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 (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.

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.

SSR analysis of cultivated groundnut (Arachis hypogaea L.) germplasm resistant to rust and late leaf spot diseases

Cultivated groundnut (Arachis hypogaea L.) is an agronomically and economically important oilseed crop grown extensively throughout the semi-arid tropics of Asia, Africa and Latin America. Rust (Puccinia arachidis) and late leaf spot (LLS, Phaseoisariopsis personata) are among the major diseases causing significant yield loss in groundnut. The development of varieties with high levels of resistance has been constrained by adaptation of disease isolates to resistance sources and incomplete resistance in resistant sources. Despite the wide range of morphological diversity observed in the cultivated groundnut gene pool, molecular marker analyses have thus far been unable to detect a parallel level of genetic diversity. However, the recent development of simple sequence repeat (SSR) markers presents new opportunities for molecular diversity analysis of cultivate groundnut. The current study was conducted to identify diverse disease resistant germplasm for the development of mapping populations and for their introduction into breeding programs. Twenty-three SSRs were screened across 22 groundnut genotypes with differing levels of resistance to rust and LLS. Overall, 135 alleles across 23 loci were observed in the 22 genotypes screened. Twelve of the 23 SSRs (52%) showed a high level of polymorphism, with PIC values ≥0.5. This is the first report detecting such high levels of genetic polymorphism in cultivated groundnut. Multi-dimensional scaling and cluster analyses revealed three well-separated groups of genotypes. Locus by locus AMOVA and Kruskal-Wallis one-way ANOVA identified candidate SSR loci that may be valuable for mapping rust and LLS resistance. The molecular diversity analysis presented here provides valuable information for groundnut breeders designing strategies for incorporating and pyramiding rust and late leaf spot resistances and for molecular biologists wishing to create recombinant inbred line populations to map these traits.

Plant hosts of the phytoplasmas and rickettsia-like-organisms associated with strawberry lethal yellows and green petal diseases

Candidatus Phytoplasma australiense (Ca. P. australiense) is associated with the plant diseases strawberry lethal yellows (SLY), strawberry green petal (SGP), papaya dieback (PDB), Australian grapevine yellows (AGY) and Phormium yellow leaf (PYL; New Zealand). Strawberry lethal yellows disease is also associated with a rickettsia-like-organism (RLO) or infrequently with the tomato big bud (TBB) phytoplasma, the latter being associated with a wide range of plant diseases throughout Australia. In contrast, the RLO has been identified only in association with SLY disease, and Ca. P. australiense has been detected only in a limited number of plant host species. The aim of this study was to identify plant hosts that are possible reservoirs of Ca. P. australiense and the SLY RLO. Thirty-one plant species from south-east Queensland were observed with disease between 2001 and 2003 and, of these, 18 species tested positive using phytoplasma-specific primers. The RLO was detected in diseased Jacksonia scoparia and Modiola caroliniana samples collected at Stanthorpe. The TBB phytoplasma was detected in 16 different plant species and Ca. P. australiense Australian grapevine yellows strain was detected in six species. The TBB phytoplasma was detected in plants collected at Nambour, Stanthorpe, Warwick and Brisbane. Ca. P. australiense was detected in plants collected at Nambour, Stanthorpe, Gatton and Allora. All four phytoplasmas were detected in diseased Gomphocarpus physocarpus plants collected at Toowoomba, Allora, Nambour and Gatton. These results indicated that the vector(s) of Ca. P. australiense are distributed throughout south-east Queensland and the diversity of phytoplasmas detected in G. physocarpus suggests it is a feeding source for phytoplasma insect vectors or it has a broad susceptibility to a range of phytoplasmas.

Rickettsia-like-organisms and phytoplasmas associated with diseases in Australian strawberries

Strawberry lethal yellows (SLY) disease in Australia is associated with the phytoplasmas Candidatus Phytoplasma australiense and tomato big bud, and a rickettsia-like-organism (RLO). Ca. P. australiense is also associated with strawberry green petal (SGP) disease. This study investigated the strength of the association of the different agents with SLY disease. We also documented the location of SLY or SGP plants, and measured whether they were RLO or phytoplasma positive. Symptomatic strawberry plants collected from south-east Queensland (Australia) between January 2000 and October 2002 were screened by PCR for both phytoplasmas and the RLO. Two previously unreported disease symptoms termed severe fruit distortion (SFD) and strawberry leaves from fruit (SLF) were observed during this study but there was no clear association between these symptoms and phytoplasmas or the RLO. Only two SGP diseased plants were observed and collected, compared with 363 plants with SLY disease symptoms. Of the 363 SLY samples, 117 tested positive for the RLO, 67 tested positive for Ca. P. australiense AGY strain and 11 plants tested positive for Ca. P. australiense PYL variant strain. On runner production farms at Stanthorpe, Queensland the RLO was detected in SLY diseased plants more frequently than for the phytoplasmas. On fruit production farms on the Sunshine Coast, Queensland, Ca. P. australiense was detected in SLY disease plants more frequently than the RLO.

Sustained genetic control of wheat rust diseases in north-eastern Australia

Control of wheat rusts in north-eastern Australia has been based on resistance breeding since the early 1920s. It has been an enduring journey of discovery, disappointment, and achievement, which has culminated in a pool of knowledge and expertise upon which today's plant breeders can efficiently target durable resistance to the major rust diseases. This paper outlines significant advances in genetic control of rusts in the region, with particular emphasis on the invaluable role played by the University of Sydney rust control program and its influence on wheat breeding in the region and throughout Australia. This paper is part of ‘Global Landscapes in Cereal Rust Control’, see Aust. J. Agric. Res. Vol. 58, no. 6.

Expression of intracellular calcium signalling genes in cattle skin during tick infestation

It is widely acknowledged that changes in intracellular calcium ion (Ca2+) concentration provide dynamic signals that control a plethora of cellular processes, including triggering and mediating host defence mechanisms. In this study, quantitative real-time PCR was used to analyse gene expression of 14 Ca2+ signalling proteins in skin obtained from high tick-resistant (HR) and low tick-resistant (LR) cattle following artificial challenge with cattle tick (Rhipicephalus (Boophilus) microplus). Up-regulation of numerous genes was observed in both HR and LR skin following tick challenge, however substantially higher transcription activation was found in HR tissue. The elevated expression in HR skin of specific Ca2+ signalling genes such as AHNAK, CASQ, IL2, NFAT2CIP and PLCG1 may be related to host resistance. Our data suggest that Ca2+ and its associated proteins might play an important role in host response to ticks and that further investigation is warranted.

Recovery of intravenously infused chromium EDTA and lithium sulphate in the urine of cattle and their use as markers to measure urine volume

A series of metabolism experiments investigated the recovery of continuous-, intravenously infused chromium complexed with ethylenediamine tetra-acetic acid (CrEDTA) and lithium sulphate in the urine of cattle with a view to using the markers to estimate urine and metabolite output in grazing cattle. The recovery of Cr in urine from these infusions was similar (90%) in metabolism trials when cattle consumed three very contrasting diets: high-grain formulated pellet, lucerne hay (Medicago sativa) or low-quality native grass hay (predominantly Heteropogon contortus). By contrast, Li recovery in urine averaged 46.3 +/- 0.40% and 72.6 +/- 0.43% for native pasture and lucerne hays, respectively, but was not constant across days. There was negligible transfer of Cr from CrEDTA in blood serum to the rumen or faeces, whereas appreciable quantities of infused Li were found in both. The ratio of urine volume estimated by spot samples and marker dilution of Cr, to urine volume measured gravimetrically, was 1.05. In grazing studies using rumen-fistulated (RF) steers grazing seven different tropical and temperate grass and legume pastures, the ratio of concentrations of purine derivatives (PD) to Cr in spot samples of urine was shown to vary diurnally in the range of 49% to 157% of the average 24 h value. This finding indicated the need for regular sampling of urine to achieve an accurate average value for the PD: Cr ratio in urine for use in estimating urinary PD excretion and hence microbial protein production in the rumen. It was concluded that continuous, intravenous infusion of CrEDTA resulted in a constant recovery of Cr in the urine of cattle across diets and, provided an intensive sampling regime was followed to account for diurnal variation, it would be suitable as a marker to estimate urine volume and urinary output of PD in grazing cattle.

