An unusual splice defect in the mitofusin 2 gene (MFN2) is associated with degenerative axonopathy in Tyrolean Grey cattle

Tyrolean Grey cattle represent a local breed with a population size of approximately 5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.

[Tremorgenic syndrome in a cattle herd after feeding silage contaminated with A. clavatus];;Tremorgenes Syndrom in einem Rinderbestand nach Verfütterung einer mit A. clavatus befallenen Silage

The clinical signs, pathological and laboratory findings of cattle suffering from a tremorgenic syndrome are described. Animals on a farm with a total of 22 cows, 18 heifers and 9 calves were fed mouldy grass and spent malt-grain silage. Five heifers were affected with muscular tremor, hyperexcitability and hypersensitivity. They were ataxic or in sternal recumbency, while their appetite remained normal. Haematology and blood chemistry in two heifers as well as cerebrospinal fluid from one sick animal were unremarkable. The pathological examination of one animal brought no macroscopic changes to light. Histological examination, however, revealed the degeneration of motor neurones in the midbrain, brain stem and spinal cord. Analysis of a silage sample provided evidence of the presence of Aspergillus clavatus, a mould capable of producing neurotoxic tremorgenic mycotoxins. Epidemiology, clinical findings, pathology and microbiological examination suggest that the five cattle were suffering from neuromycotoxicosis.

Fecal carriage of extended-spectrum beta-lactamase-producing Enterobacteriaceae in swine and cattle at slaughter in Switzerland

During the past decade, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae have become a matter of great concern in human medicine. ESBL-producing strains are found in the community, not just in hospital-associated patients, which raises a question about possible reservoirs. Recent studies describe the occurrence of ESBL-producing Enterobacteriaceae in meat, fish, and raw milk; therefore, the impact of food animals as reservoirs for and disseminators of such strains into the food production chain must be assessed. In this pilot study, fecal samples of 59 pigs and 64 cattle were investigated to determine the occurrence of ESBL-producing Enterobacteriaceae in farm animals at slaughter in Switzerland. Presumptive-positive colonies on Brilliance ESBL agar were subjected to identification and antibiotic susceptibility testing including the disc diffusion method and E-test ESBL strips. As many as 15.2% of the porcine and 17.1% of the bovine samples, predominantly from calves, yielded ESBL producers. Of the 21 isolated strains, 20 were Escherichia coli, and one was Citrobacter youngae. PCR analysis revealed that 18 strains including C. youngae produced CTX-M group 1 ESBLs, and three strains carried genes encoding for CTX-M group 9 enzymes. In addition, eight isolates were PCR positive for TEM beta-lactamase, but no bla(SHV) genes were detected. Pulsed-field gel electrophoresis showed a high genetic diversity within the strains. The relatively high rates of occurrence of ESBLproducing strains in food animals and the high genetic diversity among these strains indicate that there is an established reservoir of these organisms in farm animals. Further studies are necessary to assess future trends.

Klf4 transcription factor is expressed in the cytoplasm of prostate cancer cells

BACKGROUND: Cancer initiation and progression might be driven by small populations of cells endowed with stem cell-like properties. Here we comparatively addressed the expression of genes encoding putative stemness regulators including c-Myc, Klf4, Nanog, Oct4A and Sox2 genes in benign prostatic hyperplasia (BPH) and prostate cancer (PCA). METHODS: Fifty-eight PCA and thirty-nine BPH tissues samples were used for gene expression analysis, as evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of specific Klf4 isoforms was tested by conventional PCR. Klf4 specific antibodies were used for protein detection in a tissue microarray including 404 prostate samples. RESULTS: Nanog, Oct4A and Sox2 genes were comparably expressed in BPH and PCA samples, whereas c-Myc and Klf4 genes were expressed to significantly higher extents in PCA than in BPH specimens. Immunohistochemical studies revealed that Klf4 protein is detectable in a large majority of epithelial prostatic cells, irrespective of malignant transformation. However, in PCA, a predominantly cytoplasmic location was observed, consistent with the expression of a differentially spliced Klf4α isoform. CONCLUSION: Klf4 is highly expressed at gene and protein level in BPH and PCA tissues but a cytoplasmic location of the specific gene product is predominantly detectable in malignant cells. Klf4 location might be of critical relevance to steer its functions during oncogenesis.

