Comparison of 2 PCR assays on environmental samples cultured for Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic, contagious, and incurable enteric disease of ruminants. An in-house IS900 PCR assay validated for MAP detection in sheep has been shown to have a higher sensitivity than a commercial PCR and fecal culture. We have now compared the performance of this in-house IS900 PCR assay with a commercial ISMap02 PCR assay for the detection of MAP DNA in bovine dairy farm environmental samples. We purposefully selected 30 culture-positive, 62 culture-negative, and 62 non-interpretable environmental samples. We applied the IS900 PCR assay directly to the frozen inoculum of these samples. Inocula were incubated in an automated system, and growth was confirmed by an acid-fast bacilli stain and the IS900 PCR assay. Among culture-positive samples before incubation, the IS900 PCR assay yielded significantly more positive results than the ISMap02 PCR assay; however, among culture-negative samples, the IS900 PCR assay yielded positive results both before and after incubation. The ISMap02 PCR assay did not flag positively among the culture-negative samples either before or after incubation. The IS900 PCR assay is a sensitive method that can be used to detect MAP DNA in environmental samples before incubation. The ISMap02 PCR assay is a specific method used to detect MAP DNA in environmental samples both before and after incubation.

Role of CCR3 in respiratory syncytial virus infection of airway epithelial cells.

Respiratory syncytial virus (RSV) infection is the principal cause of severe lower respiratory tract disease and accounts for a significant risk for developing asthma later in life. Clinical studies have shown an increase in airway responsiveness and a concomitant Th2 response in the lungs of RSV-infected patients. These indications suggest that RSV may modulate aspects of the immune response to promote virus replication. Here, we show that CCR3 facilitates RSV infection of airway epithelial cells, an effect that was inhibited by eotaxin-1/CCL11 or upon CCR3 gene silencing. Mechanistically, cellular entry of RSV is mediated by binding of the viral G protein to CCR3 and selective chemotaxis of Th2 cells and eosinophils. In vivo, mice lacking CCR3 display a significant reduction in RSV infection, airway inflammation, and mucus production. Overall, RSV G protein-CCR3 interaction may participate in pulmonary infection and inflammation by enhancing eosinophils' recruitment and less potent antiviral Th2 cells.

Bayesian accuracy estimates of environmental sampling for determining herd paratuberculosis infection status and its association with the within-herd individual fecal culture prevalence in Québec dairies.

The objectives of this retrospective analysis were to: 1) estimate the diagnostic sensitivity (Se) and specificity (Sp) of bacterial culture of environmental samples for determining Mycobacterium avium subsp. paratuberculosis (MAP) infection status in Québec dairies, using a Bayesian Latent Class Model (BLCM); and 2) determine the association between the number of positive environmental samples and the individual fecal culture (IFC) apparent and true MAP within-herd prevalence. Environmental and individual fecal samples were collected from 87 commercial dairy herds participating in previous research projects. Environmental samples included two composite samples of 20 g collected from different locations within each of the following sites: an area where manure from the majority of adult cattle accumulates, a manure storage area and another site of manure accumulation chosen by the veterinarian. Samples were cultured using the MGIT Para TB culture liquid media and the BACTEC MGIT 960 system. The Se and Sp of environmental sampling were estimated using a one-test-one-population BLCM. Herds were considered positive for environmental sampling if at least one out of the six samples collected was positive. The apparent and true IFC within-herd MAP prevalence estimates for each herd were obtained using a two-stage cluster BLCM, then merged in a single dataset with the environmental sample results. The association between the within-herd MAP prevalence results (apparent and true), and the number of positive environmental samples was assessed using a zero-inflated negative binomial (ZINB) model. In all BLCMs, median posterior estimates and 95 % Bayesian credible intervals (BCI) were obtained with OpenBUGS statistical freeware. Se and Sp of environmental sampling were 43.7 % (95 % BCI: 32.5-55.5) and 96.2 % (95 % BCI: 84.2-99.8), respectively. Overall, the number of positive environmental samples increased with the apparent and true MAP within-herd prevalence. The true prevalence was higher than the apparent prevalence for a given number of positive environmental samples. The probability of not observing a positive environmental sample decreased with the prevalence. Despite its imperfect accuracy, environmental sampling is an inexpensive and non-invasive sampling method to determine MAP infection status in tie-stall herds that can be used as a proxy to estimate the true within-herd prevalence. The absence of positive environmental samples in a single sampling visit is likely an indicator of a very low within-herd prevalence rather than being MAP exempt.

