Ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing protein (ASB) 15 alters differentiation of mouse C2C12 myoblasts and phosphorylation of mitogen-activated protein kinase and Akt

Ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 is a novel ASB gene family member predominantly expressed in skeletal muscle. We have previously reported that overexpression of ASB15 delays differentiation and alters protein turnover in mouse C(2)C(12) myoblasts. However, the extent of ASB15 regulation of differentiation and molecular pathways underlying this activity are unknown. The extracellular signal-regulated kinase (Erk) 1/2 and phosphatidylinositol-3 kinase-Akt (PI3K/Akt; Akt is also known as protein kinase B) signaling pathways have a role in skeletal muscle growth. Activation (phosphorylation) of the Erk1/2 signaling pathway promotes proliferation, whereas activation of the PI3K/Akt signaling pathway promotes myoblast differentiation. Accordingly, we tested the hypothesis that ASB15 controls myoblast differentiation through its regulation of these kinases. Stably transfected myoblasts overexpressing ASB15 (ASB15+) demonstrated decreased differentiation, whereas attenuation of ASB15 expression (ASB15-) increased differentiation. However, ASB15+ cells had less abundance of the phosphorylated mitogen-activated protein kinase (active) form, despite decreased differentiation relative to control myoblasts (ASB15Con). The mitogen-activated protein kinase kinase inhibitor, U0126, effectively decreased mitogen-activated protein kinase phosphorylation and stimulated differentiation in ASB15- and ASB15Con cells. However, inhibition of the Erk1/2 pathway was unable to overcome the inhibitory effect of overexpressing ASB15 on differentiation (ASB15+), suggesting that the Erk1/2 pathway is likely not the predominant mediator of ASB15 activity on differentiation. Expression of ASB15 also altered phosphorylation of the PI3K/Akt pathway, as ASB15+ and ASB15- cells had decreased and increased Akt phosphorylation, respectively. These data were consistent with observed differences in differentiation. Administration of IGF-I, a PI3K/Akt activator, in ASB15+ was able to partially override the previously observed phenotype of delayed differentiation, whereas administration of the PI3K/ Akt inhibitor, LY294002, decreased phosphorylation of Akt and differentiation of all cell lines similar to the untreated ASB15+ myoblasts. These results provide initial evidence that ASB15 has a role in early myoblast differentiation and that its effects may be mediated in part by the PI3K/Akt signal transduction pathway.     

Effect of breed of cattle on transmission rate and innate resistance to infection with Babesia bovis and B bigemina transmitted by Boophilus microplus

OBJECTIVE: To assess the effect of breed of cattle on the transmission rates of and innate resistance to Babesia bovis and B bigemina parasites transmitted by Boophilus microplus ticks. DESIGN: Groups of 56 purebred B indicus and 52 B indicus cross B taurus (50%, F1 generation) steers were placed in a paddock seeded with and also naturally infested with B microplus which were the progeny of females ticks fed on B taurus cattle specifically infected with a virulent isolate of B bovis. The cattle were placed in the infested paddock 50 days after seeding had started. PROCEDURE: Cattle were inspected from horseback daily for 50 days. Clinically ill cattle were brought to yards and assessed by monitoring fever, depression of packed-cell volume, parasitaemia and severity of clinical signs. Any animals that met preset criteria were treated for babesiosis. Blood samples were collected from all cattle on day 28, 35 and 42 after exposure and antibodies to Babesia spp and packed cell volume measured. RESULTS: All steers, except for one crossbred, seroconverted to B bovis and B bigemina by day 35 and 75% of the crossbred steers showed a maximum depression in packed cell volume of more than 15% due to infection with Babesia spp compared with only 36% of the B indicus group. Ten of the 52 crossbreds and 1 of the 56 B indicus steers showed severe clinical signs. Two of the crossbreds required treatment of which one died 2 weeks after initial treatment. CONCLUSIONS: Pure-bred B indicus cattle have a high degree of resistance to babesiosis, but crossbred cattle are sufficiently susceptible to warrant the use of preventive measures such as vaccination. Transmission rates of B bovis and B bigemina to B indicus and crossbred cattle previously unexposed to B microplus were the same.     

