ABSTRACT
American foulbrood, because of its virulence and worldwide spread, is currently one of the most dangerous diseases of honey bees. Quick diagnosis of this disease is therefore vitally important. For its successful eradication, however, all the hives in the region must be tested. This is time consuming and costly. Therefore, a fast and sensitive method of detecting American foulbrood is needed. Here we present a method that significantly reduces the number of tests needed by combining batches of samples from different hives. The results of this method were verified by testing each sample. A simulation study was used to compare the efficiency of the new method with testing all the samples and to develop a decision tool for determining when best to use the new method. The method is suitable for testing large numbers of samples (over 100) when the incidence of the disease is low (10% or less).
Subject(s)
Bees/microbiology , Nucleic Acids/analysis , Paenibacillus/isolation & purification , Polymerase Chain Reaction/methods , Spores, Bacterial/isolation & purification , Animals , Computer Simulation , Decision Support Techniques , Paenibacillus/physiology , Polymerase Chain Reaction/economics , Sensitivity and Specificity , Spores, Bacterial/physiologyABSTRACT
BACKGROUND: Chemical communication in mammals involves globular lipocalins that protect and transport pheromones during their passage out of the body. Efficient communication via this protein - pheromone complex is essential for triggering multiple responses including aggression, mate choice, copulatory behaviour, and onset and synchronization of oestrus. The roles of lipocalins in communication were studied in many organisms and especially in mice (i.e. Mus musculus domesticus) which excrete Major Urinary Proteins (Mup) in excessive amounts in saliva and urine. Other mammals, however, often lack the genes for Mups or their expression is very low. Therefore, we aimed at characterization of candidate lipocalins in Myodes glareolus which are potentially linked to chemical communication. One of them is Aphrodisin which is a unique lipocalin that was previously described from hamster vaginal discharge and is known to carry pheromones stimulating copulatory behaviour in males. RESULTS: Here we show that Aphrodisin-like proteins exist in other species, belong to a group of Odorant Binding Proteins (Obp), and contrary to the expression of Aphrodisin only in hamster genital tract and parotid glands of females, we have detected these transcripts in both sexes of M. glareolus with the expression confirmed in various tissues including prostate, prepucial and salivary glands, liver and uterus. On the level of mRNA, we have detected three different gene variants. To assess their relevance for chemical communication we investigated the occurrence of particular proteins in saliva, urine and vaginal discharge. On the protein level we confirmed the presence of Obp2 and Obp3 in both saliva and urine. Appropriate bands in the range of 17-20 kDa from vaginal discharge were, however, beyond the MS detection limits. CONCLUSION: Our results demonstrate that three novel Obps (Obp1, Obp2, and Obp3) are predominant lipocalins in Myodes urine and saliva. On the protein level we have detected further variants and thus we assume that similarly as Major Urinary Proteins in mice, these proteins may be important in chemical communication in this Cricetid rodent.
Subject(s)
Arvicolinae/genetics , Lipocalins/genetics , Receptors, Odorant/genetics , Amino Acid Sequence , Animals , Electrophoresis, Gel, Two-Dimensional , Female , Gene Expression Profiling , Lipocalins/metabolism , Male , Molecular Sequence Data , Pheromones/genetics , Proteins/genetics , Receptors, Odorant/metabolism , Saliva/chemistry , Sequence Alignment , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tandem Mass Spectrometry , Urine/chemistry , Vagina/chemistry , Vagina/metabolismABSTRACT
The objective of this work was to create a fast and sensitive method of detecting Paenibacillus larvae from beehive debris based on PCR that does not require long-lasting cultivation steps. Various methods of extracting spores from beehive debris were compared: the original method of extraction of spores into toluene, and alternative spore extraction methods into Tween 80, into water, into isopropanol and into 95% ethanol. Isolation of DNA from various spore extractions was evaluated too. Best results were provided by isolation of DNA using the QIAamp DNA Mini Kit, without heat treatment. DNA of spores was detected by PCR from 0.25 g of beeswax debris, with the detected titer of 10(5) in 1g according to the cultivation tests.
