The Germ Theory Here we show Janet Tam, of the Ontario Beekeeper s Assn. Tech Trans-

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1 sing the microscope, Pasteur discovered living organisms small enough to go undetected in food and water, but harmful enough to kill as fast as any poison. He did not originate the germ theory of disease, but he was able to prove that illness can be caused by microorganisms and he developed methods for rendering them harmless. Everyone knows that milk is pasteurized, but fewer know that this process was invented by Pasteur to make it possible to store food safely for later use. Even fewer know about Pasteur s role saving the silk industry in France and Italy. Though the production of silk originated in China, by the 1850s France was producing ten per cent of the world s silk. But a terrible contagious disease took hold, nearly wiping out the industry in France, spreading to Italy, Spain and devastating the German production by The Germ Theory It seemed clear that a germ must be responsible, and Louis Pasteur was able to show that it was characterized by the presence of oval shaped bodies visible under the microscope. These were found in not only the larva, but the eggs and moths as well. By obtaining eggs free from these spores, growers were able to restock after destroying the infected moths and larvae. Pasteur tried to promote the diagnosis of the disease by farmers, but they were dubious of science and microscopes until Pasteur employed his daughter Zizi to demonstrate the ease by which the disease causing organisms could be identified. Following the discovery that diseasecausing germs could be killed by heat, researchers showed that certain chemicals could also sterilize without harming the person or animal that was affected by the germs. One of the first disinfectants was pure alcohol, which is still in widespread use as a topical antibacterial. Other chemicals used to disinfect include carbolic acid, acetic acid, hydrogen peroxide and so on. Not until well into the 20th Century were true antibiotics discovered, beginning with penicillin and blossoming into the whole range of disease control chemicals we now have at our disposal. Of course, the mode of action of these agents is that they poison the microbes, so they are not without risk to the host as well. Here we show Janet Tam, of the Ontario Beekeeper s Assn. Tech Transfer Program, demonstrating how to collect and prepare bee samples to diagnose for Nosema spores. (Photo by Peter Loring Borst) August

2 is characteristic of the Isle of Wight disease, the only essential feature being the death of large numbers of bees within or without the hive. By this time Nosema had been found in the United States, Australia, Brazil, Canada, England, Germany, and Switzerland. Nosema apis was considered to be a serious problem in the spring, preventing the colonies from building up and even killing them outright. As a result, serious effort was begun to find a way to control it or eradicate it altogether. Following the work of Pasteur, it was shown that the spores could be killed by the application of heat. Exposing them to hot water (135 to 138 F) for ten minutes was adequate to kill them. However, this is very near the temperature that melts beeswax (144 to 147 F), so combs cannot easily be disinfected by heat without ruining them. A Nosema workshop was held June 9, 2011 at SUNY Morrisville, NY. The presenters were: Janet Tam, Paul Cappy (NY State Apiculturist), Peter Borst and Mike Johnston. (Photo by Peter Loring Borst) Nosema spores as shown by Ingmar Fries (From Diseases of Asian Honeybees, by Ingmar Fries, used by permission of author and the publisher, Springer) The disease that was wiping out silkworms was traced to a tiny organism called a microsporidian. It was named Nosema bombycis, since it affected the silk moth larvae whose latin name is Bombis mori. Much was learned about microsporidians in the years that it took to find the cause of the devastation of the silk worm industry. These parasites were found to be single celled organisms with a very unique form and life cycle. At first, Nosema was classified as a protozoan ( first animal ). More recently, it was determined that microsporidians like Nosema are not tiny animals, but fungi. Inside the one celled body is an incredibly long structure called the polar tube. They can insert this tube into living cells and inject them with sporoplasm. This would be amazing enough, but since the tube is inside the microsporidian, it has a way of turning itself inside out. This would be like turning a garden hose inside out, which you can easily 774 picture as an impossible task! Nosema and the Isle of Wight It was soon discovered that there were microsporidian species infecting many insects and animals as well. By the end of the 19th Century over fifty different ones had been identified. And, in 1909 Enoch Zander named Nosema apis, which is now known to be a serious pest of the honey bee. This newly discovered microbe was almost