Effect of pollen extract supplementation on the varroatosis tolerance of honey bee (Apis mellifera) larvae reared in vitro.

As the main source of lipids and proteins in honey bees, pollen is a major nutrient provider involved in development and health and has been studied for tolerance stimulation against pathogens and parasites. In the case of Varroa destructor Anderson & Trueman (Acari, Mesostigmata: Varroidae) parasitization, the lack of a complete laboratory system to rear both the bee larva and the acarian parasite limited the studies concerning larval nutrition effects on the bee tolerance and resistance against varroatosis. Due to the development of this complete rearing protocol, we managed to feed young honey bee larvae with pollen supplemented solutions and to study the effect on their later development under parasitism conditions. In our experimental conditions, pollen influences neither the deformity rate, nor the survival of bees both parasitized and unparasitized. However, pollen extract supplementation seems to significantly impact the weight of the spinning bee larvae without having an effect on the physiological weight loss during pupation, so the differences found at the larval stage remain the same as at emergence. Varroa has a deleterious effect on bee pupae and led to a steady increase of the physiological weight loss experienced during metamorphosis. Interestingly, this ponderal loss associated with Varroa parasitization seems to be reduced in the polyfloral pollen supplementation condition. Altogether, this work is to our knowledge the first to study in laboratory conditions the impact of larval nutrition on the tolerance to parasitism. A diverse pollen diet may be beneficial to the bees' tolerance against V. destructor parasitism.

Impact of the Phoretic Phase on Reproduction and Damage Caused by Varroa destructor (Anderson and Trueman) to Its Host, the European Honey Bee (Apis mellifera L.).

Varroa destructor is a parasitic mite of the honeybee that causes thousands of colony losses worldwide. The parasite cycle is composed of a phoretic and a reproductive phase. During the former, mites stay on adult bees, mostly on nurses, to feed on hemolymph. During the latter, the parasites enter brood cells and reproduce. We investigated if the type of bees on which Varroa stays during the phoretic phase and if the duration of this stay influenced the reproductive success of the parasite and the damage caused to bees. For that purpose, we used an in vitro rearing method developed in our laboratory to assess egg laying rate and the presence and number of fully molted daughters. The expression level of two Varroa vitellogenin genes (VdVg1 and VdVg2), known to vary throughout reproduction, was also quantified. Results showed that the status of the bees or time spent during the phoretic phase impacts neither reproduction parameters nor the Varroa vitellogenin genes levels of expression. However, we correlated these parameters to the gene expression and demonstrated that daughters expressed the vitellogenin genes at lower levels than their mother. Regarding the damage to bees, the data indicated that a longer stay on adult bees during the phoretic phase resulted in more frequent physical deformity in newborn bees. We showed that those mites carry more viral loads of the Deformed Wing Virus and hence trigger more frequently overt infections. This study provides new perspectives towards a better understanding of the Varroa-honeybee interactions.

Assessment of immunogenicity and protective efficacy of Microsporum canis secreted components coupled to monophosphoryl lipid-A adjuvant in a vaccine study using guinea pigs.

Microsporum canis is the most common dermatophyte in pets and is of zoonotic importance but currently there is no effective vaccine available to prevent dermatophytosis. The aim of this work was to assess the immunogenicity and protective efficacy of secreted components (SC) from M. canis adjuvanted with the monophosphoryl lipid-A (MPLA), in a vaccine study using the guinea pig as an experimental model. Animals were vaccinated with either the SC adjuvanted with the MPLA, the MPLA adjuvant alone or PBS three times at two-week intervals, until 42 days prior to M. canis infection. A blind evaluation of dermatophytosis symptoms development and fungal persistence in skin was monitored weekly. The antibody response towards the SC and the levels of Interferon (IFN)γ and Interleukin-4 expressed in peripheral blood mononuclear cells were assessed along or at the end of the study period respectively. The animals that received MPLA had a significantly lower clinical score than those inoculated with PBS. However, no significant difference was observed between the guinea pigs vaccinated with the SC adjuvanted with the MPLA and those having received MPLA alone. The results also showed that vaccination induced a strong antibody response towards the SC and an increase in IFNγ mRNA level. Our results show that the MPLA adjuvant used in this vaccine study can induce per se a partial protection against a M. canis infection. Although they induce a delayed-type hypersensitivity reaction in guinea pigs, the SC do not confer a protection under the present experimental conditions.

