Hiring intentions and remuneration of veterinary practitioners in western Canada.

All private veterinary practices in western Canada (N = 1333) were surveyed during the SARS-CoV-2 pandemic (January to November 2020) to generate data on the demographics of the profession, and to quantify past and present hiring intentions (demand) as well as remuneration for veterinary associates. The response rate was 39.5% (526/1333), 186 of which had hired at least one full- (FT) or part-time (PT) associate within the 12-month period preceding the completion of the survey. When extrapolated to the practices that did not respond (nonresponders), as many as 471 practices may have hired an associate within the previous 12 mo. The median (mean) annual remuneration paid to FT associates was $90 000 ($91 730). The median number of months it took to hire an associate did not vary by province (P = 0.52); however, it did vary by practice type (P <0.0001): companion animal practice, 3.0 mo; food animal practice, 8.0 mo; and mixed animal practice, 12.0 mo. At the time of the survey, 232 of the 526 (44.1%) responding practices were currently seeking to fill 281 vacancies, representing 274 full-time equivalents (FTE). If extrapolated to the nonresponders, the total number of vacant FTE positions could have been as high as 694. The median (mean) annual wage offered for a FT associate was $87 500 ($88 940), which did not differ by province (P = 0.14) or practice type (P = 0.22). The results of this study support anecdotal reports of a shortage of private veterinary practitioners in western Canada.

Intentions d'embauche et rémunération des vétérinaires praticiens dans l'Ouest canadien. Tous les cabinets vétérinaires privés de l'Ouest canadien (N = 1333) ont été interrogés pendant la pandémie de SARS-CoV-2 (janvier à novembre 2020) afin de générer des données sur la démographie de la profession et de quantifier les intentions d'embauche passées et présentes (demande) ainsi que rémunération des associés vétérinaires. Le taux de réponse était de 39,5 % (526/1333), dont 186 avaient embauché au moins un associé à temps plein (FT) ou à temps partiel (PT) au cours de la période de 12 mois précédant la fin de l'enquête. Lorsqu'ils sont extrapolés aux pratiques qui n'ont pas répondu (non-répondants), jusqu'à 471 pratiques peuvent avoir embauché un associé au cours des 12 derniers mois. La rémunération annuelle médiane (moyenne) versée aux associés de FT était de 90 000 $ (91 730 $). Le nombre de mois qu'il a fallu pour embaucher un associé ne variait pas selon la province (P = 0,52); cependant, elle variait selon le type de pratique (P <0,0001) : pratique des animaux de compagnie, 3,0 mois; pratique des animaux destinés à l'alimentation, 8,0 mois; et pratique animale mixte, 12,0 mois. Au moment de l'enquête, 232 des 526 cabinets répondants (44,1 %) cherchaient actuellement à pourvoir 281 postes vacants, représentant 274 équivalents temps plein (ETP). Si extrapolé aux non-répondants, le nombre total de postes vacants en ETP aurait pu atteindre 694. Le salaire annuel médian (moyen) offert pour un associé à temps plein était de 87 500 $ (88 940 $), ce qui ne différait pas selon la province (P = 0,14) ou type de pratique (P = 0,22). Les résultats de cette étude appuient les rapports anecdotiques d'une pénurie de vétérinaires praticiens privés dans l'Ouest canadien.(Traduit par Dr Serge Messier).

Demographic survey of private veterinary practices in western Canada.

A workforce survey of private veterinary practices in western Canada was conducted in 2020. Data were obtained on 526 practices (response rate = 39.5%) and 1445 individual veterinary practitioners. Overall, 68.4% of practitioners identified as female, with 4 times as many females as males comprising the youngest age cohorts (26 to 35 y) of the profession. The majority of practices (67.9%) were companion animal, followed by mixed animal (21.9%) and food animal (10.2%). Most females (77.2%) and males (57.8%) were engaged in companion animal practice, whereas 23.5% of males and 6.0% of females were food animal practitioners. During an average work week, practitioners devoted 77.4% of practice time to small animals, 15.1% to food animals, and 7.5% to equine animals. A greater proportion of males (75.2%) versus females (63.2%) worked on a full-time equivalent basis (P < 0.001). Whereas males were 1.7 times (95% CI = 1.3 to 2.3; P < 0.001) more likely to be practice owners than females, 54.5% of females were owners. Practice ownership was lower than in previous surveys, a trend that may have long-term implications with respect to the corporatization of the veterinary profession.

