Evidence of transplacental transmission of equine piroplasms Theileria equi and Babesia caballi in an Italian breed mare.

Equine piroplasmosis (EP) is a vector borne disease caused by apicomplexans protists Babesia caballi (Nuttal et Strickland, 1910) and Theileria equi (Laveran, 1901). Carrier mares may transmit the infection transplacental resulting in neonatal piroplasmosis or abortions. This event has been described for T. equi by several authors over the world, but no evidence for B. caballi has been reported in Europe. In this study, vertical transmission for both parasites in an Italian breed mare has been confirmed using molecular and microscopic tools. Transplacental transmission is an underestimated problem mainly in endemic areas as it not only contributes to the spread and maintenance of the infection, but also produces significant economic losses.

Epidemiology of the Microsporidium Nosema ceranae in Four Mediterranean Countries.

Nosema ceranae is a highly prevalent intracellular parasite of honey bees' midgut worldwide. This Microsporidium was monitored during a long-term study to evaluate the infection at apiary and intra-colony levels in six apiaries in four Mediterranean countries (France, Israel, Portugal, and Spain). Parameters on colony strength, honey production, beekeeping management, and climate were also recorded. Except for São Miguel (Azores, Portugal), all apiaries were positive for N. ceranae, with the lowest prevalence in mainland France and the highest intra-colony infection in Israel. A negative correlation between intra-colony infection and colony strength was observed in Spain and mainland Portugal. In these two apiaries, the queen replacement also influenced the infection levels. The highest colony losses occurred in mainland France and Spain, although they did not correlate with the Nosema infection levels, as parasitism was low in France and high in Spain. These results suggest that both the effects and the level of N. ceranae infection depends on location and beekeeping conditions. Further studies on host-parasite coevolution, and perhaps the interactions with other pathogens and the role of honey bee genetics, could assist in understanding the difference between nosemosis disease and infection, to develop appropriate strategies for its control.

World association for the advancement of veterinary parasitology (WAAVP): Third edition of guideline for evaluating the efficacy of equine anthelmintics.

This guideline have been developed to assist in the design, execution, and interpretation of studies to assess the efficacy of anthelmintic drugs against internal parasites of equines, including nematodes, cestodes, and larval instars of Gasterophilus spp. The design and execution of critical and controlled studies are outlined, and their advantages and disadvantages are discussed. Unique considerations for specific target parasites are included. Information is also provided on selection of animals, procedures for randomization, housing, feeding, dosage titration, dosage confirmation and field studies, record keeping and necropsy procedures. Finally, this document includes guidance for group size determination and statistical analysis of study results. This guideline should assist investigators in the evaluation of anthelmintic drugs in horses by using comparable and standardized procedures in studies with appropriate numbers of animals.

Residual Tau-Fluvalinate in Honey Bee Colonies Is Coupled with Evidence for Selection for Varroa destructor Resistance to Pyrethroids.

Varroa destructor is considered one of the most devastating parasites of the honey bee, Apis mellifera, and a major problem for the beekeeping industry. Currently, the main method to control Varroa mites is the application of drugs that contain different acaricides as active ingredients. The pyrethroid tau-fluvalinate is one of the acaricides most widely used in beekeeping due to its efficacy and low toxicity to bees. However, the intensive and repetitive application of this compound produces a selective pressure that, when maintained over time, contributes to the emergence of resistant mites in the honey bee colonies, compromising the acaricidal treatments efficacy. Here we studied the presence of tau-fluvalinate residues in hives and the evolution of genetic resistance to this acaricide in Varroa mites from honey bee colonies that received no pyrethroid treatment in the previous four years. Our data revealed the widespread and persistent tau-fluvalinate contamination of beeswax and beebread in hives, an overall increase of the pyrethroid resistance allele frequency and a generalized excess of resistant mites relative to Hardy-Weinberg equilibrium expectations. These results suggest that tau-fluvalinate contamination in the hives may seriously compromise the efficacy of pyrethroid-based mite control methods.

