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Letters; Guts, nosema, and trypanosomes.


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The ‘Cororapa’ phenomenon is something everyone’s talking about; I’m just thinking about it (still). Let’s start with some basic biology, and apologies to anyone offended by my loose use of the technical terms and simpleton-ness!

 

The adult honey bee digestive tract is made up of three main regions, the oesophagus, crop and its proventriculus (‘fore-gut’), the ventriculus itself (‘mid-gut’, analogous to the stomach), and the ileum and rectum (‘hind-gut’). In a healthy ‘bee the microbial community that inhabits the digestive tract gets more abundant as we travel from the crop to the rectum. It is practically non-existent in the foregut, sparse in the mid-gut, and abundant in the hindgut. These regions of course do different things and produce different environments that different microbes inhabit, good microbes and bad microbes.

 

In the mid-gut there are normally few ‘inhabitants’. Food entering the ‘stomach’ is enclosed in a special bag, a membrane secreted by cells lining the ventriculus. Many insects do this (but not all) and it’s thought that this protects the lining of the ventriculus from abrasion and tends to keep harmful things in. The ventriculus wall is covered in a jelly-like layer, under that is another layer of germ cells that constantly produces digestive enzymes contained inside soluble granules within the cells. These cells are shed into the ‘jelly’ layer where they burst, liberating the granules which dissolve allowing the enzymes to digest the food. The membranous bag (called the peritrophic membrane) containing the food is selectively porous, the enzymes can get in and the food nutrients can get out.

 

In a bee (any kind) infected by nosema (any kind!) the main effect is that the cells producing the cells containing digestive granules are destroyed – the cell is invaded, the organism reproduces as millions of spores and the cell bursts. The bee can no longer assimilate any food and dies. It appears as though each nosema can also spread to other, but different, tissues in the body. For example, Nosema.apis has been found in muscle cells (perhaps causing the characteristic ‘crawling’); Nosema.cerenae hasn’t been found in muscle but has been found in the fat body. That’s important (if it’s right) because the fat body produces proteins (antibacterial peptides or AMPs) that make up part of the bee’s immune system. Sick bees often fly away to die, it's part of a 'colony level' immune system, and that could mean they 'disappear'.

 

Further down into the hind-gut in the ileum bees use a different kind of ‘barrier’ to protect themselves. Unlike the mid-gut this is lined with a cuticle and covered in layers of bacteria living in a ‘biofilm’. The presence of these bacteria is, if you like, a living antibiotic, defending their niche from all comers and, in the process, protecting the bee. The bee is also able to produce AMPs from the cells lining the ‘tube’ that assists in resisting ‘foreigners’.

 

Bees that harbour the trypanosomes Crithidia or Lotmaria have a layer of the parasites where these commensural bacteria should be. We don’t know what the effect of that is, it was once thought it damaged the lining layer but that could be something else. They could have no effect. In normal circumstances bumble bees and honey bees are known to survive this parasite, they have been ‘enemies’ for a while and the bee's defences and tolerance has evolved over a long period of time so that trypanosome infestations don’t normally get out of hand. It may be that in the case of co-infections of both nosema and lotmaria that N.ceranae completely occupies the bee’s immune system (for example, so AMPs are not produced) and at the same time the change in the mid-gut ecology disrupts the ‘good’ bacteria living in the hind-gut and allows the trypanosome pest to run rampant. A bee has really no prior experience of a N.ceranae infection so instead of an efficient, measured response we see a vast escalation where it tries out everything it has and leaves nothing in reserve for anything else.

 

There has been some effort to try and control at least one half of the problem – the nosema half. The antibiotic fumagillin (sold as Fumadil B) inhibits an enzyme we shall call MetAP2. Most multicellular organisms possess genes for two MetAP types, MetAP1 and MetAP2, and require one or the other to survive. They are involved in the ‘signalling’ that allows cells to divide and reproduce. Microsporidia like Nosema however do not have an MetAP1 gene, so by targeting their MetAP2 we can supress, but not eliminate, a microsporidian infection. Fumadil B has been used for years to ‘hold’ Nosema apis infections until the bees are able to eliminate are the spores for the infection, I used to use it myself.

