Epidemiology of Nosema spp. and the effect of indoor and outdoor wintering on honey bee colony population and survival in the Canadian Prairies.

The epidemiology of Nosema spp. in honey bees, Apis mellifera, may be affected by winter conditions as cold temperatures and differing wintering methods (indoor and outdoor) provide varying levels of temperature stress and defecation flight opportunities. Across the Canadian Prairies, including Alberta, the length and severity of winter vary among geographic locations. This study investigates the seasonal pattern of Nosema abundance in two Alberta locations using indoor and outdoor wintering methods and its impact on bee population, survival, and commercial viability. This study found that N. ceranae had a distinct seasonal pattern in Alberta, with high spore abundance in spring, declining to low levels in the summer and fall. The results showed that fall Nosema monitoring might not be the best indicator of treatment needs or future colony health outcomes. There was no clear pattern for differences in N. ceranae abundance by location or wintering method. However, wintering method affected survival with colonies wintered indoors having lower mortality and more rapid spring population build-up than outdoor-wintered colonies. The results suggest that the existing Nosema threshold should be reinvestigated with wintering method in mind to provide more favorable outcomes for beekeepers. Average Nosema abundance in the spring was a significant predictor of end-of-study winter colony mortality, highlighting the importance of spring Nosema monitoring and treatments.

Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR.

BACKGROUND: The microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen. OBJECTIVES: The present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees. METHODS: A procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration. RESULTS: UR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 104 spores/bee, and the stable detection level was ≥ 2.40 × 105 spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 104 copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min. CONCLUSIONS: UR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.

Development and optimization of a TaqMan assay for Nosema bombycis, causative agent of pébrine disease in Bombyx mori silkworm, based on the ß-tubulin gene.

"Pébrine" is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the ß-tubulin gene in the pébrine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 × 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure.

Prevalence of Nosema species infections in Apis cerana japonica and Apis mellifera honeybees in the Tohoku region of Japan.

We investigated here, the prevalence of Nosema microsporidia infections in the honeybees, Apis cerana japonica and Apis mellifera, in the Tohoku region of Japan. We detected Nosema ceranae DNA in 14 (2.8%) of 509 A. cerana japonica and in 34 (21.9%) of 155 A. mellifera honeybees from Aomori, Iwate, Akita, Yamagata, and Fukushima prefectures. Nosema apis DNA was undetectable in A. cerana japonica and A. mellifera. The unidentifiable Nosema species that genetically differed from N. apis, N. ceranae, and N. neumanni in terms of small subunit (SSU) rDNA, large subunit rDNA, and internal transcribed spacer sequences was identified in 105 (20.6%) of 509 A. cerana japonica and in 1 (0.6%) of 155 A. mellifera honeybees, and from Iwate prefecture. A phylogenetic tree based on SSU rDNA sequences showed that the Nosema sp. belonged to the same clade as N. thomsoni detected in moth and solitary bees in North America and N. pieriae found in cabbage butterfly in Turkey, which have not hitherto been detected in honeybees. The morphological characteristics of the spores should be analyzed to enable species identification of the Nosema sp.

An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens.

Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on a panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.

Matrix-assisted laser desorption/ionization mass spectrometry biotyping, an approach for deciphering and assessing the identity of the honeybee pathogen Nosema.

