Potential of root nodule nonrhizobial endophytic bacteria for growth promotion of Lotus corniculatus L. and Dactylis glomerata L.

AIMS: This research aimed to isolate and characterize nonrhizobial endophytic bacteria from root nodules of Medicago sativa L. and Lotus corniculatus L. with plant growth-promoting characteristics and to test its activity in a pot experiment with acid soil. METHODS AND RESULTS: Out of 44 nonrhizobial isolates, the majority exhibited indole-3-acetic acid (IAA) production; 29 produced siderophores, few isolates performed phosphate solubilization and/or produced lytic enzymes, while 30% of isolates showed notable antifungal activity. The most promising strains were identified as members of Bacillus, Pseudomonas and Serratia genera, based on 16S rRNA. Bacillus megaterium DZK1BH exhibited the overall best attributes for plant growth promotion and positively influenced the growth of L. corniculatus and Dactylis glomerata. CONCLUSIONS: Root nodule endophytic B. megaterium DZK1BH could potentially be used as a biofertilizer for growing L. corniculatus L. and D. glomerata L. in acid soils, while Bacillus mojavensis L3 is a candidate for further antifungal potential investigation. SIGNIFICANCE OF IMPACT OF THE STUDY: The use of root nodule endophytic bacteria with PGP traits may find its future application in organic agriculture, as their utilization could decrease the use of chemical fertilizers and pesticides and simultaneously promote plant growth, especially in soils with low production quality.

Plasmodium chabaudi-infected mice spleen response to synthesized silver nanoparticles from Indigofera oblongifolia extract.

Malaria is a worldwide serious-threatening infectious disease caused by Plasmodium and the parasite resistance to antimalarial drugs has confirmed a significant obstacle to novel therapeutic antimalarial drugs. In this article, we assessed the antioxidant and anti-inflammatory activity of nanoparticles prepared from Indigofera oblongifolia extract (AgNPs) against the infection with Plasmodium chabaudi caused in mice spleen. AgNPs could significantly suppress the parasitaemia caused by the parasite to approximately 98% on day 7 postinfection with P. chabaudi and could improve the histopathological induced spleen damage. Also, AgNPs were able to increase the capsule thickness of the infected mice spleen. In addition, the AgNPs functioned as an antioxidant agent that affects the change in glutathione, nitric oxide and catalase levels in the spleen. Moreover spleen IL1ß, IL-6 and TNF-α-mRNA expression was regulated by AgNPs administration to the infected mice. These results indicated the anti-oxidant and the anti-inflammatory protective role of AgNPs against P. chabaudi-induced spleen injury.

Raspberry leaf blotch emaravirus in Bosnia and Herzegovina: population structure and systemic movement.

Raspberry leaf blotch virus (RLBV) is the putative agent of the homonymous disease and even though Bosnia and Herzegovina is a major producer worldwide there is no report of the virus presence in the country. We studied the virus population structure and assessed its ability to move systemically. RLBV is widespread in production areas and has a homogeneous population structure; leading to the hypothesis that the primary mode of dissemination is propagation material. The ability of the virus to move systemically eliminates propagation of root cuttings as a viable option to obtain RLBV-free plants, leaving RT-PCR screening as the better option to propagate RLBV- free plants in the absence of clean-up facilities or certification programs in the country.

Association between use of proton pump inhibitors and non-typhoidal salmonellosis identified following investigation into an outbreak of Salmonella Mikawasima in the UK, 2013.

In November 2013, national public health agencies in England and Scotland identified an increase in laboratory-confirmed Salmonella Mikawasima. The role of proton pump inhibitors (PPIs) as a risk factor for salmonellosis is unclear; we therefore captured information on PPI usage as part of our outbreak investigation. We conducted a case-control study, comparing each case with two controls. Adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were estimated using multivariable logistic regression. Thirty-nine of 61 eligible cases were included in the study. The median age of cases was 45 years; 56% were female. Of these, 33% were admitted to hospital and 31% reported taking PPIs. We identified an association between PPIs and non-typhoidal salmonellosis (aOR 8·8, 95% CI 2·0-38·3). There is increasing evidence supporting the existence of an association between salmonellosis and PPIs; however, biological studies are needed to understand the effect of PPIs in the pathogenesis of Salmonella. We recommend future outbreak studies investigate PPI usage to strengthen evidence on the relevance of PPIs in Salmonella infection. These findings should be used to support the development of guidelines for patients and prescribers on the risk of gastrointestinal infection and PPI usage.

