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.

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).

Protection of Neonatal Broiler Chickens Following in ovo Delivery of Oligodeoxynucleotides Containing CpG Motifs (CpG-ODN) Formulated with Carbon Nanotubes or Liposomes.

Unformulated oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to stimulate the innate immune system against a variety of bacterial, viral, and protozoan infections in a variety of vertebrate species. We have previously shown that in ovo delivery of unformulated CpG-ODN was able to significantly protect neonatal broiler chickens against Escherichia coli or Salmonella Typhimurium infections. The objectives of this study were to examine the safety and immunoprotective effects of CpG-ODN formulated with 2 types of carbon nanotubes (CNTs) or 2 types of lipid-surfactant (LSC) delivery systems in neonatal broilers against E. coli septicemia. Embryonated eggs, which had been incubated for 18 days, received either 50 µg of CNT-CpG-ODN, 50 µg of LSC-CpG-ODN, 50 µg of unformulated CpG-ODN, or saline. Four days after exposure to CpG-ODN (day 1 posthatch), 1 x 10(4) or 1 x 10(5) colony-forming units of a virulent strain of E. coli isolated from a turkey with septicemia were inoculated subcutaneously in the neck. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for 8 days following challenge with E. coli. Bacterial isolations and pathologic observations were conducted immediately after birds were dead or euthanatized. The survival rate of birds in groups receiving saline following E. coli infection was 20% to 30%. In contrast, birds receiving CpG-ODN formulations had a significantly higher survival rate of 60% to 80% (P < 0.01). Bacterial loads and clinical scores were significantly lower (P < 0.05) in groups treated with CNT- or LSC-CpG-ODN compared to the groups receiving CpG-ODN or saline. Moreover, there is no evidence of any adverse effects of these formulations in any organs or in growth rates of birds until 42 days of age. This is the first time that CpG-ODN formulated with CNT and LSC have been demonstrated to have an immunomodulatory effect against an E. coli infection in neonatal broiler chickens following in ovo delivery.

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.

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.

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.

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.

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.

Inactivated and live bivalent fowl adenovirus (FAdV8b + FAdV11) breeder vaccines provide broad-spectrum protection in chicks against inclusion body hepatitis (IBH).

Fowl adenovirus (FAdV) is comprised of five species (A to E) and 12 serotypes (1-7, 8a, 8b, 9-11). Inclusion body hepatitis (IBH) is caused by FAdV-7, 8a, 8b (species E) and FAdV-2 and 11 (species D). Commercial vaccines against IBH are not available in Canada. Autogenous FAdV broiler breeder vaccines are now used in some areas where outbreaks of IBH are occurring. The objective of this study was to evaluate the efficacy of a bivalent (species D and E) live and an inactivated FAdV broiler breeder vaccine in protecting broiler chicks against IBH through maternal antibody (MtAb) transfer. FAdV seronegative broiler breeders (n = 300/group) received either a live or inactivated bivalent (FAdV-8b-SK + FAdV-11-1047) vaccine. The live vaccine (1 × 104 TCID50 of each virus/bird) was given orally once at 16 weeks of age and the inactivated vaccine (1 × 106TCID50 of each virus + 20% Emulsigen D) was given intramuscularly at 16 and 19 weeks of age. Controls (n = 150) were given saline orally. The inactivated vaccine group was boosted 3 weeks later with the same vaccine. Neutralizing antibodies (NAb) in sera (n = 10) were detected at 19, 22, 30 and 48 weeks of age. NAb were able to neutralize various FAdV serotypes within species D and E. Mean NAb were similar in the both live and killed vaccine groups at 19, 30 and 48 weeks and ranged from 2.4 to 3.7 log10. Approximately 26 ±â€¯7% of MtAbs were passively transferred through eggs to day-old chicks. Progeny challenged with a lethal dose (1 × 107 TCID50/bird intramuscularly) of FAdV-8b-SK, FAdV-11-1047, or FAdV-2-685 (n = 90/group) at 14 days post-hatch (dph) showed 98-100% protection in broiler chicks to homologous or heterologous FAdV challenges. Our data suggests that a bivalent live and an inactivated FAdV vaccine are equally effective and have the potential for the control of IBH.

Intrapulmonary Delivery of CpG-ODN Microdroplets Provides Protection Against Escherichia coli Septicemia in Neonatal Broiler Chickens.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine phosphodiester guanine (CpG) motifs (CpG-ODN) are effective immunostimulatory agents against a variety of viral, bacterial, and protozoan diseases in different animals including poultry. We have recently demonstrated that in ovo injection of CpG-ODN confers protection in neonatal chickens against bacterial septicemias. The objective of this study was to investigate the effectiveness of needle-free intrapulmonary (IPL) delivery of CpG-ODN microdroplets against Escherichia coli infection in neonatal chicks. In the present study, we used 880 chicks in total keeping 40 chicks per group. Chicks were delivered CpG-ODN or saline by IPL at the day 1 of hatch. Three days later, chicks were challenged with two doses (1 × 104 CFU, n = 20 or 1 × 105 CFU, n = 20) of E. coli. Chicks treated with CpG-ODN by the IPL route had significantly lower clinical signs and bacterial load compared to the group treated with saline ( P < 0.05). CpG-ODN-treated groups were significantly protected against E. coli septicemia. We observed dose- and exposure time-dependent immunoprotective effects of IPL CpG-ODN in chicks. We found that IPL delivery of CpG-ODN can induce protective immunity as early as 6 hr that remains effective at least until day 5 post-treatment. Moreover, there were no adverse effects of IPL delivery of CpG-ODN on growth or mortality up to 42 days of age. Based on these findings, it can be suggested that CpG-ODN delivery by IPL route can be a promising alternative to antibiotics for inducing protective immunity in chicks during the critical first week of neonatal life.

