Assessment of real-time PCR as a method for determining the presence of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in different Solanaceae cultivars

Real-time PCR was used to detect and quantify Verticillium dahliae and to assess the susceptibility of four Capsicum annuum cultivars (Luesia, Padrón, SCM331 and PI201234) and the Capsicum chinense cv. C118 to this pathogen. The symptoms which developed after infection included stunting and yellowing, and were more acute in the cv. SCM331, which also suffered defoliation in later stages of the disease and in C118, which suffered severe stunting. Quantification of the pathogen DNA in roots 23 and 34 days post-inoculation (dpi) revealed that there were significantly higher amounts of Verticillium dahliae DNA in C118 than in the other cultivars, followed by SCM331, Padrón and PI201234. The lowest amounts of fungal DNA in roots were found in Luesia. In hypocotyls, the highest amounts of fungal DNA were found in SCM331, while Luesia, Padrón and PI201234 had much lower amounts, and C118 had intermediate levels. When a compatible versus an incompatible system was studied, using the near-isogenic tomato lines LA3030 (susceptible) and LA3038 (resistant to V. dahliae), we were able to detect fungal DNA in both lines. As expected, the fungus/plant DNA ratio was lower in LA3038 than in LA3030 and it decreased with time in LA3038. The amount of Verticillium dahliae DNA in the roots of LA3030 remained constant between days 23 and 34 post-inoculation, but increased 10-fold in collars. Finally, when real-time PCR was applied as a diagnostic method to samples from pepper plants, soil and water collected from farms in northwest Spain, we were able to detect V. dahliae DNA in these samples even when symptoms of the disease were not evident.     

Sulphur accumulation after Verticillium dahliae infection of two pepper cultivars differing in degree of resistance

Elemental sulphur levels, sulphur localization in stems, and levels of sulphate, glutathione and cysteine were studied in pepper ( Capsicum annuum ) cvs Yolo Wonder (higher resistance) and Luesia (lower resistance) after inoculation with Verticillium dahliae , the cause of vascular wilt. Accumulation of elemental sulphur (S⁰) was first detected 10 days after inoculation in Yolo Wonder (mean S⁰ level 7·3  µ g g⁻¹ DW), and 15 days after inoculation in Luesia (mean S⁰ level 3·3  µ g g⁻¹ DW). The maximum level was reached 21 days after inoculation in Yolo Wonder (14·1  µ g g⁻¹ DW). In control plants, elemental sulphur was not detected. SEM-EDX (scanning electron microscopy-energy dispersive X-ray microanalysis) indicated that the sulphur was not restricted to a specific location, but was dispersed throughout the vascular tissue. Sulphate levels showed a decline at the end of the experiment in inoculated plants, possibly related to the increase in sulphur levels seen in the two cultivars. The differences in sulphate levels between the two cultivars may be due to faster sulphate breakdown in cv. Yolo Wonder.     

Sensitivity of fungi isolated from onychomycosis to Eugenia cariophyllata essential oil and eugenol

The antifungal activity of Eugenia cariophyllata essential oil and eugenol, its major constituent, on fungal strains isolated from onychomycosis was evaluated. The natural products presented prominent antifungal action with MIC of 1% and 4%, respectively.     

A survey of feeding,management and faecal pH of Thoroughbred racehorses in the North Island of New Zealand

AIM: To identify feeding and management variables associated with variation in faecal pH within a population of intensively managed Thoroughbred racehorses in New Zealand.

METHODS: A cross-sectional survey was conducted of 16 racehorse trainers in the North Island of New Zealand. Interviews were conducted at the trainers' stables to obtain information on feeding and management of horses, and faecal samples were collected and faecal pH measured.

