Risk‐spreading larviposition behaviour of female nose bot flies (Cephenemyia) attacking black‐tailed deer

While baited deer models were under observation nine Cephenemyia jellisoni Townsend (Diptera: Oestridae) females and seven C. apicata Bennett & Sabrosky engaged in a risk‐spreading larviposition behaviour by larvipositing on models only once and then flying away. Additionally, analysis of 225 unobserved larvipostions in which larvae were trapped in adhesive on the muzzles of deer models showed that 94% of C. apicata and 95% of C. jellisoni larviposited on a model only once. The number of single larvipositions was highly significant for both species. The principal adaptive significance of such risk‐spreading larviposition behaviour is that it spreads the reproductive output of a female among many hosts, and in years when adult eclosion and survival rates are low, it ensures that the larvae of the few surviving females will be distributed among a maximum number of hosts. Several other benefits of such behaviour also are discussed.     

Host Specific Cephenemyia of Deer Repelled by Non-Host Odors

Females of Cephenemyia apicata and C. jellisoni made significantly fewer (P?<?0.05) larvipositions on baited models of black-tailed deer paired with a camouflaged sheep than on similar models operated without a paired sheep. Models were baited with CO₂ and 1-octen-3-ol. In the absence of natural sheep odors these host specific flies were attracted to and larviposited on sheep models baited with CO₂ and octenol and to CO₂-baited odorless models of cows and horses. These results indicate that odors associated with sheep repelled host-seeking Cephenemyia females from baited deer models. Under natural conditions repellent odors of non-host species may assist in maintaining host specificity of these Cephenemyia species and in flies not wasting time and energy responding to non-host odors.     

Larviposition by nasopharyngeal bot fly parasites of Columbian black-tailed deer: a correction

Previous reports of Cephenemyia jellisoni Townsend (Diptera: Oestridae) larvipositing onto the lips/lower muzzle of deer, with larvae invading via the mouth, are shown to be erroneous. Additional studies with deer models baited with CO2, 1-octen-3-ol and Deer Trail Scent, and muzzle and nostrils treated with insect adhesive, revealed that only C. apicata Bennett & Sabrosky larviposited onto the lips/lower muzzle; C. jellisoni, by contrast, larviposited into the nostrils. Larval depositions were associated with females of both species observed attacking models. Females of both species also were found stuck on adhesive-treated, baited models not attended by observers. During several seasons of exposure, such models received 89 C. jellisoni larvipositions into the nostrils and 87 C. apicata larvipositions onto the lips/lower muzzle. In laboratory experiments nearly all larvae of both species remained stuck in adhesive within 1 mm or less of where they were deposited.     

Invasion of black-tailed deer by nose bot fly larvae (Diptera: Oestridae: Oestrinae)

Invasion of black-tailed deer (Odocoileus hemionus columbianus) by larvae of the nose bot flies (Cephenemyia apicata and C. jellisoni) was investigated by obssrving their expulsion by larviparous females and their subsequent activity on the host. The drying uterine fluid encasing larvae ( = larval packet) delays desiccation, ensures adhesion to hairs, and immediately dissolves upon contact with saliva. Contrary to the widely accepted nasal mode of invasion, larvae placed on muzzles of deer crawl ventrally toward the upper lip, enter the mouth, and then crawl caudally along the hard palate or tongue toward the throat. Hair located between the muzzle and nostril prevents larvae from entering the nostrils. A natural per os mode of invasion, heretofore unrecognized, is proposed. This is initiated by: (1) females depositing larval packets on the muzzle of deer, or (2) deer licking larval packets from contaminated areas around the muzzle. The positive thermotropism of larvae is compatible with such a per os mode of entry into the host.     