Genotypic diversity in field isolates of Babesia bovis from cattle with babesiosis after vaccination

Objective To determine whether particular genotypes of Babesia bovis were common to field isolates obtained from cattle properties in Queensland where the B bovis vaccine had apparently failed.
Design A comparative study of polymerase chain reaction genotypes in different populations of B bovis .
Procedure Two polymerase chain reaction assays were applied to analyse DNA extracts of B bovis vaccine (K, T and Dixie strains) and 27 field isolates from 24 properties where disease outbreaks had occurred despite the use of the vaccine. To evaluate the stability of the genotypes identified, 11 of the field isolates were inoculated into experimental cattle that had either been previously vaccinated with T strain or not vaccinated.
Results No particular genotype of B bovis was responsible for the problems observed in previously vaccinated herds. None of the isolates had genotypes identical to the vaccine strains used. No geographic trends among the genotypes were observed. Isolates that originated from the same property also had different genotypes. Blood passage of the 11 field isolates in either previously vaccinated or nonvaccinated cattle did not alter the original genotype.
Conclusion No particular genotypes identified by the Bv80 and BvVA1 polymerase chain reaction assays could be associated with vaccine failures.     

The effect of Psoroptes ovis infestation on ovine epidermal barrier function

Sheep scab is an intensively pruritic, exudative and allergic dermatitis of sheep caused by the ectoparasitic mite Psoroptes ovis. The purpose of the present study was to investigate the effect of P. ovis infestation on different components of the ovine epidermal barrier within the first 24 hours post-infestation (hpi). To achieve this, the expression of epidermal differentiation complex (EDC) genes and epidermal barrier proteins, the nature and severity of epidermal pathology and transepidermal water loss (TEWL) were evaluated.By 1 hpi a significant dermal polymorphonuclear infiltrate and a significant increase in TEWL with maximal mean TEWL (598.67 g/m²h) were observed. Epidermal pathology involving intra-epidermal pustulation, loss of epidermal architecture and damage to the basement membrane was seen by 3 hpi. Filaggrin and loricrin protein levels in the stratum corneum declined significantly in the first 24 hpi and qPCR validation confirmed the decrease in expression of the key EDC genes involucrin, filaggrin and loricrin observed by microarray analysis, with 5.8-fold, 4.5-fold and 80-fold decreases, respectively by 24 hpi.The present study has demonstrated that early P. ovis infestation disrupts the ovine epidermal barrier causing significant alterations in the expression of critical barrier components, epidermal pathology, and TEWL. Many of these features have also been documented in human and canine atopic dermatitis suggesting that sheep scab may provide a model for the elucidation of events occurring in the early phases of atopic sensitisation.     

Two major ruminant acute phase proteins,haptoglobin and serum amyloid A,as serum biomarkers during active sheep scab infestation

Two ruminant acute phase proteins (APPs), haptoglobin (Hp) and serum amyloid A (SAA), were evaluated as serum biomarkers (BMs) for sheep scab–a highly contagious ectoparasitic disease caused by the mite Psoroptes ovis, which is a major welfare and production threat worldwide. The levels of both APPs increased in serum following experimental infestation of sheep with P. ovis, becoming statistically significantly elevated from pre-infestation levels at 4 weeks post-infestation. Following successful treatment of infested sheep with an endectocide, Hp and SAA serum levels declined rapidly, with half lives of less than 3 days. In contrast, serum IgG levels which specifically bound the P. ovis-derived diagnostic antigen Pso o 2 had a half-life of 56 days. Taking into account pre-infestation serum levels, rapidity of response to infestation and test sensitivity at the estimated optimum cut-off values, SAA was the more discriminatory marker. These studies illustrated the potential of SAA and Hp to indicate current sheep scab infestation status and to augment the existing Pso o 2 serological assay to give disease-specific indications of both infestation and successful treatment.     