 

Now if you’re following closely you’ll have realised that we, being multicellular, also use the same enzymes and so many jurisdictions, not being particularly keen on putting people at risk, have banned or discouraged the use of the drug. In New Zealand I think technically it was just not re-registered rather than banned, and its use is still probably permitted in circumstances like queen rearing where there is little risk of it in the food chain. There are antibiotics that have no place in human medicine and it would be interesting to see if they had an effect on the nosemas, but that’s for another day.

 

Clearly bees are also multicellular and so they too face a risk, so what we do is decide that death by nosema is the greater of the two evils and use the lowest dose we can. Nevertheless, bees feeding on syrup dosed with fumagillin have been shown to have shortened life spans, presumably because their bodies won’t repair properly if cell division is inhibited, but it has proved to be a useful tool over the years and saved many colonies.

 

These days people have been trying it with N.ceranae infections, but here it gets very messy. The results have been very mixed, from no effect to total success to made severely worse. The reason for this appears to be because control of the dose is, um, challenging, and because ceranae appears to be less susceptible. Many things affect fumagillin concentration in hives post-treatment, including hive size, population, temperature, nectar flow and so on. There is often a treatment gap of months or more which guarantees that the antibiotic will degrade to low concentrations. Unlike a chemical on a strip, you can’t take it out before that happens. To quote from the main study;

 

“N. ceranae spore production recovered at a higher fumagillin concentration than N. apis. At lower fumagillin concentrations, significantly more infective N. ceranae spores were produced in treated bees than in untreated infected bees. Protein profiles of bees fed fumagillin confirmed our hypothesis that fumagillin affects bee physiology at concentrations that no longer suppress N. ceranae. Use of fumagillin may increase the prevalence of N. ceranae and is potentially a factor in replacement of N. apis by N. ceranae in US apiaries.”

 

I don’t’ have much to say about what might help prevent these infections in the first place and keep colonies healthy so they can resist them themselves. In the absence of Fumadil B I would be persuaded to artificially swarm an infected colony onto clean comb (foundation or ‘refreshed’ with acetic acid), not much of a ‘commercial’ solution I know. That way I’d separate the infected adult foragers, hope to clean them up, and preserve my hopefully uninfected brood with a new queen that might out-lay the problem. It works with N.apis. I would also try to improve as far as possible the general ‘gut health’ by moving the bees somewhere healthy with lots of pollen sources; I have no faith in so-called ‘probiotics’ and not much faith in some the feed additives, although my guess they are probably on the right track. I have always thought fixing up disease is an ‘ambulance at the bottom of the cliff’ approach and I’d like more focus on what makes colonies healthy instead of on what make them sick.

C’est la vie.

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In the context of what I said about fumagillin this looks quite interesting. I can't read it (yet) it's pay-walled, pity. Maybe someone else here can.

 

Research Highlights

We tested whether oxalic acid controls the honey bee parasite Nosema ceranae.

In laboratory, oral applications hindered the increase rate of artificial infections.

In field, two topic administrations decreased prevalence in young and old bees.

Increased prevalence and overwintering failure was observed in untreated colonies.

We conclude that oxalic acid can be used to control N. ceranae infections.

 

Paper Abstract

Nosema ceranae is a honey bee pathogen parasitizing the ventricular epithelium and potentially causing colony death. The effect of 0.25 M oxalic acid solution administered to the bees in form of sugar syrup was determined in laboratory and field trials. The spore numbers in a 8-d laboratory experiment were significantly lower when AO was administered (treated: 11.86 +/− 0.94 s.e. x 10^6; untreated: 30.64 +/− 0.31 s.e. x 10^6). When administered in autumn to free flying colonies twice, 3 week apart, the infection prevalence decreased in young (reduction percentage of 53.8% +/− 6.5 s.e.) and old bees (percentage reduction of 44.4% +/− 6.0 s.e.). Meanwhile increased prevalence in all the controls was detected (young and old bees: percent increase 45.7% +/− 22.8 s.e. and 10.2% +/− 5.9 s.e., respectively). While all the treated colonies overwintered correctly, the untreated ones did not (3 out of 5 were dead).

 

In the absence of commercial products approved in several countries to control nosemosis, oxalic acid syrup appears promising in the development of alternative management strategies.

