Induction of trained immunity in broiler chickens following delivery of oligodeoxynucleotide containing CpG motifs to protect against Escherichia coli septicemia.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) can promote antimicrobial immunity in chickens by enriching immune compartments and activating immune cells. Innate memory, or trained immunity, has been demonstrated in humans and mice, featuring the absence of specificity to the initial stimulus and subsequently cross-protection against pathogens. We hypothesize that CpG-ODN can induce trained immunity in chickens. We delivered single or multiple administrations of CpG-ODN to birds and mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis of peripheral blood mononuclear cells were quantified using Seahorse XFp. Next, chickens were administered with CpG-ODN twice at 1 and 4 day of age and challenged with Escherichia coli at 27 days of age. The CpG-ODN administered groups had significantly higher mitochondrial OXPHOS until 21 days of age while cellular glycolysis gradually declined by 14 days of age. The group administered with CpG-ODN twice at 1 and 4 days of age had significantly higher survival, lower clinical score and bacterial load following challenge with E. coli at 27 d of age. This study demonstrated the induction of trained immunity in broiler chickens following administration of CpG-ODN twice during the first 4 days of age to protect birds against E. coli septicemia at 27 days of age.

Oligodeoxynucleotides containing CpG motifs upregulate bactericidal activities of heterophils and enhance immunoprotection of neonatal broiler chickens against Salmonella Typhimurium septicemia.

In the past, we demonstrated that oligodeoxynucleotides containing CpG motifs (CpG-ODN) mimicking bacterial DNA, stimulate the innate immune system of neonatal broiler chickens and protect them against Escherichia coli and Salmonella Typhimurium (S. Typhimurium) septicemia. The first line of innate immune defense mechanism is formed by heterophils and plays a critical protective role against bacterial septicemia in avian species. Therefore, the objectives of this study were 1) to explore the kinetics of CpG-ODN mediated antibacterial mechanisms of heterophils following single or twice administration of CpG-ODN in neonatal broiler chickens and 2) to investigate the kinetics of the immunoprotective efficacy of single versus twice administration of CpG-ODN against S. Typhimurium septicemia. In this study, we successfully developed and optimized flow cytometry-based assays to measure phagocytosis, oxidative burst, and degranulation activity of heterophils. Birds that received CpG-ODN had significantly increased (p < 0.05) phagocytosis, oxidative burst, and degranulation activity of heterophils as early as 24 h following CpG-ODN administration. Twice administration of CpG-ODN significantly increased the phagocytosis activity of heterophils. In addition, our newly developed CD107a based flow cytometry assay demonstrated a significantly higher degranulation activity of heterophils following twice than single administration of CpG-ODN. However, the oxidative burst activity of heterophils was not significantly different between birds that received CpG-ODN only once or twice. Furthermore, delivery of CpG-ODN twice increased immunoprotection against S. Typhimurium septicemia compared to once but the difference was not statistically significant. In conclusion, we demonstrated enhanced bactericidal activity of heterophils after administration of CpG-ODN to neonatal broiler chickens. Further investigations will be required to identify other activated innate immune cells and the specific molecular pathways associated with the CpG-ODN mediated activation of heterophils.

Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model.

BACKGROUND: Necrotic enteritis (NE) is a severe intestinal infection that affects both humans and poultry. It is caused by the bacterium Clostridium perfringens (CP), but the precise mechanisms underlying the disease pathogenesis remain elusive. This study aims to develop an NE broiler chicken model, explore the impact of the microbiome on NE pathogenesis, and study the virulence of CP isolates with different toxin gene combinations. METHODS: This study established an animal disease model for NE in broiler chickens. The methodology encompassed inducing abrupt protein changes and immunosuppression in the first experiment, and in the second, challenging chickens with CP isolates containing various toxin genes. NE was evaluated through gross and histopathological scoring of the jejunum. Subsequently, jejunal contents were collected from these birds for microbiome analysis via 16S rRNA amplicon sequencing, followed by sequence analysis to investigate microbial diversity and abundance, employing different bioinformatic approaches. RESULTS: Our findings reveal that CP infection, combined with an abrupt increase in dietary protein concentration and/or infection with the immunosuppressive variant infectious bursal disease virus (vIBDV), predisposed birds to NE development. We observed a significant decrease (p < 0.0001) in the abundance of Lactobacillus and Romboutsia genera in the jejunum, accompanied by a notable increase (p < 0.0001) in Clostridium and Escherichia. Jejunal microbial dysbiosis and severe NE lesions were particularly evident in birds infected with CP isolates containing cpa, netB, tpeL, and cpb2 toxin genes, compared to CP isolates with other toxin gene combinations. Notably, birds that did not develop clinical or subclinical NE following CP infection exhibited a significantly higher (p < 0.0001) level of Romboutsia. These findings shed light on the complex interplay between CP infection, the gut microbiome, and NE pathogenesis in broiler chickens. CONCLUSION: Our study establishes that dysbiosis within the jejunal microbiome serves as a reliable biomarker for detecting subclinical and clinical NE in broiler chicken models. Additionally, we identify the potential of the genera Romboutsia and Lactobacillus as promising candidates for probiotic development, offering effective alternatives to antibiotics in NE prevention and control.

