The role of RTX toxins in host specificity of animal pathogenic Pasteurellaceae

RTX toxins are bacterial pore-forming toxins that are particularly abundant among pathogenic species of Pasteurellaceae, in which they play a major role in virulence. RTX toxins of several primary pathogens of the family of Pasteurellaceae are directly involved in causing necrotic lesions in the target organs. Many RTX toxins are known as haemolysins because they lyse erythrocytes in vitro, an effect that is non-specific, but which serves as a useful marker in bacteriological identification and as an easily measurable signal in vitro in experimental studies. More recent studies have shown that the specific targets of most RTX toxins are leukocytes, with RTX toxins binding to the corresponding beta-subunit (CD18) of beta2 integrins and then exerting cytotoxic activity. After uptake by the target cell, at sub-lytic concentrations, some RTX toxins are transported to mitochondria and induce apoptosis. For several RTX toxins the binding to CD18 has been shown to be host specific and this seems to be the basis for the host range specificity of these RTX toxins. Observations on two very closely related species of the Pasteurellaceae family, Actinobacillus suis, a porcine pathogen particularly affecting suckling pigs, and Actinobacillus equuli subsp. haemolytica, which causes pyosepticaemia in new-born foals (sleepy foal disease), have revealed that they express different RTX toxins, named ApxI/II and Aqx, respectively. These RTX toxins are specifically cytotoxic for porcine and equine leukocytes, respectively. Furthermore, the ApxI and Aqx toxins of these species, when expressed in an isogenetic background in Escherichia coli, are specifically cytotoxic for leukocytes of their respective hosts. These data indicate the determinative role of RTX toxins in host specificity of pathogenic species of Pasteurellaceae.

Macroevolution of insect–plant associations: The relevance of host biogeography to host affiliation

Identifying the factors that have promoted host shifts by phytophagous insects at a macroevolutionary scale is critical to understanding the associations between plants and insects. We used molecular phylogenies of the beetle genus Blepharida and its host genus Bursera to test whether these insects have been using hosts with widely overlapping ranges over evolutionary time. We also quantified the importance of host range coincidence relative to host chemistry and host phylogenetic relatedness. Overall, the evolution of host use of these insects has not been among hosts that are geographically similar. Host chemistry is the factor that best explains their macroevolutionary patterns of host use. Interestingly, one exceptional polyphagous species has shifted among geographically close chemically dissimilar plants.

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

BACKGROUND Mycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission. RESULTS 164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900--restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property. CONCLUSION The results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

Comparative Analysis of Genus Bifidobacterium: Insight into its Host Adaptation

Bifidobacteria is amongst one of the health promoting bacteria. The role of this important probiotic genera can be elucidated by understanding its genome. Comparative analysis of the whole genus of these bacteria can reveal their adaptation to a diverse host range. This study comprises of four research projects. In the first study, a reference library for genus Bifidobacterium was prepared. The core genes in each genus were selected based on a newly proposed statistical definition of core genome. Comparative analysis of Bifidobacterium with another probiotic genus Lactobacillus revealed the metabolic characteristics of genus Bifidobacterium. The second study investigated the immunomodulatory role of a B. bifidum strain TMC3115. The analysis of TMC3115 provided insights into its extracellular structures which might have their role in host interaction and immunomodulation. The study highlighted the variability among these genomes just not on species level but also on strain level in terms of host interaction. The last two studies aim to inspect the relationship between bifidobacteria and its host diet. Bifidobacteria, are both host- and niche-specific. Such adaptation of bifidobacterial species is considered relevant to the intestinal microecosystem and hosts’ oligosaccharides. Many species should have co-evolved with their hosts, but the phylogeny of Bifidobacterium is dissimilar to that of host animals. The discrepancy could be linked to the niche-specific evolution due to hosts’ dietary carbohydrates. The distribution of carbohydrate-active enzymes, in particular glycoside hydrolases (GHs) that metabolize unique oligosaccharides was examined. When bifidobacterial species were classified by their distribution of GH genes, five groups arose according to their hosts’ feeding behaviour. The distribution of GH genes was only weakly associated with the phylogeny of the host animals or with genomic features such as genome size. Thus, the hosts’ dietary pattern is the key determinant of the distribution and evolution of GH genes.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp aurantifolii

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.

