Marek's disease in chicken farms from Northwest Ethiopia: gross pathology, virus isolation, and molecular characterization.

Marek's disease virus (MDV) is a highly contagious, immunosuppressive, and oncogenic chicken pathogen causing marek's disease (MD). In this outbreak-based study, 70 dual-purpose chickens that originated from poultry farms in Northwest Ethiopia and suspected of MD were sampled for pathological and virological study from January 2020 to June 2020. Clinically, affected chickens showed inappetence, dyspnea, depression, shrunken combs, and paralysis of legs, wings, and neck, and death. Pathologically, single or multiple greyish white to yellow tumor-like nodular lesions of various size were appreciated in visceral organs. In addition, splenomegaly, hepatomegaly, renomegaly, and sciatic nerve enlargement were observed. Twenty-seven (27) pooled clinical samples i.e. 7 pooled spleen samples and 20 pooled feathers samples were aseptically collected. Confluent monolayer of Chicken Embryo Fibroblast cells was inoculated with a suspension of pathological samples. Of this, MDV-suggestive cytopathic effects were recorded in 5 (71.42%) and 17 (85%) pooled spleen and feather samples respectively. Molecular confirmation of pathogenic MDV was conducted using conventional PCR amplifying 318 bp of ICP4 gene of MDV-1, of which, 40.9% (9/22) tested positive. In addition, 5 PCR-positive samples from various farms were sequenced further confirming the identity of MDV. The ICP4 partial gene sequences were submitted to GenBank with the following accession numbers: OP485106, OP485107, OP485108, OP485109, and OP485110. Comparative phylogenetics showed, two of the isolates from the same site, Metema, seem to be clonal complexes forming distinct cluster. The other three isolates, two from Merawi and one from Debretabor, appear to represent distinct genotypes although the isolate from Debretabor is closer to the Metema clonal complex. On the other hand, the isolates from Merawi appeared genetically far related to the rest of the 3 isolates and clustered with Indian MDV strains included in the analysis. This study presented the first molecular evidence of MDV in chicken farms from Northwest Ethiopia. Biosecurity measures should strictly be implemented to hinder the spread of the virus. Nationwide studies on molecular characteristics of MDV isolates, their pathotypes, and estimation of the economic impact associated with the disease may help justify production and use of MD vaccines within the country.

Serological Evidence of Infectious Laryngotracheitis Infection and Associated Risk Factors in Chickens in Northwestern Ethiopia.

Infectious laryngotracheitis (ILT) is a disease of high economic consequence to the poultry sector. Gallid herpesvirus 1 (GaHV-1), a.k.a infectious laryngotracheitis virus (ILTV), under the genus Iltovirus, and the family Herpesviridae, is the agent responsible for the disease. Despite the clinical signs on the field suggestive of ILT, it has long been considered nonexistent and a disease of no concern in Ethiopia. A cross-sectional study was conducted from November 2020 to June 2021 in three selected zones of the Amhara region (Central Gondar, South Gondar, and West Gojjam zones), Ethiopia, with the objective of estimating the seroprevalence of ILTV in chickens and identifying and quantifying associated risk factors. A total of 768 serum samples were collected using multistage cluster sampling and assayed for anti-ILTV antibodies using indirect ELISA. A questionnaire survey was used to identify the potential risk factors. Of the 768 samples, 454 (59.1%, 95% CI: 0.56-0.63) tested positive for anti-ILTV antibodies. Mixed-effect logistic regression analysis of potential risk factors showed that local breeds of chicken were less likely to be seropositive than exotic breeds (OR: 0.38, 95% CI: 0.24-0.61). In addition, factors such as using local feed source (OR: 6.53, 95% CI: 1.77-24.04), rearing chickens extensively (OR: 1.97, 95% CI: 0.78-5.02), mixing of different batches of chicken (OR: 14.51, 95% CI: 3.35-62.77), careless disposal of litter (OR: 1.62, 95% CI: 0.49-4.37), lack of house disinfection (OR: 11.05, 95% CI: 4.09-47.95), lack of farm protective footwear and clothing (OR: 20.85, 95% CI: 5.40-80.45), and careless disposal of dead chicken bodies had all been associated with increased seropositivity to ILTV. Therefore, implementation of biosecurity measures is highly recommended to control and prevent the spread of ILTV. Furthermore, molecular confirmation and characterization of the virus from ILT suggestive cases should be considered to justify the use of ILT vaccines.

