Differences in physiological and biochemical responses to short-term flooding among the three avocado (Persea americana Mill.) races.

Avocado (P. americana Mill.) trees are classified into three botanical races, Mexican (M), Guatemalan (G), and West Indian (WI), each distinguished by their geographical centers of origin. While avocados are considered highly sensitive to flooding stress, comparative responses of the different races to short-term flooding are not known. This study assessed the differences in physiological and biochemical responses among clonal, non-grafted avocado cultivars of each race to short-term (2-3 days) flooding. In two separate experiments, each with different cultivars of each race, container-grown trees were divided into two treatments: 1) flooded and 2) non-flooded. Net CO2 assimilation (A), stomatal conductance (gs), and transpiration (Tr) were measured periodically over time beginning the day before treatments were imposed, through the flooding period, and during a recovery period (after unflooding). At the end of the experiments, concentrations of sugars in leaves, stems, and roots, and reactive oxygen species (ROS), antioxidants, and osmolytes in leaves and roots were determined. Guatemalan trees were more sensitive to short-term flooding than M or WI trees based on decreased A, gs, and Tr and survival of flooded trees. Guatemalan trees generally had less partitioning of sugars, particularly mannoheptulose, to the roots of flooded compared to non-flooded trees. Principal component analysis showed distinct clustering of flooded trees by race based on ROS and antioxidant profiles. Thus, differential partitioning of sugars and ROS and antioxidant responses to flooding among races may explain the greater flooding sensitivity of G trees compared to M and WI trees.

Spectral light distribution affects photosynthesis, leaf reflective indices, antioxidant activity and growth of Vanillaplanifolia.

Vanilla planifolia is an obligate sciophyte (shade plant) with crassulacean acid metabolism (CAM) photosynthesis. Plants were grown for 12 months under black, blue, green, or red photoselective shade netting (PSN) to alter the spectral light distribution impacting the plants. Light wavelengths were measured in each treatment and plants were assessed for photosynthetic characteristics, leaf chlorophyll index (LCI), maximum quantum yield of photosystem II, leaf reflectance indices, leaf area, growth, antioxidant enzymes, lipid peroxidation, reactive oxygen species (ROS), and osmolyte content. Plants grown under red PSN had a higher quantity of red and far-red light and had greater nocturnal net CO2 assimilation (NocA), leaf area and leaf dry weight than plants in the other treatments. Plants grown under blue PSN had a higher quantity of blue light, resulting in a higher LCI and maximum quantum yield than plants in the other treatments. Plants grown under the red and blue PSN had increased leaf spectral reflectance indices compared to plants in the other treatments, which resulted in the highest levels of antioxidant scavenging enzymes, ascorbic acid (AsA), proline, and glycine betaine, and the lowest levels of H2O2. These findings demonstrate that increasing light in the red and far-red or blue portions of the spectrum by using PSN alters the photosynthetic and/or antioxidant responses of V. planifolia and increasing red and far-red light by using red PSN can also accelerate plant growth, possibly due to higher photosynthesis.

Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type oppF1 influences its growth characteristics.

The Mycoplasma synoviae (MS) vaccine strain MS-H harbours a frameshift mutation in oppF1 (oligopeptide permease transporter) which results in expression of a truncated OppF1. The effect of this mutation on growth and attenuation of the MS-H is unknown. In this study, the impact of the mutation on the vaccine phenotype was investigated in vitro by introducing a wild-type copy of oppF1 gene in the MS-H genome. Wild-type oppF1 was cloned under the vlhA promoter into an oriC vector carrying a tetracycline resistance gene. MS-H was successfully transformed with the final construct pMS-oppF1-tetM or with a similar vector lacking oppF1 coding sequence (pMS-tetM). The MS-H transformed with pMS-oppF1-tetM exhibited smaller colony size than MS-H transformed with pMS-tetM. Monospecific rabbit sera against C-terminus of OppF1 detected bands of expected size for full-length OppF1 in the 86079/7NS parental strain of MS-H and the MS-H transformed with pMS-oppF1-tetM, but not in MS-H and MS-H transformed with pMS-tetM. Comparison of the growth curve of MS-H transformants harvested from media with/without tetracycline was conducted using vlhA Q-PCR which revealed that MS-H transformed with pMS-tetM had a higher growth rate than MS-H transformed with pMS-oppF1-tetM in the media with/without tetracycline. Lastly, the whole genome sequencing of MS-H transformed with pMS-oppF1-tetM (passage 27) showed that the chromosomal copy of the mutated oppF1 had been replaced with a wild-type version of the gene. This study reveals that the truncation of oppF1 impacts on growth characteristics of the MS-H and provides insight into the molecular pathogenesis of MS and perhaps broader mycoplasma species.RESEARCH HIGHLIGHTS The full-length OppF1 was expressed in Mycoplasma synoviae MS-H vaccine.Truncation of oppF1 impacts on growth characteristics of the MS-H.Chromosomal copy of the mutated oppF1 in MS-H was replaced with wild-type oppF1.

