Taxonomy and pathogenicity of <Emphasis Type="Italic">Leptographium</Emphasis> species associated with <Emphasis Type="Italic">Ips subelongatus</Emphasis> infestations of <Emphasis Type="Italic">Larix</Emphasis> spp. in northern China,including two new species

The larch bark beetle (Ips subelongatus), which occurs in larch plantations over a vast area of eastern Asia, infects both dying and fallen trees. When its population reaches a high density, the beetle may also infect healthy trees, resulting in tree decline and, eventually, death. Leptographium spp., in both their sexual and asexual states, are mainly associated with conifer-infesting bark beetles; some species are important tree pathogens. The aims of this study were to identify the Leptographium spp. associated with I. subelongatus infestations of Larix spp. in northern China and to examine their pathogenicity towards the tree. Morphological studies and phylogenetic approaches based on multilocus DNA sequence data (ITS2- partial r28S, partial β-tubulin, and EF-1α gene regions) showed that three Leptographium species occur in association with I. subelongatus in the areas investigated: Leptographium taigense, which is recorded in China for the first time, and two new species, namely L. innermongolicum sp. nov. and L. zhangii sp. nov. Leptographium innermongolicum is closely related to L. taigense, whereas L. zhangii belongs to the Grosmannia piceaperda species complex. The pathogenicity of these Leptographium species towards mature Larix spp. was tested by stem inoculation in forests. All inoculations only resulted in small lesions on the inner bark; therefore, the three Leptographium species were not considered to be pathogenic.     

Immunohistochemical Cross-Reactivity Between <Emphasis Type="Italic">Paracoccidioides</Emphasis> sp. from Dolphins and <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis>

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins’ sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Infectious Diseases in Free-Ranging Blonde Capuchins, <Emphasis Type="Italic">Sapajus flavius,</Emphasis> in Brazil

The main threats to primates worldwide are the degradation, fragmentation, and loss of their habitats; hunting (especially for bushmeat); and illegal trade. For many species, the most important threat is forest fragmentation, resulting in small populations that are restricted to isolated forest patches. In this situation, primates are particularly vulnerable to disease. The Endangered blonde capuchin (Sapajus flavius) is now restricted to a few forest patches in Northeast Brazil. We investigated the occurrence of parasites and bacterial diseases in one of three free-ranging groups of S. flavius in a small forest patch in Paraíba state, Northeast Brazil. We tested for antibodies against Leishmania spp., Trypanosoma cruzi, Toxoplasma gondii, Leptospira spp. (24 strains), and Brucella spp.. We used molecular analysis to detect Plasmodium spp., and evaluated blood smears for the presence of hemoparasites. All individuals tested negative for Leptospira spp. and B. abortus, but 8 of 48 (16%) presented antibodies for both Leishmania spp. and T. cruzi. We identified antibodies to T. gondii in 12% of the individuals tested. Plasmodium brasilianum infection was present in 4% of the individuals tested, and blood smears showed microfilariae parasites in 46% of the individuals tested. The occurrence of these infectious diseases in S. flavius may pose a significant threat in terms of reduced recruitment and poor survival rates, and an understanding of the influence of pathogens is crucial for the management of small populations of primates.     

Reprogramming <Emphasis Type="Italic">Halomonas</Emphasis> for industrial production of chemicals

Halomonas spp. are able to grow under a high salt concentration at alkali pH, they are able to resist contamination by other microbes. Development of Halomonas spp. as platform production strains for the next-generation industrial biotechnology (NGIB) is intensively studied. Among Halomonas spp., Halomonas bluephagenesis is the best studied one with available engineering tools and methods to reprogram it for production of various polyhydroxyalkanoates, proteins, and chemicals. Due to its contamination resistance, H. bluephagenesis can be grown under open and continuous processes not just in the labs but also in at least 1000 L fermentor scale. It is expected that NGIB based on Halomonas spp. be able to engineer for production of increasing number of products in a competitive manner.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

qPCR estimates of <Emphasis Type="Italic">Babesia bovis</Emphasis> and <Emphasis Type="Italic">Babesia bigemina</Emphasis> infection levels in beef cattle and <Emphasis Type="Italic">Rhipicephalus microplus</Emphasis> larvae

Babesia spp. are tick-transmitted intraerythrocytic apicomplexan parasites that infect wild and domestic animals. Babesia bovis and B. bigemina are endemic and responsible for enormous economic losses to the livestock industry in most of the Brazilian territory, wherein the tick Rhipicephalus microplus is the unique vector. Better understanding of epidemiology and parasite–host interactions may improve the tools for disease control and genetic management for selection of resistant animals. This study aimed to detect, quantify and measure the correlation between B. bigemina and B. bovis infection levels in bovine blood and into tick, by absolute quantification of hemoparasite DNA using qPCR. Blood bovine samples and larvae pools from 10 engorged R. microplus females were collected from each Canchim heifers (5/8 Charolais?+?3/8 zebu, n?=?36). All evaluated samples were positive for both Babesia species tested. Correlations of B. bovis and B. bigemina levels between cattle and tick host were 0.58 and 0.66, respectively. These high positive correlation coefficients indicate that parasitemia load in the bovine may be dependent on or may determine the parasitemia load in the ticks.     