Survival and transmission of potato virus Y, pepino mosaic virus, and potato spindle tuber viroid in water.

Hydroponic systems and intensive irrigation are used widely in horticulture and thus have the potential for rapid spread of water-transmissible plant pathogens. Numerous plant viruses have been reported to occur in aqueous environments, although information on their survival and transmission is minimal, due mainly to the lack of effective detection methods and to the complexity of the required transmission experiments. We have assessed the role of water as a source of plant infection using three mechanically transmissible plant pathogens that constitute a serious threat to tomato and potato production: pepino mosaic virus (PepMV), potato virus Y (PVY), and potato spindle tuber viroid (PSTVd). PepMV remains infectious in water at 20 ± 4°C for up to 3 weeks, PVY (NTN strain) for up to 1 week, and PSTVd for up to 7 weeks. Experiments using a hydroponic system show that PepMV (Ch2 genotype) and PVY (NTN strain) can be released from plant roots into the nutrient solution and can infect healthy plants through their roots, ultimately spreading to the green parts, where they can be detected after a few months. In addition, tubers developed on plants grown in substrate watered with PSTVd-infested water were confirmed to be the source of viroid infection. Our data indicate that although well-known pathways of virus spread are more rapid than water-mediated infection, like insect or mechanical transmission through leaves, water is a route that provides a significant bridge for rapid virus/viroid spread. Consequently, water should be taken into account in future epidemiology and risk assessment studies.

Impact of age on viral kinetics of peginterferon alfa-2a/ribavirin in chronic hepatitis C: final analysis from the PROPHESYS cohort.

The population of patients with chronic hepatitis C viral infection is ageing; however, elderly, hepatitis C-infected patients are understudied and less frequently treated. This subanalysis of data from the multinational PROPHESYS study examined associations between age (≤65 vs >65 years), on-treatment virological response and sustained virological response (SVR) in patients treated with peginterferon alfa-2a (40KD)/ribavirin in accordance with local licences. PROPHESYS comprised three cohorts studied in 19 countries according to country-specific legal and regulatory requirements. This subanalysis includes treatment-naive HCV mono-infected patients assigned to receive peginterferon alfa-2a (40KD)/ribavirin, with 6276 individuals aged ≤65 years and 349 aged >65 years. Rapid virological response (RVR) rates by Week 4 were consistently lower in older genotype (G) 1 (21.6% vs 27.2% in younger patients), G2 (80.7% vs 85.1%) and G3 (60.0% vs 74.2%) patients. SVR rates were significantly lower (29.8% vs 43.0%) and relapse rates significantly higher (43.1% vs 26.7%) in older G1 patients (P = 0.0002 vs ≤65 years). In contrast, SVR and relapse rates were similar in G2 and G3 patients regardless of age. The positive predictive value of RVR for SVR was comparable in older and younger G1 patients (66.7% vs 68.6%, respectively) and higher in older G2 (80.7% vs 75.6%) and G3 (77.8% vs 66.8%) patients. Virological response rates are generally lower in elderly CHC patients, and RVR is a reliable positive predictor of SVR in patients >65 years.

First Report of Stolbur Phytoplasma Associated with Maize Redness Disease of Maize in Bosnia and Herzegovina.

