Propagation Of Pathogens Research Articles

Different antiviral effects of IFNα and IFNβ in an HBV mouse model

Interferons α and β (IFNα and IFNβ) are type I interferons produced by the host to control pathogen propagation. However, only a minority of chronic hepatitis B (CHB) patients generate a sustained response after treatment with recombinant IFNα. The anti-HBV effect of IFNβ and the underlying mechanism are not well-understood. Here, we compared the antiviral activities of IFNα and IFNβ by application of IFNα or IFNβ expression plasmids using the well-established HBV hydrodynamic injection (HI) mouse model. Injection of IFNα expression plasmid could significantly reduce HBV serum markers including HBsAg, HBeAg and HBV DNA as well as the number of HBcAg positive cells in the liver, while IFNβ showed only a weak inhibition of HBV replication. In contrast to IFNβ, IFNα resulted in elevated expression levels of IFN stimulated genes (ISGs) as well as the proinflammatory cytokine interleukin 6 (IL6) in the liver. Moreover, IFNβ treated mice showed higher expression levels of the anti-inflammatory cytokines IL10 and TGFβ in the liver compared to IFNα. Our results demonstrated that both IFNα and IFNβ exert antiviral activities against HBV in HI mouse model, but IFNα is more effective than IFNβ.

SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1.

ABSTRACTAspects of intrinsic antiviral immunity are mediated by promyelocytic leukemia nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase protein inhibitor of activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation. IMPORTANCE Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against infection. PML-NB constituent proteins mediate aspects of intrinsic immunity to restrict herpes simplex virus 1 (HSV-1) as well as other viruses. These proteins repress viral replication through mechanisms that rely on SUMO signaling. However, the participating SUMOylation enzymes are not known. We identify the SUMO ligase PIAS1 as a constituent PML-NB antiviral protein. This finding distinguishes a SUMO ligase that may mediate signaling events important in PML-NB-mediated intrinsic immunity. Moreover, this research complements the recent identification of PIAS4 as an intrinsic antiviral factor, supporting a role for PIAS proteins as both positive and negative regulators of host immunity to virus infection.

Ecological study on antimicrobial-resistant zoonotic bacteria transmitted by flies in cattle farms.

Flies were qualitatively and quantitatively monitored on both livestock animals and the surrounding environment to investigate their role as a potential carrier for antimicrobial-resistant bacteria of zoonotic importance in cattle farms. This was done by the use of visual observations and animal photography; meanwhile, in the surrounding environment, flies were collected using sticky cards and then microscopically identified. Representative fly samples were cultured for bacterial isolation, biochemical identification, and then tested against common 12 antibiotics. The total average of dipterous flies in examined farms was 400.42 ± 6.2. Culicoides biting midges were the most common existing species (70.01%) followed by house flies, stable flies, and mosquitoes (18.31, 7.74, and 3.91%, respectively) at X (2) = 9.0, P < 0.05. The most predominant bacterial isolates were Escherichia coli (22.6%), Staphylococcus aureus and Enterobacter (17.3% each), coagulase-negative Staphylococci (CNS) (14.7%), Klebsiella sp. (8%), Salmonella spp. (6.7%), and Shigella spp. and Proteus spp. (6.7% each). The tested bacterial isolates were resistant to variant antibiotics used. S. aureus exhibited 100% resistance to colistine. However, E. coli revealed 92.9 and 78.6% resistance against tetracycline and colistine, respectively. Both Salmonella spp. and Shigella spp. were 100% resistant to penicillin, and Klebsiella sp. had 100% resistance to tetracycline. In conclusion, Culicoides biting midges and house flies could be considered as a potential carrier for multi-drug-resistant bacteria of zoonotic importance. Furthermore, cows' environment has an essential role in propagation and wide spread of antimicrobial-resistant bacterial pathogens.

Pathogen Propagation Model with Superinfection in Vegetatively Propagated Plants on Lattice Space.

