Table of Contents and AbstractsApril 2017, Vol. 81, No. 2


Detection of genome, antigen, and antibodies in oral fluids from pigs infected with foot-and-mouth disease virus

Chandrika Senthilkumaran, Ming Yang, Hilary Bittner, Aruna Ambagala, Oliver Lung, Jeffrey Zimmerman, Luis G. Giménez-Lirola, Charles Nfon (page 82)

Virus nucleic acids and antibody response to pathogens can be measured using swine oral fluids (OFs). Detection of foot-and-mouth disease virus (FMDV) genome in swine OFs has previously been demonstrated. Virus isolation and viral antigen detection are additional confirmatory assays for diagnosing FMDV, but these methods have not been evaluated using swine OF. The objectives of this study were to further validate the molecular detection of FMDV in oral fluids, evaluate antigen detection and FMDV isolation from swine OFs, and develop an assay for isotypic anti-FMDV antibody detection in OFs. Ribonucleic acid (RNA) from FMDV was detected in OFs from experimentally infected pigs by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) from 1 day post-infection (dpi) to 21 dpi. Foot-and-mouth disease virus (FMDV) was isolated from OFs at 1 to 5 dpi. Additionally, FMDV antigens were detected in OFs from 1 to 6 dpi using a lateral flow immunochromatographic strip test (LFIST), which is a rapid pen-side test, and from 2 to 3 dpi using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA). Furthermore, FMDV-specific immunoglobulin A (IgA) was detected in OFs using an isotype-specific indirect ELISA starting at dpi 14. These results further demonstrated the potential use of oral fluids for detecting FMDV genome, live virus, and viral antigens, as well as for quantifying mucosal IgA antibody response.

Application of direct polymerase chain reaction assays for Campylobacter fetus subsp. venerealis and Tritrichomonas foetus to screen preputial samples from breeding bulls in cow-calf herds in western Canada

Cheryl L. Waldner, Sarah Parker, Karen M. Gesy, Taryn Waugh, Emily Lanigan, John R. Campbell (page 91)

The primary objectives of this study were to estimate the prevalence of Campylobacter fetus subsp. venerealis (Cfv) and Tritrichomonas foetus in breeding bulls from a sentinel cohort of cow-calf herds in western Canada and to estimate the association between positive test status and non-pregnancy. The final objective was to evaluate the application of these tests when: i) screening bulls in the absence of a recognized problem with reproductive performance, and ii) testing for diagnosis of poor pregnancy rates. The crude apparent bull prevalence for Cfv was 1.1% [95% confidence interval (CI): 0.5% to 2.1%; 8/735] and herd prevalence was 2.6% (95% CI: 0.3% to 9.0%; 2/78). The crude apparent bull prevalence for T. foetus was < 0.001% (95% CI: 0.0% to 0.5%; 0/735) and herd prevalence was < 0.001% (95% CI: 0.0% to 4.6%; 0/78). Cows from herds where at least 1 bull was test positive for Cfv were 2.35 times more likely (95% CI: 1.01% to 5.48%; P = 0.047) to not be pregnant than those with no positive bulls. Polymerase chain reaction (PCR) testing of preputial material collected into phosphate-buffered saline (PBS) was recommended for screening for T. foetus when the pre-test probability of infection was > 1%. The same test for Cfv was not recommended for screening moderate- and low-risk herds due to the high risk of false positives. Tests for both T. foetus and Cfv can be used to investigate herds with reproductive problems when also ruling out other risk factors. Regardless of the type of test used, however, 3 negative tests are required to rule out infection in high-risk situations.