Maize redness (MR), caused by stolbur phytoplasma (16SrXII-A, 'Candidatus phytoplasma solani') and vectored by the cixiid planthopper Reptalus panzeri (Löw), is a severe and emerging disease of maize in southeastern Europe (2). Symptoms of MR include midrib, leaf, and stalk reddening, followed by desiccation of the entire plant, abnormal ear development, and incomplete kernel set. MR may cause significant economic losses (2). During 2010, 2011, and 2012, the presence of MR-like symptoms on maize accompanied by significant yield losses were frequently observed in maize fields in the Semberija region of northeastern Bosnia and Herzegovina. From mid-June to early July, potential vectors were collected using mouth-aspirators from maize plants in fields at three locations in the Semberija region where MR-like symptoms were previously observed. At the end of July, symptomatic maize plants were collected from six fields in the same region for phytoplasma identification. In addition, we sampled asymptomatic johnsongrass (Sorghum halepense L.), bindweed (Convolvulus arvensis L.), and volunteer wheat (Triticum aestivum L.) in areas adjacent to maize fields with MR-like symptoms, as potential phytoplasma reservoirs (2,3). A total of 49 plants (38 maize, 6 johnsongrass, 3 bindweed, and 2 wheat) and 43 R. panzeri were tested for the presence of stolbur phytoplasma. Leaves of four maize seedlings, grown in insect-proof greenhouse conditions, were used as controls. Total DNA was extracted from roots of each plant and R. panzeri using the CTAB methods (2). Initial phytoplasma detection was conducted on 16S rRNA gene using nested PCR assay with phytoplasma universal primers P1/P7 and F2n/R2 (4). Subsequently, all phytoplasma positive samples were retested employing stolbur-specific Stol11 protocol with the f2r/f3r2 primer set (1). Molecular characterization of identified phytoplasmas was performed by PCR-RFLP analysis of the tuf gene (3) and by sequence analyses of the 16S rRNA nested PCR products (GenBank Accession No. KC852868). All samples that tested positive on 16S rRNA gene using phytoplasma generic primers gave positive reaction in assays with stolbur-specific primers. Stolbur phytoplasma was identified in 36 of 49 plant samples (34 of 38 symptomatic maize plants and in 2 of 6 johnsongrass) and in 2 of 43 R. panzeri individuals. None of the control plants, bindweed, or wheat samples were positive for the presence of any phytoplasma. Tuf gene RFLP analyses enabled affiliation of all isolates to the stolbur type tuf-b. Comparison of the 16S rRNA sequence using BLAST analyses further confirmed identification of the phytoplasmas as being 'Candidatus phytoplasma solani.' The obtained sequence showed 100% identity with 'Candidatus phytoplasma solani' from corn in Serbia (JQ730750). These data clearly demonstrated association of stolbur phytoplasma with MR symptoms on maize in Semberija, which represents the first report of the MR disease and stolbur phytoplasma in maize, R. panzeri, and johnsongrass in Bosnia and Herzegovina. In the Semberija region, maize-wheat crop rotation is a traditional practice, which is a key factor for MR occurrence and persistence (2). References: (1) D. Clair et al. Vitis 42:151, 2003. (2) J. Jovic et al. Phytopathology 99:1053, 2009. (3) M. Langer and M. Maixner. Vitis 43, 191, 2004. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.

Outbreak of Shiga toxin-producing E. coli O157 associated with consumption of watercress, United Kingdom, August to September 2013.

An increase in the number of cases of Shiga toxin-producing Escherichia coli O157 PT 2 stx2 infection was reported in the United Kingdom on 9 September 2013. Of the 19 cases, 13 were interviewed, of which 10 reported consuming watercress purchased from one retailer. The retailer recalled pre-packed bagged salads containing watercress on 12 September. The descriptive epidemiology was supported by a case­case study performed after control measures were implemented.

The comparison of chronic hepatitis C treatment outcome between intravenous drug users and non-intravenous drug users.

UNLABELLED: Despite the fact that the majority of prevalent and incident cases of HCV are associated with intravenous drug use (IVDU), these patients have largely been excluded from HCV care. The aim of this study was to examine the treatment outcome of chronic hepatitis C in IVDUs compared to non-IVDUs. PATIENTS AND METHODS: Patients with chronic hepatitis C (CHC) who initiated and completed combination antiviral therapy with pegilated interferon and ribavirin, at the Hepatology Department of the University Hospital for Infectious and Tropical Diseases in Belgrade, were retrospectively analyzed. The study included a series of 254 patients of which 100 (39.4%) were former IVDU. RESULTS: Sustained virological response (SVR) was recorded in a total of 172 patients (67.7%). The analyses of the favorable treatment outcome, regarding particular viral genotypes, revealed that among those with genotype 1 and/or 4, including patients with genotype 1 recombinants with genotype 3, SVR was achieved in 114 (63.3%), while it was almost equally distributed between subgroups of former IVDU and all others (P=0.079). Among patients infected with HCV genotypes 2 and/or 3 the SVR rate was as high as 86.6%. CONCLUSION: IVDU with CHC infection should be treated with standard combination antiviral therapy for CHC, since the success rate is equal or even better than in non-IVDU patients.

