Table of Contents and AbstractsApril 2018, Vol. 82, No. 2


In vitro study of matrix metalloproteinases 1, 2, 9, 13 and serum amyloid A mRNAs expression in equine fibroblast-like synoviocytes treated with doxycycline

Samaneh Ghasemi, Kamran Sardari, Pezhman Mirshokraei, Hossein Hassanpour (page 82)

Application of synthetic matrix metalloproteinases (MMPs) inhibitors, such as doxycycline is one of the possible therapeutic options for osteoarthritis. However, little is known about the protective mechanism of doxycycline in equine models on MMPs inhibitors as well as on serum amyloid A (SAA) gene expression. This study investigated the effects of doxycycline on mRNA expression of MMP-1, MMP-2, MMP-9, MMP-13, and SAA of equine fibroblast-like synoviocytes (FLSs). The FLSs were established from synovial fluids of clinically normal metacarpophalangeal joints of 6 skeletally mature horses. The cells were treated with either 10 or 100 µg/mL of doxycycline for 48 h. The mRNA expression of MMP-1, MMP-2, MMP-9, MMP-13, and SAA were assessed using real-time polymerase chain reaction (PCR). Treatment with doxycycline resulted in significantly decreased mRNA expression of MMP-1 in FLSs at both concentrations (P = 0.001). No significant differences were detected among groups for MMP-2, MMP-9, and MMP-13 (P > 0.05). Only a tendency towards a decrease in mRNA expression level of SAA in the presence of doxycycline could be detected. Doxycycline inhibits MMP-1 gene expression at the transcript level. These findings indicate that doxycycline can protect the articular environment through inhibition of MMP-1 at transcript level.

Using a computer simulation model to examine the impact of biosecurity measures during a facility-level outbreak of equine influenza

Kelsey L. Spence, Terri L. O'Sullivan, Zvonimir Poljak, Amy L. Greer (page 89)

On-farm biosecurity measures are an important part of a control plan to minimize the introduction and spread of infectious diseases, such as equine influenza, in an equine facility. It can be challenging, however, to evaluate the efficacy of biosecurity measures under field conditions. We used an agent-based computer simulation model to describe the impact of: i) preventive vaccination; ii) reduced horse-to-horse contact; and iii) a combination of vaccination and reduced contact during an outbreak of equine influenza in a simulated horse facility. The model demonstrated that the most effective intervention was a combination of a high proportion of recently vaccinated horses and a substantial reduction in horse-to-horse contact once equine influenza had been identified in the facility. This study highlights the importance of compliance when implementing biosecurity measures, such as facility-level infection control practices, on horse farms.

Comparison between cerebrospinal fluid and serum lactate concentrations in neurologic dogs with and without structural intracranial disease

Leontine Benedicenti, Giacomo Gianotti, Evelyn M. Galban (page 97)

The objectives of this study were to investigate the relationship between cerebrospinal fluid lactate and serum concentrations in dogs with clinical signs of central nervous system disease and to establish if cerebrospinal fluid lactate (CSF) concentrations are higher in dogs with structural intracranial disease (Group Pos-MRI) compared to dogs that have clinical signs of intracranial disease but no structural brain disease (Group Neg-MRI) based on magnetic resonance imaging (MRI) findings. Using a prospective study canine blood and cerebrospinal fluid were collected in 24 dogs with neurological signs after undergoing brain MRI. Dogs were divided in 2 groups. No significant difference between serum lactate (1.57 ± 0.9 mmol/L) and CSF lactate concentration (1.34 ± 0.3 mmol/L) was detected. There was a direct correlation between CSF and serum lactate concentration (R = 0.731; P = 0.01). No significant difference was found in CSF lactate concentration between the 2 groups of dogs (P = 0.13).