 

Antonio Nanetti, Cristina Rodriguez-García, Aránzazu Meana, Raquel Martín-Hernández, Mariano Higes, Effect of oxalic acid on Nosema ceranae infection. Research in Veterinary Science. In Press, Accepted Manuscript Available online 4 August 2015 Copyright© 2015 Published by Elsevier Ltd. doi:10.1016/j.rvsc.2015.08.003

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Thanks Dave

I do believe that a varied diet is a large part to managing the problem. It can be likened to a human living on pies, yes it can be done for an extended period of time but eventually the body will show and express signs that is is failing. You can take all the supplements available but it isnt anywhere as good as taking the essentials by way of a balanced diet.

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Nice review Dave. The disappearing bees is interesting - part of the 'colony immunity' with the bees leaving the hive to die - but then's there also the N ceranae affecting the homing ability of the bees as well (further decline in numbers).

 

I will have to check files for where N. ceranae has been detected. I know there was one report which was later published as contamination.

 

There are a couple of informal studies being done with various oils to see if they can help purge the nosemas from the bees - will be interesting

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There are a couple of informal studies being done with various oils to see if they can help purge the nosemas from the bees - will be interesting

Can you look at the oxalic paper (Elsevier) @JohnF ? I don't have a registration - too poor :) That might be interesting too, particularly as it is becoming more widely used for varroa.

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Been a long time since I did stats, but Fisher's Exact Test gives a p -value greater than 0.05 but less than 0.10 (0.083)

 

So is the difference with oxalic due to chance ?

I haven't read the paper, but respond to your question above.

 

Statistics are all about confidence levels - how confident we can be that an effect seen in a experiment is a "treatment effect" as opposed to random chance. These statistical models recognise that we can never truly be 100% certain, but that some data is surer than others.

 

A p value <0.01 is generally regarded as very strong evidence that the effect seen is a treatment effect. I.e we can be 99% sure that this is a treatment effect. P value between 0.01 and 0.05 is generally still regarded as fairly strong evidence. Between 0.05 and 0.10 is often disputed, but in biological science, a result in this area is often seen as a very strong indication of a treatment effect. This takes into account that biological systems are complicated with many variables affecting the system at once.

 

The definitions above are basically my own (but with some training :)). For me, given the concerns around N ceranae, 0.083 is good enough for me to take interest...

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Not an uncommon position to take :)

 

If only it was always that easy to read. But failure to take stats into account means we may miss an opportunity to change what we do - or worse still, we may change what we do, based on a treatment result due to "chance"

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I think the paper is interesting but the number of hives is very small, so the work needs to be repeated to confirm the effectiveness of oa in what may be a very serious organism. So my concern is that if bk's use it and it really doesn't work, then years of progress may be lost.

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I've had a chance to look at the paper, so here's a precis,with a bit of editorial.

 

The study came about after Romee Van der Zee happened to notice Nosema ceranae infected colonies recovering following oxalic acid treatments against varroa infestations. To investigate this the scientists tried a couple of things;

 

In the laboratory the abdomens of one hundred flying bees from each of three colonies were used to provide a sample of nosema spores. They checked the desired species was present using PCR (the study was conducted in Spain), pooled the samples, and counted the spores with a microscope and haemocytometer. This was used for 'dose' or 'treatment', as below.

 

From three other colonies where they had established there was no nosema they pulled and incubated brood frames so that the emerging bees could be caged for five days and fed sugar syrup with a pollen substitute. Half were fed with the nosema dose in syrup,the other half just had syrup. They formed one group of infected bees fed syrup, one of infected bees fed the oxalic treatment, and one group of uninfected bees fed syrup. They then took three samples from each group a day later, six days later, and eight days later, and both qualitatively and quantitatively checked for nosema just as they had for the original spore sample.

 

In addition, in an apiary, they equalized and randomized ten colonies that did not display visible symptoms of the common diseases, including nosema, but established with PCR that Nosema ceranae was present. They then made two groups of five, sampled bees, and using a syringe dribbled a measured solution, treated one with an oxalic-laced syrup and one with plain syrup. This was repeated two weeks later, and a week after that a second, post-treatment sample of bees was collected from both the entrance and the brood frames. At three months the colonies were evaluated for brood and population.