Effect of Amblyomma cajennense Ticks on the Immune Response of BALB/c Mice and Horses

This work evaluated the effect of the Amblyomma cajennense tick on the immune response of BALB/c mice and on horse lymph node cell proliferation. We observed that mice do not develop resistance to nymphs of this tick species and that lymphocyte proliferation of this host is inhibited by tick saliva, nymphal extract, or infestations. Horse lymph node cell proliferation is inhibited by tick saliva as well. Mice lymphocytes under the effect of tick saliva, nymphal extract, or infestations display a predominantly. p Th-2 cytokine production pattern. Observed results partially explain this tick`s disease vectoring capacity and broad host range.

Genetic structure and biology of Xylella fastidiosa strains causing disease in citrus and coffee in Brazil

Xylella fastidiosa is a vector-borne, plant-pathogenic bacterium that causes disease in citrus (citrus variegated chlorosis [CVC]) and coffee (coffee leaf scorch [CLS]) plants in Brazil. CVC and CLS occur sympatrically and share leafhopper vectors; thus, determining whether X. fastidiosa isolates can be dispersed from one crop to another and cause disease is of epidemiological importance. We sought to clarify the genetic and biological relationships between CVC- and CLS-causing X. fastidiosa isolates. We used cross-inoculation bioassays and microsatellite and multilocus sequence typing (MLST) approaches to determine the host range and genetic structure of 26 CVC and 20 CLS isolates collected from different regions in Brazil. Our results show that citrus and coffee X. fastidiosa isolates are biologically distinct. Cross-inoculation tests showed that isolates causing CVC and CLS in the field were able to colonize citrus and coffee plants, respectively, but not the other host, indicating biological isolation between the strains. The microsatellite analysis separated most X. fastidiosa populations tested on the basis of the host plant from which they were isolated. However, recombination among isolates was detected and a lack of congruency among phylogenetic trees was observed for the loci used in the MLST scheme. Altogether, our study indicates that CVC and CLS are caused by two biologically distinct strains of X. fastidiosa that have diverged but are genetically homogenized by frequent recombination.

Wolbachia: why these bacteria are important to genome research

Intracellular bacteria of the genus Wolbachia were first discovered in mosquitoes in the 1920s. Their superficial similarity to pathogenic rickettsia initially raised interest in them as potential human pathogens. However, injection experiments with mice showed that they were non-pathogenic, and they were subsequently classified as symbionts of insects. Until the 1970s, Wolbachia was considered to infect a limited number of species of mosquitoes. It is now clear that Wolbachia is an extremely common intracellular agent of invertebrates, infecting nearly all the major groups of arthropods and other terrestrial invertebrates. Its wide host range and abundance can be attributed partly to the unusual phenotypes it exerts on the host it infects. These include the induction of parthenogenesis (the production of female offspring from unmated mothers) in certain insects, the feminization of genetic male crustaceans to functional phenotypic females, and the failure of fertilization in hosts when males and females have a different infection status (cytoplasmic incompatibility). All of these phenotypes favor maternal transmission of the intracellular Wolbachia. In the last year, Wolbachia has also been shown to be a widespread symbiont of filarial nematodes. It appears that Wolbachia is needed by the adult worm for normal fertility, indicating that Wolbachia is behaving like a classic mutualist in this case. This discovery exemplifies that the extent of the host range of Wolbachia and its associated phenotypes is still far from fully understood.

