Development and standardization of a monoclonal antibody-based rapid flow-through immunoassay for the detection of Aphanomyces invadans in the field

A monoclonal antibody-based flow-through immunoassay (FTA) was developed using a nitrocellulose membrane placed on the top of adsorbent pads enclosed in a plastic cassette with a test zone at the center. The FTA could be completed within 10 min. Clear purple dots against a white background indicated the presence of Aphanomyces (A.) invadans. The FTA limit of detection was 7 µg/mL for A. invadans compared to 56 µg/mL for the immunodot. FTA and polymerase chain reaction (PCR) could detect A. invadans in fish tissue homogenates at a 10^-11 dilution compared to a 10^-8 dilution by immunodot. In fish suffering from natural cases of epizootic ulcerative syndrome (EUS) collected from Mangalore, India, FTA and PCR could detect A. invadans in 100% of the samples compared to 89.04% detected by immunodot. FTA reagents were stable and produced expected results for 4 months when stored at 4~8℃. This rapid test could serve as simple and cost-effective on-site screening tool to detect A. invadans in fish from EUS outbreak areas and in ports during the shipment of live or frozen fish.     

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.     

二温式PCR检测对虾白斑综合征病毒

本研究设计了一对能扩增大小为306bp对虾白斑综合征病毒(WSSV)某段基因的特异性引物，优化建立了能快速检测WSSV的二温式PCR，在对包括105份临床样品、10份SPF南美白对虾组织样品和其他对虾病害病原在内的样品检测结果中，有65份临床样品呈现WSSV阳性，而10份SPF南美白对虾组织样品和其他对虾病害病原的PCR结果为阴性。该二温式PCR最低能检测到1pg的WSSV感染对虾组织样品总DNA。这些结果表明，该PCR具有高度的特异性和敏染性。     

Right‐sided laryngeal hemiplegia and Horner's syndrome in a horse

This study describes right laryngeal hemiplegia (LH) and right‐sided Horner's syndrome (HS) in a horse. The average temperature of the face was 3.5°C higher on the right compared with the left side, as determined by thermographic imaging. The syndrome occurred following an episode of right mid‐cervical cellulitis due to inadvertent perijugular deposition of gentamicin.     

Immunohistochemical and qPCR determination of the expression and serum level of anti‐Müllerian hormone in pre‐pubertal,intact and ovarian remnant syndrome detected bitches

The aim of this study was to determine the serum concentrations, ovarian presence and expression of anti‐Müllerian hormone (AMH) in pre‐pubertal, bitches with signs of ovarian remnant syndrome (ORS) and intact bitches. In addition, we aimed to verify the suitability of serum AMH concentrations for diagnostic purposes in sterilized bitches and/or in suspected cases of ORS in the field of veterinary medicine. For this purpose, 36 healthy female dogs divided into six groups: proestrus, oestrus, dioestrus, anoestrus, pre‐pubertal and ORS. Serum AMH concentrations were determined by electrochemiluminescence immunoassay, and ovarian presence and distribution of AMH was confirmed by immunohistochemical and qPCR techniques. According to the results of qPCR, while the expression values of AMH were at the highest concentrations in the proestrus and oestrus, there was a statistically significant decrease in these values at the later stages of the cycle (p < 0.05). According to hormone analysis, the serum AMH values of the ORS group had decreased significantly compared with the proestrus and oestrus (p < 0.05). Although serum AMH levels of ORS group were increased compared with anestrus and pre‐pubertal groups, this increase was statistically non‐significant (p > 0.05). Immunohistochemically, AMH expression was first observed in the granulosa cells of primordial follicles in folliculogenesis. Expression values were the highest in the proestrous and oestrus groups, but values from bitches in later stages of the cycle were statistically significant decrease in comparison with these groups (p < 0.05). As a result, AMH concentration and expression were found to be higher in proestrus and oestrus than in other periods (p < 0.05). In addition, the measurable level of AMH concentration in bitches with ORS is an indication that it can be used in the diagnosis of ORS.     

