CJVR - October 2022, Vol. 86, No. 4



Assessment of seasonality of rotavirus PCR detection in swine from Ontario and Quebec between 2016–2020 using submissions to a diagnostic laboratory

Hoc Tran, Robert Friendship, Davor Ojkic, Zvonimir Poljak (page 241)

The goal of this study was to determine if seasonality of rotavirus A, B, and C infection is present in Ontario and Quebec swine herds by investigating submissions to a diagnostic laboratory. Samples (N = 1557) within 755 case submissions from Canadian swine herds between 2016 and 2020 were tested for rotaviruses A, B, and C using a real-time polymerase-chain reaction assay and described. Data from Ontario and Quebec were additionally analyzed using boxplots, 6-week rolling averages, time-series decomposition, and negative binomial regression models. Percentage positivity of submissions for rotaviruses A, B, and C were discovered to be highest in nursery/weaner (n = 100, 94.0%, 60.0%, 80.0%) and grower/finisher (n = 13, 84.6%, 46.2%, 61.5%) pigs and lowest in gilt/sow (n = 45, 68.9%, 20.0%, 40.0%) and suckling pigs (n = 102, 67.6%, 10.8%, 38.2%), respectively. The most common combination of rotavirus at the sample level was AC (n = 252, 17%) and ABC (n = 175, 23.2%) at the submission level. Percent positivity for rotavirus A, B, and C across all Canadian provinces included in the study were 69.9%, 32.6%, and 53.1%, respectively. Descriptive analysis suggested little to no evidence of seasonal patterns, although a spike in November was seen in the monthly total submissions and monthly total positive submissions. Statistically, the overall month effect could not be identified as statistically significant (P > 0.05) for any of the evaluated submission counts. Overall, there was no evidence supporting seasonality of rotavirus within Ontario and Quebec swine herds between 2016 and 2020.

Macleaya cordata extract modulates inflammation via inhibition of the NF-κB and MAPK signaling pathways in porcine alveolar macrophages induced by Glaesserella parasuis

Ze Zeng, Huaqi Zhang, Ganbei Gui, Jie Luo, Shanshan Liu (page 254)

Glässer’s disease in pigs is associated with infection by Glaesserella parasuis and is characterized by pneumonia-like symptoms, fibrinous polyserositis, polyarthritis, and meningitis. Macleaya cordata, a commonly used traditional Chinese medication, has been shown to have anti-inflammatory, antiviral, antioxidative, antimicrobial, insecticidal, and antitumor properties. However, the anti-inflammatory effects of M. cordata on G. parasuis stimulation are still poorly understood. This study explored the anti-inflammatory effects and mechanisms of M. cordata extract on G. parasuis-induced inflammatory responses, via the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, in porcine alveolar macrophages (PAMs). Porcine alveolar macrophages, when stimulated with G. parasuis, initiated transcription of interleukin (IL)-1α, IL-1β, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α). Furthermore, p65, IκBα, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) phosphorylation were upregulated via the NF-κB and MAPK signaling pathways. However, treatment with M. cordata extract inhibited transcription of IL-1α, IL-1β, IL-6, IL-8, and TNF-α and reduced p65, IκBα, p38, ERK, and JNK phosphorylation, by inhibiting activation of the NF-κB and MAPK signaling pathways in PAMs induced by G. parasuis. These findings reveal that M. cordata extract can reverse the inflammatory effect initiated by G. parasuis in vitro and that it possesses significant immunosuppression activity; thus, it may offer a novel strategy for controlling and treating G. parasuis infection.

Development of optimized protocol for culturing African swine fever virus field isolates in MA104 cells

Hyeok-il Kwon, Duy Tien Do, Hung Van Vo, Seung-Chul Lee, Min Ho Kim, Dung Thi Thuy Nguyen, Tan Minh Tran, Quang Tin Vinh Le, Tram Thi Ngoc Ngo, Nam Minh Nguyen, Joo Young Lee, Toan Tat Nguyen (page 261)

