Sunday, May 31, 2015

More on PCR

In the posting below - [link] we gave an overview of the recent Warwick University paper, SE3280, commissioned by Defra in 2012 at a cost of £467,353. We note that sadly, no mention of it or its stunning results have made it into the press.
And considering the indecent haste with which the badger vaccination mischief - [link] of 74 per cent efficacy, bounced around the airwaves, only to be retracted quietly in subsequent months, that is a damned disgrace shame.

 So we will cut and paste from the paper - link] which can be accessed by clicking on Final Report on the previous link. The study set out to explore a non invasive method of identifying diseased badger groups:
. "Controlling disease spread through UK cattle herds is a significant challenge as the European badger (Meles meles) has been highlighted as a wildlife reservoir that may be a significant source of continued re-infection. Determining the disease prevalence and TB status of badger populations is a demanding challenge and currently requires direct interaction with individual animals through expensive and labour intensive trapping and testing regimes. This report describes a robust and reliable non-invasive quantitative polymerase chain reaction (qPCR) assay designed to detect the presence of M. bovis DNA in badger faecal samples."
Those samples were collected from Woodchester Park and matched with cultures and blood assays from cage trapped animals, over a long period of time.
" .. badger faecal samples were obtained from 12 social groups of badgers at Woodchester Park in Gloucestershire (the site of a long-term study on TB in badgers), with a recent history of trapped badgers having positive TB test results. Samples were taken throughout the year in a cross sectional style, contemporary with trapping efforts, while two additional intensive sampling periods were undertaken during spring and autumn. In addition, samples obtained directly from trapped badgers, were directly analysed by qPCR to compare against the culture status as a benchmark. "

The results were given in the paper as follows:
When comparing qPCR on faeces taken from trapped badgers with culture, the qPCR assay exhibited a sensitivity of 100% (95% CI: 30.8-100%) and a specificity of 95.7% (95% CI: 90.3-98.6).
"qPCR results varied by season, with spring and autumn exhibiting 100% and 80% sensitivity respectively against the combined trapped badger diagnostics for the same season. The degree of infection within a social group (trapped badger diagnostics) was strongly correlated with the degree of shedding as determined by faecal qPCR"
By taking samples over a long period of time, the Warwick team ascertained that the best results came from the most highly infected groups and that samples taken in spring gave the most robust results. They explain:
"We determined the optimum sampling strategy to be 20 samples taken over a 2 day period with a few days interval in the spring or early summer. With up to 20 samples from social groups taken across May and June, we had 100% agreement with the suite of other diagnostic tests in terms of identifying positive groups"
The paper explains that this study builds on the rigorous exploration of the contents of badger latrines in Defra project SE 3231 as a non invasive method of ascertaining infective status:
"It is a direct follow on from the rigorous ring trial (Defra project SE3231), during which Warwick University, the AHVLA and VISAVET processed faecal samples from 15 latrines from putative bTB negative badger social groups (Cambridgeshire and Bedfordshire) and 15 latrines from putative positive social groups using real-time PCR (qPCR). All putative negative samples were found to be negative by all labs, two putative positive latrines were positive in all labs and one putative positive latrine was positive in one lab.

The probability of a false positive result for the two latrines detected in common is less than 3􏰄10-9. The methodology has been further optimised such that samples containing a ten-fold lower bacterial cell count can be detected as positive, resulting in considerably increased sensitivity (see Final report SE3231 Fig 11). The test that has been developed is implemented at the social group level, rather than the individual sample."
Also involved in this work, are the Republic of Ireland, which is providing post mortem samples to further quantify qPCR results. This work has found that respiratory shedding can also be identified using faecal samples.
"Other research at Warwick University, with qPCR of faeces and culture performed in parallel on samples taken from badgers in areas in the Republic of Ireland with high levels of TB breakdown in cattle, indicates that faecal shedding is a good proxy for respiratory shedding.

The qPCR test for faeces detected all badgers shown to be also shedding via the tracheal route (n=7). The qPCR and detection of M. bovis in tissue by culture were not significantly different, with a high level of correlation in detection by both methodologies in animals with severe disease progression (Travis et al. manuscript in preparation)."
The aim of the project was to enhance the detection capabilities of qPCR in the field.
"The main aim of this project was to maximise the sensitivity of the qPCR test for applied field detection of M. bovis in badger faeces through optimisation of latrine sampling strategies. A ten-fold increase in the limit of detection has been applied compared with the previous DEFRA project."
Sampling over a long period gave the following results:
The qPCR bacterial load data [] shows that particular social groups are disproportionately responsible for shedding large numbers of M. bovis bacilli into the environment.