Many clonal plants have two reproductive patterns, seed propagation and vegetative propagation. By vegetative propagation, plants reproduce the genetically identical offspring with a low mortality, because resources are supplied from the other individuals through interconnected ramets at vegetative-propagated offspring. However, the ramets transport not only resources but also systemic pathogen. Pathogens evolve to establish and spread widely within the plant population. The superinfection, which is defined as the ability that an established pathogen spreads widely by infecting to already-infected individuals with other strains of a pathogen, is important to the evolution of pathogens. We examine the dynamics of plant reproduction and pathogen propagation considering spatial structure and the effect of superinfection on genetic diversity of pathogen by analysis of several models, 1-strain and multiple-strain models, on two-dimensional square lattice. In the analysis of 1-strain model, we derive equilibrium value by mean-field approximation and pair approximation, and its local stability by Routh-Hurwitz stability criterion. In the multiple-strain models, we analyze the dynamics by numerical simulation of mean-field approximation, pair approximation and Monte Carlo simulation. Through the analyses, we show the effect of parameter values to dynamics of models, such as transition of dominant strain of pathogen, competition between plants and pathogens and density of individuals. As a result, (i) The strain with intermediate cost becomes dominant when both superinfection rate and growth rate are low. (ii) The competition between plants and pathogens occurs in the phase of coexistence of various strains by pair approximation and Monte Carlo simulation. (iii) Too high growth rate leads to the decrease of plant population in all models. (iv) Pathogens are easy to maintain their genetic diversity with low superinfection rate. However, if they do not superinfect, the maintenance becomes difficult. (v) When growth rate of plant is low, individuals are very influenced by distant individuals.

Market analyses of livestock trade networks to inform the prevention of joint economic and epidemiological risks.

Conventional epidemiological studies of infections spreading through trade networks, e.g. via livestock movements, generally show that central large-size holdings (hubs) should be preferentially surveyed and controlled in order to reduce epidemic spread. However, epidemiological strategies alone may not be economically optimal when costs of control are factored in together with risks of market disruption from targeting core holdings in a supply chain. Using extensive data on animal movements in supply chains for cattle and swine in France, we introduce a method to identify effective strategies for preventing outbreaks with limited budgets while minimizing the risk of market disruptions. Our method involves the categorization of holdings based on position along the supply chain and degree of market share. Our analyses suggest that trade has a higher risk of propagating epidemics through cattle networks, which are dominated by exchanges involving wholesalers, than for swine. We assess the effectiveness of contrasting interventions from the perspectives of regulators and the market, using percolation analysis. We show that preferentially targeting minor, non-central agents can outperform targeting of hubs when the costs to stakeholders and the risks of market disturbance are considered. Our study highlights the importance of assessing joint economic–epidemiological risks in networks underlying pathogen propagation and trade.

Diagnostic methods for detecting fungal pathogens on vegetable seeds

Early diagnosis of seedborne fungal pathogens is particularly important, as often, infected seeds appear symptomless; seed diagnosis can avoid uncontrolled propagation of pathogens through long‐distance exchange of such material. This will prevent economic losses and unnecessary use of fungicides, so reducing costs and the introduction of toxic substances into the environment. Traditional techniques for detection of seedborne fungi are based on incubation and grow‐out methods. Although these are frequently used because of their simplicity of application, they are time‐consuming, require mycological skills, and are sometimes not sensitive enough to low levels of seed infection. Recently, new identification techniques, based on DNA analysis, have been applied and are very efficient due to high sensitivity and specificity. The most common technique is conventional PCR, while other recent techniques include nested PCR, to obviate low levels of target pathogens, multiplex PCR, to detect several pathogens simultaneously, real‐time PCR, to quantify fungi on seeds, and magnetic‐capture hybridization PCR. The main drawbacks of molecular methods are the inability to distinguish between vital and non‐vital inocula, and the difficulty in obtaining quality DNA template, due to PCR inhibitors in seeds. To reduce inhibitory effects, several modified PCR protocols, such as loop‐mediated isothermal amplification, and non‐destructive testing methods have been developed. Loop‐mediated isothermal amplification and next‐generation sequencing have been widely applied in nucleic acid analysis and, given the numerous advantages provided, their application can be substantially extended in the future for detection of fungal pathogens in seeds.