Non-antimicrobial approaches at drying-off for treating and preventing intramammary infections in dairy cows. Part 1. Meta-analyses of efficacy of using an internal teat sealant without a concomitant antimicrobial treatment.

Use of antimicrobial approaches at drying-off for preventing new intramammary infections (IMI) during the dry period in dairy cows could be replaced by non-antimicrobial approaches. Such approaches would be of interest not only for organic but also for conventional dairy producers. The objective of the current review was to quantify the effect of non-antimicrobial internal teat sealant (ITS)-based approaches at drying-off for treating and preventing IMI, when compared with no treatment or with an antimicrobial-based approach. The protocol for this review was published before initiating the review. A total of 18 trials from 16 articles could be used to investigate the effect of an ITS-based approach. With the available results, we conclude with a high level of confidence that non-antimicrobial ITS-based dry-off approaches are efficient for preventing new IMI during the dry period when compared with no treatment, and would reduce risk of new IMI by 52%. Moreover, we are relatively confident that a bismuth subnitrate-based ITS performed better than an antimicrobial for preventing new IMI during the dry period (a risk reduction of 23%). Similarly, we are relatively confident that an ITS-based approach would only slightly or not at all reduce the prevalence of IMI at calving compared with untreated quarters.

Estimating diagnostic accuracy of fecal culture in liquid media for the detection of Mycobacterium avium subsp. paratuberculosis infections in Québec dairy cows: A latent class model.

A latent class model fit within a Bayesian framework was used to estimate the sensitivity and specificity of individual fecal culture (IFC) in liquid medium (Para TB culture liquid medium and BACTEC MGIT 960 system) for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in Québec dairy cows. As a secondary objective, the within-herd paratuberculosis prevalence was estimated. A dataset including 21 commercial Québec dairy herds participating in previous research projects was retrospectively analyzed. In total, 1386 adult cows on which both IFC and serum-ELISA were available were included. The selected latent class model assumed conditional dependence between the tests. Non-informative priors for IFC accuracy and paratuberculosis prevalence were used while informative priors, obtained from the literature, were used for serum-ELISA accuracy. The WinBUGS statistical freeware was used to obtain posterior estimates (medians and 95% Bayesian credibility intervals (95% BCI)) for each parameter. The sensitivity and specificity estimates for IFC were 34.4% (95% BCI: 20.3-66.1) and 99.5% (95% BCI: 98.6-100), respectively. Sensitivity and specificity for serum-ELISA were 27.3% (95% BCI: 18.1-38.3) and 97.4% (95% BCI: 96.6-98.0). Median paratuberculosis within herd prevalence was estimated to be 0.3% (0-3.3). In conclusion, a higher sensitivity of IFC compared to serum-ELISA was observed both in the unconditional and conditional dependent models. Since the sensitivity of both IFC and serum-ELISA was relatively low, conditional dependence between the tests is more likely in the true disease positive animals. We hypothesize that conditional dependence arises because an unmeasured covariate influences the performance of both tests among disease positive animals causing both tests to incorrectly misclassify the animal as negative. One limitation of this study was the very low within herd prevalence of the participant herds.

Risk factors associated with Mycobacterium avium subsp. paratuberculosis herd status in Québec dairy herds.

Paratuberculosis is a chronic and contagious enteric disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Control of paratuberculosis is justified given the associated economic losses and the potential role of MAP in Crohn's disease in humans. Management practices that limit exposure of susceptible animals to MAP are more effective at reducing disease prevalence than testing and culling infected cows. The objective of this retrospective case-control study was to study the association between management practices and MAP status in dairy herds in Québec, Canada. A total of 26 case herds (MAP had been isolated from at least 1 environmental sample in each herd) and 91 control herds (no clinical cases of paratuberculosis and negative on 2 consecutive yearly environmental samplings) were selected among herds enrolled in the Québec Voluntary Paratuberculosis Control Program. A risk assessment questionnaire, completed at enrolment, was available for the selected herds. Culture of MAP was achieved using liquid media and the BACTEC 960 detection system. Multivariable logistic regression was used to evaluate the association between selected risk factors and MAP herd status. Herd size (OR = 1.17; 95% CI: 1.02-1.33) and proportion of cows purchased per year in the last 5 years (OR = 5.44; 95% CI: 1.23-23.98) were significantly associated with a positive MAP herd status. The management risk factors identified in the present study are in accord with previous studies. Management practices aiming to prevent the introduction of new animals into the herd and to reduce the contact of newborn calves with adult animals or their feces are key elements to minimize MAP introduction and transmission into a herd. These elements should be prioritized in control programs.