Clinical and pathological findings associated with congenital hypovitaminosis A in extensively grazed beef cattle

To determine the cause of exceptionally high mortality (41.4%) in perinatal calves on a beef cattle property 50 km south-west of Julia Creek in north-western Queensland. Investigations were based on clinical assessment of affected calves and laboratory analysis of pre- and postmortem specimens taken from 12 calves aged from 6 to 36 h of age. Associations between gross and histopathological findings and biochemical analyses conducted on serum and tissue samples were examined in relation to clinical observations. Clinical signs varied, but commonly included mild to severe ataxia, difficulty finding a teat and sucking, blindness (partial or complete, as judged by avoidance of obstacles) and depression with prominent drooping of the head. Gross and histopathological findings included herniation of the cerebellar vermis through the foramen magnum, squamous metaplasia of interlobular ducts in the parotid salivary glands and Wallerian degeneration of the optic nerves. Biochemical analysis of serum and liver samples available from four of the calves revealed low or undetectable levels of both vitamin A and vitamin E. Although vitamin E is known to have a sparing effect on vitamin A, the role (if any) played by deficiency of this vitamin was uncertain. The combination of clinical signs, postmortem findings, histopathological features and biochemical findings indicate that gestational vitamin A deficiency was highly likely to have been an important contributor to perinatal calf mortalities in this herd.

Sulfur-associated polioencephalomalacia in cattle grazing plants in the Family Brassicaceae

Polioencephalomalacia was diagnosed histologically in cattle from two herds on the Darling Downs, Queensland, during July-August 2007. In the first incident, 8 of 20 18-month-old Aberdeen Angus steers died while grazing pastures comprising 60% Sisymbrium irio (London rocket) and 40% Capsella bursapastoris (shepherd's purse). In the second incident, 2 of 150 mixed-breed adult cattle died, and another was successfully treated with thiamine, while grazing a pasture comprising almost 100% Raphanus raphanistrum (wild radish). Affected cattle were either found dead or comatose or were seen apparently blind and head-pressing in some cases. For both incidents, plant and water assays were used to calculate the total dietary sulfur content in dry matter as 0.62% and 1.01% respectively, both exceeding the recommended 0.5% for cattle eating more than 40% forage. Blood and tissue assays for lead were negative in both cases. No access to thiaminase, concentrated sodium ion or extrinsic hydrogen sulfide sources were identified in either incident. Below-median late summer and autumn rainfall followed by above-median unseasonal winter rainfall promoted weed growth at the expense of wholesome pasture species before these incidents.

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides

Resistance against synthetic pyrethroid (SP) products for the control of cattle ticks in Australia was detected in the field in 1984, within a very short time of commercial introduction. We have identified a mutation in the domain II S4-5 linker of the para-sodium channel that is associated with resistance to SPs in the cattle tick Rhipicephalus (Boophilus) microplus from Australia. The cytosine to adenine mutation at position 190 in the R. microplus sequence AF134216, results in an amino acid substitution from leucine in the susceptible strain to isoleucine in the resistant strain. A similar mutation has been shown to confer SP resistance in the whitefly, Bemisia tabaci, but has not been described previously in ticks. A diagnostic quantitative PCR assay has been developed using allele-specific Taqman® minor groove-binding (MGB) probes. Using the assay to screen field and laboratory populations of ticks showed that homozygote allelic frequencies correlated highly with the survival percentage at the discriminating concentration of cypermethrin.

Managing for rainfall variability: effect of grazing strategy on cattle production in a dry tropical savanna

Rainfall variability is a challenge to sustainable and pro. table cattle production in northern Australia. Strategies recommended to manage for rainfall variability, like light or variable stocking, are not widely adopted. This is due partly to the perception that sustainability and profitability are incompatible. A large, long-term grazing trial was initiated in 1997 in north Queensland, Australia, to test the effect of different grazing strategies on cattle production. These strategies are: (i) constant light stocking (LSR) at long-term carrying capacity (LTCC); (ii) constant heavy stocking (HSR) at twice LTCC; (iii) rotational wet-season spelling (R/Spell) at 1.5 LTCC; (iv) variable stocking (VAR), with stocking rates adjusted in May based on available pasture; and (v) a Southern Oscillation Index (SOI) variable strategy, with stocking rates adjusted in November, based on available pasture and SOI seasonal forecasts. Animal performance varied markedly over the 10 years for which data is presented, due to pronounced differences in rainfall and pasture availability. Nonetheless, lighter stocking at or about LTCC consistently gave the best individual liveweight gain (LWG), condition score and skeletal growth; mean LWG per annum was thus highest in the LSR (113 kg), intermediate in the R/Spell (104 kg) and lowest in the HSR(86 kg). MeanLWGwas 106 kg in the VAR and 103 kg in the SOI but, in all years, the relative performance of these strategies was dependent upon the stocking rate applied. After 2 years on the trial, steers from lightly stocked strategies were 60-100 kg heavier and received appreciable carcass price premiums at the meatworks compared to those under heavy stocking. In contrast, LWG per unit area was greatest at stocking rates of about twice LTCC; mean LWG/ha was thus greatest in the HSR (21 kg/ha), but this strategy required drought feeding in four of the 10 years and was unsustainable. Although LWG/ha was lower in the LSR (mean 14 kg/ha), or in strategies that reduced stocking rates in dry years like the VAR(mean 18 kg/ha) and SOI (mean 17 kg/ha), these strategies did not require drought feeding and appeared sustainable. The R/Spell strategy (mean 16 kg/ha) was compromised by an ill-timed fire, but also performed satisfactorily. The present results provide important evidence challenging the assumption that sustainable management in a variable environment is unprofitable. Further research is required to fully quantify the long-term effects of these strategies on land condition and profitability and to extrapolate the results to breeder performance at the property level.

