Investigation of Marek’s disease virus from chickens in central Ethiopia

Marek’s disease (MD) is a lymphoproliferative and neuropathic disease of domestic chickens and less commonly, turkeys and quails, caused by a highly contagious, cell-associated, oncogenic herpesvirus. In Ethiopia, MD is believed to be introduced with importation of exotic and crossbred to improve the poultry production and has been reported to be a potential threat to the poultry sector both in backyard and commercial farming systems. This study was aimed at isolation and molecular analysis of MD virus isolates circulating in chicken population in the central part of Ethiopia where commercial farms are populated. From September 2013 to January 2014, clinical and post-mortem examination were conducted on diseased chickens suspected of MD virus infection. Representative spleen and feather follicle samples were collected following sterile procedure, and infectious virus isolation was performed using primary chicken fibroblast cell culture. Cell culture inoculated with suspension of pathological samples developed characteristic MD virus cytopathic effect of rounding of the cells and small plaques. Further analysis of the virus was conducted by conventional PCR amplifying the ICP4 gene fragment from eleven tissue samples using MD virus specific primers. PCR products were further sequenced and analyzed. Nucleotide sequence similarity search of the local isolates resulted a high degree of sequence similarity with Gallid Herpes virus type 2 strain (Marek’s disease virus type 1, JN034558). To our knowledge, the present study is the first report conducted on virus isolation and molecular characterization of MD virus isolates circulated in Ethiopia. Eleven ICP4-like gene fragment (318 bp) sequences generated in the present study were uploaded in the public database (KU842366–76). Further research on virus isolation, genetic characterization, and infection dynamics is recommended targeting chickens of all age groups reared in different agro-ecological zones under different production system.

     

Microhabitat use, not temperature, regulates intensity of Gyrodactylus cichlidarum long-term infection on farmed tilapia--are parasites evading competition or immunity?

Gyrodactylids (Monogenea) are ectoparasites of fish, some of which negatively affect commercially valuable fishes. Temperature strongly regulates population dynamics of these viviparous flatworms in farmed and wild fish populations, with most gyrodactylid species showing positive temperature-abundance associations. In agreement with epidemiological theory, numerous laboratory studies demonstrate that these parasites cannot persist in confined fish populations without periodic introduction of susceptible hosts. Extinction of gyrodactylid populations is due to host immunity, which develops in several fish species. In this one-year study, we followed populations of the recognized pathogen Gyrodactylus cichlidarum infecting four genetic groups of confined tilapia (wild type Nile tilapia Oreochromis niloticus niloticus, red O. n. niloticus, Mozambique tilapia O. mossambicus and a red synthetic population called Pargo-UNAM) kept under farming conditions and subject to natural environmental fluctuations. Based on the antecedents given, we postulated the following three hypotheses: (1) parasite abundance will be regulated by water temperature; (2) parasites will induce host mortality, particularly during periods of rapid infrapopulation growth; and (3) gyrodactylid populations will eventually become extinct on confined fish hosts. We disproved the three hypotheses: (1) parasite numbers fluctuated independently of temperature but were associated to changes in microhabitat use; (2) although gyrodactylid populations exhibited considerable growth, no evidence was found of negative effects on the hosts; and (3) infections persisted for one year on confined fish. Microhabitat use changed over time, with most worms apparently migrating anteriorly from the caudal fin and ending on the pectoral fins. Gyrodactylid populations followed similar trajectories in all fish, aggregating and dispersing repeatedly. Several instances were found where increased parasite dispersion coincided with increased intensity of infection; as well as the opposite, where increased aggregation coincided with parasite population declines. Three alternative explanations could account for these observations: that parasites (1) experience differential mortality on different anatomical regions of the fish; (2) migrate to avoid intraspecific competition; and (3) migrate to escape localized immune responses induced by infection. Our data do not allow us to demonstrate which of these alternatives is correct, so we discuss the merits of each. We provide circumstantial evidence in support of the third explanation, because as shown in other fish host-gyrodactylid interactions where immune responses have been characterized, in this study worms progressively moved away from fins with high mucus cell density to those with low density - what would be anticipated if immune defenses occur and reach the fish surface through mucus.     