Comparative genomic analyses of Mycoplasma synoviae vaccine strain MS-H and its wild-type parent strain 86079/7NS: implications for the identification of virulence factors and applications in diagnosis of M. synoviae.

Mycoplasma synoviae is an economically important avian pathogen worldwide, causing respiratory disease, infectious synovitis, airsacculitis and eggshell apex abnormalities in commercial chickens. Despite the widespread use of MS-H as a live attenuated vaccine over the past two decades, the precise molecular basis for loss of virulence in this vaccine is not yet fully understood. To address this, the whole genome sequence of the vaccine parent strain, 86079/7NS, was obtained and compared to that of the MS-H vaccine. Except for the vlhA expressed region, both genomes were nearly identical. Thirty-two single nucleotide polymorphisms (SNPs) were identified in MS-H, including 11 non-synonymous mutations that were predicted, by bioinformatics analysis, to have changed the secondary structure of the deduced proteins. One of these mutations caused truncation of the oppF-1 gene, which encodes the ATP-binding protein of an oligopeptide permease transporter. Overall, the attenuation of MS-H strain may be caused by the cumulative and complex effects of several mutations. The SNPs identified in MS-H were further analyzed by comparing the MS-H and 86079/7NS sequences with the strains WVU-1853 and MS53. In the genomic regions conserved between all strains, 30 SNPs were found to be unique to MS-H lineage. These results have provided a foundation for developing novel biomarkers for the detection of virulence in M. synoviae and also for designing new genotyping assays for discrimination of MS-H from field strains.

Seroprevalence and risk factors of avian influenza H9 virus among poultry professionals in Rawalpindi, Pakistan.

BACKGROUND: Avian influenza H9 is endemic in commercial and backyard poultry in Pakistan and is a serious occupational health hazard to industry workers. This study aimed to determine the seroprevalence of avian influenza H9 infection in people working with poultry in Rawalpindi, Pakistan and assess the measures they took to protect themselves from infection. METHODS: A cross-sectional study was conducted from December 2016 to May 2017 of 419 people working with poultry in Rawalpindi Division, including farm workers, vaccinators, field veterinarians, butchers and staff working in diagnostic laboratories. Potential participants were randomly approached and gave written consent to participate. Data were collected using a standardized questionnaire and serum samples were processed to detect H9 antibodies using the haemagglutination inhibition test. RESULTS: Of the 419 participants, 406 (96.9%) were male. The mean age of the participants was 36.4 (SD 10.86) years. A total of 332 participants agreed to a blood test, 167 of whom were positive for A(H9) antibodies, giving an overall seroprevalence of 50.3%. Laboratory staff had the highest seroprevalence (100%) and veterinarians the lowest (38.5%). Vaccinators, butchers and farm workers had a seroprevalence of 83.3%, 52.4% and 45.5% respectively. Personals who used facemasks had significantly lower (P<0.002) seroprevalence (29.6%) than those who never used them (90.6%). Similarly, those who always used gloves and washed their hands with soap had a seroprevalence of 32.8% compared with 89.0% in those who never took these precautions. Of the participants who handled antigens, 92.3% were seropositive. CONCLUSION: Laboratory staff and vaccinators are exposed to viral cultures and influenza vaccines respectively which may explain their high seroprevalence.

Exopolysaccharides producing rhizobacteria and their role in plant growth and drought tolerance.