Enquête démographique sur les cabinets vétérinaires privés dans l'Ouest canadien. Une enquête sur la main-d'oeuvre des cabinets vétérinaires privés dans l'Ouest canadien a été menée en 2020. Des données ont été obtenues sur 526 cabinets (taux de réponse = 39,5 %) et 1445 praticiens vétérinaires individuels. Dans l'ensemble, 68,4 % des praticiens se sont identifiés comme des femmes, avec quatre fois plus de femmes que d'hommes parmi les cohortes d'âge les plus jeunes (26 à 35 ans) de la profession. La majorité des pratiques (67,9 %) étaient chez les animaux de compagnie, suivis des pratiques mixtes (21,9 %) et chez les animaux de rente (10,2 %). La plupart des femmes (77,2 %) et des hommes (57,8 %) travaillaient en pratique des animaux de compagnie, tandis que 23,5 % des hommes et 6,0 % des femmes étaient en pratique des animaux de rente. Au cours d'une semaine de travail moyenne, les praticiens ont consacré 77,4 % de leur temps de pratique aux petits animaux, 15,1 % aux animaux de rente et 7,5 % aux équidés. Une plus grande proportion d'hommes (75,2 %) que de femmes (63,2 %) travaillaient en équivalent temps plein (P < 0,001). Alors que les hommes étaient 1,7 fois (IC à 95 % = 1,3 à 2,3; P < 0,001) plus susceptibles d'être propriétaires d'un cabinet que les femmes, 54,5 % des femmes étaient propriétaires. La propriété de la pratique était plus faible que dans les enquêtes précédentes, une tendance qui peut avoir des implications à long terme en ce qui concerne la corporisation de la profession vétérinaire.(Traduit par Dr Serge Messier).

Depletion of pulmonary intravascular macrophages rescues inflammation-induced delayed neutrophil apoptosis in horses.

The objective of this study was to determine the effect of pulmonary intravascular macrophage depletion on systemic inflammation and ex vivo neutrophil apoptosis using an experimental model of intestinal ischemia and reperfusion injury in horses. Neutrophils were isolated before and after surgery from horses that were randomized to three treatment groups, namely, sham celiotomy (CEL, n = 4), intestinal ischemia and reperfusion (IR, n = 6), and intestinal ischemia and reperfusion with gadolinium chloride treatment to deplete pulmonary intravascular macrophages (PIMs, IRGC, n = 6). Neutrophil apoptosis was assessed with Annexin V and propidium iodide staining quantified with flow cytometry and caspase-3, caspase-8, and caspase-9 activities in neutrophil lysates. All horses experienced a systemic inflammatory response following surgery. Following surgery, ex vivo neutrophil apoptosis was significantly delayed after 12 or 24 h in culture, except in IRGC horses (12 h: CEL: P = 0.03, IR: P = 0.05, IRGC: P = 0.2; 24 h: CEL: P = 0.001, IR: P = 0.004, IRGC: P = 0.3). Caspase-3, caspase-8, and caspase-9 activities were significantly reduced in neutrophils isolated after surgery and cultured for 12 h in IR horses, but not in IRGC horses (IR caspase-3: P = 0.002, IR caspase-8: P = 0.002, IR caspase-9: P = 0.04). Serum TNF-α concentration was increased in IRGC horses for 6-18 h following jejunal ischemia. Following surgery, ex vivo equine neutrophil apoptosis was delayed via downregulation of caspase activity, which was ameliorated by PIM depletion potentially via upregulation of TNF-α.

A Bovine Enteric Mycobacterium Infection Model to Analyze Parenteral Vaccine-Induced Mucosal Immunity and Accelerate Vaccine Discovery.

