SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells

We have identified two genes, smedwi-1 and smedwi-2, expressed in the dividing adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. Both genes encode proteins that belong to the Argonaute/PIWI protein family and that share highest homology with those proteins defined by Drosophila PIWI. RNA interference (RNAi) of smedwi-2 blocks regeneration, even though neoblasts are present, irradiation-sensitive, and capable of proliferating in response to wounding; smedwi-2(RNAi) neoblast progeny migrate to sites of cell turnover but, unlike normal cells, fail at replacing aged tissue. We suggest that SMEDWI-2 functions within dividing neoblasts to support the generation of cells that promote regeneration and homeostasis.     

Identification of bovine viral diarrhea virus type 1 in yaks (Bos poephagus grunniens) in the Himalayan region

Since cattle are widely infected by bovine viral diarrhea virus (BVDV) in India, we searched for pestivirus infection in yaks. Of 71 pure and crossbred yaks from Himalayan region, pestivirus antigen was detected by Ag-ELISA in three animals. Pestivirus in leukocyte and cell culture isolated virus samples originating from positive yaks was also confirmed by RT-PCR using panpestivirus specific primers selected from 5'-untranslated region (5' UTR). The 5' UTR, N(pro) and E2 regions were sequenced and used for genetic typing. Phylogenetic analysis revealed that pestiviruses detected in three Himalayan yaks were similar genetically, belonging to BVDV-1. Antigenic characterisation of yak pestivirus also confirmed the typing as BVDV-1. This is the first report on the identification of BVDV type 1 in yaks.     

Adenosine A2A receptor agonists inhibit lipopolysaccharide-induced production of tumor necrosis factor-alpha by equine monocytes

Adenosine is an endogenous nucleoside that regulates many physiological processes by activating one or more adenosine receptor subtypes, namely A1, A2A, A2B and A3. The results of previous studies indicate that adenosine analogues inhibit lipopolysaccharide (LPS)-induced production of reactive oxygen species (ROS) by equine neutrophils primarily through activation of A2A receptors. Because peripheral blood monocytes produce cytokines that are responsible for many of the deleterious effects of LPS, the current study was performed to evaluate the effects of an array of novel adenosine receptor agonists on LPS-induced production of tumor necrosis factor-alpha (TNF-alpha), and to assess the selectively of these agonists for equine adenosine A2A over the A1 receptor. Radioligand binding studies performed with equine tissues expressing adenosine A1 and A2A receptor subtypes yielded a rank order of affinity for the equine A2A receptor of ATL307>ATL309 approximately ATL310 approximately ATL313>ATL202 approximately ATL361 approximately ATL376>ATL372>CGS21680>NECA. Co-incubation of equine peripheral blood monocytes with LPS and these agonists resulted in inhibition of TNF-alpha production with a rank order of potency that strongly correlated with their binding affinities for equine adenosine A2A receptors. Results of experiments performed with one of the adenosine receptor agonists (ATL313) and selective adenosine receptor antagonists confirmed that inhibition of LPS-induced production of TNF-alpha occurred via stimulation of A2A receptors. Although incubation of monocytes with IB-MECA, a compound purported to act as an adenosine A3 receptor agonist, reduced LPS-induced TNF-alpha production, this effect of IB-MECA was inhibited by the A2A selective antagonist ZM241385 but not by the A3 receptor antagonist MRS1220. These results indicate that the adenosine receptor subtype responsible for regulation of LPS-induced cytokine production by equine monocytes is the A2A receptor. To address the signal transduction mechanism responsible for the anti-inflammatory effects of ATL313 in equine monocytes, production of cAMP was compared in the presence and absence of either the adenosine A2A receptor antagonist ZM241385 or the adenosine A2B receptor antagonist MRS1706. In the absence of the antagonists, ATL313 increased production of cAMP; ZM241385 inhibited this effect of ATL313, whereas MRS1706 did not. Furthermore, incubation of monocytes with either the stable analogue of cAMP, dibutyryl cAMP, or forskolin, an activator of adenylyl cyclase, also inhibited LPS-induced production of TNF-alpha production by equine monocytes. Collectively, the results of the current study indicate that adenosine analogues inhibit LPS-induced production of TNF-alpha by equine monocytes primarily via activation of adenosine A2A receptors and do so in a cAMP-dependent manner. The results of this study indicate that stable adenosine analogues that are selective for adenosine A2A receptors may be suitable for development as anti-inflammatory drugs in horses.     

Selected Aspects of Advanced Porcine Reproductive Technology

In vitro fertilization (IVF) of in vitro matured (IVM) oocytes in pigs has become the most popular method of studying gametogenesis and embryogenesis in this species. Furthermore, because of recent advances in in vitro culture (IVC) of IVM–IVF embryos, in vitro production (IVP) of embryos now enables us to generate viable embryos as successfully as for in vivo -derived embryos and with less cost and in less time. These technologies contribute not only to developments in reproductive physiology and agriculture but also to the conservation of porcine genetic resources and the production of cloned or genetically modified pigs. However, in IVP, there still remains the problem of abnormal ploidy, which is caused by performing procedures under non-physiological conditions. In recent years, unique technologies such as intracytoplasmic sperm injection (ICSI) or xenografting of gonadal tissue into immunodeficient experimental animals have been developed to help conserve gamete resources. These technologies combined with IVP are expected to be useful for the conservation of gametes from important genetic resources. Here, we discuss the developmental ability and normality of porcine IVP embryos and also the utilization of ICSI and xenografting in advancing biotechnology in pigs.     

