Table of Contents and AbstractsJanuary 2018, Vol. 82, No.1


Safety evaluation of a new anxiolytic product containing botanicals Souroubea spp. and Platanus spp. in dogs

Aleksandar Masic, Rui Liu, Kristen Simkus, Jeff Wilson, John Baker, Pablo Sanchez, Ammar Saleem, Cory C. Harris, Tony Durst, John T. Arnason (page 3)

Separation anxiety and noise aversion are common behavioral problems in dogs. They elicit fear responses such as cowering, seeking out the owner, and attempting to escape. This can result in property damage, injury to the dog, and disruption of the owner-pet bond, possibly leading to pet abandonment or euthanasia. A novel botanical anxiolytic product was evaluated for safety in dogs as the target animal species. Its intended use is for the treatment and prevention of anxiety and noise aversion in dogs. It contains a defined mixture of Souroubea spp. vine and Platanus spp. bark, delivering the active principle, betulinic acid, at a recommended dose of 1 mg/kg body weight (BW). In the current target animal safety study, 16 healthy male beagle dogs were administered either a placebo or the newly formulated botanical tablets at 0.5×, 2.5×, or 5× the recommended dose (1 mg/kg BW) over 28 d. The dogs were monitored for occurrence of any systemic or local adverse events. In the investigation presented here, there were no clinically significant adverse effects following treatment, as determined by clinical observations, physical examinations, BW, hematology, clinical biochemistry, and urinalysis. Pharmacokinetic analysis demonstrated that the concentration of betulinic acid in serum was below 0.020 µg/mL in treated animals. Under the conditions of these studies, the formulated blend of S. sympetala and P. occidentalis, when administered up to 5× the intended dose for 28 consecutive d, showed no adverse effects on the health of dogs.

Development and optimization of a cell-associated challenge model for Mycoplasma hyorhinis in 7-week-old cesarean-derived, colostrum-deprived pigs

Brian Martinson, F. Chris Minion, Dianna Jordan (page 12)

Mycoplasma hyorhinis (MHR) causes polyserositis and lameness in grower pigs. While herd-specific vaccines for this bacterium are being marketed, there are currently no licensed, commercially available vaccines for MHR. The objective of this study was to develop a challenge model in cesarean-derived, colostrum-deprived (CDCD) pigs using cell-associated MHR that results in both severe pericarditis and lameness, in order to evaluate suitable vaccine candidates. We investigated administering MHR to 7-week-old pigs over 3 d using 3 different routes compared to administering MHR on a single day using 1 of 3 routes. Pigs were monitored for 21 d for signs of lameness and well-being. At the end of the study, pigs were examined for evidence of polyserositis and arthritis associated with Mycoplasma. Results indicate that clinical manifestation of disease depended more on the route of administration than on the total dose given. A single intravenous (IV) administration of MHR resulted in extensive polyserositis, while a single intranasal (IN) administration showed little to no signs of disease. A single intraperitoneal (IP) administration did not induce the same level of polyserositis as observed in the IV group, but did result in an increased incidence of lameness. Furthermore, pigs administered MHR by IP (Day 0), IV (Day 1), and IN (Day 2) on 3 consecutive days showed a more robust disease manifestation, which resulted in both polyserositis and lameness. Optimization of this group showed that elimination of the 3rd-day IN challenge had no detrimental effect on clinical outcomes. The consecutive day administration of cell-associated MHR will allow polyserositis and lameness to be simultaneously evaluated in future vaccine trials.

Comparison of antimicrobial resistance genes in feedlots and urban wastewater

Alicia G. Beukers, Rahat Zaheer, Shaun R. Cook, Alexandre V. Chaves, Michael P. Ward, Lisa Tymensen, Paul S. Morley, Sherry Hannon, Calvin W. Booker, Ron R. Read, Tim A. McAllister (page 24)

The use of antibiotics in livestock production in North America and possible association with elevated abundance of detectable antimicrobial resistance genes (ARG) is a growing concern. Real-time, quantitative PCR (RT-qPCR) was used to determine the relative abundance and diversity of ARG in fecal composite and catch basin samples from 4 beef feedlots in Alberta. Samples from a surrounding waterway and municipal wastewater treatment plants were also included to compare the ARG profile of urban environments and fresh water with that of feedlots. The relative abundance of 18 resistance genes across 5 antibiotic families including sulfonamides, tetracyclines, macrolides, fluoroquinolones, and β-lactams was examined. Sulfonamide, fluoroquinolone, and β-lactam resistance genes predominated in wastewater treatment samples, while tetracycline resistance genes predominated in cattle fecal composite samples. These results reflect the types of antibiotic that are used in cattle versus humans, but other factors such as co-selection of ARG and variation in the composition of bacterial communities associated with these samples may also play a role.

