Glanders and the risk for its introduction through the international movement of horses

Glanders is the contagious zoonotic disease caused by infection with Burkholderia mallei. It affects primarily horses, donkeys and mules. The disease was eradicated from large areas of the Western world in the early 20th century, but, over the last 10–20 years, has emerged and re‐emerged in areas in which it was previously unknown or had been eradicated. Although glanders was previously thought to manifest in only acute or chronic presentations, it now appears that B. mallei can produce latent infections similar to those caused by Burkholderia pseudomallei. These latent infections may or may not be detectable by current diagnostic tests. The diagnostic test currently recommended by the World Organisation for Animal Health (Office International des Epizooties [OIE]) for international trade in equids is the complement fixation test (CFT). This test has been shown to have varying sensitivities and specificities depending on the antigen and methodology used. False positives are problematic for the horse‐owner and veterinary authority, whereas false negatives may allow the reintroduction of B. mallei into B. mallei‐free areas. These gaps in knowledge of the epidemiology of glanders, and weaknesses in its diagnosis, coupled with the increased movement of equids, indicate that infection with B. mallei remains a major risk in the context of international movement of equids.     

EVALUATION OF QUANTITATIVE THYROID SCINTIGRAPHY FOR DIAGNOSIS AND STAGING OF DISEASE SEVERITY IN CATS WITH HYPERTHYROIDISM: COMPARISON OF THE PERCENT THYROIDAL UPTAKE OF PERTECHNETATE TO THYROID‐TO‐SALIVARY RATIO AND THYROID‐TO‐BACKGROUND RATIOS

Thyroid scintigraphy is commonly used for evaluation of cats with hyperthyroidism, with the thyroid‐to‐salivary ratio (T/S) being the most common method to quantify the degree of thyroid activity and disease. Calculation of thyroid‐to‐background ratios (T/B) or percent thyroidal uptake of ^99mTcO^?₄ (TcTU) has only been reported in a few studies. The purpose of this prospective, cross‐sectional study was to evaluate a number of quantitative scintigraphic indices as diagnostic tests for hyperthyroidism, including the T/S, three different T/B, TcTU, and estimated thyroid volume. Of 524 cats referred to our clinic for evaluation of suspected hyperthyroidism, the diagnosis was confirmed (n = 504) or excluded (n = 20) based on results of a serum thyroid panel consisting of thyroxine (T₄), triiodothyronine (T₃), free T₄ (fT₄), and thyroid‐stimulating hormone (TSH) concentrations. In the hyperthyroid cats, median values for TcTU, T/S, and three T/B ratios were all significantly higher (P < 0.001) than values in euthyroid suspect cats or clinically normal cats. All scintigraphic parameters were relatively sensitive and specific as diagnostic tests for hyperthyroidism, but the T/S ratio had the highest test accuracy. The T/S ratio correlated strongly with the TcTU (r = 0.85). However, the TcTU had a higher and more significant correlation (P < 0.01) with serum T₄ (r = 0.76 vs. 0.64), T₃ (r = 0.77 vs. 0.64), and estimated thyroid volume (r = 0.62 vs. 0.38). Overall, calculation of TcTU is an accurate diagnostic test, but also appears to be the best parameter to predict the functional volume and metabolic activity of the feline adenomatous thyroid gland.     

Clinical Evaluation of a Peptide‐ELISA based upon N‐terminal B‐cell Epitope of the VapA Protein for Diagnosis of Rhodococcus equi Pneumonia in Foals