RATIONALE: The microsporidia are obligate intracellular pathogenic fungi that parasitize a wide range of invertebrate and vertebrate hosts and have important impacts on health, food security and the economy. In this paper, we focus on Nosema ceranae and N. apis, which chronically infect the digestive tract of honeybees, altering their physiology and lifespan. METHODS: We applied matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for rapid molecular profiling of extracts of Nosema spores in order to identify the species and the geographical origin, and assess the viability status of Nosema microsporidia in conjunction with a flow cytometric approach. Pure solutions of spores were prepared for flow cytometric analysis and MALDI-MS profiling. A mechanical extraction of viable or heat-killed Nosema spores was conducted to obtain mass fingerprints of peptides/proteins for samples of microsporidia from different geographical origins (MBO.NC01, MBO.NC02 and MBO.NA01). RESULTS: A distinction in the peptide/protein profiles between two isolates with different geographical origins was observed. Mass fingerprints of viable and experimentally killed spores were also clearly distinguishable, regardless of Nosema species. Finally, using our computational models on the different Nosema species, we were able to classify five independent isolates of Nosema microsporidia. CONCLUSIONS: We have shown that MALDI-MS is a rapid, cost-effective and simple method for identifying Nosema species. We demonstrated that MALDI Biotyping could represent a valuable surveillance tool of nosemosis in apiaries for sanitary services and beekeepers.

Identification and characterization of three microsporidian genera concurrently infecting a silkworm, Bombyx mori, in Brazil.

Microsporidia are important entomopathogens known for infecting insects such as the silkworm (Bombyx mori) thus impairing global silk production. This study aimed to identify and characterize the microsporidia isolated from a diseased larva of silkworm, collected from a sericulture farm in southern Brazil. Identification was performed by phylogenetic analysis of the nucleotide sequences of the SSU rRNA genes. Characterization was performed by analyzing spore sizes, tissue tropism, internal and external symptoms, and pathogenicity against B. mori. Microsporidia belonging to three different genera were identified, namely, Endoreticulatus, Nosema and Tubulinosema. After inoculation of the mixed spores of the microsporidian isolates into B. mori larvae, a high prevalence of Tubulinosema spp. was observed. This isolate showed high prevalence on the silk glands and a late mortality, initially of around 10% until the 20th day post-inoculation but reaching 91.5% upon pupation. Therefore, we demonstrated that Tubulinosema spp. causes chronic infection with slow pathogenicity. We identified for the first time three different microsporidians concurrently infecting B. mori in Brazil. Tubulinosema is of particular interest because of its potential threat to silk production; it affects the formation of silk glands in B. mori while not presenting distinguishable external symptoms or causing the immediate death of these insects. Further studies focusing on this species, mainly regarding its life cycle within the host and the sublethal effects of surviving individuals, demonstrate the importance of describing it as a new species and improving the characterization of the disease in order to prevent its spread.

Black queen cell virus and Nosema ceranae coinfection in Africanized honey bees from southern Brazil / Coinfecção por vírus da realeira negra (BQCV) e Nosema ceranae em abelhas africanizadas no sul do Brasil

Bees are fundamental in several aspects, especially in relation to plant biodiversity and pollination. Recently, immense losses are being faced in the number of Brazilian colonies, mainly in southern states of the country, which has a strong beekeeping activity. There are indications that, among the reasons for the losses, pathogens that affect the health of bees may be involved. Among them, the microsporidium Nosema and the black queen cell virus (BQCV) stand out for their prevalence. In this study, 92 colonies of 17 apiaries from southern Brazil were evaluated for infection by Nosema ceranae, Nosema apis and BQCV. Nucleic acid extractions and cDNA synthesis were performed from adult bee samples, followed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and multiplex PCR. Eight BQCV positive samples were subjected to sequencing. The results showed that N. ceranae and BQCV are circulating in the Southern region of the country, which may be the reason for the loss of colonies. N. apis was not found. N. ceranae was found in 57.6% (53/92) of the colonies and BQCV in 32.6% (30/92). Co-infection was found in 25% (23/92) of the colonies studied, a factor that is suggested to be reducing the hosts' longevity due to the synergistic action of the pathogens. The samples submitted to sequencing indicated similarity of 96.8 to 100% between them, in addition to strong similarity with sequences from Asia, United States, Germany and Peru. This study reports the circulation of N. ceranae and BQCV in apiaries in southern Brazil, in addition to being the first phylogenetic analysis of the Brazilian BQCV sequence.(AU)