Ocular distribution and toxicity of intravitreal injection of triamcinolone acetonide in normal equine eyes

Objective  To determine ocular distribution and toxicity of a single injection of intravitreal triamcinolone acetonide (TA) in normal horses.
Animals studied  Six adult horses, donated to North Carolina State University.
Procedures  Six horses were injected intravitreally with either 10, 20, or 40 mg ( n  = 2 each) of TA. The opposite eye of each horse was injected with balanced salt solution (BSS). Ocular toxicity was assessed by biomicroscopy, tonometry, indirect ophthalmoscopy, and electroretinogram. Aqueous humor (AH), vitreous humor (VH), and plasma samples were collected. Horses were euthanized 7 or 21 days after injection and eyes enucleated for histopathology. TA concentrations in AH, VH, and plasma were measured by HPLC.
Results  Three control eyes and one TA eye developed inflammation after injection or collection of AH. Positive bacterial cultures ( Corynebacterium spp., Staphylococcus spp., and Streptococcus spp.) were obtained from three of these eyes. Other than transient corneal edema in TA injected eyes, which resolved by 7 days after injection, no other changes were observed. TA crystals were visible within the vitreous body. No evidence of TA toxic effect was noted on histopathology. TA was detected in all AH and VH samples from treated eyes following injection. Drug was not detected in the plasma.
Conclusions  There was no evidence of overt toxicity from intravitreal TA in normal horses and a single intravitreal injection resulted in TA ocular levels for 21 days. However, the risk for bacterial infections with intravitreal injection or anterior chamber aspirations in horses is high. Use of topical and systemic antibiotics after injection is recommended.     

Monthly model for genetic evaluation of laying hens. II. Random regression

1. We investigated the use of monthly production records for genetic evaluation of laying hens, derived from a test day model with random regression in dairy cattle and compared it with other models. 2. Records of 6450 hens, daughters of 180 sires and 1335 dams, were analysed using a model with restricted maximum likelihood (REML): traits considered were monthly and cumulative egg production. Five models were studied: (1) random regression with covariates derived from the regression of Ali and Schaeffer (Canadian Journal of Animal Science, 67: 637-644, 1987) (RRMAS), (2) random regression with covariates derived from quartic polynomial (RRMP4), (3) fixed regression with covariates derived from Ali and Schaeffer (FRM), (4) multiple trait (MTM) and (5) cumulative (CM). 3. The models were compared on the basis of Spearman rank correlations of individual breeding values and sire breeding values estimated from subsets of full-sib split data. The hens (about 10% per generation) which ranked highest on their estimated breeding values from different models were compared phenotypically with their full records. 4. The estimates of heritability resulting from RRMP4 were biased upward from the estimates obtained from MTM, so this model was discarded. The heritabilities for monthly productions from RRMAS and MTM showed a similar pattern. They were high for the 1st month of production, decreased to their lowest value at about month 5 of production and increased again to the end of lay. 5. Spearman rank correlations between animal breeding values estimated by monthly models (RRMAS, FRM and MTM) were high, between 0.91 and 0.98, whereas those between estimates of monthly models and CM were lower, from 0.85 to 0.87. The correlations estimated either from intermittent months of measurements (odd vs even months) or full records were generally high, from 0.93 to 0.99. Information from odd months of production could be sufficient for cost-efficient recording schemes. The RRMAS generally had the highest correlation of sire breeding values between subsets of full-sib records, followed by MTM, RM and CM. Monthly models selected hens with higher productivity than the cumulative model. 6. In conclusion, genetic evaluation based on monthly production may be better than using cumulative production and RRMAS appeared to be the best among the models tested here.     

Detection of genomic DNA of the crayfish plague fungus Aphanomyces astaci (Oomycete) in clinical samples by PCR

A diagnostic procedure, based on a polymerase chain reaction method (PCR) was developed to detect infection of crayfish with the Oomycete Aphanomyces astaci. A set of oligonucleotide primers was designed to specifically amplify A. astaci DNA in the ITS region surrounding the 5.8S rDNA gene. The PCR amplifies a 115 bp amplicon. The specificity of the primers was demonstrated by testing on 27 A. astaci strains and against 20 non-A. astaci Oomycetes and 5 fungal species. Most of the non-A. astaci Oomycete or fungal species included in the study are either known parasites of freshwater crayfish cuticle or can be found in their natural environment. Specificity was also tested against crayfish tissue and some known parasites and bacteria infecting crayfish.

A protocol for the extraction of A. astaci DNA from infected crayfish tissue was developed. The optimised method allows the detection of two genome equivalents of purified A. astaci genomic DNA.

The method was tested on noble crayfish (Astacus astacus), artificially infected with A. astaci. Detection of A. astaci was possible at the very first time of sampling, which was 2 days after the beginning of spore exposure.