Efficacy of an accelerated hydrogen peroxide disinfectant to inactivate porcine epidemic diarrhea virus in swine feces on metal surfaces

Derald J. Holtkamp, Jacqueline Myers, Paul R. Thomas, Locke A. Karriker, Alejandro Ramirez, Jianqiang Zhang, Chong Wang (page 100)

In May of 2013, porcine epidemic diarrhea virus (PEDV) was detected in swine for the first time in North America. It spread rapidly, in part due to contaminated livestock trailers. The objective of this study was to test the efficacy of an accelerated hydrogen peroxide disinfectant for inactivating PEDV in the presence of feces on metal surfaces, such as those found in livestock trailers. Three-week-old barrows were inoculated intragastrically with 5 mL of PEDV-negative feces for the negative control, 5 mL of untreated PEDV-positive feces for the positive control, and 5 mL or 10 mL of PEDV-positive feces that was subjected to treatment with a 1:16 or 1:32 concentrations of accelerated hydrogen peroxide disinfectant for a contact time of 30 min at 20°C. These pigs served as a bioassay to determine the infectivity of virus following treatment. Rectal swabs collected from the inoculated pigs on days 3 and 7 post-inoculation were tested by using PEDV-specific real-time reverse transcription polymerase chain reaction and the proportion of pigs in each group that became infected with PEDV was assessed. None of the pigs used for the bioassay in the 4 treatment groups and the negative control group became infected with PEDV, which was significantly different from the positive control group (P < 0.05) in which all pigs were infected. The results suggest that the application of the accelerated hydrogen peroxide under these conditions was sufficient to inactivate the virus in feces found on metal surfaces.

Concurrent vaccination of boars with type 1 and type 2 porcine reproductive and respiratory syndrome virus (PRRSV) reduces seminal shedding of type 1 and type 2 PRRSV

Jiwoon Jeong, Changhoon Park, Ikjae Kang, Su-Jin Park, Chanhee Chae (page 108)

The objective of the present study was to determine the effect of concurrent vaccination of boars with type 1 and type 2 porcine reproductive and respiratory syndrome virus (PRRSV) on seminal shedding of both genotypes. The boars tolerated well concurrent administration of 2 commercial PRRSV vaccines, and no adverse reactions were observed. No interference in the humoral immune response (measured as the level of anti-PRRSV antibodies) or the cell-mediated immune response (measured as the level of PRRSV-specific interferon-γ-secreting cells) was observed after concurrent administration compared with single administration of the same vaccines. Concurrent vaccination significantly reduced the load of type 1 and type 2 PRRSV in blood and semen after singular (type 1 or type 2) and dual (type 1 and type 2) PRRSV challenge, and it did not significantly affect the efficacy of each vaccine. The results demonstrate that concurrent vaccination of boars with type 1 and type 2 PRRSV reduces shedding of both genotypes in semen.

Effect of probiotic Lactobacillus murinus LbP2 on clinical parameters of dogs with distemper-associated diarrhea

Luis Delucchi, Martín Fraga, Pablo Zunino (page 118)

The objective of this study was to assess the effect of the probiotic Lactobacillus murinus native strain (LbP2) on general clinical parameters of dogs with distemper-associated diarrhea. Two groups of dogs over 60 d of age with distemper and diarrhea were used in the study, which was done at the Animal Hospital of the Veterinary Faculty of the University of Uruguay, Montevideo, Uruguay. The dogs were treated orally each day for 5 d with the probiotic or with a placebo (vehicle without bacteria). Clinical parameters were assessed and scored according to a system specially designed for this study. Blood parameters were also measured. Administration of the probiotic significantly improved the clinical score of the patients, whereas administration of the placebo did not. Stool output, fecal consistency, mental status, and appetite all improved in the probiotic-treated dogs. These results support previous findings of beneficial effects with the probiotic L. murinus LbP2 in dogs. Thus, combined with other therapeutic measures, probiotic treatment appears to be promising for the management of canine distemper-associated diarrhea.