Seroprevalence and associated risk factors of equine herpesvirus type-1/-4 in selected districts of Northwest Amhara, Ethiopia.

This study aimed to estimate the prevalence, determine the distribution, and identify the epidemiological risk factors of EHV-1/-4 infections in selected districts of Northwest Amhara Region. 460 serum samples were collected from equines using multistage cluster sampling technique, and a competitive Enzyme-linked immunosorbent assay (cELISA) was performed. Various risk factors for the occurrence of EHV-1/-4 were considered. Statistical analysis was performed using R version 4.3.1. 65.9% (303) equids were tested positive for antibodies against EHV-1/-4. Based on district, the highest prevalence was recorded in Wogera (86.1%), while the lowest was in Debark (47.4%). There was a significant difference (p <0.05; 95% CI: 1.1067993-3.682843) in the prevalence of EHV-1/-4 among species and donkeys are 2.019 times more likely to get an EHV infection than horses. The prevalence of EHV-1/-4 was highest in equids with the age of 3-8 years and lowest in < 3 years, and the difference was statistically significant (p <0.05; 95% CI: 1.9812042-6.771820). Statistically significant variation (p <0.05; 95% CI: 1.1173822-2.684013) was also observed between sex of equids in which females had 1.73 times higher chance to get EHV infection than males. Higher prevalence was found in lactating equids (81.6%), followed by pregnant equids (74.6%), and dry equids (66.4%). Generally, this study indicated a high and wide distribution of EHV-1/-4 infection in the study area, which needs due attention. Devising strategies to prevent and minimize the spread and occurrence of the infection is crucial.

Application of structural equation modelling to inform best management strategies for Marek's disease in Amhara region, Ethiopia.

Marek's disease, a highly contagious and an economically significant oncogenic and paralytic viral diseases of poultry, is becoming a serious problem in Ethiopia's poultry sector. The aim of the study was to examine the relationship between risk factors and their contribution to develop risk with the intentions to implement MD control measures in the different chicken production systems of Ethiopia using the SEM framework. A questionnaire was designed based on the framework and each model constructed was measured using a set of rating scale items. Thus, a sample size of 200 farmers from different production systems were chosen for the data collection. From the analysis, Cornbrash's Alpha (coefficient of reliability) based on the average inter-item correlations were evaluated for each parameter. The result showed that when litter management goes up by 1, the number of sick goes down by 37.575, the number of staff goes up by 1, the number of sick goes down by 7.63, litter management goes up by 1, the number of deaths goes down by 2.505, flock size goes up by 1, the number of deaths goes down by 0.007 than the rest of the activities. The result of this structural equation modeling finding indicates that the data fit the model well (χ2 = 0.201, RMSEA = 0.000, CFI = 1.00, TLI = 1.496, Degrees of freedom = 2) and the model was appropriated. In conclusion, flock size, litter management and number of staff activities have more impact on the numbers of sick, drops in egg production and the number of deaths. Therefore, practicing regular awareness creation for producers regarding management techniques is recommended.

A Preliminary Investigation on a Commercial Ovine Pasteurellosis Vaccine Using Clinical and Pathological Endpoints.