Comparison of a competitive inhibition ELISA and the card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in cattle

OBJECTIVE: To compare a recently developed recombinant MSP-5 competitive inhibition ELISA with a card agglutination test for detection of antibodies to Anaplasma marginale and Anaplasma centrale in Australian cattle. MATERIALS AND METHODS: The ELISA was compared with the card agglutination test using 208 sera from cattle in Anaplasma-free herds, 86 sera from cattle experimentally infected with A marginale or A centrale and 757 sera from cattle in areas endemic for A marginale. RESULTS: The specificity of the ELISA, based on testing 208 sera from cattle in Anaplasma-free areas, was 99.5%, and the sensitivities for detection of antibodies to A marginale and A centrale in sera from the experimentally infected cattle were 98.0% and 100%, respectively. For the same sets of sera, the specificity of the card agglutination test was 98.6% and the sensitivities for detection of antibodies to A marginale and A centrale were 98.0% and 100%, respectively. For the 757 sera collected from cattle in areas endemic for A marginale, the agreement between the ELISA and the card agglutination test depended on the positive threshold selected for the ELISA. The maximum achievable agreement was 91.5% (kappa = 0.73; 95% confidence interval 0.66, 0.79). CONCLUSION: We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs.     

Y are you not pregnant: identification of Y chromosome segments in female cattle with decreased reproductive efficiency

Reproductive efficiency is of economic importance in commercial beef cattle production, since failure to achieve pregnancy reduces the number of calves marketed. Identification of genetic markers with predictive merit for reproductive success would facilitate early selection of females and avoid inefficiencies associated with sub-fertile cows. To identify regions of the genome harboring variation affecting reproductive success, we applied a genome-wide association approach based on the >700,000 SNP marker assay. To include the largest number of individuals possible under the available budget, cows from several populations were assigned to extremes for reproductive efficiency, and DNA was pooled within population and phenotype before genotyping. Surprisingly, pools prepared from DNA of low reproductive cattle returned fluorescence intensity data intermediate between fertile females and males for SNP mapped to the Y chromosome (i.e., male sex chromosome). The presence of Y-associated material in low reproductive heifers or cows was confirmed by Y-directed PCR, which revealed that 21 to 29% of females in the low reproductive category were positive by a Y chromosome PCR test normally used to sex embryos. The presence of the Y chromosome anomaly was further confirmed with application of additional Y-specific PCR amplicons, indicating the likelihood of the presence of some portion of male sex chromosome in female cattle in various beef cattle herds across the U.S. Discovery of this Y anomaly in low reproductive females may make an important contribution to management of reproductive failures in beef cattle operations.     

Gold nanoelectrodes of varied size: transition to molecule-like charging

A transition from metal-like double-layer capacitive charging to redox-like charging was observed in electrochemical ensemble Coulomb staircase experiments on solutions of gold nanoparticles of varied core size. The monodisperse gold nanoparticles are stabilized by short-chain alkanethiolate monolayers and have 8 to 38 kilodaltons core mass (1.1 to 1.9 nanometers in diameter). Larger cores display Coulomb staircase responses consistent with double-layer charging of metal-electrolyte interfaces, whereas smaller core nanoparticles exhibit redox chemical character, including a large central gap. The change in behavior is consistent with new near-infrared spectroscopic data showing an emerging gap between the highest occupied and lowest unoccupied orbitals of 0.4 to 0.9 electron volt.     

Studies on failure of T strain live Babesia bovis vaccine

SUMMARY Field investigations of the protection afforded by the Australian live Babesia bovis vaccine used in the early 1990s (T strain) revealed inadequate vaccine-induced protection in certain herds. Vaccination/challenge trials using 207 experimental cattle were conducted to evaluate the protection afforded by T strain B bovis against field isolates from these herds. The trials investigated whether isolates that could ‘break-through’ T strain immunity were present in the field, the ability or inability of specific cattle to develop protective immunity after vaccination with T strain and the effect of attenuation and maintenance procedures on the immunogenicity of T strain. The results showed that B bovis parasites present early in the process of attenuation of T strain were more protective than those remaining late in the process. They also showed that cattle from properties experiencing vaccine failures were less protected by T strain vaccination than Bos taurus cattle randomly selected from the general population if vaccinated with highly attenuated T strain. A hypothesis is offered to explain these findings .