Food provisioning alters infection dynamics in populations of a wild rodent

While pathogens are often assumed to limit the growth of wildlife populations, experimental evidence for their effects is rare. A lack of food resources has been suggested to enhance the negative effects of pathogen infection on host populations, but this theory has received little investigation. We conducted a replicated two-factor enclosure experiment, with introduction of the bacterium Bordetella bronchiseptica and food supplementation, to evaluate the individual and interactive effects of pathogen infection and food availability on vole populations during a boreal winter. We show that prior to bacteria introduction, vole populations were limited by food availability. Bordetella bronchiseptica introduction then reduced population growth and abundance, but contrary to predictions, primarily in food supplemented populations. Infection prevalence and pathological changes in vole lungs were most common in food supplemented populations, and are likely to have resulted from increased congregation and bacteria transmission around feeding stations. Bordetella bronchiseptica-infected lungs often showed protozoan co-infection (consistent with Hepatozoon erhardovae), together with more severe inflammatory changes. Using a multidisciplinary approach, this study demonstrates a complex picture of interactions and underlying mechanisms, leading to population-level effects. Our results highlight the potential for food provisioning to markedly influence disease processes in wildlife mammal populations.     

A rapid method for capture and identification of immunogenic proteins in Bordetella pertussis enriched membranes fractions: a fast-track strategy applicable to other microorganisms

Mass spectrometry (MS) coupled with 1-D and 2-D electrophoresis can be utilized to detect and identify immunogenic proteins, but these methods are laborious and time-consuming. We describe an alternative, simple, rapid gel-free strategy to identify multiple immunogenic proteins from Bordetella pertussis (Bp). It couples immunoprecipitation to nano liquid chromatography- tandem mass spectrometry (IP-nLC-MS/MS) and is significantly both time- and labor-saving. We developed a gel-free magnetic bead-based immunoprecipitation (IP) method using different NP-40/PBS concentrations in which solubilized proteins of Bp Tohama I membrane fractions were precipitated with polyclonal rabbit anti-Bp whole cell immune sera. Immune complexes were analyzed by MS and Scaffold analysis (> 95% protein identification probability). Total immunoproteins identified were 50, 63 and 49 for 0.90%, 0.45% and 0.22% NP-40/PBS buffer concentrations respectively. Known Bp proteins identified included pertactin, serotype 2 fimbrial subunit and filamentous hemagglutinin. As proof of concept that this gel-free protein immunoprecipitation method enabled the capture of multiple immunogenic proteins, IP samples were also analyzed by SDS-PAGE and immunoblotting. Bypassing gels and subjecting immunoprecipitated proteins directly to MS is a simple and rapid antigen identification method with relatively high throughput. IP-nLC-MS/MS provides a novel alternative approach for current methods used for the identification of immunogenic proteins.     

Oligomeric behavior of Bordetella pertussis adenylate cyclase toxin in solution

Adenylate cyclase (AC) toxin from Bordetella pertussis inserts into eukaryotic cells, producing intracellular cAMP, as well as hemolysis and cytotoxicity. Concentration dependence of hemolysis suggests oligomers as the functional unit and inactive deletion mutants permit partial restoration of intoxication and/or hemolysis, when added in pairs [M. Iwaki, A. Ullmann, P. Sebo, Mol. Microbiol. 17 (1995) 1015-1024], suggesting dimerization/oligomerization. Using affinity co-precipitation and fluorescence resonance energy transfer (FRET), we demonstrate specific self-association of AC toxin molecules in solution. Flag-tagged AC toxin mixed with biotinylated-AC toxin, followed by streptavidin beads, yields both forms of the toxin. FRET measurements of toxin, labeled with different fluorophores, demonstrate association in solution, requiring post-translational acylation, but not calcium. AC toxin mixed with DeltaR, an inactive mutant, results in enhancement of hemolysis over that with wild type alone, suggesting that oligomers are functional. Dimers and perhaps higher molecular mass forms of AC toxin occur in solution in a manner that is relevant to toxin action.     

Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin

The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.     

Immunosuppressive effect of Cryptosporidium baileyi infection on vaccination against avian infectious bronchitis in chicks

Two-day-old commercial chicks were inoculated orally with 2 × 10⁶ oocysts of Cryptosporidium baileyi and vaccinated with 10^3.5 EID₅₀/head of a commercially available avian infectious bronchitis (IB) live virus vaccine at 4 and 14 days following inoculation. Chicks infected with C. baileyi were shown to have an immunosuppressive effect on IB virus. It is concluded that infection with the protozoon in early life may increase their susceptibility to IB.     

The effect of ecological and temporal factors on the composition of Bartonella infection in rodents and their fleas

The composition of Bartonella infection was explored in wild Gerbillus andersoni rodents and their Synosternus cleopatrae fleas. Rodent blood samples and fleas were collected in two periods (two different seasons; 4 months apart) from juveniles and adult hosts, and their bartonellae lineages were identified by a 454-pyrosequencing analysis targeting a specific Bartonella citrate synthase gene (gltA) fragment. The rate of Bartonella spp. co-infection was estimated and the assemblage and distribution of bartonellae lineages across the samples with respect to ecological and phylogenetic distance similarities were analyzed. Moreover, environmental factors that could explain potential differences between samples were investigated. Out of the 91 bartonellae-positive samples, 89% were found to be co-infected with more than two phylogenetically distant Bartonella genotypes and additional closely related (but distinguishable) variants. These bartonellae lineages were distributed in a non-random manner, and a negative interaction between lineages was discovered. Interestingly, the overall composition of those infections greatly varied among samples. This variability was partially explained by factors, such as type of sample (blood versus fleas), flea sex and period of collection. This investigation sheds light on the patterns of Bartonella infection and the organization of Bartonella lineages in fleas and rodents in nature.     

Validation of a rodent model of source memory

Source memory represents the origin (source) of information. Recently, we proposed that rats (Rattus norvegicus) remember the source of information. However, an alternative to source memory is the possibility that rats selectively encoded some, but not all, information rather than retrieving an episodic memory. We directly tested this ‘encoding failure’ hypothesis. Here, we show that rats remember the source of information, under conditions that cannot be attributed to encoding failure. Moreover, source memory lasted at least seven days but was no longer present 14 days after studying. Our findings suggest that long-lasting source memory may be modelled in non-humans. Our model should facilitate attempts to elucidate the biological underpinnings of source memory impairments in human memory disorders such as Alzheimer''s disease.     

Polymorphisms at the innate immune receptor TLR2 are associated with Borrelia infection in a wild rodent population

The discovery of the key role of Toll-like receptors (TLRs) in initiating innate immune responses and modulating adaptive immunity has revolutionized our understanding of vertebrate defence against pathogens. Yet, despite their central role in pathogen recognition and defence initiation, there is little information on how variation in TLRs influences disease susceptibility in natural populations. Here, we assessed the extent of naturally occurring polymorphisms at TLR2 in wild bank voles (Myodes glareolus) and tested for associations between TLR2 variants and infection with Borrelia afzelii, a common tick-transmitted pathogen in rodents and one of the causative agents of human Lyme disease. Bank voles in our population had 15 different TLR2 haplotypes (10 different haplotypes at the amino acid level), which grouped in three well-separated clusters. In a large-scale capture–mark–recapture study, we show that voles carrying TLR2 haplotypes of one particular cluster (TLR2_c2) were almost three times less likely to be Borrelia infected than animals carrying other haplotypes. Moreover, neutrality tests suggested that TLR2 has been under positive selection. This is, to our knowledge, the first demonstration of an association between TLR polymorphism and parasitism in wildlife, and a striking example that genetic variation at innate immune receptors can have a large impact on host resistance.     