Workflow of Lotmaria passim isolation: Experimental infection with a low-passage strain causes higher honeybee mortality rates than the PRA-403 reference strain.

The impact of trypanosomatid parasites on honeybee health may represent a major threat to bee colonies worldwide. However, few axenic isolates have been generated to date and with no details on cell culture passages, a parameter that could influence parasite virulence. To address this question, a trypanosomatid isolation protocol was developed and a new strain was obtained, named L. passim C1. Using experimental infection of worker honeybees, we compared the virulence and mortality rates of the ATCC PRA-403 reference strain and C1 strain, the latter showing higher virulence from 10 days post-infection onward. This study highlights the impact of cell culture passages on the pathogenicity of L. passim in honeybees, providing new evidence of its negative effects on honeybee health.

Experimental evidence of harmful effects of Crithidia mellificae and Lotmaria passim on honey bees.

The trypanosomatids Crithidia mellificae and Lotmaria passim are very prevalent in honey bee colonies and potentially contribute to colony losses that currently represent a serious threat to honey bees. However, potential pathogenicity of these trypanosomatids remains unclear and since studies of infection are scarce, there is little information about the virulence of their different morphotypes. Hence, we first cultured C. mellificae and L. passim (ATCC reference strains) in six different culture media to analyse their growth rates and to obtain potentially infective morphotypes. Both C. mellificae and L. passim grew in five of the media tested, with the exception of M199. These trypanosomatids multiplied fastest in BHI medium, in which they reached a stationary phase after around 96 h of growth. Honey bees inoculated with either Crithidia or Lotmaria died faster than control bees and their mortality was highest when they were inoculated with 96 h cultured L. passim. Histological and Electron Microscopy analyses revealed flagellated morphotypes of Crithidia and Lotmaria in the lumen of the ileum, and adherent non-flagellated L. passim morphotypes covering the epithelium, although no lesions were evident. These data indicate that parasitic forms of these trypanosomatids obtained from the early stationary growth phase infect honey bees. Therefore, efficient infection can be achieved to study their intra-host development and to assess the potential pathogenicity of these trypanosomatids.

Age and Method of Inoculation Influence the Infection of Worker Honey Bees (Apis mellifera) by Nosema ceranae.

The microsporidian parasite Nosema ceranae is a highly prevalent, global honey bee pathogen. Apis mellifera is considered to be a relatively recent host for this microsporidia, which raises questions as to how it affects its host's physiology, behavior and longevity, both at the individual and colony level. As such, honey bees were inoculated with fresh purified spores of this pathogen, both individually (Group A) or collectively (Group B) and they were studied from 0 to 15 days post-emergence (p.e.) to evaluate the effect of bee age and the method of inoculation at 7 days post-infection. The level of infection was analyzed individually by qPCR by measuring the relative amount of the N. ceranae polar tubule protein 3 (PTP3) gene. The results show that the bee's age and the method of infection directly influence parasite load, and thus, early disease development. Significant differences were found regarding bee age at the time of infection, whereby the youngest bees (new-born and 1 day p.e.) developed the highest parasite load, with this load decreasing dramatically in bees infected at 2 days p.e. before increasing again in bees infected at 3-4 days p.e. The parasite load in bees infected when older than 4 days p.e. diminished as they aged. When the age cohort data was pooled and grouped according to the method of infection, a significantly higher mean concentration and lower variation in N. ceranae infection was evident in Group A, indicating greater variation in experimental infection when spores were administered collectively to bees through their food. In summary, these data indicate that both biological and experimental factors should be taken into consideration when comparing data published in the literature.

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.

Nosema ceranae in Apis mellifera: a 12 years postdetection perspective.