Modified live infectious bursal disease virus (IBDV) vaccine delays infection of neonatal broiler chickens with variant IBDV compared to turkey herpesvirus (HVT)-IBDV vectored vaccine.

Chickens are commonly processed around 35-45days of age in broiler chicken industry hence; diseases that occur at a young age are of paramount economic importance. Early age infection with infectious bursal disease virus (IBDV) results in long-lasting immunosuppression and profound economic losses. To our knowledge, this is the first study comparing the protection efficacy of modified live (MdLV) IBDV and herpesvirus turkey (HVT)-IBDV vaccines against early age variant IBDV (varIBDV) infection in chicks. Experiments were carried out in IBDV maternal antibody (MtAb) positive chicks (n=330), divided into 6 groups (n=50-60/group), namely Group 1 (saline), Group 2 (saline+varIBDV), Group 3 (HVT-IBDV), Group 4 (HVT-IBDV+varIBDV), Group 5 (MdLV) and Group 6 (MdLV+varIBDV). HVT-IBDV vaccination was given via the in ovo route to 18-day-old embryonated eggs. MdLV was administered via the subcutaneous route in day-old broilers. Group 2, Group 4 and Group 6 were orally challenged with varIBDV (SK-09, 3×103 EID50) at day 6 post-hatch. IBDV seroconversion, bursal weight to body weight ratio (BBW) and bursal histopathology were assessed at 19 and 35days of age. Histopathological examination at day 19 revealed that varIBDV-SK09 challenge caused severe bursal atrophy and lower BBW in HVT-IBDV but not in MdLV vaccinated chicks. However by day 35, all challenged groups showed bursal atrophy and seroconversion. Interestingly, RT-qPCR analysis after varIBDV-SK09 challenge demonstrated an early (9days of age) and significantly high viral load (∼5744 folds) in HVT-IBDV vaccinated group vs unvaccinated challenged group (∼2.25 folds). Furthermore, flow cytometry analysis revealed inhibition of cytotoxic CD8+ T-cell response (CD44-downregulation) and decreased splenic lymphocytes counts in chicks after HVT-IBDV vaccination. Overall, our data suggest that MdLV delays varIBDV pathogenesis, whereas, HVT-IBDV vaccine is potentially immunosuppressive, which may increase the risk of early age varIBDV infection in broilers.

Increased Incidence of Enterococcal Infection in Nonviable Broiler Chicken Embryos in Western Canadian Hatcheries as Detected by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry.

The emergence of enterococcal infections in neonatal broiler chickens in the poultry industry has become common in many countries, including Canada. The objective of this study was to examine the bacterial infections in nonviable broiler chicken embryos in three western Canadian poultry hatcheries using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The pattern of embryo mortality that occurred during incubation and the breakout analysis results were similar in all three hatcheries. The majority of embryo mortality occurred during the late stage of incubation (35.08%), followed by the early stage of incubation (15.35%). The breakout analysis showed that 65.82% of swabs had at least one type of bacterial growth while 34.17% of swabs were negative for bacterial isolation. Of those 65.82% swabs with bacterial growth, 34.3% of swabs yielded a mixed bacterial population while 31.52% yielded one type of bacterial growth. The frequency of bacterial isolation from hatch debris (60%-75%) increased with the age of broiler breeders. MALDI-TOF MS was able to provide genus-level identification of 83.13% of isolates among all bacterial types isolated. MALDI-TOF MS identified Enterococcus and Escherichia coli isolates with 97.18% and 100% accuracy at species level, respectively, whereas Staphylococcus species were identified with 62.59% accuracy. The congruence between MALDI-TOF MS identification and 16S rRNA or cpn60 universal gene target sequencing was 100% or 90%, respectively. Of all bacteria isolated, Enterococcus species (29.71%) were the most prevalent, followed by E. coli (19.46%). About 56% of E. coli-infected samples were coinfected with Enterococcus species. Among all Enterococcus species isolated, Enterococcus faecalis (79.58%) was the most prevalent, followed by Enterococcus faecium (8.1%). Overall, our study showed that Enterococcus-associated embryo mortality was predominant in all three hatcheries investigated and suggests that MALDI-TOF MS technology can be applied to identify bacteria such as Enterococcus species isolated from poultry.