Drought stress not only effect the population and activities of microorganisms inhabiting the rhizosphere but also various physiological and biochemical process in plants that is, photosynthesis, respiration, translocation, uptake of ions, carbohydrates, and nutrient metabolism. Plant growth promoting rhizobacteria (PGPR) and their exopolysaccharides (EPS) showed profound effects on plant growth and drought tolerance. Reactions of bacteria to drought stress at various organizational levels are different which depends on intensity of stress, duration, species, and growth stage. PGPR could be effectively utilized in developing strategies to facilitate water conservation strategies of plants. They have the ability to improve plant growth directly by enhancing level of phytohormones, siderophore, biofilm, and exopolysaccharides production and by increasing the nutrient availability in the rhizosphere or indirectly by protecting plants from pathogen attack. EPS producing bacteria are capable to maintain higher soil moisture content and growth of plants even under severe dried sandy soils. The evidence of survival of rhizobacteria under low moisture content obtained from the fact of rhizobacterial occurrence in the soil of desert and effective nodule formation in desert soil. Beside this, EPS produced by PGPR form rhizosheath around the roots and thus protect the plant roots from desiccation for a longer period of time. Important role exhibited by exopolysaccharides includes, protection from desiccation, microbial aggregation, plant-microbe interaction, surface attachment, bioremediation and its use by many industries for stabilizing, thickening, coagulating, gelling, suspending and for film forming. Plants inoculated with EPS-producing bacteria showed higher accumulation of proline, sugars, and free amino acids under water deficit stress. Biofilms formed by the PGPR around the roots are made up of bacterial populations or bacterial communities that encased inside the polymeric extracellular matrix formed by bacteria itself, they adhered to the external surfaces that contain sufficient moisture. It is concluded that the application of PGPR in combination with their EPS is a promising measure to combat drought stress thus, increasing global food security.

A Challenging Case of Retroperitoneal Abscess in a Post-Partum Crohn's Disease Patient.

BACKGROUND Crohn's disease (CD) is an inflammatory bowel disease affecting approximately 1 in 3000 people in the United States. Since the inflammation of CD is transmural, patients are at risk for fistula and abscess formation. Retroperitoneal abscesses are one type of which physicians must be aware. CASE REPORT We present the case of a 29-year-old woman with CD who complained of right hip and flank pain that began when she was 6-months pregnant. After delivery, she continued to complain of severe right flank pain and was admitted to the hospital 1 month later. CT scan imaging revealed a complicated retroperitoneal and right flank abscess, possibly due to a colonic intramural fistula. She developed severe acute necrotizing soft-tissue infection requiring 13 days of intensive care. She required debridement of the necrotizing infection of the right flank, drainage of the abscess, and washout for intraperitoneal sepsis. The patient tolerated the procedures well and was discharged 1 month later. CONCLUSIONS Given that the clinical manifestation of retroperitoneal fistula with abscess is insidious and its formation is less common than intraperitoneal abscesses, we hope healthcare providers learn from this case to avoid morbidity and mortality. When presented with a pregnant CD patient complaining of nonspecific abdominal symptoms, providers should consider fistulization and/or abscess formation. The option to evaluate pregnant patients using noninvasive methods, such as ultrasound or low-dose CT scan, can decrease radiation exposure to the fetus and prevent delays in diagnosis and treatment.

Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type obg influencing its growth characteristics.

The temperature-sensitive (ts+) Mycoplasma synoviae vaccine strain MS-H harbors a non-synonymous mutation which results in Glycine to Arginine substitution at position 123 in the highly conserved glycine-rich motif of Obg-fold in the GTP-binding protein Obg. In-silico analysis of the wild-type and mutant Obgs of M. synoviae has indicated that this amino acid substitution affects structure of the protein, potentially leading to abrogation of Obg function in vivo. Present study was conducted to develop the first expression vector for M. synoviae and to investigate the potential effect(s) of complementation of MS-H vaccine with the wild-type obg from 86079/7NS, the parent strain of MS-H. An oriC vector, pKS-VOTL, harboring the 86079/7NS obg gene, downstream of the variable lipoprotein haemagglutinin (vlhA) gene promoter, also cloned from 86079/7NS, was used to transform MS-H. The plasmid was localised at the chromosomal oriC locus of MS-H without any detectable integration at the chromosomal obg locus. Analysis of the MS-H transformants revealed abundant obg transcripts as well as Obg protein, when compared to the MS-H transformed with a similar vector, pMAS-LoriC, lacking obg coding sequence. The MS-H transformants complemented with wild-type Obg maintained their original temperature-sensitivity phenotype (consistent with MS-H vaccine) but, when compared to the MS-H transformed with pMAS-LoriC, had significantly higher (p < 0.05) growth rate and viability at the permissive (33°C) and non-permissive temperature (39.5°C), respectively. Analysis of Obg expression in MS-H and its wild-type parent strain revealed comparatively lower levels of Obg in MS-H. These results indicate that not only the mutation in Obg, but also the level of Obg expression, can confer functional abnormalities in the bacterial host. Furthermore, with the construction of first expression vector for M. synoviae, this study has set foundation for the development of recombinant vaccine(s) based on MS-H.

