Immunohistochemical detection of scrapie prion proteins in clinically normal sheep in Pennsylvania.

Following diagnosis of scrapie in a clinically suspect Suffolk sheep, 7 clinically normal flockmates were purchased by the Pennsylvania Department of Agriculture to determine their scrapie status using an immunohistochemical procedure. Two of the 7 euthanized healthy sheep had positive immunohistochemical staining of the prion protein of scrapie (PrP-Sc) in their brains, nictitating membranes, and tonsils. The PrP-Sc was localized in the areas of the brain where, histopathologically, there was neurodegeneration and astrocytosis. The PrP-Sc occurred within germinal centers of the affected nictitating membranes and tonsils and was located in the cytoplasm of the dendrite-like cells, lymphoid cells, and macrophages. These results confirm that immunohistochemical examination of the nictitating membrane can be used as a screen for the presence of scrapie infection in clinically normal sheep at a capable veterinary diagnostic laboratory. In sheep with a PrP-Sc-positive nictitating membrane, the diagnosis of scrapie should be confirmed by histopathology and immunohistochemical examination of the brain following necropsy. Following full validation, immunohistochemistry assays for detection of PrP-Sc in nictitating membrane lymphoid tissues can improve the effectiveness of the scrapie control and eradication program by allowing diagnosis of the disease in sheep before the appearance of clinical signs.     

Catalog of Micro-Tom tomato responses to common fungal, bacterial, and viral pathogens

Lycopersicon esculentum cultivar Micro-Tom is a miniature tomato with many advantages for studies of the molecular biology and physiology of plants. To evaluate the suitability of Micro-Tom as a host plant for the study of pathogenesis, Micro-Tom plants were inoculated with 16 well-known fungal, bacterial, and viral pathogens of tomato. Athelia rolfsii, Botryotinia fuckeliana, Oidium sp., Phytophthora infestans, and Sclerotinia sclerotiorum caused typical symptoms and sporulated abundantly on Micro-Tom. Micro-Tom was resistant to Alternaria alternata, Corynespora cassiicola, and Fusarium oxysporum. When Micro-Tom was inoculated with 17 isolates of Ralstonia solanacearum, many isolates induced wilt symptoms. Agrobacterium tumefaciens also was pathogenic, causing crown galls on stem tissue after needle prick inoculation. In Micro-Tom sprayed with Pseudomonas syringae pv. tomato, P. s. pv. tabaci, or P. s. pv. glycinea, bacterial populations did not increase, and yellow lesions appeared only on leaves sprayed with P. s. pv. tomato. Tomato mosaic virus, Tomato aspermy virus, and Cucumber mosaic virus systemically infected Micro-Tom, which developed symptoms characteristic of other cultivars of tomato after infection with the respective virus. These results indicated that Micro-Tom was generally susceptible to most of the important tomato pathogens and developed typical symptoms, whereas certain pathogens were restricted by either hypersensitive resistance or nonhost resistance on Micro-Tom. Therefore, an assortment of Micro-Tom–pathogen systems should provide excellent models for studying the mechanism of susceptible and resistant interactions between plants and pathogens.     

Effects of Dietary Supplementation of Barodon on Growth Performance,Innate Immunity and Disease Resistance of Juvenile Olive Flounder,Paralichthys olivaceus,Against Streptococcus iniae

This study was conducted to evaluate the effects of dietary supplementation of Barodon, an anionic alkali mineral complex, on growth, feed utilization, humoral innate immunity and disease resistance of olive flounder. A basal experimental diet was used as a control and supplemented with 0.1, 0.2, 0.3, 0.4, or 0.5% Barodon. Triplicate groups of fish (26.4 ± 0.2 g) were fed one of the diets to apparent satiation twice daily for 10 wk. The growth performance was enhanced (P < 0.05) linearly and quadratically in fish fed diets containing Barodon compared with that in fish fed the control. Feed utilization was significantly improved by Barodon supplementation. Serum lysozyme and antiprotease activities were increased quadratically in Barodon fed groups. Also, significantly higher superoxide dismutase activity was found in Barodon‐fed fish. Dietary supplementation of 0.1–0.3% Barodon resulted in significant enhancement of fish disease resistance against Streptococcus iniae. The findings in this study indicate that dietary supplementation of Barodon can enhance growth, feed utilization, innate immunity, and disease resistance of olive flounder and that the optimum level seems to be 0.1% in diets.     

Chitosan nanoparticles enhance developmental competence of in vitro-matured porcine oocytes

Oxidative stress is inevitable as it is derived from the handling, culturing, inherent metabolic activities and medium supplementation of embryos. This study was performed to investigate the protective effect of chitosan nanoparticles (CNPs) on oxidative damage in porcine oocytes. For this purpose, cumulus–oocyte complexes (COCs) derived from porcine slaughterhouse ovaries were exposed to different concentrations of CNPs (0, 10, 25 and 50 µg/ml) during in vitro maturation (IVM). Oocytes treated with 25 µg/ml CNPs showed significantly higher levels of GSH, along with a significant reduction in ROS levels compared to control, CNPs10 and CNPs50 groups. In parthenogenetic embryo production, the maturation rate was significantly higher in the CNPs25 group than that in the control and all other treated groups. In addition, when compared to the CNPs50 and control groups, CNPs25-treated oocytes showed significantly higher cleavage and blastocyst development rates. The highest concentration of CNPs reduced the total cell number and ratio of ICM: TE cells in parthenogenetic embryos, suggesting that there is a threshold where benefits are lost if exceeded. In cloned embryos, the CNPs25 group, as compared to all other treated groups, showed significantly higher maturation and cleavage rates. Furthermore, the blastocyst development rate in the CNPs25-treated group was significantly higher than that in the CNPs50-treated group, as was the total cell number. Moreover, we found that cloned embryos derived from the CNPs25-treated group showed significantly higher expression levels of Pou5f1, Dppa2, and Ndp52il genes, compared with those of the control and other treated groups. Our results demonstrated that 25 µg/ml CNPs treatment during IVM improves the developmental competence of porcine oocytes by reducing oxidative stress.     

