CVMA | Table of Contents and Abstracts

Table of Contents and AbstractsOctober 2020, Vol. 84, No. 4


Integrin alpha-v/beta3 expression in equine lungs and jejunum

Nguyen Phuong Khanh Le, Volker Gerdts, Baljit Singh (page 245)

Integrin alpha-v/beta3 (αvβ3) recognizes arginine-glycine-aspartic acid (RGD) sequences and has important functions in cell adhesion, signaling, and survival. However, the expression of integrin αvβ3 in the equine lungs and jejunum is not well understood. The objective of this study was to explore the hitherto unknown expression of integrin αvβ3 in the lungs and jejuna of the horse using light and electron immunocytochemistry. Immunohistochemistry showed integrin αvβ3 on the epithelium, the immune cells in Peyer’s patches, the smooth muscle, and the endothelium of equine jejuna. In equine lungs, we recognized integrin αvβ3 on the endothelium of blood vessels, the alveolar septa, the bronchial lymph nodes, and the cartilages, although the expression of integrin αvβ3 was weak on the epithelium of bronchioles. In conclusion, these are the first data to show the expression of integrin αvβ3 in equine lungs and jejuna.

Clinical significance of Proteus mirabilis bacteriuria in dogs, risk factors and antimicrobial susceptibility

Magali Decôme, Benoît Cuq, Julie-Hélène Fairbrother, Laure Gatel, Bérénice Conversy (page 252)

The objectives of this study were to describe the in vitro antimicrobial susceptibility and clinical significance of Proteus mirabilis in canine bacteriuria and to identify the risk factors associated with P. mirabilis urinary tract infections. This is a retrospective observational study of 48 P. mirabilis-positive canine urinary cultures. Only 22 of the 48 P. mirabilis isolates (45.8%) were non-susceptible to at least one tested antimicrobial. Most P. mirabilis isolates (98%) were susceptible to enrofloxacin, 93.7% to amoxicillin/clavulanic acid, and 85.4% to ampicillin, cephalothin, and trimethoprim-sulfamethoxazole. Five multidrug-resistant isolates were detected (10.4%). A significant increase in antimicrobial resistance was observed over the study period. Positive P. mirabilis cultures were associated with bacterial cystitis in 36 of 39 dogs (92.3%), pyelonephritis in 2 of 39 dogs (5.1%), and one dog had both bacterial cystitis and pyelonephritis (2.5%). There was no subclinical bacteriuria. Most urinary tract infections were complicated as risk factors were identified in 37 of 39 dogs (94.8%). The most commonly identified risk factors were the presence of a contaminated peri-vulvar area with urine/feces or a hypoplastic vulva. To conclude, P. mirabilis bacteriuria was associated with upper and lower urinary tract infections in this study and was found more frequently in complicated bacterial cystitis. Multidrug-resistant isolates and increased P. mirabilis antimicrobial resistance have been identified over the last 10 years, but most isolates remain susceptible to first-line antimicrobials such as amoxicillin and trimethoprim-sulfamethoxazole.

Stability of canine urine samples under different storage conditions

Stephan Neumann, Kim Fechner, Claus-Peter Czerny (page 259)

The stability of canine urine samples is essential when the samples cannot be analyzed immediately. The objective of this study was to investigate the stability of canine urine samples at room temperature and under refrigerated conditions. Samples from 20 dogs were collected, divided, and stored at 4°C and 20°C. The samples were examined up to 48 h after collection for specific gravity, pH, protein, bilirubin, glucose, ketones, and sediment and at 4 h and 24 h for bacterial growth. Specific gravity and all chemistry parameters were stable for a minimum of 48 h in 90% of samples. The sediment was stable, apart from crystals. The bacterial growth of 3 bacterial species tested in vitro, as well as the clinical samples, was mostly constant over 24 h at the refrigerated temperature. In urine samples stored at room temperature, the total number of aerobic growing bacteria was increasing. The results of our study showed that routinely measured parameters were stable in unpreserved urine for a minimum of 4 h and up to 48 h in most cases. If it is not possible to culture urine immediately, it is recommended that urine samples be stored at 4°C for a period of up to 24 h.

Evaluation of red blood cell profiles in dogs with heartworm disease

Su-Jung Kim, Sang-Il Suh, Changbaig Hyun (page 265)

Recent studies have found that anemia and anisocytosis are precipitating factors for certain heart diseases in dogs. This study evaluated the prevalence and correlation of anemia and red blood cell distribution width (RDW) in dogs with heartworm disease (HWD). The study population consisted of 20 healthy control dogs and 86 dogs with HWD: 28 dogs with no clinical signs or pulmonary hypertension (Group 1), 42 dogs with mild clinical signs but no pulmonary hypertension (Group 2), and 16 dogs with severe clinical signs and pulmonary hypertension (Group 3). Along with echocardiographic interrogation of pulmonary hypertension, red blood cell (RBC) profiles were evaluated, including RDW. The total number of red blood cells (tRBCs), hematocrit (HCT), and hemoglobin (HGB) concentration was significantly lower in Group 3 dogs compared to control dogs (P < 0.05), while the RDW was significantly higher in Group 3 dogs than in control dogs (P < 0.05). The RDW was closely correlated to other RBC profiles and the presence of pulmonary hypertension (P < 0.05). The severity of tricuspid regurgitant gradient (TRG) was closely correlated with Hb and tRBC (P < 0.05), but not with the RDW and reticulocyte count. This study finding indicated that anemia and anisocytosis are common complications in dogs with severe clinical signs and pulmonary hypertension caused by heartworm disease (HWD). It would therefore be beneficial for clinicians to routinely check red blood cell (RBC) profiles, including RDW, in order to monitor the progression of heartworm disease in dogs.