BVD eradication in Switzerland - a new approach

Diverse concepts for BVD eradication or control have been applied in several countries with varying success. Results of previous studies conducted in Switzerland have shown that the prevalence of antibody-positive animals is high and that BVDV is widespread in the country causing serious economic losses. A new approach to eradicate BVD in the cattle population in Switzerland was chosen. It consists in testing the whole Swiss cattle population for virus detection in a short period of time, without initial antibody screening. Identified persistently infected (PI) animals have to be slaughtered, and new herd infections should be avoided by movement restrictions. Ear-notches are collected using special tags for labeling the animals, and are analyzed using ELISA or rtRT-PCR methods. Confirmatory tests if needed are performed on blood samples using rtRT-PCR. The eradication program is divided into four phases: (1) Pre-pasturing phase: all young bovines going to transhumance in summer have to be negative tested before. (2) Initial phase: all non-tested bovines have to be tested. Movement restrictions are effective at the same time. (3) Calves phase: all newborn calves have to be tested. (4) Surveillance phase: several strategies will be compared using a modeling approach. After the pre-pasture phase already 595,230 animals (37% of the livestock) have been tested within four months. A prevalence of 1.1% of PIs was observed. The average age of infected animals is 403 days compared to 794 days for non-infected animals, with the oldest PI-animal being over 11 years old. On average PI-animals are slaughtered within 18 days after the last positive result. The pre-pasture phase has shown that sampling and testing a high number of animals in a short time is challenging but possible. The next phase will deal with double the number of animals in a similar time frame. The coordination between all partners as well as the collaboration of farmers is the key factor for ensuring the success of the program.

"Self" and "nonself" manipulation of interferon defense during persistent infection: bovine viral diarrhea virus resists alpha/beta interferon without blocking antiviral activity against unrelated viruses replicating in its host cells

Bovine viral diarrhea virus (BVDV), together with Classical swine fever virus (CSFV) and Border disease virus (BDV) of sheep, belongs to the genus Pestivirus of the Flaviviridae. BVDV is either cytopathic (cp) or noncytopathic (ncp), as defined by its effect on cultured cells. Infection of pregnant animals with the ncp biotype may lead to the birth of persistently infected calves that are immunotolerant to the infecting viral strain. In addition to evading the adaptive immune system, BVDV evades key mechanisms of innate immunity. Previously, we showed that ncp BVDV inhibits the induction of apoptosis and alpha/beta interferon (IFN-alpha/beta) synthesis by double-stranded RNA (dsRNA). Here, we report that (i) both ncp and cp BVDV block the induction by dsRNA of the Mx protein (which can also be induced in the absence of IFN signaling); (ii) neither biotype blocks the activity of IFN; and (iii) once infection is established, BVDV is largely resistant to the activity of IFN-alpha/beta but (iv) does not interfere with the establishment of an antiviral state induced by IFN-alpha/beta against unrelated viruses. The results of our study suggest that, in persistent infection, BVDV is able to evade a central element of innate immunity directed against itself without generally compromising its activity against unrelated viruses ("nonself") that may replicate in cells infected with ncp BVDV. This highly selective "self" and "nonself" model of evasion of the interferon defense system may be a key element in the success of persistent infection in addition to immunotolerance initiated by the early time point of fetal infection.

