Emerging Infectious Diseases of Chelonians: An Update.

Chelonians are increasingly challenged by anthropogenic threats and disease. This article summarizes recent literature and clinical experiences regarding 4 emerging infectious diseases in turtles and tortoises: ranaviruses, cryptosporidiosis, intranuclear coccodiosis of Testudines, and Emydomyces testavorans.

Transcriptional responses in honey bee larvae infected with chalkbrood fungus.

BACKGROUND: Diseases and other stress factors working synergistically weaken honey bee health and may play a major role in the losses of bee populations in recent years. Among a large number of bee diseases, chalkbrood has been on the rise. We present here the experimental identification of honey bee genes that are differentially expressed in response to infection of honey bee larvae with the chalkbrood fungus, Ascosphaera apis. RESULTS: We used cDNA-AFLP Technology to profile transcripts in infected and uninfected bee larvae. From 64 primer combinations, over 7,400 transcriptionally-derived fragments were obtained A total of 98 reproducible polymorphic cDNA-AFLP fragments were excised and sequenced, followed by quantitative real-time RT-PCR (qRT-PCR) analysis of these and additional samples.We have identified a number of differentially-regulated transcripts that are implicated in general mechanisms of stress adaptation, including energy metabolism and protein transport. One of the most interesting differentially-regulated transcripts is for a chitinase-like enzyme that may be linked to anti-fungal activities in the honey bee larvae, similarly to gut and fat-body specific chitinases found in mosquitoes and the red flour beetle. Surprisingly, we did not find many components of the well-characterized NF-kappaB intracellular signaling pathways to be differentially-regulated using the cDNA-AFLP approach. Therefore, utilizing qRT-PCR, we probed some of the immune related genes to determine whether the lack of up-regulation of their transcripts in our analysis can be attributed to lack of immune activation or to limitations of the cDNA-AFLP approach. CONCLUSIONS: Using a combination of cDNA-AFLP and qRT-PCR analyses, we were able to determine several key transcriptional events that constitute the overall effort in the honey bee larvae to fight natural fungal infection. Honey bee transcripts identified in this study are involved in critical functions related to transcriptional regulation, apoptotic degradation of ubiquitinated proteins, nutritional regulation, and RNA processing. We found that immune regulation of the anti-fungal responses in honey bee involves highly coordinated activation of both NF-kappaB signaling pathways, leading to production of anti-microbial peptides. Significantly, activation of immune responses in the infected bee larvae was associated with down-regulation of major storage proteins, leading to depletion of nutritional resources.

Long-term storage of Ascosphaera aggregata and Ascosphaera apis, pathogens of the leafcutting bee (Megachile rotundata) and the honey bee (Apis mellifera).

Survival rates of Ascosphaera aggregata and Ascosphaera apis over the course of a year were tested using different storage treatments. For spores, the storage methods tested were freeze-drying and ultra-low temperatures, and for hyphae, freeze-drying, agar slants, and two methods of ultra-low temperatures. Spores of A. aggregata and A. apis stored well at -80 degrees C and after freeze-drying. A. aggregata hyphae did not store well under any of the methods tested while A. apis hyphae survived well using cryopreservation. Spores produced from cryopreserved A. apis hyphae were infective. Long-term storage of these two important fungal bee diseases is thus possible.

Morphology and development of the reticuloperidial ascomata of Auxarthron conjugatum.

Light and electron microscopy showed that the reticuloperidium of thick-walled hyphae, characteristic of the mature ascoma of Auxarthron conjugaturn, originated from branches that grew from the broad, gyre-like hyphal loops making up the ascomatal initials. Within the developing peridium, short, acropetally proliferating chains of prototunicate asci each arose from a single crozier and matured from base to tip. The walls of young asci were two-layered but evanesced as they matured with the outer layer dissolving before the inner one. Distal asci in some chains retained the inner wall, detached from adjacent asci by septum schizolysis and when transferred to fresh media produced germ tubes and mycelium. Ultraviolet epifluorescent staining with a DNA intercalator (Hoechst) indicated that these spore-like asci probably contained diploid nuclei. In normal asci, ascospores had an inner, electron lucent primary wall and a three-layered secondary wall. The deposition pattern of the middle layer of the secondary wall created the distinctive array of pits and ridges characteristic of the ascospores in this taxon. The production of ascospores, spore-like asci and arthroconidia, along with the tendency of ascospores to adhere in a mass, is interpreted as contributing to the reproductive flexibility and inoculum potential of A. conjugatum. In all respects the ascomata of A. conjugatum differed substantially from the morphologically similar taxon, Myxotrichum arcticum. These findings underscore the benefit of using DNA-based phylogenies in concert with cytological and ultrastructural observations for exploring selective pressures behind homoplasious characters and revealing novel structural features.