Evaluation of a PCR assay on overgrown individual fecal samples cultured for Mycobacterium avium subsp. paratuberculosis.

Microbial overgrowth can interfere with Mycobacterium avium subsp. paratuberculosis (MAP) growth and detection. We estimated the percentage of positive samples by PCR performed on the incubated media of individual fecal samples classified as non-interpretable (NI) by bacteriologic culture of liquid media. A total of 262 liquid cultures declared NI and 88 samples declared negative were included in the study. MAP DNA was detected in 7 NI samples (2.7%; 95% CI: 1.1-5.4%) and in 1 negative sample (1.1%; 95% CI: 0.3-6.2%). The PCR allowed the detection of MAP-positive samples that had been missed in the initial bacteriologic culture. However, the benefit of these few additional positive results must be weighed against the additional costs incurred. Using PCR to classify overgrown cultures optimizes the detection process and eliminates the NI outcome.

Determining bovine viral diarrhea and infectious bovine rhinotracheitis infections in dairy cattle using precolostral blood.

The objective of this study was to determine if precolostral blood samples are useful to detect apparent fetal infections with bovine viral diarrhea (BVD) and infectious bovine rhinotracheitis (IBR) viruses. A convenience sample of 317 sera from 50 Canadian herds was used in the study. Antibody level was measured using 2 commercial IBR and BVD ELISA kits. Precolostral status of sera was confirmed on 304 samples using serum gamma-glutamyl transferase activity. Postcolostral serum samples yielded a higher proportion of positive results to IBR (OR = 86; 95% CI: 17.8 to 415.7) and BVD (OR = 199.3; 95% CI: 41.7 to 952.3) than did precolostral samples. All positive precolostral serum samples (n = 7 of 304) originated from calves born to vaccinated cows. Postcolostral positive serum samples (n = 11 of 13) originated mostly (60%) from calves born to non-vaccinated cows. Precolostral serum sampling can detect apparent fetal infections in a herd.

Utilisation du serum précolostral pour le dépistage de la diarrhée virale bovine (BVD) et rhinotracheite infectieuse bovine (IBR) dans les troupeaux laitiers. L'objectif de cette étude était d'évaluer l'utilité du prélèvement de sérum précolostral de nouveaux nés pour détecter des infections foetales apparentes par IBR et BVD dans un troupeau. Un échantillonnage de convenance de 317 sérums, prélevés de 50 troupeaux canadiens, a été utilisé. Les niveaux d'anticorps des sérums ont été mesurés en utilisant 2 trousses ELISA (IBR et BVD). Le statut précolostral a été confirmé pour 304 échantillons par la mesure de l'activité sérique des gamma glutamyl transférases. Une plus grande proportion de résultats positifs à IBR (RC = 86; IC 95%: 17,8 à 415,7) et BVD (RC = 199,3; IC 95 %: 41,7 à 952,3) a été observée parmi les échantillons postcolostraux que parmi les précolostraux. Tous les échantillons précolostraux positifs (n = 7/304) provenaient de veaux nés de mères vaccinées. Les échantillons postcolostraux positifs (n = 11/13) étaient majoritairement (60 %) prélevés à partir de veaux nés de mères non vaccinées. Le prélèvement de sérum précolostal peut détecter des infections foetales apparentes dans les troupeaux.(Traduit par les auteurs).

Evaluation of a PCR assay on overgrown environmental samples cultured for Mycobacterium avium subsp. paratuberculosis.

