Table of Contents and AbstractsOctober 2017, Vol. 81, No.4


Reduced activities of thiamine-dependent and cytochrome c oxidase enzymes in cerebral cortex of cattle affected by sulfur-induced polioencephalomalacia

Samat Amat, Steve Hendrick, Igor Moshynskyy, Elemir Simko (page 242)

Sulfur-induced polioencephalomalacia (PEM) is an important disease affecting cattle in certain geographical regions. However, the pathogenesis of brain damage is not completely understood. We previously observed that excess dietary sulfur may influence thiamine status and altered thiamine metabolism may be involved in the pathogenesis of sulfur-induced PEM in cattle. In this study, we evaluated the activities of thiamine-dependent enzymes [α-ketogluterate dehydrogenase (α-KGDH) and pyruvate dehydrogenase (PDH)] and cytochrome c oxidase (COX) in the cerebral cortex of sulfur-induced PEM-affected cattle (n = 9) and clinically normal cattle (n = 8, each group) exposed to low or high dietary sulfur [LS = 0.30% versus HS = 0.67% sulfur on a dry matter (DM) basis]. Enzyme activities in PEM brains were measured from the brain tissue regions and examined using ultraviolent (UV) light illumination to show fluorescence or non-fluorescence regions. No gross changes under regular or UV light, or histopathological changes indicative of PEM were detected in the brains of cattle exposed to LS or HS diets. The PDH, α-KGDH, and COX activities did not differ between LS and HS brains, but all enzymes showed significantly lower (P < 0.05) activities in UV-positive region of PEM brains compared with LS and HS brains. The UV-negative regions of PEM brain had similar PDH activities to LS and HS brains, but the activities of α-KGDH and COX were significantly lower than in LS and HS brains. The results of this study suggest that reduced enzyme activities of brain PHD, α-KGDH, and COX are associated with the pathogenesis of sulfur-induced PEM.

Xylazine infusion in isoflurane-anesthetized and ventilated healthy horses: Effects on cardiovascular parameters and intestinal perfusion

Klaus Hopster, Liza Wittenberg-Voges, Sabine B.R. Kästner (page 249)

To investigate the effects of a xylazine infusion during isoflurane anesthesia on global perfusion parameters and gastrointestinal oxygenation and microperfusion, 8 adult warmblood horses were sedated with xylazine and anesthesia induced with midazolam and ketamine. Horses were mechanically ventilated during anesthesia. After 3 h of stable isoflurane anesthesia (FEIso 1.3 Vol %), a xylazine infusion with 1 mg/kg body weight (BW) per hour was started for 1 h and then stopped. Before, during, and after xylazine infusion, heart rate (HR), arterial blood pressure (MAP), cardiac output (CO), central venous pressure (CVP), and pulmonary artery pressure (PAP) were measured and systemic vascular resistance (SVR) was calculated. Arterial blood gases were taken and oxygen delivery (DO2) and alveolar dead space (VDalv) were calculated. Further intestinal oxygen and microperfusion were measured using white light spectroscopy and laser Doppler flowmetry. Surface probes were placed via median laparotomy on the stomach, the jejunum, and the colon. Wilcoxon rank-sum test was used to compare values over time (P < 0.05).

During xylazine infusion, MAP, CVP, PAP, SVR, and VDalv increased significantly, whereas CO, DO2, and intestinal microperfusion decreased. Intestinal oxygenation remained unchanged. All parameters returned to pre-xylazine values within 1 h after stopping xylazine infusion.

A xylazine infusion during constant isoflurane anesthesia in horses impairs global and intestinal perfusion without changing tissue oxygenation in normoxic healthy horses. Further studies are necessary, however, to evaluate whether a possible reduction of isoflurane concentration by xylazine infusion will ameliorate these negative effects.