RESULTS: Ninety-seven percent of the horses surveyed were confined in an area ≤5 × 5 m for ≥12 h/day. Trainer's age, number of years they had trained horses, age and gender of horses, weeks in race training, racing class, frequency of feeding, bedding type, and exercise workload had no effect on mean faecal pH. Acidic faecal pH (pH ≤6.32) was associated with stables with ≤12 horses, and trainers at stables with ≤12 horses offered more concentrate feed than those at stables with >12 horses. Acidic faecal pH was associated with trainers who offered 4 kg of grain as the only form of concentrate fed, or offered ≤2.25 kg hay/day. Horses that displayed stable vices had less acidic faecal pH than horses that did not display stable vices, viz pH 6.70 (standard error of the mean (SEM) 0.135) vs 6.43 (SEM 0.029) (p=0.04).

CONCLUSIONS AND CLINICAL RELEVANCE: Racehorse management in New Zealand is similar to that observed in other major racing countries. Trainers with ≤12 horses fed more concentrates and their horses had lower faecal pH than those of trainers with >12 horses. Irrespective of management system, it appears important to provide at least 2.25 kg of hay/day ad libitum, to buffer hindgut acidosis associated with diets high in soluble carbohydrate.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 3. In vivo ultrasonographic assessment of the cross-sectional area and echogenicity of the superficial digital flexor tendon

AIM: To determine if the superficial digital flexor tendon (SDFT) of young Thoroughbred horses changed in size and echogenicity in association with early race training.

METHODS: Cross-sectional area (CSA) and echogenicity were determined ultrasonographically at five levels of the SDFT of the forelimbs of 2-year-old fillies (n=14), corresponding to 4, 8, 12, 16 and 20 cm distal to the accessory carpal bone (DACB). Measurements were made before and after a 13-week period in which a trained group of seven horses was compared with another group of seven untrained horses.

RESULTS: Level below the accessory carpal bone had a significant effect on CSA and Level 8 was smaller than all other levels except Level 12, while Level 12 was smaller than Levels 4 and 20 but not different from Levels 8 and 16. There was a significant interaction between level and time due to effects observed at Level 8. The CSA at Level 8 measured pre-training was different from that of Levels 4 and 20 in both pre- and post-training groups (p<0.05), but when measured post-training was not different from any other measurement. There was also a significant interaction between treatment group and time. There was no difference between CSA for the untrained and trained groups at the pre-training observation (p=0.9), but post-training the CSA (pooled over all levels) in trained horses was significantly larger than that of the untrained horses both post-training (p=0.019) and pre-training (p=0.034), and was not different from the pretraining CSA recorded in the trained group (p=0.29). Treatment group had no effect on echogenicity (p=0.43), while echogenicity was less at the end of the trial in both trained and untrained horses (p<0.001).

CONCLUSIONS: Early training for racing was associated with an increase in mean CSA of the SDFT. Other factors such as age and maturity may play a role in limiting this increase.     

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 1. Study design,and clinical,nutritional, radiological and histological observations

AIMS: This is the first in a series of papers reporting studies in 2-year-old Thoroughbred racehorses that aimed to determine the response of musculoskeletal tissues to early training on grass and sand racetracks. In this paper, the experimental set-up of the whole study is described, and nutritional, workload, and clinical, radiographic and pathological outcomes are reported, including semi-quantitative assessment of macroscopic changes in articular cartilage.

METHODS: The study group comprised 14 two-year-old Thoroughbred fillies reared entirely at pasture. Of these, seven were selected by a licensed racehorse trainer to undergo a 4-week period of initial training in which they were taught to accept saddle and rider, followed by a 13-week period of flatrace training at a racetrack (Weeks 1–13); the other seven fillies were confined to large grass enclosures and were not trained. Nutrient, including macro- and trace-element intakes were estimated. Distances cantered or galloped and average velocities were quantified for the trained horses. All horses were observed daily, weighed approximately weekly, and underwent a clinical lameness examination at Weeks 5, 9 and 13. Distal forelimbs were radiographed prior to Week 1, during Weeks 7–8, and again at the end of the study, when macroscopic changes in articular cartilage of the proximal surface of the proximal phalanx were also scored after staining with India ink.