Culture of Mycobacterium avium subsp. paratuberculosis (MAP) is the definitive antemortem test method for paratuberculosis. Microbial overgrowth is a challenge for MAP culture, as it complicates, delays, and increases the cost of the process. Additionally, herd status determination is impeded when noninterpretable (NI) results are obtained. The performance of PCR is comparable to fecal culture, thus it may be a complementary detection tool to classify NI samples. Our study aimed to determine if MAP DNA can be identified by PCR performed on NI environmental samples and to evaluate the performance of PCR before and after the culture of these samples in liquid media. A total of 154 environmental samples (62 NI, 62 negative, and 30 positive) were analyzed by PCR before being incubated in an automated system. Growth was confirmed by acid-fast bacilli stain and then the same PCR method was again applied on incubated samples, regardless of culture and stain results. Change in MAP DNA after incubation was assessed by converting the PCR quantification cycle (Cq) values into fold change using the 2-ΔCq method (ΔCq = Cq after culture - Cq before culture). A total of 1.6% (standard error [SE] = 1.6) of the NI environmental samples had detectable MAP DNA. The PCR had a significantly better performance when applied after culture than before culture (p = 0.004). After culture, a 66-fold change (SE = 17.1) in MAP DNA was observed on average. Performing a PCR on NI samples improves MAP culturing. The PCR method used in our study is a reliable and consistent method to classify NI environmental samples.

Detection of Mycobacterium avium subspecies paratuberculosis in tie-stall dairy herds using a standardized environmental sampling technique and targeted pooled samples.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of Johne's disease, a chronic contagious enteritis of ruminants that causes major economic losses. Several studies, most involving large free-stall herds, have found environmental sampling to be a suitable method for detecting MAP-infected herds. In eastern Canada, where small tie-stall herds are predominant, certain conditions and management practices may influence the survival and transmission of MAP and recovery (isolation). Our objective was to estimate the performance of a standardized environmental and targeted pooled sampling technique for the detection of MAP-infected tie-stall dairy herds. Twenty-four farms (19 MAP-infected and 5 non-infected) were enrolled, but only 20 were visited twice in the same year, to collect 7 environmental samples and 2 pooled samples (sick cows and cows with poor body condition). Concurrent individual sampling of all adult cows in the herds was also carried out. Isolation of MAP was achieved using the MGIT Para TB culture media and the BACTEC 960 detection system. Overall, MAP was isolated in 7% of the environmental cultures. The sensitivity of the environmental culture was 44% [95% confidence interval (CI): 20% to 70%] when combining results from 2 different herd visits and 32% (95% CI: 13% to 57%) when results from only 1 random herd visit were used. The best sampling strategy was to combine samples from the manure pit, gutter, sick cows, and cows with poor body condition. The standardized environmental sampling technique and the targeted pooled samples presented in this study is an alternative sampling strategy to costly individual cultures for detecting MAP-infected tie-stall dairies. Repeated samplings may improve the detection of MAP-infected herds.

Mycobacterium avium ssp. paratuberculosis (MAP) est l'agent étiologique de la maladie de Johne, une entérite chronique contagieuse des ruminants et responsable d'importantes pertes économiques. Plusieurs études, la plupart réalisées dans des grands troupeaux en stabulation libre, ont démontré que la technique de culture de prélèvements de l'environnement est appropriée pour la détection des troupeaux infectés par MAP. Dans l'est du Canada où prédominent les petits troupeaux en stabulation entravée, certaines conditions et pratiques de régie pourraient avoir un impact sur la survie, la transmission et l'isolement de MAP. Notre objectif était d'estimer la performance d'une technique standardisée de culture de prélèvements de l'environnement combinée à des échantillons groupés ciblés pour la détection des troupeaux laitiers en stabulation entravée infectés par MAP. Vingt-quatre troupeaux (19 infectés et 5 non infectés) ont été enrôlés, mais seulement 20 troupeaux ont été visités 2 fois dans la même année pour y prélever 7 échantillons de l'environnement et 2 échantillons groupés (vaches malades et vaches maigres). Des échantillons individuels de toutes les vaches dans le troupeau ont été également prélevés. L'isolement de MAP a été réalisé en utilisant le milieu de culture MGIT ParaTB et le système de détection BACTEC 960. Globalement, MAP a été isolée dans 7 % des cultures de l'environnement. La sensibilité de la technique était de 44 % (IC 95 % : 20 % à 70 %) en combinant le résultat des 2 visites et de 32 % (IC 95 % : 13 % à 57 %) en utilisant aléatoirement le résultat d'une seule visite. La meilleure stratégie d'échantillonnage était la combinaison des échantillons de la fosse, de l'écureur, du groupe de vaches malades et du groupe de vaches maigres. La technique standardisée de prélèvements de l'environnement combinée aux échantillons groupés ciblés présentée dans cette étude est une alternative économique à la culture individuelle pour détecter des troupeaux laitiers infectés par MAP. La répétition des prélèvements pourrait contribuer à améliorer la détection des troupeaux infectés par MAP.(Traduit par les auteurs).