Development of a multivalent adjuvanted inactivated vaccine against variant arthrotropic avian reoviruses.

Variant avian reoviruses (ARVs) are economically important emerging pathogens of poultry, which mainly affect young broiler chickens and cause significant production losses. Currently, there are no effective commercial vaccines available for control and prevention of emerging variant ARVs. In this study, monovalent inactivated adjuvated (20% Emulsigen D) broiler breeder vaccines containing antigens from ARV genotype cluster (C) group -2, -4, -5, or -6, and a multivalent vaccine containing antigens from all the four indicated genotypic cluster groups were developed and evaluated for their efficacy in protecting broiler progenies against homologous or heterologous ARV challenge. The use of monovalent or multivalent inactivated vaccines in a prime-boost immunization strategy induced the production of ARV specific antibodies in broiler breeders. The maternal antibodies were effectively transferred to broiler progenies. Broiler progenies obtained from immunized breeders demonstrated milder clinical symptoms and reduced gross and histopathological lesions after homologous ARV challenge. More severe gross and histological lesions were observed in challenged progenies from unvaccinated broiler breeders. However, cross protection was not observed when either of the monovalent-vaccine groups were challenged with a heterologous virus. In addition, the progenies from the unvaccinated ARV challenged control or heterologous ARV challenged vaccinated groups had significantly reduced body weight gain (p < 0.01) than the unchallenged-control, challenged-multivalent, or homologous ARV-challenged monovalent vaccine groups. However, homologous ARV challenged progenies in the multivalent or monovalent vaccine groups had similar body weight gain as the control unchallenged group with significantly reduced viral load (p < 0.01) in the gastrocnemius tendon tissue. This study indicates that broad-spectrum protection of broiler progenies from variant ARV infections is feasible through the development of multivalent vaccines after proper characterization, selection and incorporation of multiple antigens based on circulating ARV genotypes in targeted regions.

Detection of Chlamydia abortus in aborted chorioallantoises of horses from Western Canada.

Chlamydiae are reported to cause abortion in several species, however the association between Chlamydia sp. and equine abortions is poorly understood. A zoonotic transfer event of C. psittaci from aborted equine tissues in Australia has emphasized the need to better understand the prevalence of this pathogen in equine populations. The prevalence of chlamydia in equine abortions in North America has not been investigated thoroughly. We examined 99 formalin-fixed, paraffin-embedded placental samples submitted between 2009 and 2020 from equine abortions in Western Canada using chlamydia-specific 16S rRNA conventional PCR testing; 26 of 99 submissions tested positive for chlamydial DNA. Most of these submissions (n = 17) had no final diagnosis noted on their original pathology reports. DNA sequencing identified 22 of the 26 cases as C. abortus; 21 of the 22 C. abortus-positive samples were positive on chlamydial immunohistochemistry. These findings contrast with studies in Europe that found a low prevalence of chlamydiae using similar methodology. The high prevalence of the potentially zoonotic C. abortus identified in our study suggests that more substantial biosecurity protocols may be warranted for equine foaling, abortion, and stillbirth in Western Canada to prevent zoonotic transfer of the pathogen.

Surveillance of Babesia odocoilei in wild and farmed cervid populations of Saskatchewan, Canada.

Objective: To investigate whether Babesia odocoilei could be detected from farmed and wild cervid diagnostic submissions prior to its first reported occurrence in Saskatchewan. Procedure: Polymerase chain reaction (PCR) for B. odocoilei was used to survey 85 fresh-frozen samples and 112 formalin-fixed, paraffin-embedded samples from Saskatchewan cervids submitted for necropsy between 2000 and 2014. Results: The PCR was positive for B. odocoilei in 1/84 white-tailed deer, 1/41 moose, 0/37 mule deer, and 1/35 elk. The positive elk was from a farmed herd, but the remaining 2 positive samples were from wild cervids. The positive moose sample was the earliest confirmed infection, dating back to 2008. Therefore, 1.5% of the study population tested positive over the 14-year period. Conclusion: There were low numbers of cervids infected with B. odocoilei in the study population. Clinical relevance: Babesiosis should be included as a differential diagnosis for disease in susceptible cervids when clinical signs are compatible; however, a lack of suggestive clinical signs or necropsy findings does not preclude infection. Thus, monitoring prevalence of the disease within Saskatchewan (and Canada) will likely require targeted surveillance.

