Viral and bacterial agents associated with experimental transmission of infectious proventriculitis of broiler chickens.

Proventriculitis of broilers can be reproduced by oral inoculation of day-old chicks with a proventricular homogenate from affected 3-wk-old broilers. The objective of the following studies was to isolate from this homogenate viral and bacterial isolates that could produce proventriculitis. A monoclonal antibody to infectious bursal disease virus (IBDV) was used to precipitate virus from the homogenate. A primary chicken digestive tract cell culture system was also used to isolate virus from a 0.2-microm filtrate of the homogenate, and a bacterium was also isolated from the homogenate. In trial 1, day-old birds were orally inoculated with either proventriculus homogenate or monoclonal antibody immunoprecipitated IBDV (MAB-IBDV). At 4, 7, 14, and 21 days postinfection (PI), 12 birds from each treatment group were subjected to necropsy. In trial 2, day-old birds were orally inoculated with either infectious proventriculus homogenate, suspect virus isolated in cell culture and propagated in embryo livers and spleens, or a bacterial isolate. Twelve birds from each treatment were subjected to necropsy at days 7, 14, 21, and 28 PI. In trial 3, treatments were maintained in negative pressure isolation chambers, and an additional treatment included virus plus bacterial isolate. Twenty-four birds from each treatment were subjected to necropsy at day 21 PI. In trial 1, infectious homogenate decreased body weight and relative gizzard weights at 4, 7, 14, and 21 days PI. Proventriculus relative weight was increased at days 7, 14, and 21 PI, and proventriculus lesion scores were increased at days 14 and 21 PI. Bursa/spleen weight ratios were decreased at day 14, and feed conversion was increased at days 4 and 21. The MAB-IBDV treatment decreased proventriculus and gizzard relative weights at day 4 PI, increased proventriculus lesion scores and bursa/spleen weight ratios at day 14, and decreased heterophil/lymphocyte ratios at day 21. In trial 2, all infected birds had significantly higher mean relative proventriculus weights at 21 days PI and had higher 4-wk mean proventriculus scores as compared with both control groups. In trial 3, birds treated with homogenate and birds treated with both suspect virus and the bacterial isolate had significantly higher proventriculus lesion scores; higher relative weights of proventriculus, gizzard, liver, and heart; lower body weights; and lower relative bursa weights compared with the saline control group. These studies suggest that infectious proventriculitis has a complex etiology involving both viral and bacterial infection.     

First Established Pregnancies in Mediterranean Italian Buffaloes (Bubalus bubalis) Following Deposition of Sexed Spermatozoa near the Utero Tubal Junction

At the time of AI following Ovsynch protocol, a total of 51 buffaloes were randomly divided in a first group (n = 30) subjected to conventional AI into the uterine body with 20 million non-sex sorted frozen-thawed spermatozoa, while a second group (n = 21) was inseminated near the utero-tubal junction (UTJ) ipsilateral to the ovary carrying the preovulatory follicle with 2.5 million live (4 million total) sex-sorted frozen-thawed spermatozoa. The semen used for flowcytometric sorting was collected and processed on a farm in Italy, and then shipped to a laboratory in Germany. Eleven buffaloes were inseminated with X-chromosome bearing spermatozoa and 10 with Y-chromosome bearing spermatozoa. Conception rates after conventional and UTJ inseminations were 43.3% (n = 13) and 42.8% (n = 9) respectively (p = 0.97). Eight of the nine foetuses obtained after insemination with sexed spermatozoa corresponded to the sex as predicted by the cell sorting procedure (five male and four female foetuses by ultrasound vs six male and three female foetuses by cell sorting). In conclusion, for the first time buffalo semen has been successfully subjected to procedures for flowcytometric sperm sorting and freezing. Low doses of sexed spermatozoa have been deposited near the UTJ giving conception rates similar to those of conventional AI with full dose.     

IMAGING DIAGNOSIS—MULTIMODALITY FINDINGS IN AN ADULT DOG WITH PRIMARY SARCOMA OF THE PULMONARY ARTERY AND MYOCARDIAL METASTASES

Intravascular pulmonary artery sarcomas in combination with myocardial metastasis are rare in dogs. We describe the radiographic, echocardiographic, and electrocardiographic‐gated (ECG‐gated) computed tomographic angiography (CTA) findings in a dog with pulmonary artery sarcoma. All imaging studies demonstrated severe main pulmonary artery enlargement. Echocardiography and ECG‐gated CTA revealed a mass occluding the lumen of the right pulmonary artery. In addition, CTA revealed focal left ventricular myocardial contrast enhancement and parenchymal lung changes. Postmortem examination confirmed the presence of a large thrombus associated with arteriosclerosis and an intravascular sarcoma in the right pulmonary artery with metastases to the myocardium, lungs and brain.     