Morphological Observations and Fatty Acid Composition of Indoor-Cultivated Cordyceps sinensis at a High-Altitude Laboratory on Sejila Mountain,Tibet

Cordyceps sinensis, a caterpillar entomopathogenic fungus-host larva complex, is a rare medicinal herb found in the Qinghai-Tibetan Plateau and its surrounding high-altitude areas. The alternation of generations in the life cycle, whatever the fungus or its host insect, requires special growth conditions. However, it is difficult to simulate the growth conditions of C. sinensis, which hinders its artificial cultivation. In this work, the life cycle from the host larva to C. sinensis was observed in an indoor-cultivation laboratory at 4,200 m a.s.l. on Sejila Mountain, Tibet. Comparative examinations between indoor-cultivated and wild C. sinensis demonstrated that the indoor-cultivated C. sinensis preferred to germinate multiple long, slim stromata at diverse positions on dead larvae, including but not limited to their heads. Their fatty acid composition shows a significant difference in the levels of polyunsaturated fatty acids (PUFAs). In indoor-cultivated C. sinensis, PUFAs constituted 24.59% and 49.43%, respectively, of neutral and polar lipids; meanwhile, in wild C. sinensis, PUFAs represented 34.34% and 61.25% of neutral and polar lipids, respectively. These observations and fatty acid data suggest that environmental factors, particularly temperature, soil pressure and light intensity, strongly affect the growth of C. sinensis. Our new findings may provide important information for improving techniques for the large-scale artificial cultivation of C. sinensis.     

A Murine Model of Candida glabrata Vaginitis Shows No Evidence of an Inflammatory Immunopathogenic Response

Candida glabrata is the second most common organism isolated from women with vulvovaginal candidiasis (VVC), particularly in women with uncontrolled diabetes mellitus. However, mechanisms involved in the pathogenesis of C. glabrata-associated VVC are unknown and have not been studied at any depth in animal models. The objective of this study was to evaluate host responses to infection following efforts to optimize a murine model of C. glabrata VVC. For this, various designs were evaluated for consistent experimental vaginal colonization (i.e., type 1 and type 2 diabetic mice, exogenous estrogen, varying inocula, and co-infection with C. albicans). Upon model optimization, vaginal fungal burden and polymorphonuclear neutrophil (PMN) recruitment were assessed longitudinally over 21 days post-inoculation, together with vaginal concentrations of IL-1β, S100A8 alarmin, lactate dehydrogenase (LDH), and in vivo biofilm formation. Consistent and sustained vaginal colonization with C. glabrata was achieved in estrogenized streptozotocin-induced type 1 diabetic mice. Vaginal PMN infiltration was consistently low, with IL-1β, S100A8, and LDH concentrations similar to uninoculated mice. Biofilm formation was not detected in vivo, and co-infection with C. albicans did not induce synergistic immunopathogenic effects. This data suggests that experimental vaginal colonization of C. glabrata is not associated with an inflammatory immunopathogenic response or biofilm formation.     

Metformin treatment of diverse Caenorhabditis species reveals the importance of genetic background in longevity and healthspan extension outcomes

Metformin, the most commonly prescribed anti‐diabetes medication, has multiple reported health benefits, including lowering the risks of cardiovascular disease and cancer, improving cognitive function with age, extending survival in diabetic patients, and, in several animal models, promoting youthful physiology and lifespan. Due to its longevity and health effects, metformin is now the focus of the first proposed clinical trial of an anti‐aging drug—the Targeting Aging with Metformin (TAME) program. Genetic variation will likely influence outcomes when studying metformin health effects in human populations. To test for metformin impact in diverse genetic backgrounds, we measured lifespan and healthspan effects of metformin treatment in three Caenorhabditis species representing genetic variability greater than that between mice and humans. We show that metformin increases median survival in three C. elegans strains, but not in C. briggsae and C. tropicalis strains. In C. briggsae, metformin either has no impact on survival or decreases lifespan. In C. tropicalis, metformin decreases median survival in a dose‐dependent manner. We show that metformin prolongs the period of youthful vigor in all C. elegans strains and in two C. briggsae strains, but that metformin has a negative impact on the locomotion of C. tropicalis strains. Our data demonstrate that metformin can be a robust promoter of healthy aging across different genetic backgrounds, but that genetic variation can determine whether metformin has positive, neutral, or negative lifespan/healthspan impact. These results underscore the importance of tailoring treatment to individuals when testing for metformin health benefits in diverse human populations.     

