Katherine Aronstein, Research Molecular Biologist, USDA/ARS
Those of us working with bees have experienced significant changes in beekeeping practices in recent years. It seems that difficulties of keeping bees healthy has transformed routine management into an epic struggle for colonies’ survival. Some of the problems can be explained by the introduction and spread of new diseases and microbes that have become resistant to drug treatments. We recently described one of these mechanisms for drug resistance in AFB bacteria (Murray and Aronstein, 2006). The increased use of synthetic insecticides inside and outside of beehives has not helped either. Combinations of different pesticides (even when they are not highly toxic to bees when used alone) can produce unintended results by affecting the overall health and immune response of bees, making them susceptible to a variety of diseases and stressors (Reed et al., 2009).
Threatened with losing essential pollinators, the US Congress has approved increased levels of bee research funding to discover and mitigate the causes of bee decline. Did bees experience the effect of a new yet un-identified factor (e.g., disease, pesticide etc) or is this the same old problem showing its ugly and exaggerated forms? This problem is now a major focus of several investigations. Scientists are looking at the root cause of the Colony Collapse Disorder (CCD) syndrome, searching for new diseases, harmful chemicals or a combination of these factors which could inflict stress on bee colonies pushing them over the edge for recovery. Many of these urgent questions will be addressed in our new multi-institutional USDA Coordinated Agricultural Project (CAP) by systematically analyzing bees, pollen and wax samples collected from stationary apiaries.
Among multiple suspects identified so far, bee viruses and a microscopic Nosema parasite have attracted the most attention in the press, and rightly so. Most of these are intracellular parasites which are undetectable by visual colony examination. When bees are finally showing sign of the disease, it is for the most part too late to save the colony since most of the bees are infected and dying.
Nosema apis has been known to occur in the United States since at least the 1950s, but its presence in bees has been a matter of mixed concern. When infected bees were found crawling in front of the colonies leaving yellow strips of diarrhea, they were normally treated with antibiotics (Higes et al., 2009), and that took care of the problem. Then why is it that scientists now suspect Nosema in recent losses of bees? Some scientists even point to Nosema as the primary reason for CCD (Higes et al., 2009). Apparently, a new species of Nosema (N. ceranae) is now widely spread in the U.S. and around the world, silently replacing N. apis. Little was made of this discovery in 1996; but concern was reawakened in 2005 when bees in Asia were observed suffering from the disease. In 2006 N. ceranae was detected in Europe causing heavy loses of colonies in Spain, France, Germany and Switzerland (Higes, et al., 2006). Since this new species is not readily detectable in the apiary, infection goes unnoticed for a long time. Bees could be infected for weeks and not show clinical signs of the disease. The microscopic identification of this new species is also a challenge since both N. apis and N. ceranae spores look similar when observed under a microscope. Therefore, for species identification, bee samples are normally sent to a laboratory capable of DNA amplification. In the laboratory, scientists can determine genetic differences between the two species of Nosema using PCR. Since N. ceranae infected colonies can die much faster, survival of the colonies depends on a timely detection and treatment of the disease. However, precious time is lost waiting for lab results. Therefore, development of a rapid and simple identification tool could save an entire apiary and prevent disease epidemics.
The idea behind this tool is not new, being based on the principles of immunology (antibody-protein interaction). The tool is often designed in a dipstick format for easy use in the field or home. Although antibody-based dipstick tests involve sophisticated technology derived through research and development, the actual products are user-friendly and packaged as a kit of reagents. Such methods have been developed for the detection of medically important diseases and their insect vectors and the detection of HIV in human blood. Perhaps the most familiar use is the home pregnancy test.
By adapting this technology to beekeeping needs, it will be possible to develop a simple and error-free method for the detection of the Nosema infection in bee samples. This research is currently conducted at the Weslaco Honey Bee Research Unit (USDA/ARS) as part of the CAP project. The final product, a dipstick assay for the detection of Nosema spores in bee samples, will be developed in collaboration with private biotechnology companies. Some of the companies have already received our proposal.
