Rapidly quantitative detection of Nosema ceranae in honeybees using ultra-rapid real-time quantitative PCR

BackgroundThe microsporidian parasite Nosema ceranae is a global problem in honeybee populations and is known to cause winter mortality. A sensitive and rapid tool for stable quantitative detection is necessary to establish further research related to the diagnosis, prevention, and treatment of this pathogen.ObjectivesThe present study aimed to develop a quantitative method that incorporates ultra-rapid real-time quantitative polymerase chain reaction (UR-qPCR) for the rapid enumeration of N. ceranae in infected bees.MethodsA procedure for UR-qPCR detection of N. ceranae was developed, and the advantages of molecular detection were evaluated in comparison with microscopic enumeration.ResultsUR-qPCR was more sensitive than microscopic enumeration for detecting two copies of N. ceranae DNA and 24 spores per bee. Meanwhile, the limit of detection by microscopy was 2.40 × 10⁴ spores/bee, and the stable detection level was ≥ 2.40 × 10⁵ spores/bee. The results of N. ceranae calculations from the infected honeybees and purified spores by UR-qPCR showed that the DNA copy number was approximately 8-fold higher than the spore count. Additionally, honeybees infected with N. ceranae with 2.74 × 10⁴ copies of N. ceranae DNA were incapable of detection by microscopy. The results of quantitative analysis using UR-qPCR were accomplished within 20 min.ConclusionsUR-qPCR is expected to be the most rapid molecular method for Nosema detection and has been developed for diagnosing nosemosis at low levels of infection.     

The effects of maxillary nerve block,ethmoidal nerve block and their combination on cardiopulmonary responses to nasal stimulation in anesthetized Beagle dogs

ObjectiveTo describe an approach for ethmoidal nerve block (E_BLOCK) and to compare the effects of a maxillary nerve block (M_BLOCK), E_BLOCK and their combination (M-E_BLOCK) on heart rate (HR), systolic (SAP), mean (MAP), diastolic (DAP) arterial pressures and respiratory rate (f_R) during nasal stimulation in dogs.Study designProspective, blinded, randomized, crossover placebo-controlled study.AnimalsBeagle dogs (five cadavers, nine live dogs), with a median (interquartile range) weight of 10.5 (10.3–11.0) kg.MethodsThe accuracy of iohexol injections (each 1 mL) at the maxillary and ethmoidal foramina in cadavers was evaluated using computed tomography. Then, anesthetized dogs were administered four bilateral treatments separated by 1 week, saline or 2% lidocaine 1 mL per injection: injections of saline at the maxillary and ethmoidal foramina (Control), injections of lidocaine at the maxillary foramina and saline at the ethmoidal foramina (M_BLOCK), injections of saline at the maxillary foramina and lidocaine at the ethmoidal foramina (E_BLOCK) and injections of lidocaine at all foramina (M-E_BLOCK). The ventral nasal meatus was bilaterally stimulated using cotton swabs, and HR, SAP, MAP, DAP and f_R were continuously recorded. Values for each variable were compared before and after stimulation using Wilcoxon signed-rank test. Changes in variables among treatments were analyzed using Mann–Whitney U and Kruskal–Wallis tests (p ≤ 0.05).ResultsComputed tomography revealed iohexol distribution around the openings of the target foramina in all cadavers. In living dogs, HR, SAP, MAP, DAP and f_R significantly increased after stimulation within each treatment (p < 0.03). Physiologic responses were significantly attenuated, but not absent, in the M-E_BLOCK [HR (p = 0.019), SAP, MAP, DAP and f_R (all p ≤ 0.001)] compared with those in the Control.Conclusions and clinical relevanceConcurrent injections of lidocaine at the maxillary and ethmoidal foramina attenuated HR, arterial pressure and f_R responses to nasal stimulation in Beagle dogs.     