Objectif: Déterminer si Babesia odocoilei pouvait être détectée dans les soumissions de diagnostic de cervidés d'élevage et sauvages avant sa première occurrence signalée en Saskatchewan. Procédure: La réaction d'amplification en chaîne par la polymérase (PCR) pour B. odocoilei a été utilisée pour étudier 85 échantillons fraîchement congelés et 112 échantillons fixés au formol et inclus en paraffine de cervidés de la Saskatchewan soumis à l'autopsie entre 2000 et 2014. Résultats: La PCR était positive pour B. odocoilei chez 1/84 cerf de Virginie, 1/41 orignal, 0/37 cerf mulet et 1/35 wapiti. Le wapiti positif provenait d'un troupeau d'élevage, mais les deux autres échantillons positifs provenaient de cervidés sauvages. L'échantillon d'orignal positif était la première infection confirmée, remontant à 2008. Par conséquent, 1,5 % de la population étudiée a été testée positive au cours de la période de 14 ans. Conclusion: Il y avait un faible nombre de cervidés infectés par B. odocoilei dans la population étudiée. Pertinence clinique: La babésiose devrait être incluse comme diagnostic différentiel de maladie chez les cervidés sensibles lorsque les signes cliniques sont compatibles; cependant, l'absence de signes cliniques évocateurs ou de résultats d'autopsie n'exclut pas l'infection. Ainsi, la surveillance de la prévalence de la maladie en Saskatchewan (et au Canada) nécessitera probablement une surveillance ciblée.(Traduit par Dr Serge Messier).

Exposure of embryonating eggs to Enterococcus faecalis and Escherichia coli potentiates E. coli pathogenicity and increases mortality of neonatal chickens.

Enterococci and Escherichia coli are opportunistic pathogens of poultry and are associated with embryo and neonatal chick mortality. We have recently demonstrated that 56% of dead broiler chicken embryos in commercial hatcheries in western Canada were due to the coinfection of Enterococcus species and E. coli. The objective of this study was to investigate the host-pathogen interactions of Enterococcus faecalis and E. coli in developing chicken embryos. Embryonating eggs at 12 d of incubation were dipped in a solution of E. faecalis and/or E. coli for 30 s to expose the eggshell to study the migration and colonization of E. faecalis and E. coli in the internal organs of chicken embryos and subsequent neonatal chicken mortality following hatch. A multidrug-resistant E. faecalis isolate from a dead chicken embryo and an E. faecalis isolate from a case of yolk sac infection were able to colonize the internal organs of chicken embryos rapidly compared to an E. faecalis isolate from a healthy chicken without affecting viability or hatchability of embryos. Although E. faecalis colonized internal organs of chicken embryos, no evidence of inflammation of these organs nor the expression of virulence genes of E. faecalis was observed. Although E. faecalis and E. coli alone did not affect the viability of embryos, a significantly high neonatal chicken mortality (27%) was observed following exposure of embryos to both E. faecalis and E. coli. Upregulation of IL-1 and CXCR4 was evident 48 h before peak mortality of neonatal chickens; this could suggest a possible link of cytokine dysregulation to increased mortality in coinfected neonatal chickens. However, further studies are warranted to investigate this issue vis-à-vis coinfection with E. faecalis and E. coli in chicken embryos and neonatal chickens.

Comparison of Therapeutic Antibiotics, Probiotics, and Synthetic CpG-ODNs for Protective Efficacy Against Escherichia coli Lethal Infection and Impact on the Immune System in Neonatal Broiler Chickens.

The poultry industry needs alternatives to antibiotics, as there are growing public concerns about the emergence of antimicrobial resistance owing to antimicrobial use in animal production. We have reported that the administration of neonatal chicks with synthetic DNA oligodeoxynucleotides containing unmethylated cytosine guanine dinucleotide (CpG) motifs (CpG-ODN) can protect against bacterial pathogens in chickens. The objective of this study was to compare the immunoprotective effects of CpG-ODN and probiotics against Escherichia coli infection vs. commonly used therapeutic antibiotics. Day-old broiler chicks were divided into five groups (n = 35/group; 30 for the challenge experiment and 5 for the flow cytometry analysis). The chicks in Group 1 received a single dose of CpG-ODN by the intramuscular route on day 4 (D4) posthatch (PH), and Group 2 received drinking water (DW) with a probiotic product (D1-D15 PH, DW). The Group 3 chicks received tetracycline antibiotics during D9-D13 in DW; the Group 4 chicks got sodium sulfamethazine on D9, D10, and D15 PH in DW; and the Group 5 chicks were administered intramuscular (IM) saline D4 PH, DW. We challenged all the groups (n = 30/group) with E. coli (1 × 105 or 1 × 106 colony-forming units/bird) on D8 PH through the subcutaneous route. Our data demonstrated that the CpG-ODNs, but not the probiotics, could protect neonatal broiler chickens against lethal E. coli septicemia, as would the tetracycline or sodium sulfamethazine. The flow cytometry analysis (n = 5/group) revealed enrichment of immune cells in the CpG-ODN group and a marked decrease in macrophages and T-cell numbers in antibiotics-treated groups, indicating immunosuppressive effects. Our data showed that, like therapeutic antibiotics, CpG-ODNs reduced clinical signs, decreased bacterial loads, and induced protection in chicks against E. coli septicemia. Unlike therapeutic antibiotics-induced immunosuppressive effects, CpG-ODN caused immune enrichment by increasing chicken immune cells recruitment. Furthermore, this study highlights that, although therapeutic antibiotics can treat bacterial infections, the ensuing immunosuppressive effects may negatively impact the overall chicken health.