 

The result of the lab test was that the uninfected bees just fed the syrup still had no spores. The infected groups showed an exponential increase in spore count, by day eight the treated group with an average of around 10 million spores, but the untreated group with around 30 million.

 

In the apiary field trial, of the untreated hives 3 were lost, in the treated group all survived, although their condition was poor. The hives started with 85% or so of older bees infected and about 30% of the young bees infected. By November the proportion of young bees infected in the untreated hives had increased by more than 40%, and the increase in the number of old infected bees was about 10%. In the treated hives the proportion of young infected bees had decreased by more than 50% and the number of infected old bees decreased by more than 40%. The amount of brood in the all the surviving colonies looked much the same, but the bee population was much higher in all the treated colonies, with more than six frames covered. The two remaining untreated colonies were on two or three frames.

 

The authors note that this is rather preliminary work, especially with respect to the dose administered which seems to have been a bit of a guess at the time, and the carrier (sugar syrup). According to them the bees do not ingest the syrup (much); perhaps they should if it is to be effective. They also wonder if part of the reason for variation in the effect of ceranae that has been observed may be because beekeepers were unwittingly treating, as a 'by-kill' of their varroa treatment.

 

Taken together the results of lab and field work suggest to me that there might be something in Van der Zee's original observation, and that OA could have some effect on the severity of cernae infections. The methodology adopted looks okay, and certainly could be replicated by other trial work; more quantitative assays would be helpful, but the science here isn't particularly difficult or resource hungry. Not bad for a first attempt. The paper has very little say about how oxalic might work, suggesting the acidification of the ventriculus lumen might be the mechanism, but "the aptitude to chelating metallic captions" mentioned might have lost something in translation. The problem of how we diagnose a ceranae infection remains, and prophylactic treatment is out of the question. There is the worry that even this treatment will add to the burden on an already stretched bee immune system, but so far we little to no idea how to combat N. ceranae infections and there is not much chance of the bees surviving one on their own. Just perhaps, in my artificial swarm (see above) I'd treat the broodless bees with OA. It also left me wondering about the 'ceranae does't like the cold' idea. Oxalic does and its use is higher in cold regions where brood all but disappears. Do we see ceranae in regions like Germany or Scandinavia where oxalic has been used routinely? What information do we have about ceranae in New Zealand? Anecdotes anyone?

 

And my thanks to @Otto

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Thanks Dave, very interesting information. I did wonder about the bees ingesting syrup with that kind of concentration of OA. I think they don't like that. We know that honeys can have OA in it but much lower rates. Not sure how it can work, maybe low concentrations (attractive to bees) can have an impact, if any of this helps slow nosema ceranae.

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Personally I'm a big believer in iodine (best antibacterial agent yet) Hence I always mix a little with my sugar syrup and always a little salt which works as an electrolyte as its also an alkalising mineral. Its what I do for my kids when they have a crook guts so I do it for my bees. I believe preventions better than cure.

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Isn't this ripping action how Oxalic works ? Mites are softer bodied than bees and get abraded by the crystals ?

No. The best guess is that OA interferes with electron transport in mitochondria.

 

Personally I'm a big believer in iodine (best antibacterial agent yet)

But we want bacteria with our bees. It keeps them alive. Bacteria are good.

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My understanding is the condensed vapour crystals are really sharp and physically rip the gut wall. Isn't this ripping action how Oxalic works ? Mites are softer bodied than bees and get abraded by the crystals ?

I got blasted in the face with Hot OA and spent 5 days in Greenlane intensive care.

The OA Crystals embedded themselves in both eyes and the acid burnt all the skin off the eyes down to within a few Microns of the Cornea blood vessels

A team of specialists put me back together using High purity Ascorbic Acid, Sodium Citrate, Steroids and lubricants dosed topically every 30 minutes 24 hours a day.

My eyesight is now better than it ever was and at my last follow up consultation last week the specialist said in disbelief

"No one reads that line on the chart, no one"

Except me.

So the long and short of it is that OA probably isnt going to hurt Bees.

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No. The best guess is that OA interferes with electron transport in mitochondria.

 

 

But we want bacteria with our bees. It keeps them alive. Bacteria are good.

that right ..good bacteria are good and iodine wont hu1et them as it denatures proteins with certain proteins/amminos in the cell wall. Im not saying feed bucket loads but enough to keep the immune system up.
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