Mycobacterial diseases of cattle are responsible for considerable production losses worldwide. In addition to their importance in animals, these infections offer a nuanced approach to understanding persistent mycobacterial infection in native host species. Mycobacteriumavium ssp. paratuberculosis (MAP) is an enteric pathogen that establishes a persistent, asymptomatic infection in the small intestine. Difficulty in reproducing infection in surrogate animal models and limited understanding of mucosal immune responses that control enteric infection in the natural host have been major barriers to MAP vaccine development. We previously developed a reproducible challenge model to establish a consistent MAP infection using surgically isolated intestinal segments prepared in neonatal calves. In the current study, we evaluated whether intestinal segments could be used to screen parenteral vaccines that alter mucosal immune responses to MAP infection. Using Silirum® - a commercial MAP bacterin - we demonstrate that intestinal segments provide a platform for assessing vaccine efficacy within a relatively rapid period of 28 days post-infection. Significant differences between vaccinates and non-vaccinates could be detected using quantitative metrics including bacterial burden in intestinal tissue, MAP shedding into the intestinal lumen, and vaccine-induced mucosal immune responses. Comparing vaccine-induced responses in mucosal leukocytes isolated from the site of enteric infection versus blood leukocytes revealed substantial inconsistences between these immune compartments. Moreover, parenteral vaccination with Silirum did not induce equal levels of protection throughout the small intestine. Significant control of MAP infection was observed in the continuous but not the discrete Peyer's patches. Analysis of these regional mucosal immune responses revealed novel correlates of immune protection associated with reduced infection that included an increased frequency of CD335+ innate lymphoid cells, and increased expression of IL21 and IL27. Thus, intestinal segments provide a novel model to accelerate vaccine screening and discovery by testing vaccines directly in the natural host and provides a unique opportunity to interrogate mucosal immune responses to mycobacterial infections.

Regional Dichotomy in Enteric Mucosal Immune Responses to a Persistent Mycobacterium avium ssp. paratuberculosis Infection.

Chronic enteric Mycobacterium avium ssp. paratuberculosis (MAP) infections are endemic in ruminants globally resulting in significant production losses. The mucosal immune responses occurring at the site of infection, specifically in Peyer's patches (PP), are not well-understood. The ruminant small intestine possesses two functionally distinct PPs. Discrete PPs function as mucosal immune induction sites and a single continuous PP, in the terminal small intestine, functions as a primary lymphoid tissue for B cell repertoire diversification. We investigated whether MAP infection of discrete vs. continuous PPs resulted in the induction of significantly different pathogen-specific immune responses and persistence of MAP infection. Surgically isolated intestinal segments in neonatal calves were used to target MAP infection to individual PPs. At 12 months post-infection, MAP persisted in continuous PP (n = 4), but was significantly reduced (p = 0.046) in discrete PP (n = 5). RNA-seq analysis revealed control of MAP infection in discrete PP was associated with extensive transcriptomic changes (1,707 differentially expressed genes) but MAP persistent in continuous PP elicited few host responses (4 differentially expressed genes). Cytokine gene expression in tissue and MAP-specific recall responses by mucosal immune cells isolated from PP, lamina propria and mesenteric lymph node revealed interleukin (IL)22 and IL27 as unique correlates of protection associated with decreased MAP infection in discrete PP. This study provides the first description of mucosal immune responses occurring in bovine discrete jejunal PPs and reveals that a significant reduction in MAP infection is associated with specific cytokine responses. Conversely, MAP infection persists in the continuous ileal PP with minimal perturbation of host immune responses. These data reveal a marked dichotomy in host-MAP interactions within the two functionally distinct PPs of the small intestine and identifies mucosal immune responses associated with the control of a mycobacterial infection in the natural host.

Case-control study of risk factors for pasture-and endocrinopathy-associated laminitis in North American horses.

OBJECTIVE To investigate risk factors for the development of pasture- and endocrinopathy-associated laminitis (PEAL) in horses and ponies in North America. DESIGN Case-control study. ANIMALS 199 horses with incident cases of PEAL and 351 horses from 2 control populations (healthy horses [n = 198] and horses with lameness not caused by laminitis [153]) that were evaluated in North America between January 2012 and December 2015 by veterinarian members of the American Association of Equine Practitioners. PROCEDURES North American members of the American Association of Equine Practitioners were contacted to participate in the study, and participating veterinarians provided historical data on incident cases of PEAL, each matched with a healthy control and a lameness control. Conditional logistic regression analysis was used to compare data on PEAL-affected horses with data on horses from each set of controls. RESULTS Horses with an obese body condition (ie, body condition score ≥ 7), generalized or regional adiposity (alone or in combination), preexisting endocrinopathy, or recent (within 30 days) glucocorticoid administration had increased odds of developing PEAL, compared with horses that did not have these findings. CONCLUSIONS AND CLINICAL RELEVANCE The present study identified several risk factors for PEAL that may assist not only in managing and preventing this form of laminitis, but also in guiding future research into its pathogenesis.

