Mutants of rabies viruses in skunks: immune response and pathogenicity.

In studies to develop an oral rabies vaccine for wildlife, the immune response to and pathogenicity of two types of mutants of rabies viruses were examined. Forty-five small plaque mutants were selected from cultures of ERA rabies virus treated with 8-azaguanine or 5-fluorouracil and tested for pathogenicity in mice. Two of these mutants AZA 1 and AZA 2 (low pathogenicity in mice) were given to skunks by oral (bait), intestinal (endoscope) and intramuscular routes. Additionally, challenge virus standard (CVS) rabies virus and mutants of this and ERA rabies virus (CVS 3766 and 3713, and ERA 3629) that were resistant to neutralization by specific antiglycoprotein monoclonal antibodies (and apathogenic in mice) were tested by various routes in skunks. Skunks given AZA 1 and AZA 2 were challenged at three months postinoculation with street rabies virus. After oral administration, there were very low rates of seroconversion with AZA 1 and AZA 2 and on challenge only 2/7 given AZA 1 and 1/8 given AZA 2 survived. None of the skunks given the other mutants orally seroconverted. AZA 2 produced a high rate of seroconversion (8/8) by the intestinal route and all challenged skunks in this group survived (7/7). All skunks vaccinated intramuscularly with AZA 1 (4/4) or AZA 2 (4/4) developed high levels of rabies neutralizing antibodies and survived challenge. The mutant CVS 3766, while apathogenic when given intracerebrally to adult mice, was consistently pathogenic by this route (and intranasally) in skunks. These results demonstrate that skunks are highly resistant to oral immunization by live rabies virus vaccines and that pathogenicity (by intracerebral route) of the mutant CVS 3766 is markedly different in mice and skunks.     

Enzyme-linked immunosorbent assays for detecting antibody to Rickettsia australis in sera of various animal species

New endemic areas of spotted fever-like rickettsial disease have been found in south-eastern Australia (Gippsland, Victoria and Flinders Island, Tasmania). The rickettsia responsible is currently unknown although it may be Rickettsia australis. To investigate serological evidence of rickettsial exposure in various wild animal species, a competitive ELISA was developed which detected antibodies to R. australis. It was based on inhibition of an indirect ELISA detecting antibody to R. australis in guinea pig sera. Pre- and post-infection sera from 2 dogs, 2 rabbits, 5 mice and 6 rats, experimentally infected with R. australis, were tested by competitive ELISA. The results showed that all pre-infection sera were negative and all post-infection sera positive for antibody to R. australis. To test the utility of the competitive ELISA for detecting natural rickettsial infection in non-laboratory animals, 51 dog sera, negative for rickettsial antibody by immunofluorescence (IF) and 20 IF positive dog sera (collected from various locations on the east coast of Australia) were tested. Compared to the IF test the competitive ELISA was 90% sensitive and 96% specific. This new test has potential for detecting antibody to R. australis in the sera of different wild animal species.     

High dietary calcium to phosphorus ratio and alkali-forming potential as factors promoting silica urolithiasis in sheep

Sheep were used to study factors previously found to promote silica urolithiasis in a rat model. In addition to high silica, these dietary factors included elevated calcium, a high calcium to phosphorus ratio and alkali-forming effects. Wether lambs had ad libitum access to a diet of 50% of grass hay and 50% ground oats plus supplement. Diet analysis was 3.4% total SiO2, .29% calcium, .25% phosphorus, 11.3% CP and 28% ADF. Treatments (40 lambs/treatment) consisted of a control (C), limestone to increase dietary calcium to .6% (L), L + 1% sodium bicarbonate (LS) and L + 1% ammonium chloride (LA). After a 91-d experimental period followed by a 56-d postexperimental finishing period, silica kidney deposits were found in all treatments, and SiO2 made up 74% to 97% of the urolithic ash. Kidney urolith incidences in the four treatments were C, 7/40; L, 12/40; LS, 20/40; and LA, 9/40. A higher urolith incidence in LS (LS vs C, P less than .05) and a trend toward a higher incidence in L (L vs C, P less than .2), accompanied by elevated urine pH (L = LS greater than C greater than LA, P less than .01), lend support to the concept that high-silica diets having high calcium to phosphorus ratios and alkali-forming potentials contribute to silica urolithiasis.     