Roles of the crp and sipB genes of Salmonella enterica serovar Typhimurium in protective efficacy and immune responses to vaccination in mice

Songbiao Chen, Chengshui Liao, Chunjie Zhang, Xiangchao Cheng (page 102)

Salmonella enterica serovar Typhimurium has a wide host range and is capable of causing infections ranging from severe gastroenteritis to systemic infection in humans. To determine if attenuated S. Typhimurium strains can serve as safe and effective oral vaccines to prevent typhoid fever, the biologic characteristics of crp and sipB deletion mutants were evaluated. Previous studies had found that the crp and sipB genes are related to Salmonella pathogenicity. In this study, cytotoxicity, protective efficacy, and immune responses of the host were analyzed. Our previous data had shown a significance decrease in virulence for the crp and sipB mutants compared with a wild-type strain. The current study confirmed this finding in HeLa cells and showed that the crp mutant was significantly less cytotoxic (P < 0.05) than the sipB mutant. Mice vaccinated with the crp mutant showed significantly better protection after challenge with the wild-type strain (P < 0.05) and significantly greater responses in serum IgG (P < 0.01) and secretory IgA (P < 0.05) compared with the mice vaccinated with the sipB mutant (P < 0.05). Our results indicate that the crp mutant has the potential to be a vaccine candidate and is safe in mice.

Venous blood gases, plasma biochemistry, and hematology of wild-caught common chameleons (Chamaeleo chamaeleon)

David Eshar, Melanie Ammersbach, Boaz Shacham, Gad Katzir, Hugues Beaufrère (page 106)

The purpose of this study was to determine a wide range of selected hematologic, venous blood gases, and plasma biochemistry analytes in common chameleons (Chamaeleo chamaeleon). Blood samples were collected from the ventral tail vein of 41 common chameleons to determine reference intervals for 30 different blood analytes. The calcium-to-phosphorus ratio, packed cell volume (PCV), refractometric total solids (TS), blood cell counts, and differentials were also determined. The microscopic evaluation of blood smears revealed inclusion bodies in monocytes in 7 of the samples. Females showed significantly higher values of plasma proteins and calcium and cholesterol concentrations and males showed significantly higher values of aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) plasma concentrations. Significant differences were found between similar analytes determined by different testing methodologies in the PCV/hematocrit, electrolytes (sodium, potassium), and plasma proteins [TS, total protein (TP) and albumin]. Blood analytes determined in this study can provide baseline data that may be useful when evaluating the health status of common chameleons, taking into consideration the potential effects of gender and the type of analyzer used.

26S proteasome and insulin-like growth factor-1 in serum of dogs suffering from malignant tumours

Ingrid Gerke, Franz-Josef Kaup, Stephan Neumann (page 115)

Studies in humans have shown that the ubiquitin-proteasome pathway and the insulin-like growth factor axis are involved in carcinogenesis, thus, components of these systems might be useful as prognostic markers and constitute potential therapeutic targets. In veterinary medicine, only a few studies exist on this topic. Here, serum concentrations of 26S proteasome (26SP) and insulin-like growth factor-1 (IGF-1) were measured by canine enzyme-linked immunosorbent assay (ELISA) in 43 dogs suffering from malignant tumors and 21 clinically normal dogs (control group). Relationships with tumor size, survival time, body condition score (BCS), and tumor entity were assessed. The median 26SP concentration in the tumor group was non-significantly higher than in the control group. However, dogs with mammary carcinomas displayed significantly increased 26SP levels compared to the control group and dogs with tumor size less than 5 cm showed significantly increased 26SP concentrations compared to dogs with larger tumors and control dogs. 26SP concentrations were not correlated to survival time or BCS. No significant difference in IGF-1 levels was found between the tumor group and the control group; however, IGF-1 concentrations displayed a larger range of values in the tumor group. Dogs with tumors greater than 5 cm showed significantly higher IGF-1 levels than dogs with smaller tumors. The IGF-1 concentrations were positively correlated to survival time, but no correlation with BCS was found. Consequently, serum 26SP concentrations seem to be increased in some dogs suffering from malignant tumors, especially in dogs with mammary carcinoma and smaller tumors. Increased serum IGF-1 concentrations could be an indication of large tumors and a poor prognosis.