Role of toll-like receptor 4 and caspase-3, -8, and -9 in lipopolysaccharide-induced delay of apoptosis in equine neutrophils.

OBJECTIVE To evaluate the effect of lipopolysaccharide (LPS) on apoptosis of equine neutrophils in vitro. SAMPLE Venous blood samples from 40 adult horses. PROCEDURES Neutrophils were isolated from blood samples and cultured with or without LPS from Escherichia coli O55:B5 for 12 or 24 hours. Neutrophil apoptosis was assessed by use of cytologic examination, annexin V and propidium iodide staining quantified with flow cytometry, coincubation with inducers of intrinsic and extrinsic apoptosis or a toll-like receptor (TLR) 4 inhibitor, and measurement of caspase-3, -8, and -9 activities. RESULTS Treatment with LPS resulted in a significant delay in apoptosis after incubation for 12 and 24 hours (neutrophils from blood samples of 40 horses). There was a significant correlation between increases in LPS dose and decreases in apoptosis after incubation for 24 hours (3 experiments, each of which involved neutrophils obtained from the same 3 horses at 3 separate times). Caspase-9 activity, but not caspase-3 or -8 activity, was significantly reduced in LPS-treated neutrophils after incubation for 12 hours (neutrophils from blood samples of 17 horses). Treatment with a TLR4 inhibitor or intrinsic and extrinsic inducers of apoptosis prevented LPS-delayed apoptosis. CONCLUSIONS AND CLINICAL RELEVANCE LPS treatment delayed apoptosis of equine neutrophils in vitro for up to 24 hours in a dose-dependent manner by alteration of the intrinsic pathway of apoptosis and was dependent on TLR4 signaling. Increased neutrophil life span may contribute to the development of a systemic inflammatory response syndrome in endotoxemic horses.

Pain and adverse behavior in declawed cats.

Objectives The aim of this study was to assess the impact of onychectomy (declawing) upon subsequent development of back pain and unwanted behavior in cohorts of treated and control cats housed in two different locations. Methods This was a retrospective cohort study. In total, there was 137 declawed and 137 non-declawed cats, of which 176 were owned cats (88 declawed, 88 non-declawed) and 98 were shelter cats (49 declawed and 49 non-declawed). All cats were physically examined for signs of pain and barbering. The previous 2 years of medical history were reviewed for documented unwanted behavior such as inappropriate elimination and biting with minimal provocation and aggression. All declawed cats were radiographed for distal limb abnormalities, including P3 (third phalanx) bone fragments. The associations of declaw surgery with the outcomes of interest were examined using χ2 analysis, two sample t-tests and manual, backwards, stepwise logistic regression. Results Significant increases in the odds of back pain (odds ratio [OR] 2.9), periuria/perichezia (OR 7.2), biting (OR 4.5) and barbering (OR 3.06) occurred in declawed compared with control cats. Of the 137 declawed cats, 86 (63%) showed radiographic evidence of residual P3 fragments. The odds of back pain (OR 2.66), periuria/perichezia (OR 2.52) and aggression (OR 8.9) were significantly increased in declawed cats with retained P3 fragments compared with those declawed cats without. Optimal surgical technique, with removal of P3 in its entirety, was associated with fewer adverse outcomes and lower odds of these outcomes, but operated animals remained at increased odds of biting (OR 3.0) and undesirable habits of elimination (OR 4.0) compared with non-surgical controls. Conclusions and relevance Declawing cats increases the risk of unwanted behaviors and may increase risk for developing back pain. Evidence of inadequate surgical technique was common in the study population. Among declawed cats, retained P3 fragments further increased the risk of developing back pain and adverse behaviors. The use of optimal surgical technique does not eliminate the risk of adverse behavior subsequent to onychectomy.