As abelhas mostram-se fundamentais em diversos aspectos, especialmente com relação à biodiversidade de plantas e polinização. Recentemente, estão sendo enfrentadas imensas perdas no número de colônias brasileiras, principalmente nos estados do sul do país, com forte atividade apícola. Há indicativos de que, dentre as razões para as perdas, possam estar envolvidos patógenos que afetam a saúde das abelhas. Dentre eles, o microsporídio Nosema e o vírus da realeira negra (BQCV) destacam-se pela prevalência. Neste estudo, foram avaliadas 92 colônias, de 17 apiários do sul do Brasil, a respeito da infecção por Nosema ceranae, Nosema apis e BQCV. Foram realizadas extrações de ácidos nucleicos e síntese de cDNA a partir de amostras de abelhas adultas, seguidos de Reação em Cadeia da Polimerase-Transcriptase Reversa (RT-PCR). Oito amostras positivas para BQCV foram submetidas a sequenciamento. Os resultados mostraram que N. ceranae e BQCV estão circulando na região sul do país, podendo ser a razão para as perdas de colônias. N. apis não foi encontrado. N. ceranae foi encontrado em 57.6% (53/92) das colônias e BQCV em 32.6% (30/92). Foi encontrada coinfecção por ambos em 25% (23/92) das colônias estudadas, fator que sugere a diminuição da longevidade do hospedeiro por ação sinérgica dos patógenos. As amostras submetidas ao sequenciamento indicaram similaridade de 96.8 a 100% entre elas, além de forte similaridade com sequências da Ásia, Estados Unidos, Alemanha e Peru. Este estudo relata a circulação de N. ceranae e BQCV nos apiários do sul do Brasil, além de ser a primeira análise filogenética da sequência do BQCV brasileiro.(AU)

Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees.

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae-infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

Nationwide Screening for Bee Viruses and Parasites in Belgian Honey Bees.

The health of honey bees is threatened by multiple factors, including viruses and parasites. We screened 557 honey bee (Apis mellifera) colonies from 155 beekeepers distributed all over Belgium to determine the prevalence of seven widespread viruses and two parasites (Varroa sp. and Nosema sp.). Deformed wing virus B (DWV-B), black queen cell virus (BQCV), and sacbrood virus (SBV) were highly prevalent and detected by real-time RT-PCR in more than 95% of the colonies. Acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV) and deformed wing virus A (DWV-A) were prevalent to a lower extent (between 18 and 29%). Most viruses were only present at low or moderate viral loads. Nevertheless, about 50% of the colonies harbored at least one virus at high viral load (>107 genome copies/bee). Varroa mites and Nosema sp. were found in 81.5% and 59.7% of the honey bee colonies, respectively, and all Nosema were identified as Nosema ceranae by real time PCR. Interestingly, we found a significant correlation between the number of Varroa mites and DWV-B viral load. To determine the combined effect of these and other factors on honey bee health in Belgium, a follow up of colonies over multiple years is necessary.

A new isolate of Nosema fumiferanae (Microsporidia: Nosematidae) from the date moth Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839 (Lepidoptera: Pyralidae).

In this study, a microsporidian pathogen of the date moth (Apomyelois (Ectomyelois) ceratoniae, Zeller, 1839) also known as the carob moth, is described based on light microscopy, ultrastructural characteristics and comparative molecular analysis. The pathogen infects the gut and hemolymph of A. ceratoniae. All development stages are in direct contact with the host cell cytoplasm. Fresh spores with nuclei arranged in a diplokaryon are oval and measured 3.29 ± 0.23 µm (4.18-3.03 µm, n = 200) in length and 1.91 ± 0.23 µm (2.98-1.66 µm, n = 200) in width. Spores stained with Giemsa's stain measured 3.11 ± 0.31 µm (3.72-2.41 µm, n = 150) in length and 1.76 ± 0.23 µm (2.16-1.25 µm, n = 150) in width. Spores have an isofilar polar filament with 10-12 coils. An 1110 bp long alignment of the current microsporidium showed an SSU rRNA gene difference of only 0.0009, corresponding to >99.91% sequence similarity with Nosema fumiferanae, while RPB1 gene sequences were 98.03% similar within an alignment of 969 bp. All morphological, ultrastructural and molecular features indicate that the microsporidian pathogen of A. ceratoniae is the new isolate of the N. fumiferanae and is named here as Nosema fumiferanae TY61.