Genetic analysis of syndactyly in German Holstein cattle

Congenital syndactyly with a variable number of affected feet was observed in eight black and white German Holstein calves. Analysis of the pedigree data revealed that all affected individuals could be traced back to a single founder. The pedigree was consistent with monogenic autosomal recessive inheritance and variable expressivity. Bovine syndactyly or "mulefoot" has been previously shown to map on the telomeric end of bovine chromosome 15 and we performed PCR genotyping of microsatellite markers spanning 27 cM of this chromosomal region to test the new cases for genetic linkage with the phenotype. The haplotype segregation confirmed the suggested inheritance pattern of the mulefoot mutation in this family and markers RM004, BM848 and BMS820 showed significant linkage to the phenotype. The results confirmed the chromosomal location of the mulefoot gene in this pedigree. Furthermore the study demonstrated that although marker testing has been available for nearly a decade the use of mulefoot carriers in cattle breeding remains uncontrolled. The presented family provides a resource for positional cloning of the causative mutation.

Ruminal drinkers: endocrine and metabolic status and effects of suckling from a nipple instead of drinking from a bucket

In ruminal drinkers (RD) ingested milk is transported into the rumen and not into the abomasum. Because this is followed by changes in digestibility and absorption, we have tested whether this is associated with postprandial metabolic and endocrine changes. Unweaned, bucket-fed calves (one RD, two controls) were studied on seven farms. On d 1, after metabolic and endocrine 12-h profiles were studied, RD and one control calf were fed for 10 d by nipple, whereas the other control calf was fed by bucket. On d 11, metabolic and endocrine 12-h profiles were again studied. On d 1, mean plasma concentrations of glucose, triglycerides, urea, insulin, insulin-like growth factor-1 (IGF-1), 3,5,3'-triiodothyronine (T3), thyroxine (T4) and leptin were significantly different between RD and controls, whereas mean concentrations of non-esterified fatty acids (NEFA), total protein, albumin, and glucagon did not differ significantly among groups. In RD concentrations of glucose, NEFA, insulin, growth hormone, IGF-1, and T4 were higher, and of urea were lower on d 11 than on d 1. Glucose and insulin concentrations increased postprandially in healthy calves on d 1, but barely in RD and remained lower than in controls, and there was no rise of NEFA and triglyceride concentrations on d 1 after the initial postprandial decrease in RD, in contrast to controls. But on d 11 postprandial responses of these four traits were similar in RD and controls and urea and T4 concentrations on d 11 became normalized. However, glucose and T3 concentrations in RD on d 11 were still lower than in one or both control groups. In conclusion, various metabolic and endocrine traits in RD differed from healthy controls. Drinking by floating nipple instead of drinking from bucket for 10 d normalized several metabolic and endocrine traits in RD.

Simulating the impact of four control strategies on the population dynamics of Neospora caninum infection in Swiss dairy cattle

A dynamic deterministic simulation model was developed to assess the impact of different putative control strategies on the seroprevalence of Neospora caninum in female Swiss dairy cattle. The model structure comprised compartments of "susceptible" and "infected" animals (SI-model) and the cattle population was divided into 12 age classes. A reference model (Model 1) was developed to simulate the current (status quo) situation (present seroprevalence in Switzerland 12%), taking into account available demographic and seroprevalence data of Switzerland. Model 1 was modified to represent four putative control strategies: testing and culling of seropositive animals (Model 2), discontinued breeding with offspring from seropositive cows (Model 3), chemotherapeutic treatment of calves from seropositive cows (Model 4), and vaccination of susceptible and infected animals (Model 5). Models 2-4 considered different sub-scenarios with regard to the frequency of diagnostic testing. Multivariable Monte Carlo sensitivity analysis was used to assess the impact of uncertainty in input parameters. A policy of annual testing and culling of all seropositive cattle in the population reduced the seroprevalence effectively and rapidly from 12% to <1% in the first year of simulation. The control strategies with discontinued breeding with offspring from all seropositive cows, chemotherapy of calves and vaccination of all cattle reduced the prevalence more slowly than culling but were still very effective (reduction of prevalence below 2% within 11, 23 and 3 years of simulation, respectively). However, sensitivity analyses revealed that the effectiveness of these strategies depended strongly on the quality of the input parameters used, such as the horizontal and vertical transmission factors, the sensitivity of the diagnostic test and the efficacy of medication and vaccination. Finally, all models confirmed that it was not possible to completely eradicate N. caninum as long as the horizontal transmission process was not interrupted.