Cattle Immunized with a Recombinant Subunit Vaccine Formulation Exhibits a Trend towards Protection against Histophilus somni Bacterial Challenge.

Histophilosis, a mucosal and septicemic infection of cattle is caused by the Gram negative pathogen Histophilus somni (H. somni). As existing vaccines against H. somni infection have shown to be of limited efficacy, we used a reverse vaccinology approach to identify new vaccine candidates. Three groups (B, C, D) of cattle were immunized with subunit vaccines and a control group (group A) was vaccinated with adjuvant alone. All four groups were challenged with H. somni. The results demonstrate that there was no significant difference in clinical signs, joint lesions, weight change or rectal temperature between any of the vaccinated groups (B,C,D) vs the control group A. However, the trend to protection was greatest for group C vaccinates. The group C vaccine was a pool of six recombinant proteins. Serum antibody responses determined using ELISA showed significantly higher titers for group C, with P values ranging from < 0.0148 to < 0.0002, than group A. Even though serum antibody titers in group B (5 out of 6 antigens) and group D were significantly higher compared to group A, they exerted less of a trend towards protection. In conclusion, the vaccine used in group C exhibits a trend towards protective immunity in cattle and would be a good candidate for further analysis to determine which proteins were responsible for the trend towards protection.

Recombinant Mycoplasma mycoides proteins elicit protective immune responses against contagious bovine pleuropneumonia.

Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a devastating respiratory disease mainly affecting cattle in sub-Saharan Africa. The current vaccines are based on live-attenuated Mmm strains and present problems with temperature stability, duration of immunity and adverse reactions, thus new vaccines are needed to overcome these issues. We used a reverse vaccinology approach to identify 66 Mmm potential vaccine candidates. The selection and grouping of the antigens was based on the presence of specific antibodies in sera from CBPP-positive animals. The antigens were used to immunize male Boran cattle (Bos indicus) followed by a challenge with the Mmm strain Afadé. Two of the groups immunized with five proteins each showed protection after the Mmm challenge (Groups A and C; P<0.05) and in one group (Group C) Mmm could not be cultured from lung specimens. A third group (Group N) showed a reduced number of animals with lesions and the cultures for Mmm were also negative. While immunization with some of the antigens conferred protection, others may have increased immune-related pathology. This is the first report that Mmm recombinant proteins have been successfully used to formulate a prototype vaccine and these results pave the way for the development of a novel commercial vaccine.

Administration of Poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) and Avian Beta Defensin as Adjuvants in Inactivated Inclusion Body Hepatitis Virus and its Hexon Protein-Based Experimental Vaccine Formulations in Chickens.

Inclusion body hepatitis (IBH) is one of the major infectious diseases adversely affecting the poultry industry of the United States and Canada. Currently, no effective and safe vaccine is available for the control of IBH virus (IBHV) infection in chickens. However, based on the excellent safety and immunogenic profiles of experimental veterinary vaccines developed with the use of new generation adjuvants, we hypothesized that characterization of vaccine formulations containing inactivated IBHV or its capsid protein hexon as antigens, along with poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) and avian beta defensin 2 (ABD2) as vaccine adjuvants, will be helpful in development of an effective and safe vaccine formulation for IBH. Our data demonstrated that experimental administration of vaccine formulations containing inactivated IBHV and a mixture of PCEP with or without ABD2 as an adjuvant induced significantly higher antibody responses compared with other vaccine formulations, while hexon protein-based vaccine formulations showed relatively lower levels of antibody responses. Thus, a vaccine formulation containing inactivated IBHV with PCEP or a mixture of PCEP and ABD2 (with a reduced dosage of PCEP) as an adjuvant may serve as a potential vaccine candidate. However, in order to overcome the risks associated with whole virus inactivated vaccines, characterization of additional viral capsid proteins, including fiber protein and penton of IBHV along with hexon protein in combination with more new generation adjuvants, will be helpful in further improvements of vaccines against IBHV infection.

Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells.

Shiga toxin-producing Escherichia coli (STEC) serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS) to secrete effector proteins (T3SPs) that result in the formation of attaching and effacing (A/E) lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes.

Analysis of immune responses to recombinant proteins from strains of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia.