Cross-infectivity of honey and bumble bee-associated parasites across three bee families.

Recent declines of wild pollinators and infections in honey, bumble and other bee species have raised concerns about pathogen spillover from managed honey and bumble bees to other pollinators. Parasites of honey and bumble bees include trypanosomatids and microsporidia that often exhibit low host specificity, suggesting potential for spillover to co-occurring bees via shared floral resources. However, experimental tests of trypanosomatid and microsporidial cross-infectivity outside of managed honey and bumble bees are scarce. To characterize potential cross-infectivity of honey and bumble bee-associated parasites, we inoculated three trypanosomatids and one microsporidian into five potential hosts - including four managed species - from the apid, halictid and megachilid bee families. We found evidence of cross-infection by the trypanosomatids Crithidia bombi and C. mellificae, with evidence for replication in 3/5 and 3/4 host species, respectively. These include the first reports of experimental C. bombi infection in Megachile rotundata and Osmia lignaria, and C. mellificae infection in O. lignaria and Halictus ligatus. Although inability to control amounts inoculated in O. lignaria and H. ligatus hindered estimates of parasite replication, our findings suggest a broad host range in these trypanosomatids, and underscore the need to quantify disease-mediated threats of managed social bees to sympatric pollinators.

Occurrence of virus, microsporidia, and pesticide residues in three species of stingless bees (Apidae: Meliponini) in the field.

Bees are important pollinators whose population has declined due to several factors, including infections by parasites and pathogens. Resource sharing may play a role in the dispersal dynamics of pathogens among bees. This study evaluated the occurrence of viruses (DWV, BQCV, ABPV, IAPV, KBV, and CBPV) and microsporidia (Nosema ceranae and Nosema apis) that infect Apis mellifera, as well as pesticide residues in the stingless bees Nannotrigona testaceicornis, Tetragonisca angustula, and Tetragona elongata sharing the same foraging area with A. mellifera. Stingless bees were obtained from 10 nests (two of N. testaceicornis, five of T. angustula, and three of T. elongata) which were kept in the field for 1 year and analyzed for the occurrence of pathogens. Spores of N. ceranae were detected in stingless bees but were not found in their midgut, which indicates that these bees are not affected, but may be vectors of the microsporidium. Viruses were found in 23.4% of stingless bees samples. APBV was the most prevalent virus (10.8%) followed by DWV and BQCV (both in 5.1% of samples). We detected glyphosate and its metabolites in small amounts in all samples. The highest occurrence of N. ceranae spores and viruses was found in autumn-winter and may be related to both the higher frequency of bee defecation into the colony and the low food resources available in the field, which increases the sharing of plant species among the stingless bees and honey bees. This study shows the simultaneous occurrence of viruses and spores of the microsporidium N. ceranae in asymptomatic stingless bees, which suggest that these bees may be vectors of pathogens.

European Foulbrood in stingless bees (Apidae: Meliponini) in Brazil: Old disease, renewed threat.