A Probably Minor Role for Land-Applied Goat Manure in the Transmission of Coxiella burnetii to Humans in the 2007–2010 Dutch Q Fever Outbreak

In 2007, Q fever started to become a major public health problem in the Netherlands, with small ruminants as most probable source. In order to reduce environmental contamination, control measures for manure were implemented because of the assumption that manure was highly contaminated with Coxiella burnetii. The aims of this study were 1) to clarify the role of C. burnetii contaminated manure from dairy goat farms in the transmission of C. burnetii to humans, 2) to assess the impact of manure storage on temperature profiles in dunghills, and 3) to calculate the decimal reduction time of the Nine Mile RSA 493 reference strain of C. burnetii under experimental conditions in different matrices. For these purposes, records on distribution of manure from case and control herds were mapped and a potential relation to incidences of human Q fever was investigated. Additionally, temperatures in two dunghills were measured and related to heat resistance of C. burnetii. Results of negative binomial regression showed no significant association between the incidence of human Q fever cases and the source of manure. Temperature measurements in the core and shell of dunghills on two farms were above 40°C for at least ten consecutive days which would result in a strong reduction of C. burnetii over time. Our findings indicate that there is no relationship between incidence of human Q fever and land applied manure from dairy goat farms with an abortion wave caused by C. burnetii. Temperature measurements in dunghills on two farms with C. burnetii shedding dairy goat herds further support the very limited role of goat manure as a transmission route during the Dutch human Q fever outbreak. It is very likely that the composting process within a dunghill will result in a clear reduction in the number of viable C. burnetii.     

Detection of Cryptosporidium parvum Oocysts on Fresh Produce Using DNA Aptamers

There are currently no standard methods for the detection of Cryptosporidium spp., or other protozoan parasites, in foods, and existing methods are often inadequate, with low and variable recovery efficiencies. Food testing is difficult due to the low concentrations of parasites, the difficulty in eluting parasites from some foods, the lack of enrichment methods, and the presence of PCR inhibitors. The main objectives of the present study were to obtain DNA aptamers binding to the oocyst wall of C. parvum, and to use the aptamers to detect the presence of this parasite in foods. DNA aptamers were selected against C. parvum oocysts using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Ten rounds of selection led to the discovery of 14 aptamer clones with high affinities for C. parvum oocysts. For detecting parasite-bound aptamers, a simple electrochemical sensor was employed, which used a gold nanoparticle-modified screen-printed carbon electrode. This aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and the anti- C. parvum aptamer. Square wave voltammetry was employed to quantitate C. parvum in the range of 150 to 800 oocysts, with a detection limit of approximately 100 oocysts. The high sensitivity and specificity of the developed aptasensor suggests that this novel method is very promising for the detection and identification of C. parvum oocysts on spiked fresh fruits, as compared to conventional methods such as microscopy and PCR.     

The effects of shell size and coil orientation on reproduction in female hermit crabs, Clibanarius vittatus

We investigated the effects of shell coil orientation and shell size on reproduction in field populations of the hermit crab, Clibanarius vittatus. Females were collected in the intertidal in Beaufort, NC. Shell parameters were measured and size (cephalothorax length) and reproductive status were determined for 70 females occupying Busycon shells. Crabs were categorized as berried (eggs on the pleopods), mature ovaries, or non-reproductive (no eggs). For berried females, the number of eggs was recorded. By offering a separate group of females access to empty shells, it was possible to calculate optimal shell size and the deficit in shell size for field-collected animals.Females that were berried were in shells closer to the optimal shell size than females with mature ovaries, both for shell weight and shell volume. And females with mature ovaries were in shells that were closer to the optimal size than females that were non-reproductive. For both categories of females without eggs on the pleopods, the majority of females were in shells that were too big (in weight and internal volume). While the percentage of berried females did not differ between dextral (Busycon carica) and sinistral (Busycon sinistrum) shells, the non-reproductive females had a much smaller deficit in volume in sinistral shells compared to dextral shells. For berried females, there was no relationship between the magnitude of their shell deficit and the number of eggs carried. Our results suggest that reproduction is inhibited when females occupy shells sufficiently greater than the optimal shell size.     

