidney even if it is there in some birds.


Clinical details

1/ The virus has an incubation period of 3 – 7 days. Birds start to become unwell 3 – 7 days after they have been exposed to the virus.


2/ Initially, birds develop a hunched posture, fluff their feathers and become reluctant to move. They then develop a green mucoid diarrhoea and start to vomit.


3/ Deaths start 12 – 24 hours after the first birds become unwell. Deaths peak on day 4 and stop usually by day 7. In lofts in Melbourne, mortality rates have ranged from 6 to 36 % with an average of 22%. However, in some of the worst affected lofts in Sydney, mortality rates have been up to 50%. Approximately seven days after the onset of symptoms most birds in the loft appear to regain their health and appear clinically normal, however, the occasional death can be expected to occur for several weeks after the outbreak due to complications associated with recovery in individual birds,( such as adhesions to adjacent organs and secondary bacterial infections ).


4/ During the outbreak, every bird in the loft is thought to become infected, however, more extensive testing of larger numbers of birds needs to be done to confirm this. Not all birds become unwell. Some birds continue to behave normally. Others display symptoms that may be mild to severe, and then recover. However, in many lofts, a significant proportion of birds that develop symptoms die. Terminally, these birds develop a hunched posture and become dyspneic. Their mucous membranes are congested and cyanotic. The glottis is rounded on inspiration. Many terminal birds become sternally recumbent. During the outbreak food intake is reduced within the loft by approximately 50%.


5/ During infection, the virus penetrates the bowel wall and presumably is carried in the bloodstream to the liver and occasionally the kidneys. In the liver, the virus causes massive necrosis of hepatocytes. The primary cause of death is viral hepatic necrosis. Secondary causes of death include dehydration and hypothermia.


6/ Surviving birds from infected lofts become carriers of the virus. Testing ( by cloacal Rota PCR ) has shown that surviving birds from infected lofts shed the virus in their faeces for 9 to 12 weeks and are infectious to previously non-infected birds during this time. In testing done at the MBVC, 13 of 14 birds were positive on cloacal swab 9 weeks post infection while 10 of 10 were negative on cloacal swab at 13 weeks. Two birds tested by liver Rota PCR at 9 weeks post infection were positive while two tested at 15 weeks were both negative. These results suggest that the carrier state persists for 9-12 weeks, however, they are at odds with the spread of the virus from WA to Victoria via the auction at Kyabram about 5-6 months after the initial outbreak in WA had died down. It may be that, where most birds stop shedding by 9-12 weeks, as detected by cloacal PCR, some birds carry the virus for much longer. Under natural conditions evidence suggests shedding may last up to 6 months in at least some birds. It has been suggested that the auction was like a natural experiment, which measured the duration of infection in a different and in some respects more realistic way, perhaps with the stress of transport contributing to the renewal of shedding. As larger numbers of birds are tested the duration of the carrier state and nature of shedding will become clearer


8/ It is thought that wild birds, in particular Streptopelia spp. (lace neck ) doves, and also flies, may transmit the virus. This, however, needs to be validated. To date, the only wild, free-flying birds that the virus has been detected in are feral pigeons. However, very few birds other than pigeons have been presented for testing. One of our biggest missing pieces of information is how the virus spreads locally from loft to loft in nearby suburbs.


9/ Anecdotally, it appears that the virus is not particularly robust in the environment and does not survive under ambient conditions for more than 2- 3weeks. Deliberate attempts to infect birds with the faeces of recovered birds failed if more than 3 weeks had passed since collection.


Persistence of immunity: the Western Australian experience


It appears that some recovered birds from previously infected lofts are vulnerable to re-infection after about 10 months.  Initially, it was not known if recovered birds could catch the disease again or, if they could, at what time they would become vulnerable to reinfection. Further work needs to be done but the experience in Western Australia gives us an indication.


Fresh cases of Rota virus were diagnosed in two previously infected lofts in Western Australia on 26 March 2017. Both lofts had had Rota virus during the previous outbreak of May/ June 2016. The fanciers explained that their birds were again showing symptoms similar to those displayed during the earlier Rota virus outbreak and that some birds had already died. Histopathology and a Rota PCR were done to confirm the diagnosis. One loft had 90 race birds of which about 20 died. Of these, most were young birds bred since the initial outbreak while three were old birds that raced during 2016 after surviving the outbreak. In the second loft two of 100 died with about 10 % of birds showing mild depression, diarrhoea and vomiting.


The PRF of Western Australia commenced their 2017 racing season in June.  Within 4 weeks of starting the season, changes to the race season had to be made in order to accommodate the effect of Rota virus, ( racing continued but championship points were not allocated). Approximately 40% of competitors lofts had become infected with Rota virus. It appeared that the young birds, when mixing in the race baskets with birds from other lofts for the first time, had been exposed to Rota virus. The most likely source of the exposure was older birds that had survived the previous year’s outbreak and were carriers of the virus. As all fanciers with unwell birds had previously had Rota in their lofts in 2016, it seems that, although it is possible for some immunity to be passed from parents to their youngsters during breeding, it is insufficient to prevent disease reliably. Compared to the previous year only small numbers (less than 10 %) of birds in each loft became obviously unwell and even lower numbers died. The majority of birds that died were youngsters. Presumably some immunity passes from recovered birds, which are subsequently bred from, to their youngsters. As these young ones mature this passively acquired immunity gradually fades making them increasingly vulnerable to clinical disease. Partial immunity can modify the severity of symptoms. As in the year before, after about 4 weeks most birds in most lofts had recovered and racing was resumed and the season completed.