Investigation of bacterial microorganisms in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Beijing, China

Objective To determine the bacterial microorganisms in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Beijing, China. The effect of breed, sex and age of dogs and season on the presence or absence of bacteria in the conjunctival sac of clinically normal dogs was evaluated. Sample population This investigation included 240 healthy dogs, 27 dogs with unilateral corneal ulcer and one dog with bilateral corneal ulcer. Procedure The 480 samples from healthy dogs and the 29 samples from dogs with ulcerative keratitis were incubated in an aerobic and 5% CO₂ environment at 37 °C for 48 h. Logistic regression analysis was performed. Statistical significance was set at P < 0.01. Results Of 480 normal eyes, Staphylococcus spp. were the most frequently isolated organisms (40.29%). Neisseria spp. (11.47%) were the next most frequently isolated organisms, followed by Corynebacterium spp. (9.4%). Of 29 eyes with ulcerative keratitis, Staphylococcus spp. were also the most frequently isolated bacteria (47.06%). Streptococcus spp. (12.94%) and Pseudomonas spp. (8.24%) were the second and third, respectively. Season (P = 0.002) was a significant factor influencing presence or absence of bacterial microorganisms in the conjunctival sac of normal dogs in Beijing, China, while the effects of breed (P = 0.095), sex (P = 0.588) and age (P = 0.866) of dogs were insignificant. Conclusion Staphylococcus spp. were the most frequently isolated organisms, and S. intermedius predominated in the conjunctival sac of clinically normal dogs and dogs with ulcerative keratitis in Beijing, China. The likelihood of detecting bacteria depends on the season.     

UV‐light and dietary vitamin D and their effects on ionized calcium and 25‐OH‐D plasma concentrations in captive gentoo penguins (Pygoscelis papua)

In this study, the effect of ultraviolet (UV) light and dietary vitamin D on calcium metabolism in permanently indoor‐housed gentoo penguins (Pygoscelis papua ) was investigated. The study consisted of three periods, each completed with blood samples to analyse plasma concentrations of 25‐OH‐D, 1,25‐(OH)₂‐D, ionized (iCa) and total calcium (tCa). During the first study period (D), animals were housed under routine conditions without UV‐light and fed a diet of different fish species, supplemented with 1,000 IU vitamin D per animal and day. The following study period (Baseline) of 28‐day duration consisted of the same diet without any vitamin D supplementation and without UV‐light. During the study period (UVB) artificial UV‐light was added for 3 weeks. The vitamin D content of fish was measured by high‐performance liquid chromatography. It varied between fish species and between facilities, ranging from no measurable content in capelin (Mallotus villosus ) to 7,340 IU vitamin D/kg original matter (OM) in herring (Clupea spp). The average dietary vitamin D content was 311 IU/kg OM at facility 1 and 6,325 IU/kg OM at facility 2, resulting in a vitamin D intake per animal and day without supplementation of 130 IU (25.5 IU/kg body weight BW) and 2,454 IU (438.2 IU/kg BW) respectively. The supplementation of vitamin D elevated significantly the plasma concentrations of 25‐OH‐D by an intraindividual difference of 15 (range ?2 to 59) nmol/L and tCa by 0.1 (0.0–0.3) mmol/L only at facility 2. The exposure to UV‐light raised the blood concentrations of tCa at facility 2 by 0.15 (0.1–0.2) mmol/L, and of iCa and tCa for females at facility 1 by 0.23 (0.13–0.41) mmol/L and 1.8 (1.1–2.5) mmol/L respectively. No significant influence of the study periods (D) and (UVB) was found for the concentrations of 1,25‐(OH)₂‐D at both facilities.     