How does it work?
A kit will contain multiple strips of cellulose, each of which is intended for a single use. After crushing a bee or the dissected guts from several bees in a vial with reagent, a strip of cellulose is inserted in the homogenate. A few moments later, either a single or a double band will appear. A negative result is indicated if only one blue band (control) is visible (see (-), Fig. 1A). A positive result is indicated if two bands are visible, one blue for the control and the other red indicating the target Nosema protein (see (+), Fig. 1A).
Nosema ceranae antibodies (Ab) are incorporated into the cellulose strip and serve as a “red flag” and an anchor for the immunogen (a Nosema protein). The immunological reaction is based on a very strong bond that develops between Nosema Ab and the Nosema protein which was originally used for the development of this Ab (Fig. 1B). When Nosema spores are added to the reaction mix, the Ab will bind to the Nosema protein and form a very strong bond. If this reaction is coupled with color detection using secondary antibodies (Detector Ab), the appearance of two color bands (control and Nosema-specific) indicate the presence of Nosema spores. The appearance of a single color band (control) indicates negative results.
Since the basic idea behind the test is not new, many before us have attempted to develop such tests. However, a poor quality of antibodies, a lack of specificity, and a low level of sensitivity are the main difficulties that have prevented the successful development of a quick test. Antibodies are usually produced by injecting a foreign protein (immunogen) into animals such as goats, rabbits, rats, or mice. The animals’ immune system then detects a foreign invasion and responds by producing antibodies. Clearly, the quality of the antibodies can make an immunological assay a success or a failure. How well antibodies detect the original immunogen depends on its type and purity.
When immunogen is produced in the laboratory, the time, effort, and resources required for its synthesis can be substantial. It is challenging to make a pure immunogen. Contaminating molecules can serve as secondary immunogens, resulting in antibodies lacking specificity. We decided to avoid this mistake by choosing a novel way to develop antibodies, the so-called Genomic Antibody Technology (GAT) (Fig. 2). The use of this technology is a completely new way of thinking about immunogens. Using GAT technology, a piece of circular DNA (plasmid) containing the DNA sequence encoding the protein of interest (Nosema in our case) is injected into the animal. A pure protein is then produced directly in the animal (and by the animal), thereby bypassing the normal lengthy protein production procedure in the laboratory. In-vivo expressed protein is recognized by the animal as a foreign invader which, in turn, triggers production and release of antibodies in the animal’s blood.
One difficulty of using GAT technology is that it requires prior knowledge of the protein sequences. That is a serious obstacle unless the pathogen’s genome has been sequenced. Fortunately, both Nosema genomes are being sequenced by the USDA. We were able to identify a target protein sequence located on the Nosema ceranae spore wall (the DNA sequence provided courtesy of Dr. Jay Evans, Beltsville Honey Bee Research Laboratory, USDA/ARS). We are now testing our new Nosema ceranae antibodies on bee samples. So far our tests show a high Ab sensitivity that can detect Nosema spores in crude bee homogenates at a 1 : 5000 dilution, similar to commercially produced Abs. We are also testing the minimal amount of spores that can be detected by the test. Using routine gel-based laboratory methods we determined that our new Abs can detect one infected bee among one thousand non-infected bees. This level of sensitivity will allow for the detection of very low rates of infection in bee colonies.
Who will be able to use this test?
The test is not intended to replace current methods used in research laboratories. There is no need to replace high throughput technology designed for processing large numbers of samples. Instead, it will help beekeepers, hobbyists as well as commercial beekeepers to detect and monitor the progression of the disease in the field. It will help beekeepers make educated decisions about disease management. Most impotently, this new tool will (1) encourage reduced use of antibiotics since it will discourage unnecessary treatments, (2) give regulators new decision-making tools in regard to inter-state and international bee movement, and (3) give producers of queens and package bees a means to detect and monitor Nosema levels in their production colonies. Disclaimer "Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture."