Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

Background The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype. Results We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying. Conclusion We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects

Comparison of Culture and a Novel 5′ Taq Nuclease Assay for Direct Detection of Campylobacter fetus subsp. venerealis in Clinical Specimens from Cattle

A Campylobacter fetus subsp. venerealis-specific 5′ Taq nuclease PCR assay using a 3′ minor groove binder-DNA probe (TaqMan MGB) was developed based on a subspecies-specific fragment of unknown identity (S. Hum, K. Quinn, J. Brunner, and S. L. On, Aust. Vet. J. 75:827-831, 1997). The assay specifically detected four C. fetus subsp. venerealis strains with no observed cross-reaction with C. fetus subsp. fetus-related Campylobacter species or other bovine venereal microflora. The 5′ Taq nuclease assay detected approximately one single cell compared to 100 and 10 cells in the conventional PCR assay and 2,500 and 25,000 cells from selective culture from inoculated smegma and mucus, respectively. The respective detection limits following the enrichments from smegma and mucus were 5,000 and 50 cells/inoculum for the conventional PCR compared to 500 and 50 cells/inoculum for the 5′ Taq nuclease assay. Field sampling confirmed the sensitivity and the specificity of the 5′ Taq nuclease assay by detecting an additional 40 bulls that were not detected by culture. Urine-inoculated samples demonstrated comparable detection of C. fetus subsp. venerealis by both culture and the 5′ Taq nuclease assay; however, urine was found to be less effective than smegma for bull sampling. Three infected bulls were tested repetitively to compare sampling tools, and the bull rasper proved to be the most suitable, as evidenced by the improved ease of specimen collection and the consistent detection of higher levels of C. fetus subsp. venerealis. The 5′ Taq nuclease assay demonstrates a statistically significant association with culture (χ(2) = 29.8; P < 0.001) and significant improvements for the detection of C. fetus subsp. venerealis-infected animals from crude clinical extracts following prolonged transport

The tolerance of steers (Bos taurus) to sorghum ergot (Claviceps africana) in a feedlot during the cooler months in subtropical Queensland

Two experiments tested the tolerance of steers (Bos taurus) to sorghum ergot (Claviceps africana) during cooler months in south-east Queensland. Sorghum grain containing 2.8% ergot and 28 mg/kg ergot alkaloids (84% dihydroergosine, 10% dihydroelymoclavine, 6% festuclavine) was incorporated into feedlot rations. In a previous study in summer–autumn, ergot (1.1–4.4 mg alkaloids/kg ration) severely reduced performance in steers when the temperature–humidity index (THI; dry bulb temperature °C + 0.36 dew-point temperature °C + 41.2) was ~70, whereas a THI of ~79 was tolerated by steers fed ergot-free rations. Experiment 1 was conducted in winter–spring, with rations containing 0, 2.8, 5.6, 8.2 or 11.2 mg ergot alkaloids/kg ration. All ergot inclusions depressed feed intake (14% average reduction) and growth rate (34% average reduction), even when the weekly average daily THI was less than 65. Rectal temperatures were occasionally elevated in ergot-fed steers (P 0.05) in the group receiving 4.3 mg/kg alkaloid, compared with Controls. Plasma prolactin concentrations were depressed, relative to the Controls, by dietary alkaloid inclusion greater than 1.1 mg/kg, with alkaloid intake of 4.3 mg/kg causing the greatest reduction (P 65)

The tolerance of steers (Bos taurus) to sorghum ergot (Claviceps africana) in a feedlot during the cooler months in subtropical Queensland

Two experiments tested the tolerance of steers (Bos taurus) to sorghum ergot (Claviceps africana) during cooler months in south-east Queensland. Sorghum grain containing 2.8% ergot and 28 mg/kg ergot alkaloids (84% dihydroergosine, 10% dihydroelymoclavine, 6% festuclavine) was incorporated into feedlot rations. In a previous study in summer-autumn, ergot (1.1-4.4 mg alkaloids/kg ration) severely reduced performance in steers when the temperature-humidity index (THI; dry bulb temperature °C + 0.36 dew-point temperature °C + 41.2) was ∼70, whereas a THI of ∼79 was tolerated by steers fed ergot-free rations. Experiment 1 was conducted in winter-spring, with rations containing 0, 2.8, 5.6, 8.2 or 11.2 mg ergot alkaloids/kg ration. All ergot inclusions depressed feed intake (14% average reduction) and growth rate (34% average reduction), even when the weekly average daily THI was less than 65. Rectal temperatures were occasionally elevated in ergot-fed steers (P 65)

Quality of handling and holding yard environment, and beef cattle temperament: 2. Consequences for stress and productivity