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [³H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM ; B_max 22·5 pmol mg protein⁻¹) were detected that do not bind nicotinic compounds specifically. The estimated IC₅₀ values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [³H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC₅₀ values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM , 0·8(±0·2) μM , 2·8(±0·6) μM and 78(±1·9) μM , respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [³H]QNB were found. Muscarinic antagonists selectively displaced [³H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC₅₀ 2·13(±1·02) μM ) among different subtype-specific mAChR antagonists (4-DAMP had IC₅₀ 49·9(±9·13) μM and methoctramine had IC₅₀ 121(±14·2) μM ) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC₅₀ 71·3(±19·6) μM ) and carbachol (IC₅₀ 253(±87·1) μM ) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.     

Genome-wide association study for spot blotch resistance in Afghan wheat germplasm

Spot blotch (SB), caused by Bipolaris sorokiniana, is a devastating disease of wheat globally, especially in South Asia and South America. Understanding the genetics of resistance to SB is important for developing breeding strategies to improve resistance. A panel of 301 genotypes from Afghanistan was phenotyped over two crop seasons using a mixture of virulent B. sorokiniana isolates and genotyped using DArTSeq to obtain genome-wide markers. Fifty genotypes (16.6%) showed disease scores less than the resistant control. Principal component analysis using the genotypic data clustered the genotypes into five different groups. Among models used for genome-wide association mapping, the multilocus mixed model, and fixed and random model circulating probability unification algorithms were most effective in identifying significant marker-trait associations (MTA). Twenty-five MTAs at p ≤ .001 were identified on chromosomes 1A, 1B, 1D, 2B, 2D, 3A, 3B, 4A, 5A, 5B, 6A, 7A, and 7D, indicating the quantitative nature of resistance to SB. Phenotypic variation explained by these markers ranged from 2.0% to 17.7%, and genomic regions on the chromosomes 1D, 2D, 3A, 3B, 4A, 5A, and 5B coincided with loci identified in previous studies. Three single nucleotide polymorphism (SNP) markers on chromosomes 1B (SNP 1113207) and 5A (SNPs 5411867 and 998276) were significant in both crop seasons as well as in the combined analysis across seasons. Marker 5411867 is close to Vrn-A1, shown to be associated with SB in previous studies. Furthermore, among known SB resistance genes, Sb2 on chromosome 5B was predicted to be significant in this panel.     

Effect of allele combinations at Ppd-1 loci on durum wheat grain filling at contrasting latitudes

Flowering time is the most critical developmental stage in wheat, as it determines environmental conditions during grain filling. Thirty-five spring durum genotypes carrying all known allele variants at Ppd-1 loci were evaluated in fully irrigated field experiments for three years at latitudes of 41°N (Spain), 27°N (northern Mexico) and 19°N (southern Mexico). Relationships between weight of central grains of main spikes (W) and thermal time from flowering to maturity were described by a logistic equation. Differences in flowering time between the allele combination causing the earliest (GS100/Ppd-B1a) and the latest (Ppd-A1b/Ppd-B1a) flowering were 7, 20 and 18 days in Spain, northern Mexico and southern Mexico, respectively. Flowering delay drastically reduced the mean grain filling rate (R) and W at all sites. At autumn-sowing sites, an increase of 1°C in mean temperature during the first half of the grain filling period decreased W by 5.2 mg per grain. At these sites, W was strongly dependent on R. At the spring-sowing site (southern Mexico), W depended on both R and grain filling duration. Our results suggest that incorporating the allele combinations GS100/Ppd-B1a and GS105/Ppd-B1a (alleles conferring photoperiod insensitivity) in newly released varieties can reduce the negative effects of climate change on grain filling at the studied latitudes.     