immediately declared to be the cause of the mysterious Isle of Wight Disease, which had devastated bees on that island, and which spread to England. In a case remarkably similar to the current bee die off, Isle of Wight Disease was first attributed to Nosema, then to the tracheal mite. A 1919 USDA Pamphlet on Nosema Disease states: Bullamore and Malden, of England, after studying the symptoms of the disease, arrived at the conclusion that no one symptom Nosema, circa 1919 Our 1919 pamphlet described the outward symptoms of Nosema: Weakness, especially in the spring of the year, should cause a suspicion that the disease is present. The suspicion is strengthened if in such a colony the brood in general is normal, if the adult bees are not noticeably different in outward appearance or behavior from bees of healthy colonies, if the queen is present and if stores are abundant. The standard diagnosis for Nosema apis was to remove the stomach from the bee and note its color. While the colony symptoms may justify a very strong suspicion that the disease is present, an examination of the stomachs from adult bees of the colony is necessary in making a definite diagnosis. If the stomach upon removal appears swollen and lighter in color than a healthy one, Nosema infection may be suspected; if it is chalk white and easily torn, infection is very probable; should the tissues of the organ when crushed be milky in appearance, infection is practically certain. The method for examining the bees for Nosema spores is essentially the same as the method used by Louis Pasteur s daughter Zizi. The pamphlet states plainly that Nosema spores are so distinct as to be not likely confused with anything else. Evidently, Zander was the one who first associated diarrhea with Nosema. He went so far as to declare it a sure sign of Nosema disease, even though it was often not present. This connection has persisted in many beekeepers minds up to the present day, despite the fact that diarrhea and Nosema can occur quite independently of each other. Probably, the two occur together when the bees have been confined for long periods in winter. Studies were done in Ithaca, NY around 1920, and reported in the Journal of Parasitology. They stated that the presence of intercellular parasites in insects is extremely common, almost universal. In the case of the honey bee, Nosema was discovered present in the bees of every country where they were examined, and was the primary para- American Bee Journal

3 site in bees, although some researchers claimed to have seen others. It was thought that there could be another, unnamed microsporidian. The life cycle of Nosema As with many parasites, the cycle begins with the bee swallowing the spores. These can be present in food or water. The spore germinates like a seed and sends out the bizarre polar filament, which injects itself into the bee s cell. Through this tube, the Nosema organism sends live cells (sporoplasm) which reproduce in the host cells, and develop into new spores. When a cell is filled with spores, it bursts open and spreads the disease throughout the bee. Millions of these spores are passed via feces to other bees. Nosema is most frequently found in the worker bees, although it also infects the queen and the drones. There are no reliable outward symptoms, and the disease can progress unnoticed until the bees begin to die. Symptoms such as crawling, inability to fly, distended abdomen, etc., are seen with other disorders and so cannot be considered reliable indications of Nosema. Therefore, examination under a microscope remains the only sure method diagnosing the presence of Nosema. Nosema apis has long been associated with poor overwintering and slow buildup in the Spring. A sharp spike in spore numbers was typically noticed in April, reaching up to 2.5 million spores. As the weather improved and old bees were replaced, the spore count normally fell off very quickly. Nosema ceranae has a completely different seasonal progression. Nosema ceranae In 1994, during a study being done in China on the infectivity of N. apis on the Asian honey bee (Apis cerana) a new species of Nosema was discovered. Little attention was paid to it at the time since it was assumed that it only affected the Asian bee. More than ten years passed before N. ceranae was found to infect European bees located in Taiwan, as well. The examination of preserved bee specimens revealed that this parasite has been in the US at least as far back as Spanish researchers, concerned about colony collapse in Spain, discovered that N. ceranae was widespread in that country by Robert J. Paxton wrote: Whether A. cerana was its only host, or its principal host, or a source of cross-species infection for A. mellifera, is less clear. This is because sampling of other Asiatic honey bee species or other potential Asian host (bee or other insect) species has not been undertaken for N. ceranae. In addition, its recent discovery in Argentinean bumble bees suggests that it may have a wider host range in