Non-antibiotic approaches at drying-off for treating and preventing intramammary infections: a protocol for a systematic review and meta-analysis.

Intramammary infection (IMI) treatment and prevention at drying-off is one of the leading causes for using antimicrobials on dairy farms. The objective of the current paper is to describe the protocol used for conducting a systematic review of the literature on non-antibiotic strategies that can be used on dairy cows at dry off to treat and prevent IMI. Relevant literature will be identified using a combination of database search strategies and iterative screening of references. To be included in the review, articles will have to: (1) be published after 1969; (2) be written in English, French, or Spanish; (3) use a study design such as a controlled trial, an observational study, or an experimental study conducted in vivo; (4) be conducted on commercial dairy cows; (5) investigate a non-antibiotic intervention used at dry off; and finally, (6) report on a relevant mastitis outcome. Titles and abstracts, then full articles will be reviewed for inclusion. Specific data will be extracted and risk of bias will be assessed for all included articles. The planned systematic review will be the first to colligate, in a coherent whole, studies investigating non-antibiotic strategies for treating and preventing IMI at drying-off.

A systematic review of risk factors associated with the introduction of Mycobacterium avium spp. paratuberculosis (MAP) into dairy herds.

The objective of this study was to systematically collect and appraise the scientific evidence related to risk factors associated with the introduction of Mycobacterium avium spp. paratuberculosis (MAP) into a herd of cattle. An electronic search was conducted to collect relevant references addressing 2 specific questions: are i) purchasing/introduction of cattle into a herd, and ii) presence of wildlife or domestic animals, risk factors for the introduction of MAP into a herd? The screening was based on titles and abstracts and selected studies were fully analyzed. Seventeen manuscripts published between 1996 and 2011 were ultimately analyzed. Unit of interest was mainly the herd (n = 17). The specific description of the risk factors studied varied between studies. The principal study design was cross-sectional (n = 15). The review indicated that purchase/introduction of animals was an important risk factor and that the importance of wildlife or other domestic species as a mechanism for transmission into a cattle herd was not measurable.

Revue systématique sur les facteurs de risque associés à l'introduction deMycobacterium aviumspp.paratuberculosis(MAP) dans les troupeaux laitiers. L'objectif était la collecte et l'évaluation systématique des preuves scientifiques liées à des facteurs de risque associés à l'introduction de Mycobacterium avium spp. paratuberculosis (MAP) dans un troupeau.Une recherche électronique a été réalisée pour trier des publications afin de répondre à 2 questions : Est-ce que i) l'achat/introduction de bovins dans un troupeau, et ii) la présence d'animaux sauvages et domestiques sont des facteurs de risque pour l'introduction de MAP dans un troupeau. La sélection a été effectuée en se basant sur les titres, les résumés, et les études choisies ont été entièrement analysés.Dix-sept manuscrits publiés entre 1996 et 2011 ont été sélectionnés. L'unité d'intérêt a principalement été le troupeau (n = 17). Les descriptions des facteurs de risque étudiés variaient d'une étude à l'autre. La majorité des études étaient de type transversal (n = 15).L'achat d'animaux est un facteur de risque bien documenté confirmant l'introduction de la paratuberculose dans le troupeau. Toutefois, malgré la présence de MAP dans les populations domestiques et sauvages à proximité du bétail, l'importance de ce facteur de risque dans la transmission à un troupeau n'était pas mesurable.(Traduit par les auteurs).

Immunostimulatory properties of a novel adjuvant administered with inactivated influenza virus vaccine.

The immunopotentiating activity of a new delivery system was investigated comparatively to Alhydrogel adjuvant, as an antiviral inactivated vaccine after one injection. The efficiency of the new formulation (BioMed) was evaluated with an inactivated porcine strain of influenza (A/Sw/IN/1726/88 H1N1) in the pig model. The first assessment criteria was the follow-up of selected immunological parameters such as, antibody levels, lymphoproliferation, double positive CD4+CD8+ T lymphocytes and cytokine production (IL-2, IL-4, IFN-gamma). The second criteria was the estimate of the protection level of animals exposed to a homologous challenge of 50 PID50 one month after a single immunizing or control injection. In the BioMed group of animals, 4 pigs (out of 6) were free of macroscopic lesion, while lesions could be seen in all individuals of other groups and virus was isolated in only one animal, whereas all other animals of other groups had virus in their lungs. This better protection of BioMed animals seems to be correlated mainly with higher levels of antibodies and to a lesser extent with a slightly better CMI response and probably with the production of memory CD4+CD8+ T cells.

Increased expression of Th2-associated chemokines in bullous pemphigoid disease. Role of eosinophils in the production and release of these chemokines.

Bullous pemphigoid is an inflammatory disease of the skin associated with eosinophil infiltration and the presence of high levels of Th2 cytokines in the associated blister fluid. Little is known about the contribution of chemokines in this disease. We found that eotaxin and MCP-4 mRNA and immunoreactivity were expressed in all biopsies of BP patients and were mainly localized to the epidermis and eosinophils. The expression of eotaxin and MCP-4 was enhanced in eosinophils following IL-5 treatment. Subsequent stimulation of IL-5-primed eosinophils with Ig-immune complexes, results in increase secretion of eotaxin and MCP-4 in the supernatants. Using immunostaining, these two chemokines were localized to the granules of eosinophils. BF was found to contain chemotactic activity for eosinophils, neutrophils and T cells. The chemotactic effect of BF for eosinophils was more effective when eosinophils were stimulated with IL-5 or IL-4. We also found that the levels of Th(2)-associated chemokines (eotaxin and MCP-4) in BF were significantly higher than the Th(1)-associated chemokines (MIP-1beta and IP-10). This was consistent with the increased chemotaxis of polarized Th(2) cells toward BF, when compared to Th(1)-differentiated T cells. Our results support the involvement of Th(2)-associated chemokines in the pathogenesis of BP disease.

Human airway smooth muscle cells express the high affinity receptor for IgE (Fc epsilon RI): a critical role of Fc epsilon RI in human airway smooth muscle cell function.

Several reports suggest that activated airway smooth muscle (ASM) cells are capable of generating various proinflammatory mediators, including cytokines and chemokines. However, little is known about the mechanism involved in this process. In this regard, we have examined the expression and the role of the high affinity IgE receptor (Fc epsilonRI) by ASM cells. Human ASM cells were found to constitutively express transcripts coding for alpha, beta, and gamma subunits of Fc epsilonRI. Flow cytometry and Western blot analysis confirmed the expression of Fc epsilonRI alpha-chain protein. Interestingly, Fc epsilonRI alpha-chain immunoreactivity was also demonstrated in smooth muscle within bronchial biopsies of asthmatic subjects. Cross-linking of Fc epsilonRI induced mobilization of free calcium in ASM cells, one of the critical signals to trigger smooth muscle contraction. Furthermore, cultured ASM cells released IL-4, IL-13, IL-5, and eotaxin but not IFN-gamma, when sensitized with IgE followed by anti-IgE Ab cross-linking. The addition of anti-Fc epsilonRI alpha-chain Abs directed against IgE binding site inhibited this release. Taken together, these results suggest a potential new and important mechanism by which ASM cells may participate in airway inflammation and bronchoconstriction associated with allergic asthma.

CCR3 expression and function in asthmatic airway smooth muscle cells.

Asthma is characterized by an increase in airway smooth muscle mass and a decreased distance between the smooth muscle layer and the epithelium. Furthermore, there is evidence to indicate that airway smooth muscle cells (ASMC) express a wide variety of receptors involved in the immune response. The aims of this study were to examine the expression of CCR3 on ASMC, to compare this expression between asthmatic and nonasthmatic subjects, and to determine the implications of CCR3 expression in the migration of ASMC. We first demonstrated that ASMC constitutively express CCR3 at both mRNA and protein levels. Interestingly, TNF-alpha increases ASMC surface expression of CCR3 from 33 to 74%. Furthermore, using FACS analysis, we found that ASMC CCR3 is expressed to a greater degree in asthmatic vs control subjects (95 vs 75%). Functionality of the receptor was demonstrated by calcium assay; the addition of CCR3 ligand eotaxin to ASMC resulted in an increase in intracellular calcium production. Interestingly, ASMC was seen to demonstrate a positive chemotactic response to eotaxin. Indeed, ASMC significantly migrated toward 100 ng/ml eotaxin (2.2-fold increase, compared with control). In conclusion, the expression of CCR3 by ASMC is increased in asthmatics, and our data show that a CCR3 ligand such as eotaxin induces migration of ASMC in vitro. These results may suggest that eotaxin could be involved in the increased smooth muscle mass observed in asthmatics through the activation of CCR3.

