February 2014 has brought us yet new published research which Red Squirrel groups are claiming is evidence of grey squirrels carrying SQPV. When you look at the detail ("Squirrelpox Virus: Assessing Prevalence, Transmission and Environmental Degradation", Plusone Journal, Feb 2014), you can see that there is little additional information or conclusion from the previous publication by the same authors ("Squirrelpox virus in Northern Ireland: quantifying the risk to red squirrels", 2012). It appears to use much the same data, comes to pretty similar conclusions, and generally varies by a little more writing and a few additional illustrations.
But given there seems to be so little knowledge of the situation in Red Squirrel groups, and given we always like to keep our readers informed so that they can argue the points when faced with them, we thought we would provide a roundup of the available science as it stands.
Introduction
There are four stages at which any squirrel can be: No sign of SQPV at all, infected with SQPV, infected with SQPV and showing resistance (antibodies), and lastly showing antibodies but no SQPV.
This is just like having the flu - you are healthy, you then get the flu, you then develop immunity to that strain of the flu and cure yourself, and then you show antibodies for that strain of the flu, but you are no longer infectious. People who have a weaker immune system will get the flu worse, and for longer, and be more liable to infect others, while people with healthy immune systems might hardly show a clinical symptom, nor be infectious for long at all, but will clearly show the antibodies if their blood is tested.
Blood is tested using a process known as ELISA, which detects the antibodies present in the blood. It is unable to show whether there is any active viral DNA within the blood or tissue, but simply shows that a squirrel has been in contact with the disease at some point previously. It could well be fighting off the disease at present, or it could equally have been cured and non-infectious for a long period of time. Either way, antibodies in themselves are absolutely no guarantee that a squirrel is infected, infectious or likely to be a carrier.
This was expressed well in the 2012 publication identified above:
"To date, ELISA has been the only method used in epidemiological studies for the detection of SQPV. However, this represents an incomplete picture of pox occurrence because it tests only for the presence of antibodies (an indicator of past infection) and cannot provide data on current infection rates. Information on both the presence of virus particles (as assessed by detection of viral DNA) and their concentrations in red and grey squirrel populations is necessary to understand the spread of the disease."
So, if you wish to decide whether a squirrel is genuinely infected with SQPV, then you need to actually find that viral DNA. Scientists use a method known as Quantitive real-time PCR ("qPCR" for short) to attempt to do this. This is by no means 100% accurate, like most things in biosciences, but it can at least give an indication of whether viral DNA is present, and some sort of guide to the level of viral DNA present (often referred to as "viral load"). Out of interest, one of the laboratory companies which carries this sort of work out in the UK state on their own website:
"We would recommend that you use three replicates for each sample. Duplicate readings are the bare minimum however occasionally larger deviations are seen - especially where very low expression levels are seen, triplicate readings makes it much more likely that usable data will be obtained throughout."
So wherever you see qPCR being used to indicate low viral load, or on limited samples, it is worth keeping those words in your mind at all times!
History
It is quite clear from research in the first half of the 20th century that SQPV was endemic in the red squirrel. A paper by Middleton in 1930 clearly indicated clinical symptoms of the disease in red squirrels, and further noted that these symptoms were seen in red squirrels which had not come into contact with grey squirrels.
Of course, this begs the question of where the disease came from. There is a temptation to blame grey squirrels as bringing it in to the UK, given they were introduced in certain specific areas, but they could not account for cases of the disease right across the country in the red squirrel population. It is of course possible that diseases are endemic - otherwise how would we have a different poxvirus affecting red squirrels in Spain, and yet another poxvirus affecting grey squirrels in the USA? Neither of these locations blame imported animals for those strains of poxvirus.
One thing of possible interest is that SQPV (the UK strain of parapoxvirus in squirrels) has significant genomic similarity to Orf virus found in sheep. It could be that the virus morphed into SQPV as it jumped species. But there is currently no direct evidence of that, so it is simply an interesting point to note, and gives one possible explanation for the origin of the disease in the UK.