Efficacy comparison of commercial porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae monovalent and bivalent vaccines against a dual challenge

Siyeon Yang, Su-Jin Park, Taehwan Oh, Hyejean Cho, Chanhee Chae (page 272)

The objective of this study was to compare the efficacy of commercially available porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae vaccines. A total of 80 pigs was randomly divided into 6 treatment groups; 4 of the groups each received a different vaccine as well as a dual challenge. The remaining 2 groups were used as controls, 1 of which also received a dual challenge. Two of the 4 groups of pigs were administered 2 monovalent vaccines (designated as either monovalent vaccine A or B) of PCV2 at 7 days old and of M. hyopneumoniae at 21 days old. The remaining 2 vaccinated groups of pigs received a bivalent vaccine (designated as either bivalent vaccine A or B) of PCV2 and M. hyopneumoniae at 21 days old. All 4 vaccinated groups were challenged with M. hyopneumoniae at 42 days old [−14 d post-challenge (dpc)], followed by a PCV2d challenge at 56 days old (0 dpc). All 4 vaccinated/challenged groups displayed a reduction in clinical signs, PCV2d viremia, nasal shedding of M. hyopneumoniae, and lung lesions compared with pigs in the unvaccinated and challenged groups. Vaccination and challenge improved growth performance and increased the immunologic responses (M. hyopneumoniae- and PCV2-specific antibodies and interferon-γ-secreting cells) when compared to pigs in the unvaccinated/challenged groups. Pigs in groups vaccinated with either a monovalent or bivalent vaccine A treatment and challenge produced a larger amount of M. hyopneumoniae- and PCV2d-specific interferon-γ-secreting cells within the pigs and simultaneously reduced the nasal shedding of M. hyopneumoniae and PCV2d viremia compared with groups vaccinated with either a monovalent or bivalent vaccine B treatment and challenge. Both the bivalent vaccines and the respective monovalent vaccines were efficacious against a dual challenge of M. hyopneumoniae and PCV2d.

ß-glucan from Saccharomyces cerevisiae is involved in immunostimulation of ovine ruminal explants

Man Zhang, Xin Jin, Gui-fang Cao, Yin-feng Yang (page 283)

In this study, we investigated whether β-glucan from Saccharomyces cerevisiae exerts beneficial effects on mucosal immunity in an ovine ruminal explant (ORE) model. Once the ORE model was established, viability was assessed through histological change, E-cadherin expression, CK-18 and Ki-67 distribution. Then, the OREs were co-cultured with β-glucan, following which, gene and protein expression levels of sheep β-defensin-1 (SBD-1), pro-inflammatory interleukin (IL)-6, and anti-inflammatory IL-10 were detected using quantitative real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). Hematoxylin & eosin staining, qPCR, and immunohistochemistry showed that the overall ORE structure was intact after 96 hours in culture, but explants cultured for more than 24 hours showed epithelial degradation. Therefore, we performed the follow-up test within 24 hours. qPCR and ELISA revealed that the gene and protein expression levels of SBD-1, IL-6, and IL-10 in the OREs significantly increased (P < 0.05) after treatment with β-glucan compared with controls. This study identified the feasibility and optimal conditions of ORE culture and demonstrated that β-glucan activates SBD-1, IL-6, and IL-10 secretion in OREs to promote mucosal immunity.

Effect of Achyranthes japonica Nakai extract on immunity and anti-inflammation in dogs

Gun-Hwi Lee, Kyung-A. Hwang, Ji-Houn Kang, Kyung-Chul Choi (page 294)

Achyranthes japonica Nakai (A. japonica) is a medicinal herb found widely distributed throughout Korea. The biological activities of A. japonica are well-documented and include anti-fungal, anti-inflammatory, and immunity enhancement. The objective of the present study was to investigate the immune-related activities of A. japonica extract in dogs. The extract was acquired by ethanol extraction and purified by filtration. To examine the effect of A. japonica extract on immune cell viability, human lymphocytes, such as Jurkat T-cells and Ramos B-cells, were exposed to the extract. After treatment with the extract, the number of Ramos B-cells was increased, whereas Jurkat T-cells remained unaffected. Griess assay revealed decreased nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated mouse macrophage Raw 264.7 cells after exposure to A. japonica extract. To evaluate the in-vivo effect in dogs, feed containing A. japonica extract was provided to 8 dogs for 2 months. Blood samples were collected before, during, and after consumption of the feed. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples and the number of T-cells and B-cells were assessed using flow cytometry with anti-dog fluorescein isothiocyanate (FITC)-conjugated CD3 and anti-dog phycoerythrin (PE)-conjugated CD21 antibodies, respectively. We observed a significant increase in the average number of B-cells in the PBMCs during ingestion of the feed containing A. japonica. In addition, enzyme-linked immunosorbent assay (ELISA) revealed a decrease in the levels of tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, in 3 out of 8 dogs and increased levels of interleukin-10 (IL-10), an anti-inflammatory cytokine, in 4 out of 8 dogs. Taken together, we believe that these changes indicate that A. japonica extract is beneficial in improving the immunity of dogs by stimulating B-cells and inducing production of anti-inflammatory responses.