Stingless bees (Apidae: Meliponini) are a group of bees with vestigial stings showing a high level of social organization. They are important pollinators in tropical and subtropical regions, and, in the last decades, stingless beekeeping has increased rapidly in Brazil. Bee-collected pollen and honey of Apis mellifera can be an important source of disease when used as supplements to feed stingless bee colonies, a common and increasing practice adopted by stingless beekeepers. Here, we aimed to investigate the presence of pathogens commonly found in honey bees in diseased colonies of Melipona species in Espírito Santo and São Paulo States, Southeast Brazil. We detected, for the first time, the bacterium Melissococcus plutonius and symptoms of European foulbrood in Melipona spp., associated with brood death and colony losses in some cases. In addition, we tested for the presence of the bacterium Paenibacillus larvae and the fungus Aschosphaera apis, as well as the six more common honey bee viruses in Brazil (BQCV, ABPV, DWV, KBV, IAPV, CBPV) and the microsporidia Nosema apis and Nosema ceranae. However, only one sample of brood was infected with N. ceranae and all other pathogens, with the exception of Melissococcus plutonius, were absent in the analyzed brood. Lastly, we looked for toxic pollen in all food fed to diseased colonies, but none was present.

Direct and sensitive detection of a microsporidian parasite of bumblebees using loop-mediated isothermal amplification (LAMP).

The reduction of bumblebee populations has been reported in the last decades, and the microsporidian parasite Nosema bombi is considered as one of the factors contributing to such reduction. Although the decline of bee populations affects both wild plants and human food supply, the effects of Nosema spp. infections are not known because it is difficult to obtain infective spores from wild bees due to their low prevalence. Microscopical observation of fecal samples or midgut homogenates and/or PCR are generally used for N. bombi detection. However, the germination rate of microsporidian spore declines if they are kept at 4 °C for a long time or frozen. It is therefore crucial to minimize the diagnosis and isolation time of infective spores from field-collected samples. Therefore, we performed a loop-mediated isothermal amplification (LAMP) assay for the direct detection of N. bombi in bumblebee midgut homogenates. Using this method, we could detect N. bombi from individuals from which it was visible under the microscope and directly from wild individuals.

A specific molecular label for identifying mature Nosema bombycis spores.

Microsporidia are a fascinating phylum of obligate intracellular pathogens with unique infection processes and complicated life cycles. Microsporidian life cycles can be divided roughly into intracellular and extracellular stages. Currently, research on their life cycles were mainly explored by morphology because there are few molecular markers available with which to distinguish the different life stages. In this study, we generated H20, a monoclonal antibody (MAb) to label mature spores of Nosema bombycis. Immunofluorescence assays showed that the target protein of H20, which is highly stable and was barely affected by alkali and sodium dodecyl sulfate (SDS) treatments, was located on the mature spore surface. Western blot analysis showed that spore wall protein 26 (SWP26) was the likely target of H20. This MAb can specifically identify mature spores in a complex biological sample based on immunological detection of the parasite.

Rapid detection of silkworm microsporidia by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Microsporidia cause the disease pébrine in silkworm and are known to be detrimental to sericulture and beekeeping. The microsporidian species Nosema bombycis was rapidly identified in silkworm (Bombyx mori) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Four types of microsporidian spores purified from infected silkworm could be distinguished based on the differences in their mass fingerprints. Microsporidia growing in a silkworm larva were also identified based on their mass spectra after rapid separation using filtration and centrifugation for 30 min.

The levels of natural Nosema spp. infection in Apis mellifera iberiensis brood stages.

Nosema ceranae is the most prevalent endoparasite of Apis mellifera iberiensis and it is a major health problem for bees worldwide. The infective capacity of N. ceranae has been demonstrated experimentally in honey bee brood, however no data are available about its prevalence in brood under natural conditions. Thus, brood combs from 10 different hives were analyzed over two consecutive years, taking samples before and after winter. A total of 1433 larvae/pupae were analyzed individually and N. ceranae (3.53%) was the microsporidian most frequently detected, as opposed to Nosema apis (0.42%) which was more frequently detected in conjunction with N. ceranae (0.71%). The active multiplication of both microsporidians was confirmed by the expression (real-time-PCR) of the N. ceranae polar tube protein 3 gene and/or the N. apis RNA polymerase II gene in 24% of the brood samples positive for Nosema spp. Both genes are related to microsporidian multiplication. As such, N. ceranae multiplication was confirmed in 1.06% of the samples, while N. apis multiplication was only observed in co-infections with N. ceranae (0.07%). Brood cells were analyzed for the presence of Nosema spp., as those are the immediate environment where the brood stages develop. The brood samples infected by Nosema spp. were in brood cells in which that microsporidians were not detected, while brood cells positive for N. ceranae hosted brood stages that were not apparently infected, indicating that this is unlikely to be the main pathway of infection. Finally, the colonies with brood infected by N. ceranae showed higher levels (numbers) of infected adult bees, although the differences were not significant before (P = 0.260), during (P = 0.055) or after (P = 0.056) brood sampling. These results show that N. ceranae is a bee parasite ubiquitous to all members of the colony, irrespective of the age of the bee. It is also of veterinary interest and should be considered when studying the epidemiology of the disease.