Association between weight change during initial chemotherapy and clinical outcome in dogs with multicentric lymphoma

The majority of the known prognostic factors in dogs with lymphoma have been evaluated before treatment commences or at the time of diagnosis. Prognostic factors evaluated during the initial phase of treatment are less described but may provide important clinical information. In this retrospective study, 82 canine lymphoma patients were categorized according to the weight change between diagnosis and after 5 weeks of chemotherapy. Dogs that gained greater than 5% or lost greater than 5% of initial body weight were categorized as increased‐ or decreased‐weight groups, respectively. Those in which weight changed less than 5% were categorized as the maintained‐weight group. The median progression‐free survival (PFS) in the increased‐weight group, maintained‐weight group and decreased‐weight group was 226, 256 and 129 days, respectively. The decreased‐weight group had significantly shorter PFS than the increased and maintained groups (P = .023, P = .003, respectively). The median survival time (ST) in the increased‐weight group, maintained‐weight group and decreased‐weight group was 320, 339 and 222 days, respectively. There was no significant difference in ST among the three groups (P = .128). In Cox‐regression results, weight change group and initial body weight were significant risk factors associated to PFS (P = .007, P = .001, respectively) while only patient's initial body weight was a significant risk factor to ST (P = .013). In conclusion, evaluation of initial body weight and weight changes over time can provide valuable information regarding PFS and ST in dogs with multicentric lymphoma.     

Immunogenic properties of Landrace pigs selected for resistance to mycoplasma pneumonia of swine

Mycoplasma pneumonia of swine (MPS) lung lesions and immunogenic properties were compared between a Landrace line that was genetically selected for reduced incidence of pulmonary MPS lesions, and a non‐selected Landrace line. The MPS‐selected Landrace line showed significantly lower degrees of pulmonary MPS lesions compared with the non‐selected Landrace line. When changes in immunity before and after vaccination were compared, the percentage of B cells in the peripheral blood of the MPS‐selected Landrace line was significantly lower than that of the non‐selected line. Furthermore, the concentration of growth hormone and the mitogen activity of peripheral blood mononuclear cells in the MPS‐selected Landrace line showed significantly (P < 0.05) lower increases after vaccination than the non‐selected line. Conversely, the concentration of peripheral blood interferon (IFN)‐γ and salivary immunoglobulin A (IgA) after Mycoplasma hyopneumoniae vaccination was significantly higher in the MPS‐selected Landrace line than in the non‐selected line. Gene expression of toll‐like receptor (TLR)2 and TLR4 was significantly higher in the MPS‐selected Landrace line in immune tissues, with the exception of the hilar lymph nodes. The present results suggest that peripheral blood IFN‐γ, salivary IgA TLR2, and TLR4 are important immunological factors influencing the development of MPS lesions.     

Utilization of digital differential display to identify differentially expressed genes related to rumen development

This study aimed to identify the genes associated with the development of the rumen epithelium by screening for candidate genes by digital differential display (DDD) in silico. Using DDD in NCBI's UniGene database, expressed sequence tag (EST)‐based gene expression profiles were analyzed in rumen, reticulum, omasum, abomasum and other tissues in cattle. One hundred and ten candidate genes with high expression in the rumen were derived from a library of all tissues. The expression levels of 11 genes in all candidate genes were analyzed in the rumen, reticulum, omasum and abomasum of nine Japanese Black male calves (5‐week‐old pre‐weaning: n = 3; 15‐week‐old weaned calves: n = 6). Among the 11 genes, only 3‐hydroxy‐3‐methylglutaryl‐CoA synthase 2 (HMGCS2), aldo‐keto reductase family 1, member C1‐like (AKR1C1), and fatty acid binding protein 3 (FABP3) showed significant changes in the levels of gene expression in the rumen between the pre‐ and post‐weaning of calves. These results indicate that DDD analysis in silico can be useful for screening candidate genes related to rumen development, and that the changes in expression levels of three genes in the rumen may have been caused by weaning, aging or both. © 2015 Japanese Society of Animal Science     