The genome equivalents ranged from 1x103 to 4x105 per gram (N.B. 10-100s of genome equivalents are considered to represent 1 cfu)[22]. There is a variation in the cumulative load between social groups, as shown in the bubble plot; a small number of social groups appear to be responsible for most of bacteria shed and therefore potentially represent the greatest risk for onward transmission."
That mention of '1 cfu' reminded us of previous research - [link] which found just how little bacteria is needed to infect a calf.

So, the sampling:
"The state of infection in the social group affects the likelihood of detection: as expected, heavily infected social groups were identified positive with less intensive sampling regimes. The data clearly shows that by sampling in periods of peak badger activity, the chance of detecting a social group as positive with a fixed number of samples increases.

Spring is again clearly shown as the optimal sampling season, with the seven social groups with the highest prevalence of infection detected with 95% probability in 17 or less samples. In autumn 23 or less samples would detect the seven most infected social groups with 95% probability. In spring or autumn, all social groups could be detected positive with 95% certainty within 40 samples"
And the results:
"We have determined an optimal sampling strategy for latrine faecal qPCR testing, which when applied in the field demonstrated a 100% sensitivity, 100% specificity"
"Sampling should occur in the spring or early summer with up to 20 samples taken from each social group across two days with a few days interval."
"The faecal qPCR test has been shown to be robust and reliable with no significant difference observed between results obtained from two centres at the social group level."
And cost?
We would envisage the processing of the initial samples occurring in batches of five samples until either at least one sample was positive or all 20 samples were returned as negative

. To determine a sample as positive, a second and third replicate of that sample must be extracted. A social group will be considered positive if at least one sample is positive on at least one of the confirmatory re- extraction.

This gives a false positive rate of 1%.  The cost for a social group per season would range between £81.30 and £208.20 depending on when a positive sample was detected.

So, in a nutshell, Owen Paterson's qPCR project appears to be able to identify groups of infectious badgers, upspilling zTuberculosis  into the environment. Sensitivity and specificity is 100 per cent and the cost of this non-invasive technology is around £200 per group sampled.

So why no publicity?
Why were Defra giving false information to the secretary of State, about its capabilities?
And why are Defra and assorted fellow travelers so against identifying these highly infected time bombs?

The reason we think is that if a social group of badgers was so identified and APHA failed to act on that information, then as we have said many times, litigation for victims would not be a possibility but a certainty.

Far better to bury this work, and hope it stays buried. Keep killing sentinel tested cattle and ignore the message this canary is offering.

Wednesday, May 27, 2015

PCR - Prof. Wellington and SE3280

As farmers, we want eventually to target tuberculosis where it found, rather than a bureaucratic, high profile sweep of wildlife. But circumstances (and dead cattle, sheep, pigs, alpacas and cats) dictate that while levels of zoonotic tuberculosis in its wild life hosts - badgers - are so high, then an interim population reduction is working well. And we would encourage farmers to take the opportunity this new government has offered, without the Viva supported drag anchor of the LibDims - [link] and get on with it.

But what then? We can't keep having bureaucratic area culls, and many would prefer to aim their fire at diseased badgers, rather than all badgers. And so we come full circle to the PCR study, SE3280 which last year was said not to be delivering expected results - [link]

On May 14th, Professor Wellington gave a lecture at Liverpool University, discussing the work, which can be viewed on this link

There are some fairly sweeping and totally contradictory statements concerning cattle and zTB in that clip, but she did mention sheep and cats. Nevertheless the core issues of identifying diseased groups of badgers, as opposed to healthy ones is addressed. And appears to have been successful. Professor Wellington indicated that 20 samples over 2 days in spring or autumn is the optimum for the qPCR screen, and at around £10 each, the cost is not astronomical.

 It would appear that infection levels in spring and autumn are very high as social groups shake out their old and young, and re group. This presents a danger period for cattle, if one assumes that badgers can be kept out of cattle yards in the winter.

The complete paper is "The Diagnostic Potential of Real Time PCR: Proportion of badgers clinical - Travis et al" 2014  Click the link for Final Report on this LINK
Key points we noted are:
 * The degree of infection [identified by culture, gamma IFN and StakPac bloods] strongly correlated with the degree of shedding as determined by faecal qPCR.
 * Sensitivety was 100 per cent
 * Specificity 95.7 per cent

And finally:

"We suggest that a small number of social groups may be responsible for the majority of m.bovis shed in the environment and therefore present the highest risk of inward transmission".
Above is a screen grab from Prof. Wellington's film clip, during her excursions to sample faeces from the inhabitants of Woodchester Park, and the level of infectivity in the various badger groups.

