Isolation and molecular characterisation of Neospora caninum in cattle in New Zealand

AIM: To isolate Neospora caninum from the brains of naturally infected cattle and use molecular techniques to characterise the isolates.

METHODS: Neospora caninum tachyzoites were isolated in Vero cell culture from the brains of a cow and two calves. The isolates were characterised using polymerase chain reaction (PCR) methods, DNA sequencing, an immunofluorescent anti-body test (IFAT), transmission electron microscopy (TEM), and immunohistochemistry (IHC). The brains of the three cattle were subjected to histopathological examination. A pathogenicity study was conducted in 120 BALB/c mice.

RESULTS: Neospora caninum tachyzoites were isolated from all three cases and first observed in vitro between 14 and 17 days post-inoculation. Parasites were sub-cultured and maintained in Vero cell culture for more than 6 months. PCR products were generated for all three isolates, using two different primers. Sequencing of the PCR products and a subsequent BLAST search identified the isolates as N. caninum. In addition, the isolates tested positive using IFAT and IHC, and ultrastructure revealed by TEM was characteristic of N. caninum. Histopathological examination revealed lesions characteristic of N. caninum in 1/3 brains. In the pathogenicity study using BALB/c mice, the mortality rate was 3–7%.

CONCLUSION: This was the first successful isolation of N. caninum in New Zealand confirmed using molecular characterisation tests.     

The distribution of copper,vitamin B12 and zinc in the livers of sheep

Six livers from one-year-old sheep were each sampled at 27 different sites and the concentrations of copper, vitamin B,, and zinc determined for each sample. The distribution of these components was uniform throughout the liver, indicating that analysis of a single liver biopsy sample would give an accurate assessment of their hepatic concentration.     

Fertilization success of the New Zealand geoduck,Panopea zelandica: Effects of sperm concentration,gamete age and contact time

Valuable aquaculture and fisheries industries for the North American geoduck, Panopea generosa, have driven interest in developing similar ventures for other geoduck species including P. zelandica from New Zealand. However, little is known about the fertilization kinetics of this species, or the conditions under which the amount and quality of larvae can be maximized. We determined the effects of sperm concentration, gamete age and contact time on the fertilization success of P. zelandica using an extended‐Vogel‐Czihak‐Chang‐Wolf (EVCCW) model. The model provided a good fit to laboratory data when applied to individual contact times. For a contact time of 10 s, optimal fertilization was achieved at concentrations of approximately 10⁴ sperm μL^?1, but this decreased to as little as 10² sperm μL^?1 for contact times of several minutes. Optimal fertilization was always <100% (max. observed 70%) and the proportion of fertilized eggs decreased rapidly at sperm concentrations above the optimal. According to the model this was due to a slow block to polyspermy. If commercial hatchery facilities ensure that broodstock are in ripe condition, and use sperm <30‐min old, then optimal fertilization can be expected at sperm densities of 10²–10³ sperm μL^?1 at contact times of 5–10 min.     

Molecular identification and characterization of the intervening sequences (IVSs) within 23S ribosomal RNA (rRNA) genes of Taylorella asinigenitalis isolated in France

In the helix 25 region, 32 French Taylorella asinigenitalis isolates carried at least one 23S rRNA gene not containing intervening sequences (IVSs). No IVSs in the region were identified in three isolates and the other remaining 29 isolates carried one or more IVSs (UCD-1(T)IVS1A, UCD-1(T)IVS1B and UK-1IVS1B) described already and two new kinds of IVS (TaIVS1C and TaIVS1D). In the helix 45 region, no T. asinigenitalis isolates not carrying any IVSs were identified. UK-1IVS2B was identified in the region from 26 isolates. Five new kinds of IVSs (TaIVS2D, E, F, G and H) occurred in the region in the 13 isolates. Distinctly different tandem repeat units (RS48 and RS32 and RS-A, -B and -C) were evident in both regions, respectively, from the French (n=32) and American (n=3) T. asinigenitalis isolates. Thus, several different kinds of tandem repeat units and their combinations in IVSs in both regions within the gene were shown in 32 French isolates.     