First Report of Citrus tristeza virus in Citrus Orchards in Bosnia and Herzegovina.

The citrus growing area in Bosnia and Herzegovina (B&H) is limited to the confluence of the river Neretva, which is close to the Adriatic coastal region. Approximately 6 ha are grown in the country. Mandarins (Citrus reticulata Blanco) and lemons (Citrus limon L.) grafted on trifoliate orange (Poncirus trifoliata (L.) Raf.) are the most cultivated species. In June 2012, 25 samples were collected from individual trees from three locations in Herzegovina district of B&H (Mostar, Capljina, and Ljubuski). Samples of different Citrus spp. (C. reticulata Blanco, C. aurantium L., C. limon L., C. sinensis (L.) Osbeck, P. trifoliata (L.) Raf., and Fortunella margarita Lour) and varieties were collected from infield plants, commercial citrus orchards, and a nursery. Out of 25, 10 citrus trees exhibited leaves chlorosis, whereas as all others were apparently symptomless. Double antibody sandwich (DAS)-ELISA test, using commercial kit from the DSMZ, Germany (product code AS-0988), was carried out to confirm the presence of Citrus tristeza virus (CTV). In addition, further analyses were performed using reverse transcription (RT)-PCR targeting the coat protein gene (2). CTV was detected in 8 out of the 25 tested samples with DAS-ELISA, whereas CTV was detected in 14 samples by RT-PCR. Being grafted on P. trifoliata rootstock, no typical CTV symptoms in the field were observed on the CTV-infected trees; interestingly, the lab analyses evidenced the CTV presence in all inspected locations of the Herzegovina district. To our knowledge, this is the first report of CTV in Bosnia and Herzegovina; nevertheless, the virus presence is also reported from neighboring countries Croatia (1) and Montenegro (3). The PCR products of four samples were additionally analyzed by sequencing. The preliminary results by sequencing of the coat protein gene of four selected CTV isolates (Accessions HF947341, HF947342, HF947343, and HF947347) showed 99% nucleotide identity with the CTV resistance breaking isolates from Montenegro (FR871866) and Croatia (EU579422). Although a very small number of samples were tested in this study, CTV appears to be widely distributed in the citrus orchards of the country. This could be related to the traditional use of tolerant P. trifoliata rootstock that prevents the development of the tristeza decline as well as to the virus isolates present in the region, which appear not to cause another economically devastating CTV disease such as stem pitting. Further research will be dedicated to the biological properties of the genetic variability of these identified CTV isolates and the assessment of potential aphid vectors. References: (1) S. Cerni et al. Plant Dis. 89:342, 2005. (2) M. E. Hilf et al. Options Méditerranéennes B 65:89, 2009. (3) T. Papic et al. Plant Dis. 89:434, 2005.

Testosterone response of hepatic gene expression in female mice having acquired testosterone-unresponsive immunity to Plasmodium chabaudi malaria.

Blood-stage malaria of Plasmodium chabaudi is characterized by its responsiveness to testosterone (T): T suppresses development of protective immunity, whereas once acquired immunity is T-unresponsive. Here, we have analyzed the liver, a T target and lymphoid organ with anti-malaria activity, for its T-responsiveness of gene expression in immune mice. Using Affymetrix microarray technology, in combination with quantitative RT-PCR, we have identified (i) T-unresponsive expression of newly acquired mRNAs encoding diverse sequences of IgG- and IgM-antibodies, (ii) 24 genes whose expression has become T-unresponsive including those encoding the T(H)2 response promoting EHMT2 and the erythrocyte membrane protein band 7.2 STOM, (iii) T-unresponsive expression of mRNAs for the cytokines IL-1ß, IL-6, TNFα, and IFNγ, as well as iNOS, which are even not inducible by infection, and (iv) 35 genes retaining their T-responsiveness, which include those encoding the infection-inducible acute phase proteins SAA1, SAA2, and ORM2 as well as those of liver metabolism which encode the T-downregulated female-prevalent enzymes CYP2B9, CYP2B13, CYP3A41, CYP7A1, and SULT2A2 and the T-upregulated male-prevalent enzymes CYP2D9, CYP7B1, UGT2B1, HSD3B2, HSD3B5, respectively. The mRNA of the latter T-metabolizing enzyme is even 5-fold increased by T, suggesting a decrease in the effective T concentrations in the liver of immune mice. Collectively, our data suggest that the liver, which has acquired a selective T-unresponsiveness of gene expression, contributes to the acquired T-unresponsive, antibody-mediated protective immunity to blood-stage malaria of P. chabaudi.