The goal of this study was to identify a candidate commercial cell line for the replication of African swine fever virus (ASFV) by comparing several available cell lines with various medium factors. In the sensitivity test of cells, MA104 and MARC-145 had strong potential for ASFV replication. Next, MA104 cells were used to compare the adaptation of ASFV obtained from tissue homogenates and blood samples in various infectious media. At the 10th passage, the ASFV obtained from the blood sample had a significantly higher viral load than that obtained from the tissue sample (P = 0.000), exhibiting a mean cycle threshold (Ct) value = 20.39 ± 1.99 compared with 25.36 ± 2.11. For blood samples, ASFV grew on infectious medium B more robustly than on infectious medium A (P = 0.006), corresponding to a Ct value = 19.58 ± 2.10 versus 21.20 ± 1.47. African swine fever virus originating from blood specimens continued to multiply gradually and peaked in the 15th passage, exhibiting a Ct value = 14.36 ± 0.22 in infectious medium B and a Ct value = 15.42 ± 0.14 in infectious medium A. When ASFV was cultured from tissue homogenates, however, there was no difference (P = 0.062) in ASFV growth between infectious media A and B. A model was developed to enhance ASFV replication through adaptation to MA104 cells. The lack of mutation at the genetic segments encoding p72, p54, p30, and the central hypervariable region (CVR) in serial culture passages is important in increasing the probability of maintaining immunogenicity when developing a vaccine candidate.

Relationship of persistent lymphocytosis, antibody titers, and proviral load with expression of interleukin-12, interferon-γ, interleukin-2, interleukin-4, interleukin-10, and transforming growth factor-β in cows infected with bovine leukemia virus from a high-prevalence dairy complex

Ernesto Marin-Flamand, Diana Michele Araiza-Hernandez, Alejandro Vargas-Ruiz, Ignacio Carlos Rangel-Rodríguez, Lilia A. González-Tapia, Hugo Ramírez-Álvarez, Ruperto Javier Hernández-Balderas, Lucía Angélica García-Camacho (page 269)

Bovine leukemia virus (BLV) subclinical infection promotes persistent lymphocytosis (PL), which is related to susceptibility and progression to lymphoma. Moreover, lymphocyte counts directly correlate with BLV antibody titers and proviral load, and cell immune responses are considered atypical due to immune suppression. In order to determine the relationship of PL, antibody titers, and proviral load with interleukin (IL)-12, interferon (IFN)-γ, IL-2, IL-4, IL-10, and transforming growth factor (TGF)-β expression in a 3-month interval, 58 cows were selected (30 BLV+ and 28 BLV) from a high-prevalence dairy herd to complete 3 monthly blood samplings for the assessment of PL, BLV antibody titers, BLV proviral load, and IL-12, IFN-γ, IL-2, IL-4, IL-10, and TGF-β expression. At sampling conclusion, the BLV-infected cows were grouped according to PL, BLV proviral load, and BLV antibody titers as follows: BLV+PL+ (n = 16) and BLV+PL (n = 14); high proviral load (HPL) (n = 18) and low proviral load (LPL) (n = 13); high antibody titers (HAT) (n = 17) and low antibody titers (LAT) (n = 14). The BLV+PL+ cows showed significantly higher proviral load and antibody titers than the BLV+PL group; however, the former suggested spread presumably unrelated to lymphoma outcome, because HPL was observed in PL cows in the last sampling. Consistent with the data, a higher antibody response strongly indicated BLV susceptibility since it was linked to PL+ occurrence and a cytokine profile compatible with immune suppression. Furthermore, a reversion to lower antibody titers was observed in cows with HPL far ahead of time, most likely due to long-term immune suppression. In addition, high expression of IL-10 and TGF-β was associated with reduced IL-12, IFN-γ, IL-2, and IL-4 expression alongside PL, HAT, and HPL in BLV-infected cows, suggesting an IL-10- and TGF-β-induced immune suppression. The IL-10 expression was increasing throughout, implying disease progression, as described. In conclusion, the proliferative expansion of lymphocytes known as PL might enhance a regulatory-rich cell population (Bregs and/or Tregs) that secretes IL-10 and TGF-β, leading to immune suppression. Further studies must be conducted regarding the types of regulatory cells involved in BLV-induced immune suppression.