Effects of drought and the presence of the 1BL/1RS translocation on grain vitreosity, hardness and protein content in winter wheat

Grain texture is an important component of end-use quality in wheat. The effects of water availability on the components of texture; vitreosity, determined using a Light Transflectance meter (LTm), grain hardness measured using the single-kernel characterisation system (SKCS), and protein content, were studied in field experiments of winter wheat in the UK in 2001/2002 and 2002/2003. Experiments were grown on a drought prone soil and employed a mapping population of 46 doubled haploid (DH) lines and their parents, Beaver (+1BL/1RS, soft wheat) and Soissons (1B, hard wheat). The results showed that drought increased hardness in both seasons, but the effect was never sufficient to move a line from the soft class into the hard class. Puroindoline (PIN)-a:b peak height ratio explained ca. 78% of the variation in hardness, and drought also appeared to increase the amounts of PINs in the grain. Minor quantitative trait loci (QTLs) were found for hardness on chromosomes 2A, 2D, 3A and 6D, also associated with QTLs for PINs. Vitreosity also increased in response to drought in both seasons. Variation in vitreosity explained 7–11% of the overall variation in texture within a hardness class, with hardness increasing on average by 2.2 SKCS units for each 10% increase in the proportion of vitreous grains. The relationship between vitreosity and protein content was poor, despite the fact that protein content also increased in response to drought. Minor QTLs associated with both protein content and vitreosity were found on chromosomes 1B, 4D and 5D. A minor QTL for vitreosity was also found on chromosome 2D. However, there appeared to be no direct relationship between alleles at the Ha locus, the gene which controls the difference between hard and soft wheats, and vitreosity. A positive relationship between the presence of the 1BL/1RS translocation and the proportion of vitreous grains was identified, suggesting that vitreosity was strongly linked to changes in protein quality.     

The efficacy of intraruminal pellets composed of elemental selenium and iron in sheep

Abstract

Commercial selenium pellets, manufactured after CSIRO workers drew attention to the significance of grain size on the rate of release of selenium, were tested in 27 sheep grazing a low-selenium New Zealand pasture. The pellets were shown by microscopy to contain mainly 10–20 µm particles of selenium, often agglomerated into larger lumps.

There was a considerable variation in the length of time pellets maintained blood selenium levels above the deficiency level of 250 nmol/? (20 µg/?). Whereas four animals given pellets had blood levels below 250 nmol/? after only 343 days, two animals had levels of 375 and 400 nmol/? after 651 days when the level in control sheep was 125±32 nmol/?. The pellets were recovered from all but one animal and had varying degrees of surface coating which was assumed to be mainly calcium phosphate.

Two pellets recovered from sheep at 386 and 484 days, when blood selenium levels were 175 and 1813 nmol/? respectively, were sectioned and examined by light and electron microscopy. Both pellets still contained unreacted selenium but differed in the degree of surface coating. The pellet recovered at 386 days had a solid and continuous coating whereas the coating on the pellet recovered at 484 days was not continuous and consisted of an open lattice of interlocking needles.

It appears that it is the extent of this coating which limits the effective life of the pellet in sheep.     

A proposed new approach and protocol to defining mineral deficiencies using reference curves. Cobalt deficiency in young sheep is used as a model

The most conclusive way of determining whether animals are deficient in a trace element is to measure production responses to supplementation in a field trial. However the opportunity, expertise and resources necessary to run such trials are not always readily available and there is often a considerable delay in reaching a diagnosis. Provided the degree of a production response can be closely related to a tissue level of the element or its metabolite then analysis of tissue samples can replace the need for field trials. The paper uses data from a series of cobalt liveweight response trials with lambs to outline a proposed methodology for constructing response curves which, for any specified level of Vitamin B12 in serum, can be used to determine (a) the expected liveweight response to supplementation, and (b) the probability of getting a response at least as great as some given level eg. that considered sufficient to just cover the costs of rectifying the deficiency. A protocol for future production response trials is described. It is planned that all appropriate production response trial data will be used to derive ;response; and ;probability of response; curves for use in diagnosing cobalt, selenium and copper deficiencies in sheep and cattle. It is suggested that the methodology could be applied in many biological systems involving deficiencies.