Efficacy and pharmacokinetics of bupivacaine with epinephrine or dexmedetomidine after intraperitoneal administration in cats undergoing ovariohysterectomy

Javier Benito, Beatriz Monteiro, Francis Beaudry, Paulo Steagall (page 124)

The aim of this study was to determine the efficacy and pharmacokinetics of bupivacaine in combination with epinephrine or dexmedetomidine after intraperitoneal administration in cats undergoing ovariohysterectomy. Sixteen healthy adult cats (3.3 ± 0.6 kg) were included in a prospective, randomized, masked clinical trial after obtaining owners' consent. Anesthetic protocol included buprenorphine-propofol-isoflurane. Meloxicam [0.2 mg/kg body weight (BW)] was administered subcutaneously before surgery. Cats were randomly divided into 2 groups to receive 1 of 2 treatments. Intraperitoneal bupivacaine 0.25% (2 mg/kg BW) was administered with epinephrine (BE group; 2 µg/kg BW) or dexmedetomidine (BD group; 1 µg/kg BW) before ovariohysterectomy (n = 8/group). A catheter was placed in the jugular vein for blood sampling. Blood samples were collected for up to 8 h after bupivacaine was administered. Plasma concentrations and pharmacokinetics of bupivacaine were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS) and non-compartmental model, respectively. Pain was evaluated using the UNESP-Botucatu multidimensional composite pain scale (MCPS), the Glasgow composite feline pain scale (GPS), and a dynamic visual analog scale up to 8 h after extubation. Rescue analgesia was provided with buprenorphine if MCPS was ≥ 6. Repeated measures linear models were used for analysis of pain and sedation scores (P < 0.05). Maximum bupivacaine plasma concentrations (Cmax) for BE and BD were 1155 ± 168 ng/mL and 1678 ± 364 ng/mL (P = 0.29) at 67 ± 13 min (Tmax) and 123 ± 59 min (P = 0.17), respectively. Pharmacokinetic parameters and pain scores were not different between treatments (P > 0.05). One cat in the BE group received rescue analgesia (P = 0.30). Intraperitoneal bupivacaine with epinephrine or dexmedetomidine produced concentrations below toxic levels and similar analgesic effects. It is therefore safe to administer these drug combinations in cats undergoing ovariohysterectomy.

H9N2 avian influenza virus retained low pathogenicity after serial passage in chickens

Akinlolu Jegede, Qigao Fu, Yohannes Berhane, Min Lin, Ashok Kumar, Jiewen Guan (page 131)

The H9N2 strains of avian influenza viruses (AIVs) circulate worldwide in poultry and cause sporadic infection in humans. To better understand the evolution of these viruses while circulating in poultry, an H9N2 chicken isolate was passaged 19 times in chickens via aerosol inoculation. Whole-genome sequencing showed that the viruses from the initial stock and those after the 8th and 19th passages (P0, P8, and P19) all had the same monobasic cleavage site in the hemagglutinin (HA), typical for viruses of low pathogenicity. However, at position 226 of the HA protein the ratio of glutamine (which favors avian-type receptor binding) to leucine (which favors mammalian-type receptor binding) decreased from 54:46 in P0, to 87:13 in P8, and then 0:100 in P19. In chickens exposed to aerosols of P0, P8, or P19, replication of the viruses was similar and mainly limited to the respiratory tract. None of the infected chickens showed any clinical signs. Over the 19 passages the viruses maintained relatively stable infectivity but gradually lost lethality to chicken embryos. According to the hemagglutination inactivation assay, P8 was slightly and P19 significantly (P < 0.05) less thermostable than P0. Collectively, after 19 passages in chickens the H9N2 AIVs retained low pathogenicity with a positive selection of L226 in the HA. These findings suggest that H9N2 viruses might acquire mammalian specificity after asymptomatic circulation in avian species.

Time course of Salmonella shedding and antibody response in naturally infected pigs during grower-finisher stage

Saranya Nair, Abdolvahab Farzan, Terry L. O'Sullivan, Robert M. Friendship (page 139)

A longitudinal trial was conducted to determine the course of Salmonella shedding and antibody response in naturally infected grower-finisher pigs. Ten-week-old pigs (n = 45) were transferred from a farm with history of salmonellosis and housed at a research facility. Weekly fecal samples (weeks 1 to 11) as well as tissue samples at slaughter were cultured for Salmonella. Serum samples were tested for presence of Salmonella antibody by enzyme-linked immunosorbent assay (ELISA). Data were analyzed using a multilevel mixed-effects logistic regression model. Over 10 wk, 91% and 9% of pigs shed Salmonella ≤ 4 and > 5 times, respectively. The estimated median of Salmonella shedding duration was 3 to 4 wk but some pigs shed Salmonella for up to 8 wk. Salmonella shedding increased 1 wk post-arrival but followed a decreasing pattern afterwards up to week 11 (P < 0.05). Salmonella isolates (n = 29), which were recovered from 18 pigs at different occasions, were S. Typhimurium (28%), S. Livingstone (21%), S. Infantis (14%), S. Montevideo (7%), S. Benfica (3%), S. Amsterdam (3%), S. Senftenberg (17%), and S. I:Rough-O (7%). Of 11 pigs from which the first and last isolates were serotyped, 10 pigs were reinfected with a different serotype. At slaughter, Salmonella was isolated from 7 pigs, of which 5 (71%) had not tested positive for at least 7 wk prior to slaughter. Antibody response peaked 4 wk after the peak of Salmonella infection; Salmonella shedding reduced as antibody response elevated (P < 0.05). These findings indicate that pigs may shed Salmonella into the mid-point of the grower-finisher stage and may be reinfected with different serotypes.