Differentiation of soil properties related to the spatial association of wheat roots and soil macropores

Under certain soil conditions, e.g. hardsetting clay B-horizons of South-Eastern Australia, wheat plants do not perform as well as would be expected given measurements of bulk soil attributes. In such soils, measurement indicates that a large proportion (80%) of roots are preferentially located in the soil within 1 mm of macropores. This paper addresses the question of whether there are biological and soil chemical effects concomitant with this observed spatial relationship. The properties of soil manually dissected from the 1-3 mm wide region surrounding macropores, the macropore sheath, were compared to those that are measured in a conventional manner on the bulk soil. Field specimens of two different soil materials were dissected to examine biological differentiation. To ascertain whether the macropore sheath soil differs from rhizosphere soil, wheat was grown in structured and repacked cores under laboratory conditions. The macropore sheath soil contained more microbial biomass per unit mass than both the bulk soil and the rhizosphere. The bacterial population in the macropore sheath was able to utilise a wider range of carbon substrates and to a greater extent than the bacterial population in the corresponding bulk soil. These differences between the macropore sheath and bulk soil were almost non-existent in the repacked cores. Evidence for larger numbers of propagules of the broad host range fungus Pythium in the macropore sheath soil were also obtained.

Canine Distemper Virus in a Crab-eating Fox (Cerdocyon thous) in Brazil: Case Report and Phylogenetic Analyses

Although canine distemper is enzootic worldwide and has a wide host range, there are no reports of canine distemper virus there are no reports of canine distemper virus provide information on virus phylogeny and histopathologic lesions. The objective of this study is report and describe canine distemper in a crab-eating fox (C. thous), with a focus oil the phylogeny of the virus strain and the histopathologic lesions in the animal.

Damage level of the two-spotted spider mite Tetranychus urticae Koch (acari: tetranychidae) in soybeans

Among phytophagous spider mites, the two-spotted spider mite Tetranychus urticae Koch, 1836 is one of the most important agricultural pests, not only because of the damage it causes, but also because it has a wide host range, infesting many commercial crops such as leafy greens, cotton, beans, and soybeans, among others. This study was carried out in a greenhouse of the Faculdade de Ciências Agrárias (FCA) of the Universidade Federal da Grande Dourados (UFGD), located in the city of Dourados, state of Mato Grosso do Sul. The experiment was arranged in a randomized block design with 5 treatments and 4 replicates. The treatments consisted of 5 levels in percentage of chlorotic symptoms (indicating mite damage): 0%, 25%, 50%, 75%, and 100%. All of the characteristics evaluated, except for number of pods per plant, the number of seeds per plant, the total weight (productivity), and the weight of 1000 seeds, were significantly influenced by the different levels of chlorotic symptoms. The economic damage level for the two-spotted spider mite Tetranychus urticae, according to the equation y = 66.63-0.51 x, based on the price of US$ 11.00 per bag of soybeans and a control cost of US$ 16.00, would be 15.80% chlorotic symptoms. At a price of US$ 29.00 per bag with the same control cost, the economic damage level would be 13% of chlorotic symptoms.

Ecology of phlebotomine sand flies (Diptera: Psychodidae)in a focus of Leishmania (Viannia) brasiliensis in northeastern Colombia

The phlebotomine sand fly fauna of two coffee plantations in a Leishmania-endemic area of Norte de Santander, Colombia was studied. Regular insect collections using a variety of methods were made for three and a half years. Information was obtained on diurnal resting sites, host range and seasonal abundance for 17 species, of wich five (Lutzomyia spinicrassa, Lu. serrana,Lu. shannoni, Lu. ovallesi and Lu. gomezi) were far more numerous than the others, anthropophilic and present throughout the year. The behaviour of these and the remaining 12 species is discussed in relation to their potential role in transmission of Leishmania (Viannia) brasiliensis in the area.

Chagas disease and human migration

Human Chagas disease is a purely accidental occurrence. As humans came into contact with the natural foci of infection might then have become infected as a single addition to the already extensive host range of Trypanosoma cruzi that includes other primates. Thus began a process of adaptation and domiciliation to human habitations through which the vectors had direct access to abundant food as well as protection from climatic changes and predators. Our work deals with the extraction and specific amplification by polymerase chain reaction of T. cruzi DNA obtained from mummified human tissues and the positive diagnosis of Chagas disease in a series of 4,000-year-old Pre-Hispanic human mummies from the northern coast of Chile. The area has been inhabited at least for 7,000 years, first by hunters, fishers and gatherers, and then gradually by more permanent settlements. The studied specimens belonged to the Chinchorro culture, a people inhabiting the area now occupied by the modern city of Arica. These were essentially fishers with a complex religious ideology, which accounts for the preservation of their dead in the way of mummified bodies, further enhanced by the extremely dry conditions of the desert. Chinchorro mummies are, perhaps, the oldest preserved bodies known to date.

