A real-time polymerase chain reaction assay for the detection of Mycoplasma agalactiae

AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene.

METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay.

RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 10⁴ cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%.

CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae.     

Dynamics of the free-living stages of sheep intestinal parasites on pasture in the North Island of New Zealand. 1. Patterns of seasonal development

Abstract

AIM: To describe the seasonal pattern of development of third-stage infective larvae (L3) from eggs of Teladorsagia (=Ostertagia) circumcincta, Trichostrongylus colubriformis and Haemonchus contortus on pasture in the North Island of New Zealand.

METHODS: Sheep faeces containing known numbers of eggs of all three nematode species were deposited on, or buried in, pasture plots at three sites, viz coastal Manawatu, Upper Hutt Valley, and East Cape hill country. Development was measured by recovering L3 from faeces, herbage and soil 28–31 days after deposition on 13–18 occasions, between January 2005 and July 2006. Analysis of the number of larvae recovered used a mixed model including number of eggs deposited, weight of faeces recovered (an assumed indicator of earthworm activity), site, contamination date, and position of deposited faeces, i.e. on the surface or buried.

RESULTS: There was a significant effect of contamination date on development of all three species, with maximum numbers ofL3 developing between late spring (November) and early autumn (March), and minimum numbers in June and July. There were large differences between species, with H. contortus exhibiting a long period (April to October) where development was close to zero, whereas T. circumcincta developed to some extent all year round. Development of T. colubriformis was intermediate between the other two species.

Burying faeces containing nematode eggs increased the number of L3 recovered compared with surface deposition (p≤0.001), although there were a small number of exceptions involving only T. colubriformis. The weight of faeces recovered at harvest, which was assumed to be an indication of earthworm activity, was correlated with the number of L3 recovered for all species (p<0.001). In a separate analysis, earthworms were assumed tohave been active if <5 g faeces remained at harvest. Where this occurred, the number of L3 of T. colubriformis and T.circumcincta recovered was reduced by 56% and 58%, respectively (p<0.001).

CONCLUSIONS: A marked seasonal pattern of development was observed for all three species, with the most larvae developing in spring-early autumn and the least in winter. This seasonal pattern was most pronounced in H. contortus and least obvious in T. circumcincta. Burying faeces containing eggs generally resulted in more L3 being recovered, whilst the apparent activity of earthworms resulted in fewer larvae being recovered.     

Development and spatial distribution of the free-living stages of Teladorsagia circumcincta and Trichostrongylus colubriformis on pasture: A pilot study

Abstract

AIMS: To measure the development of Teladorsagia (=Ostertagia) circumcincta and Trichostrongylus colubriformis eggs to third-stage infective larvae (L3) at different times of the year. Also, to measure the spatial distribution of L3 across herbage, soil and faeces, in order to assess whether spatial issues could be important in larval dynamics on pasture.

METHODS: Field plots were contaminated with sheep faeces containing approximately 20,000 eggs of each of T. circumcincta and T. colubriformis on five separate occasions, viz 01 December 1996 (summer), 18 March 1998 (autumn), 17 June 1998 (winter), 15 October 1998 (spring), and 23 July 1999 (winter). Replicate plots (n=10) were harvested at intervals for up to 12 months after deposition of faeces, and the number and distribution of L3 were measured. Larvae were sampled from faeces (where these remained), herbage, and three soil zones to a depth of 145 mm.

RESULTS: There were large differences between contamination dates in the percentage of eggs that developed to L3. For both species the highest percentage development was for eggs deposited in December (7.8% and 25.9% for T. circumcincta and T. colubriformis, respectively) and the lowest for June (0.4% and 0.03% T. circumcincta and T. colubriformis, respectively). Development in winter was often delayed, and this was always associated with a low yield of larvae, probably due to compounding mortalities associated with long periods of exposure to low temperatures.

The relative distribution of L3 present on herbage, in faeces or in the soil varied between sampling times. However, overall the most L3 were recovered from soil (74% and 66% for T.circumcincta and T. colubriformis, respectively, averaged over all samples), and the lowest recoveries were from the herbage.

CONCLUSIONS: Although the data are limited, the results indicated that the highest percentage of eggs developed to infective larvae in summer and only minimal development occurred in winter. The data do not support the view that substantial contamination of pastures with sheep parasites occurs over winter. Large numbers of larvae were recovered from soil, which indicates that, assuming they can subsequently migrate onto herbage, soil is a potentially important reservoir ofinfective larvae in New Zealand. Therefore, the spatial distribution of L3 on pasture may affect both the dynamics and transmission of parasite populations. Further work on both these issues is warranted.     

A laser ablation method for the synthesis of crystalline semiconductor nanowires

A method combining laser ablation cluster formation and vapor-liquid-solid (VLS) growth was developed for the synthesis of semiconductor nanowires. In this process, laser ablation was used to prepare nanometer-diameter catalyst clusters that define the size of wires produced by VLS growth. This approach was used to prepare bulk quantities of uniform single-crystal silicon and germanium nanowires with diameters of 6 to 20 and 3 to 9 nanometers, respectively, and lengths ranging from 1 to 30 micrometers. Studies carried out with different conditions and catalyst materials confirmed the central details of the growth mechanism and suggest that well-established phase diagrams can be used to predict rationally catalyst materials and growth conditions for the preparation of nanowires.     

Resident forum abstractsEVALUATION OF THROMBOELASTOGRAPHY (TEG) IN NORMAL CATS

Objective: To establish normal parameters of thromboelastography (TEG) in healthy adult cats. Background: Thromboelastography (TEG) is an in vitro test of coagulation that has been shown to be useful in humans, dogs and select species to identify and quantify alterations of hemostasis (e.g., hypercoagulable and hypocoagulable states). It has also been demonstrated to be useful in monitoring effects of anticoagulant therapies. This test has not been evaluated in cats. Methods: Blood was collected from 25 clinically normal cats by venipuncture using a 21 gauge×3 1/2 inch butterfly catheter and syringe for medial saphenous or jugular venipuncture. A single 1.8 mL sample in 3.8% Sodium Citrate (9:1) was collected from each cat. Recalcified whole blood was analyzed 30 minutes following collection with the TEG® 5000 analyzer (Haemoscope, Niles, IL). Analysis temperature was 37.6°C. TEG parameters recorded included: R‐value (represents initial fibrin formation), K (time from R to standard fixed measure of clot firmness which represents contributions of platelets and fibrinogen), maximum amplitude (MA; represents absolute clot strength), and alpha angle (α; the slope of TEG tracing which represents rate of clot formation). The coagulation index (CI) was derived from the formula generated for humans to provide an overall assessment of whether the sample was hyper‐ or hypocoagulable. Results: Values for the 25 normal cat samples are reported as mean ±2 standard deviations. R=2.97; 1.23–4.72; K=1.54, 0.38–2.71; α=70.70, 57.76–83.65; MA=58.50, 45.26–71.74 and CI=2.27, 0.07–4.46. Compared to historical information obtained on normal dogs, cats have significantly shorter R and K and larger α, MA and CI. Conclusions: TEG does have reproducible performance when used to evaluate coagulation status in normal cats. Compared to dogs, normal cats favor a hypercoagulable state. Species‐specific normal values are necessary for interpretation of TEG results. This test bears potential value for use in future experimental and clinical work to investigate hemostasis in cats receiving anticoagulant therapies or in cats suffering from diseases such as cardiomyopathy which are thought to be associated with altered coagulation status.