A total of 227 field samples from naturally exposed foals aged between 3 weeks and 6 months were used in an evaluation of a peptide‐based enzyme‐linked immunosorbent assay (ELISA) for diagnosis of Rhodococcus equi infection. A biotinylated peptide derived from the virulence‐associated protein A (VapA) of R. equi, a horse pathogen, was synthesized and designated as PN11‐14. The peptide corresponds to the N‐terminal B‐cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein. Based upon a serum immunoglobulin (Ig)G titre of 512 as a positive cut‐off value for the R. equi infection, the ELISA provided the overall sensitivity of 47.62%, specificity of 69.67% and an accuracy of 59.47% with a positive predictive value of 57.47% for true R. equi pneumonia. The assay was improved by detecting VapA‐specific IgGb antibodies against N‐terminal B‐cell epitope of the VapA protein rather than IgG antibodies. The VapA‐IgGb ELISA showed the overall sensitivity of 70.47%, specificity of 72.13% and accuracy of 71.36% with a positive predictive value of 68.52%. Diagnosis of R. equi disease in 6‐week‐old foals showed that the VapA‐IgGb ELISA provided an increasing trend (P = 0.0572) in sensitivity of 82.4% in comparison with the VapA‐IgG ELISA which showed the sensitivity of 58.8%. However, differences in specificity of both tests were statistically insignificant (P = 0.357) as analysed by the McNemar test. These results indicated that detection of VapA‐specific IgGb antibodies may be a better predictor of R. equi disease in foals.     

Development of a simple IgE‐independent anaphylactic model using buckwheat antigen and B‐type CpG oligodeoxynucleotide from Streptococcus thermophilus

We developed a severe anaphylactic model in mice using buckwheat antigen and B‐type CpG‐oligodeoxynucleotides (CpG‐ODNs) from Streptococcus thermophilus genome. In typical systemic anaphylaxis models, animals are challenged with large quantity of antigens via an intravenous (i.v.) route. Here, we showed a simple anaphylactic shock after challenge via intraperitoneal (i.p.) route. The i.p. method is simpler than i.v. administration and has a lower risk for failure. To generate this anaphylactic model, 5‐week‐old female BALB/c mice were first i.p. sensitized with buckwheat antigen mixed with B‐type CpG‐ODN. After 2 weeks, mice were challenged with antigen to induce anaphylactic shock, which was evaluated by scoring the severity symptoms and measuring serum levels of various proteins and splenic cell producing cytokines. Immunoglobulin (Ig)G_2a production and interferon‐γ positive cells were markedly increased in mice immunized with antigen mixed with B‐type CpG‐ODN, whereas serum IgE levels were decreased by B‐type CpG‐ODN. We also examined the effects of various ODNs (A, B and C‐type CpG‐ODNs) and antigens (buckwheat, α‐casein, β‐lactoglobulin and ovalbumin) on anaphylactic severity, and found that the combination of buckwheat and B‐type CpG‐ODN induced the most intense anaphylactic shock. This model is expected to contribute to the study of the prevention of anaphylactic shock.     

Prognostic significance of hypermethylation of death‐associated protein kinase (DAPK) gene CpG island in dogs with high‐grade B‐cell lymphoma

Death‐associated protein kinase (DAPK) is a serine/threonine kinase and a tumour suppressor gene. Diffuse large B‐cell lymphomas with inactivated DAPK through hypermethylation of a CpG island is known to result in a biologically aggressive phenotype in humans. This retrospective study was carried out to analyse the prognostic significance of DAPK CpG island hypermethylation in canine lymphoma. We hypothesized that DAPK CpG island hypermethylation can be a negative prognostic indicator in dogs with nodal high‐grade B‐cell lymphoma. Forty‐seven dogs with high‐grade B‐cell lymphoma, according to the updated Kiel classification, were evaluated after being treated with a CHOP (vincristine, cyclophosphamide, doxorubicin and prednisolone)‐based chemotherapy protocol. The methylation status of the DAPK CpG island was examined by methylation‐specific PCR. Progression‐free survival (PFS) and overall survival (OS) were compared using the Kaplan‐Meier analysis and log‐rank test. The cox proportional hazard regression model was used to evaluate the effect of multiple variables. Hypermethylation of the DAPK CpG island was detected in 21 of the 47 dogs. The PFS and OS in dogs with the hypermethylation (median: 220 and 266 days, respectively) were significantly shorter than those of dogs without hypermethylation (median: 301 and 412 days, respectively) (PFS, P = .036; OS, P = .007). In the multivariate analysis, hypermethylation of the DAPK CpG island remained an independent prognostic factor in predicting shortened PFS (P = .047) and OS (P = .021) as well as clinical substage b. Overall, hypermethylation of the DAPK CpG island was a negative prognostic factor in canine high‐grade B‐cell lymphoma.     