Evaluation of the efficacy of a novel porcine circovirus type 2 synthetic peptide vaccine

Jiwoon Jeong, Changhoon Park, Seeun Kim, Su-Jin Park, Ikjae Kang, Kee Hwan Park, Chanhee Chae (page 146)

A novel porcine circovirus type 2 (PCV2) peptide vaccine comprised of a consensus capsid (Cap) protein domain encoded by open reading frame 2 was developed to control PCV2 infection. The efficacy of the vaccine was evaluated against a commercial baculovirus-expressed recombinant PCV2 subunit vaccine based on the Cap protein. The amino acid sequence of this Cap protein was designed based on the alignment of amino acid sequences from different isolates from Europe, North America, and Asia. The vaccine was evaluated in either phosphate-buffered saline or adjuvanted with aluminum hydroxide, cobalt oxide, or liposome. Overall the PCV2 peptide vaccine was less efficacious against PCV2 challenge compared with the commercial PCV2 vaccine. The peptide vaccine was the most efficacious when liposome was used as an adjuvant, significantly (P < 0.05) reducing viremia while increasing the levels of neutralizing antibodies and interferon-γ secreting cells. This suggests, in the presence of liposome, the peptide vaccine was able to elicit both humoral and cellular immune responses.

Brief Communications

Effect of a commercially available fish-based dog food enriched with nutraceuticals on hip and elbow dysplasia in growing Labrador retrievers

Sabrina Manfredi, Francesco Di Ianni, Nicola Di Girolamo, Sergio Canello, Giacomo Gnudi, Gianandrea Guidetti, Francesca Miduri, Martina Fabbi, Eleonora Daga, Enrico Parmigiani, Sara Centenaro, Antonella Volta (page 154)

The aim of this study was to evaluate the prevalence of hip and elbow dysplasia in a group of growing Labrador retrievers fed a fish-based diet enriched with nutraceuticals with chondroprotective properties. The puppies ranged from 3 to 12 mo of age and were divided into 2 groups, each fed a different diet. The control diet consisted of a high quality, chicken-based dog food, while the test diet was a fish-based dog food, enriched with nutraceuticals. Hip and elbow joints were radiographed and scored at 6 and 12 mo of age. Overall, 42 dogs completed the study. At 12 mo of age, no differences were found between the groups in the prevalence of hip and elbow dysplasia, although dogs fed the fish-based food enriched with nutraceuticals had a less severe grade of osteoarthritis at 12 mo. It was concluded that the fish-based diet with nutraceuticals did have beneficial effects on the development of severe osteoarthritis.

Peak vertical force in a stabilized canine cranial cruciate deficient stifle model: A one-year follow-up

Bertrand Lussier, Alexandre Gagnon, Maxim Moreau, Jean-Pierre Pelletier, Éric Troncy (page 159)

This study aimed to describe the peak vertical force (PVF) over a 1-year period in a stabilized canine cranial cruciate deficient stifle model. Our hypothesis was that PVF would be restored to Baseline (intact) at the end of the follow-up. Fifteen (> 20 kg) mixed-breed dogs were included in this study. Cranial cruciate ligament was transected on Day (D) 0 followed by lateral suture stabilization at D28. Peak vertical force was acquired at D−1, D14, D26, D91, D210 and D357. When compared to Baseline, the PVF was significantly decreased at D14, D26, and D91. Values at D210 and D357 were not statistically different to Baseline. This study suggests a return to normal baseline peak vertical force in a canine cranial cruciate deficient stifle model when lateral suture stabilization has been performed 28 days after surgical transection.