Comparación de antibióticos terapéuticos, probióticos y CpG-ODN sintéticos en su eficacia protectora contra la infección letal por Escherichia coli y el impacto en el sistema inmunológico en pollos de engorde recién eclosionados. La industria avícola necesita alternativas a los antibióticos ya que existe una creciente preocupación pública sobre la aparición de resistencia a los antimicrobianos debido a su uso en la producción animal. Se ha informado que la administración de oligodesoxinucleótidos de ADN sintético que contienen motivos de dinucleótidos de citosina guanina (CpG) no metilados (CpG-ODN) a pollitos recién eclosionados puede proteger contra patógenos bacterianos en pollos. El objetivo de este estudio fue comparar los efectos inmunoprotectores de CpG-ODN y de los probióticos contra la infección por Escherichia coli frente a los antibióticos terapéuticos de uso común. Los pollos de engorde de un día se dividieron en cinco grupos (n = 35/grupo; 30 para el experimento de desafío y 5 para análisis de citometría de flujo). Los pollitos del Grupo 1 recibieron una dosis única de CpG-ODN por vía intramuscular el día 4 (D4) después de la eclosión (PH), y el Grupo 2 recibió agua potable (DW) con un producto probiótico del día uno al quince después de la eclosion en agua de bebida. Los pollitos del Grupo 3 recibieron tetraciclina durante los días nueve a trece (D9­D13) en agua de bebida (DW9; los pollitos del Grupo 4 recibieron sulfametazina de sodio en los días nueve, diez y 15 (D9, D10 y D15) después de la eclosion en agua de bebida; ya los pollitos del Grupo 5 se les administró solución salina intramuscular (IM) al día cuatro después de la eclosión en agua de bebida. Se desafiaron todos los grupos (n = 30/grupo) con E. coli (1 × 105 o 1 × 106 unidades formadoras de colonias/ave) en el día ocho después de la eclosión por vía subcutánea. Nuestros datos demostraron que los CpG-ODN, pero no los probióticos, pudieron proteger a los pollos de engorde recién eclosionados contra la septicemia letal por E. coli, al igual que la tetraciclina o la sulfametazina sódica. El análisis de citometría de flujo (n = 5/grupo) reveló un enriquecimiento de células inmunes en el grupo CpG-ODN y una marcada disminución en el número de macrófagos y células T en los grupos tratados con antibióticos, lo que indica efectos inmunosupresores. Nuestros datos mostraron que, al igual que los antibióticos terapéuticos, los CpG-ODN redujeron los signos clínicos, disminuyeron las cargas bacterianas e indujeron protección en los pollitos contra la septicemia por E. coli. A diferencia de los efectos inmunosupresores inducidos por antibióticos terapéuticos, los CpG-ODN provocaron un enriquecimiento inmunitario al aumentar el reclutamiento de células inmunitarias de pollo. Además, este estudio destaca que, aunque los antibióticos terapéuticos pueden tratar las infecciones bacterianas, los efectos inmunosupresores resultantes pueden tener un impacto negativo en la salud general de los pollos.

Virulence of Emerging Arthrotropic Avian Reoviruses Correlates With Their Ability to Activate and Traffic Interferon-γ Producing Cytotoxic CD8+ T Cells Into Gastrocnemius Tendon.

Newly emerging arthrotropic avian reoviruses (ARVs) are genetically divergent, antigenically heterogeneous, and economically costly. Nevertheless, the mechanism of emerging ARV-induced disease pathogenesis and potential differences in virulence between virus genotypes have not been adequately addressed. In this study, the life cycle of ARV, including the formation of cytoplasmic ARV neo-organelles, paracrystalline structures, and virus release mechanisms, were characterized in the infected host cell by transmission electron microscopy (TEM). In addition, progressive changes in the structure of infected cells were investigated by time-lapse and field emission scanning electron (FE-SE) microscopy. ARVs from the four genotypic cluster groups included in the study caused gross and microscopic lesions in the infected birds. Marked infiltration of γδT cells, CD4+ and CD8+ T lymphocytes were observed in ARV infected tendon tissues starting day 3 post-infection. The ARV variant from genotype cluster-2 triggered significantly high trafficking of IFN-γ producing CD8+ T lymphocytes in tendon tissues and concomitantly showed high morbidity and severe disease manifestations. In contrast, the ARV variant from genotype cluster-4 was less virulent, caused milder disease, and accompanied less infiltration of IFN-γ producing CD8+ T cells. Interestingly, when we blunted antiviral immune responses using clodronate liposomes (which depletes antigen-presenting cells) or cyclosporin (which inhibits cytokine production that regulates T-cell proliferation), significantly lower IFN-γ producing CD8+ T cells infiltrated into tendon tissues, resulting in reduced tendon tissues apoptosis and milder disease manifestations. In summary, these data suggest that the degree of ARV virulence and tenosynovitis/arthritis are potentially directly associated with the ability of the virus to traffic massive infiltration of cytotoxic CD8+ T cells into the infected tissues. Moreover, the ability to traffic cytotoxic CD8+ T cells into infected tendon tissues and the severity of tenosynovitis differ between variants from different ARV genotype cluster groups. However, more than one virus isolate per genotype group needs to be tested to further confirm the association of pathogenicity with genotype. These findings can be used to further examine the interaction of viral and cellular pathways which are essential for the pathogenesis of the disease at the molecular level and to develop effective disease control strategies.