Lactobacillus casei regulates differentiation of Th17/Treg cells to reduce intestinal inflammation in mice

Kai Wang, Hao Dong, Yu Qi, Zhihua Pei, Shushuai Yi, Xiaojie Yang, Yanli Zhao, Fanxing Meng, Shouping Yu, Tiezhong Zhou, Guixue Hu (page 122)

In order to study the ability of Lactobacillus casei to ameliorate murine enteritis, 18 mice were randomly divided into 3 groups: the enteritis group, intervention group, and control group. The interleukin (IL)-6 and transforming growth factor-β (TGF)-β content in mouse peripheral blood and duodenum was detected using an enzyme-linked immunosorbent assay (ELISA). The number of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) and CD4+IL-17A+ Th17 cells in the mesenteric lymph nodes (MLN) and spleen were detected using flow cytometry, and quantitative reverse transcription polymerase chain reaction (PCR) and western blot analysis were used to measure Foxp3 and retinoid-related orphan receptor-γ (RORγt) mRNA and protein expression in the MLN. Histological changes in the duodenum were observed. Results indicate that in the intervention group, IL-6 content in mouse peripheral blood and duodenum was significantly lower than in the enteritis group (P < 0.05), while TGF-β content was significantly increased compared to the enteritis group (P < 0.05). For the intervention group, the percentages of CD4+CD25+Foxp3+ Tregs in spleen and MLN were increased (P < 0.05), while the percentages of CD4+IL-17A+ Th17 cells were decreased compared to the enteritis group (P < 0.05). The expression of Foxp3 mRNA and protein in the intervention group was higher than in the enteritis group, while RORγt mRNA and protein were significantly lower (P < 0.05). After mice in the enteritis group were treated with L. casei, duodenal inflammation was relieved. This study demonstrated that L. casei could have possible implications for the enterotoxigenic Escherichia coli (ETEC) induced intestinal inflammation by regulating the ratio imbalance of Th17/Treg cells.

Inflammatory bowel disease affects density of nitrergic nerve fibers in the mucosal layer of the canine gastrointestinal tract

Andrzej Rychlik, Slawomir Gonkowski, Marcin Nowicki, Jaroslaw Calka (page 129)

The objective of this study was to determine the effect of inflammatory bowel disease (IBD) on the density of nitrergic nerve fibers in the mucosal layer of different sections of the gastrointestinal tract of dogs. Twenty-eight German shepherd hybrid dogs of both sexes, weighing from 15 to 25 kg and aged 6 to 10 y, were studied. The dogs were divided into 4 groups with 7 animals in each group: healthy animals, as well as dogs suffering from mild, moderate, and severe IBD. Immunoreactivity to neuronal isoform of nitric oxide synthase, which is a marker of nitrergic neurons, in samples of the mucosal layer in the duodenum, jejunum, and descending colon was studied using the single immunofluorescence method and the number of nerve fibers was evaluated in each observation field. The obtained results showed that IBD causes an increase in the number of nitrergic nerve fibers in all intestinal segments studied and these changes are directly proportional to the intensity of the disease process. These observations may be useful in diagnostic evaluation of the stage of canine inflammatory bowel disease in veterinary practice. The pathological mechanisms of these observed changes and the specific reasons for them are still not completely explained, however, and further study is required.

Assessment of regional left ventricular systolic function by strain imaging echocardiography in phenotypically normal and abnormal Maine coon cats tested for the A31P mutation in the MYBPC3 gene

Arine Pellegrino, Alexandre G.T. Daniel, Guilherme G. Pereira, Paula H. Itikawa, Maria Helena M.A. Larsson (page 137)

Myocardial dysfunction occurs in cats with hypertrophic cardiomyopathy (HCM), but little is known about the early stages of the disease. Strain imaging echocardiography is a method that enables the quantitative assessment of myocardial function and deformity, allowing the characterization of systolic dysfunction. The objective of this study was to assess systolic function using strain imaging echocardiography in Maine coon cats genetically tested for the A31P mutation in the MYBPC3 gene, with and without ventricular hypertrophy. For this purpose, 57 Maine coon cats of both genders, with an unknown status regarding the mutation at inclusion, were included prospectively and evaluated by conventional and strain imaging echocardiography. Comparisons were made among cats without hypertrophy (n = 45), suspect cats (n = 7), and cats with hypertrophic cardiomyopathy (n = 5), and also between the heterozygous for the mutation group (n = 26) and the negative for the mutation group (n = 28). Finally, in the group of phenotypically normal cats, heterozygous cats carrying the mutation were compared to cats without the mutation. Strain values were compared among the groups (blinded prospective study). While echocardiography demonstrated normal contractility, strain values (middle of the septum) were lower in HCM cats. Strain values (base of anterior wall of the left ventricle) were lower in heterozygous than in negative cats, even before hypertrophy. Negative correlation was observed between some values of myocardial strain and thickness. While strain imaging echocardiography was able to detect systolic abnormalities, despite apparent normality on conventional echocardiography, it was not able to identify cats that carry the A31P mutation in the MYBPC3 gene. Strain imaging echocardiography could be a useful tool, however, for detecting systolic alterations in HCM cats with an apparently normal systolic function or for detecting alterations in normal carriers of the MYBPC3 gene mutation.