Staining of heterochromatin bands and the determination of rye chromosomes in triticale (wheat × rye hybrid)

Summary Fifteen lines of triticale were studied using the C-banding staining method. A typical, complete chromosomal complement was found in 11 lines, while the remaining lines demonstrated some variations. A slight modification in the technique is discussed.Supported in part by CSRS/USDA Grant No. 801-15-03A.     

Cool Season C3-Grasses (Wheat,Rye, Barley,and Oats) Differ in Shoot: Root Ratio When Applied With Different NPK Sources

Shoot:root (S:R) response in four winter C₃-grasses (cereals) viz. wheat (Triticum aestivum L.), rye (Secale cereale L.), barley (Hordeum vulgare L.) and oats (Avena sativa L.) was investigated under eight different NPK sources (S₁ = 20-20-20, S₂ = 20-27-5, S₃ = 7-22-8, S₄ = 10-10-10-20S, S₅ = 11-15-11, S₆ = 31-11-11, S₇ = 24-8-16, and S₈ = 19-6-12) in pot experiment at Dryland Agriculture Institute, West Texas A&M University, Canyon, Texas, USA during winter 2009–2010. The experiment was performed in completely randomized design (CRD) with three repeats. The objective of this experiment was to investigate whether the S:R of winter cereals differ or not when applied with different NPK sources. Considerable variations in the shoot: root was noticed in the four grasses at different growth stages, i.e., 30, 60 and 90 days after emergence (DAE) when applied with different NPK fertilizers sources. Wheat had the highest S:R of 3.4 at 30 days after emergence (DAE), barley at 60 DAE (6.5), while oats at 90 DAE (3.9). Among the NPK sources, crops had the highest S:R of 3.6, 6.2 and 3.7 when applied with S₃, S₂, and S₆ at 30, 60 and 90 DAE, respectively. Shoot to root ratio showed positive relationship with increase in shoot weight and negative relationship with increase in root weight. The NPK fertilizer S₆ (31: 11: 11), an acid loving fertilizer had negative effects on the shoot and root development of cool season cereals, but among these cereals under study, barley and oats roots were affected more than wheat and rye. It was concluded from this present study, that the four cool season cereals responded differently in terms of shoot: root to different NPK fertilizers at different growth stages.     

A duplex-PCR bioassay to detect a Trichoderma virens biocontrol isolate in non-sterile soil

This paper describes the identification and utilisation of a sequence-characterised amplified region (SCAR) marker specific for the Trichoderma virens biocontrol isolate GV4. The marker was developed from a RAPD-PCR amplification product unique to isolate GV4. When used as a hybridisation probe in Southern blot analysis, it hybridised to the DNA of the species T. virens alone and not to that of other Trichoderma species or closely related genera Gliocladium and Verticillium. The marker also produced a GV4-specific RFLP, distinguishing it from other T. virens isolates when probed to blots with HindII, BamHI or PstI genomic DNA digests. Primers designed from the sequence of the RAPD marker produced a diagnostic amplification product of 346 bp for GV4 alone, distinguishing it from all other test isolates. With the exception of one, test isolates did not produce an amplification product with the SCAR primers. The exception was a single Verticillium psalliotae isolate (ICMP5509) that produced a product of approx. 400 bp that was easily distinguished from the 346 bp product of GV4. The reliability of the SCAR-based diagnostic test was further improved with the introduction of a positive PCR reaction control to each test, achieved by converting the test to a duplex PCR system. Two universal primers flanking the two ITS and the 5.8S region of the ribosomal gene complex were introduced to each reaction to provide a test for PCR reaction inhibitors to eliminate false negatives in the diagnosis. Amplification of this multi-copy genomic region did not reduce diagnostic sensitivity of the single copy SCAR marker. To further increase the sensitivity of detecting GV4 propagules while maintaining a fast sample assessment assay, soil was amended with cornmeal, as a nutrient source, and a mix of antibiotics to favour Trichoderma growth. The soil mix was subsequently incubated for 5 d before total DNA was extracted. Under these conditions, the duplex soil PCR assay detected GV4 down to a concentration of 10 spores g⁻¹ soil in non-sterile agricultural field soil. This study is the first to report the use of a duplex-PCR diagnostic bioassay for a species within the Hypocrea/Trichoderma genus.     