Induction of Freezing Tolerance by the Application of Hydrogen Peroxide and Salicylic Acid as Tuber-Dip or Canopy Spraying in <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L. Plants

Low temperature stress is a current challenge to plants that is associated with climate change. In plants, exposure to extreme temperatures is followed by the accumulation of reactive oxygen species, such as hydrogen peroxide (H₂O₂), leading to oxidative stress. Salicylic acid (SA) and H₂O₂ mediate the tolerance responses to stress and have been reported to induce freezing tolerance in potato microplants. The objectives of the present investigation were (1) to evaluate the short- and long-term effects of H₂O₂ and SA treatments on freezing tolerance in potato (Solanum tuberosum L.) plants grown from tubers and (2) to analyse the relationship between catalase (CAT) activity and H₂O₂ concentration associated with freezing tolerance responses. We observed the lowest freezing survival rates in 45-day-old potato plants (cv. Granate) compared to younger plants. The two treatments consisted of (1) the tuber-dip (long-term) treatment in which sprouted minitubers were saturated for 1 h in SA 10^?5 M or H₂O₂ 1 mM and planted in soil under greenhouse conditions and (2) the crop-spray (short-term) treatment in which plants 5–8 cm high were sprayed twice a week with SA 10^?5 M or H₂O₂ 1 mM until 45 days of age. In all treatments, 45-day-old plants were then exposed to ? 6?±?1 °C for 4 h. The survival rate was measured 15 days after freezing. CAT and H₂O₂ measurements were performed 1 h before and after the freezing treatment. The results showed that SA and H₂O₂ induced freezing tolerance in both the short- and long-term treatments. Survival was significantly higher in SA- and H₂O₂-treated plants than in control plants. In both the long- and short-term treatments this higher survival was associated with lower internal H₂O₂ concentrations after freezing compared with control plants and decreasing oxidative stress. SA and H₂O₂ induced different levels of CAT activity after freezing compared to that found in the control plants in the long- and the short-term treatments. These results suggest the SA and H₂O₂ function in independent pathways in terms of their induction of freezing tolerance that depends on the method the treatment was applied, by spraying the canopy or by immersion of the sprouted seed tuber.     

A versatile method for assessing pathogenicity of Hymenoscyphus fraxineus to ash foliage

We describe a method for inoculating rachises of Fraxinus excelsior (European or common ash) with Hymenoscyphus fraxineus, which is faster than previous methods and allows associated foliar symptoms to be assessed on replicate leaves. A total of ten ash seedlings were inoculated with five isolates of H. fraxineus and lesion development assessed over four weeks. A five‐point disease progress scale of symptom development was developed from no lesion (0), lesion on rachis (1), “pre‐top dead,” with curling of distal leaflets and bending of the rachis (2), top dead, with wilting and death of distal leaflets (3) to leaf abscission (4). The method revealed variation in aggressiveness of H. fraxinus isolates and may be suitable for assessing the resistance of F. excelsior and other Fraxinus species to dieback. The in vitro growth rate of H. fraxineus isolates was highly correlated with both disease progress and the length of rachis lesions on susceptible plants, indicating that it can be used as a preliminary step in selecting isolates with high aggressiveness for use in resistance screening.     