Risk factors causing the development of infection and inflammation of the lower urinary tract

Persistence as well as high survival rate and resistance of microorganisms belong to the key reasons promoting symptoms of lower urinary tract diseases. They can stay active for a long time and can substantially deteriorate the life quality of patients suffering from chronic infections and inflammations in the wall of the urinary bladder even after the eradication of the pathogen. Recent studies in the field of clinical microbiology reflect serious qualitative and quantitative changes in the infection incidence structure, which is mainly related to changes in the bacterial pathogenicity. Pathogenicity island genes control the synthesis of different adhesins, invasins, hemolysins and toxins (hemolysin, cytotoxic necrotizing factor type 1, etc.) as well as iron ion absorption system, which are essential for the pathogen propagation and activity in tissues. The determination of genetic determinants will provide a new insight into the bacterial evolution process and will help to understand the reason of prompt formation and propagation of virulent strains as well as fast development of the infection and inflammation in the lower urinary tract, which may explain different severity of the pathology and symptoms of the lower urinary tract.

Green leaf volatiles: biosynthesis, biological functions and their applications in biotechnology.

Plants have evolved numerous constitutive and inducible defence mechanisms to cope with biotic and abiotic stresses. These stresses induce the expression of various genes to activate defence-related pathways that result in the release of defence chemicals. One of these defence mechanisms is the oxylipin pathway, which produces jasmonates, divinylethers and green leaf volatiles (GLVs) through the peroxidation of polyunsaturated fatty acids (PUFAs). GLVs have recently emerged as key players in plant defence, plant-plant interactions and plant-insect interactions. Some GLVs inhibit the growth and propagation of plant pathogens, including bacteria, viruses and fungi. In certain cases, GLVs released from plants under herbivore attack can serve as aerial messengers to neighbouring plants and to attract parasitic or parasitoid enemies of the herbivores. The plants that perceive these volatile signals are primed and can then adapt in preparation for the upcoming challenges. Due to their 'green note' odour, GLVs impart aromas and flavours to many natural foods, such as vegetables and fruits, and therefore, they can be exploited in industrial biotechnology. The aim of this study was to review the progress and recent developments in research on the oxylipin pathway, with a specific focus on the biosynthesis and biological functions of GLVs and their applications in industrial biotechnology.

Do pathogen effectors play peek-a-boo?

Epigenetics is a burgeoning field of research and many organisms are found to regulate genes in a transient (“above genetics”) way due to (temporarily) decorating DNA or proteins that associate with DNA. This can change transcription of affected genes and since such DNA modifications are reversible, the expression potential over time and generations can therefore be extremely variable. This allows many organisms to readily adapt and experiment with changes to the environment they are living in or challenges they are facing, such as pathogens (Iwasaki and Paszkowski, 2014). This research topic deals mainly with epigenetic mechanisms of plant responses but in the field of plant pathology, recent experimental data illustrate the involvement of epigenetic changes in both plant host and their pathogens (e.g., Luna et al., 2012; Qutob et al., 2013; Yu et al., 2013; Soyer et al., 2014). Indeed, pathogen pressure is very much an environmental cue that will induce epigenetic changes in plant hosts, but, in response, pathogens seem equipped to respond in kind. The interaction between many pathogens and their hosts is modulated by large suites of effectors, often small proteins that are secreted by pathogens and end up in host tissues, in the apoplast or cytoplasm. They play various roles supporting the infection and the pathogen's propagation, such as suppressing host defense responses (Giraldo and Valent, 2013). Naturally, hosts have evolved mechanisms that use such effectors or their actions as cues to mount a counter defense to stop the pathogen in its track. Such cues are often perceived by resistance gene products, the sentries that are part of the host surveillance system and whose activation triggers the defense responses. In a tit-for-tat, the pathogen evolves to prevent the offending effector from being expressed and this can be done in a variety of ways: mutating its recognition, deletion from the genome, or preventing its expression. Especially the latter option is attractive since the coding information would still be available and the effector could potentially be “recycled” later on if the pressure is relieved, that is, if the host with the resistance gene that recognized the offending effector activity is gone from the environment. In the very timely perspective by Gijzen et al. (2014), the authors elaborate on the last scenario and work from their own laboratory gives an example of how an offending effector becomes silenced in a pathogen as to regain virulence and that this effect persists over many generations (Qutob et al., 2013). Interestingly, many effectors have been found embedded in regions with large numbers of transposable elements. Transposable elements have been dubbed “genome modifiers” and have been shown to affect gene expression while themselves being very prone to epigenetic control (Fedoroff, 2012). Intriguing findings indeed suggest the possible control of the expression of fungal effector genes embedded in epigenetically-controlled transposable element-rich chromatin (Soyer et al., 2014). The perspective Gijzen et al. highlights several recent publications that point to possible mechanisms by which such transient changes in effector expression may occur. It offers an explanation for several older literature reports in which such variable and apparently reversible virulences (avirulences) were observed both in the laboratory and in the field. In addition, and as a warning, this perspective discusses the consequences of these findings for the current development of diagnostic assays based solely on simple DNA analysis and the error rates this would produce; the idea for the need for more-sophisticated assays is floated. Finally, the question is posed whether this phenomenon is more wide-spread than currently appreciated, a prospect that makes the re-assessment of such diagnostic assays even more dire.