Introduction: In this study we aimed to provide preliminary evidence on the safety and efficacy of the currently used ovine pasteurellosis vaccine in Ethiopia using clinical and pathological endpoints. Methods: Twenty, conventionally reared, apparently healthy, seronegative male lambs, were randomly classified into two groups of 10 animals as "vaccinated-challenged" and "unvaccinated-challenged controls". The first group received 1 mL of the licensed Pasteurella multocida biotype A based vaccine subcutaneously while the second group received phosphate-buffered saline as a placebo. Following vaccination, lambs were monitored for one month for potential vaccine adverse reactions. Five weeks postvaccination, all lambs were immunosuppressed using dexamethasone, and intratracheally challenged with 5.2×109 CFU/mL live Mannheimia haemolytica A1 (clinical isolates). Then, all lambs were followed up for eight days for clinical examination and necropsied on the ninth day postchallenge for pathological investigation. Results: There were no safety issues recorded during the study. In terms of clinical signs, lambs developed fever, depression, mucoid bilateral oculonasal discharge, coughing and sneezing regardless of their vaccination status. Fisher's exact test between vaccination status and each clinical sign showed a statistically insignificant association (p>0.05). The main pathological findings in both groups were pulmonary congestion, atelectasis, emphysema, and suppurative bronchopneumonia. Consolidation lung lesion score of +1 (5/10 of vaccinated, 6/10 of unvaccinated) and +2 (3/10 of vaccinated, 4/10 of unvaccinated) were recorded in a statistically indifferent manner among both vaccinated and nonvaccinated groups (p>0.05). Discussion and Conclusion: Collectively, the results suggested that the vaccine posed no safety concern and presumably lacks protective efficacy against local isolates. However, the study did not analyze antibody titer and their functionality using serum bactericidal assays. Further confirmatory studies could provide more evidence on the vaccine efficacy. Safety should further be assessed in a field setting involving a large number of animals to enable detection of rare vaccine adverse events.

Serotyping and Seroprevalence of Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi and Assessment of Determinants of Ovine Pasteurellosis in West Amhara Sub-region, Ethiopia.

A cross-sectional study was undertaken in four (4) districts of the West Amhara sub-region of Ethiopia with the aim of assessing the diversity and distribution of serotypes of Pasteurella species, their seroprevalence, and associated risk factors, and knowledge, attitude, and practice of farmers toward ovine pasteurellosis. A total of 600 sheep sera were collected using multistage cluster sampling. Each sample was examined for the presence of six (6) serotype-specific antibodies using an indirect haemagglutination test. We are reporting a higher seroprevalence of 90.17% (541/600) in which all seropositive animals were shown to have been co-infected with multiple serotypes. Individual serotype prevalence showed that serotype A7 has the highest prevalence of 77.83% followed by A2 (74.33%), T15 (64%), T4 (62%), PA (60%), and A1 (39.17%). In this study, being female [odds ratio (OR): 2.45, 95% CI (1.09-5.52), p = 0.031] and living in high altitude areas [OR: 20.29, 95% CI (2.54-161.95), p = 0.004] were found to be significantly associated with sero-positivity. A questionnaire survey (n = 384) employed in a face-to-face interview was used to assess the knowledge, attitude, and practice of farmers related to ovine pasteurellosis. Accordingly, the majority (72.4%) of respondents had an inadequate knowledge level of the disease. The proportion of farmers with a favorable attitude and good practices toward the disease was 50.26 and 77.6%, respectively. This study is highly indicative that ovine pasteurellosis is a ubiquitous disease in the study area challenging the sheep production sector. The existence of diverse serotypes reported to lack cross-protective immunity is likely to explain why the current vaccination practice with the mono-serotype Pasteurella multocida biotype A vaccine is not providing adequate protection against outbreaks of the disease. Prioritization of one or more serotypes for inclusion in a multivalent vaccine should be dictated by the abundance and distribution of a particular serotype, its clinical importance, and its resultant economic impact. Furthermore, training farmers on key aspects of the disease is vital in the implementation of effective disease management strategies through a participatory approach. Data from the remaining regions of the country could help realize the development of an effective vaccine that works best at the national level.

Isolation and Molecular Detection of Pasteurellosis from Pneumonic Sheep in Selected Areas of Amhara Region, Ethiopia: An Implication for Designing Effective Ovine Pasteurellosis Vaccine.

Introduction: Pneumonic pasteurellosis mainly caused by bacterial species of Mannheimia, Pasteurella, and Bibersteinia causes a significant financial loss to the sheep production sector through reduced productivity and high mortality. There is a dearth of information on the major agents involved in the disease in the Amhara region, Ethiopia. Therefore, the aim of this study was to isolate and molecularly confirm Mannheimia, Pasteurella, and Bibersteinia from nasal swabs of sheep suspected of pneumonic pasteurellosis in selected areas of the Amhara region. Methods: Isolation and phenotypic characterization were performed using microbiological and biochemical testing according to standard methods. Molecular confirmation of isolates was done through amplification of virulence associated genes, PHSAA and Rpt2, of Mannheimia hemolyticausing multiplex PCR. Results: Accordingly, 46 out of 141 (32.62%) samples were presumably identified as M. hemolytica with no Pasteurella multocida and Bibersteinia trehalosi. Seven (n=7) out of the 46 isolates tested positive for either of the two virulence genes. Discussion and conclusion: The finding of this study is indicative that M. hemolytica is the main bacteria linked with pneumonic pasteurellosis in the study area which suggests the need to develop a polyvalent vaccine including strains of M. hemolytica or its antigenic determinants. However, the role of other bacterial, viral, and parasitic agents in the cases investigated should also be considered.

Comparative Safety, Immunogenicity, and Efficacy of CEF Cell-Based and DF-1 Cell Line Adapted Infectious Bursal Disease Vaccines in Specific-Pathogen-Free Chickens.

Infectious bursal disease (IBD) is an immunosuppressive and economically important disease of young chickens caused by infectious bursal disease virus (IBDV). The National Veterinary Institute (Bishoftu, Ethiopia) produces intermediate IBDV vaccine using primary chicken embryo fibroblast (CEF) cells, a method with technical and economical cumbersome. This study assessed the safety, immunogenicity, and efficacy of DF-1 cell line-adapted IBDV LC-75 vaccine strain in reference to the CEF-based vaccine. Confluent monolayer of DF-1 cells was infected with IBDV and cells with cytopathic effects were passaged until 3rd passage. Viral growth was confirmed using a one-step RT-PCR targeting IBDV VP2 gene. Viral titer increased from 1st passage through 3rd passage. Safety was assessed in 30 specific-pathogen-free chickens (15 chickens/group) injected with 10-fold field dose of each vaccine intraocularly and monitored for 21 days. For immunogenicity and efficacy, 60 specific-pathogen-free chickens were grouped into 3 (20 chickens/group). First and 2nd group received DF-1 cell and CEF-based IBDV vaccines, respectively. The 3rd group served as unvaccinated control. Antibody response was measured using iELISA. Chickens were challenged 4 weeks postvaccination with very virulent IBDV (vvIBDV) intraocularly and followed-up for 10 days. Vaccination did not cause any adverse reactions during the 21 days of follow-up. In addition, both vaccines induced higher antibody titer 14 and 24 days-post-vaccination as compared to unvaccinated controls (p < 0.05). Moreover, DF-1 and CEF-based IBDV LC-75 vaccines rendered a complete protection against vvIBDV. Contrarily, morbidity and mortality in unvaccinated chickens was 50% and 30%, respectively. The results indicated that DF-1 and CEF cell-based IBDV vaccines are comparably immunogenic and efficacious. Therefore, DF-1 cell-line can be considered an affordable and convenient alternative to the CEF-based approach. The suitability of DF-1 cells to grow other IBDV strains and safety of these vaccines on bursa of Fabricius should further be investigated.