Comparison of 3 vaccination strategies against porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, and porcine circovirus type 2 on a 3 pathogen challenge model

Jiwoon Jeong, Ikjae Kang, Seeun Kim, Kee Hwan Park, Changhoon Park, Chanhee Chae (page 39)

The objective of this study was to compare clinical, microbiologic, immunologic, and pathologic parameters in pigs each concurrently administered porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae, and porcine circovirus type 2 (PCV2) vaccine from 1 of 2 commercial sources at 21 days of age and challenged with field strains of each of the 3 pathogens. Pigs were challenged with PRRSV and M. hyopneumoniae at 42 days of age (−14 days post-challenge, dpc) followed by a challenge with PCV2 at 56 days of age (0 dpc). Significant differences were observed between vaccinated challenged and unvaccinated challenged groups in clinical (average daily gain and clinical signs), microbiologic (viremia and nasal shedding), immunologic (antibodies and interferon-γ secreting cells), and pathologic (lesions) outcomes. Significant differences were observed among the 3 vaccinated challenged groups in microbiologic (nasal shedding of M. hyopneumoniae and viremia of PCV2) and immunologic (M. hyopneumoniae- and PCV2-specific interferon-γ secreting cells) outcomes. The vaccination regimen for PRRSV vaccine, M. hyopneumoniae vaccine, and PCV2 vaccine is efficacious for controlling triple challenge with PRRSV, M. hyopneumoniae, and PCV2 from weaning to finishing period.

Biological characteristics and conjugated antigens of ClfA A-FnBPA and CP5 in Staphylococcus aureus

Tiansen Li, Meiling Huang, Zhanhui Song, Hui Zhang, Chuangfu Chen (page 48)

To obtain immunogenic conjugate antigens, adipic acid dihydrazide (ADH), as a bridge, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDAC), as a coupling agent, were used to conjugate the purified fusion protein, clumping factor A-fibronectin binding protein ClfA A-FnBPA, and type 5 capsular polysaccharide (CP5). The conjugates were mixed with an adjuvant, and mice were immunized 3 times and challenged with Staphylococcus aureus 1 week later. Antibody titers were determined by indirect enzyme-linked immunosorbent assay (ELISA). At 14 days after the first immunization, antibodies against the purified protein and conjugate were detected; after 28 days, antibody levels increased; and a week after the third immunization, antibody levels continued to increase. However, the conjugate antibody titers were higher than those of the purified protein during the study, and no IgG antibodies against purified CP5 were detected during the entire experiment. The protection rate increased to 90% in the conjugate group, indicating that the conjugate imparts a relatively higher protective efficacy than the purified protein and purified CP5.

Comparison of the oral and rectal mucosal and colonic serosal microcirculations of healthy, anesthetized horses

Philip J. Kieffer, Jarred M. Williams, Molly K. Shepard, Steeve Giguère, Kira L. Epstein (page 55)

The objectives of the study were to: i) determine baseline microvascular perfusion indices (MPI) and assess their repeatability in healthy horses under general anesthesia, and ii) compare the MPIs of 3 microvascular beds (oral mucosa, colonic serosa, and rectal mucosa). Healthy adult horses were anesthetized and sidestream dark field microscopy was used to collect video loops of the oral mucosa, rectal mucosa, and colonic serosa under normotensive conditions without cardiovascular support drugs; videos were later analyzed to produce MPIs. Baseline MPI values were determined for each site, which included the total vessel density (TVD), perfused vessel density (PVD), portion perfused vessels (PPV), and microcirculatory flow index (MFI). Differences in MPIs between microvascular beds were not statistically significant. Repeatability of the measurements varied for each MPI. In particular, the site of sampling had a profound effect on the repeatability of the PPV measurements and should be considered in future studies.