It was only in 1981 that Scott et al identified the virus specifically as a parapoxvirus. It was initially nicknamed SPPV (Squirrel ParaPoxVirus), but McInnes et al suggested in 2006 that it did not fit the class of a parapoxvirus, and suggested it should be considered an unidentified genus within the poxvirus family. As such, it was suggested the change of name to SQPV (squirrelpox virus).
Since that time, there has been little work beyond researching the specifics of the disease. There has been some attempts to look at the interactions between red and grey squirrels, but the difficulty has consistently been that there are simply too many variables.
In the last couple of years we have started to see research papers beginning to look into the route of transmission of SQPV between squirrels which until this time has been little more than hearsay. The first of any interest was a BMC Veterinary Research report in 2010 where Atkin et al looked at the effectiveness of qPCR in detecting viral DNA of SQPV. It stated very little, but hinted at the possibilities for extending this research to study the likes of ectoparasites as possible routes of transmission. Then in 2012, and now a little extended in 2014, we have qPCR actually being put to use to try and analyse real-world populations of red and grey squirrels.
2014 Research
The latest research aimed to look at three main factors:
- assess the antibodies and viral DNA presence for SQPV in red and grey squirrels
- analyse saliva, urine, faeces and ectoparasites from SQPV positive squirrels to seek viral DNA (possible routes of transmission)
- analyse virus degradation rates under different environmental conditions (how long viral DNA can last outwith a squirrel body)
Squirrels used in the research included:
- grey squirrels trapped between March and June 2012
- culled grey squirrels supplied by outside agencies, killed between April 2010 and June 2012 and frozen as samples.
- red squirrels which have died naturally, or been killed by predation, vehicle collision etc.
- red squirrels believed to be SQPV positive and killed as part of disease control strategies
In total, they studied 208 grey squirrels and 40 red squirrels. These squirrels represented 37 individual forests within 17 localities. Of the total 248 squirrels, 11 could not be attributed to being culled in a specific season, so were discounted. That left 237 squirrels of which 143 were known to have died in the spring, 57 in the summer, 4 in the autumn and 33 in the winter, although all 248 were used for some parts of the research.
So looking at these areas of the research in turn:
i) assess the antibodies and viral DNA presence for SQPV in red and grey squirrels
In total, 54 squirrels (around 22%) of 248 tested positive for SQPV antibodies using ELISA. This included 53 grey squirrels, and just 1 red squirrel. This simply shows that 22% of all red and grey squirrels tested had been exposed to the SQPV virus in some way.
Of course, having hopefully read the introduction, you will be aware that ELISA is only part of the story. The researchers went on to use qPCR to analyse the viral DNA present in both the red and grey squirrels. This was on a reduced set of 243 squirrels and showed a total of 23 squirrels (10%) that showed viral DNA.
Now, of course, you could be forgiven for thinking that around half the squirrels testing positive under ELISA were demonstrated to show viral DNA, and that therefore, while ELISA might be an unreliable method of demonstrating viral DNA, it at least could give a fair indication of the presence of viral DNA.
However, when you overlay the 54 ELISA squirrels with the 23 qPCR squirrels, you find an overlap of just 4 squirrels (three grey and one red) which showed both antibodies and viral DNA present. So in actual fact, what had been found was that only 7% of squirrels shown to have antibodies actually show any sign of viral DNA. These four squirrels (3 grey, 1 red) were in the act of fighting off the disease when they died or were killed.
Now we know from the results that 20 grey squirrels sampled positive under qPCR, which means that 3 red squirrels sampled positive as well (23 in total). As such, we have 17 grey squirrels and 2 red squirrels which had died showing viral DNA but no immune response, which means they either were unable to mount an immune response, or they had become infected very shortly before they died or were killed.