Quantitative assessment of muscle in dogs using a vertebral epaxial muscle score

Lisa M. Freeman, James Sutherland-Smith, Lori R. Prantil, Amy F. Sato, John E. Rush, Bruce A. Barton (page 255)

Muscle loss associated with disease (cachexia) or with aging (sarcopenia) is common in dogs, but clinically relevant methods for quantifying muscle loss are needed. We previously validated an ultrasound method of quantifying muscle size in dogs in a single breed. The goal of this study was to assess the variability and reproducibility of the Vertebral Epaxial Muscle Score (VEMS) in other dog breeds. Static ultrasound images were obtained from 38 healthy, neutered dogs of 5 different breeds between 1- and 5-years-old. The maximal transverse right epaxial muscle height and area at the level of the 13th thoracic vertebra (T13) were measured. Length of the 4th thoracic vertebra (T4) was measured from thoracic radiography. Ratios of the muscle height and area to vertebral length (height/T4 and area/T4, respectively) were calculated to account for differences in body size among breeds. Reproducibility testing was performed on 2 dogs of each breed (26% of the total) to determine intra- and inter-investigator reproducibility, as well as intra-class correlation. Mean height/T4 = 1.02 ± 0.18 and mean area/T4 = 3.32 ± 1.68. There was no significant difference for height/T4 (P = 0.10) among breeds, but breeds were significantly different in area/T4 (P < 0.001). Intra-class correlation ranged from 0.80 to 0.99. Testing showed better reproducibility for height/T4 compared to area/T4. The VEMS using height/T4 was valid and reproducible for healthy dogs of different sizes and body conformations. Studies assessing this technique in dogs with congestive heart failure and other diseases associated with muscle loss are warranted.

Equine pituitary pars intermedia dysfunction: An international survey of veterinarians' approach to diagnosis, management, and estimated prevalence

James L. Carmalt, Cheryl L. Waldner, Andrew L. Allen (page 261)

The objectives of the present study were to determine if diagnosis and treatment of equine pituitary pars intermedia dysfunction (PPID) vary by geographic region and to report the prevalence of PPID in horses as observed by veterinarians across locations. An online questionnaire was developed for veterinarians who treat horses. Veterinary associations, especially equine specialty subgroups, were contacted and a survey link was sent to members of each organization. Generalized linear models were used to examine whether the method of diagnosis and treatment of this condition, as well as its reported prevalence, differed by geographic region. Veterinarians from 426 separate clinics in 20 countries returned surveys. Diagnosis of PPID varied by region, but was usually based on clinical signs and an adjunct endocrine test. Horses with PPID were treated medically by 63% of veterinarians and 75% of these used pergolide mesylate as treatment. The median prevalence estimated was 1% and this did not differ by geographic location. Half the veterinarians were caring for 5 or more animals with PPID. Overall, diagnostic approach differed in geographic regions. In general, European veterinarians were more likely than those in North America to diagnose PPID based on clinical signs alone, without using an adjunct laboratory test. Veterinarians reported that cost and management responsibilities were their clients' primary concerns associated with the long-term treatment of this disease, which indicates a need for additional treatment options for PPID.

Evaluation of a welfare assessment tool to examine practices for preventing, recognizing, and managing pain at companion-animal veterinary clinics

Lauren C. Dawson, Cate E. Dewey, Elizabeth A. Stone, Cornelia I. Mosley, Michele T. Guerin, Lee Niel (page 270)