Current contagious bovine pleuropneumonia (CBPP) vaccines are based on live-attenuated strains of Mycoplasma mycoides subsp. mycoides (Mmm). These vaccines have shortcomings in terms of efficacy, duration of immunity and in some cases show severe side effects at the inoculation site; hence the need to develop new vaccines to combat the disease. Reverse vaccinology approaches were used and identified 66 candidate Mycoplasma proteins using available Mmm genome data. These proteins were ranked by their ability to be recognized by serum from CBPP-positive cattle and thereafter used to inoculate naïve cattle. We report here the inoculation of cattle with recombinant proteins and the subsequent humoral and T-cell-mediated immune responses to these proteins and conclude that a subset of these proteins are candidate molecules for recombinant protein-based subunit vaccines for CBPP control.

Large animal models for vaccine development and testing.

The development of human vaccines continues to rely on the use of animals for research. Regulatory authorities require novel vaccine candidates to undergo preclinical assessment in animal models before being permitted to enter the clinical phase in human subjects. Substantial progress has been made in recent years in reducing and replacing the number of animals used for preclinical vaccine research through the use of bioinformatics and computational biology to design new vaccine candidates. However, the ultimate goal of a new vaccine is to instruct the immune system to elicit an effective immune response against the pathogen of interest, and no alternatives to live animal use currently exist for evaluation of this response. Studies identifying the mechanisms of immune protection; determining the optimal route and formulation of vaccines; establishing the duration and onset of immunity, as well as the safety and efficacy of new vaccines, must be performed in a living system. Importantly, no single animal model provides all the information required for advancing a new vaccine through the preclinical stage, and research over the last two decades has highlighted that large animals more accurately predict vaccine outcome in humans than do other models. Here we review the advantages and disadvantages of large animal models for human vaccine development and demonstrate that much of the success in bringing a new vaccine to market depends on choosing the most appropriate animal model for preclinical testing.

Bistable expression of CsgD in Salmonella enterica serovar Typhimurium connects virulence to persistence.

Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment.

CpG-ODNs induced changes in cytokine/chemokines genes expression associated with suppression of infectious bronchitis virus replication in chicken lungs.

The process of virus replication in host cells is greatly influenced by the set of cytokines, chemokines and antiviral substances activated as a result of host-virus interaction. Alteration of cytokines profiles through manipulation of the innate immune system by innate immune stimulants may be helpful in inhibiting virus replication in otherwise permissive cells. The aim of present studies was to characterize innate immune responses capable of inhibiting infectious bronchitis virus (IBV) replication in chicken lungs after in ovo administration of CpG ODN. In our experiments, CpG ODN 2007 or PBS solution was injected on 18th embryonic day (ED) via the chorioallontoic route. CpG ODN and PBS inoculated embryos were challenged with virulent IBV on the 19th ED. Lung tissue samples from experimental chicks were analysed for cytokines/chemokines gene expression at 24h, 48h, and 72h, post infection. Our data showed significant differential up-regulation of IFN-γ, IL-8 (CXCLi2) and MIP-1ß genes and suppression of IL-6 gene expression being associated with inhibition of IBV replication in lungs tissue retrieved from embryos pre-treated with CpG ODN. It is expected that understanding of the innate immune modulation of target tissues by the virus and innate immune stimulants will be helpful in identification of valuable targets for development of novel, safe, effective and economical control strategies against IBV infection in chickens.

Morphometric examination of the equine adult and foal lung.

To understand the mechanisms of airway inflammation associated with equine diseases such as Rhodococcus equi infection, we must identify baseline "normal" structural characteristics of the horse lung. To develop a detailed understanding of the morphology of the horse lung, we adapted and applied stereological methods to the lungs from healthy adult horses (N = 4) and 1-day (N = 5) and 30-day (N = 5) old foals. The left lung was fixed in situ by intrabronchial instillation of glutaraldehyde/paraformaldehyde fixative at 25 cm H2 O column and sampled using a fractionator design followed by embedding in glycol methacrylate. The lung was characterized into parenchyma and non-parenchyma, where median parenchymal density was 81.0% in 1-day-old foals, 84.4% in 30-day-old foals and 93.7% in adult lungs. The median volume density of alveolar airspace per lung was 45.9% in 1-day-old, 55.5% in 30-day and 66.9% in adult horse lungs. The median alveolar surface area increased with age, from 205.3 m(2) , 258.2 m(2) , and 629.9 m(2) in 1-day-old foals, 30-day-old foals, and adults, respectively. While the median alveolar surface density decreased with age, the mean linear intercept (mean free distance within acinar airspaces) increased with age. Alveolar surface area was greater than endothelial surface area within each lung. The ratio between alveolar and endothelial surface density remains unchanged with age. The median endothelium surface area was 106.2 m(2) in 1-day, 147.5 m(2) in 30-day, and 430 m(2) in adult lungs. The data suggest the foal lung is functionally developed and postnatal lung development and remodelling is driven by alveolar expansion paralleled with angiogenesis.

