Production of IFN-γ in feline whole blood after incubation with potential T-cell epitopes of the nucleocapsid protein of feline coronavirus

Interferon gamma (IFN-γ) plays an important role in cell mediated responses against mutated feline coronavirus strains (FCoV) involved in the pathogenesis of feline infectious peritonitis (FIP). The aim of this study was to establish a combined in silico and in vitro approach to assess feline leukocyte production of IFN-γ in response to selected peptides of the nucleocapside protein (N) of FCoVs. To this aim, we designed, through a bioinformatic approach, 8 potentially immunogenic peptides from the protein N corresponding to sequences of residues 14, 182, 198 detected only in FCoVs from FIP cats (virulent strains), only in FCoVs from healthy cats (avirulent strains) and both in FIP and in healthy cats (mixed strains). The peptides or a sham solution were incubated with whole blood from 16 cats (7 healthy and 9 with chronic diseases other than FIP) and IFN-γ concentration was measured on plasma using an ELISA system. RT-PCR expression of IFN-γ mRNA was also evaluated after incubation of the peptides or a sham solution with whole blood from 4 clinically healthy cats. The mean plasma concentration of IFN-γ in samples incubated with peptides decreased and the expression of IFN-γmRNA did not change compared with the sham solution, except for some cats with chronic diseases (which probably have a "pre-activated" immune response). These cats responded to "avirulent" or "mixed" peptides by increasing the concentration of IFN-γ and the expression of IFN-γ mRNA. The combined approach employed in this study allowed us to identify potentially immunogenic peptides of FCoV N protein that can modulate the production of IFN-γ especially in cats with a "pre-activated" cell mediated response.     

Identification and genotyping of feline infectious peritonitis-associated single nucleotide polymorphisms in the feline interferon-γ gene

Feline infectious peritonitis (FIP) is an immune-mediated, highly lethal disease caused by feline coronavirus (FCoV) infection. Currently, no protective vaccine or effective treatment for the disease is available. Studies have found that some cats survive the challenge of virulent FCoV isolates. Since cellular immunity is thought to be critical in preventing FIP and because diseased cats often show a significant decrease in interferon-γ (IFN-γ) production, we investigated whether single nucleotide polymorphisms (SNP) in the feline IFN-γ gene (fIFNG) are associated with the outcome of infection. A total of 82 asymptomatic and 63 FIP cats were analyzed, and 16 SNP were identified in intron 1 of fIFNG. Among these SNP, the fFING + 428 T allele was shown to be a FIP-resistant allele (p = 0.03), and the heterozygous genotypes 01C/T and +408C/T were found to be FIP-susceptible factors (p = 0.004). Furthermore, an fIFNG + 428 resistant allele also showed a clear correlation with the plasma level of IFN-γ in FIP cats. For the identification of these three FIP-related SNP, genotyping methods were established using amplification refractory mutation system PCR (ARMS-PCR) and restriction fragment length polymorphisms (RFLP), and the different genotypes could easily be identified without sequencing. The identification of additional FIP-related SNP will allow the selection of resistant cats and decrease the morbidity of the cat population to FIP.     

Identification of CAX gene family and expression profile analysis of response to abiotic stress in alfalfa北大核心CSCD

Ca²⁺/H⁺反向转运蛋白（CAX）是一类重要的跨膜转运蛋白,在调控植物Ca²⁺平衡、抵抗非生物胁迫和转运重金属离子等方面具有重要作用。利用生物信息学方法在全基因组水平对紫花苜蓿CAX基因家族进行了鉴定,并对其理化性质、结构特征、系统进化关系、顺式作用元件、染色体定位等进行了分析。结果表明,在紫花苜蓿全基因组中共筛选鉴定出15个MsCAX基因,分布于紫花苜蓿15条染色体上,发生22对基因片段重复事件,编码367~460个氨基酸,等电点为5.2~6.5,且均表现为疏水性蛋白。系统进化关系分析结果表明,MsCAXs分为2个亚家族,同一亚家族成员具有相似的基因结构、保守基序和跨膜结构域数量。MsCAXs启动子区域存在光响应性、激素反应性和胁迫响应元件。利用qRT-PCR分析了6个MsCAXs基因在非生物胁迫下的表达模式,结果表明,在干旱和低温胁迫下,6个MsCAX基因均显著下调表达,在盐和盐碱胁迫下,3个MsCAX基因上调表达。说明在不同的非生物胁迫下,MsCAXs基因表现出不同的表达模式。研究结果为进一步探索紫花苜蓿CAX基因家族的功能提供了参考。     

The occurrence of the feline “Candidatus Mycoplasma haemominutum” in dog in China confirmed by sequence-based analysis of ribosomal DNA

In the present study, polymerase chain reaction (PCR) assay was used to detect haemoplasmas (haemotropic bacteria) in 40 clinically healthy pet dogs in Foshan city, Guangdong Province, China, and one dog was found positive. Comparison of its 16S ribosomal DNA (rDNA) sequence with relevant sequences showed that the isolated haemoplasma had greater sequence identity to feline species “Candidatus Mycoplasma haemominutum” (99%) than to “Candidatus Mycoplasma haematoparvum” (95%). This result, for the first time, indicates the presence of the feline “Candidatus Mycoplasma haemominutum” in Chinese dogs and it represents the first survey of its kind in China by using PCR assay. The results indicated that dog may represent one of the hosts for the feline “Candidatus Mycoplasma haemominutum”.     