The 17 grey squirrels are of particular interest, because we have culling activity taking place in areas where red squirrels are thought to be at risk, and we're finding grey squirrels who are demonstrated to be freshly contracting the disease (we know that they'd mount an immune response very quickly were they not to have been culled). As such, these grey squirrels are moving into areas where reds are likely to be, and contracting the disease. This flies in the face of popular conservation "wisdom" that says the greys are carriers and spreading the disease. If anything, this speaks of a situation where reds are dying of the disease, and greys are moving in to replace them as it happens, contracting the disease and recovering quickly, as the majority of grey squirrels either show antibodies or no sign of sqpv contact. This shows that those that come into contact with SQPV recover quickly, and therefore do not infect the majority of other grey squirrels, or else there would be a lot higher seroprevalence than is seen.
The second point of interest is that for a disease that is said by conservationists to be such significant blight to red squirrels, it is surprising that only 3 red squirrels were found to be affected by SQPV.
And the final point of interest is that viral loads were found to be very much lower in grey squirrels which tested positive under both tests than those red squirrels similarly testing positive. Results for greys were an average of 8,370v/ml while for red squirrels it was 21,032,512v/ml. This means that the blood of red squirrels was 2,512 times more infected than the blood of the grey squirrels, and in the test of lips, reds were shown to be nearly 50,000 times more infected than greys. So it is quite clear that if you take a red squirrel and a grey squirrel both testing positive under ELISA and qPCR, the red squirrel is going to be vastly more infectious than the grey squirrel. Literally thousands of times more infectious.
One quote from the conclusion of the report made for particularly worrying reading, and shows that over-active conservationists can be a dangerous problem:
"However, it is somewhat disconcerting that some of the red squirrels testing negative were classed as suspected infected, and consequently culled by local authorities prior to being sent to the laboratory for analysis. This suggests that the mechanisms for identifying potential infected live animals is inaccurate (see also [10]) and that culling on this basis alone could, in fact, put extra pressure on already threatened populations. It also highlights the importance of knowing the infection status of sympatric squirrel populations before decisions are taken to cull."
ii) analyse saliva, urine, faeces and ectoparasites from SQPV positive squirrels to seek viral DNA (possible routes of transmission)
- Saliva
All of the qPCR positive grey squirrels were shown to be negative for viral DNA within saliva. The single red squirrel which was shown to be positive demonstrated an extraordinarily high level of viral DNA however. A level of 29,549,584v/ml was seen - higher even than the average viral level in the bloodstream for an infected red squirrel. This demonstrates that an infected red squirrel is very likely to be able to spread the disease via saliva. This perhaps indicates one of the great dangers of feeders, which encourage red squirrels to share food in close proximity.
- Urine
Tests of urine extracted from 38 grey squirrels (none was extracted from red squirrels) indicated two grey squirrels showed viral DNA at low levels of 1,040v/ml and 880v/ml respectively in the urine. Strangely however, all 38 indicated no viral DNA in lip tissue or bloodstream tests. It raises the question of whether the tests are in some way flawed or inaccurate, or whether a grey squirrel could actually demonstrate no viral DNA within its body, but produce viral DNA in its urine. How that might ever happen is one open to significant future research. Perhaps infected water going straight through the body? And if that could happen in a squirrel, how many other animals could produce infected urine, but show no viral DNA within their body?
It is worth noting that no red squirrel urine was able to be extracted for testing.
- Faeces
All faeces from qPCR positive grey squirrels appeared normal and were negative for viral DNA. In contrast, the infected red squirrel displayed signs of diarrhoea (liquid in appearance), which was black in colour (presence of blood?). It showed a viral DNA level of 18,434v/ml which meant faeces from an infected red squirrel could well be infectious.
- Ectoparasites
Fleas, ticks and mites were found on 153 of the squirrels, with the remaining 93 showing no signs of ectoparasites giving a total of 246. Another two could not be examined due to their condition (total of 248). Ectoparasites were tested and were only ever found to have viral DNA if the squirrel they were found on tested positive for viral DNA.