Detection of the Severe Acute Respiratory Syndrome‐Related Coronavirus and Alphacoronavirus in the Bat Population of Taiwan

Bats have been demonstrated to be natural reservoirs of severe acute respiratory syndrome coronavirus (SARS CoV) and Middle East respiratory syndrome (MERS) CoV. Faecal samples from 248 individuals of 20 bat species were tested for partial RNA‐dependent RNA polymerase gene of CoV and 57 faecal samples from eight bat species were tested positive. The highest detection rate of 44% for Scotophilus kuhlii, followed by 30% for Rhinolophus monoceros. Significantly higher detection rates of coronaviral RNA were found in female bats and Scotophilus kuhlii roosting in palm trees. Phylogenetic analysis classified the positive samples into SARS‐related (SARSr) CoV, Scotophilus bat CoV 512 close to those from China and Philippines, and Miniopterus bat CoV 1A‐related lineages. Coronaviral RNA was also detected in bat guano from Scotophilus kuhlii and Myotis formosus flavus on the ground and had potential risk for human exposure. Diverse bat CoV with zoonotic potential could be introduced by migratory bats and maintained in the endemic bat population in Taiwan.     

对虾白斑综合征病毒单抗介导间接ELISA的建立

从白斑综合征病毒（WSSV）青岛株感染的克氏原螯虾（Canbarus proclarkii）中提纯病毒，用纯化病毒免疫BALB／c小鼠，采用细胞融合法获得4株阳性杂交瘤细胞，分别命名为1B1、1E4、4E6和4E5。4株单抗均为IgM。4E5株单抗在免疫转印中与37500左右的病毒蛋白条带呈阳性反应。用此株单抗作一抗，建立检测病毒蛋白的间接ELISA。该方法用于人工感染WSSV的螯虾组织样品中病毒的检测，48h后即有阳性检出，而正常螯虾组织均呈阴性。     

l‐carnitine and fat type in the maternal diet during gestation and lactation modify the fatty acid composition and expression of lipid metabolism‐related genes in piglets

This study examined the influence of adding different amounts of maternal dietary l ‐carnitine and two fat types on fatty acid (FA) composition and the expression of lipid metabolism‐related genes in piglets. The experiment was designed as a 2 × 2 factorial with two fat types (3.5% soyabean oil, SO, and 3.5% fish oil, FO) and two levels of l ‐carnitine (0 and 100 mg/kg) added to the sows' diets. A higher proportion of n‐3 polyunsaturated fatty acids (PUFA) and a lower ratio of n‐6/n‐3 PUFA in sow milk and piglet tissues were observed in the FO groups than in the SO groups. Adding l ‐carnitine increased the proportion of C16:1 in sow milk and decreased n‐3 PUFA in piglet subcutaneous fat. Hepatic peroxisome proliferator‐activated receptor α (PPAR‐α) was more abundantly expressed in piglets from the FO groups than from the SO groups (p < 0.05), whereas stearoyl‐CoA‐desaturase (SCD), sterol regulatory element binding protein‐1 (SREBP1) and ?6‐desaturase (D6D) genes were less expressed in the FO groups compared with piglets from the SO groups. The expression of fatty acid synthase (FAS) genes was decreased in the SO groups with l ‐carnitine compared to that of the other dietary treatments. No differences among dietary treatments were observed with regard to the expression of acetyl‐CoA carboxylase (ACC). In conclusion, FO and l ‐carnitine supplementation in sows affect FA composition and hepatic gene expression in piglets.     

2010年黑龙江猪高热病流行病学调查

2010年黑龙江省多个地区暴发猪“高热病”,为明确其病因及流行特征,对来自大庆市、齐齐哈尔市、佳木斯市,绥化市等地区送检的43例病例(70头病死猪),应用病原学、血清学和PCR方法进行诊断,对确诊的病例进行流行病学回顾性研究.结果表明,小养殖户或小型猪场发病率较高,发病高峰期集中在9～12月份,发病日龄集中在1～3月龄仔猪(54.16％);病死猪以败血症为主,43例病例中猪链球菌病23例( 53.49％),猪瘟8例(18.60％),副猪嗜血杆菌病8例(18.60％),猪繁殖与呼吸综合征6例(13.95％),圆环病毒病4例(9.30％),混合感染发病率高达58.13％.以上结果表明,猪“高热病”病因复杂多样,防制工作任重而道远.