Human‐mediated Foot‐and‐mouth Disease Epidemic Dispersal: Disease and Vector Clusters

Disease clusters were retrospectively explored at national level using a geo‐referenced dataset from the 2001 Uruguayan Foot‐and‐Mouth Disease (FMD) epidemic. Disease location and time (first 11 epidemic weeks) were analysed across 250 counties (of which 160 were infected), without and with control for human mobility related factors (human population and road densities). The null hypothesis of random disease distribution over space and/or time was assessed with: (i) purely temporal; (ii) purely spatial; and (iii) space/time tests. At least within epidemic weeks 2 and 6, a principal disease cluster was observed in 33 contiguous counties (P < 0.01). Two secondary clusters, located at >100 km from each other, were also observed (P < 0.01). The purely spatial test that controlled for human population density identified two non‐contiguous clusters (P < 0.01). Space and time analysis also revealed the same 33 counties as members of the principal cluster, of which 31 were also clustered when human population was controlled (P < 0.01). No clusters were reported by the spatial test when road density was assessed. The hypothesis that human mobility related factors autocorrelate with disease was empirically supported by two pieces of information: (i) removal of human population/road densities eliminated >93.9% of the counties included in the principal disease cluster; and (ii) statistically significant correlations (P < 0.05) were observed in the first three epidemic weeks between road density and the number of cases. Clusters where human population density was associated with 47% greater number of cases/sq. km than that of the principal cluster indicated possible roles as disease vectors (vector clusters). Selective control policy in vector clusters is recommended. Periodic (i.e. weekly) cluster and correlation analyses of both disease and other covariates may facilitate disease surveillance and help design space‐specific control policy.     

β‐carotene attenuates lipopolysaccharide‐induced inflammation via inhibition of the NF‐κB,JAK2/STAT3 and JNK/p38 MAPK signaling pathways in macrophages

β‐carotene is one of the most abundant carotenoids, has potential anti‐inflammatory effect, it has been reported that β‐carotene could suppress LPS‐induced inflammatory responses by inhibiting nuclear factor kappa B (NF‐κB) translocation, but the more detailed molecular mechanisms underlying the anti‐inflammatory action of β‐carotene remain to be fully understood. In this study, we investigated the influence of β‐carotene on the activation of JAK2/STAT3, MAPK, and NF‐κB signaling pathway induced by LPS in RAW264.7 cells and peritoneal macrophages. Cells were treated with different concentrations of β‐carotene for 3 hr after LPS treatment for 24 hr. The mRNA expression and the release of IL‐1β, IL‐6, and TNF‐α were evaluated by RT‐PCR and ELISA, and the level of signaling proteins of JAK2/STAT3, MAPK, and NF‐κB signaling pathway were detected by Western blot. The results showed that β‐carotene significantly suppressed (p < 0.05) LPS‐induced release of IL‐1β, IL‐6, and TNF‐α and their mRNA expression. LPS‐induced JAK2/STAT3, IκB/NF‐κB p65, JNK/p38 MAPK signal activation were significantly attenuated (p < 0.05) by β‐carotene in a dose‐dependent manner. In conclusion, β‐carotene could attenuate LPS‐induced inflammation via inhibition of the NF‐κB, JAK2/STAT3, and JNK/p38 MAPK signaling pathways in macrophages.     