Coincidence of v-raf murine sarcoma viral oncogene homolog B mutation (V595E) with phosphorylated v-raf murine sarcoma viral oncogene homolog B in urothelial carcinoma in dogs

Hirofumi Yamasaki, Yosuke Uematsu, Yuhei Hayashi, Masao Yamashita, Meina Tei, Kazuyuki Uchida, Kenichiro Ono, Hidehiro Hirao (page 286)

Expression of phosphorylated v-raf murine sarcoma viral oncogene homolog B (pBRAF) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) were investigated in urothelial carcinoma (UC) in dogs with or without the BRAF gene mutation (V595E). Among the 10 cases of UC with V595E (−), cytoplasmic immunoreactivity against pBRAF of neoplastic cells was reported in 8, with 7 displaying moderate reactivity and 1 displaying intense reactivity. Nuclear immunoreactivity against pBRAF was detected in 5 cases; however, these reactivities were non-specific, due to pBRAF being limited in the cytoplasm. In addition, positive cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells was detected in 7 cases and nuclear immunoreactivity against ERK1/2 was detected in 6 cases. Among the 13 cases of UC with V595E (+), cytoplasmic immunoreactivity against pBRAF of neoplastic cells was detected in all 13 cases and nuclear immunoreactivity against pBRAF was detected in 10 cases; however, the nuclear immunoreactivity was non-specific. Cytoplasmic immunoreactivity against pERK1/2 of neoplastic cells was detected in all 13 cases and nuclear immunoreactivity against pERK1/2 was also detected in all cases. As nuclear pERK1/2 indicates a progressive signaling process in the mitogen-activated protein kinase pathway, V595E (+) UC might be in its growing stage. Probable phosphorylated sites of pBRAF at Thr598/Ser601, detected in this study, are major and essential sites of the upstream rat sarcoma viral oncogene homolog (RAS) signaling pathway. In human cancers, the BRAF mutation never coincides with oncogenic RAS. To our knowledge, this is the first report on the simultaneous occurrence of the BRAF mutation (V595E) and pBRAF expression (at Thr598/Ser601) in dogs with UC with V595E (+).

Anatomical landmarks for a proximal paravertebral thoracolumbar block in sheep: Cadaver and in-vivo study

Perla Velazquez-Delgado, Eduardo Gutierrez-Blanco, Antonio Ortega-Pacheco, Jose Leonardo Guillermo-Cordero, Brighton T. Dzikiti, Alexander Valverde (page 294)

The objective of this study was to describe the anatomy of the spinal nerves, specifically the last thoracic nerve (T13) and the first to third lumbar nerves (L1 to L3), in order to safely carry out an accurate proximal paravertebral block (PPVB) in sheep. This study consisted of 2 phases. In Phase 1, 7 sheep cadavers were dissected to identify the path and relevant anatomical landmarks of spinal nerves T13 and L1 to L3. In Phase 2, 2 healthy sheep received bilateral injections of 0.35 mL/kg body weight (BW) for each hemithoracolumbar area (0.088 mL/kg BW per nerve) of a dye-lidocaine solution (50:50) using a PPVB approach and then assessed for 15 min for signs of systemic and local effects of lidocaine. After euthanasia, the infiltrated area was dissected to assess the spread of the dye. Successful nerve staining (> 2 cm in length), macroscopic evidence of intraneural/intravascular injection, and spread to the epidural space and the abdominal cavity were recorded. In Phase 1, each branch of all nerves was easily identified and located using the caudal aspect of the spinous apophysis and the lateral edge of the transverse process of the respective vertebrae. An overlap was observed between the costoabdominal (T13), the iliohypogastric (L1), and ilioinguinal (L2) nerves. In Phase 2, all nerves were stained at least 2 cm from the injection site. There was no diffusion of the dye into the epidural space or abdominal cavity. In conclusion, using the anatomical landmarks described specifically for sheep, the PPVB provided accurate perineural distribution of the injected dye-lidocaine solution, which could result in clinical analgesia of the flank.

Correlation of hepatic copper levels, rhodanine scores and histological diagnosis in archived canine liver samples

Ashish Gupta, Ahmad Al-Dissi (page 300)