Evaluation of the vital staining method for Lacazia loboi through the experimental inoculation of BALB/c mice.

The viability of the currently unculturable fungal pathogen Lacazia loboi can be determined by means of fluorescein diacetate-ethidium bromide (FD-EB) staining. This technique can be used in experimental study of the mycosis, in attempts to cultivate the fungus and in attempts to gauge the success of therapies. In the present study, the potential applications of FD-EB vital staining were studied using a proposed murine experimental model of lobomycosis. BALB/c mice were inoculated in the footpads with an L. loboi suspension that appeared in FD-EB staining to have lost viability after being held for 15 days at room temperature, whereas a control group of mice was inoculated with apparently viable fungi. The animals were killed after 1, 2, 4, 6, 8, 11 and 13 months. Both inoculated footpads were excised, one for determination of viability and the other for histological examination. In the group injected with nonviable material, no active infection was noted; inoculation sites showed small quantities of macrophage-laden infiltrate and no viable fungal cells. In the control group, the infection progressed with exuberant infiltrates surrounding copious fungal growth, most of which consisted of cells staining as viable in FD-EB. These results suggest that the FD-EB vital staining is a sensitive and specific method that can reliably be used for viability determination in L. loboi.

Evaluation of the vital staining method for Lacazia loboi through the experimental inoculation of BALB/c mice

The viability of the currently unculturable fungal pathogen Lacazia loboi can be determined by means of fluorescein diacetate-ethidium bromide (FD-EB) staining. This technique can be used in experimental study of the mycosis, in attempts to cultivate the fungus and in attempts to gauge the success of therapies. In the present study, the potential applications of FD-EB vital staining were studied using a proposed murine experimental model of lobomycosis. BALB/c mice were inoculated in the footpads with an L. loboi suspension that appeared in FD-EB staining to have lost viability after being held for 15 days at room temperature, whereas a control group of mice was inoculated with apparently viable fungi. The animals were killed after 1, 2, 4, 6, 8, 11 and 13 months. Both inoculated footpads were excised, one for determination of viability and the other for histological examination. In the group injected with nonviable material, no active infection was noted; inoculation sites showed small quantities of macrophage-laden infiltrate and no viable fungal cells. In the control group, the infection progressed with exuberant infiltrates surrounding copious fungal growth, most of which consisted of cells staining as viable in FD-EB. These results suggest that the FD-EB vital staining is a sensitive and specific method that can reliably be used for viability determination in L. loboi

Arachnomyces kanei (anamorph Onychocola kanei) sp. nov., from human nails.

Five isolates of a slow-growing cycloheximide resistant hyphomycetous fungus were obtained from nail specimens and investigated for their relationship to Onychocola canadensis (teleomorph Arachnomyces nodosetosus), a known agent of onychomycosis. In one patient diagnosed with superficial white onychomycosis, etiology was confirmed by a nail sample showing atypical filaments in direct microscopy, and by a follow-up specimen yielding cultures of the same fungus. A case of mixed infection with Aspergillus sydowii was also confirmed after examination of cultures grown from three successive microscopic-positive hallux nail specimens. For other isolates, etiological significance could not be confirmed by repeat sampling or results of direct microscopy were negative or unknown. Mating experiments yielded setose ascomata containing smooth oblate ascospores typical of Arachnomyces species. Phylogenetic analysis of ITS 2 region sequences support the conspecificity of the isolates and their placement within the genus. A. kanei sp. nov. (anamorph O. kanei sp. nov.) is described.

[Effects of temperature, relative humidity and pH on germination of chalkbrood fungus, Ascosphaera apis spore].

Studies on the effects of environmental temperature, relative humidity and pH-value on the germination of Ascosphaera apis spore at the stages of activation, enlargement and germ-tube production showed that the germination was found to be independent of temperature within the range of 15-40 degrees C was and 25-40 degrees C, respectively at the stage of activation and enlargement, but closely correlated with the temperature within the range of 25-37 degrees C at the stage of germ-tube production, with the optimum range of 31-35 degrees C. Relative humidity below 80% inhibited spore germination. pH value within the range of 5-7.8 did not affect the spore germination significantly, but pH < 5 reduced the enlargement and germ-tube production drastically. The results indicated that A. apis is a highly specialized pathogen for the life in honeybee larvae.