Application of single‐step genomic best linear unbiased prediction with a multiple‐lactation random regression test‐day model for Japanese Holsteins

This study aimed to evaluate a validation reliability of single‐step genomic best linear unbiased prediction (ssGBLUP) with a multiple‐lactation random regression test‐day model and investigate an effect of adding genotyped cows on the reliability. Two data sets for test‐day records from the first three lactations were used: full data from February 1975 to December 2015 (60 850 534 records from 2 853 810 cows) and reduced data cut off in 2011 (53 091 066 records from 2 502 307 cows). We used marker genotypes of 4480 bulls and 608 cows. Genomic enhanced breeding values (GEBV) of 305‐day milk yield in all the lactations were estimated for at least 535 young bulls using two marker data sets: bull genotypes only and both bulls and cows genotypes. The realized reliability (R²) from linear regression analysis was used as an indicator of validation reliability. Using only genotyped bulls, R² was ranged from 0.41 to 0.46 and it was always higher than parent averages. The very similar R² were observed when genotyped cows were added. An application of ssGBLUP to a multiple‐lactation random regression model is feasible and adding a limited number of genotyped cows has no significant effect on reliability of GEBV for genotyped bulls.     

Serodiagnosis of Burkholderia mallei infections in horses: state-of-the-art and perspectives

Burkholderia mallei causes glanders or farcy in solipeds, a disease that must be reported to the OIE (Office International des Epizooties, Paris, France). The number of reported outbreaks has increased steadily during the last decade. Serodiagnosis is hampered by the considerable number of false-positives and -negatives of the internationally prescribed tests. The major problem leading to low sensitivity and specificity of complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e. crude preparations of whole cells. Future perspectives for the development and evaluation of serological test kits using well-characterized single antigens are discussed in the light of recent molecular research on B. mallei and the closely related saprozoonotic agent B. pseudomallei.     

Recognition of a B‐cell Epitope of the VapA Protein of Rhodococcus equi in Newborn and Experimentally Infected Foals

The aim of this study was to evaluate the usefulness of the previously identified B‐cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein of Rhodococcus equi and its association with R. equi pneumonia. A modified peptide designated PN11‐14 corresponding to the epitope was recognized by all sera from experimentally infected foals with virulent R. equi ATCC103⁺ containing the virulence plasmid but not by its plasmid‐cured derivative ATCC103^? strain. Marked levels of VapA‐specific immunoglobulin (Ig)G were detected in all sera from the ATCC103⁺ infected foals at 2 weeks after the infection. One control animal had high titres as determined by the peptide enzyme‐linked immunosorbent assay (ELISA), indicating the ELISA may not absolutely differentiate between foals with R. equi pneumonia and healthy exposed foals in farms where the prevalence of disease is high. However, numbers of animals used were small. Further evaluation of the peptide ELISA with field samples is necessary to determine whether the assay is diagnostically useful. This study showed that levels of passive transfer of maternal IgG antibodies to the epitope in newborn foals could be measured. Interestingly, the maternally derived antibodies were found to significantly (P < 0.05 by Student's t‐test) decline 2 weeks after birth. Seroconversion against naturally occurring VapA expressing R. equi could be detected in some foals at 4 weeks of age. Antibodies to the epitope peaked and were significantly (P < 0.05) greater in foals aged between 6 and 8 weeks. These results indicated that the peptide ELISA could be used to monitor anti‐VapA antibodies in foals, particularly those at the age of 4–6 weeks. It is possible that the ELISA may be of some use as a diagnostic test on farms where R. equi is non‐endemic. Further studies using large number of field samples are needed to verify this assumption.     

Anti‐microbial Susceptibility of Streptococcus spp. Isolated from Bovine Mastitis in Argentina

The in vitro susceptibility to penicillin G, erythromycin and clindamycin was determined by the disc diffusion test and by E‐test for a total of 47 streptococcal strains (three Streptococcus uberis, 36 Streptococcus agalactiae, eight Streptococcus dysgalactiae spp. dysgalactiae) isolated from bovine intramammary infections in Argentina. Moreover, resistance phenotypes of erythromycin‐resistant streptococcal isolates was characterized. MIC₉₀ of penicillin G, erythromycin and clindamycin for S. agalactiae were 0.75, 8.0 and 12.0 μg/ml respectively. Resistance to erythromycin and clindamycin was detected in 13 (27.6%) and 12 (25.5%) isolates respectively. No isolate was resistant to penicillin G. Resistance against macrolides, lincosamides and streptogramin B (MLS_B) represented by the constitutive MLS_B phenotype was present in 11 (23.4%) erythromycin‐resistant isolates and two isolates (4.3%) expressed the M phenotype. The inducible MLS_B phenotype was not identified. Results suggest that beta‐lactams are the first‐line antibiotics when treating streptococcal udder infections; however, the continuous monitoring of the antibiotic resistance is essential, as the emergence of resistant strains has become a growing concern on the therapy of bovine mastitis.     