Animal treading stimulates denitrification in soil under pasture

The effects of animal treading on denitrification in a mixed ryegrass-clover pasture were studied. A single treading event of moderate or severe intensity was applied in plots during spring by using dairy cows at varying stocking rates (4.5 cows 100 m⁻² for 1.5 or 2.5 h, respectively). Treading caused a significant short-term 21 days) increase in denitrification. Denitrification rates reached a maximum of 52 g N₂O-N ha⁻¹ day⁻¹ at 8 days after severe treading compared to 2.3 g N₂O-N ha⁻¹ day⁻¹ under nil treading. Thereafter, denitrification rates declined, and were similar to non-trodden control plots after 28 days. Soil aeration, was significantly reduced by treading as expressed by water-filled porosity. In addition, soil NH₄⁺-N and NO₃⁻-N concentrations were also increased by treading. We propose that the underlying processes involved in increasing denitrification under treading were two-fold. Firstly, treading caused a temporary (e.g. 3 days after treading) reduction in soil aeration through soil physical damage, and secondly, reduced soil N utilisation prompted by reduced plant growth led to increased soil NH₄⁺-N and NO₃⁻-N availability. This study shows that treading, without the influence of other grazing animal factors (e.g. excretion), can cause a large short-term stimulation of denitrification in grass-clover pastures.     

Measurement of microbial sulfur in soil

A technique was developed to estimate the amount of sulfur held by the microbial population in soil. This method involves lysing the microbial cells with chloroform and measuring the S released to 10 mm CaCl₂ or 0.1 m NaHCO₃ extracts. Calculations of biomass-S were made from the experimentally determined proportion of cell S released. To determine this proportion, two species each of bacteria and fungi, which were grown at three concentrations of S, were added to the soil and treated with CHCl₃. The average values of 34.6% with CaCl₂ and 41.2% with NaHCO₃ were calculated for the proportion of microbial S extracted following CHCl₃ treatment. Biomass-S was found to represent approximately 2.3% of the total S in the soil.     

Biodiversity of mannose-specific lectins within Narcissus species

Mannose-specific lectins (MSLs) were isolated from the bulbs of 27 species of wild Spanish Narcissi and compared to the commercially available MSL from daffodil (Narcissus pseudonarcissus, NPA). Molecular weight analysis showed the monomers of all the MSLs were at, or around, 12.5 kD. Haemagglutination assays showed that the MSLs exhibited activities at up to four times greater than that displayed by NPA and other MSLs derived from other species such as Galanthus nivalus (snowdrop) and Allium ursinum (ramson). Elution profiles from ion exchange chromatography exhibited similarities for species within the same taxonomic section suggesting that this method could aid in species classification. Further analysis by isoelectric focusing showed many isolectins are present in vivo and that even within a single peak from ion exchange chromatography there are numerous isolectins present. The basis of the isolectin heterogeneity is suggested to reside in the tetraploidy (sometime triploidy) nature of Narcissus genes.     

An illustrated assessment key for foliage blight of potatoes

Summary A series of drawings illustrates Malcolmson's (1976) 1–9 scoring scale of categories of foliage blight.