Nosema ceranae is a hot topic in honey bee health as reflected by numerous papers published every year. This review presents an update of the knowledge generated in the last 12 years in the field of N. ceranae research, addressing the routes of transmission, population structure and genetic diversity. This includes description of how the infection modifies the honey bee's metabolism, the immune response and other vital functions. The effects on individual honey bees will have a direct impact on the colony by leading to losses in the adult's population. The absence of clear clinical signs could keep the infection unnoticed by the beekeeper for long periods. The influence of the environmental conditions, beekeeping practices, bee genetics and the interaction with pesticides and other pathogens will have a direct influence on the prognosis of the disease. This review is approached from the point of view of the Mediterranean countries where the professional beekeeping has a high representation and where this pathogen is reported as an important threat.

Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis.

Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host's cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite's survival within the cell.

Permanent prevalence of Nosema ceranae in honey bees (Apis mellifera) in Hungary.

Nosema ceranae is present in honey bee (Apis mellifera L.) colonies worldwide. Studies on the comparative virulence of N. ceranae and N. apis showed significant differences in individual mortality, and the prevalence of N. ceranae seems to be predominant in both the continental and the Mediterranean climate regions. This study attempted to monitor the geographical and seasonal distribution of these two Nosema species in Hungary, using a simple laboratory method. The distribution of N. ceranae and N. apis infection rates along all seasons was homogeneous (P = 0.57). In co-infected samples, the intensity of N. ceranae infection was always significantly higher than that of N. apis infection (P < 0.001). The infection rate of infected bees in exterior samples was higher than in interior samples in each season; however, the differences were not statistically significant. The species N. ceranae had been present in Hungary already in 2004. Statistical analysis of data shows that the infection level is best represented by sampling exterior bees to establish the proportion of infected bees rather than by determining the mean spore count.

Effect of oxalic acid on Nosema ceranae infection.

Nosema ceranae is a honey bee pathogen parasitizing the ventricular epithelium and potentially causing colony death. The effect of 0.25 M oxalic acid solution administered to the bees in the form of sugar syrup was determined in laboratory and field trials. The spore numbers in an 8-day laboratory experiment were significantly lower when AO was administered (treated: 11.86 ± 0.94 s.e. × 10^6; untreated: 30.64 ± 0.31 s.e.x10^6). When administered in autumn to free flying colonies twice, 3 weeks apart, the infection prevalence decreased in young (relative reduction of 53.8% ± 6.5 s.e.) and old bees (relative reduction of 44.4% ± 6.0 s.e.). Meanwhile increased prevalence in all the controls was detected (young and old bees: relative increase of 45.7% ± 22.8 s.e. and 10.2% ± 5.9 s.e., respectively). While all the treated colonies overwintered correctly, the untreated ones did not (3 out of 5 were dead). In the absence of commercial products approved in several countries to control nosemosis, oxalic acid syrup appears promising in the development of alternative management strategies.

New multiplex PCR method for the simultaneous diagnosis of the three known species of equine tapeworm.

Although several techniques exist for the detection of equine tapeworms in serum and feces, the differential diagnosis of tapeworm infection is usually based on postmortem findings and the morphological identification of eggs in feces. In this study, a multiplex polymerase chain reaction (PCR)-based method for the simultaneuos detection of Anoplocephala magna, Anoplocephala perfoliata and Anoplocephaloides mamillana has been developed and validated. The method simultaneously amplifies hypervariable SSUrRNA gene regions in the three tapeworm species in a single reaction using three pairs of primers, which exclusively amplify the internal transcribed spacer 2 (ITS-2) in each target gene. The method was tested on three types of sample: (a) 1/10, 1/100, 1/500, 1/1000, 1/2000 and 1/5000 dilutions of 70 ng of genomic DNA of the three tapeworm species, (b) DNA extracted from negative aliquots of sediments of negative control fecal samples spiked with 500, 200, 100, 50 and 10 eggs (only for A. magna and A. perfoliata; no A. mamillana eggs available) and (c) DNA extracted from 80, 50, 40, 30, 10 and 1 egg per 2 µl of PCR reaction mix (only for A. magna and A. perfoliata; no A. mamillana eggs available). No amplification was observed against the DNA of Gasterophilus intestinalis, Parascaris equorum and Strongylus vulgaris. The multiplex PCR method emerged as specific for the three tapeworms and was able to identify as few as 50 eggs per fecal sample and as little as 0.7 ng of control genomic DNA obtained from the three species. The method proposed is able to differentiate infections caused by the two most frequent species A. magna or A. perfoliata when the eggs are present in feces and is also able to detect mixed infections by the three cestode species.