Immunogenic properties and mycoplasmal pneumonia of swine (MPS) lung lesions in Large White pigs selected for higher peripheral blood immune capacity

Immunogenic properties and mycoplasmal pneumonia of swine (MPS) lung lesions were compared between the immunity‐selected Large White line and the non‐selected Large White line. The selected Large White line showed a higher level of pulmonary MPS lesions compared with the non‐selected Large White line. Subsequent to vaccination, the percentage of natural killer cells and T cells (CD3⁺CD4⁺CD8^‐ and CD3⁺CD4^?CD8⁺ T cells) were significantly increased in the non‐selected line but remained unchanged in the immunity‐selected Large White line. Secretion of Mycoplasma hyopneumoniae vaccine‐specific immunoblogulin G and phagocyte activity in peripheral blood were significantly higher in the immunity‐selected Large White line than in the non‐selected line. Expression of interleukin (IL)‐4 and IL‐6 messenger RNA in hilar lymph nodes was significantly lower in the immunity‐selected Large White line than in the non‐selected line. However, expression of IL‐10 in all immune tissues was significantly higher in the immunity‐selected Large White line. These results suggest that the selection for high immunity was not effective in increasing resistance to MPS lung lesions.     

Increased humoral antibody response of foot-and-mouth disease virus vaccine in growing pigs pre-treated with poly-γ-glutamic acid

This study was conducted to determine if humoral antibody response of foot-and-mouth disease (FMD) vaccine improved in 8-week-old growing pigs born to well-vaccinated sows pre-treated with 60 mg of poly-γ-glutamic acid (γ-PGA) three days before vaccination. Antibody against FMD virus serotype O was measured 0, 2, 4 and 6 weeks post-vaccination, using a PrioCHECK FMDV type O ELISA kit. The results showed that positive antibody reactions against FMDV serotype O antigen among a component of the vaccine significantly increased in response to pre-injection with γ-PGA.     

Hepatitis E virus as an emerging zoonotic pathogen

Hepatitis E outbreaks are a serious public health concern in developing countries. The disease causes acute infections, primarily in young adults. The mortality rate is approximately 2%; however, it can exceed 20% in pregnant women in some regions in India. The causative agent, hepatitis E virus (HEV), has been isolated from several animal species, including pigs. HEV genotypes 3 and 4 have been isolated from both humans and animals, and are recognized as zoonotic pathogens. Seroprevalence studies in animals and humans indirectly suggest that HEV infections occur worldwide. The virus is primarily transmitted to humans via undercooked animal meats in developed countries. Moreover, transfusion- and transplantation-mediated HEV infections have recently been reported. This review summarizes the general characteristics of hepatitis E, HEV infection status in animals and humans, the zoonotic transmission modes of HEV, and HEV vaccine development status.     

Apparent ileal digestibility of nutrients and amino acids in soybean meal,fish meal,spray‐dried plasma protein and fermented soybean meal to weaned pigs

This study sought to determine whether fermentation could increase apparent ileal digestibility (AID) of dry matter (DM), nitrogen (N), energy (E) and amino acids (AA) in fermented soybean meal (FSBM) greater than that of soybean meal (SBM) in weaned pigs. Four weaned pigs (10.00 ± 0.30 kg) were surgically equipped with T‐cannulas and randomly followed a 4 × 4 Latin square design of treatments (SBM, FSBM, fish meal and spray‐dried plasma protein). Overall, the fermentation process was able to reduce the amount of anti‐nutritional factors (ANF), including trypsin inhibitors, raffinose and stachyose, in the FSBM diet, which were significantly reduced by 39.4, 92.2, and 92.9%, respectively, as compared to the SBM diet. As a consequence of ANF reduction in FSBM, the AID of DM, N and E as well as AA was significantly greater with FSBM than SBM. Taken all together, the fermentation process improved the nutritional quality of SBM, due to ANF reduction, leading to improvement of digestibility of AA. As such, FSBM can be potentially used as a specialized feed ingredient, especially for young animal diets in an attempt to reduce diet costs.