Selective breeding and characterization of a black mealworm strain of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae)

Larvae of Tenebrio molitor Linnaeus are edible insects and are approved as a food ingredient in Korea. They are typically yellow; however, rare black larvae have been found in breeding boxes at insect farms. It is not clear whether black larvae represent a different species that invaded and hybridized with the yellow larvae of T. molitor or whether T. molitor shows intraspecific color variation. In this study, we characterized and identified black larvae for applications in industrial fields as well as accurate breeding and management. First, in a comparative analysis, we did not detect differences in the morphological characteristics of yellow and black larvae and adults, with the exception of larval body color. For accurate species identification, molecular analyses (p-distances and neighbor-joining) were performed based on partial COI sequences of 33 yellow and seven black larvae. Genetic divergence between yellow and black larvae ranged from 0.0% to 2.1%, revealing intraspecific variation. A neighbor-joining analysis strongly supported the classification of the two morphs as a single species. Black larvae were separated from yellow larvae and maintained by selective breeding. As a result, black larvae were completely fixed in the F2 generation (F1 = 96% and F2 = 100%). Yellow and black larvae showed no significant differences in developmental characteristics and fecundity. These findings improve our understanding of diversity within an important edible insect species and contribute to quality assurance in the food industry based on clear species identification.     

Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans

Streptococcus mutans and Candida albicans are found together in the oral biofilms on dental surfaces, but little is known about the ecological interactions between these species. Here, we studied the effects of S. mutans UA159 on the growth and pathogencity of C. albicans. Initially, the effects of S. mutans on the biofilm formation and morphogenesis of C. albicans were tested in vitro. Next, we investigate the influence of S. mutans on pathogenicity of C. albicans using in vivo host models, in which the experimental candidiasis was induced in G. mellonella larvae and analyzed by survival curves, C. albicans count in hemolymph, and quantification of hyphae in the host tissues. In all the tests, we evaluated the direct effects of S. mutans cells, as well as the indirect effects of the subproducts secreted by this microorganism using a bacterial culture filtrate. The in vitro analysis showed that S. mutans cells favored biofilm formation by C. albicans. However, a reduction in biofilm viable cells and inhibition of hyphal growth was observed when C. albicans was in contact with the S. mutans culture filtrate. In the in vivo study, injection of S. mutans cells or S. mutans culture filtrate into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, a reduction in hyphal formation was observed in larval tissues when C. albicans was associated with S. mutans culture filtrate. These findings suggest that S. mutans can secrete subproducts capable to inhibit the biofilm formation, morphogenesis and pathogenicity of C. albicans, attenuating the experimental candidiasis in G. mellonella model.     

Occupational Exposure to Swine,Poultry, and Cattle and Antibody Biomarkers of Campylobacter jejuni Exposure and Autoimmune Peripheral Neuropathy

Introduction

Foodborne Campylobacter jejuni infection has been associated with an increased risk of autoimmune peripheral neuropathy, but risks of occupational exposure to C. jejuni have received less attention. This study compared anti-C. jejuni IgA, IgG, and IgM antibody levels, as well as the likelihood of testing positive for any of five anti-ganglioside autoantibodies, between animal farmers and non-farmers. Anti-C. jejuni antibody levels were also compared between farmers with different animal herd or flock sizes. The relationship between anti-C. jejuni antibody levels and detection of anti-ganglioside autoantibodies was also assessed.

