A novel TaqMan ® assay for Nosema ceranae quantification in honey bee, based on the protein coding gene Hsp70

Cilia, Giovanni; Cabbri, Riccardo; Maiorana, Giacomo; Cardaio, Ilaria; Dall’Olio, Raffaele; Nanetti, Antonio

doi:10.1016/j.ejop.2018.01.007

Cited by 25 publications

(31 citation statements)

References 31 publications

(31 reference statements)

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“…The increasing worldwide prevalence of N. ceranae in the past decade, and, conversely, a decrease in the N. apis prevalence (even the absence of this parasite) in some regions suggests that N. ceranae might be displacing N. apis [6,26,67]. However, while in Southern Europe, especially in the Mediterranean countries like Spain, Italy, Israel, Greece, and Turkey, N. ceranae has been the dominant species for 10 years [6,25,37,49], in Northern Europe (Ireland, Sweden, Norway, and Germany), N. apis is still the predominant species [27,30,38]. For example, in Sweden, the majority of bee colonies (89%) were infected only with N. apis, and in other bee colonies, coinfection was identified [38].…”

Section: Discussionmentioning

confidence: 99%

“…Conflicting results from different studies can be attributed to many factors, such as the biological characteristics of honey bees (caste, age of the bees, commercially and traditionally managed bees) [31,32], the genetic diversity of honey bees, bee subspecies and lineages [24,[33][34][35], climatic and environmental differences [7,18,36], beekeeping practices [10], as well as diagnostic methods [37] and research conditions (number of analyzed bees, time and method of sampling, natural population research or experiment) [31,38,39]. Nosema species can only be confirmed using molecular methods [39], which can have different levels of resolution, for example, the single-copy Hsp70 gene method qPCR detects a lower amount of N. ceranae copies compared to the multicopy 16S rRNA gene method [37]. When conducting experimental infection studies, the laboratory results may be affected by specific conditions such as the duration of the experiment, the temperature and humidity, the method of infection, number of bees in the cages, the type of their diet, etc.…”

Section: Introductionmentioning

confidence: 99%

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Prevalence of the Microsporidian Nosema spp. in Honey Bee Populations (Apis mellifera) in Some Ecological Regions of North Asia

Ostroverkhova

Konusova

Кучер

et al. 2020

Veterinary Sciences

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Two species of microsporidia, Nosema apis and Nosema ceranae, are obligate intracellular parasites that are widespread in the world and cause the infectious disease (Nosemosis) of the Western honey bee Apis mellifera. Information on the prevalence and distribution of Nosema species in North Asia conditions is scarce. The main aim of the present study is to determine the prevalence of Nosema spp. (Nosemosis) in honey bees inhabiting some inland regions of North Asia (Western and Eastern Siberia, Altai Territory, Russia, and northeastern part of Kazakhstan). The objective of the paper is also to assess the influence of climatic factors on the spread of N. ceranae. Eighty apiaries in four ecological regions of North Asia (southern taiga, sub-taiga zone, forest steppe, and mountain taiga forests) were investigated with regard to distribution, prevalence, and diversity of Nosema infection in honey bees using duplex-PCR. Nosema infected bees were found in 65% apiaries of ecoregions studied, and coinfection was predominant (36.3% of Nosema-positive apiaries). Both N. apis and N. ceranae occur across subarctic and warm summer continental climates, but while N. apis predominates in the former, N. ceranae is more predominant in the latter. No statistically significant differences in Nosema distribution were identified in various climatic zones. In the sub-taiga zone (subarctic climate), low presence of colonies with pure N. ceranae and a significantly higher proportion of coinfection apiaries were revealed. Long-term epidemiological study of Nosema spp. prevalence in the sub-taiga zone showed a surprising percentage increase of Nosema-positive apiaries from 46.2% to 74.1% during 2012–2017. From 2012 to 2015, N. apis became a predominant species, but in 2016–2017, the coinfection was mainly detected. In conclusion, the results of this investigation showed that N. ceranae is widespread in all study ecoregions of North Asia where it exists in combination with the N. apis, but there is no replacement of N. apis by N. ceranae in the studied bee populations.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Prevalence of the Microsporidian Nosema spp. in Honey Bee Populations (Apis mellifera) in Some Ecological Regions of North Asia