Neospora caninum immunoblotting improves serodiagnosis of bovine neosporosis

Neospora caninum ranges among the major causes of infectious abortion in cattle worldwide. The present study was designed to improve the serodiagnostic tools by complementing a conventional ELISA with a highly sensitive and species-specific N. caninum immunoblot. To evaluate this test combination, sera from several groups of cows were tested. The first group, consisting of experimentally infected calves, showed that immunoblot antibody reactivities were detectable 1 to 3 days earlier than those found in ELISA. The first immunodominant bands that appeared were a 29-kDa (NcSAG1) and a 36-kDa (NcSRS2) antigen. Other groups, based upon naturally infected cattle, were used to compare the diagnostic sensitivity of ELISA and immunoblotting. Overall, N. caninum immunoblotting exhibited a higher sensitivity (98%) than ELISA (87%). Conversely, immunoblotting also confirm in two other cases, true transient negativation in some animals. In general, banding patterns and band staining intensity correlated to the semiquantitative ELISA findings. On the other hand, the banding pattern could not be used to discriminate between sera from animals with a recent abortion and those of cows with latent N. caninum infection. We also addressed putative cross-reactions due to infection with Toxoplasma gondii. Sera from animals with a serologically proven T. gondii infection were either clearly negative by Neospora immunoblotting or they yielded a specific immunoblot antibody profile indicating a double infection with N. caninum. Sera from animals with positive findings in both Toxoplasma and Neospora ELISA thus provided dichotomic results in the immunoblot by allowing to confirm or to rule out the specificity of the antibody reaction in Neospora ELISA. Altogether, our findings demonstrate that N. caninum immunoblotting is a very sensitive and specific complementary tool to improve the serology for N. caninum infections in cattle.

Ontogenesis of mRNA levels and binding sites of hepatic alpha-adrenoceptors in young cattle

Catecholamines affect hepatic glucose production through (alpha- and beta2-) adrenoceptors (AR). We studied mRNA abundance and binding of hepatic alpha-AR in pre-term (P0) calves and in full-term calves at day 0 (F0), day 5 (F5) and day 159 (F159) to test the hypothesis that gene expression and numbers of hepatic alpha-AR in calves are influenced by age and associated with beta2-AR and selected traits of glucose metabolism. mRNA levels of alpha1- and alpha2-AR were measured by real time RT-PCR. alpha1- and alpha2-AR numbers (maximal binding, Bmax) were determined by saturation binding of (3H)-prazosin and (3H)-RX821002, respectively. alpha1- and alpha2-AR subtypes were evaluated by competitive binding. alpha1A-AR mRNA levels were lower in P0 than in F0, F5 and F159 and alpha(2AD)-AR mRNA levels were lower in F159 than in P0, F0 and F5, while alpha2C-AR mRNA levels increased from P0 and F0 to F5 and F159. Bmax of alpha1-AR increased from P0 to F5, then decreased in F159. Bmax of alpha2-AR decreased from F0 to F159. Bmax of alpha1-AR was positively associated with mRNA levels of alpha1A-AR (r = 0.7), Bmax of beta2-AR (r = 0.5) and negatively with hepatic glycogen content (r = -0.6). Bmax of alpha2-AR was negatively associated with Bmax of beta2-AR (r = -0.4). In conclusion, mRNA levels and binding sites of alpha1- and alpha2-AR in calves exhibited developmental changes and were negatively associated with hepatic glycogen content.