A foodborne outbreak of Salmonella Bareilly in the United Kingdom, 2010.

We report the preliminary findings of the investigation of an outbreak of foodborne Salmonella Bareilly. Between August and November 2010, there were 231 laboratory-confirmed reports of S. Bareilly in the United Kingdom. A case­control study showed that consumption of bean sprouts was significantly associated with illness. The investigation concluded that raising public awareness to ensure the correct preparation of raw bean sprouts during cooking was the principal means of preventing further cases.

Distribution of HBV genotypes, subgenotypes and HBsAg subtypes among chronically infected patients in Serbia.

Hepatitis B virus (HBV) has been classified into eight genotypes (some of them further divided into two or more subgenotypes) and nine HBsAg subtypes, distinctly distributed geographically. The aim of this study was to gain insight into the distribution of HBV genotypes, subgenotypes and HBsAg subtypes among HBV chronically infected patients in Serbia, since there were no previously published data on this subject. Eighty-nine plasma samples that gave a positive result in a nested PCR were included for genotype identification. Genotyping was performed by direct sequencing of the part of the S/pol gene, and the HBsAg subtype was deduced from the HBsAg sequence. Two HBV genotypes, A and D, were encountered in Serbia, with genotype D (D - 82%, A - 18%) and subgenotype D3 (47.9%) being prevalent. Genotype D isolates had three assigned subtypes (ayw2, ayw3, ayw4), with ayw2 found to be the most prevalent (ayw2 - 53.4%, ayw3 - 43.8%, ayw4 - 1.4%). Genotype A isolates belonged to the A2 subgenotype and the HBsAg subtype adw2, as expected for samples from European population. The results correspond to country's geographical position, being in close proximity to the Mediterranean basin and on the main route between the Middle East and Central Europe.

[Primary liver cancer mortality in Belgrade population]. / Mortalitet od primarnih malignih tumora jetre u populaciji Beograda.

The aim of this analysis was to estimate death rates for primary liver cancer (PLC) and their secular trends in the population of Belgrade during the period 1982-2001. Mortality data (number of deaths caused by PLC) in Belgrade were obtained from the Belgrade Municipal Institute of Statistics (unpublished data). Death rates per 100,000 population were adjusted by direct method using world population as a standard. Regression coefficient was assessed by Fisher's test., Average annual age-adjusted mortality rate for PLC in Belgrade was 5.7/100,000 (95%CI-confidence interval 5.3-6.0), for males 7.5/100,000 (95%CI 7.0-7.9), and for females 4.2/100,000 (95%CI 3.8-4.7). The lowest mortality for PLC was in the age up to 45 (bellow 1/100,000) for both sexes, and the highest values were registered in the age group over 75, 71.1/100,000 (95%CI 62.2-84.0) for males, and 52.9/100,000 (95%CI 45.6-60.3) for females. Mortality trends for PLC in Belgrade in the period 1982-2001 showed increasing tendency in males (y=7.079+0.039x, p=0.331) and decreasing tendency in females (y=4.747-0.048x, p=0.199).

Salivary immunoglobulin A and G subclasses in HIV positive patients.

Many of the opportunistic infections in HIV are related to the mucous membranes, but the protection of oral mucosal compartment mediated by IgA and IgG subclasses in saliva is still not well documented. Therefore, the aim of this study was to investigate the status of salivary IgA and IgG subclass in HIV positive patients. Levels of IgG1, IgG2, IgG3 and IgG4 were determined by ELISA and IgA1 and IgA2 by radial immunodiffusion in the resting whole saliva of 33 HIV patients and 21 HIV-seronegative healthy controls and were expressed in mg/L (IgA) and ug/L (IgG) of saliva. Salivary IgA2 was significantly reduced in the HIV positive patients (p<0.0009) when compared to the healthy controls, but no differences in salivary IgA1 level between HIV patients and healthy controls were found. On the other hand, salivary IgG1, IgG2, IgG3 and IgG4 subclasses were increased (p<0.0009) in HIV patients in comparison to the healthy controls. We can conclude that inspite of IgA2 deficiency which was found in studied HIV positive patients, mucosal antibody responses are quite normal and might not predispose development of oral opportunistic infections.