IN SITU HYBRIDIZATION AND VIRUS CHARACTERIZATION OF SKUNK ADENOVIRUS IN NORTH AMERICAN WILDLIFE REVEALS MULTISYSTEMIC INFECTIONS IN A BROAD RANGE OF HOSTS.

Skunk adenovirus-1 (SkAdV-1) has been reported infecting several North American wildlife species; however, lesions associated with disease have not yet been completely characterized, particularly in porcupines. We describe and characterize the tissue distribution and lesions associated with SkAdV-1 infection in 24 wildlife diagnostic cases submitted between 2015 and 2020, including 16 North American porcupines (Erethizon dorsatum), three striped skunks (Mephitis mephitis), and five raccoons (Procyon lotor), which constitute a new host species. The most common lesion in all species was severe necrotizing bronchopneumonia with (n=12) or without (n=10) interstitial involvement. Intranuclear inclusion bodies were common in respiratory epithelium (n=21) and less often in renal tubular (n=6) and biliary epithelium (n=1). Several cases (n=4) had secondary bacterial infections, including Bordetella bronchiseptica, Pasteurella multocida, and Streptococcus zooepidemicus. In situ hybridization in porcupine (n=6), raccoon (n=1), and skunk (n=1) revealed SkAdV-1 DNA in multiple tissue types, including lung, trachea, turbinates, liver, kidney, lymph node, and brain, and multiple cell types including epithelial, endothelial, and mesothelial cells. These findings were consistent across species. Comparison of viral genomes from a porcupine and a raccoon with that originally isolated from a skunk demonstrated DNA point mutations affecting several viral genes, including the fiber protein gene. Our findings show the spectrum of disease associated with SkAdV-1 infection in a broad host range of wildlife species.

Species-specific PCR assay for the detection of Babesia odocoilei.

We developed a PCR assay for the detection of Babesia odocoilei based on the 18S rRNA gene. Multiple specimens of B. odocoilei were examined, and the assay consistently produced a small specific PCR product of 306 bp. The PCR assay was also challenged with DNA from 13 other Babesia species and 2 Theileria species, originating from 10 different host species; however, nonspecific DNA amplification and multiple banding patterns were observed, and the amplicon banding patterns varied between different isolates of the same species. Sensitivity was determined to be 6.4 pg of DNA, and an estimated 0.0001% parasitism. This assay can be utilized for species-specific differential detection of B. odocoilei.

CpG-ODN induced antimicrobial immunity in neonatal chicks involves a substantial shift in serum metabolic profiles.

Synthetic CpG-ODNs can promote antimicrobial immunity in neonatal chicks by enriching immune compartments and activating immune cells. Activated immune cells undergo profound metabolic changes to meet cellular biosynthesis and energy demands and facilitate the signaling processes. We hypothesize that CpG-ODNs induced immune activation can change the host's metabolic demands in neonatal chicks. Here, we used NMR-based metabolomics to explore the potential of immuno-metabolic interactions in the orchestration of CpG-ODN-induced antimicrobial immunity. We administered CpG-ODNs to day-old broiler chicks via intrapulmonary (IPL) and intramuscular (IM) routes. A negative control group was administered IPL distilled water (DW). In each group (n = 60), chicks (n = 40) were challenged with a lethal dose of Escherichia coli, two days post-CpG-ODN administration. CpG-ODN administered chicks had significantly higher survival (P < 0.05), significantly lower cumulative clinical scores (P < 0.05), and lower bacterial loads (P < 0.05) compared to the DW control group. In parallel experiments, we compared NMR-based serum metabolomic profiles in neonatal chicks (n = 20/group, 24 h post-treatment) treated with IM versus IPL CpG-ODNs or distilled water (DW) control. Serum metabolomics revealed that IM administration of CpG-ODN resulted in a highly significant and consistent decrease in amino acids, purines, betaine, choline, acetate, and a slight decrease in glucose. IPL CpG-ODN treatment resulted in a similar decrease in purines and choline but less extensive decrease in amino acids, a stronger decrease in acetate, and a considerable increase in 2-hydroxybutyrate, 3-hydroxybutyrate, formic acid and a mild increase in TCA cycle intermediates (all P < 0.05 after FDR adjustment). These perturbations in pathways associated with energy production, amino acid metabolism and nucleotide synthesis, most probably reflect increased uptake of nutrients to the cells, to support cell proliferation triggered by the innate immune response. Our study revealed for the first time that CpG-ODNs change the metabolomic landscape to establish antimicrobial immunity in neonatal chicks. The metabolites highlighted in the present study can help future targeted studies to better understand immunometabolic interactions and pinpoint the key molecules or pathways contributing to immunity.

Prevalence of Equus caballus Papillomavirus Type-2 Infection and Seropositivity in Asymptomatic Western Canadian Horses.