Failure of a VapA/CpG oligodeoxynucleotide vaccine to protect foals against experimental Rhocococcus equi pneumonia despite induction of VapA-specific antibody and interferon-γ response.

We evaluated the immunogenic and protective potential of a recombinant VapA/CpG oligodeoxynucleotide (ODN) 2395 vaccine in neonatal foals undergoing experimental Rhodococcus equi challenge. Foals (n = 8) were vaccinated by intramuscular injection on days 1 and 15 of the study; control foals (n = 7) received a phosphate-buffered saline (PBS) solution. All foals were challenged by intrabronchial administration of 5 × 106 R. equi 103⁺ on day 29. Bronchoalveolar lavages were done on days 15, 29, and 36 and total cell count, differential cell count, rVapA-stimulated cell proliferation and interferon (IFN)-γ mRNA expression determined. Clinical examination, complete blood (cell) counts, serology for VapA-specific antibodies, and culture of nasal and fecal swabs were done on days 1, 15, 29, 36, 43, and 50. Foals were humanely euthanized on day 50 and severity of pneumonia scored on a 4-point scale. Vaccination resulted in a significant increase in VapA-specific immunoglobulin (Ig) production, with total IgG and IgG(T) being increased by day 15. Expression of VapA-specific IFN-γ mRNA by BAL cells was increased in the vaccinated foals following challenge. Postmortem lung severity scores did not differ between groups. Two foals shed virulent R. equi in feces; however, real-time polymerase chain reaction (PCR) revealed the isolates to be different from the challenge strain.

Nous avons évalué le potentiel immunogène et protecteur d'un vaccin recombinant VapA/oligodéoxynucléotide CpG (ODN) 2395 chez des poulains nouveau-nés soumis à une infection défi par Rhodococcus equi. Les poulains (n = 8) étaient vaccinés par voie intramusculaire aux jours 1 et 15 de l'étude; les poulains témoins (n = 7) ont reçu une injection d'une solution de saline tamponnée (PBS). Tous les poulains ont été challengés par administration intra-bronchique de 5 × 106R. equi 103+ au jour 29. Des lavages broncho-alvéolaires (LBA) ont été effectués aux jours 15, 29 et 36 et on détermina le nombre total de cellules, un dénombrement cellulaire différentiel, la prolifération des cellules rVapA stimulées et l'expression d'ARNm de l'interféron (IFN)-γ. Un examen clinique, des comptages cellulaires sanguins complets, une analyse sérologique pour détecter les anticorps spécifiques contre VapA, et une culture d'écouvillons nasal et fécal ont été effectués aux jours 1, 15, 29, 36, 43 et 50. Les poulains ont été euthanasiés au jour 50 et la sévérité de la pneumonie notée sur une échelle de 4 points. La vaccination a causé une augmentation significative de la production d'immunoglobulines (Ig) spécifiquement dirigées contre VapA, les quantités totales d'IgG et d'IgG(T) ayant augmentées au jour 15. L'expression d'ARNm de l'IFN-γ spécifique au VapA par les cellules des LBA était augmentée chez les poulains vaccinés suite au challenge. Aucune différence ne fut notée dans les pointages de sévérité des lésions pulmonaires lors des examens post-mortem. Deux poulains excrétaient du R. equi virulent dans leurs fèces; toutefois, l'analyse par réaction d'amplification en chaîne par la polymérase (PCR) a démontré que ces isolats étaient différents de la souche utilisée pour le challenge.(Traduit par Docteur Serge Messier).