Lethal and sub-lethal effects of thymol on honeybee (Apis mellifera) larvae reared in vitro.

BACKGROUND: Thymol offers an attractive alternative to synthetic chemicals to keep Varroa under control. However, thymol accumulates in bee products and is suspected of having adverse effects on colonies and especially on larvae. In this study, we investigated the effects of acute and chronic exposure to thymol on larvae reared in vitro with contaminated food and compared results to the theoretical larval exposure based on the amount of pollen and honey consumed by larvae during their development. RESULTS: The laboratory assays reveal that, first, the 48 h-LD50 of thymol introduced into larval food is 0.044 mg larva(-1) . Second, the 6 day-LC50 is 700 mg kg(-1) food. A significant decrease of larval survival and mass occurred from 500 mg thymol kg(-1) food (P < 0.0001). Finally, vitellogenin expression, which reached a maximum at the fifth instar larvae, is delayed for individuals exposed to 50 mg thymol kg(-1) food (P < 0.0006). That is 10 times higher than the theoretical level of exposure. CONCLUSION: Based on the level of thymol residue found in honey and pollen, these results suggest that the contamination of food by thymol represents no notable risk for the early-developing larvae.

Artificial feeding of Varroa destructor through a chitosan membrane: a tool for studying the host-microparasite relationship.

Rearing pests or parasites of very small size in the absence of their living host is a challenge for behavioural, physiological and pathological studies. For feeding Varroa destructor, an ectoparasitic mite of Apis mellifera, a confinement space with a membrane separating the nutritive solution and the space was designed. The mite measures less than 2 mm and bears a perforating apparatus with a length of 15 µm. The membrane, an essential element of the chamber, has a thickness of 0.1 µm, and is made of chitosan. It closes one face of the individual confinement chamber and allows piercing and the ingestion of the nutritive solution. Factors inducing feeding can be applied on the inner walls or on the membrane. In the particular case of Varroa, the highest percentages of feeding mites are obtained by addition of host haemolymph to the nutritive solution, suggesting the kairomonal role of haemolymph in addition to its nutritional one. The membrane concept can be easily applied to several mites or other micro-pests.

Feline polymorphonuclear neutrophils produce pro-inflammatory cytokines following exposure to Microsporum canis.

The mechanisms involved in the establishment of the specific immune response against dermatophytes remain unknown. Polymorphonuclear neutrophils (PMNs) are recruited early during the infection process and participate in the elimination of dermatophytes. They could therefore be involved in the induction of the immune response during dermatophytoses by producing specific cytokines. The aim of this work was to assess the in vitro cytokine production by feline PMNs exposed to living arthroconidia from the dermatophyte species Microsporum canis or stimulated with either a secreted or a structural component of M. canis, the latter consisting of heat-killed arthroconidia. The levels of specific cytokines produced by PMNs were determined by capture ELISA and/or quantitative RT-PCR. Results showed that PMNs secrete TNFα, IL-1ß and IL-8 following exposure to M. canis living arthroconidia and stimulation with both a secreted component and heat-killed arthroconidia. The level of IL-8 mRNA was also increased in PMNs stimulated with M. canis living arthroconidia. In conclusion, infective M. canis arthroconidia induce the production of pro-inflammatory cytokines by feline PMNs that can be activated either by secreted or structural fungal components. Our results suggest that these granulocytes are involved in the initiation of the immune response against M. canis.

Fungalysin and dipeptidyl-peptidase gene transcription in Microsporum canis strains isolated from symptomatic and asymptomatic cats.