Molecular identification of blast resistance genes in rice landraces from northeastern India

Rice blast disease caused by the fungus Magnaporthe oryzae is one of the most devastating diseases causing huge losses worldwide. In the present study, major blast resistance genes were investigated in landraces originating from northeastern India. Based on phenotypic evaluation, 288 landraces were classified into three distinct groups: resistant (75), moderately resistant (127) and susceptible (86). The genetic frequencies of the 18 major blast resistance genes were between 6.2% and 27.4%, with only two genotypes possessing a maximum of nine blast resistance genes. The cluster and population structure analysis grouped the landraces into two groups. Through principal coordinate analysis, the scatter plots partitioned the resistant and moderately resistant landraces into different groups. Analysis of molecular variance showed maximum (96%) diversity within populations and least (4%) diversity between populations. Association analysis identified six markers, CRG4_2, RM72, tk59-2, pi21_79-3, RM1233 and RM6648, that are significantly associated with blast disease and explained a phenotypic variance of 1.1–6.5%. The associated genes could be used in marker-assisted rice breeding programmes for gene pyramiding to develop rice varietal resistance against blast disease. The present study represents a valuable blast resistance genetic resource that could be used for identification of new R genes, donors for blast resistance, and genomic studies.     

养猪生产中后代性别的预选——发展现状和应用前景

动物精子的性别可通过流式精子分选仪和DNA标识进行鉴定,而利用鉴定性别的精子(即性控精子)并借助人工授精技术或其它授精技术产生的后代,在过去5年中估计已多达30000个(其中大多数是母牛)。有关文献资料证明,能有效地区分X精子和Y精子的唯一标记物是精子染色体中的DNA。众所周知,目前世界各地采用的方法是Beltsville精子分选技术,该技术根据X精子群和Y精子群中DNA相对含量上的差异,用荧光染液(Hoechst33342染液)标记精子,随后利用流式细胞仪分选经荧光标记的精子,从而达到分离精子的目的。目前,X精子或Y精子正常的生产速度是每小时1500万个,该项技术已在家畜、实验动物和动物园动物中应用,如果将该技术应用在人上,在预选后代性别比例上可达90%～95%的成功率。因动物品种不同,性控精子在动物体内的授精部位也不相同。常规的人工授精技术、宫内授精技术、输卵管内授精技术、用于胚胎移植的体外受精(in-vitro fertilization,IVF)技术或子宫角深部授精技术均能有效地使动物怀孕,至于利用哪种技术进行性控精子的精则取决于动物品种。尽管所有动物都能获得高纯度的分选精子,但是在实际生产中利用低剂量精子还难以让母猪怀孕。子宫角深部授精技术每次授精0.5～1.00亿个性控精子已能产生可喜的效果:利用特制的输精管,输入常规人工授精所需精子量的五十分之一的性控精子,足以使动物怀孕。性控精子通过常规的授精技术能够被猪成功利用前,还需重新设计输精管,同时输精的次数和每次授精时精子数量也需作进一步的研究。分选精子的低温保存技术已被牛人工授精普遍采用。尽管已能利用冷冻的性别分选精子生产小猪,但是性别分选精子经冷冻和解冻处理后在常规生产中的应用还未达到最理想的效果。本文将讨论猪精子性别分选的最新研究成果及其发展趋势,并重点探讨将性别分选技术应用于养猪生产中必须对其进行必要的技术开发。     

Depletion study and estimation of withdrawal periods for florfenicol and florfenicol amine in pacu (Piaractus mesopotamicus)

The intensive production of farmed fish is at a global all‐time high, and the control of bacteria proliferation in fish farms requires the frequent use of antimicrobials. This practice raises important environmental concerns related to the emergence of antimicrobial‐resistant bacteria strains. Only a few antimicrobial drugs have been approved for use in aquaculture, one of which is florfenicol. This work studies the depletion and withdrawal period of florfenicol and its main metabolite, florfenicol amine, in pacu (Piaractus mesopotamicus), a neotropical characin widely farmed in the southern hemisphere. Juvenile pacu (average weight of 724 g) were stocked in a closed‐loop laboratory system with controlled water temperature (25.8°C), and were fed for 10 consecutive days with a diet containing an intended dose of 10 mg/florfenicol per kg bw. Muscle and skin tissues were collected at 1, 3, 6, 8, 10, 12 and 16 days post‐treatment, and florfenicol and florfenicol amine were quantified using a validated ultra‐high performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS) method. The limits of quantitation for florfenicol and florfenicol amine were 10 ng/g in muscle and 50 ng/g in skin. Considering a maximum residue limit of 1000 ng/g for the sum of florfenicol and florfenicol amine in muscle with skin in natural proportions a withdrawal period of 5 days (water temperature 25.8°C) or 129 degree days was calculated on the basis of the upper limit of the one‐sided 95% confidence interval for the 99^th percentile derived from the residue depletion study.