Role of motAB in adherence and internalization in polarized Caco-2 cells and in cecal colonization of Campylobacter jejuni.

Campylobacter jejuni, a gram-negative motile bacterium commonly found in the chicken gastrointestinal tract, is one of the leading causes of bacterial gastroenteritis in humans worldwide. An intact and functional flagellum is important for C. jejuni virulence and colonization. To understand the role of C. jejuni motility in adherence and internalization in polarized Caco-2 cells and in cecal colonization of chickens we constructed a C. jejuni NCTC11168 V1 deltamotAB mutant. The motAB genes code for the flagellar motor, which enables the rotation of the flagellum. The nonmotile deltamotAB mutant expressed a full-length flagellum, which allowed us to differentiate between the roles of full-length flagella and motility in the ability of C. jejuni to colonize. To study the adherence and invasion abilities of the C. jejuni deltamotAB mutant we chose to use polarized Caco-2 cells, which are thought to be more representative of in vivo intestinal cell architecture and function. Although the C. jejuni deltamotAB mutant adhered significantly better than the wild type to the Caco-2 cells, we observed a significant reduction in the ability to invade the cells. In this study we obtained evidence that the flagellar rotation triggers C. jejuni invasion into polarized Caco-2 cells and we believe that C. jejuni is propelled into the cell with a drill-like rotation. The deltamotAB mutant was also tested for its colonization potential in a 1-day-old chicken model. The nonmotile C. jejuni deltamotAB mutant was not able to colonize any birds at days 3 and 7, suggesting that motility is essential for C. jejuni colonization.

Analyses of lipid rafts, Toll-like receptors 2 and 4, and cytokines in foals vaccinated with Virulence Associated Protein A/CpG oligonucleotide vaccine against Rhodococcus equi.

Rhodococcus equi establishes long-term pulmonary infection, survives in phagolysosomes of alveolar macrophages and causes pneumonia in foals. The failure of the foal to clear R. equi bacteria is believed to be due to its inability to produce IFN-γ and defects in Toll-like receptor(TLR) signaling. Lipid rafts sequester immune receptors such as TLRs and facilitate efficient cell signaling and therefore, a deficiency in accumulation of receptors in lipid rafts may result in failure to activate. We tested whether a Virulence Associated Protein A (VapA)/CpG vaccine against R. equi would impact the production of IL-10, IFN-γ and TNF-α in lung tissue and fluid samples, alter expression of TLR2 and TLR4 and alter their association with the lipid rafts in broncho-alveolar lavage (BAL) cells. Eight foals, 1­6 days of age, were vaccinated against R. equi followed by a booster at day 14 and challenged with R. equi (5 x 10(6) CFU/ml;10 ml) on day 28. This group was termed "vaccinated pre-challenge" before the infection and "vaccinated post-challenge" after the infection. A second group of foals (n = 7) was not vaccinated but challenged with R. equi on day 28 of the study. This group was termed "non-vaccinated pre-challenge" and after infection with R. equi was named "non-vaccinated post-challenged. We report adaptation of previous protocols to isolate plasma membrane fractions from BAL cells and identification of lipid raft fractions based on the presence of flotillin-1 and GM-1 and absence of transferrin receptor. TLR2 and TLR4 were restricted to plasma membrane fractions 7­9 of alveolar cells collected from vaccinated foals before and after the challenge. Western blots showed that vaccinated post-challenge foals had higher expression of TLR2 in their lung tissues compared to non-vaccinated pre-challenge foals. TNF- concentration was higher in BAL fluid collected from the vaccinated compared to the non-vaccinated foals on day 28. Lung tissue extracts collected on day 49 from the non-vaccinated R. equi challenged foals showed higher expression of IL-10 compared to the vaccinated-challenged foals. However, there were no differences among the groups with respect to the concentration of IFN-γ in BAL fluid or lung tissue extracts. Taken together, we modified previous protocols to isolate plasma membrane fractions from BAL cells of foals and report that the vaccination with a VapA/CPG vaccine increases association of TLR2 and TLR4 with lipid raft fractions and alters expression of TNF-α and IL-10. The data point to a subtle effect of vaccination on the association of TLR2 and TLR4 with lipid rafts in BAL cells.