All parasites (fleas and ticks, but no mites) found on infected red squirrel were found to be positive for viral DNA. In contrast, while fleas, ticks and mites were all found on infected grey squirrels, ticks and mites were not found to be positive for viral DNA. Further, only 27% of fleas tested positive for viral DNA from grey squirrels.
Looking at viral loads, the average for fleas from grey squirrels an incredibly low 25 virus particles per ectoparasite (v/e). This was in contrast to red squirrels where fleas showed on average 2,875v/e, ticks 2,162v/e and "other" (assumed to be mites?) at 25,683v/e. In other words, a flea from an infected red squirrel is liable to be 115 times more infectious than a flea from an infected grey squirrel, and taking into account the fact that nearly three quarters of fleas from infected grey squirrels show no sign of viral DNA, it is clear that fleas represent a vastly greater danger of infection if found to be from red squirrels. Add to that the infection rates in ticks and mites which are not an issue for grey squirrels, and you have a picture of just how infectious a red squirrel is liable to be in contrast to a grey squirrel, when taking into account ectoparasites as a possible route of transmission.
iii) analyse virus degradation rates under different environmental conditions (how long viral DNA can last outwith a squirrel body)
Scab tissue from an infected red squirrel was taken and divided into 1mg pieces, 90 in total. 15 were taken for each of 6 experimental groups. These groups consisted of three temperatures (5, 15 and 25 degrees Celsius), each of which was held in wet and dry conditions for the 30 day period of the experiment. These samples were then analysed to see the number of degraded and intact virus particles. This was to see how temperature and dampness would affect the survival of a particle outside a squirrel body.
The results of this test were not exactly hugely conclusive. In specific dampness/temperature combinations, more degradation was seen, but it did not create any sort of dampness or temperature trend. The forces at work are clearly a little more complex than just a straight change in relation to temperature or dampness alone. The one notable trend was that a period of dry warm weather (25 degrees) showed significantly more intact virus particles than any other of the 6 conditions, so a dry summer could be a more dangerous time for spread of disease potentially.
Conclusion
So we see from this analysis of the recent research paper that ELISA (and therefore the presence of antibodies within a squirrel bloodstream) can not be relied upon as an indication of a squirrel being infectious, or a carrier. This is important, as it is a surprisingly common misconception.
It was also particularly surprising that around 17 grey squirrels were culled in the period between becoming infected and mounting an immune response (which is not a long period of time with a grey squirrel). This begs the question of whether trapping is being done in areas where grey squirrels are moving in and becoming infected (because they clearly were not infected until very shortly before being killed).
We do however see that even infected grey squirrels are vastly lower in viral load than their red cousins. As such, they are far less likely to be infectious than red squirrels are.
Further, we see that they are dramatically less likely to transmit the disease via bodily fluids (urine being the unusual and rather strange result).
Where ectoparasites are concerned, we also see that only a quarter of fleas (and no other ectoparasites) were found to have some level of viral DNA on infected grey squirrels. This level was consistently lower than that seen in ectoparasites from red squirrels. It does beg the question as to whether these ectoparasites received the viral DNA from the grey squirrel, or whether the grey squirrel received the viral DNA from the ectoparasite. If grey squirrels were liable to transmit the viral DNA to ectoparasites, would we not expect all ectoparasites to show viral DNA, in the way that we see in red squirrels?
The overall picture appears to be of grey squirrels becoming infected, rather than infecting, and then recovering quickly, thereby keeping the overall level of the disease relatively low in the population. Viral loads are far lower in blood and lip tissue, transmission via bodily fluids is almost non-existent and ectoparasites showed either no viral DNA or low viral DNA when found on infected grey squirrels, which does suggest transmission to the grey squirrel rather than the other way round.
As such, there is very significant doubt as to whether grey squirrels can be considered a reservoir species for SQPV, and therefore culling on this basis should be reviewed.
Continue: Facts about Grey Squirrels damaging trees >
Native by Birth - Condemned by Origin