Synthetic blends of volatile, phytopathogen-induced odorants can be used to manipulate vector behavior

Volatile organic compounds (VOCs) are emitted from all plants and these VOCs are important means of communication between plants and insects. It has been documented that pathogen infections alter VOC profiles rendering infected plants more attractive to specific vectors transmitting these pathogens than uninfected plants, thus potentially aiding in pathogen propagation. Mimicking these chemical cues might enable insect attraction away from the plant or disruption of host finding behavior of the vector. However, the practical implications have not been fully explored. We used citrus, Diaphorina citri and huanglongbing (HLB) as a model host-vector-disease system because HLB threatens citrus production worldwide and is similar to other critical diseases of food crops, such as Zebra Chip affecting potato. We formulated a synthetic chemical blend using selected HLB-specific biomarker compounds, and tested the blend with the Attenu assay system for chemosensory proteins. The Attenu assay system is a procedure that identifies interactions between insect chemosensory proteins and their ligands. We found that an equimolar mixture of compounds mimicking the volatile profile of HLB-infected citrus bound chemosensory proteins. Further investigation of this blend in laboratory behavioral assays resulted in development of a synthetic lure that was more attractive to D. citri than natural citrus tree volatiles. This strategy could provide a new route to produce chemical lures for vector population control for a variety of plant and/or animal systems and it may result in the development of a practical lure for monitoring vectors of disease, such as D. citri.

Plant mitochondria under pathogen attack: A sigh of relief or a last breath?

Plants constitute excellent sources for pathogen nutrition and survival. To fight against pathogen attack, higher plants have developed a sophisticated immune system responsible for pathogen recognition and activation of downstream defense responses. After pathogen perception, mitochondria play an important role in the defense strategy of the plant cell, integrating and amplifying diverse signals such as salicylic acid, nitric oxide, reactive oxygen species (ROS) or pathogen elicitors. Signals perceived by mitochondria usually impact on their normal function, destabilizing the organelle, generating changes in respiration, membrane potential and ROS production. At this stage, mitochondria produce several signals influencing the redox state of the cell and promoting changes in the expression of nuclear genes by mitochondrial retrograde regulation. At more advanced stages, they promote programmed cell death in order to avoid pathogen propagation to the whole plant. Recent evidence indicates that plants and pathogens have evolved mechanisms to modulate the immune response by acting on mitochondrial functions. In this review, we summarize knowledge about the involvement of mitochondria in different aspects of the response of plants to pathogen attack.

A stochastic agent-based model of pathogen propagation in dynamic multi-relational social networks.

We describe a general framework for modeling and stochastic simulation of epidemics in realistic dynamic social networks, which incorporates heterogeneity in the types of individuals, types of interconnecting risk-bearing relationships, and types of pathogens transmitted across them. Dynamism is supported through arrival and departure processes, continuous restructuring of risk relationships, and changes to pathogen infectiousness, as mandated by natural history; dynamism is regulated through constraints on the local agency of individual nodes and their risk behaviors, while simulation trajectories are validated using system-wide metrics. To illustrate its utility, we present a case study that applies the proposed framework towards a simulation of HIV in artificial networks of intravenous drug users (IDUs) modeled using data collected in the Social Factors for HIV Risk survey.

Chlamydia trachomatis promotes 3T3 cell differentiation into adipocytes.