RESULTS: Dietary intakes met or exceeded recommended requirements for all nutrients except sodium, which was low in the trained horses. Bodyweight increased throughout the study in the untrained horses, and increased until Week 7 and then decreased slightly in the trained horses. Mean velocity data were used to define three stages of the training programme: Stage 1 comprised canter in Weeks 1–4; Stage 2 comprised canter in Weeks 5–8; and Stage 3 comprised canter in Weeks 9–13 and galloping twice weekly. Four of seven horses completed training. These covered a mean distance of 179.2 km at mean velocities (excluding gallops) of 7.63 m/sec (SD 0.58), 8.99 m/sec (SD 0.56), and 8.43 m/sec (SD 0.74) for Stages 1–3, respectively, and galloped 4.45 km at 14.4 (SD 0.1) m/sec. The three horses that did not complete training became lame in Weeks 9, 10 and 11, and covered 147.9 km at velocities of 7.38 m/sec (SD 0.44), 8.88 m/sec (SD 0.33) and 8.43 m/sec (SD 0.59) and galloped 2.1 km. Overall, slight or intermittent lameness in trained horses was noted on 76/655 (12%) of horse observation days. Swelling was evident on 284/655 (41%) of horse observation days in the metacarpophalangeal (MCPJ) and metatarsophalangeal (MTPJ) joints (92%), palmar metacarpal tendon region (7%) or carpus (1%). Swelling of the MCPJ or MTPJ was not associated with obvious lameness. Radiographic changes were minor and no gross lesions in bone or tendon tissue were evident except for one case of dorsal metacarpal disease. Post mortem, the cartilage of some MCPJ and MTPJ had obvious wear lines and high lesion scores, which were not consistently related to clinical evidence of pain, lameness or joint swelling. Mean lesion scores were not significantly different between the MCPJ and MTPJ, or between trained and untrained horses.

CONCLUSIONS: Workload can be readily quantified in racehorses under semi-commercial training conditions. Obvious lesions in cartilage of the MCPJ or MTPJ were present in some trained and some untrained horses and not consistently associated with clinical evidence of lameness, joint swelling or change in other connective tissues.

CLINICAL RELEVANCE: Workload data in racing horses are likely to be highly relevant for studying the pathogenesis of changes in bone, tendon and cartilage during training, for training management and for risk analysis in racehorse populations. Although obvious cartilage lesions produced little clinical effect, such lesions have previously been shown to be progressive and to prejudice athletic capability. Detection of such occult lesions in young horses will require more sophisticated detection methods.     

Early Pregnancy Detection of Iraqi Riverine Buffalo (Bubalus bubalis) Using the BioPRYN Enzyme‐Linked Immunosorbent Assay for PSPB and the Progesterone Assay

This study was undertaken to detect pregnancy in Iraqi riverine buffalo (Bubalus bubalis) using three different methods (rectal palpation, plasma progesterone concentration and detection of the presence of pregnancy‐specific protein B (PSPB) with the BioPRYN^® enzyme‐linked immunosorbent assay (ELISA) test. The aim of the study was to identify the most sensitive, early and accurate method for detecting pregnancy. Twenty‐two female riverine buffalo that were 6.0 ± 0.93 years old were used. Four blood samples per buffalo were taken via jugular venipuncture at days 22–24, 32–34, 42–44 and 58–61 post‐mating (PM) to measure the progesterone concentration (ng/ml) and to detect the presence of plasma PSPB. The rectal palpation method was employed to evaluate all buffalo on days 42–44 and 58–61 PM. The BioPRYN^® test differed (p < 0.01) from the other tests with earlier accuracy for detecting pregnant and non‐pregnant buffalo. Eighty‐eight percent of pregnant and 76.9% of non‐pregnant buffalo were distinguished early (days 22–24 PM) using BioPRYN^® and plasma PSPB‐ELISA level (2.09 ± 0.12 ng/ml) in relation to 66.7% and 53.9% detected using the progesterone assay at similar days (4.30 ± 0.40 ng/ml). In conclusion, these results described, for the first time, the early and accurate pregnancy detection of water riverine buffalo using BioPRYN^® technology and provided the plasma levels of PSPB using an ELISA test. These findings will improve the reproductive and productive efficiency of Iraqi riverine buffalo by adapting the recent management and reproductive strategies in Iraq and in the world.