Ostroverkhova

Konusova

Кучер

et al. 2020

Veterinary Sciences

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“…Two aliquots from each DNA extract were taken and analyzed separately in duplicate by qPCR with primers and probes specific for N. ceranae , and respectively designed on sequences of the 16S rRNA [ 36 ] and Hsp70 [ 37 ] genes ( Table 1 ). For each target gene, a total reaction volume of 15 μL was prepared using 2x QuantiTect Probe PCR Master Mix (Qiagen, Hilden, Germany), forward and reverse primers (2 μM), forward and reverse probes (500 nM) and 3 μL DNA extract.…”

Section: Methodsmentioning

confidence: 99%

“…The standard curve was generated by amplifying the serially diluted plasmidas in a duplex qPCR assay. The real-time PCR assay was performed on a Rotorgene Corbett 6000 (Corbett Research, Sydney, Australia) following the amplification and quantification protocols for either gene sequence [ 36 , 37 ]. All the analyses were conducted with two technical replicates for each target gene.…”

Section: Methodsmentioning

confidence: 99%

Effect of Api-Bioxal® and ApiHerb® Treatments against Nosema ceranae Infection in Apis mellifera Investigated by Two qPCR Methods

Cilia

Garrido²,

Bonetto

et al. 2020

Veterinary Sciences

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Nosema ceranae is a worldwide distributed midgut parasite of western honey bees, leading to dwindling colonies and their collapse. As a treatment, only fumagillin is available, causing issues like resistance and hampered bee physiology. This study aimed to evaluate ApiHerb® and Api-Bioxal® as treatments against N. ceranae. The efficacy was tested using two qPCR methods based on the 16S rRNA and Hsp70 genes. In addition, these methods were compared for their aptitude for the quantification of the infection. For this, 19 colonies were selected based on the presence of N. ceranae infections. The colonies were divided into three groups: treated with ApiHerb, Api-Bioxal with previous queen caging and an untreated control. All colonies were sampled pre- and post-treatment. The bees were analyzed individually and in duplicate with both qPCR methods. All bees in the pre-treatment tested positive for N. ceranae. Both treatments reduced the abundance of N. ceranae, but ApiHerb also decreased the prevalence of infected bees. Analysis with the 16S rRNA method resulted in several orders of magnitude more copies than analysis with the Hsp70 method. We conclude that both products are suitable candidates for N. ceranae treatment. From our analysis, the qPCR method based on the Hsp70 gene results as more apt for the exact quantification of N. ceranae as is needed for the development of veterinary medicinal products.

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“…The use of microscopic and biomolecular analysis techniques applied to melissopalynology allowed to carry out an evaluation on the pollen composition of some honey commercialized and declared unifloral. For each DNA sample to be analyzed, through real time PCR, different mixes are prepared, each containing specific primers for a specific botanical species (Laube et al, 2010;Schievano et al, 2013;Cilia et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

Melissopalinological study of Sicilian honey by morphological and molecoular approach

Vancheri¹,

Tetamo²,

Reale³

et al. 2019

Biodiversity Journal

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With the Legislative Decree n.179 of 21 May 2004 on the unifloral honey's characteristics and with the CEE regulation 2081/92 of DOP denomination (from protect origin), scientific investigations in the agri-food sector started to occupy a prominent position with increasing importance. This area of investigation has been considerably deepened, becoming fundamental in order to consider this term valid, as it is also fundamental for investigation on production chain and denomination, made on various types of products. The morpho-genetic characterization analysis of pollen from honeys, made in the present work, lands itself well to this purpose; with the aim of ascertaining the validity of the wording of "unifloral" applied to some products from the beekeeping industry. Since the implications to which the investigations in the agri-food sector lead to cover not only the purely scientific-agronomic, but also the legal field, it follows the importance of an increasingly scientifically accurate methodology that also allows a faster processing of the sample. The protocol commonly used for pollen characterization of honeys is based on the visual recognition of the pollen present in the sample, their count and statistical analysis of the data obtained. Our method, using Real Time PCR technology, allows a qualitative and quantitative analysis of the pollen species inherent in the sample, thus allowing a fast and accurate analysis of the data that lends itself well to assist the classical research based on visual recognition of pollen.

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A novel TaqMan ® assay for Nosema ceranae quantification in honey bee, based on the protein coding gene Hsp70

Cited by 25 publications

References 31 publications

Prevalence of the Microsporidian Nosema spp. in Honey Bee Populations (Apis mellifera) in Some Ecological Regions of North Asia

Prevalence of the Microsporidian Nosema spp. in Honey Bee Populations (Apis mellifera) in Some Ecological Regions of North Asia

Effect of Api-Bioxal® and ApiHerb® Treatments against Nosema ceranae Infection in Apis mellifera Investigated by Two qPCR Methods

Melissopalinological study of Sicilian honey by morphological and molecoular approach

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