Equus caballus papillomavirus type 2 (EcPV-2) has been recognized as a potential cause of a subset of genital squamous cell carcinomas (SCCs) in horses. In the current study, we measured EcPV-2 seropositivity in 50 healthy horses from Western Canada, and these were compared to a herd of horses with known EcPV-2 exposure. Second, the presence of EcPV-2 DNA was measured using EcPV-2-specific PCR (polymerase chain reaction), performed on a variety of tissues collected at necropsy from 70 horses that lacked any history, gross, or histologic evidence of neoplasia or papillomavirus-associated disease. EcPV-2-specific RNA in situ hybridization (R-ISH) was performed on PCR-positive samples to identify the specific tissues infected. The prevalence of asymptomatic infection with EcPV-2 in Western Canadian horses was 20/70 (29%). Exposure to EcPV-2 as measured by seropositivity was 18/50 (36%). EcPV-2 positivity by anatomic location, as measured by R-ISH, was as follows: penis 10/29 (35%), vulva 5/34 (15%), eyelid 8/68 (12%), oral mucosa 7/65 (11%), skin from muzzle 7/68 (10%), and retropharyngeal lymph node 2/64 (3%). The youngest horses with EcPV-2 infection, based on PCR, were fetuses, suggesting for the first time that vertical transmission of EcPV-2 occurs in horses. The current study observed an increased prevalence of EcPV-2 as compared to previous studies. We suggest that this difference is due to our use of biopsies in place of superficial swabs. We propose that EcPV-2 infection in asymptomatic horses is more common than previously reported and that the virus' role in equine genital SCCs may be more complex than originally thought.

Prevalence and Prognostic Impact of Equus caballus Papillomavirus Type 2 Infection in Equine Squamous Cell Carcinomas in Western Canadian Horses.

Equus caballus papillomavirus type-2 (EcPV-2) has been proposed as a causal factor in equine genital squamous cell carcinoma (SCC). This study had 2 objectives: first, calculate the frequency of papillomavirus (PV) and EcPV-2 infection in papillomas, carcinomas in situ (CIS), and SCCs in Western Canadian horses; and second, determine if EcPV-2 status of equine SCCs is associated with overall survival (OS). EcPV-2 status of 115 archived tissue samples, spanning 6 years, was determined using broad spectrum (MY09/11) and EcPV-2-specific polymerase chain reaction (PCR) assays, EcPV-2-E6/E7 chromogenic RNA in situ hybridization (R-ISH), and amplicon sequencing. A retrospective survey gathered data on history, outcome, breeding, treatment, and rationales of referring veterinarians when managing PV-associated diseases. Histologic grade and completeness of surgical margins of SCCs were also considered. EcPV-2 DNA was identified in 10/58 (17%) SCC, 8/27 (30%) papillomas, 0/5 CIS, and 0/11 lesions identified as "other." Overall, 18/101 (18%) of these lesions were positive for EcPV-2. EcPV-2 was identified in 10/35 (29%) SCCs arising from genital tissues but in 0/22 SCCs from other locations. There was no association between breeding history and EcPV-2 status of genital SCCs. EcPV-2 status of genital SCCs was not associated with OS (P = .76). The strongest negative predictors of OS were a lack of treatment (P < .01) and recurrence post-treatment (P < .01). Weaker predictors of OS included older age at time of diagnosis (P = .02). Completeness of margins at surgical excision, concurrent disease, treatment type, anatomic location of the SCC (anogenital vs other), and histologic grade of the SCC did not influence OS (P > .1).

The dynamics of molecular evolution of emerging avian reoviruses through accumulation of point mutations and genetic re-assortment.

In the last decade, the emergence of variant strains of avian reovirus (ARV) has caused enormous economic impact in the poultry industry across Canada and USA. ARVs are non-enveloped viruses with ten segments of double-stranded RNA genome. So far, only six genotyping cluster groups are identified worldwide based on sequence analysis of the σC protein encoded by the S1 segment. In this study, we performed deep next generation whole-genome sequencing and analysis of twelve purified ARVs isolated from Saskatchewan, Canada. The viruses represent different genotyping cluster. A genome-wide sequence divergence of up to 25 per cent was observed between the virus isolates with a comparable and contrasting evolutionary history. The proportion of synonymous single-nucleotide variations (sSNVs) was higher than the non-synonymous (ns) SNVs across all the genomic segments. Genomic segment S1 was the most variable as compared with the other genes followed by segment M2. Evidence of positive episodic/diversifying selection was observed at different codon positions in the σC protein sequence, which is the genetic marker for the classification of ARV genotypes. In addition, the N-terminus of σC protein had a persuasive diversifying selection, which was not detected in other genomic segments. We identified only four ARV genotypes based on the most variable σC gene sequence. However, a different pattern of phylogenetic clustering was observed with concatenated whole-genome sequences. Together with the accumulation of point mutations, multiple re-assortment events appeared as mechanisms of ARV evolution. For the first time, we determined the mean rate of molecular evolution of ARVs, which was computed as 2.3 × 10-3 substitution/site/year. In addition, widespread geographic intermixing of ARVs was observed between Canada and USA, and between different countries of the world. In conclusion, the study provides a comprehensive analysis of the complete genome of different genotyping clusters of ARVs including their molecular rate of evolution and spatial distribution. The new findings in this study can be utilized for the development of effective vaccines and other control strategies against ARV-induced arthritis/tenosynovitis in the poultry industry worldwide.