Genome analysis of Mycoplasma synoviae strain MS-H, the most common M. synoviae strain with a worldwide distribution.

BACKGROUND: The bacterial pathogen Mycoplasma synoviae can cause subclinical respiratory disease, synovitis, airsacculitis and reproductive tract disease in poultry and is a major cause of economic loss worldwide. The M. synoviae strain MS-H was developed by chemical mutagenesis of an Australian isolate and has been used as a live attenuated vaccine in many countries over the past two decades. As a result it may now be the most prevalent strain of M. synoviae globally. Differentiation of the MS-H vaccine from local field strains is important for epidemiological investigations and is often required for registration of the vaccine. RESULTS: The complete genomic sequence of the MS-H strain was determined using a combination of Illumina and Nanopore methods and compared to WVU-1853, the M. synoviae type strain isolated in the USA 30 years before the parent strain of MS-H, and MS53, a more recent isolate from Brazil. The vaccine strain genome had a slightly larger number of pseudogenes than the two other strains and contained a unique 55 kb chromosomal inversion partially affecting a putative genomic island. Variations in gene content were also noted, including a deoxyribose-phosphate aldolase (deoC) fragment and an ATP-dependent DNA helicase gene found only in MS-H. Some of these sequences may have been acquired horizontally from other avian mycoplasma species. CONCLUSIONS: MS-H was somewhat more similar to WVU-1853 than to MS53. The genome sequence of MS-H will enable identification of vaccine-specific genetic markers for use as diagnostic and epidemiological tools to better control M. synoviae.

Tritrophic interactions between parasitoids and cereal aphids are mediated by nitrogen fertilizer.

Host plant nutritional quality can directly and indirectly affect the third trophic levels. The aphid-parasitoid relationship provides an ideal system to investigate tritrophic interactions (as the parasitoids are completely dependent for their development upon their hosts) and assess the bottom up forces operating at different concentrations of nitrogen applications. The effects of varying nitrogen fertilizer on the performance of Aphidius colemani (V.) reared on Sitobion avenae (F.) and Aphidius rhopalosiphi (D.) reared on Rhopalosiphum padi (L.) were measured. Parasitism and percent emergence of parasitoids were positively affected by nitrogen fertilizer treatments while developmental duration (egg, larval, and pupal stages) was not affected by increasing nitrogen inputs. In males and females of both parasitoid species, adult longevity increased with the increasing nitrogen fertilizer. Hind tibia length and mummy weight of both parasitoid species increased with nitrogen fertilizer concentrations, as a result of larger aphids. This study showed that nitrogen application to the soil can have important consequences for aboveground multitrophic interactions.

Development of an oriC vector for use in Mycoplasma synoviae.

Mycoplasma synoviae, an important poultry pathogen, belonging to the class Mollicutes, causes airsacculitis, synovitis, decreased egg production and produces significant economic losses. Efforts to determine M. synoviae virulence factors and their role in pathogenicity require suitable tools for genetic manipulation of this pathogen. This study describes, for the first time, the identification and cloning of the origin of replication (oriC) of M. synoviae to develop a replicable oriC vector for this mycoplasma. Shuttle vectors containing different putative oriC regions along with tetracycline resistance gene tetM were constructed to transform M. synoviae. An oriC vector, pMAS-LoriC, harbouring the complete dnaA gene along with upstream and downstream DnaA boxes, successfully transformed M. synoviae at an average transformation frequency of 1.07×10(-8) transformants per colony-forming unit (CFU), and remained freely replicating as well as integrated at the chromosomal oriC. Plasmid copy number for pMAS-LoriC was estimated to be 62±29 (average±SD) per cell. This study also provided evidence of the occurrence of homologous recombination and the functionality of the heterologous tetM determinant in M. synoviae. The transformation technique and the oriC vector developed in this study have the potential to be used in targeted gene disruption, gene complementation and expression studies in this organism.

High-resolution melting-curve analysis of obg gene to differentiate the temperature-sensitive Mycoplasma synoviae vaccine strain MS-H from non-temperature-sensitive strains.