Saturday, May 16, 2015

HRH Prince Charles and the need for a badger cull.

This week, the letters - [link] which HRH Prince Charles wrote to ministers a decade ago have been made public, and on agriculture in general and bTB in particular, they make for interesting reading.

 In March 2005, the Prince wrote to the then Prime Minister, Tony Bliar to express his concern over the increasing pile of dead cattle and lack of any control on eradicating zoonotic tuberculosis from its wildlife reservoir, badgers.

Political Editor,  of the   Farmers Guardian - [link] , Alistair Driver has the overview, and Bliar's reply.

HRH Prince Charles,  pictured here with Ayrshire dairy cattle,  confirming a recent meeting, pointed out  to Bliar : 

"You said that you were aware of the recent study in the republic of Ireland which proved that badger culling was effective in ridding cattle of TB - in Donegal for instance, by the fifth year of the trial there was a 96 per cent reduction in cattle infections in the 'badger removal' area."
And he emphasises the need for urgent action:
"I do urge you to look again at introducing a proper cull of badgers where it is necessary. I for one, cannot understand how the "Badger lobby" seem to mind not at all about the slaughter of thousands of expensive cattle, and yet object to a managed cull of an over-population of badgers - to me, this is intellectually dishonest."
We couldn't agree more Sir. And a week later, on March 30th 2005, Prime Minister Bliar replied thus:
"You raised the issue of bovine TB and the link to badgers. The Irish trials have indeed changed everything here, and I know Ben Bradshaw acknowledges this."
Acknowledge it he most certainly did, with answers to the carefully crafted 538 Parliamentary Questions, tabled to the Ministry of Agriculture's office in 2004 and which form the basis of this site.

And then the fragrant Ben, waved a couple of fingers at Owen Paterson who had tabled the questions, and said words to the effect "OK, we know it's badgers ..... but whaddya going to do about it?"

Ten years later,  the answer, as we have found out to our cost,  was over 300,000 dead cattle, thousands of dead alpacas, goats, sheep, pigs and domestic pets, and on badgers? Absolutely nothing at all.

Which gives support to the comment that if some politician's lips are moving, they're lying.

Saturday, May 02, 2015

Research v. Research

The more we read these 'research' papers, some good, many weak and now some repeat performances, the more we understand that on many occasions, a conclusion is reached ahead of any work. And thus a paper can be quoted to fit almost any conclusion.

Take radio collars attached to badgers and to cattle sharing the same pastures. Work was done on this in GB in 2009. We reported it in this posting - [link] The conclusion of that paper was that contact was much more frequent than was thought.

The results showed that a single badger (V59) had recordable contacts with 5 of the 13 cattle. Inter-group contact between the two badger social groups was recorded, mainly in September.
Six proximity data loggers (two badger loggers and four cattle loggers) recorded 103 and 32 inter-species interactions respectively (Tables 3 & 4). Overall, two Valley badgers and five cattle were implicated in inter-specific contacts, with the two badgers contacting all of the five cattle. All five cattle were in the top eight for CI rankings in cattle, with four out of the five amongst the top five.
So just two badgers recorded 103 inter species interactions? (Inter species = contact with cattle) and the authors reckon Defra should test the cattle more regularly? Amazing conclusions.

But now a different set of collars were attached to some Northern Irish cattle and badgers - [link] with somewhat different results.Or maybe the parameters were set differently.
Researcher Dr O'Mahoney enthused: “Proximity collars are a new and exciting technology, which allow a hitherto unprecedented level of data to be obtained on interactions between animals.
Not really 'new'. It's been done before, but we digress. The study found no direct contacts and concluded:

This study occurred in one area of Northern Ireland over a relatively short period of time, so whilst direct interactions between cattle and badgers were not recorded, that does not necessarily mean that interactions never occur.

However, it does support the increasing evidence that such contact is likely to be at a very low level, but still may be important if infected animals are involved.

Nothing like stating the blindingly obvious, is there?

 However,  New Zealand came to a totally different conclusion, using sedated possums and a video camera [ [link]

 And they recorded the interaction on a video of how cattle react to a sedated possum - [link]

They nuzzle it, lick it, smell it and roll it around.

And if that possum happened to be a half dead badger???????????????????

What happens if a group of these..

..comes in contact with one of these?

As a cattle farmer, you live in fear of your next TB test, that's what.