There is experimental and clinical evidence showing that some viral and bacterial pathogens are linked to the accumulation of excessive body fat and obesity. The aim of the study was to investigate the ability of C. trachomatis to propagate in the pre-adipocyte cell line and induce its differentiation into fat cells. 3T3 L1 pre-adipocytes or McCoy cells were plated and infected with C. trachomatis. The cell monolayers were further studied by immunofluorescent and quantitative RT-PCR methods. C. trachomatis can efficiently propagate in 3T3 L1 cells, a mouse pre-adipocyte cell line. The morphological characteristics of chlamydial growth revealed in 3T3 L1 cells with the monoclonal chlamydial MOMP-specific antibody resembled those seen in McCoy cells, a classic cell line used for chlamydial research. The number of chlamydial 16S rRNA copies detectable in the lysates of McCoy and 3T3 cells infected with C. trachomatis was almost identical, suggesting similar efficiency of pathogen propagation in both cell lines. Moreover, there was a significant increase in aP2 mRNA transcript levels as well as moderate induction of SCD-1 mRNA in the total RNA extracted from the infected 3T3 L1 cells 48 h following the pathogen inoculation. The increased expression of the adipogenic markers was also accompanied by lipid droplet accumulation in the C. trachomatis infected 3T3 L1 cells, suggesting their transformation into differentiated adipocytes. The direct effect of the pathogen on fat cell progenitors observed in this work may explain abnormal fat deposition at the sites of chronic inflammation caused by C. trachomatis.

Intramolecular Donor Strand Complementation in the E. coli Type 1 Pilus Subunit FimA Explains the Existence of FimA Monomers As Off-Pathway Products of Pilus Assembly That Inhibit Host Cell Apoptosis

Type 1 pili are filamentous organelles mediating the attachment of uropathogenic Escherichia coli to epithelial cells of host organisms. The helical pilus rod consists of up to 3000 copies of the main structural subunit FimA that interact via donor strand complementation, where the incomplete Ig-like fold of FimA is completed by insertion of the N-terminal extension (donor strand) of the following FimA subunit. Recently, it was shown that FimA also exists in a monomeric, assembly-incompetent form and that FimA monomers act as inhibitors of apoptosis in infected host cells. Here we present the NMR structure of monomeric wild-type FimA with its natural N-terminal donor strand complementing the Ig fold. Compared to FimA subunits in the assembled pilus, intramolecular self-complementation in the monomer stabilizes the FimA fold with significantly less interactions, and the natural FimA donor strand is inserted in the opposite orientation. In addition, we show that a motif of two glycine residues in the FimA donor strand, separated by five residues, is the prerequisite of the alternative, parallel donor strand insertion mechanism in the FimA monomer and that this motif is preserved in FimA homologs of many enteroinvasive pathogens. We conclude that FimA is a unique case of a protein with alternative, functionally relevant folding possibilities, with the FimA polymer forming the highly stable pilus rod and the FimA monomer promoting pathogen propagation by apoptosis suppression of infected epithelial target cells.

Glutamate Receptor-Like Channel3.3 Is Involved in Mediating Glutathione-Triggered Cytosolic Calcium Transients, Transcriptional Changes, and Innate Immunity Responses in Arabidopsis

The tripeptide reduced glutathione (GSH; γ-glutamate [Glu]-cysteine [Cys]-glycine) is a major endogenous antioxidant in both animal and plant cells. It also functions as a neurotransmitter mediating communication among neurons in the central nervous system of animals through modulating specific ionotropic Glu receptors (GLRs) in the membrane. Little is known about such signaling roles in plant cells. Here, we report that transient rises in cytosolic calcium triggered by exogenous GSH in Arabidopsis (Arabidopsis thaliana) leaves were sensitive to GLR antagonists and abolished in loss-of-function atglr3.3 mutants. Like the GSH biosynthesis-defective mutant PHYTOALEXIN DEFICIENT2, atglr3.3 showed enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Pathogen-induced defense marker gene expression was also decreased in atglr3.3 mutants. Twenty-seven percent of genes that were rapidly responsive to GSH treatment of seedlings were defense genes, most of which were dependent on functional AtGLR3.3, while GSH suppressed pathogen propagation through the AtGLR3.3-dependent pathway. Eight previously identified putative AtGLR3.3 ligands, GSH, oxidized glutathione, alanine, asparagine, Cys, Glu, glycine, and serine, all elicited the AtGLR3.3-dependent cytosolic calcium transients, but only GSH and Cys induced the defense response, with the Glu-induced AtGLR3.3-dependent transcription response being much less apparent than that triggered by GSH. Together, these observations suggest that AtGLR3.3 is required for several signaling effects mediated by extracellular GSH, even though these effects may not be causally related.