Molecular phylogenetic analysis of key <Emphasis Type="Italic">Jatropha</Emphasis> species inferred from nrDNA ITS and chloroplast (<Emphasis Type="Italic">trn</Emphasis>L-F and <Emphasis Type="Italic">rbc</Emphasis>L) sequences

The genus Jatropha (Euphorbiaceae) contains species that are of significant economic and ornamental value. However, Jatropha breeding material is rather limited due to incomplete information regarding phylogenetic relationships among germplasm resources. Phylogenetic analyses were performed based on the internal transcribed spacer of nuclear ribosomal DNA (nrDNA ITS), two chloroplast regions (trnL-F and rbcL), and the combined (ITS+trnL-F+rbcL) dataset among twenty-five specimens representing six key Jatropha species. Phylogenetic relationships of Jatropha were well resolved between subgenus Curcas and subgenus Jatropha, and demonstrated the intermediate position of section Polymorphae among sections of both subgenera. Jatropha curcas and J. integerrima demonstrated a close phylogenetic relationship. The molecular data agreed with the morphological classification that recognized J. multifida and J. podagrica in sec. Peltatae. The distinct intraspecific divergence that occurred in J. curcas could be attributed to restricted gene flow caused by geographical isolation and different ecological conditions. Phylograms produced with trnL-F and rbcL sequence data suggested slow rates of sequence divergence among Jatropha spp., while the ITS gene tree had good resolution suggesting high genetic variation of ITS among Jatropha species.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

First record of the tick <Emphasis Type="Italic">Ixodes</Emphasis> (<Emphasis Type="Italic">Pholeoixodes</Emphasis>) <Emphasis Type="Italic">kaiseri</Emphasis> in Turkey

Nymphs and larvae belonging to Ixodes spp. were collected from a red fox in Turkey. The ticks were identified morphologically and molecularly (16S rDNA PCR and phylogenetic analysis) as I. kaiseri. Sequence and phylogenetic analyses show that our I. kaiseri isolate is very similar to I. kaiseri isolates collected from Germany, Serbia, Romania, and Hungary. Therefore, the existence of I. kaiseri has been demonstrated for the first time in Turkey. More studies relating to the regional distribution and vectorial competence of I. kaiseri are needed.     

<Emphasis Type="Italic">Sordaria fimicola</Emphasis>-like ascomycete isolated from <Emphasis Type="Italic">Pinus coulteri</Emphasis> needles in Slovakia

This is the first report of Sordaria fimicola-like ascomycete which was encountered during a diversity study of injured tissues of coulter pine in Slovakia. The fungus was identified as Sordaria fimicola by morphological analyses. Sequence analysis of internal transcribed spacer region (ITS) showed that the fungus is highly related to the ITS sequences of several S. fimicola isolates documenting wide ecological valence and geographical distribution of S. fimicola-like ascomycetes.     

Identification of <Emphasis Type="Italic">O</Emphasis>-GlcNAcylated proteins in <Emphasis Type="Italic">Plasmodium falciparum</Emphasis>

Background

Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans.

Methods

The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS).

Results

While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins.

Conclusions

This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.

     

Goats in the city: prevalence of <Emphasis Type="Italic">Giardia duodenalis</Emphasis> and <Emphasis Type="Italic">Cryptosporidium</Emphasis> spp. in extensively reared goats in northern India

Background

Various characteristics of goats mean they are highly suitable livestock for backyard rearing by people with limited resources. They are a popular livestock choice in India, where they are often kept to supplement an already scarce income. In these settings, hygiene and sanitation standards tend to be low, and weakens the interface between humans and animals, thus reducing the barrier between them and thereby increasing the likelihood that zoonotic and anthroponotic infections will occur.

Results

This study reports an investigation of the occurrence of Cryptosporidium spp. and Giardia duodenalis in goats being reared in different settings in urban and peri-urban areas in northern India, and addressed the zoonotic potential of these important protozoan parasites shed from goats living close to humans. The overall prevalence of G. duodenalis was 33.8 and 0.5% for Cryptosporidium spp.; the relatively low prevalence of cryptosporidiosis may reflect that most samples were derived from adult animals. The prevalence of G. duodenalis excretion was found to be similar to that reported in other studies. However, although other studies have reported a predominance of non-zoonotic Assemblage E in goats, in this study potentially zoonotic Assemblages predominated [Assemblage A (36%) and Assemblage B (32%)].

Conclusions

The results of this study indicate that in this area where goats and humans are living in close proximity, there may be sharing of intestinal parasites, which can be detrimental for both host species.

     

Size structures and comparative phenology of syntopic populations of <Emphasis Type="Italic">Hirudo verbana</Emphasis> and <Emphasis Type="Italic">Hirudo medicinalis</Emphasis> in eastern Ukraine

Ranges of different species of medicinal leeches (Hirudo spp.) are nearly mutually exclusive with very few areas of overlapping. This biogeographical pattern suggests different ecological requirements for the two species. However, two habitats in Hungary and Ukraine where populations of H. verbana and H. medicinalis coexist were found. The aim of this research was to reveal differences in size structures and phenologies between the two species in a steppe pond with two populations. The study was conducted from March to September in 2009 and 2010 in Horila Dolyna, Kharkiv Region. Leeches were collected several times during the season, weighed and released. The abundance of H. verbana was always higher. The Mann-Whitney U-test and the Wilcoxon test were applied. Significant differences between sequential samples within the species were found. The large weight classes of H. verbana emerged by the end of summer and caused a higher variance in contrast to H. medicinalis. This suggests the higher growth rate and the more r-strategic biology of H. verbana in comparison with the largely K-strategic H. medicinalis.