A two year national surveillance for Aethina tumida reflects its absence in Spain.

BACKGROUND: The Small Hive Beetle (SHB) is considered one of the major threats to the long-term sustainability and economic success of honey bee colonies in Europe. The risk of introduction into the EU had been reported as moderate to high. Indeed, it has been recently reported an outbreak in the south of Italy. Here, the presence of Aethina tumida in beekeeping farms in Spain was evaluated using a previously described qPCR protocol. FINDINGS: When hive debris from 398 colonies (collected in 2010 and 2011) was analysed, grouped by region, SHB were not detected in any of the samples, making it unnecessary to analyse the samples individually. CONCLUSION: The SHB free-status is shown. This epidemiological surveillance would appear to be useful to detect the possible future entry of this pathogen.

Holistic screening of collapsing honey bee colonies in Spain: a case study.

BACKGROUND: Here we present a holistic screening of collapsing colonies from three professional apiaries in Spain. Colonies with typical honey bee depopulation symptoms were selected for multiple possible factors to reveal the causes of collapse. RESULTS: Omnipresent were Nosema ceranae and Lake Sinai Virus. Moderate prevalences were found for Black Queen Cell Virus and trypanosomatids, whereas Deformed Wing Virus, Aphid Lethal Paralysis Virus strain Brookings and neogregarines were rarely detected. Other viruses, Nosema apis, Acarapis woodi and Varroa destructor were not detected. Palinologic study of pollen demonstrated that all colonies were foraging on wild vegetation. Consequently, the pesticide residue analysis was negative for neonicotinoids. The genetic analysis of trypanosomatids GAPDH gene, showed that there is a large genetic distance between Crithidia mellificae ATCC30254, an authenticated cell strain since 1974, and the rest of the presumed C. mellificae sequences obtained in our study or published. This means that the latter group corresponds to a highly differentiated taxon that should be renamed accordingly. CONCLUSION: The results of this study demonstrate that the drivers of colony collapse may differ between geographic regions with different environmental conditions, or with different beekeeping and agricultural practices. The role of other pathogens in colony collapse has to bee studied in future, especially trypanosomatids and neogregarines. Beside their pathological effect on honey bees, classification and taxonomy of these protozoan parasites should also be clarified.

Coprologically diagnosing Anoplocephala perfoliata in the presence of A. magna.

Current copro-diagnostic tests for Anoplocephala perfoliata show high variation in their sensitivity and given the morphological similarity of Anoplocephala spp. eggs, this could be related to the presence of Anoplocephala magna alone or co-existing with A. perfoliata. In the present study, coprology was significantly more sensitive (p<0.01) at detecting A. magna than A. perfoliata. This difference was independent of the parasite burden and was greater when testing was limited to horses with mature or gravid tapeworms. A. magna infection was strongly linked to young horses (≤ 2 years). The eggs of A. magna are smaller. Using 15 and 70 µm cut-offs for oncosphere diameter and the major shell bisector length, respectively, the eggs of A. perfoliata were identified with 100% sensitivity, 97% specificity and 98% sensitivity, 84% specificity. The use of these two morphometric variables would therefore be useful for the copro-identification of A. perfoliata in countries where both species coexist.

Screening alternative therapies to control Nosemosis type C in honey bee (Apis mellifera iberiensis) colonies.