Qualitative risk assessment for the endemisation of Dirofilaria repens in the state of Brandenburg (Germany) based on temperature-dependent vector competence

Climate change with an increase in average temperature may be responsible for propagation of temperature-dependent vectors and/or vector-associated pathogens in regions that were previously not endemic. The analysis of climate data of Brandenburg state (Germany) had showed that the extrinsic development of Dirofilaria repens, the causative agent of canine subcutaneous dirofilariosis, was possible from the years 2001 to 2012. This finding, associated to the movement of infected dogs and their relocation from endemic European areas to Germany makes possible a rapid endemization of the infection and highlight the risk for human health, being D. repens a frequent cause of zoonotic infections.

Healthy sheep that differ in scrapie associated PRNP genotypes exhibit significant differences of expression pattern associated with immune response and cell-to-cell signalling in retropharyngeal lymph nodes

The present study was conducted to test the hypothesis whether prion protein gene (PRNP) associated scrapie susceptibility is connected with physiological changes in tissue involved in pathogen uptake, migration and propagation. Jejunum, ileal Peyer's patches, retropharyngeal lymph nodes, brain stem and liver of healthy and non scrapie-infected sheep with PRNP genotypes representing the scrapie risk class R1 (scrapie-resistant) and R5 (scrapie-susceptible), respectively, were comparatively analysed by microarray technology and quantitative reverse transcriptase polymerase chain reaction (RT qPCR). Significantly higher expression levels of genes involved in immune response and cell communication pathways in retropharyngeal lymph nodes of R1 sheep in comparison with R5 animals strongly suggest PRNP associated physiological processes with impact as an early barrier in pathogen defence.Equal expression patterns in brain stem suggest no physiological differences in brain of healthy R1 and R5 animals. In addition, similar expression pattern in liver indicates that there are no transcriptional differences in genes of the hepatic energy metabolism between animals of scrapie classes R1 and R5.

Development of an immunosensor assay for detection of haptoglobin in mastitic milk

Detection of subclinical mastitis in dairy cows is important, as it represents a major economic challenge for the dairy industry worldwide owing to propagation of mastitis-causing pathogens and to long-term reduction in milk yield and quality. Haptoglobin (Hp) is one of the most sensitive acute phase proteins in milk during udder inflammation and as an indicator of mastitis. The aim of this study was to develop a rapid and sensitive immunosensor assay for measuring Hp concentration in mastitic milk. The immunosensor was constructed by immobilizing anti-bovine Hp antibody on a gold electrode through gold nanoparticles fabricated on self-assembled L-cysteine layers. The immunosensor assay was used to measure Hp concentration in 20 milk samples positive for bacteria with a somatic cell count > 5 × 10(5) cells/mL from cows without clinical signs of mastitis. Results were compared with those obtained using a commercial ELISA kit. Reproducibility of Hp measurement and stability after storage for 20 days were good for the immunosensor assay. Measurement of Hp was linear over a range of 15-100 mg/L, with a limit of detection of 0.63 mg/L. Agreement between results obtained with the immunosensor and ELISA methods was satisfactory as analyzed using the Wilcoxon signed-rank test (Z = -1.739, P = .073). An immunosensor assay for measuring Hp in milk provided rapid results and was easy to perform, facilitating its potential use in the field for the diagnosis of subclinical mastitis once a cutoff value for Hp concentration is established.

Chikungunya virus impacts the diversity of symbiotic bacteria in mosquito vector

Mosquitoes transmit numerous arboviruses including dengue and chikungunya virus (CHIKV). In recent years, mosquito species Aedes albopictus has expanded in the Indian Ocean region and was the principal vector of chikungunya outbreaks in La Reunion and neighbouring islands in 2005 and 2006. Vector-associated bacteria have recently been found to interact with transmitted pathogens. For instance, Wolbachia modulates the replication of viruses or parasites. However, there has been no systematic evaluation of the diversity of the entire bacterial populations within mosquito individuals particularly in relation to virus invasion. Here, we investigated the effect of CHIKV infection on the whole bacterial community of Ae.albopictus. Taxonomic microarrays and quantitative PCR showed that members of Alpha- and Gammaproteobacteria phyla, as well as Bacteroidetes, responded to CHIKV infection. The abundance of bacteria from the Enterobacteriaceae family increased with CHIKV infection, whereas the abundance of known insect endosymbionts like Wolbachia and Blattabacterium decreased. Our results clearly link the pathogen propagation with changes in the dynamics of the bacterial community, suggesting that cooperation or competition occurs within the host, which may in turn affect the mosquito traits like vector competence.