Successful prevention, recognition, and treatment of pain are integral to ensuring veterinary patient welfare. A canine and feline welfare assessment tool, incorporating verbal interviews with veterinarians using open-ended questions, was developed to assess pain management practices that safeguard and improve patient welfare. The tool was evaluated in 30 companion- and mixed-animal veterinary clinics in Ontario in order to assess its reliability, feasibility, and validity, while also benchmarking current practices. Responses were analyzed according to a scoring scheme developed based on published literature and expert opinion. Based on weighted kappa statistics, interview scoring had substantial inter-observer (Kw = 0.83, 0.73) and near-perfect intra-observer (Kw = 0.92) agreement, which suggests that the tool reliably collects information about pain management practices. Interviews were completed at all recruited clinics, which indicates high feasibility for the methods. Validity could not be assessed, as participants were reluctant to share information about analgesic administration from their clinical records. Descriptive results indicated areas for which many veterinarians are acting in accordance with best practices for pain management, such as pre-emptive and post-surgical analgesia for ovariohysterectomy patients, and post-surgical care instructions. Areas that offer opportunity for enhancement were also highlighted, e.g., training veterinary staff to recognize signs of pain and duration of analgesia in ovariohysterectomy patients after discharge. Overall, based on this limited sample, most veterinarians appear to be effectively managing their patients' pain, although areas with opportunity for enhancement were also identified. Further research is needed to assess trends in a broader sample of participants.

Morphologic and molecular (28S rDNA) characterization of Dactylogyrus spp. in Cyprinus carpio and Ctenopharyngodon idella in Mashhad, Iran

Amin Ahmadi, Hassan Borji, Abolghasem Naghibi, Mohammad Reza Nasiri, Hassan Sharifiyazdi (page 280)

Dactylogyrids are a group of monogenean parasites that have a high species intensity on the gills of cyprinid fish. In this study, 89 common carp (Cyprinus carpio) and 25 grass carp (Ctenopharyngodon idella) were collected from Mashhad, in northeastern Iran. A total of 31 Dactylogyrus specimens, 20 and 11 specimens from the gills of common carp and grass carp, respectively, were collected and studied by morphologic analysis and molecular analysis based on 28S rDNA. Four lineages were revealed: D. anchoratus, D. extensus, D. lamellatus, and a new species. Phylogenetic analysis showed that 2 species, namely D. extensus and D. lamellatus, were identical to those previously reported. In addition, nucleotide sequencing showed the greatest homology (93.01%) of D. anchoratus to be with a species registered as D. inexpectatus in the Basic Local Alignment Search Tool database. The new Dactylogyrus species formed a distinct clade of its own in the phylogram. From the morphologic, molecular, and phylogenetic evidence, we propose that this isolate is a single new species within the genus Dactylogyrus. Further phylogenetic analysis, however, including the incorporation of additional molecular targets, is required to infer relationships among species in the Dactylogyrus genus.

Histological and functional characterizations of the digital cushion in Quarter horses

Babak Faramarzi, Linnea Lantz, Dongbin Lee, Wael Khamas (page 285)

The digital cushion (DC) plays a role in absorbing and dampening forces applied to the foot and therefore supports internal structures such as navicular bone; yet, its architecture is not well-known. The goal of this study was to characterize the microanatomical structure of the DC in horses with clinically sound hooves. Both forefeet from the cadavers of 12 adult Quarter horses were cut and sectioned and samples of the following 4 regions of the DC were obtained: axial proximal (AxProx), axial distal (AxDis), abaxial lateral (AbxLat), and abaxial medial (AbxMed). The samples were processed and stained with hematoxylin and eosin, Masson's trichrome, and Weigert's elastic stains. On each slide, 2 central 3- × 3-mm areas were microscopically assessed and all measurements were done within the 9-mm2 area. The number of detected collagen bundles, nerve fascicles, vessels, and the diameter of wall thickness and lumen of blood vessels were measured. Elastic fiber profiles were categorized based on relative density of elastic fibers detected in the field. The percentage of samples in which chondrocytes and adipose tissues were either present or absent was calculated. Significant structural differences were identified among the 4 regions of the DC. The AxDis region contained more collagen bundles (P < 0.0001) and less elastic fiber profiles than the AxProx region (P < 0.0001). The AxDis also contained more collagen bundles than the AbxMed and AbxLat (P < 0.0001) regions. Our findings provide insight into the structure of the DC of mature Quarter horses. The structural differences in the various regions of the DC are presumably related to the different functional properties of those regions; yet more research is warranted.

Ultrasonography as a complementary diagnostic method for evaluating the skin of healthy cats

Giordana Zanna, Eric Zini, Fabia Scarampella, Anna Attanasi, Silvana Arrighi, Edoardo Auriemma (page 292)

Ultrasonography is not often used in feline dermatology. The purpose of this study was to assess the usefulness and applicability of ultrasonography for skin evaluation in 21 clinically healthy cats. Ultrasonographic examination was conducted in 4 cutaneous regions (frontal, dorsal neck, sacral, and abdominal) using an 18-MHz linear-sequential-array transducer. Findings were assessed using histomorphometric analysis of skin samples set as reference standards. Morphologic evaluation, thickness measurements, measurement variability, and comparison between regions and genders were carried out. The ultrasonographic pattern of feline skin was characterized by 3 distinct layers of different echogenicity and echostructure. Skin was thickest at the dorsal neck region and thinnest at the abdominal region. Skin at the frontal region and dorsal neck region was thicker in males. Variability was < 10% in all regions. No apparent correspondence was found between ultrasonographic and histometric measurements of skin thickness. Collectively, these findings suggest that ultrasonography is a simple, noninvasive, and reproducible technique that allows cutaneous layers to be identified and accurately measures skin thickness in cats.

Protective efficacy of a Salmonella Typhimurium ghost vaccine candidate constructed with a recombinant lysozyme–PMAP36 fusion protein in a murine model

Ja Young Moon, So Young Kim, Won Kyong Kim, Zhili Rao, Jung Hee Park, Ji Young Mun, Boram Kim, Hyo Sun Choi, Jin Hur (page 297)

A Salmonella Typhimurium ghost vaccine was constructed with the use of a recombinant fusion protein consisting of lysozyme and porcine myeloid antimicrobial peptide 36 expressed by the Escherichia coli overexpression system. After confirmation of its effectiveness by transmission electron microscopy the vaccine was evaluated in a murine model. Of the 60 BALB/c mice equally divided into 4 groups, group A mice were intramuscularly inoculated with 100 µL of sterile phosphate-buffered saline, and the mice in groups B, C, and D were intramuscularly inoculated with approximately 1.0 × 104, 1.0 × 105, or 1.0 × 106 cells of the S. Typhimurium ghost vaccine, respectively, in 100-µL amounts. The serum IgG titers against S. Typhimurium outer membrane proteins were significantly higher in groups B to D than in group A, as were the concentrations of interleukin-10 and interferon gamma in supernatants of harvested splenocytes. After challenge with wild-type S. Typhimurium, all the vaccinated groups showed significant protection compared with group A, notably perfect protection in groups C and D. Overall, these results show that intramuscular vaccination with 1.0 × 105 cells of this ghost vaccine candidate provided efficient protection against systemic infection with virulent S. Typhimurium.

Brief Communications

Identification of Mycobacterium avium subspecies paratuberculosis strains isolated from dairy goats and dairy sheep in Ontario, Canada

Cathy A. Bauman, Andria Jones-Bitton, Christina Ahlstrom, Lucy Mutharia, Jeroen De Buck, Jocelyn Jansen, David Kelton, Paula Menzies (page 304)

The main objective of this study was to identify the circulating strains of Mycobacterium avium subspecies paratuberculosis (Map) in fecal isolates obtained from dairy goat (N = 29 farms) and dairy sheep (N = 21 farms) populations in Ontario, Canada. Further subtyping was performed to determine if there was adequate diversity between strains that could be used to establish Map transmission patterns. Type C was the dominant strain of Map isolates (95.2%) identified in dairy goats (n = 21). Sub-typing of the Type C strains, based on variable number tandem repeats (VNTR) and mycobacterial interspersed repetitive units, identified 3 VNTR types: INMV 1 (n = 10), INMV 2 (n = 10), and a type not previously identified (n = 1). Only 2 sheep isolates could be identified; both were Type S, sub-type III. Current typing methods demonstrate little Map diversity in the dairy goat population and are therefore of limited use to investigate infection patterns.

A recombinase polymerase amplification-based assay for rapid detection of African swine fever virus

Jianchang Wang, Jinfeng Wang, Yunyun Geng, Wanzhe Yuan (page 308)

A recombinase polymerase amplification (RPA)-based method was developed for rapid and specific detection of African swine fever virus (ASFV), the etiologic agent of African swine fever, a devastating disease of swine. Primers and the exo probe targeting the conserved region of the P72 gene of ASFV were designed and the reaction was run on the Genie III scanner device. Using recombinant plasmid DNA containing the P72 gene as template, we showed that the amplified product could be detected in less than 10 min and that the detection limit was 102 copies DNA/reaction [same detection limit as real-time polymerase chain reaction (PCR)]. The RPA assay did not cross-detect CSFV, PCV-2, PRV, PRRSV, or FMDV, common viruses seen in pigs. Tests of recombinant plasmid-spiked serum samples revealed that RPA and real-time PCR had the same diagnostic rate. The RPA assay, which is simple, cost-effective, and fast, is a promising alternative to real-time PCR for ASFV detection.

Seroprevalence of Mycoplasma suis infection in pigs in eastern China as estimated by a blocking enzyme-linked immunosorbent assay

Liang Zhongyang, Zhang Jiansong, Shen Yijuan, Xia Yuting, Li Yufeng, Xu Jiarong (page 313)

Porcine infectious anemia caused by Mycoplasma suis is a global disease and results in serious economic losses. To determine the prevalence of M. suis infection in eastern China, a cross-sectional serologic study was conducted with 3458 porcine serum samples randomly obtained from January 2014 to August 2016. The samples were tested with a blocking enzyme-linked immunosorbent assay developed in our laboratory. The seroprevalence was 33.3% in the complete sample set and was 25.9%, 37.8%, and 37.8% in 2014, 2015, and 2016, respectively. The seroprevalence was distinctly higher in summer (39.9%) and autumn (42.0%) than in spring (28.9%) and winter (23.3%). Shanghai was the region with the highest seroprevalence (54.2%) and Jiangsu the region with the lowest (23.0%). The seroprevalence was markedly higher in boars (47.1%), multiparous sows (47.0%), and replacement gilts (39.2%) than in piglets (24.2%), fattening pigs (17.2%), and nursery pigs (12.5%). These data demonstrate that the prevalence of M. suis infection is increasing yearly in eastern China.

The role of active transport in the transcellular movement of the peripheral a2-adrenoceptor antagonist, MK-467: An in vitro pilot study

Rachel Bennett, Mari Palviainen, Marikki Peltoniemi, Lauri Vuorilehto, Mika Scheinin, Marja Raekallio, Outi Vainio (page 318)

MK-467 is a peripherally acting α2-adrenoceptor antagonist due to its low lipid solubility and poor penetration of the blood-brain barrier (BBB). The aim of this study was to assess whether MK-467 could be a substrate of an active efflux transport mechanism. Using Madin-Darby Canine Kidney cells (MDCKII) and MDCKII cells transfected with the human multidrug resistance gene 1, drug transport was assessed in apical-basolateral and basolateral-apical directions. MK-467 was studied at 2 concentrations: 200 and 1000 ng/mL. Samples for analysis were taken at 15, 30, 45, 60, and 90 min after drug application. Drug concentrations were measured using liquid chromatography and mass spectrometry. MK-467 showed no apparent permeability in the apical-basolateral direction, transport in the basolateral-apical direction occurred in both cell lines. Efflux ratios were not calculated. However, MK-467 appeared to undergo active cellular transport. The identity of the transporter requires further investigation.