The liver is the main storage site for copper. Excess copper accumulation, however, is a risk factor for the development of chronic hepatitis in dogs. Mass spectrometry or rhodanine staining are frequently used methods to assess copper levels in the liver. The association was studied between analytic hepatic copper levels and rhodanine scores in archived canine formalin-fixed-paraffin-embedded liver sections from 2014 to 2021 with various diagnoses. Thirty-six (N = 36) liver samples with analytic interpretation of toxic (n = 12), high normal (n = 17), and normal (n = 7) copper levels were selected for the study. Rhodanine staining for each of these samples was graded (scale: 1 to 5), and the association was determined between actual liver copper levels and rhodanine scores and histological diagnoses (chronic hepatitis or other diagnoses). The analytic copper level and rhodanine scores were significantly higher (P < 0.05) in samples designated as toxic compared to normal. There was a significant association between hepatic copper levels and rhodanine scores (P < 0.05). Rhodanine score, but not the actual liver copper levels were significantly (P < 0.05) associated with chronic hepatitis versus other diagnoses. Rhodanine scores of ≥ 1.89 were statistically significant predictors of chronic hepatitis. It was concluded from this study that actual liver copper levels are positively associated with rhodanine scores and rhodanine scores can be a useful predictor of chronic hepatitis.

Chlorhexidine shampooing of dogs the night before elective surgery: Are human recommendations applicable to veterinary medicine?

Ömer Coskun, Sivert Viskjer (page 306)

The objective of this study was to investigate the effect of the night before surgery chlorhexidine shampooing on skin bacterial colony-forming units (CFU) in dogs. Twenty-five dogs had the right hindleg washed with chlorhexidine gluconate solution the night before sampling, the untreated left hindleg was used as a control. Colony-forming units were counted from 150 agar plates, 75 from each side. Median CFU on the treated side and the control side after clipping was 11 and 50, respectively (P = 0.01). Samples obtained after scrubbing the skin with CHXG, and after the final disinfection with alcohol showed no difference in CFU between sides. The “night before” chlorhexidine wash effectively decimated the skin surface bacterial CFU, but this effect was only evident after clipping. After the routine preoperative chlorhexidine scrubbing and alcohol disinfection no beneficial effects were proven.

Clinical findings and patient outcomes following surgical treatment of chronic gastrointestinal foreign body obstructions in dogs and cats: 72 cases (2010–2020)

Tiffany Kan, Rebecka S. Hess, Dana L. Clarke (page 311)

The objective of this study was to characterize clinicopathologic factors and outcomes for dogs and cats with chronic small intestinal foreign body obstructions (CFBO). Medical records of 72 dogs and cats diagnosed with CFBO between 2010 to 2020 were reviewed for duration of clinical signs, pre-surgical and intraoperative findings, complications, and outcomes. A chronic foreign body was defined as clinical signs, or the observation of foreign material ingestion, at least 7 days prior to presentation. Twenty-two (31%) patients had a small intestinal resection and anastomosis (SIRA) and were more likely to have longer duration of clinical signs (P = 0.01). Eleven (15%) patients developed major post-operative complications. Sixty-eight (94%) patients survived to follow-up. Although all patients that did not survive (n = 4, 100%) had a SIRA, patients with CFBO had a high survival rate. Therefore, clients should not be deterred from pursuing surgical intervention.

Short Communications

Kaolin activation of recalcified citrated whole blood in a point-of-care viscoelastic coagulation test

Amy M. Molitoris, Armelle M. deLaforcade, Alexandra Pfaff, Elizabeth A. Rozanski (page 316)

The viscoelastic coagulation monitor (VCM) is described as a point-of-care analyzer relying on activation of fresh whole blood (FWB) via contact between 2 glass plates. Kaolin is used as an activator in thromboelastography to reduce variability and shorten clotting times.

The goal of this study was to compare VCM results from kaolin-activated, recalcified citrated samples with that from FWB. The VCM testing was performed using FWB and kaolin-activated, recalcified citrated samples. The VCM results were recorded for clot time (CT; seconds), clot formation time (CFT; seconds), alpha (degree), amplitude at 10 and 20 minutes (A10 and A20; VCM units), maximum clot firmness (MCF; VCM units), and lysis index (LI; %). Values were compared using a t-test or Wilcoxon-Mann-Whitney test, with a P-value < 0.05 considered significant. Variability between samples was calculated using Levene’s test.

The VCM kaolin activation resulted in significantly faster CT and CFT (P < 0.0001), higher alpha angle (P < 0.001), and higher A10 and A20 (P = 0.007, P = 0.015) compared to FWB. There was no difference in MCF, LI30, or LI45. There was no difference in variability identified.

The addition of kaolin to recalcified citrated whole blood VCM samples results in more rapid clotting of FWB alone and could be considered for clinical use in dogs.