Improved immunogenicity against HIV-1 clade C antigens by using NYVAC-C with restored replication competence

Background: In order to improve the immunogenicity of currently available non-replicating pox virus HIV vaccine vectors, NYVAC was genetically modified through re-insertion of two host range genes (K1L and C7L), resulting in restored replicative capacity in human cells. Methods: In the present study these vectors, expressing either a combination of the HIV-1 clade C antigens Env, Gag, Pol, Nef, or a combination of Gal, Pol, Nef were evaluated for safety and immunogenicity in rhesus macaques, which were immunized at weeks 0, 4 and 12 either by scarification (conventional poxvirus route of immunization), intradermal or by intramuscular injection (route used in previous vaccine studies). Results: Replication competent NYVAC-C-KC vectors induced higher HIV-specific responses, as measured by IFN-g ELISpot assay, than the replication defective NYVAC-C vectors. Application through scarification only required one immunization to induce maximum HIV-specific immune responses. This method simultaneously induced relatively lower anti-vector responses. In contrast, two to three immunizations were required when the NYVAC-C-KC vectors were given by intradermal or intramuscular injection and this method tended to generate slightly lower responses. Responses were predominantly directed against Env in the animals that received NYVAC-C-KC vectors expressing HIV-1 Env, Gag, Pol, Nef, while Gag responses were dominant in the NYVAC-C-KC HIV-1 Gag, Pol, Nef immunized animals. Conclusion: The current study demonstrates that NYVAC replication competent vectors were well tolerated and showed increased immunogenicity as compared to replication defective vectors. Further studies are needed to evaluate the most efficient route of immunization and to explore the use of these replication competent NYVAC vectors in prime/boost combination with gp120 proteinbased vaccine candidates. This study was performed within the Poxvirus T-cell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Improvement of the HIV/AIDS vaccine candidate NYVAC-C by deletion of the viral type I IFN inhibitor and/or acquisition of replication competence

Background: Recombinant viruses based on the attenuated vaccinia virus strain NYVAC are promising HIV vaccine candidates as phase I/II clinical trials have shown good safety and immunogenicity profiles. However, this NYVAC strain is non-replicating in most human cell lines and encodes viral inhibitors of the immune system. Methods: With the aim to increase the immune potency of the current NYVAC-C vector (expressing the codon optimized clade C HIV-1 genes encoding gp120 and Gag-Pol-Nef polyprotein), we have generated and characterized three NYVAC-C-based vectors by, 1) deletion of the viral type I IFN inhibitor gene (NYVAC-CdeltaB19R), 2) restoration of virus replication competence in human cells by re-inserting K1L and C7L host range genes (NYVAC-C-KC) and, 3) combination of both strategies (NYVACC- KC-deltaB19R). Results: Insertion of the KC fragment restored the replication competence of the viruses in human cells (HeLa cells and primary dermal fibroblasts and keratinocytes), increased the expression of HIV antigens by more than 3-fold compared to the non-replicating homologs, inhibited apoptosis induced by the parental NYVAC-C and retained attenuation in a newborn mouse model. In adult mice, replication-competent viruses showed a limited capacity to replicate in tissues surrounding the inoculation site (ovaries and lymph nodes). After infection of keratinocytes, PBMCs and dendritic cells these viruses induced differential modulation in specific host cell signal transduction pathways, triggering genes important in immune modulation. Conclusion: We have developed improved NYVAC-C-based vectors with enhanced HIV-1 antigen expression, with the ability to replicate in cultured human cells and partially in some tissues, with an induced expression of cellular genes relevant to immune system activation, and which trigger IFN-dependent and independent signalling pathways, while maintaining a safety phenotype. These new vectors are promising new HIV vaccine candidates. These studies were performed within the Poxvirus Tcell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.