Meta‐analysis of the prevalence of thermotolerant Campylobacter in food‐producing animals worldwide

The objective of this meta‐analysis was to summarize available information on the prevalence of thermotolerant Campylobacter (TC) in different food‐producing animals worldwide. Databases (i.e., PubMed, ScienceDirect, Scopus) were searched from 1980 to 2017 unrestricted by language. The inclusion criteria were as follows: prevalence or incidence studies, published in peer‐reviewed journals, and they must have reported the total number of animal samples studied and the number of samples that were positive for the presence of TC. When the identification of Campylobacter species was available, this information was included in the analysis. Multilevel random‐effect meta‐analysis models were fitted to estimate mean occurrence rate of TC and to compare them among different factors potentially associated with the outcome. The mean occurrence rate of TC in food‐producing animals was 0.424 (95% CI: 0.394–0.455), and the mean occurrence rate of Campylobacter jejuni and Campylobacter coli were 0.214 and 0.133, respectively. Pigs and poultry showed the highest prevalence of TC; however, there were differences in the prevalence of each Campylobacter species. Campylobacter jejuni was observed in broilers (0.322; 95% CI: 0.273–0.377) and hens (0.395; 95% CI: 0.265–0.542), while C. coli was restricted essentially in pigs (0.553; 95% CI: 0.541–0.650). The prevalence of C. jejuni in intensively bred cattle was higher (0.302; 95% CI: 0.227–0.389) than the prevalence in extensively bred cattle (0.172; 95% CI: 0.119–0.242) while the prevalence of C. coli was similar (0.051; 95% CI: 0.028–0.091 vs. 0.050; 95% CI: 0.027–0.091) in both production systems. Agar with or without blood used for the isolation of TC did not affect the prevalence observed. The method of species identification did not seem to generate differences in the prevalence of Campylobacter species. The prevalence of Campylobacter in primary food production has a strong impact on the entire agri‐food chain. National authorities must monitor the situation with the aim to establish the appropriate risk management measures.     

Immunogenicity of Murein‐associated Proteins from Temperature‐stressed Streptococcus suis Cultures

We compared immunogenicity in pigs of whole cell lysate proteins (WCP) with murein‐associated proteins (MAP) obtained from a virulent serotype 2 strain of Streptococcus (S.) suis grown at 32 or 42°C. Protein fractions were tested for their ability to induce antibodies in 3‐week‐old piglets by enzyme‐linked immunosorbent assay and Western blot analysis. We found a significant increase in the antibody levels in all sera irrespective of the preparation used for immunization. However, α‐WCP sera showed higher reactivities than α‐MAP sera, and piglets immunized with 32°C preparations (α‐32 sera) showed higher responses than those immunized with 42°C preparations (α‐42 sera). Western blot analysis revealed that α‐WCP sera in part reacted with different proteins when compared with α‐MAP sera. Furthermore, some proteins were only detected by α‐32 but not by α‐42 sera. In conclusion, the results demonstrate the immunogenicity of cell wall MAP of S. suis, and highlight the importance of considering growth conditions in the preparation of subunit vaccines.     