Microsporum canis is the main pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis in domestic carnivores. In cats, M. canis causes symptomatic or asymptomatic infection. Recent conflicting data raise the question of whether the clinical status of the infected cat (symptomatic or asymptomatic) is directly correlated to the proteolytic activity of M. canis strains. Here, the transcription of fungalysin and dipeptidyl-peptidase genes (DPP) of M. canis was compared between four strains isolated from symptomatic and asymptomatic cats during the first steps of the infection process, namely in arthroconidia, during adherence of arthroconidia to corneocytes and during early invasion of the epidermis, using a new ex vivo model made of feline epidermis. There was no detectable transcription of the fungalysin genes in arthroconidia or during the first steps of the infection process for any of the tested strains, suggesting that these proteases play a role later in the infection process. Among DPP, the DPP IV gene was the most frequently transcribed both in arthroconidia and later during infection (adherence and invasion), but no significant differences were observed between M. canis strains isolated from symptomatic and asymptomatic cats. This study shows that the clinical aspect of M. canis feline dermatophytosis depends upon factors relating to the host rather than to the proteolytic activity of the infective fungal strain.

Secreted dipeptidyl peptidases as potential virulence factors for Microsporum canis.

Dermatophytoses caused by Microsporum canis are frequently encountered in cats and dogs; they are highly contagious and readily transmissible to humans. In this study, two single genes, respectively coding for dipeptidyl peptidases IV and V (DppIV and DppV), were isolated and characterized. Both proteins share homology with serine proteases of the S9 family, some of which display properties compatible with implication in pathogenic processes. Both genes are expressed in vivo in experimentally infected guinea-pigs and in naturally infected cats, and when the fungus is grown on extracellular matrix proteins as the sole nitrogen and carbon source. DppIV and V were produced as active recombinant proteases in the yeast Pichia pastoris; the apparent molecular weight of rDppV is 83 kDa, whereas rDppIV appears as a doublet of 95 and 98 kDa. Like other members of its enzymatic subfamily, rDppIV has an unusual ability to cleave Pro-X bonds. This activity does not enhance the solubilization of keratin by fungal secreted endoproteases, and the protease probably acts solely on small soluble peptides. RDppV showed no ability to induce delayed-type hypersensitivity (DTH) skin reactions in guinea-pigs, despite the known immunogenic properties of homologous proteins.

Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes.

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis, mainly in cats and humans. The mechanisms involved in adherence of M. canis to epidermis have never been investigated. Here, a model was developed to study the adherence of M. canis to feline corneocytes through the use of a reconstructed interfollicular feline epidermis (RFE). In this model, adherence of arthroconidia to RFE was found to be time-dependent, starting at 2 h post-inoculation and still increasing at 6 h. Chymostatin, a serine protease inhibitor, inhibited M. canis adherence to RFE by 53%. Moreover, two mAbs against the keratinolytic protease subtilisin 3 (Sub3) inhibited M. canis adherence to RFE by 23%, suggesting that subtilisins, and Sub3 in particular, are involved in the adherence process.

Reconstructed interfollicular feline epidermis as a model for the screening of antifungal drugs against Microsporum canis.

A fully differentiated reconstructed interfollicular feline epidermis (RFE) was recently developed in vitro. It was shown to be relevant for the study of Microsporum canis-epidermal interactions. In this study, RFE was evaluated as a potential model for the in vitro screening of drugs against M. canis. As a preliminary step, the minimum inhibitory concentration of miconazole nitrate against M. canis IHEM 21239 grown on Sabouraud's dextrose agar was determined to be 0.3 microg mL(-1). RFE grown at the air-liquid interface was cultured for 24 h in RFE culture medium, supplemented with either miconazole (range 0.1-1 microg mL(-1)) or its solvent (dimethylsulfoxide). Then, RFE was inoculated in triplicate with 1 x 10(5 )M. canis arthroconidia and incubated for five additional days. To evaluate fungal growth, RFE was processed for routine histopathology, three serial sections being performed across the block at 100 microm intervals. No fungal growth was detected invading or on the surface of infected RFE in the presence of miconazole concentrations equal to or higher than 0.3 microg mL (final concentration in the culture medium). This study demonstrates that RFE is an adequate model for the in vitro screening of drugs against M. canis and potentially against other skin pathogens.

