Wooden hoof blocks: are we using the right wood?

AIMS: To investigate the association between the density of wooden hoof blocks and resistance to wear in pasture-based dairy herds, and to assess the density of commercially available wooden hoof blocks. METHODS: Three types of wooden hoof blocks with different densities (low, medium and high) were attached to 36 lactating dairy cows with parity ≤2 and sound locomotion (score ≤2 on a scale of 1-4). The height of wooden blocks was measured in three different regions, front, abaxial and caudal on Days 7, 11, 14, 18, 21, 25 and 28 after application. Due to the loss of low-density wooden blocks, the data for these blocks were analysed for only two measurements on Days 7 and 11. The data for medium and high-density wooden blocks were analysed from Days 7-25. A linear mixed model with repeated measures was used to analyse the repeated observations. Height, density and surface area of commercially available hoof blocks (n = 19) were measured and compared to the blocks used in this study. RESULTS: The magnitude of wear, in the front and the abaxial point of the blocks were greater in blocks made of low-density wood compared to those made of medium and high-density wood (p < 0.001). The amount of wear increased over time for all groups (p < 0.001). Wood density was negatively associated with wear and loss. Measurements of commercial wooden blocks revealed that the 13/19 (63%) had lower density and 12/19 (68%) less surface area than the wooden blocks with medium density used in this study. CONCLUSION: In this study, the density of the wood was significantly associated with the longevity of hoof blocks when applied to hooves of pasture-based dairy cows. CLINICAL RELEVANCE: The longevity of the wooden hoof blocks applied to treat lame cows plays a significant role in the healing of the claw horn lesions. The density of a wooden hoof block affects the rate of wear of the block, and this should be considered by manufacturers and those treating lame cows.

Milk acidification to control the growth of Mycoplasma bovis and Salmonella Dublin in contaminated milk.

Bacterial contamination of milk fed to calves compromises calf health. Several bacterial pathogens that infect cows, including Mycoplasma bovis and Salmonella enterica ssp. enterica serovar Dublin, are shed in milk, providing a possible route of transmission to calves. Milk acidification lowers the milk pH so that it is unsuitable for bacterial growth and survival. The objectives of this study were to (1) determine the growth of M. bovis and Salmonella Dublin in milk, and (2) evaluate the efficacy of milk acidification using a commercially available acidification agent (Salstop, Impextraco, Heist-op-den-Berg, Belgium) to control M. bovis and Salmonella Dublin survival in milk. For the first objective, 3 treatments and a positive control were prepared in 10 mL of milk and broth, respectively, and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 104 cfu/mL. Each treatment was retained at 5, 23, or 37°C with the positive control at 37°C. Aliquots were taken at 4, 8, 24, 28, 32, 48, 52, and 56 h after inoculation and transferred onto agar medium in triplicate following a 10-fold dilution series in sterile phosphate-buffered saline. All plates were incubated and colonies counted. For the second objective, 4 treatments and a positive control were prepared with 100 mL of milk and inoculated with M. bovis or Salmonella Dublin to an approximate concentration of 106 cfu/mL. With the use of Salstop, treatments were adjusted to an approximate pH of 6, 5, 4, or 3.5. The positive control was left untreated. At 1, 2, 4, 6, 8, and 24 h after treatment, triplicate aliquots were taken, the pH measured, and then the aliquots were transferred onto agar medium and into broth for enrichment. Following incubation, agar colonies were counted, while broths were plated and incubated prior to colonies being counted. All trials were repeated. Mycoplasma bovis did not grow in milk, but Salmonella Dublin proliferated. The pH of all acidification treatments remained stable for 24 h. No viable M. bovis organisms were detected at 1 h of exposure to pH 3.5 and 4 or at 8 h of exposure to pH 5. Following 24 h of exposure to pH 6 M. bovis remained viable. No viable Salmonella Dublin organisms were detected at 2 and 6 h of exposure to pH 3.5 and 4, respectively. Salmonella Dublin remained viable following 24 h of exposure to pH 5 and 6. These results demonstrate that milk acidification using Salstop is effective at eliminating viable M. bovis and Salmonella Dublin organisms in milk if the appropriate pH and exposure time are maintained.

Characterisation and mapping of gene Lr73 conferring seedling resistance to Puccinia triticina in common wheat.

KEY MESSAGE: A gene conferring seedling resistance to Puccinia triticina was mapped to chromosome 2BS in the wheat Morocco. The gene was shown to be distinct and was therefore designated Lr73. The wheat genotype Morocco, widely susceptible to isolates of Puccinia triticina, was resistant to an Australian isolate of this pathogen collected in 2004. Genetic studies established that the resistance in Morocco was also present the Australian wheat genotypes Avocet, Halberd, Harrier, Tincurrin and a selection of cultivar Warigal lacking the resistance gene Lr20. Genetic studies based on a cross with Halberd showed that the gene is dominant and located on chromosome 2BS (XwPt8760-4 cM-Lr73-1.4 cM-XwPt8235). The gene was genetically independent of the Lr13, Lr16 and Lr23 loci, also located on chromosome 2BS, indicating that it is distinct. The locus designation Lr73 was therefore assigned. On the basis of multi-pathotype tests, it is likely Lr73 is also present in the Australian wheat cultivars Clearfield STL, Federation (with Lr10), Gatcher (with Lr10 and Lr27+Lr31), Marombi (with Lr1 and Lr37), Pugsley (with Lr1 and Lr37), Spear (with Lr1), Stiletto and Tarsa (with Lr1). Gene Lr73 is unlikely to be of value in resistance breeding. However, recognising Lr73 is important to avoid its inadvertent selection in breeding programmes. Furthermore, the apparent rarity of avirulence for genes like Lr73, sometimes referred to as "fossil" resistance genes, makes them of interest in terms of the evolution of disease resistance in host plants and of virulence in the respective rust pathogens.

Development of a novel in-water vaccination protocol for DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine in adult sheep.

Intensive livestock production is associated with an increased incidence of salmonellosis. The risk of infection and the subsequent public health concern is attributed to increased pathogen exposure and disease susceptibility due to multiple stressors experienced by livestock from farm to feedlot. Traditional parenteral vaccine methods can further stress susceptible populations and cause carcass damage, adverse reactions, and resultant increased production costs. As a potential means to address these issues, in-water delivery of live attenuated vaccines affords a low cost, low-stress method for immunization of livestock populations that is not associated with the adverse handling stressors and injection reactions associated with parenteral administration. We have previously established that in-water administration of a Salmonella enterica serovar Typhimurium dam vaccine conferred significant protection in livestock. While these experimental trials hold significant promise, the ultimate measure of the vaccine will not be established until it has undergone clinical testing in the field wherein environmental and sanitary conditions are variable. Here we show that in-water administration of a S. Typhimurium dam attenuated vaccine was safe, stable, and well-tolerated in adult sheep. The dam vaccine did not alter water consumption or vaccine dosing; remained viable under a wide range of temperatures (21-37°C); did not proliferate within fecal-contaminated trough water; and was associated with minimal fecal shedding and clinical disease as a consequence of vaccination. The capacity of Salmonella dam attenuated vaccines to be delivered in drinking water to protect livestock from virulent Salmonella challenge offers an effective, economical, stressor-free Salmonella prophylaxis for intensive livestock production systems.

Protective immunity conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine when delivered in-water to sheep challenged with Salmonella enterica serovar Typhimurium.

Stimulation of acquired immunity to Salmonella in livestock is not feasible in neonates (which can be infected within 24h of birth) and is challenging in feedlots, which typically source animals from diverse locations and vendors. Induction of innate immune mechanisms through mass vaccination of animals upon arrival to feedlots is an alternative approach. Transport, environmental conditions, changes in social grouping, and further handling during feedlot assembly are significant stressors. These factors, as well as concurrent exposure to a diversity of pathogens, contribute to the risk of disease. We have shown that oral immunization of calves with a modified live Salmonella enterica serovar Typhimurium vaccine strain, which lacks the DNA adenine methylase gene (S. Typhimurium dam), attenuates the severity of clinical disease, reduces fecal shedding, and promotes clearance of salmonellae following virulent homologous and heterologous challenge. This study examines the safety and efficacy of a S. Typhimurium dam vaccine in sheep via oral delivery in drinking water (ad libitum), as a means to effectively vaccinate large groups of animals. Adult merino sheep were vaccinated in drinking water -28 days, -7 days and 24h pre and 24h post-virulent Salmonella Typhimurium challenge which was administered via the oral route. Significant attenuation of clinical disease (temperature, appetite, and attitude) and reduction in mortality and virulent Salmonella Typhimurium fecal shedding and tissue colonization was observed in animals that received the vaccine 28 and 7 days pre-challenge. Further, vaccination did not pose a risk to stock previously infected with virulent salmonellae as mortalities and clinical disease in sheep vaccinated prior to or following virulent challenge did not differ significantly from the non-vaccinated controls. The capacity of S. Typhimurium dam vaccines delivered in drinking water to protect livestock from virulent Salmonella challenge offers an effective, economical, stressor free Salmonella prophylaxis for intensive livestock production systems.

Prevalence of major enteric pathogens in Australian dairy calves with diarrhoea.

OBJECTIVE: Determine the prevalence of the major enteric pathogens in dairy and dairy beef calves with diarrhoea in Australia. DESIGN: Cross-sectional study. METHODS: Faecal samples from 84 Australian dairy and dairy beef properties (597 samples) were screened for rotavirus and coronavirus using real-time reverse transcription polymerase chain reaction, for Salmonella spp. using selective enrichment faecal culture, and for enterotoxigenic Escherichia coli (K99) and Cryptosporidium parvum using a commercial enzyme-linked immunosorbent assay. A logistic regression with random effects model was used to compare prevalence of pathogens in dairy and dairy beef operations. RESULTS: Enteric pathogens were isolated from 97.6% of outbreaks and 95.0% of samples. Rotavirus was the most common pathogen identified (477/597, 79.9%) followed by C. parvum (349/597, 58.5%), Salmonella spp. (142/597, 23.8%), coronavirus (129/597, 21.6%) and E. coli K99 (104/597, 17.4%). Multiple pathogens were identified on 96.4% of farms and from 71.0% of samples. Samples from dairy beef properties were more likely to have multiple pathogens than dairy properties (P < 0.05), whereas rotavirus and Salmonella spp. were more likely to be identified in samples collected from dairy beef than dairy properties (P < 0.05). CONCLUSION: Most outbreaks of calf diarrhoea in dairy and dairy beef operations involve multiple pathogens. Rotavirus and C. parvum were the most frequently identified pathogens across production systems. Salmonella spp. and rotavirus were more frequently identified in dairy beef operations.

Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species.

Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms-a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity.

Chromosomal location and molecular mapping of a tan spot resistance gene in the winter wheat cultivar Red Chief.

The winter wheat cultivar Red Chief has been identified as the wheat cultivar most resistant to Pyrenophora tritici-repentis (Ptr). This study was undertaken to determine the inheritance, chromosomal location and molecular mapping of a tan spot resistance gene in Red Chief. χ² analysis of the F2 segregation data of the hybrids between 21 monosomic lines of the susceptible wheat cultivar Chinese Spring and the resistant cultivar Red Chief revealed that tan spot resistance in cv. Red Chief is controlled by a single recessive gene located on chromosome 3A. Linkage analysis using SSR markers in the Red Chief/Chinese Spring F2 population showed that the tsr4 gene is clustered in the region around Xgwm2a, on the short arm of chromosome 3A. This marker has also been identified as the closest marker to the tsr3 locus on chromosome 3D in synthetic wheat lines. Validation analysis of this marker for the tsr3 and tsr4 genes using 28 resistant and 6 susceptible genotypes indicated that the 120 bp allele (the tsr3 gene) specific fragment was observed in 11 resistant genotypes, including the three synthetic lines XX41, XX45 and XX110, while the 130 bp allele was amplified only in cv. Red Chief and Dashen. Xgwm2a can be used to trace the presence of the target gene in successive backcross generations and pyramiding of the tsr3 & tsr4 genes into a commonly grown and adaptable cultivar.

Cross-protective immunity conferred by a DNA adenine methylase deficient Salmonella enterica serovar Typhimurium vaccine in calves challenged with Salmonella serovar Newport.

Intensive livestock production and management systems are associated with increased fecal-oral pathogen transmission and a resultant high prevalence of multiple Salmonella serovars in many large dairy farms and feedlots. Thus, it is imperative to develop livestock vaccines that are capable of eliciting potent states of cross-protective immunity against a diversity of serovars of a given species. Immunization with modified live Salmonella enterica serovar Typhimurium vaccine strains, that lack the DNA adenine methylase (Dam), confers cross-protective immunity in murine and avian models of typhoid fever as well as in a bovine model of salmonellosis. Here we examined whether a dam mutant Typhimurium vaccine (serogroup B) has the capacity to elicit cross-protection against a virulent challenge with an emerging, clinically relevant, and multi-drug resistant strain of serovar Newport (serogroup C2-C3) that has been associated with clinical disease in recent salmonellosis outbreaks in calves. Vaccinated animals challenged with Newport exhibited a significant attenuation of clinical disease (improved attitude scores, increased daily weight gains and reduced fever and diarrhea) and a concomitant reduction in Newport fecal shedding and colonization of mesenteric lymph nodes and lungs compared to non-vaccinated control animals. The capacity to elicit cross-protective immunity in calves suggests that dam mutant vaccines have potential application toward the prevention and control of Salmonella infection in commercial livestock production systems wherein livestock are exposed to a diversity of Salmonella serovars.

Molecular mapping of resistance genes to tan spot [Pyrenophora tritici-repentis race 1] in synthetic wheat lines.

Synthetic wheat lines (2n = 6x = 42, AABBDD), which are amphiploids developed from the hybrid between tetraploid wheat (Triticum turgidum L., 2n = 4x = 28, AABB) and Aegilops tauschii Coss. (2n = 2x = 14, DD), are important sources of resistance against tan spot of wheat caused by Pyrenophora tritici-repentis. In the present study, inheritance, allelism and genetic linkage analysis in synthetic wheat lines have been carried out. Segregation analysis of the phenotypic and molecular data in F(2:3) populations of CS/XX41, CS/XX45, and CS/XX110 has revealed a 1:2:1 segregation ratio indicating that resistance of tan spot in these synthetic lines is controlled by a single gene. Allelism tests detected no segregation for susceptibility among F(1) and F(2) plants derived from intercrosses of the resistance lines XX41, XX45 and XX110 indicating that the genes are either allelic or tightly linked. Linkage analysis using SSR markers showed that all the three genes: tsn3a in XX41, Tsn3b in XX45 and tsn3c in XX110 are clustered in the region around Xgwm2a, located on the short arm of chromosome 3D. The linked markers and genetic relationship of these genes will greatly facilitate their use in wheat breeding and deployment of cultivars resistant to tan spot.

Localization of a novel recessive powdery mildew resistance gene from common wheat line RD30 in the terminal region of chromosome 7AL.

Segregation analysis of resistance to powdery mildew in a F(2) progeny from the cross Chinese Spring (CS) x TA2682c revealed the inheritance of a dominant and a recessive powdery mildew resistance gene. Selfing of susceptible F(2) individuals allowed the establishment of a mapping population segregating exclusively for the recessive resistance gene. The extracted resistant derivative showing full resistance to each of 11 wheat powdery mildew isolates was designated RD30. Amplified fragment length polymorphism (AFLP) analysis of bulked segregants from F(3)s showing the homozygous susceptible and resistant phenotypes revealed an AFLP marker that was associated with the recessive resistance gene in repulsion phase. Following the assignment of this AFLP marker to wheat chromosome 7A by means of CS nullitetrasomics, an inspection of simple sequence repeat (SSR) loci evenly spaced along chromosome 7A showed that the recessive resistance gene maps to the distal region of chromosome 7AL. On the basis of its close linkage to the Pm1 locus, as inferred from connecting partial genetic maps of 7AL of populations CS x TA2682c and CS x Virest ( Pm1e), and its unique disease response pattern, the recessive resistance gene in RD30 was considered to be novel and tentatively designated mlRD30.

Powdery mildew resistance gene Pm22 in cultivar Virest is a member of the complex Pm1 locus in common wheat ( Triticum aestivum L. em Thell.).

The powdery mildew resistance gene Pm22, identified in the Italian wheat cultivar Virest and originally assigned to wheat chromosome 1D, was mapped to chromosome 7A with the aid of molecular markers. Mapping of common AFLP and SSR markers in two wheat crosses segregating for Pm22 and Pm1c, respectively, indicated that Pm22 is a member of the complex Pm1 locus. Pm22 also showed a pattern of resistance reaction to a differential set of Blumeria graminis f. sp. tritici isolates that was distinguishable from those from other Pm1 alleles in lines Axminster/8*Cc ( Pm1a), MocZlatka ( Pm1b), Weihenstephan Stamm M1N ( Pm1c) and Triticum spelta var. duhamelianum TRI 2258 ( Pm1d). Based on these results, the gene symbol Pm1e is proposed for the powdery mildew resistance gene in cv. Virest.

Localisation of genes for resistance against Blumeria graminis f.sp. hordei and Puccinia graminis in a cross between a barley cultivar and a wild barley (Hordeum vulgare ssp. spontaneum) line.

The aims of this investigation have been to map new (quantitative) resistance genes against powdery mildew, caused by Blumeria graminis f.sp. hordei L., and leaf rust, caused by Puccinia hordei L., in a cross between the barley ( Hordeum vulgare ssp. vulgare) cultivar "Vada" and the wild barley ( Hordeum vulgare ssp. spontaneum) line "1B-87" originating from Israel. The population consisted of 121 recombinant inbred lines. Resistance against leaf rust and powdery mildew was tested on detached leaves. The leaf rust isolate "I-80" and the powdery mildew isolate "Va-4", respectively, were used for the infection in this experiment. Moreover, powdery mildew disease severity was observed in the field at two different epidemic stages. In addition to other DNA markers, the map included 13 RGA (resistance gene analog) loci. The structure of the data demanded a non-parametric QTL-analysis. For each of the four observations, two QTLs with very high significance were localised. QTLs for resistance against powdery mildew were detected on chromosome 1H, 2H, 3H, 4H and 7H. QTLs for resistance against leaf rust were localised on 2H and 6H. Only one QTL was common for two of the powdery mildew related traits. Three of the seven QTLs were localised at the positions of the RGA-loci. Three of the five powdery mildew related QTLs are sharing their chromosomal position with known qualitative resistance genes. All detected QTLs behaved additively. Possible sources of the distorted segregation observed, the differences between the results for the different powdery mildew related traits and the relation between qualitative and quantitative resistance are discussed.

A resistance gene analog useful for targeting disease resistance genes against different pathogens on group 1S chromosomes of barley, wheat and rye.

Comparative sequence analysis of the resistance gene analog (RGA) marker locus aACT/CAA (originally found to be tightly linked to the multiallelic barley Mla cluster) from genomes of barley, wheat and rye revealed a high level of relatedness among one another and showed high similarity to a various number of NBS-LRR disease resistance proteins. Using the sequence-specific polymerase chain reaction (PCR), RGA marker aACT/CAA was mapped on group 1S chromosomes of the Triticeae and was associated with disease resistance loci. In barley and rye, the marker showed linkage to orthologous powdery mildew resistance genes Mla1 and Pm17, respectively, while in wheat linkage with a QTL against fusarium head blight (FHB) disease was determined. The use of RGA clones for R gene mapping and their role in the expression of qualitative and quantitative resistance is discussed.

Chromosomal location of AFLP markers in common wheat utilizing nulli-tetrasomic stocks.

Amplified fragment length polymorphism (AFLP) markers with a total of 256 EcoRI + ANN - MseI + CNN primer combinations were investigated employing the common wheat cultivar Triticum aestivum 'Chinese Spring.' On average, 103 fragments per primer combination were amplified, ranging from a maximum of 226 fragments to a minimum of 18 fragments. The primer combinations E + AAA - M + CNN and E + ATT - M + CNN produced very few distinct fragments. By using 15 randomly chosen EcoRI + ANN - MseI + CNN primer combinations, 928 AFLP markers were allocated to wheat chromosomes, of which 131 were assigned to specific chromosome arms. These AFLP markers were locus-specific and randomly distributed on the different chromosomes. In addition, 6 and 41 AFLP markers were simultaneously absent in two nulli-tetrasomics (NTs) of both homoeologous and non-homoeologous groups, respectively, whereas additional fragments were detected in N1BT1A, N5AT5D, and N6BT6A lines.

Allele-specific amplification of polymorphic sites for the detection of powdery mildew resistance loci in cereals.

Primers for the polymerase chain reaction (PCR) were tailored to selectively amplify RFLP marker alleles associated with resistance and susceptibility for powdery mildew in cereals. The differentiation between marker alleles for susceptible and resistant genotypes is based on the discrimination of a single nucleotide by using allele-specific oligonucleotides as PCR primers. The PCR assays developed are diagnostic for RFLP alleles at the loci MWG097 in the barley genome and Whs350 in the wheat genome. The first marker locus is closely linked to MlLa resistance in barley, while the latter is linked to Pm2 resistance locus in wheat. PCR analysis of 31 barley and 30 wheat cultivars, with some exceptions, verified the presence or absence of the resistance loci investigated. These rapid PCR-based approaches are proposed as an efficient alternative to conventional procedures for selecting powdery mildew-resistant genotypes in breeding programs.