Salmonella enterica serovar Typhimurium gene sseK3 is required for intracellular proliferation and virulence

Fuyu Du, Chengshui Liao, Yadong Yang, Chuan Yu, Xiaojie Zhang, Xiangchao Cheng, Chunjie Zhang (page 302)

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most significant zoonotic pathogens that poses a threat to humans. Previous studies have identified that Salmonella-secreted effector K3 (SseK3) is a novel translated and secreted protein of S. Typhimurium. The objective of this study was to determine whether deletion of the sseK3 gene can attenuate the virulence of S. Typhimurium. To do this, we constructed an sseK3 deletion mutant using the double-exchange allele of the suicide plasmid pRE112ΔsseK3 and assessed the virulence and intracellular proliferation of the mutant. The sseK3 deletion mutant exhibited adhesion and invasion properties similar to those of wild-type (WT) S. Typhimurium, although the virulence and intracellular proliferation of the mutant were significantly reduced compared to that of the WT strain. Furthermore, the observed increase in the median lethal dose (LD50) reflects a decrease in the pathogenicity of the sseK3 deletion mutant in a murine model. In summary, we concluded that disruption of sseK3 can attenuate the intracellular proliferation and reduce the virulence of S. Typhimurium.

Brief Communications

Mycoplasma hyopneumoniae genetic variability within swine production flows

Alyssa M. Betlach, Eduardo Fano, Amanda Sponheim, Robert Valeris-Chacin, Laura Dalquist, Randall S. Singer, Maria Pieters (page 310)

The aim of this study was to assess the genetic variability of Mycoplasma hyopneumoniae within various swine production flows. Four M. hyopneumoniae positive production flows, composed of 4 production stages, were selected for this study. Laryngeal and/or bronchial swabs were collected from each production stage within a flow, for a period of 4 months up to 3 years. A multiple-locus variable-number tandem repeat analysis was performed to assess the genetic variation of M. hyopneumoniae within and across production flows through the identification of variable-number tandem repeat (VNTR) types. A maximum of 6 M. hyopneumoniae VNTR types were identified in a single flow, in which VNTR types appeared to be flow specific. An identical VNTR type was detected across several production stages for up to 3 years. In this study, minimal M. hyopneumoniae genetic variation was evidenced within and across production flows.

A potential approach for assessing the quality of human and nonhuman adenoviral vector preparations

Ekramy E. Sayedahmed, Suresh K. Mittal (page 314)

Various types of human and nonhuman adenoviral (AdV) vectors are being used as gene delivery vectors in preclinical and clinical investigations. The objective of this study was to determine the ratio between the 2 best assays that would effectively address the variability in the titration of various AdV vectors in different cell lines and help obtain consistent results in preclinical and clinical studies using different AdV vectors. Here, we compared plaque-forming units, tissue culture infectious dose 50, focus-forming units (FFU), virus particle (VP) count, and genome copy number (GCN) of purified preparations of human AdV type C5, bovine AdV type 3, and porcine AdV type 3 to determine a correlation between infectious and noninfectious virus particles. Our results suggest that a VP:FFU or a VP:GCN ratio could accurately reflect the quality of an AdV preparation and could serve as an indicator to control batch-to-batch variability.

Molecular diagnosis using RNAscope in-situ hybridization in canine malignancies

Keijiro Shiomitsu, Sandra M. Bechtel, Patrick M. Thompson, Salvatore Frasca, Jr. (page 319)

Immunohistochemistry has been used extensively to evaluate protein expression in clinical and research settings. However, immunohistochemistry is not always successful in veterinary medicine due to the lack of reliable antibody options, poor tissue preservation, labor-intensive staining, and antigen-retrieval optimization processes. RNAscope in-situ hybridization (ISH) is a powerful technology that uses a specific sequence probe to identify targeted mRNA. In this study, we demonstrate RNAscope ISH in 4 common canine malignancies, which are traditionally diagnosed by histopathology and immunohistochemistry. Probes were designed for commonly targeted mRNA markers of neoplastic tumors; these included c-kit in mast cell tumor, microphthalmia-associated transcription factor in malignant melanoma, ionized calcium-binding adapter molecule-1 in histiocytic sarcoma, and alkaline phosphatase in osteosarcoma. A strong staining signal was obtained by these 4 targets in each canine malignancy. These results support the use of RNAscope ISH for definitive diagnosis in canine malignancies.