A comparison of in vitro relaxant responses to ipratropium bromide, β-adrenoceptor agonists and theophylline in feline bronchial smooth muscle

This study compares the potency and efficacy of different relaxant drugs including anticholinergic, β-adrenergic and methylxanthine agents on acetylcholine-contracted feline bronchi, and investigates the influence of the initial muscarinic-induced tone on bronchodilator response. Feline bronchi were removed from euthanased client-owned cats and were contracted with acetylcholine to cause either 40% or 80% of the acetylcholine maximal contraction. The efficacy and potency of bronchodilating drugs were obtained from cumulative dose-response curves with efficacy (E(max)) as the maximal relaxant response and potency (-logEC(50)) as the logarithm of the concentration of drug inducing 50% of maximal relaxation. Under low contractile tone (40%), all bronchodilators relaxed feline bronchi in a concentration-dependent manner with the following rank order of potency: formoterol>ipratropium bromide>fenoterol>isoprenaline>salbutamol≥salmeterol>theophylline. E(max) values ranged from 80% to 100% depending on the tested drug. Constriction of feline bronchi with high-dose acetylcholine (80%) caused a rightward and downward shift of the β(2)-mimetic dose-response curves. Significant decreases in -logEC(50) and E(max) values were reported for salbutamol, formoterol and salmeterol. This study provides evidence that existing classes of bronchodilators produce effective relaxation of acetylcholine-contracted feline bronchi and that airway responsiveness to β(2)-stimulants is dependent on the magnitude of the initial muscarinic-induced tone. The clinical relevance of these in vitro findings has yet to be explored in clinical trials.     

Clinicopathological findings and disease staging of feline infectious peritonitis: 51 cases from 2003 to 2009 in Taiwan

Fifty-one cats histopathologically confirmed to have been naturally infected by feline infectious peritonitis (FIP), were collected to analyse the clinical and laboratory findings and to characterise disease staging. Effusive FIP was found in 33 cats, non-effusive FIP in 12 cats, and mixed-type in six cats. Highly significant decreases in haematocrit and albumin levels and an increase in total bilirubin level were noted in both effusive and non-effusive FIP, at first presentation and before death. In serial blood examinations of the effusive group, anaemia and increases in bilirubin and aspartate aminotransferase (AST) were observed from 2 weeks to 0-3 days before death. The packed cell volume, bilirubin, AST, potassium, and sodium levels were established to predict disease staging and survival time. Cumulative points ranging from 0 to 4, 5 to 11 and excess of 12, indicate that the cat can survive for at least 2 weeks, less than 2?weeks and less than 3 days, respectively.     

A single-nucleotide polymorphism in the 3′ untranslated region of the LPIN1 gene and association analysis with performance traits in chicken

1. A single nucleotide polymorphism (SNP), c.*77C>G, was found in the 3′ UTR of the chicken LPIN1 gene by DNA sequencing. In total, 860 chickens were genotyped by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) in a F2 resource population obtained by crossing F0 Gushi chickens and Anka broilers, and the associations of this polymorphism with chicken growth, carcass, muscle fibre traits and serum biochemistry parameters were analysed.

2. Significant associations were found between the polymorphism and breast muscle fibre diameter (FDB). Comparison of the different genotypes of c.*77C>G in the F2 resource population showed that the GG genotype had significantly higher values than that of CG genotype in FDB. c.*77C>G was predicted to cause changes to multiple microRNA (miRNA) binding sites. But the total mRNA level of chicken LPIN1, LPIN1-;α and LPIN1-β in liver and muscle tissues did not show significant difference among GG, CG and CC genotypes, respectively.

3. The results suggested that chicken LPIN1 has a potential effect on muscle fibre development, but no effect on other studied traits.     

Association of a missense mutation in the luteinizing hormone/choriogonadotropin receptor gene （LHCGR） with superovulation traits in Chinese Holstein heifers

Background: Upon binding luteinizing hormone in the ovary, the luteinizing hormone/choriogonadotropin receptor (LHCGR) is necessary for follicular maturation and ovulation, as well as luteal function. We detected mutations in the LHCGR gene and evaluated their association with superovulation. Methods: Using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing, we examined polymorphisms in LHCGR and the genotypes associated with superovulation traits in 127 Chinese Holstein heifers. Results: A G/T polymorphism (ss52050737) in exon 11 was significantly associated with the total number of ova and the number of transferable embryos. Conclusions: LHCGR may be a new predictor for superovulation in Chinese Holstein heifers.     