Pathogenesis of dermatophytosis.

Despite the superficial localization of most dermatophytosis, host-fungus relationship in these infections is complex and still poorly elucidated. Though many efforts have been accomplished to characterize secreted dermatophytic proteases at the molecular level, only punctual insights have been afforded into other aspects of the pathogenesis of dermatophytosis, such as fungal adhesion, regulation of gene expression during the infection process, and immunomodulation by fungal factors. However, new genetic tools were recently developed, allowing a more rapid and high-throughput functional investigation of dermatophyte genes and the identification of new putative virulence factors. In addition, sophisticated in vitro infection models are now used and will open the way to a more comprehensive view of the interactions between these fungi and host epidermal cells, especially keratinocytes.

Immunization and dermatophytes.

PURPOSE OF REVIEW: Despite the availability of effective vaccines for certain animal species, vaccination against dermatophytosis requires improvement and further development in both animals and humans. This review provides an update on the current situation and focuses on recent advances in host-dermatophyte relationships that could have implications for future vaccination against the most prevalent of the fungal diseases. RECENT FINDINGS: Numerous dermatophytic virulence factors have recently been isolated and characterized at the molecular level, notably secreted proteases involved in the invasion of the keratin network. Their precise roles in the different steps of the infectious process and in immunopathogenesis are being studied, while all aspects of the host immune response against dermatophytes, including the innate response, are becoming increasingly documented. In addition, new molecular tools are now available for studying dermatophytes, which will accelerate research on this topic. SUMMARY: The growth of knowledge concerning all aspects of the host-dermatophyte relationship should contribute towards sound strategies for the development of effective and safe vaccines against dermatophytosis.

RNA silencing in the dermatophyte Microsporum canis.

Dermatomycoses caused by Microsporum canis are frequent in domestic animals and easily transmissible to humans. Several proteases secreted by this fungus were identified as potential virulence factors, but the construction of deficient strains is required to investigate their role in the pathogenesis of the disease. Using target genes encoding two of these proteases, a first evaluation of the utility of RNA-mediated silencing as a reverse genetic tool in dermatophytes was carried out. SUB3 and DPPIV, respectively coding for a subtilisin and a dipeptidyl peptidase, were both down-regulated, by means of two plasmid constructs designed to express an RNA hairpin that corresponds to part of their respective sequence. The degree of attenuation was evaluated by enzymatic assay of the transformants culture supernatants, and by real-time reverse transcriptase-polymerase chain reaction. Enzymatic activities and expression levels varied from less than 5% to 100% of that of control transformants obtained with plasmid without hairpin inserts. Inhibition was globally more efficient for SUB3 than for DPPIV. These results show that RNA silencing can be used for functional genomics in M. canis, and particularly to circumvent the limits and technical difficulties of conventional disruption methods.

Reconstructed interfollicular feline epidermis as a model for Microsporum canis dermatophytosis.

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis. The complexity of mechanisms involved in dermatophytic infections makes relevant in vivo studies particularly difficult to perform. The aim of this study was to develop a new in vitro model of M. canis dermatophytosis using feline fetal keratinocytes in reconstructed interfollicular epidermis, and to investigate its relevance in studying the host-pathogen relationship. Histological analysis of reconstructed interfollicular feline epidermis (RFE) revealed a fully differentiated epidermis. A proliferation assay showed replicating cells only in the basal layer, indicating that RFE is a well-stratified living tissue, leading to the formation of a horny layer. Histopathological analysis of RFE infected by M. canis arthroconidia revealed that the fungus invades the stratum corneum and produces SUB3, a keratinase implicated in the infectious process. In view of these results, an M. canis dermatophytosis model on RFE seems to be a useful tool to investigate mechanisms involved in natural M. canis feline infections.