Methods

Serum samples from 129 Agricultural Health Study swine farmers (some of whom also worked with other animals) and 46 non-farmers, all from Iowa, were analyzed for anti-C. jejuni antibodies and anti-ganglioside autoantibodies using ELISA. Information on animal exposures was assessed using questionnaire data. Anti-C. jejuni antibody levels were compared using Mann-Whitney tests and linear regression on log-transformed outcomes. Fisher’s Exact Tests and logistic regression were used to compare likelihood of positivity for anti-ganglioside autoantibodies.

Results

Farmers had significantly higher levels of anti-C. jejuni IgA (p < 0.0001) and IgG (p = 0.02) antibodies compared to non-farmers. There was no consistent pattern of anti-C. jejuni antibody levels based on animal herd or flock size. A higher percentage of farmers (21%) tested positive for anti-ganglioside autoantibodies compared to non-farmers (9%), but this difference was not statistically significant (p = 0.11). There was no significant association between anti-C. jejuni antibody levels and anti-ganglioside autoantibodies.

Conclusions

The findings provide evidence that farmers who work with animals may be at increased risk of exposure to C. jejuni. Future research should include longitudinal studies of exposures and outcomes, as well as studies of interventions to reduce exposure. Policies to reduce occupational exposure to C. jejuni should be considered.     

Pregnant sows immunized with Cryptosporidium parvum significantly reduced infection in newborn piglets challenged with C. parvum but not with C. hominis

BackgroundThe piglet is the only model to investigate the immunogenic relationship between Cryptosporidium hominis and C. parvum, the species responsible for diarrhea in humans. Despite being indistinguishable antigenically, and high genetic homology between them, they are only moderately cross protective after an active infection.Methodology/Principal findingsHere we examined the degree of passive protection conferred to piglets suckling sows immunized during pregnancy with C. parvum. After birth suckling piglets were challenged orally with either C. parvum or C. hominis at age 5 days. Animals challenged with C. parvum had significant reduction of infection rate, while piglets challenged with C. hominis showed no reduction despite high C. parvum serum and colostrum IgG and IgA antibody.Conclusions/SignificanceWe add these data to earlier studies where we described that infection derived immunity provides partial cross-protection. Together, it appears that for full protection, vaccines against human cryptosporidiosis must contain antigenic elements derived from both species.     

Zebrafish Egg Infection Model for Studying Candida albicans Adhesion Factors

Disseminated candidiasis is associated with 30–40% mortality in severely immunocompromised patients. Among the causal agents, Candida albicans is the dominant one. Various animal models have been developed for investigating gene functions in C. albicans. Zebrafish injection models have increasingly been applied in elucidating C. albicans pathogenesis because of the conserved immunity, prolific fecundity of the zebrafish and the low costs of care systems. In this study, we established a simple, noninvasive zebrafish egg bath infection model, defined its optimal conditions, and evaluated the model with various C. albicans mutant strains. The deletion of SAP6 did not have significant effect on the virulence. By contrast, the deletion of BCR1, CPH1, EFG1, or TEC1 significantly reduced the virulence under current conditions. Furthermore, all embryos survived when co-incubated with bcr1/bcr1, cph1/cph1 efg1/efg1, efg1/efg1, or tec1/tec1 mutant cells. The results indicated that our novel zebrafish model is time-saving and cost effective.     