[Modern microsurgery of the eye]. / Savremena mikrohirurgija oka.

On the basis of obtained results on the University Eye Clinic of Novi Sad, the modern aspects of the most frequent microsurgical operations in ophthalmology are presented. Cataract operations with endocapsular implantation of IOL result in improvement of visual function and daily activities in 98% of patients. Vitreoretinal surgery leads anatomical and functional improvement in 50% to 85% of all cases in correlation with pathology. Different laser microsurgical techniques are used in vascular retinal diseases, corneal refractive surgery, prevention and treatment of glaucoma, secondary cataract treatment, and treatment of diabetic retinopathy.

[The circadian rhythm of serum aldosterone in the diagnostics of primary aldosteronism]. / Dnevni ritam aldosterona u serumu u dijagnostici primarnog aldosteronizma.

Diagnosis of primary aldosteronism should be easy in patients with unprovoked hypokaliemia and hypertension. However, it is more difficult to differentiate patients with primary normokaliemic aldosteronism from those with essential hypertension with low plasma renin activity. The aim of this study was to evaluate the appropriate testing of diagnosis and differential diagnosis of primary aldosteronism. The study group consisted of 17 patients with primary aldosteronism (12 with aldosteronoma and 5 with bilateral hyperplasia) and 20 patients with essential hypertension. The finding of increased serum aldosterone level using a single measurement, without anti-hypertensive therapy and on normal sodium intake is sufficient to confirm the diagnosis of primary aldosteronism with specificity of 95% and sensitivity of 94%. Upright posture test is shown to be fairly good tool in differentiation aldosteronoma and bilateral adrenal hyperplasia. Specificity of this test is 100% and sensitivity 88%. Although primary aldosteronism is not a common cause of hypertension it is reasonable to screen for this disorder because complete cure can be provided in a large percentage of patients.

Epidemiology of hepatitis D virus (delta) infection in Yugoslavia.

In 614 HBsAg-positive Yugoslavian patients, radioimmunoassay testing for anti-delta showed the presence of this antibody in serum in 11.2%. Of the patients, 213 belonged to a risk group (i.v. drug users, hemophiliacs, hemodialysed patients and patients with posttransfusion hepatitis); a significant number of these patients (63; 29.6%) were found to have anti-delta. A second group was composed of 401 HBsAg-positive patients from the general population (patients with acute hepatitis B, with fulminant hepatitis B and patients with chronic HBV infection); delta infection was found only in six (1.5%). Immunohistochemical methods failed to demonstrate the delta antigen in the livers of 73 patients with chronic HBV infection. Testing the liver of 36 patients with fulminant hepatitis B for delta antigen demonstrated this reactivity in only one (2.8%) liver sample. Delta antigen was also found in the liver of a female patient who underwent biopsy in 1972. The results of this study suggest the HDV is not endemic in Yugoslavia; however, it is frequently found in patients at risk of blood exposure, primarily i.v. drug users.

[Diagnostic and prognostic criteria in acute non-A, non-B viral hepatitis with a prolonged course]. / Dijagnosticki i prognosticki kriterijumi u akutnom virusnom hepatitisu "ni-A-ni-Be" produzenog toka.

In a prospective three-year-long study of Non-A, Non-B infections carried out in the Institute of Infectious and Tropical Diseases in Belgrade, by a method of exclusion and in about 30% of patients' liver biopsies, 75 cases of acute viral hepatitis Non-A, Non-B were discovered. It makes only 2.29% of all cases of acute viral hepatitis diagnosed and treated in the same period in this institution. In prolonged forms of the disease (24 cases) the chronicity was shown by epidemiological data (posttransfusion hepatitis), fluctuation of transaminase and rise of gamma globulins in acute phase of the disease. Liver biopsies performed in the first four months of the disease were of no prognostic value.