This experiment assessed the effects of different quality and quantity of handling and quality of the holding yard environment on the productivity and physiological parameters indicative of stress in beef cattle. One-hundred-and-forty-four steers were given one of three human handling and yarding experiences on six occasions during a 12-month grazing period post-weaning (backgrounding): Good handling/yarding, Poor handling/yarding and Minimal handling/yarding. At the end of this phase the cattle were lot-fed for 78 days, with no handling/yarding treatments imposed, before being transported for commercial slaughter. Temperament was assessed by flight speed (FS) and a fear of humans (FOH) test, which measured the proximity to a stimulus person (ZA), the closest approach to the person (CA) and the amount the cattle moved around the test arena (TT). Mid-way through backgrounding, the Minimal treatment group was heavier than the Good, which was heavier than the Poor (mean weights 207, 201 and 194 kg, respectively; P = 0.05; LSD = 5.4), but by the end of backgrounding there was no difference between treatments and treatment did not affect liveweight during lot-feeding. At the end of backgrounding, plasma cortisol levels were significantly lower (P < 0.001) in the Good treatment group compared to the Poor and Minimal groups but at the end of lot-feeding there was no significant difference between the groups. Treatment affected plasma non-esterified fatty acid levels in backgrounding (P = 0.060) and lot-feeding (P = 0.046) with levels being higher in the Minimal than the Good and Poor groups (backgrounding: 0.52, 0.44 and 0.47 nmol/L, respectively; SE 0.02; lot-feeding: 0.46, 0.41 and 0.41 mmol/L, respectively; LSD = 0.05). Significant weak to moderate (r-value <0.50) negative correlations were found between FS and average daily gain, but there were no consistent correlations between measures from the FOH test and productivity. FS and TT were weakly positively correlated with plasma l-lactate, glucose and cortisol levels, and CA was weakly to moderately negatively correlated with l-lactate and glucose levels. The results indicate that, whilst being imposed, the Good treatment reduced stress and the Poor treatment negatively impacted on liveweight gain. Minimal handling/yarding appeared to cause the cattle to experience stress, perhaps because of the relative novelty of being handled and confined. This work also confirms previous findings that cattle with high FS have poorer liveweight gains under both pasture and feedlot conditions and FS has some value as a predictor of productivity. Correlations also indicated that agitated cattle show a heightened arousal and stress responses when being handled. Fear of humans, as assessed by ZA, CA and TT, did not adversely affect productivity

Quality of handling and holding yard environment, and beef cattle temperament: 1. Relationships with flight speed and fear of humans

Numerous tests have been used to measure beef cattle temperament, but limited research has addressed the relationship between such tests and whether temperament can be modified. One-hundred-and-forty-four steers were given one of three human handling and yarding experiences on six occasions during a 12-month grazing period post-weaning (backgrounding): Good handling/yarding, Poor handling/yarding and Minimal handling/yarding. At the end of this phase the cattle were lot-fed for 78 days, with no handling/yarding treatments imposed, before being transported for commercial slaughter. Temperament was assessed at the start of the experiment, during backgrounding and lot-feeding by flight speed (FS) and a fear of humans test, which measured the proximity to a stimulus person (zone average; ZA), the closest approach to the person (CA) and the amount the cattle moved around the test arena (total transitions; TT). During backgrounding, FS decreased for all treatments and at the end of backgrounding there was no difference between them. The rate of decline, however, was greatest in the Good group, smallest in the Minimal group with the Poor intermediate. In contrast, ZA was affected by treatment, with a greater reduction for the Good group than the others (P = 0.012). During lot-feeding, treatment did not affect FS, but all groups showed a decrease in ZA, with the greatest change in the Poor group, the least in the Good and the Minimal intermediate (P = 0.052). CA was positively correlated with ZA (r = 0.18 to 0.66) and negatively with TT (r = -0.180 to -0.659). FS was consistently correlated with TT only (r = 0.17 to 0.49). These findings suggest that FS and TT measure a similar characteristic, as do ZA and CA, but that these characteristics are different from one another, indicating that temperament is not a unitary trait, but has different facets. FS and TT measure one facet that we suggest is general agitation, whilst ZA and CA measure fear of people. Thus, the cattle became less agitated during backgrounding, but the effect was not permanently influenced by the quantity and quality of handling/yarding. However, Good handling/yarding reduced fearfulness of people. Fear of people was also reduced during lot-feeding, probably as a consequence of frequent exposure to humans in a situation that was neutral or positive for the cattle. Crow

Quality of handling and holding yard environment, and beef cattle temperament: 2. Consequences for stress and productivity