Asia than A. cerana. In the years that followed, many reports connected severe losses to this newly described organism. When colony collapse disorder (CCD) was first identified in the US, it was soon realized that the hives had high numbers of N. ceranae, and that N. apis was generally not found any more. In 2010, a report from Croatia stated the same thing, that only Nosema ceranae was present in that country. Some researchers even went so far as to claim that Nosema was the main cause of CCD, both here and abroad. In 2008, Spanish researchers published work showing exactly how Nosema can cause colony collapse in bees. Further, their work showed that the application of Fumagillin could eliminate the infection, but that it tended to recur after six months, possibly from infected equipment. The effect of Nosema on the honey bee colony Researchers in Slovenia have characterized the effects of Nosema and these include impaired protein metabolism and decreased digestive activity in the mid-gut. This reduces the amino acids available to the bee and results in smaller hypopharyngeal glands and poor quality fat buildup. Nosema disease can cause premature aging, shift in labor division, and premature foraging. Another interesting result seems to be that Nosema-infected bees fly during bad weather. It is unclear whether this is a result of poor responses and judgment or even a behavior that is intended to protect the colony by eliminating the sick bees by causing them to fly off and die. Spanish researchers stated in the May- June issue of Apidologie that: N. ceranae presents a different epidemiological pattern and pathology compared to N. apis... and is characterized by the ability to detect the disease-causing agent throughout the year. The continuous death of highly infected bees, mostly foragers, has a clear effect on colony population and productivity. However, there is a large body of evidence that Nosema infections do not cause serious losses under other climatic conditions. A report in the Apidologie of January August

4 2011 states clearly that while Nosema ceranae has completely replaced N. apis in the Balkans, the parasite causes no CCD-like 776 symptoms in the region. Similar findings are reported by the German Bee Monitoring Project. They write: The results obtained with the German bee monitoring project did not reveal any relation between infection with Nosema and winter losses, although both Nosema species are prevalent in Germany. Since no losses occurred during summer, although colonies were infected by Nosema, it can also be ruled out that infection with Nosema killed colonies between spring and autumn as described in the Spanish studies. Their studies show a correlation between winter losses and specific viruses (DWV and ABPV) but not Nosema. Fumagillin and the treatment of Nosema Antibiotics were not known before the 20th Century. In fact, infection was a leading cause of death up till then. Pasteur and Lister showed that infections could be prevented by sterilizing either with heat or antiseptic chemicals such as carbolic acid. However, once an infection had become severe, amputation was often resorted to as the sole means of preventing death in the patient. In the 1930s Bayer experimented with new drugs and soon the Sulfa drugs were in widespread use. This was followed by the development of penicillin and related substances. Their widespread adoption saved tens of thousands of lives, especially during World War II. Soon, antibiotics became commonplace and death from minor infections is rare today. In the 1950s, work was conducted to find a treatment for Nosema disease. Researchers tried antibiotics, sulfa drugs, arsenicals, and various anti-protozoan agents without success. They used a new antibiotic extracted from a common soil fungus called Aspergillus fumigatus. Ironically, this fungus itself causes serious illness in both bees and people (stonebrood in honey bees, and Aspergillosis in humans). In 1949, isolates of Aspergillus had been found to be effective against Staphylococcus bacteria, but not against polio and influenza (no wonder, since these are viruses and do not respond to antibiotics!). For many years, the use of fumagillin was recommended to be included in syrup fed to bee colonies to help them build up, especially in spring, when N. apis infections were liable to be widespread in overwintered bees. By the 1970s, however, many US beekeepers shifted to moving their whole operations to the southern states to avoid unpredictable losses and to have bees in better condition for early pollination contracts such as apples and cherries. A 1987 publication from California suggests that in addition to the use of fumagillin, preventive measures include keeping colonies strong, protected from the wind, and exposed to the maximum amount of sunshine. This advice still holds true, of course. It is probably wind and dampness that kills bees in winter more than cold. Many colonies survive very low temperatures without ill effects if they are dry and off the ground. Alternative treatments In view of the widespread occurrence of American Bee Journal