Field performance of new in vitro androgenesis-derived double haploids of barley

Although barley is extensively grown in the Peruvian highlands as a food crop, agricultural and quality performance have historically been poor. A double-haploid technique was used to obtain barley varieties better suited to this environment. Three crosses were constructed: Ya/LM94, B16/LM94 and B12/LM94. From all crosses, F₁ anthers were cultured in vitro to produce double-haploid lines. At DH₆, promising materials were evaluated in the Peruvian highlands, along with their parents and two commercial controls (PPch, UNA80). Expert agronomic criteria for the ideal barley cultivar for this region were defined and Euclidean distance was used for simultaneous selection of the recorded agricultural traits. Ya/LM94-PC27 exhibited the shortest Euclidean distance to the expert criteria (0.41), higher yield than controls, and low plant height. Periods from first rain to flowering and maturity suited growth during the rainy season. Ya/LM94-PC27 was resistant to stripe rust, and its grain protein content suited the malting-brewing industry. Hectoliter and 1,000-grain masses met or surpassed industry requirements.     

Een bladvlekkenziekte van prei,veroorzaakt door Pleospora herbarum (Pers.) Rabenh.

Summary During the rainy year of 1951 a leafspot disease of leek caused byPleospora herbarum (Pers.) Rabenh. gave serious losses in some cases.The fungus causes a soft rot, later on the oval leafspots are growing dry, brownish white. When humidity is high many perithecia appear in about 2 months (see fig.).To control the disease spraying with copper compounds is advised.     

Comparison of four rapid DNA extraction techniques for conventional polymerase chain reaction testing of three Mycobacterium spp. that affect birds

Mycobacteriosis is an avian disease that is most commonly caused by Mycobacterium avium or Mycobacterium genavense. In order to optimize molecular laboratory tests for diagnosing mycobacteriosis in birds, we compared four methods of rapid DNA extraction with isolates of M. avium, M. genavense, and Mycobacterium fortuitum. DNA extraction methods included enzymatic lysis, boiling for 30 min followed by enzymatic lysis, four cycles of freezing and thawing followed by enzymatic lysis, and bead beating followed by enzymatic lysis. The DNA yield and purity for the four methods were evaluated by spectrophotometry and compared. The bead beating with enzymatic lysis technique yielded significantly purer and higher concentrations of extracted DNA compared with other DNA extraction methods. All four methods yielded extraction products for all three organisms that were successfully amplified by polymerase chain reaction (PCR) for a fragment in the 65-kD heat shock protein gene. Subjectively, the PCR amplification products were most abundant for samples extracted by bead beating with enzymatic lysis.     

Role of bovine viral diarrhea virus biotype in the establishment of fetal infections

OBJECTIVE: To examine the role of bovine viral diarrhea virus (BVDV) biotype on the establishment of fetal infection in cattle. ANIMALS: 30 mixed-breed pregnant cows. PROCEDURE: Pregnant cows were inoculated oronasally with either i-WNADL, originating from an infectious BVDV cDNA clone of the National Animal Disease Laboratory (NADL) isolate, or the parental virus stock, termed NADL-A. RESULTS: All cows developed neutralizing antibodies to BVDV, and virus was commonly isolated from peripheral blood mononuclear cells or nasal swab specimens of NADL-A inoculated cows; however, virus was rarely isolated from specimens of i-WNADL inoculated cows. i-WNADL did not cause fetal infection, whereas all fetuses harvested from NADL-A inoculated cows at 6 weeks after inoculation had evidence of infection. Immunoblot analysis of fetal virus isolates revealed the absence of NS3, confirming a noncytopathic (NCP) biotype BVDV in the NADL-A stock. The sequence of the NCP contaminant (termed NADL-1102) and the i-WNADL genome were virtually identical, with the exception of a 270 nucleotide-long insert in the i-WNADL genome. Phylogenetic analyses revealed that NADL-1102 forms a monophyletic group with 6 other NADL genomes. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that the contaminating NCP virus in the NADL-A stock was the ancestral NADL virus, which originally infected a bovine fetus and recombined to produce a cytopathic (CP) variant. Following oronasal infection of pregnant cows, viremia and transplacental transmission of CP BVDV to the fetus is rare, compared with the high occurrence of maternal viremia and fetal infection observed with NCP BVDV.