This experiment assessed the effects of different quality and quantity of handling and quality of the holding yard environment on the productivity and physiological parameters indicative of stress in beef cattle. One-hundred-and-forty-four steers were given one of three human handling and yarding experiences on six occasions during a 12-month grazing period post-weaning (backgrounding): Good handling/yarding, Poor handling/yarding and Minimal handling/yarding. At the end of this phase the cattle were lot-fed for 78 days, with no handling/yarding treatments imposed, before being transported for commercial slaughter. Temperament was assessed by flight speed (FS) and a fear of humans (FOH) test, which measured the proximity to a stimulus person (ZA), the closest approach to the person (CA) and the amount the cattle moved around the test arena (TT). Mid-way through backgrounding, the Minimal treatment group was heavier than the Good, which was heavier than the Poor (mean weights 207, 201 and 194 kg, respectively; P = 0.05; LSD = 5.4), but by the end of backgrounding there was no difference between treatments and treatment did not affect liveweight during lot-feeding. At the end of backgrounding, plasma cortisol levels were significantly lower (P < 0.001) in the Good treatment group compared to the Poor and Minimal groups but at the end of lot-feeding there was no significant difference between the groups. Treatment affected plasma non-esterified fatty acid levels in backgrounding (P = 0.060) and lot-feeding (P = 0.046) with levels being higher in the Minimal than the Good and Poor groups (backgrounding: 0.52, 0.44 and 0.47 nmol/L, respectively; SE 0.02; lot-feeding: 0.46, 0.41 and 0.41 mmol/L, respectively; LSD = 0.05). Significant weak to moderate (r-valu

The use of anaesthesia and analgesia during dehorning and their effects on the welfare of Bos indicus cattle

In northern Australia, cattle genotypes are predominantly Bos indicus-derived and the extensive nature of the production systems mean they are commonly dehorned at 5-9 months of age. Dehorning at this age is permitted without the use of anaesthetics or analgesics. To adopt pain relief management, producers must be convinced it is effective and practical. We examined the effects on welfare of the administration of a local anaesthetic and/or a non-steroidal anti-inflammatory drug (NSAID) during the dehorning of Brahman heifers (animal ethics approval RH245/08). Weaners, 5-7 months-old, were allocated to four treatments (n=15/group): Sham dehorn (S); Dehorn only (D); Dehorn + local anaesthetic (cornual block) + NSAID (LA/NSAID); and Dehorn + NSAID (NSAID). Blood samples were taken, via jugular venipuncture, 20min before treatment then 0.5, 1, 1.5, 2, 3, 5, 7, 9, 24, 48 and 72 hours and 2, 4 and 6 weeks post-dehorning. Liveweights were recorded fortnightly for 8 weeks. Counts of behaviour during dehorning showed LA/NSAID vocalised (mean±s.e; 1.6±0.9) and struggled (1.9±0.9) less (GLM,

A systems evaluation of high-input management using fortified molasses for beef production in Australia's dry tropics

The potential of beef producers to profitably produce 500-kg steers at 2.5 years of age in northern Australia's dry tropics to meet specifications of high-value markets, using a high-input management (HIM) system was examined. HIM included targeted high levels of fortified molasses supplementation, short seasonal mating and the use of growth promotants. Using herds of 300-400 females plus steer progeny at three sites, HIM was compared at a business level to prevailing best-practice, strategic low-input management (SLIM) in which there is a relatively low usage of energy concentrates to supplement pasture intake. The data presented for each breeding-age cohort within management system at each site includes: annual pregnancy rates (range: 14-99%), time of conception, mortalities (range: 0-10%), progeny losses between confirmed pregnancy and weaning (range: 0-29%), and weaning rates (range: 14-92%) over the 2-year observation. Annual changes in weight and relative net worth were calculated for all breeding and non-breeding cohorts. Reasons for outcomes are discussed. Compared with SLIM herds, both weaning weights and annual growth were >= 30 kg higher, enabling 86-100% of HIM steers to exceed 500 kg at 2.5 years of age. Very few contemporary SLIM steers reached this target. HIM was most profitably applied to steers. Where HIM was able to achieve high pregnancy rates in yearlings, its application was recommended in females. Well managed, appropriate HIM systems increased profits by around $15/adult equivalent at prevailing beef and supplement prices. However, a 20% supplement price rise without a commensurate increase in values for young slaughter steers would generally eliminate this advantage. This study demonstrated the complexity of pro. table application of research outcomes to commercial business, even when component research suggests that specific strategies may increase growth and reproductive efficiency and/or be more pro. table. Because of the higher level of management required, higher costs and returns, and higher susceptibility to market changes and disease, HIM systems should only be applied after SLIM systems are well developed. To increase profitability, any strategy must ultimately either increase steer growth and sale values and/or enable a shift to high pregnancy rates in yearling heifers