Improving the Efficacy of Wastewater-Polishing Reed Beds

Microbiological Contaminants in Treated Wastewater Demand for high quality drinking and recreational waters rises exponentially due to global demographic growth in the human population, reinforcing an urgent need for microbiologically safe reclaimed waters (Graczyk and Lucy. 2007). Wastewater discharges are worldwide risk factors for the introduction of human pathogens into surface waters used as drinking and recreational resources. Cryptosporidium parvum, Giardia duodenalis, and human-virulent microsporidia, (i.e., Encephalitozoon intestinalis, E. hellem, E. cuniculi, and Enterocytozoon bieneusi) are waterborne enteropathogens inflicting considerable morbidity in healthy people and mortality (e.g., Cryptosporidium and microspora) in immunodeficient individuals (Savioli, et al., 2006; Weber and Bryan, 1994). Their transmissive stages, i.e., oocysts, cysts, and spores, respectively, are resistant to environmental stressors and are therefore long-lasting and relatively ubiquitous in the environment (Graczyk, et al., 1997; Matchis, et al., 2005; Wolfe,1992). These pathogens are category B biodefense agents on the NIH list. Microsporidian spores are on Contaminant Candidate List of the U.S. EPA (Nwachcuku and Gerba, 2004) because spore identification, removal, and inactivation in drinking water are technologically challenging. Surface water is not routinely monitored for these pathogens, despite evidence demonstrating environmental contamination derived from wastewater discharges (Graczyk and Lucy, 2007). Environmentally, all these pathogens have a broad zoonotic reservoir (Graczyk et al., 1997; Matchis et al., 2005; Savioli, et al., 2006). Constructed Wetland Concept Constructed wetlands of either vertical or horizontal flow are increasingly used worldwide for secondary or tertiary treatment of municipal wastewater due to minimum electric requirements and low maintenance costs (Davidson et al., 2005; Reinoso et al., 2008). The wetland concept has become an attractive wastewater treatment alternative to conventional tertiary treatment processes for: a) municipal wastewater; b) on-site domestic wastewater treatment; and c) concentrated animal feeding operations (CAFO) (Karpiscak et al., 2001). In wetlands, human-pathogenic microorganisms are physically removed and biodegraded by sedimentation (Dai and Boll, 2006; Karim et al., 2004), filtration and evapotranspiration-driven attachment to plant roots (Gerba et al., 1999; Dorsch and Veal, 2001; Weaver et al., 2003), natural die-off (Nokes et al., 2003), UV radiation, straining and sorption by the biofilm (Quinonez-Diaz et al., 2001), and protozoan predation (Stott et al., 2001). It is thought that performance of wetlands in removing human pathogens is superior to that of secondary wastewater treatment, i.e., conventional sewage sludge activation (Ulrich et al., 2005). Horizontal wetlands usually discharge to surface waters that are frequently used for recreation or drinking water production (Davidson et al., 2005). Constructed Wetland Operation In general, wastewater can be injected under the wetland surface for plug flow hydraulics (Weaver et al., 2003), or be delivered to the wetland surface for free-surface flow. Because the wastewater resides in wetlands for certain time, these areas can act as endemic sites supporting both propagation and transmission of human zoonotic pathogens (Graczyk et al., 2007). Sizing reed-bed systems for a residence time of 5 days has become a standard practice (Davidson et al., 2005; Quinonez-Diaz et al., 2001; Thurston et al., 2001), leaving plenty of time for propagation and spreading of wastewater-derived pathogens in wetland habitats via a wide variety of wildlife (Graczyk and Lucy, 2007; Graczyk et al., 2007). In addition, any temporal or permanent malfunctioning caused by clogged inlet pipes can cause hydraulic short circuits that bypass part of the filtration area in wetlands. Aims of the Project The purposes of the project were to: a) determine species of microbiological contaminants entering, residing, and leaving constructed horizontal wetlands used for tertiary treatment of municipal wastewater; and b) determine removal efficacy of Cryptosporidium oocysts, G. duodenalis cysts, and human-virulent microsporidian spore species by wetlands from secondary-treated wastewater. This title belongs to WERF Research Report Series . ISBN: 9781843395317 (eBook)