Application of ROC curve analysis to FAMACHA© evaluation of haemonchosis on two sheep farms in South Africa

Test sensitivity and specificity for the FAMACHA(?) clinical test for anaemia due to haemonchosis have previously been shown to be adequate in differentiating between heavily/less heavily infected sheep, but these properties give no objective guidance for setting the optimum threshold at which anthelmintic treatment should occur. The aim of this work was to use Receiver Operating Characteristic (ROC) curves to evaluate the diagnostic accuracy of FAMACHA(?) testing by estimating the area under the ROC curve, and to use two-graph ROC curves to decrease subjectivity in selecting treatment thresholds on two farms with contrasting management. Test diagnostic accuracy, and thus discriminating power as determined by the area under the ROC curves, ranged from "moderate to good" on the first farm, and from "moderate to high" on the second farm for haematocrit (the Gold Standard for the test) cut-offs of ≤ 22% and ≤ 19% on both farms respectively. Accuracy of classification between haematocrit cut-offs was not significantly different within farms, but did differ significantly between farms, with test accuracy being highest on the second farm at both haematocrit cut-offs (p<0.05). The results also showed the suitability of the two-graph ROC curve approach for discriminating not only between different levels of accuracy of evaluators, but also to give an indication of the so-called ROC cut point (i.e. the desired threshold level) at which animals should be treated for a given level of risk of loss. The approach appears to have the potential not only to validate the diagnostic accuracy of the test across the complete testing range (i.e. all FAMACHA(?) categories from 1 to 5), but also to compensate for such inaccuracy by allowing objective adjustment of the threshold treatment level according to the output of the two-graph ROC method.     

Can we see fade? A survey of anesthesia providers and our ability to detect partial neuromuscular block in dogs

Objective

To assess the ability to visually detect fade during train-of-four (TOF) or double burst stimulation (DBS) in anesthetized dogs recovering from nondepolarizing neuromuscular block.

Development and use of real-time PCR to detect and quantify Mycoplasma haemocanis and “Candidatus Mycoplasma haematoparvum” in dogs

Two canine haemoplasma species have been recognised to date; Mycoplasma haemocanis (Mhc), which has been associated with anaemia in splenectomised or immunocompromised dogs, and “Candidatus Mycoplasma haematoparvum” (CMhp), recently described in an anaemic splenectomised dog undergoing chemotherapy. The study aim was to develop quantitative real-time PCR assays (qPCRs) incorporating an endogenous internal control to detect Mhc and CMhp and to apply these assays to DNA samples extracted from canine blood collected in Northern Tanzania (n = 100) and from dogs presented to a Trinidadian veterinary hospital (n = 185).QPCRs specific for Mhc and CMhp were designed using 16S rRNA gene sequence data, and each was duplexed with an assay specific for canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The assays detected ≤10 copies of a sequence-specific haemoplasma plasmid per reaction and neither assay showed cross-reactivity with 10⁶ copies of the sequence-specific plasmid from the non-target canine haemoplasma species.Nineteen of the 100 Tanzanian samples (19%) were positive for Mhc alone and one (1%) was dually infected. One Trinidadian sample was negative for canine GAPDH DNA and was excluded from the study. Of the 184 remaining Trinidadian samples, nine (4.9%) were positive for Mhc alone, five (2.7%) for CMhp alone, and two (1.1%) dually infected.This is the first report of canine haemoplasma qPCR assays that use an internal control to confirm the presence of amplifiable sample DNA, and their application to prevalence studies. Mhc was the most commonly detected canine haemoplasma species.     

Postpartum reproductive failure in cattle: is the examination of gene expression in the endometrium a key to success?

High reproductive performance is required for successful management of dairy farms. After calving, especially endometritis is one of the main reasons for reproductive failure. However, subclinical endometritis remains undetected in many cases and causes a high financial loss. To elucidate the cellular processes in the endometrium, the acquisition of the gene expression will provide helpful information. In the literature, numerous cytokines and enzymes were discussed to play important roles in preparing the endometrium for implantation. This review gives an overview about our present understanding of the functions of cyclooxygenases 1 and 2 (COX-1/COX-2), tumor necrosis factor alpha (TNFalpha), the inducible nitric oxide synthase (iNOS), haptoglobin as well as the interleukins 1 and 6 (IL-1/IL-6). Their role is not only to regulate certain physiological processes in the bovine reproductive tract, but to act also as inflammatory mediators in infectious diseases. A better understanding of cellular processes may help to improve identification and treatment of postpartum reproductive failure.