CpG-ODN Induces a Dose-Dependent Enrichment of Immunological Niches in the Spleen and Lungs of Neonatal Chicks That Correlates with the Protective Immunity against Escherichia coli.

Immunoprotective function of oligodeoxynucleotides containing CpG motifs (CpG-ODN) has been demonstrated in neonatal chickens against common bacterial pathogens such as E.coli and Salmonella sp. Our recent study reported that CpG-ODN administration enriches immune compartments in neonatal chicks. However, a causal relationship between CpG-ODN-induced immune enrichment and protective mechanisms remains unestablished. In this study, we investigated in ovo administered CpG-ODN-mediated immune cell recruitment in the immunological niches in lymphoid (spleen) and nonlymphoid (lungs) organs using various doses of CpG-ODN and examined whether the immunological profiles have any correlation with immunoprotection against E.coli infection. Eighteen-day-old embryonated eggs were injected with either 5, 10, 25, and 50 µg of CpG-ODN or saline (n = ~40 per group). On the day of hatch (72 hr after CpG-ODN treatment), we collected the spleen and lungs (n = 3-4 per group) and examined the recruitment of macrophages/monocytes, their expression of MHCII and CD40, and the number of CD4+ and CD8+ T-cell subsets in the immunological niches in the spleen and lungs using flow cytometry. We observed the dose-dependent recruitment of immune cells, wherein 25 µg and 50 µg of CpG-ODN induced significant enrichment of immunological niches in both the spleen and the lungs. Four days after the CpG-ODN treatment (1-day after hatch), chicks were challenged with a virulent strain of E. coli (1 × 104 or 1 × 105 cfu, subcutaneously). Clinical outcome and mortality were monitored for 8 days postchallenge. We found that both 25 µg and 50 µg of CpG-ODN provided significant protection and reduced clinical scores compared to saline controls against E. coli infection. Overall, the present study revealed that CpG-ODNs orchestrate immunological niches in neonatal chickens in a dose-dependent manner that resulted in differential protection against E. coli infection, thus supporting a cause and effect relationship between CpG-ODN-induced immune enrichment and the antibacterial immunity.

Mucosal delivery of CpG-ODN mimicking bacterial DNA via the intrapulmonary route induces systemic antimicrobial immune responses in neonatal chicks.

The transition to antibiotic-free poultry production in the face of pathogenic threats is a very challenging task. We recently demonstrated that mucosal delivery of CpG-ODN alone by the intrapulmonary route (IPL) has potential as an effective alternative to antibiotics in neonatal chicks against Escherichia coli septicemia. How exactly mucosal delivery of CpG-ODN elicits, protective antibacterial immunity remained poorly understood. In this study, CpG-ODN or saline was delivered via the intrapulmonary route to day-old chicks (n = 80/group) using a compressor nebulizer in an acrylic chamber (1 mg/mL CpG-ODN for 15 minutes). In the first part of the study, two days after mucosal CpG-ODN delivery, 40 chicks from each group were challenged subcutaneously with 1 × 105 cfu (n = 20) or 1 × 106 cfu (n = 20) of E. coli and the mortality pattern was monitored for seven days. We found significantly higher survival, better clinical conditions and lower bacterial loads in chicks that received mucosal CpG-ODN. To explore the mechanisms behind this protective immunity, we first looked at the kinetics of the cytokine gene expression (three birds/ group/ time for 10 time-points) in the lungs and spleens. Multiplex gene analysis demonstrated a significant elevation of pro-inflammatory cytokine genes mRNA in the CpG-ODN group. Interleukin (IL)-1ß robustly upregulated many folds in the lung after CpG-ODN delivery. Lipopolysaccharide-induced tumor necrosis factor (LITAF) and IL-18 showed expression for an extended period in the lungs. Anti-inflammatory cytokine IL-10 was upregulated in both lungs and spleen, whereas IL-4 showed upregulation in the lungs. To investigate the kinetics of immune enrichment in the lungs and spleens, we performed flow cytometry, histology, and immunohistochemistry at 24, 48 and 72 hrs after CpG-ODN delivery. CpG-ODN treated lungs showed a significant enrichment with monocytes/macrophages and CD4+ and CD8+ T-cell subsets. Macrophages in CpG-ODN treated group demonstrated mature phenotypes (higher CD40 and MHCII expression). Importantly, mucosal delivery of CpG-ODN via the intrapulmonary route significantly enriched immune compartment in the spleen as well, suggesting a systemic effect in neonatal chicks. Altogether, intrapulmonary delivery of aerosolized CpG-ODN orchestrates protective immunity against E. coli septicemia by not only enhancing mucosal immunity but also the systemic immune responses.

Use of slide scrape lysates for polymerase chain reaction confirmation of disseminated Mycobacterium avium infection in a cat.

Disseminated mycobacteriosis in a 3-year-old domestic medium-haired cat was diagnosed on lymph node cytology. Slide scrape lysates from the cytology submission were used to confirm Mycobacterium avium by polymerase chain reaction (PCR) and sequencing and proved a simple technique that could be a valuable tool in veterinary diagnostics and research.