Prokaryotic expression of the extracellular domain of porcine programmed death 1 (PD-1) and its ligand PD-L1 and identification of the binding with peripheral blood mononuclear cells in vitro

Yan-Ping Zhu, Feng Yue, Yong He, Peng Li, Yuan Yang, Yu-Ting Han, Yan-Fang Zhang, Guo-Peng Sun, Dong-Guang Guo, Mei Yin, Xuan-Nian Wang (page 147)

Programmed cell death protein 1 (PD-1), a costimulatory molecule of the CD28 family, has 2 ligands, PD-L1 and PD-L2. Our previous studies showed that the expression of PD-1 and PD-L1 is up-regulated during viral infection in pigs. Extensive studies have shown that blockade of the PD-1/PD-L1 pathways by anti-PD-L1 antibody or soluble PD-1 restores exhausted T-cells in humans and mice. In the present study the extracellular domains of PD-1 and PD-L1 were used to evaluate the binding of PD-1 and PD-L1 with peripheral blood mononuclear cells (PBMCs). We amplified the cDNA encoding the extracellular domains of PD-1 and PD-L1 to construct recombinant expression plasmids and obtain soluble recombinant proteins, which were then labeled with fluorescein isothiocyanate (FITC). The His-ExPD-1 and His-ExPD-L1 recombinant proteins were expressed in the form of inclusion bodies with a relative molecular weight of 33.0 and 45.0 kDa, respectively. We then prepared polyclonal antibodies against the proteins with a multi-antiserum titer of 1:102 400. Binding of the proteins with PBMCs was evaluated by flow cytometry. The fluorescence signals of His-ExPD-1-FITC and His-ExPD-L1-FITC were greater than those for the FITC control. These results suggest that the soluble recombinant proteins may be used to prepare monoclonal antibodies to block the PD-1/PD-L1 pathway.

Brief Communications

Prevalence of small ruminant lentivirus and Mycobacterium avium subsp. paratuberculosis co-infection in Ontario dairy sheep and dairy goats

Nancy Stonos, Cathy Bauman, Paula Menzies, Sarah K. Wootton, Niel A. Karrow (page 155)

Infection with small ruminant lentiviruses (SRLV) causes a variety of chronic inflammatory conditions that limit production. Mycobacterium avium subsp. paratuberculosis (MAP) is also a major production-limiting disease of sheep and goats, which causes severe inflammation of the small intestine. Previous studies have indicated that both SRLV and MAP are widespread in small ruminants in Ontario. This study estimated the prevalence of SRLV and MAP co-infection. Serum samples that were previously tested for MAP infection were re-tested for SRLV. The apparent prevalence of co-infection was low, with 3.4% [95% confidence interval (CI): 1.9 to 5.9] and 14.3% (95% CI: 11.6 to 17.5) of sheep and goats respectively, positive for both infections. However, co-infection is widespread with 36.8% (95% CI: 19.1 to 59.1) and 71.4% (95% CI: 52.8 to 84.9) of sheep and goat farms with 1 or more co-infected animals. A significant association was found between SRLV seropositivity and MAP fecal culture (P = 0.021), suggesting that co-infected goats may be more likely to shed MAP in their feces.