Immunoglobulin G2 deficiency with transient hypogammaglobulinemia and chronic respiratory disease in a 6-month-old Holstein heifer.

A 6-month-old Holstein heifer that was nonresponsive to medical treatment was evaluated for chronic respiratory disease. Complete blood count and serum chemistry revealed neutrophilic leukocytosis and low globulin levels. Assays for bovine leukemia virus, bovine virus diarrhea, and bovine leukocyte adhesion deficiency were negative. Serum globulin subclass assays revealed transient low concentrations of immunoglobulin (Ig) G1 and IgA, persistent low IgG2, and subnormal IgM. Vaccination with 2 doses of multiple, inactived viruses induced seroconversion for most viruses. Flow cytometric analysis of blood lymphocyte subpopulation demonstrated an increase in CD5+ B-cells. Blood lymphocyte proliferation and neutrophil function tests were normal. Results of immunologic assays indicated IgG2 deficiency with transient hypogammaglobulinemia.

How to substantiate eradication of bovine brucellosis when aspecific serological reactions occur in the course of brucellosis testing.

Collaborative work was financed by the EU to develop and assess new diagnostic tools that can differentiate between bovine brucellosis and bovine infections due to Yersinia enterocolitica O:9 either in conjunction with, or as an alternative to, the classical serological, bacteriological or allergic skin tests. Sixteen heifers were experimentally infected with Brucella abortus biovar 1 (five heifers), Brucella suis biovar 2 (two heifers), Y. enterocolitica O:9 (six heifers) and Y. enterocolitica O:3 (three heifers). Four heifers, naturally infected with Y. enterocolitica O:9 that presented aspecific brucellosis serological reactions were also included in the experiment. A self-limited infection was induced in cattle by B. suis biovar 2. All the brucellosis serological tests used, i.e. the slow agglutination test (SAW), the Rose Bengal test (RB), the complement fixation test (CFT), indirect and competitive ELISA's, lacked specificity when used to analyze sera from Y. enterocolitica O:9 infected animals. A Yersinia outer membrane proteins (YOPs)-ELISA was also used and although the test is able to detect a Yersinia group infection, it provided no evidence of whether or not there is a possible brucellosis infection when dual infections are present. The brucellergen IFN-gamma test showed a lack of specificity also. The only test that was proven to be specific is the brucellergen skin test. All brucellosis serological tests, except the indirect ELISA, were limited in their ability to detect B. abortus persistently infected animals. Based on these experimental studies, a strategy was implemented as part of the year 2001 Belgian Brucellosis Eradication Program to substantiate the eradication of bovine brucellosis. Epidemiological inquiries have identified risk factors associated with aspecific serological reactions, possible transmission and infection of cattle by B. suis biovar 2 from infected wild boars; and both legal and administrative measures taken by the veterinary services. No cases of bovine brucellosis have been confirmed in Belgium since March 2000.

Phenotypic and molecular characterization of a Brucella strain isolated from a minke whale (Balaenoptera acutorostrata).

Isolation of Brucella spp. in marine mammals has been reported during the past several years. A Brucella strain from the spleen and liver of a minke whale (Balaenoptera acutorostrata) was isolated. Conventional typing methods indicated that this isolate was related to the genus Brucella but did not match the profiles of any known Brucella species or biovar. Successful PCR amplification of the Brucella rrs-rrl spacer sequence and of the insertion sequence IS6501 also indicated that the minke whale strain was related to the genus Brucella. In addition, the rrs gene of this strain shared a very high degree of nucleotide identity (>98%) with published Brucella spp. rrs sequences. However, RFLP studies using an IS6501-specific probe showed a unique profile for this strain in comparison with the profiles of the six known Brucella species. Moreover, analysis of the omp2 locus by PCR-RFLP, by Southern hybridization using omp2a- and omp2b-specific probes, and by DNA sequencing showed that the minke whale isolate possesses two copies of the omp2b gene instead of one omp2a and one omp2b gene copy or two copies of the omp2a gene described in the six known Brucella species. Thus, molecular typing methods showed that this isolate is clearly distinct from all other known Brucella species and strains. The specific molecular features of this minke whale Brucella isolate raise questions about the lineage between the Brucella strains isolated from marine mammals and the Brucella species isolated from terrestrial mammals.