5 antibiotic resistant bacteria and the tendency toward natural, non-chemical beekeeping, it is no wonder that the idea of using antibiotics as a preventive measure is met with resistance. Many beekeepers have not and will never administer fumagillin products to their hives, either due to its high cost or because they object to exposing their bees to this type of chemical. In fact, the European Union does not allow this and many other chemicals to be administered to bee colonies. Even in our country there is a strong movement away from chemicals. There have been some promising alternatives. Nozevit is a product developed in Croatia. Its use was thoroughly described in the May 2009 issue of the American Bee Journal. It is described as a natural extract of oak bark, and a rich source of tannin. The manufacturers state that tannins stick to the mucosa to form a resilient membrane and this may be the mode of action of this preparation. It is clear that there is a strong demand for non-chemical treatments for bee disorders, and preparations made from natural substances such as bark, essential oils, etc. have a widespread appeal. It should be made clear, however, that simply because a product is natural it does not therefore follow that it is safe, non-poisonous, or better. Every substance has to be evaluated on its own merits, and not on false distinctions such as natural vs. synthetic, and so on. Nature produces many of the deadliest poisons known (Amanitas, aflatoxins, strychnine; the list is long). The future of Nosema treatment Most beekeepers believe that a better bee can be developed, given resources and resolve. In Denmark, selection for Nosema resistant bees was undertaken more than 20 years ago. Per Kryger stated problems with Nosema have been greatly reduced, though he acknowledged that other factors may be at work, such as hygienic methods, better ventilation, isolating apiaries and using only Nosema-free bees when making splits. He emphasized that the necessary work and study has been done entirely by private bee breeders, who very early on adopted technological advances such as a computer-assisted method of counting spores. In the last few years there has been a lot of excitement surrounding the biological phenomenon called RNA interference (RNAi). This refers to a cellular mechanism that uses small strands of RNA (genetic code) to regulate cell functions, turn genes on and off, and most importantly it can attack foreign material such as viruses, that disrupt the cell functions. It has been found that by adding synthetically produced RNA, cell defenses can be enhanced and diseasecausing organisms can be directly targeted. In other words, the cellular response to Nosema could be strengthened. Beyond that, it is possible that specific anti-nosema RNA could be designed and delivered to the bees cells, working somewhat like a vaccine. These technologies are still in their infancy, Experimental Apiary in which the nosema-disease experiments made during the summer of 1915 were conducted. (From USDA Bulletin No. 780, in the public domain) but there is a good chance that they will become commonplace in the future, leading to a decrease in disease and parasites and at the same time, a decrease in the need for expensive and poisonous chemical controls. Selected References Bailey, L. The epidemiology and control of Nosema disease of the honey-bee Annual of Applied Biology (1955) 43 (3): Chen, Yan Ping and Zachary Y. Huang. Nosema ceranae, a newly identified pathogen of Apis mellifera in the USA and Asia Apidologie (2010) 41: Franzen, C. Microsporidia: A Review of 150 Years of Research The Open Parasitology Journal (2008) 2: Fries, Ingemar. Diseases of Asian Honeybees In: Honeybees of Asia. H.R. Hepburn, S.E. Radloff, editors (2011). Higes, Mariano. How natural infection by Nosema ceranae causes honeybee colony collapse Environmental Microbiology (2008) 10(10), Katznelson, H. and C. A. Jamieson. Control of Nosema Disease of Honeybees with Fumagillin Science, New Series (1952) 115 (2977): Klee, Julia. Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera Journal of Invertebrate Pathology (2007) 96: Kudo, R. Notes on Nosema apis Zander The Journal of Parasitology. (1920) 7(2) Paxton, Robert J. Does infection by Nosema ceranae cause Colony Collapse Disorder in honey bees (Apis mellifera)? Journal of Apicultural Research (2010) 49(1): Stevanovic, Jevrosima. Dominance of Nosema ceranae in honey bees in the Balkan countries in the absence of symptoms of colony collapse disorder Apidologie (2011) 42: White, G. F. Nosema-disease USDA Bulletin No Washington, D. C. June 12, Rd 30 1/2 Glenn, CA A. Suhre Enterprises Summer and Fall Queens Now Available For August & September! We have gentle, hygienic Italian Queens with over 20 years experience breeding queens All Major Credit Cards Accepted For Questions, or to Place an Order Please Call Adam Anytime Cell adamsuhre@live.com August