Histological structure and age-related changes in the luminal diameter of the excurrent duct system of guinea cocks (Numida meleagris) and associated changes in testosterone concentrations

Ibn Iddriss Abdul-Rahman, Ian Jeffcoate (page 60)

As little information is available on the reproductive system of guinea fowl (Numida meleagris), a study was conducted on 49 male guinea fowl to document the histological structure and developmental changes in the luminal diameter of the ducts within the excurrent duct system and associated changes in concentrations of testosterone. Age-related changes were analyzed using the Kruskal-Wallis test and medians separated by the Mann-Whitney U-test. Tubuli recti were clearly visible in the guinea fowl and the rete testes were both intracapsular and extracapsular. Regardless of age, the luminal diameter of the proximal ductuli efferentes was the largest, while that of the connecting duct was the smallest. The luminal diameter of all ducts within the epididymal region increased (P < 0.001) monthly until 20 wk of age, and then increased marginally every month thereafter. Peripheral testosterone concentrations also peaked at 20 wk of age and declined thereafter. In adult birds, the ductus deferens enlarged posteriorly, from an average of about 279 µm cranially to 678 µm caudally. Peripheral testosterone concentrations strongly and positively correlated with the luminal diameter of ducts within the excurrent duct system. The pattern of increase in the luminal diameter of all ducts followed the pattern of testosterone secretion in these birds, which indicates that testosterone concentrations may be closely related to the development of the excurrent duct system in male guinea fowl.

Brief Communications

Livestock-associated methicillin-resistant Staphylococcus aureus and Clostridium difficile in wild Norway rats (Rattus norvegicus) from Ontario swine farms

Jamie L. Rothenburger, Joyce D. Rousseau, J. Scott Weese, Claire M. Jardine (page 66)

Methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile are important human pathogens that are also carried by animals. The role of wild mammals on farms in their maintenance and transmission, however, is poorly understood. To determine if Norway rats (Rattus norvegicus) are potential carriers of these bacteria on Canadian farms, we tested 21 rats from swine farms in Ontario. The MRSA spa type t034 was isolated from 1 (4.8%) rat. This livestock-associated strain often colonizes pigs and pig farmers, suggesting that transmission among rats and pigs or environmental transmission is possible on pig farms. Clostridium difficile ribotype 078 was isolated from 1 rat from a different farm. This strain is associated with infection in piglets, calves, and humans. The identification of MRSA and C. difficile in Norway rats on farms in Canada adds to the growing knowledge about the role of rats in the ecology of these pathogens. Further studies are required to determine if rats play a part in the epidemiology of these pathogens on farms.

Development and application of a droplet digital polymerase chain reaction (ddPCR) for detection and investigation of African swine fever virus

Xulong Wu, Lu Xiao, Hua Lin, Shijie Chen, Miao Yang, Wei An, Yin Wang, Zexiao Yang, Xueping Yao, Zizhong Tang (page 70)

The aim of this study was to develop a droplet digital polymerase chain reaction (ddPCR) method to detect African swine fever virus (ASFV). The methods of ASFV real-time PCR and ddPCR were established and optimal reaction conditions were confirmed. Each method was evaluated for linearity, limit of detection, and specificity. The results indicated that ASFV ddPCR had a high degree of linearity (R2 ≥ 0.998) and specificity. The detection limit was 10 copies/reaction, which was approximately a 10-fold greater sensitivity than real-time PCR. This sensitive method could be used as an efficient molecular biology tool to diagnose ASFV, which is very important for preventing the spread of diseases across borders.

Effect of transoral tracheal wash on respiratory mechanics in dogs with respiratory disease

Meghan E. Vaught, Elizabeth A. Rozanski, Armelle M. deLaforcade (page 75)

The purpose of this study was to determine the impact of a transoral tracheal wash (TOTW) on respiratory mechanics in dogs and to describe the use of a critical care ventilator (CCV) to determine respiratory mechanics. Fourteen client-owned dogs with respiratory diseases were enrolled. Respiratory mechanics, including static compliance (Cstat) and static resistance (Rstat), were determined before and after TOTW. Pre- and post-wash results were compared, with a P-value of < 0.05 considered significant. The mean ± standard deviation (SD) value of Cstat pre-TOTW was 1.59 ± 0.94 mL/cmH2O/kg while the mean ± SD of Cstat post-TOTW was 1.29 ± 0.71 mL/cmH2O/kg (P = 0.045). The median Rstat was not significantly different pre- and post-wash. The transoral tracheal wash altered respiratory mechanics, as observed by a reduction in Cstat, presumably due to airway flooding and collapse. While no long-lasting effects were noted in these clinical patients, this effect should be considered when performing TOTW on dogs with respiratory diseases. Respiratory mechanics testing using a CCV was feasible and may be a useful clinical testing approach.