Development of near infrared analysis of faeces to estimate non-grass proportions in diets selected by cattle grazing tropical pastures

Grass (monocots) and non-grass (dicots) proportions in ruminant diets are important nutritionally because the non-grasses are usually higher in nutritive value, particularly protein, than the grasses, especially in tropical pastures. For ruminants grazing tropical pastures where the grasses are C-4 species and most non-grasses are C-3 species, the ratio of C-13/C-12 in diet and faeces, measured as delta C-13 parts per thousand, is proportional to dietary non-grass%. This paper describes the development of a faecal near infrared (NIR) spectroscopy calibration equation for predicting faecal delta C-13 from which dietary grass and non-grass proportions can be calculated. Calibration development used cattle faeces derived from diets containing only C-3 non-grass and C-4 grass components, and a series of expansion and validation steps was employed to develop robustness and predictive reliability. The final calibration equation contained 1637 samples and faecal delta C-13 range (parts per thousand) of [12.27]-[27.65]. Calibration statistics were: standard error of calibration (SEC) of 0.78, standard error of cross-validation (SECV) of 0.80, standard deviation (SD) of reference values of 3.11 and R-2 of 0.94. Validation statistics for the final calibration equation applied to 60 samples were: standard error of prediction (SEP) of 0.87, bias of -0.15, R-2 of 0.92 and RPD of 3.16. The calibration equation was also tested on faeces from diets containing C-4 non-grass species or temperate C-3 grass species. Faecal delta C-13 predictions indicated that the spectral basis of the calibration was not related to C-13/C-12 ratios per se but to consistent differences between grasses and non-grasses in chemical composition and that the differences were modified by photosynthetic pathway. Thus, although the calibration equation could not be used to make valid faecal delta C-13 predictions when the diet contained either C-3 grass or C-4 non-grass, it could be used to make useful estimates of dietary non-grass proportions. It could also be ut :sed to make useful estimates of non-grass in mixed C-3 grass/non-grass diets by applying a modified formula to calculate non-grass from predicted faecal delta C-13. The development of a robust faecal-NIR calibration equation for estimating non-grass proportions in the diets of grazing cattle demonstrated a novel and useful application of NIR spectroscopy in agriculture.

Bats and Emerging Zoonoses: Henipaviruses and SARS.

Nearly 75% of all emerging infectious diseases (EIDs) that impact or threaten human health are zoonotic. The majority have spilled from wildlife reservoirs, either directly to humans or via domestic animals. The emergence of many can be attributed to predisposing factors such as global travel, trade, agricultural expansion, deforestation habitat fragmentation, and urbanization; such factors increase the interface and or the rate of contact between human, domestic animal, and wildlife populations, thereby creating increased opportunities for spillover events to occur. Infectious disease emergence can be regarded as primarily an ecological process. The epidemiological investigation of EIDs associated with wildlife requires a trans-disciplinary approach that includes an understanding of the ecology of the wildlife species, and an understanding of human behaviours that increase risk of exposure. Investigations of the emergence of Nipah virus in Malaysia in 1999 and severe acute respiratory syndrome (SARS) in China in 2003 provide useful case studies. The emergence of Nipah virus was associated with the increased size and density of commercial pig farms and their encroachment into forested areas. The movement of pigs for sale and slaughter in turn led to the rapid spread of infection to southern peninsular Malaysia, where the high-density, largely urban pig populations facilitated transmission to humans. Identifying the factors associated with the emergence of SARS in southern China requires an understanding of the ecology of infection both in the natural reservoir and in secondary market reservoir species. A necessary extension of understanding the ecology of the reservoir is an understanding of the trade, and of the social and cultural context of wildlife consumption. Emerging infectious diseases originating from wildlife populations will continue to threaten public health. Mitigating and managing the risk requires an appreciation of the connectedness between human, livestock and wildlife health, and of the factors and processes that disrupt the balance.

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.