Oxytocin release and lactation performance in Syrian Shami cattle milked with and without suckling

Oxytocin (OT) release and lactation performance in primiparous Syrian Shami cows were evaluated in response to two different machine milking regimes. Six cows were milked in the presence of the calves (PC) and subsequently suckled, whereas six cows were exclusively machine milked without the presence of their calves (WC) until day 91 post partum. Milk yield and milk constituents were determined weekly. The degree of udder evacuation was determined by the succeeding removal of residual milk. PC released OT during the milking process, whereas in WC no OT release was detected throughout the milking process. Consequently, the residual milk fraction was much lower in PC than in WC (11% v. 58%, P<0.05) and daily milk yield until day 91 post partum was higher in PC than in WC (12.6+/-0.3 v. 7.1+/-0.4 kg, P<0.05). In conclusion, Syrian Shami cattle are not suitable to be exclusively machine milked without the presence of their calves.

Ontogeny of mRNA abundance of nuclear receptors and nuclear receptor target genes in young cattle

After birth the development of appropriate detoxification mechanisms is important. Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid receptors (RAR, RXR), and NR target genes are involved in the detoxification of exogenous and endogenous substances. We quantified abundances of hepatic mRNA of NR and several NR target genes (cytochromes, CYP; cytochrome P450 reductase, CPR; UDP-glucuronosyl transferase, UDP) in calves at different ages. Gene expression was quantified by real-time RT-PCR. Abundance of mRNA of CAR and PXR increased from low levels at birth in pre-term calves (P0) and full-term calves (F0) to higher levels in 5-day-old calves (F5) and in 159-day-old veal calves (F159), whereas mRNA levels of PPARalpha did not exhibit significant ontogenetic changes. RARbeta mRNA levels were higher in F5 and F159 than in F0, whereas no age differences were observed for RARalpha levels. Levels of RXRalpha and RXRbeta mRNA were lower in F5 than in P0 and F0. Abundance of CYP2C8 and CYP3A4 increased from low levels in P0 and F0 to higher levels in F5 and to highest levels in F159. Abundance of CPR was transiently decreased in F0 and F5 calves. Levels of UGT1A1 mRNA increased from low levels in P0 and F0 to maximal level in F5 and F159. In conclusion, mRNA levels of NR and NR target genes exhibited ontogenetic changes that are likely of importance for handling of xeno- and endobiotics with increasing age.

Congenital syndactyly in cattle: four novel mutations in the low density lipoprotein receptor-related protein 4 gene (LRP4)

BACKGROUND: Isolated syndactyly in cattle, also known as mulefoot, is inherited as an autosomal recessive trait with variable penetrance in different cattle breeds. Recently, two independent mutations in the bovine LRP4 gene have been reported as the primary cause of syndactyly in the Holstein and Angus cattle breeds. RESULTS: We confirmed the previously described LRP4 exon 33 two nucleotide substitution in most of the affected Holstein calves and revealed additional evidence for allelic heterogeneity by the identification of four new LRP4 non-synonymous point mutations co-segregating in Holstein, German Simmental and Simmental-Charolais families. CONCLUSION: We confirmed a significant role of LRP4 mutations in the pathogenesis of congenital syndactyly in cattle. The newly detected missense mutations in the LRP4 gene represent independent mutations affecting different conserved protein domains. However, the four newly described LRP4 mutations do still not explain all analyzed cases of syndactyly.

Digestive processes in ruminal drinkers characterized by means of the acetaminophen absorption test

The aim of the present study was to measure transit patterns of nutrients and the absorptive ability in ruminal drinkers (RDs) compared with healthy unweaned calves. The acetaminophen (paracetamol) absorption test was used to characterize the oroduodenal transit rate. Clinical examination and the analysis of various blood parameters provided supplementary information on digestive processes. Three unweaned bucket-fed calves (one RD and two healthy controls) each from seven Swiss dairy farms were included in the study. Measurements (tests 1 and 2) were performed twice at an interval of 10 days. Between tests, the feeding technique of the RDs and one control calf per farm was changed to feeding with a nipple instead of by bucket (without nipple). Acetaminophen appearance in the blood was delayed and reduced in RDs compared with the controls. Acid-base metabolism and several haematological and metabolic parameters differed markedly between RDs and healthy controls. The characteristics of the oroduodenal transit rate, absorptive abilities and clinical status in RDs were nearly normalised within 10 days of reconditioning.