Nosemosis type C caused by the microsporidium Nosema ceranae is one of the most widespread of the adult honey bee diseases, and due to its detrimental effects on both strength and productivity of honey bee colonies, an appropriate control of this disease is advisable. Fumagillin is the only veterinary medicament recommended by the World Organization for Animal Health (OIE) to suppress infections by Nosema, but the use of this antibiotic is prohibited in the European Union and few alternatives are available at present to control the disease. In the present study three therapeutic agents (Nosestat®, Phenyl salicylate and Vitafeed Gold®) have been tested to control N. ceranae infection in honey bee colonies, and have been compared to the use of fumagillin. None of the products tested was effective against Nosema under our experimental conditions. Low consumption of the different doses of treatments may have had a strong influence on the results obtained, highlighting the importance of this issue and emphasizing that this should be evaluated in studies to test therapeutic treatments of honey bee colonies.

The prevalence of the honeybee brood pathogens Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius in Spanish apiaries determined with a new multiplex PCR assay.

The microorganisms Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius are the three most important pathogens that affect honeybee brood. The aim of the present study was to evaluate the prevalence of these pathogens in honeybee colonies and to elucidate their role in the honeybee colony losses in Spain. In order to get it, a multiplex polymerase chain reaction (PCR) assay was developed to simultaneously amplify the16S ribosomal ribonucleic acid (rRNA) gene of P. larvae and M. plutonius, and the 5.8S rRNA gene of A. apis. The multiplex PCR assay provides a quick and specific tool that successfully detected the three infectious pathogens (P. larvae, M. plutonius and A. apis) in brood and adult honeybee samples without the need for microbiological culture. This technique was then used to evaluate the prevalence of these pathogens in Spanish honeybee colonies in 2006 and 2007, revealing our results a low prevalence of these pathogens in most of the geographic areas studied.

Apoptosis in the pathogenesis of Nosema ceranae (Microsporidia: Nosematidae) in honey bees (Apis mellifera).

Nosema ceranae is a parasite of the epithelial ventricular cells of the honey bee that belongs to the microsporidian phylum, a biological group of single-cell, spore-forming obligate intracellular parasites found in all major animal lineages. The ability of host cells to accommodate a large parasitic burden for several days suggests that these parasites subvert the normal host cells to ensure optimal environmental conditions for growth and development. Once infected, cells can counteract the invasive pathogen by initiating their own death by apoptosis as a defence strategy. To determine whether N. ceranae blocks apoptosis in infected ventricular cells, cell death was assessed in sections of the ventriculum from experimentally infected honey bees using the TUNEL assay and by immunohistochemistry for caspase-3. Ventricular epithelial cells from infected bees were larger than those in the uninfected control bees, and they contained N. ceranae at both mature and immature stages in the cytoplasm. Apoptotic nuclei were only observed in some restricted areas of the ventriculum, whereas apoptosis was typically observed throughout the epithelium in uninfected bees. Indeed, the apoptotic index was higher in uninfected versus infected ventriculi. Our results suggested that N. ceranae prevents apoptosis in epithelial cells of infected ventriculi, a mechanism possible designed to enhance parasite development.

Nosema ceranae (Microsporidia), a controversial 21st century honey bee pathogen.

The worldwide beekeeping sector has been facing a grave threat, with losses up to 100-1000 times greater than those previously reported. Despite the scale of this honey bee mortality, the causes underlying this phenomenon remain unclear, yet they are thought to be multifactorial processes. Nosema ceranae, a microsporidium recently detected in the European bee all over the world, has been implicated in the global phenomenon of colony loss, although its role remains controversial. A review of the current knowledge about this pathogen is presented focussing on discussion related with divergent results, trying to analyse the differences specially based on different methodologies applied and divisive aspects on pathology while considering a biological or veterinarian point of view. For authors, the disease produced by N. ceranae infection cannot be considered a regional problem but rather a global one, as indicated by the wide prevalence of this parasite in multiple hosts. Not only does this type of nosemosis causes a clear pathology on honeybees at both the individual and colony levels, but it also has significant effects on the production of honeybee products.