Progesterone induces mucosal immunity in a rodent model of human taeniosis by Taenia solium

More than one quarter of human world's population is exposed to intestinal helminth parasites. The Taenia solium tapeworm carrier is the main risk factor in the transmission of both human neurocysticercosis and porcine cysticercosis. Sex steroids play an important role during T. solium infection, particularly progesterone has been proposed as a key immunomodulatory hormone involved in susceptibility to human taeniosis in woman and cysticercosis in pregnant pigs. Thus, we evaluated the effect of progesterone administration upon the experimental taeniosis in golden hamsters (Mesocricetus auratus). Intact female adult hamsters were randomly divided into 3 groups: progesterone-subcutaneously treated; olive oil-treated as the vehicle group; and untreated controls. Animals were treated every other day during 4 weeks. After 2 weeks of treatment, all hamsters were orally infected with 4 viable T. solium cysticerci. After 2 weeks post infection, progesterone-treated hamsters showed reduction in adult worm recovery by 80%, compared to both vehicle-treated and non-manipulated infected animals. In contrast to control and vehicle groups, progesterone treatment diminished tapeworm length by 75% and increased proliferation rate of leukocytes from spleen and mesenteric lymph nodes of infected hamsters by 5-fold. The latter exhibited high expression levels of IL-4, IL-6 and TNF-α at the duodenal mucosa, accompanied with polymorphonuclear leukocytes infiltration. These results support that progesterone protects hamsters from the T. solium adult tapeworm establishment by improving the intestinal mucosal immunity, suggesting a potential use of analogues of this hormone as novel inductors of the gut immune response against intestinal helminth infections and probably other bowel-related disorders.     

A genetic component of resistance to fungal infection in frog embryos

The embryo has traditionally been considered to completely rely upon parental strategies to prevent threats to survival posed by predators and pathogens, such as fungi. However, recent evidence suggests that embryos may have hitherto neglected abilities to counter pathogens. Using artificial fertilization, we show that among-family variation in the number of Saprolegnia-infected eggs and embryos in the moor frog, Rana arvalis, cannot be explained by maternal effects. However, analysed as a within-females effect, sire identity had an effect on the degree of infection. Furthermore, relatively more eggs and embryos were infected when eggs were fertilized by sperm from the same, compared with a different, population. These effects were independent of variation in fertilization success. Thus, there is likely to be a significant genetic component in embryonic resistance to fungal infection in frog embryos. Early developmental stages may show more diverse defences against pathogens than has previously been acknowledged.     

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.     

The first human case of Trichinella spiralis infection in Korea

Three cases of human infection by Trichinella spiralis were first confirmed by detecting encysted larvae in the biopsied muscle in December 1997, in Korea. The patients were one 35- and two 39-year-old males residing in Kochang-gun, Kyongsangnam-do. They had a common past history of eating raw liver, spleen, blood and muscle of a badger, Meles meles melanogenys, and complained of high fever, facial and periorbital edema, and myalgia. Hematologic and biochemical examinations revealed leukocytosis and eosinophilia, and highly elevated levels of GOT, GPT, LDH and CPK. In the gastrocnemius muscle of a patient, roundly coiled nematode larvae were detected. The larvae measured 0.775-1.050 (av. 0.908) mm in length, and 0.026-0.042 (av. 0.035) mm in maximum width. The specific IgG antibody levels in three patients' sera were significantly higher when compared with those of normal controls. The patients were treated with flubendazole and albendazole for 15-30 days, and discharged at 13-34 days post-admission. From the above findings, it was confirmed that T. spiralis is present in Korea, and the badger plays a role of as the natural host.     