Gamma-Irradiated Fowl Cholera Mucosal Vaccine: Potential Vaccine Candidate for Safe and Effective Immunization of Chicken Against Fowl Cholera.

Fowl cholera (FC) caused by Pasteurella multocida is among the serious infectious diseases of poultry. Currently, formalin inactivated FC (FI-FC) vaccine is widely used in Ethiopia. However, reports of the disease complaint remain higher despite the use of the vaccine. The aim of this study was to develop and evaluate gamma-irradiated mucosal FC vaccines that can be used nationally. In a vaccination-challenge experiment, the performance of gamma-irradiated P. multocida (at 1 kGy) formulated with Montanide gel/01 PR adjuvant was evaluated at different dose rates (0.5 and 0.3 ml) and routes (intranasal, intraocular, and oral), in comparison with FI-FC vaccine in chicken. Chickens received three doses of the candidate vaccine at 3-week intervals. Sera, and trachea and crop lavage were collected to assess the antibody levels using indirect and sandwich ELISAs, respectively. Challenge exposure was conducted by inoculation at 3.5×109 CFU/ml of P. multocida biotype A intranasally 2 weeks after the last immunization. Repeated measures ANOVA test and Kaplan Meier curve analysis were used to examine for statistical significance of antibody titers and survival analysis, respectively. Sera IgG and secretory IgA titers were significantly raised after second immunization (p=0.0001). Chicken survival analysis showed that intranasal and intraocular administration of the candidate vaccine at the dose of 0.3 ml resulted in 100% protection as compared to intramuscular injection of FI-FC vaccine, which conferred 85% protection (p=0.002). In conclusion, the results of this study showed that gamma-irradiated FC mucosal vaccine is safe and protective, indicating its potential use for immunization of chicken against FC.

P10s-PADRE vaccine combined with neoadjuvant chemotherapy in ER-positive breast cancer patients induces humoral and cellular immune responses.

Breast cancer patients diagnosed with HR+/HER2- tumors face a persistent risk of distant recurrence long after completion of their treatment. Strategies to induce anti-tumor immune responses could complement standard-of-care therapies for these patients. The current study was performed to examine the feasibility, safety and immunogenicity of adding P10s-PADRE to standard-of-care chemotherapy in HR+/HER2- early-stage breast cancer patients. Twenty-five subjects were treated in a single-arm Phase Ib clinical trial. Five different immunization schedules were considered to evaluate the feasibility of eliciting an immune response. The primary immunogenicity endpoint was antibody titer. The expression of several activation markers on natural killer (NK) cells and serum concentrations of Th1/Th2 cytokines were also examined. The percentage of tumor-infiltrating lymphocytes (TILs) was determined. Antibody response was superior in schedule C where 3 weekly immunizations preceded the first dose of chemotherapy. A significant change in CD16, NKp46 and CD94 expression levels on NK cells and a rise in serum content of IFN-γ was observed after treatment. Schedule C showed an increase in TILs in residual lesions. The combination therapy is safe and immunogenic with treatment schedule C being immunologically promising. Randomized trials focused on long-term survival outcomes are needed to evaluate clinical benefits.

Thinking Cancer.

Evolutionary theories are necessarily invoked for understanding cancer development at the level of species, at the level of cells and tissues, and for developing effective therapies. It is crucial to view cancer in a Darwinian light, where the differential survival of individual cells is based on heritable variations. In the process of this somatic evolution, multicellularity controls are overridden by cancer cells, which become increasingly autonomous. Ecological epigenetics also helps understand how rogue cells that have basically the same DNA as their normal cell counterpart overcome the tissue homeostasis. As we struggle to wrap our minds around the complexity of these phenomena, we apply often times anthropomorphic terms, such as subversion, hijacking, or hacking, to describe especially the most complex among them-the interaction of tumors with the immune system. In this commentary we highlight examples of the anthropomorphic thinking of cancer and try to put into context the relative meaning of terms and the mechanisms that are oftentimes invoked to justify those terms.