Utilisation d'un lysat de grattage de lame pour la confirmation par amplification en chaîne par la polymérase d'une infection disséminée à Mycobacterium avium chez un chat. Une mycobactériose disséminée chez un chat domestique à poil moyen âgé de 3 ans a été diagnostiquée à l'aide d'une cytologie des ganglions lymphatiques. La soumission d'un lysat de grattage d'une lame provenant de la soumission de cytologie a été utilisé pour confirmer Mycobacterium avium par amplification en chaîne par la (PCR) et séquençage et elle s'est avérée une technique simple qui pourrait être un outil utile dans les diagnostics et la recherche vétérinaires.(Traduit par Isabelle Vallières).

Synthetic CpG-ODN rapidly enriches immune compartments in neonatal chicks to induce protective immunity against bacterial infections.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) induce innate immunity against bacterial infections. Despite recent advances, how CpG-ODN alone protects against bacterial infections remained elusive. Here, we report for the first time, to our knowledge, that CpG-ODN orchestrates anti-microbial protective immunity by inducing a rapid enrichment of various immune compartments in chickens. In this study, eighteen-day-old embryonated eggs were injected with either 50 µg of CpG-ODN or saline (~n = 90 per group). In the first experiment, four days after CpG-ODN treatment, chicks were challenged subcutaneously with a virulent strain of Escherichia coli (E. coli) and mortality was monitored for 8 days. We found significant protection, and reduced clinical scores in CpG-ODN treated chicks. To gain insights into mechanisms of protection induced by CpG-ODN, first we investigated cytokine expression kinetics elicited by CpG-ODN. The spleen and lung were collected from embryos or chicks (n = 3-4 per group) at 10 time points post-CpG-ODN inoculation. Multiplex gene analysis (interleukin (IL)-1, IL-4, IL-6, IL-10, IL-18, interferon (IFN)-γ, IFN-α, and lipopolysaccharide induced tumor necrosis factor (LITAF), revealed a significantly higher expression of pro-inflammatory cytokines following CpG-ODN treatment compared to the saline controls. In our study, LITAF stands out in the cytokine profiles of spleen and lungs, underscoring its role in CpG-ODN-induced protection. The third experiment was designed to examine the effects of CpG-ODN on immune cell populations in spleen, lungs, and thymus. Flow cytometry analysis was conducted at 24, 48 and 72 hrs (thymus only collected at 72 hr) after CpG-ODN administration to examine the changes in CD4+ and CD8+ T-cell subsets, monocyte/macrophage cell populations and their expression of maturation markers (CD40 and CD86). Flow cytometry data indicated a significant enrichment of macrophages, CD4+ and CD8+ T-cell subsets in both spleen and lungs of CpG-ODN treated embryos and chicks. Macrophages in spleen and lungs showed an upregulation of CD40 but not CD86, whereas thymocytes revealed significantly high CD4 and CD8 expression. Overall, the present study has demonstrated that CpG-ODN provides protection in neonatal chicks against E. coli infection not only by eliciting cytokine responses and stimulating immune cells but also through enriching immunological niches in spleen and lungs.

Broad spectrum protection of broiler chickens against inclusion body hepatitis by immunizing their broiler breeder parents with a bivalent live fowl adenovirus vaccine.

Historically, fowl adenovirus (FAdV) associated inclusion body hepatitis (IBH) was considered a secondary disease in broiler chickens associated with immunosuppression. However, we previously reported the occurrence of IBH as a primary disease in the broiler chicken industry in Canada as a result of infections with various FAdV serotypes. Therefore, the objectives of this study were to develop an immunization strategy in broiler breeders using live FAdV 11-1047 and FAdV8a-TR59 to confer homologous and heterologous protection in broiler progeny against IBH and to study the efficacy of natural exposure of naïve broiler breeders to a vaccine virus from live FAdV vaccinated birds as an immunization technique. Broiler breeders vaccinated orally with FAdV8a-TR59 (1 × 104 TCID50/bird) and FAdV11-1047 (1 × 104 TCID50/bird), FAdV8a-TR59 (1 × 106 TCID50/bird) and FAdV11-1047 (1 × 106 TCID50/bird) or FAdV8b (1 × 106 TCID50/bird) transferred substantial levels of neutralizing antibodies to their progeny. The efficacy of maternal antibodies was studied by challenging 14-day old broiler chickens with 1 × 107 TCID50 of FAdV2-685, FAdV7-x11a like, FAdV8a-TR59, FAdV8b-SK or FAdV11-1047 which are the dominant serotypes causing IBH outbreaks in Canada. Broiler chickens from the low and high dose vaccinated breeders were significantly protected against all serotypes of FAdV (P < 0.05). Comingling of unvaccinated broiler breeders with FAdV-vaccinated broiler breeders was an effective immunization technique for in-contact naïve birds. This study confirms that IBH can be effectively controlled in Canada by vaccination of broiler breeder parents with a bivalent vaccine containing live FAdV8a-TR59 and FAdV11-1047.