Temperature-sensitive (ts+) vaccine strain MS-H is the only live attenuated M. synoviae vaccine commercially available for use in poultry. With increasing use of this vaccine to control M. synoviae infections, differentiation of MS-H from field M. synoviae strains and from rarely occurring non-temperature-sensitive (ts-) MS-H revertants has become important, especially in countries where local strains are indistinguishable from MS-H by sequence analysis of variable lipoprotein haemagglutinin (vlhA) gene. Single nucleotide polymorphisms (SNPs) in the obg of MS-H have been found to associate with ts phenotype. In this study, four PCRs followed by high-resolution melting (HRM)-curve analysis of the regions encompassing these SNPs were developed and evaluated for their potential to differentiate MS-H from 36 M. synoviae strains/isolates. The nested-obg PCR-HRM differentiated ts+ MS-H vaccine not only from field M. synoviae strains/isolates but also from ts- MS-H revertants. The mean genotype confidence percentages, 96.9±3.4 and 8.8±11.2 for ts+ and ts- strains, respectively, demonstrated high differentiating power of the nested-obg PCR-HRM. Using a combination of nested-obg and obg-F3R3 PCR-HRM, 97% of the isolates/strains were typed according to their ts phenotype with all MS-H isolates typed as MS-H. A set of respiratory swabs from MS-H vaccinated specific pathogen free chickens and M. synoviae infected commercial chicken flocks were tested using obg PCR-HRM system and results were consistent with those of vlhA genotyping. The PCR-HRM system developed in this study, proved to be a rapid and reliable tool using pure M. synoviae cultures as well as direct clinical specimens.

Mutations in GTP binding protein Obg of Mycoplasma synoviae vaccine strain MS-H: implications in temperature-sensitivity phenotype.

Mycoplasma synoviae strain MS-H, developed by chemical mutagenesis of the Australian field strain 86079/7NS, is a live temperature-sensitive (ts (+)) vaccine used for control of M. synoviae infection in poultry worldwide. Genetic basis of temperature sensitivity and attenuation of MS-H has not been revealed thus far. Comparison of the complete genome sequence of MS-H, its parent strain 86079/7NS and two non-temperature sensitive (ts (-)) reisolates of MS-H revealed a mutation in a highly conserved domain of GTP binding protein Obg of MS-H, with reversion in ts (-) MS-H reisolates. Nucleotide change from G to A at position 369 of the obg gene resulted in an alteration of glycine to arginine at position 123 in Obg fold. Further analysis of the complete obg gene sequence in several MS-H reisolates revealed that a Gly123Arg substitution was associated with alteration in temperature sensitivity phenotype of MS-H. A second mutation, C to T at position 629, in obg gene was found in some of the MS-H reisolates and appeared to suppress the effects of the Gly123Arg substitution. In silico analysis of point mutations revealed that Gly123Arg has highly destabilizing effect on the MS-H Obg structure that can potentially abolish its biological functions in vivo especially at non-permissive temperature. Findings of this study implicate Obg alteration (Gly123Arg) as one of the possible causes of MS-H attenuation/temperature sensitivity and warrant further investigations into exploring the role of Obg-like proteins, an evolutionarily conserved protein from human to bacteria, in the biology of mycoplasmas.

Combination of differential growth at two different temperatures with a quantitative real-time polymerase chain reaction to determine temperature-sensitive phenotype of Mycoplasma synoviae.

Mycoplasma synoviae infections result in significant economic losses in the chicken and turkey industries. A commercially available live temperature-sensitive (ts (+)) vaccine strain MS-H has been found to be effective in controlling M. synoviae infections in commercial layer and broiler breeder farms in various countries, including Australia. Detection and differentiation of MS-H from field strains (ts (-)) and from ts (-) MS-H reisolates in vaccinated flocks is vital in routine flock status monitoring. At present microtitration is the only available technique to determine the ts phenotype of M. synoviae. This technique is time consuming and not amenable to automation. In the present study, a quantitative real-time polymerase chain reaction (Q-PCR) was combined with simultaneous culturing of M. synoviae at two different temperatures (33°C and 39.5°C) to determine the ts phenotype of 22 Australian M. synoviae strains/isolates. The M. synoviae type strain WVU-1853 was also included for comparison. A ratio of the copy numbers of the variable lipoprotein haemagglutinin (vlhA) gene at the two temperatures was calculated and a cut-off value was determined and used to delineate the ts phenotype. In all M. synoviae strains/isolates tested in this study, the ts phenotype determined using Q-PCR was in agreement with that determined using conventional microtitration. Combination of Q-PCR with differential growth at two different temperatures is a rapid, reliable and accurate technique that could be used as an effective tool in laboratories actively involved in ts phenotyping of M. synoviae strains/isolates.