Microsporidial keratitis retrospectively diagnosed by ultrastructural study of formalin-fixed paraffin-embedded corneal tissue: a case report.

BACKGROUND: The utility of formalin-fixed paraffin-embedded (FFPE) corneal tissue specimens for retrospective diagnosis of microsporidial keratitis was evaluated by transmission electron microscopy (TEM) analysis and the possible second case of microsporidial keratitis after Descemet stripping automated endothelial keratoplasty (DSAEK) was described. CASE PRESENTATION: A 68-year-old man presented with multiple crystalline opacities in the corneal stroma that progressed extremely slowly after DSAEK. Fungiflora Y staining of corneal scrapings from the affected regions revealed an oval microorganism. Topical voriconazole administration was ineffective and penetrating keratoplasty was performed. Histological and molecular analyses were carried out on the excised cornea. Ziehl-Neelsen staining revealed an acid-fast, oval organism that was visible by ultraviolet illumination after Fungiflora Y and Uvitex 2B staining, whereas periodic acid-Schiff and Grocott's staining did not yield any significant findings. Microsporidium was detected by TEM of FFPE tissue. Nosema or Vittaforma sp. was suspected as the causative microorganism by PCR of FFPE tissue and by the fact that those species are known to cause eye infection. The corneal graft has maintained transparency at 1 year and half postoperatively. CONCLUSIONS: This is the first known case of microsporidial keratitis diagnosed retrospectively by molecular and ultrastructural study of FFPE tissue, and the possible second case of microsporidial keratitis after DSAEK. Microsporidial keratitis should be considered when corneal opacity refractory to conventionally known therapy would occur after DSAEK. Our findings suggest that more microsporidial keratitis cases than have been reported to date can be identified by TEM or PCR examination of FFPE corneal specimens.

Development of a nucleic acid lateral flow strip for rapid, visual detection of Nosema bombycis in silkworm eggs.

Nosema bombycis, the pathogen of silkworm pébrine, causes enormous economic losses to sericulture. As such, quarantine of commercial silkworm eggs represents an important safeguard to the silkworm industry. Here, we established a user-friendly detection system based on a nucleic acid lateral flow strip (NAFLS) that combines polymerase chain reaction (PCR) and a colloidal gold strip. PCR primers were designed based on the sequence of LSU rDNA of N. bombycis and has favourable specificity for common microsporidian isolates in silkworms. The forward and reverse primers were labeled on the 5' end with biotin and carboxyfluorescein (FAM), respectively. Genomic DNA was extracted from egg samples and was used as a template for PCR, followed by subsequent detection by NALFS. The detection limit of purified N. bombycis genomic DNA was 1 pg, 100× more sensitive than that of agarose gel electrophoresis (AGE). Furthermore, the sensitivity of detection of simulated "infected" silkworm eggs was 10-100× higher than that of AGE. NALFS detected infection in 27 of 29 samples of silkworm eggs oviposited by female moths infected in lab; ≥2% infected eggs per batch are detected as positive, while ≥40% infected eggs per batch are required for detection by AGE. Collectively, NALFS is easy to use and has great potential for widespread use in the detection of N. bombycis in silkworm egg production.