The impact of specific and non-specific immunity on the ecology of Streptococcus pneumoniae and the implications for vaccination

More than 90 capsular serotypes of Streptococcus pneumoniae coexist despite competing for nasopharyngeal carriage and a gradient in fitness. The underlying mechanisms for this are poorly understood and make assessment of the likely population impact of vaccination challenging. We use an individual-based simulation model to generalize widely used deterministic models for pneumococcal competition and show that in these models short-term serotype-specific and serotype non-specific immunity could constitute the mechanism governing between-host competition and coexistence. We find that non-specific immunity induces between-host competition and that serotype-specific immunity limits a type''s competitive advantage and allows stable coexistence of multiple serotypes. Serotypes carried at low prevalence show high variance in carriage levels, which would result in apparent outbreaks if they were highly pathogenic. Vaccination against few serotypes can lead to elimination of the vaccine types and induces replacement by others. However, in simulations where the elimination of the targeted types is achieved only by a combination of vaccine effects and the competitive pressure of the non-vaccine types, a universal vaccine with similar-type-specific effectiveness can fail to eliminate pneumococcal carriage and offers limited herd immunity. Hence, if vaccine effects are insufficient to control the majority of serotypes at the same time, then exploiting the competitive pressure by selective vaccination can help control the most pathogenic serotypes.     

Introgressive hybridization facilitates adaptive divergence in a recent radiation of monkeyflowers

A primary goal in evolutionary biology is to identify the historical events that have facilitated the origin and spread of adaptations. When these adaptations also lead to reproductive isolation, we can learn about the evolutionary mechanisms contributing to speciation. We reveal the complex history of the gene MaMyb2 in shaping flower colour divergence within a recent radiation of monkeyflowers. In the Mimulus aurantiacus species complex, red-flowered M. a. ssp. puniceus and yellow-flowered M. a. ssp. australis are partially isolated because of differences in pollinator preferences. Phylogenetic analyses based on genome-wide variation across the complex suggest two origins of red flowers from a yellow-flowered ancestor: one in M. a. ssp. puniceus and one in M. a. ssp. flemingii. However, in both cases, red flowers are caused by cis-regulatory mutations in the gene MaMyb2. Although this could be due to distinct mutations in each lineage, we show that the red allele in M. a. ssp. puniceus did not evolve de novo or exist as standing variation in its yellow-flowered ancestor. Rather, our results suggest that a single red MaMyb2 allele evolved during the radiation of M. aurantiacus that was subsequently transferred to the yellow-flowered ancestor of M. a. ssp. puniceus via introgressive hybridization. Because gene flow is still possible among taxa, we conclude that introgressive hybridization can be a potent driver of adaptation at the early stages of divergence that can contribute to the origins of biodiversity.     

Karyology of the Atlantic forest rodent Juliomys (Cricetidae): A new karyotype from southern Brazil

Juliomys is a small rodent from the family Cricetidae which inhabits the Atlantic forest and forests from Argentina to eastern Brazil. The three species recognized so far have different karyotypes. In this paper, we describe a new karyotype with 2n = 32, FN = 48 found in Juliomys specimens from a high-altitude area in the Atlantic forest of southern Brazil. The karyotype was analyzed after G- and C-banding and silver staining of the nucleolus organizer regions (Ag-NOR) and its G-banding patterns were compared with those of the newly described species Juliomys ossitenuis (2n = 20, FN = 36). The 2n = 32 karyomorph presented peculiar features and was very different from those of the other species of the genus: J. pictipes (2n = 36, FN = 34), J. rimofrons (2n = 20, FN = 34) and J. ossitenuis (2n = 20, FN = 36). Differences were mostly due to centric and tandem fusions, pericentric inversion and loss of heterochromatin. The karyotype represents a powerful tool to differentiate Juliomys species and our data suggest that the karyotype described herein belongs to a new species.     