Adjusted method of penis fixation during boar semi-automatic semen collection aiming to reduce bacterial contamination

Semen collection has an essential role in the initial bacterial load in boar ejaculates and extended semen. The study aimed to explore the efficacy of an adjusted penis fixation in a semi-automatic collection system on reducing bacterial contamination of ejaculates in two-boar studs with different scenarios. Historically, stud A had low levels of bacterial load in raw semen, while stud B had a high level of contamination. A total of 56 mature boars had their semen collected using two methods of penis fixation: (a) Traditional: The penis was fixed directly with the artificial cervix and transferred to the adjustable clamp; (b) Adjusted: The fixation was performed with one gloved-hand, and after exteriorization, the penis was gripped using the artificial cervix with the other gloved-hand and transferred to the adjustable clamp. The bacterial load (p = .0045) and the occurrence of ejaculates >231 CFU/ml (p = .0101) were reduced in the Adjusted compared to the Traditional method. Bacterial load was reduced when using the Adjusted method in stud B (p = .0011), which showed a greater occurrence of critical factors for bacterial contamination (p ≤ .0034). The Adjusted method reduced the occurrence of ejaculates >231 CFU/ml when the preputial ostium was dirty (p = .016) and the duration of semen collection was >7 min (p = .022) compared to the Traditional method. In conclusion, the Adjusted penis fixation was efficient in reducing bacterial load of ejaculates, mainly in boar stud B, which had high contamination challenges.     

Phylogenetic analysis of Myanmar indigenous chickens using mitochondrial D-loop sequence reveals their characteristics as a genetic resource

Myanmar indigenous chickens play important roles in food, entertainment, and farm business for the people of Myanmar. In this study, complete mitochondrial D-loop sequences (1232 bp) were analyzed using 176 chickens, including three indigenous breeds, two fighting cock populations, and three indigenous populations to elucidate genetic diversity and accomplish a phylogenetic analysis of Myanmar indigenous chickens. The average haplotype and nucleotide diversities were 0.948 ± 0.009 and 0.00814 ± 0.00024, respectively, exhibiting high genetic diversity of Myanmar indigenous chickens. Sixty-four haplotypes were classified as seven haplogroups, with the majority being haplogroup F. The breeds and populations except Inbinwa had multiple maternal haplogroups, suggesting that they experienced no recent purifying selection and bottleneck events. All breeds and populations examined shared haplogroup F. When 232 sequences belonging to haplogroup F (79 from Myanmar and 153 deposited sequences from other Asian countries/region) were analyzed together, the highest genetic diversity was observed in Myanmar indigenous chickens. Furthermore, Myanmar indigenous chickens and red junglefowls were observed in the center of the star-like median-joining network of 37 F-haplotypes, suggesting that Myanmar is one of the origins of haplogroup F. These findings revealed the unique genetic characteristic of Myanmar indigenous chickens as important genetic resources.     

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.     

Evaluation of effects of olfactory and auditory stimulation on separation anxiety by salivary cortisol measurement in dogs

Separation anxiety (SA) is a serious behavioral problem in dogs. In this study, salivary cortisol was studied to determine if the owner''s odor or voice could reduce SA in dogs. Twenty-eight dogs with SA were divided into three groups: group 1 (control), group 2 (with owner''s clothes during the separation period; SP) and group 3 (a recording of the owner''s voice was played during SP). The dog''s saliva was collected after the owner and their dog were in the experimental room for 5 min (PRE). The dog was then separated from the owner for 20 min and saliva collected four times at intervals of 5 min (SP1–4). Finally, the owner was allowed back into the room to calm the dog for 5 min, after which saliva was collected (POST). Evaluation of salivary cortisol concentrations by ELISA revealed that the ratios of SP1 concentration to PRE or POST concentrations were significantly higher in group 1 than in group 2 or 3. Additionally, the concentrations of SP1–PRE and SP1–POST among groups differed significantly. These findings indicate that the owner''s odor or voice may be helpful to managing stress in dogs with SA.     

Isolation and characterization of antimicrobial-resistant Escherichia coli from national horse racetracks and private horse-riding courses in Korea

Limited information is available regarding horse-associated antimicrobial resistant (AR) Escherichia (E.) coli. This study was designed to evaluate the frequency and characterize the pattern of AR E. coli from healthy horse-associated samples. A total of 143 E. coli (4.6%) were isolated from 3,078 samples collected from three national racetracks and 14 private horse-riding courses in Korea. Thirty of the E. coli isolates (21%) showed antimicrobial resistance to at least one antimicrobial agent, and four of the AR E. coli (13.3%) were defined as multi-drug resistance. Most of the AR E. coli harbored AR genes corresponding to their antimicrobial resistance phenotypes. Four of the AR E. coli carried class 1 integrase gene (intI1), a gene associated with multi-drug resistance. Pulsed-field gel electrophoretic analysis showed no genetic relatedness among AR E. coli isolated from different facilities; however, cross-transmissions between horses or horses and environments were detected in two facilities. Although cross-transmission of AR E. coli in horses and their environments was generally low, our study suggests a risk of transmission of AR bacteria between horses and humans. Further studies are needed to evaluate the risk of possible transmission of horse-associated AR bacteria to human communities through horse riders and horse-care workers.     

Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.