FC‐30 Presence of dust mites in the environment of dust mite‐sensitized atopic dogs

Dust mites (DM) are the most common offending aeroallergens in atopic dogs. The aim of this study was to compare the DM load of households with atopic dogs (Group A, n = 8) that had positive intradermal test reactions to Dermatophagoides farinae, D. pteronyssinus, Acarus siro, Lepidoglyphus destructor and/or Tyrophagus putrescentiae to the DM load of households with nonatopic dogs (Group B, n = 4) and of nonpet households (Group C, n = 8). Group A dogs presented with perennial pruritus, were free of pathogenic mites and fleas, did not respond to an elimination diet, and fulfilled the diagnostic criteria of atopic dermatitis. All Group B dogs tested intradermally negative and had no dermatological problems. Dust samples were vacuum collected in a standardized fashion from the human (all groups) and dog mattresses (Groups A and B) or from the couch (Group C) four times, once for each season of the year. The presence of DM was assessed with a commercial test (Acarex test) and stereoscopically. At least one DM was found in all Group A houses. The DM load was not significantly different between the seasons or the three animal groups. The sensitivity of the Acarex test was significantly lower than that of stereoscopic examination (P < 0.001). In conclusion, the environmental DM load was similar between atopic and nonatopic dogs, the presence of dogs in a household didn't increase DM numbers, and stereoscopy was more sensitive than the Acarex test for the detection of DM. Funding: Self‐funded.     

Inhibitory effect of heat‐killed Lactobacillus strain on immunoglobulin E‐mediated degranulation and late‐phase immune reactions of mouse bone marrow‐derived mast cells

This study investigated the in vitro effect of Lactobacillus strains, a major group of probiotic lactic acid bacteria, on immunoglobulin E (IgE)‐ and antigen‐induced mast cell degranulation and subsequent gene expression. Bone marrow‐derived mast cells (BMMCs) from DBA/2 mice were cultured with heat‐killed Lactobacillus strains for 24 h. Some strains significantly inhibited IgE‐ and antigen‐induced β‐hexosaminidase release from BMMCs. Furthermore, Lactobacillus reuteri NBRC 15892, which exhibited the strongest inhibitory activity, significantly reduced the elevated interleukin (IL)‐4, IL‐13, tumor necrosis factor‐α, and cyclooxygenase‐2 expression levels that was induced by 1–2 h of stimulation with IgE and antigens. The suppressive effect of NBRC 15892 strain on BMMC degranulation was significantly reduced in the presence of a toll‐like receptor (TLR)2‐neutralizing antibody. In addition, downregulation of cell surface FcεRIα expression was observed after 6 h of NBRC 15892 treatment. These results suggest that some Lactobacillus strains inhibited IgE‐mediated mast cell degranulation and subsequent late‐phase reactions involving mast cells via a TLR2‐dependent mechanism with FcεRIα downregulation.     

Questionnaire‐based Analysis of Owner‐reported Scratching and Pain Signs in Cavalier King Charles Spaniels Screened for Chiari‐like Malformation and Syringomyelia

Background

Chiari‐like malformation (CM) and syringomyelia (SM) cause a pain syndrome in Cavalier King Charles spaniels (CKCS). Clinical signs are not consistently apparent on neurologic examination, and owner reporting of signs provides vital clinical history. However, owner questionnaires for this disease are not well developed.

Objectives

To develop a tool to capture owner‐reported clinical signs for use in clinical trials and to compare owner‐reported signs with the presence of pain on neurologic examination and SM on magnetic resonance imaging (MRI).

Animals

Fifty client‐owned CKCS.

Methods

Owners completed a questionnaire and pain/scratch map. Each dog underwent a neurologic examination and craniocervical magnetic resonance imaging (MRI). Questionnaire responses were developed into scores, area of shading for pain/scratch maps was measured, and consistency of responses between these tools was assessed. Owner‐reported findings were compared with neurologic examination findings and presence and severity of SM on MRI.

Results

Thirty‐three dogs were symptomatic and 17 asymptomatic; 30 had SM. The most common sign of pain was crying out when lifted (n = 11). Extent of shaded areas on maps positively correlated with questionnaire scores for pain (r² = 0.213, P = 0.006) and scratch (r² = 0.104, P = 0.089). Owner‐reported findings were not significantly associated with presence or severity of SM or neurologic examination findings. Owner‐reported lateralization of signs was significantly associated with SM lateralization (P < 0.0001).