Toxoplasmosis seroprevalence in urban rodents: a survey in Niamey,Niger

A serological survey of Toxoplasma gondii was conducted on 766 domestic and peridomestic rodents from 46 trapping sites throughout the city of Niamey, Niger. A low seroprevalence was found over the whole town with only 1.96% of the rodents found seropositive. However, differences between species were important, ranging from less than 2% in truly commensal Mastomys natalensis, Rattus rattus and Mus musculus, while garden-associated Arvicanthis niloticus displayed 9.1% of seropositive individuals. This is in line with previous studies on tropical rodents - that we reviewed here - which altogether show that Toxoplasma seroprevalence in rodent is highly variable, depending on many factors such as locality and/or species. Moreover, although we were not able to decipher statistically between habitat or species effect, such a contrast between Nile grass rats and the other rodent species points towards a potentially important role of environmental toxoplasmic infection. This would deserve to be further scrutinised since intra-city irrigated cultures are extending in Niamey, thus potentially increasing Toxoplasma circulation in this yet semi-arid region. As far as we are aware of, our study is one of the rare surveys of its kind performed in Sub-Saharan Africa and the first one ever conducted in the Sahel.     

Monitoring long-term evolutionary changes following Wolbachia introduction into a novel host: the Wolbachia popcorn infection in Drosophila simulans

Wolbachia may act as a biological control agent for pest management; in particular, the Wolbachia variant wMelPop (popcorn) shortens host longevity and may be useful for dengue suppression. However, long-term changes in the host and Wolbachia genomes can alter Wolbachia spread and/or host effects that suppress disease. Here, we investigate the phenotypic effects of wMelPop in a non-native host, Drosophila simulans, following artificial transinfection approximately 200 generations ago. Long-term rearing and maintenance of the bacteria were at 19°C in the original I-102 genetic background that was transinfected with the popcorn strain. The bacteria were then introgressed into three massbred backgrounds, and tetracycline was used to create uninfected sublines. The effect of wMelPop on longevity in this species appears to have changed; longevity was no longer reduced at 25°C in some nuclear backgrounds, reflecting different geographical origin, selection or drift, although the reduction was still evident for flies held at 30°C. Wolbachia influenced productivity and viability, and development time in some host backgrounds. These findings suggest that long-term attenuation of Wolbachia effects may compromise the effectiveness of this bacterium in pest control. They also emphasize the importance of host nuclear background on Wolbachia phenotypic effects.     

Evidence for a pathway that facilitates nitric oxide diffusion in the brain

Nitric oxide (NO) is a diffusible messenger that conveys information based on its concentration dynamics, which is dictated by the interplay between its synthesis, inactivation and diffusion. Here, we characterized NO diffusion in the rat brain in vivo. By direct sub-second measurement of NO, we determined the diffusion coefficient of NO in the rat brain cortex. The value of 2.2 × 10⁻⁵ cm²/s obtained in vivo was only 14% lower than that obtained in agarose gel (used to evaluate NO free diffusion). These results reinforce the view of NO as a fast diffusing messenger but, noticeably, the data indicates that neither NO diffusion through the brain extracellular space nor homogeneous diffusion in the tissue through brain cells can account for the similarity between NO free diffusion coefficient and that obtained in the brain. Overall, the results support that NO diffusion in brain tissue is heterogeneous, pointing to the existence of a pathway that facilitates NO diffusion, such as cell membranes and other hydrophobic structures.     

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites into host cells. Separate groups of gerbils were immunized with the purified recombinant proteins singly or in combinations and animals were then challenged with N. caninum. Following these experimental challenges, the protective efficacy of each vaccination was determined by assessing animal survival rate. All experimental groups showed protective effects of different degrees against experimental infection. The highest protection efficacy was observed for combined vaccination with NcSRS2 and NcDG1. Our results indicate that combined vaccination with the N. caninum recombinant antigens, NcSRS2 and NcDG1, induces the highest protective effect against N. caninum infection in vitro and in vivo.     

Drosophila as a model system to unravel the layers of innate immunity to infection

Innate immunity relies entirely upon germ-line encoded receptors, signalling components and effector molecules for the recognition and elimination of invading pathogens. The fruit fly Drosophila melanogaster with its powerful collection of genetic and genomic tools has been the model of choice to develop ideas about innate immunity and host-pathogen interactions. Here, we review current research in the field, encompassing all layers of defence from the role of the microbiota to systemic immune activation, and attempt to speculate on future directions and open questions.