Hormonal asynchrony and embryonic development

Luteinizing hormone (LH), progesterone and estradiol profiles in peripheral blood serum were compared among parous and nonparous females with normal, abnormal or no embryonic development. Hormonal profiles between parous and nonparous females of the same embryonic status did not differ and the data were combined. Estrous cycle length was longer (P<.05) in parous (22.3±.4 days) than nonparous females (21.0±.4 days). Females with normal developing embryos had a higher serum progesterone concentration at Days 3 and 6 and a lower ratio of estradiol to progesterone than did females with abnormal embryonic development. Females with a normal embryo had higher (P<.05) preovulatory LH peaks than females with abnormal development or no recovery of an oocyte or embryo (34.3±4.7, 11.8±6.8 and 13.3±2.5 ng/ml, respectively). The interval from onset of estrus to LH peak was 8.9±2.1, 13.7±3.7 and 13.5±6.2 hr for females with normal, abnormal or no recovery of an embryo. The lower LH peak, the longer interval from onset of estrus to LH peak, and lower progesterone concentration in peripheral blood serum in females with abnormal embryos or no recovery indicated that these females had a hormonal asynchrony. The hormonal asynchrony may produce an undesirable uterine environment for male and female gametes or embryos which resulted in fertilization failure or embryonic death. In the second experiment, more transferable embryos were obtained when superovulated females received prostaglandin F_2α (PGF_2α) intravenously rather than intramuscularly. Administering PGF₂α intravenously rather than intramuscularly may have caused the demise of the corpus luteum sooner and thereby produced a more normal uterine environment which allowed more embryos to develop normally.     

Candida glabrata Binding to Candida albicans Hyphae Enables Its Development in Oropharyngeal Candidiasis

Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata.     

The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans

Several families of crystal proteins from Bacillus thuringiensis exhibit nematicidal activity. Cry5B protein, a pore-forming toxin, has been intensively studied yielding many insights into the mode of action of crystal protein at molecular level and pathogenesis of pore-forming toxins. However, little attention was paid to Cry6A, another representative nematicidal crystal protein. Cry6A shares very low homology with Cry5B at amino acid sequence and probably acts in a distinct pathway from Cry5B and even the other main commercial crystal proteins. In the current study, we comprehensively investigated the nematicidal properties of Cry6Aa2 against the free-living soil nematode Caenorhabditis elegans and examined the physical response of C. elegans to Cry6Aa2 attack. Our results indicate that Cry6Aa2 exhibits high lethal activity to C. elegans and could cause detrimental effects on C. elegans, including obviously suppressed growth, decreased brood size, and even abnormal motility. Meanwhile, our study additionally shows that C. elegans could defend against the Cry6Aa2 toxin harmful threat through behavioral defense responses, such as reduced oral uptake and physical avoidance. In general, this study suggests that Cry6Aa2 possesses diverse nematicidal properties, which strongly indicates that Cry6Aa2 is a promising potential candidate of nematicidal agent. Moreover, this study highlights the importance of behavioral responses in defense of C. elegans for survival and demonstrates the key role of crystal protein in the interaction of B. thuringiensis–C. elegans. These findings could shed light on understanding the interaction of C. elegans with B. thuringiensis and provide a perfect model to study the role of pathogenic factor in the interaction of pathogen–host.     

Glucosylceramide Administration as a Vaccination Strategy in Mouse Models of Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen and the causative agent of the disease cryptococcosis. Cryptococcosis is initiated as a pulmonary infection and in conditions of immune deficiency disseminates to the blood stream and central nervous system, resulting in life-threatening meningoencephalitis. A number of studies have focused on the development of a vaccine against Cryptococcus, primarily utilizing protein-conjugated components of the Cryptococcus polysaccharide capsule as antigen. However, there is currently no vaccine against Cryptococcus in the clinic. Previous studies have shown that the glycosphingolipid, glucosylceramide (GlcCer), is a virulence factor in C. neoformans and antibodies against this lipid inhibit fungal growth and cell division. In the present study, we have investigated the possibility of using GlcCer as a therapeutic agent against C. neoformans infections in mouse models of cryptococcosis. GlcCer purified from a non-pathogenic fungus, Candida utilis, was administered intraperitoneally, prior to infecting mice with a lethal dose of C. neoformans. GlcCer administration prevented the dissemination of C. neoformans from the lungs to the brain and led to 60% mouse survival. GlcCer administration did not cause hepatic injury and elicited an anti-GlcCer antibody response, which was observed independent of the route of administration and the strains of mouse. Taken together, our results suggest that fungal GlcCer can protect mice against lethal doses of C. neoformans infection and can provide a viable vaccination strategy against Cryptococcus.