Conclusions

The questionnaire and maps may be useful for clinical trials. Lack of association of owner‐reported signs with SM highlights our lack of understanding of the pathophysiology of pain in this disease.     

Are B‐symptoms more reliable prognostic indicators than substage in canine nodal diffuse large B‐cell lymphoma

In humans B‐symptoms refer to systemic symptoms of lymphoma such as fever, weight loss, and night sweats and influence the prognosis of patients. In canine lymphoma, substage B is used to describe any clinical sign observed. Aim of the retrospective study was to compare the prognostic value of substage B with B‐symptoms to predict treatment response and survival in canine nodal diffuse large B‐cell lymphoma. Affected dogs treated with CHOP chemotherapy between 2008 and 2019 were included. B‐symptoms were defined by weight loss greater than 10% of normal weight, fever and the occurrence of unexplained resting tachypnoea substituted human night sweats. Substage B was defined as any symptoms but lymphadenopathy. Fifty‐five cases were included. B‐symptoms were present in 20/55 (36%) and substage B in 40/55 (74%) patients. No significant associations between B‐symptoms or substage B and weight, sex, breed, WHO stage and lymphoma grade were found. Treatment response was negatively associated with both substage B (P = .02) and B‐symptoms (P = .001). B‐symptoms significantly decreased progression free survival (PFS) (95 vs 330 days, P = .001) and lymphoma specific survival (LSS) (160 vs 462 days, P = .001). Data showed that B‐symptoms might be a more reliable prognostic indicator than substage B in canine nodal diffuse large B‐cell lymphoma. Prospective studies assessing B‐symptoms in a larger cohort of patients and in other common lymphoma types are warranted. The abstract was presented at the fourth meeting of the European Canine Lymphoma Network Group in Lugano, 22 June 2019 and published in the proceeding of the meeting on the page 26.     

The Occurrence of Pathogenic Listeria monocytogenes and Antibodies against Listeriolysin‐O in Buffaloes

The occurrence of Listeria monocytogenes in meat and milk samples, and antilisteriolysin O (ALLO) antibodies in sera of buffaloes were studied. Isolation of the pathogen was attempted from the samples by selective enrichment in University of Vermont Medium and plating onto Dominguez–Rodriguez isolation agar. The pathogenicity of the isolates was tested by Christie, Atkins, Munch Petersen test and mouse inoculation test. Of 167 meat samples 2.4 and 10.17% were positive for L. monocytogenes and Listeria sp., respectively. Of the 64 milk samples 6.25 and 26.13% were positive for L. monocytogenes and Listeria sp., respectively. A total of 284 serum samples were tested by listeriolysin O (LLO)‐based indirect enzyme‐linked immunosorbent assay of which 25.35% were found to be seropositive. The culture positivity for L. monocytogenes and detection of ALLO did not show any agreement (κ=0.035). The prevalence of pathogenic L.monocytogenes in milk and meat and the occurrence of anti‐LLO antibodies is of concern from the public health point of view.     

Variance Components of an Enzyme‐linked Immunosorbent Assay for Detection of IgG Antibodies in Milk Samples to Mycobacterium avium subspecies paratuberculosis in Dairy Cattle

Milk samples from 120 cows were tested up to 10 times in an enzyme‐linked immunosorbent assay (ELISA) for detection of antibodies to Mycobacterium avium ssp. paratuberculosis. The purpose of the study was to estimate variance components of the assay attributable to laboratory factors using mixed model theory. Because of significant interaction between the between‐run, between‐day and between‐plate variables, the ELISA‐plate variable was nested in run‐number and run‐number was nested in day‐number. The nested variable accounted for 68% of the laboratory variability (P<0.001), whereas the intra‐plate variability accounted for only 0.04% of the laboratory variability (P>0.9). Therefore, it was concluded that the intra‐plate variability could be ignored whereas the variability from the combined run‐day‐plate variable should be considered in any analyses based on the ELISA.