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Background Free-range local chickens (FRLC) farming is an important activity in Tanzania, however, they have not been well-characterized. This study aimed to phenotypically characterize three Tanzanian FRLCs and to determine their population structure. A total of 389 mature breeder chickens (324 females and 65 males) from three popular Tanzanian FRLC ecotypes (Kuchi, Morogoro-medium and Ching’wekwe) were used for the phenotypic characterization. Progenies of these chickens were utilized to assess population structure. The ecotypes were collected from four geographical zones across Tanzania: Lake, Central, Northern and Coastal zones. Body weights and linear measurements were obtained from the mature breeders, including body, neck, shanks, wingspan, chest girth, and shank girth. Descriptive statistics were utilized to characterize the chickens. Correlations between the linear measurements and differences among the means of measured linear traits between ecotypes and between sexes were assessed. A total of 1399 progeny chicks were genotyped using a chicken 600 K high density single nucleotide polymorphism (SNP) panel for determination of population structure. Results The means for most traits were significantly higher in Kuchi relative to Ching’wekwe and Morogoro-medium. However, shank length and shank girth were similar between Kuchi and Morogoro-medium females. All traits were correlated with the exception of shank girth in Morogoro-medium. Admixture analyses revealed that Morogoro-medium and Ching’wekwe clustered together as one population, separate from Kuchi. Conclusions Phenotypic traits could be used to characterize FRLCs, however, there were variations in traits among individuals within ecotypes; therefore, complementary genomic methods should be considered to improve the characterization for selective breeding.

This study was carried out to assess the response of three Ghanaian local chicken ecotypes to LaSota (lentogenic) and virulent field strains of Newcastle disease virus (NDV). Local chickens sampled from the Interior Savannah (IS), Forest (FO) and Coastal Savannah (CS) agro-ecological zones were bred and their offspring were challenged with LaSota NDV at 4 weeks of age. The LaSota challenge was replicated four times with different chicken groups. A total of 1438 chicks comprising 509 Coastal Savannah, 518 Forest and 411 Interior Savannah ecotypes were used. Pre- and post-challenge anti-NDV antibody titre levels were determined via ELISA assays. A second trial was conducted by introducing sick birds infected with virulent NDV to a flock of immunologically naïve chickens at 4 weeks old. Body weights were measured pre- and post-infection. Sex of the chickens was determined using a molecular method. In both trials, there was no significant difference among ecotypes in body weight and growth rate. In the LaSota trial, anti-NDV antibody titre did not differ by ecotype or sex. However, there was a positive linear relationship between body weight and antibody titre. In the velogenic NDV trial, survivability and lesion scores were similar among the three ecotypes. This study confirms that a relatively high dose of LaSota (NDV) challenge has no undesirable effect on Ghanaian local chicken ecotypes. All three Ghanaian local chicken ecotypes were susceptible to velogenic NDV challenge. Resistance to NDV by Ghanaian local chickens appears to be determined more by the individual's genetic makeup than by their ecotype.

Newcastle disease (ND) has a great impact on poultry health and welfare with its most virulent (velogenic) strain. In addition, issues exacerbated by the increase in global temperatures necessitates a greater understanding of the host immune response when facing a combination of biotic and abiotic stress factors in poultry production. Previous investigations have revealed that the host immune response is tissue-specific. The goal of this study was to identify genes and/or signaling pathways associated with immune response to NDV (Newcastle disease virus) in the trachea, an essential organ where NDV replicate after the infection, by profiling the tissue specific transcriptome response in two genetically distinct inbred chicken lines when exposed to both abiotic and biotic stressors. Fayoumis appear to be able to respond more effectively (lower viral titer, higher antibody levels, immune gene up-regulation) and earlier than Leghorns. Our results suggest NDV infection in Fayoumis appears to elicit proinflammatory processes, and pathways such as the inhibition of cell viability, cell proliferation of lymphocytes, and transactivation of RNA, more rapidly than in Leghorns. These differences in immune response converge at later timepoints which may indicate that Leghorns eventually regulate its immune response to infection. The profiling of the gene expression response in the trachea adds to our understanding of the chicken host response to NDV infection and heat stress on a whole genome level and provides potential candidate genes and signaling pathways for further investigation into the characterization of the time-specific and pathway specific responses in Fayoumis and Leghorns.

Heat stress results in reduced productivity, anorexia, and mortality in chickens. The objective of the study was to identify genes and signal pathways associated with heat stress and Newcastle disease virus (NDV) infection in the liver of chickens through RNA-seq analysis, using two highly inbred chicken lines (Leghorn and Fayoumi). All birds were held in the same environment until 14 days of age. On day 14, half the birds were exposed to 38 &deg, C with 50% relative humidity for 4 h, then 35 &deg, C until the end of the experiment. The remaining birds were kept at 25 &deg, C throughout the experiment. The heat-treated birds were inoculated at 21 days of age with 107 EID50 (One EID50 unit is the amount of virus that will infect 50 percent of inoculated embryos) NDV La Sota strain to investigate the effects of both heat stress and NDV infection. Physiological parameters were recorded as blood phenotypes at three stages: acute heat (AH), chronic heat (CH1), and chronic heat combined with NDV infection (CH&amp, NDV), at 4 h, 7 days, and 10 days post-initiation of heat treatment, respectively. Our previous work revealed that the heat-resilient Fayoumi line maintained a more stable acid-base balance in their blood compared to the Leghorn line. Liver samples were harvested on both AH and CH&amp, NDV to characterize the transcriptome profiles of these two inbred lines. Both genetic lines and treatments had large impact on the liver transcriptome. Fayoumi birds had more differentially expressed genes (DEGs) than Leghorn birds for both treatments. Metabolic and immune-related genes were on the DEG list, with Fayoumi having more immune-related DEGs than Leghorns, which was confirmed by gene functional enrichment analysis. Weighted correlation network analysis (WGCNA) indicated that the driver genes such as Solute Carrier Family genes could be very important for stabilizing the acid-base balance in Fayoumi birds during heat stress. Therefore, candidate genes such solute carrier family genes could be potential genetic targets that are regulated by Fayoumis to maintain physical hemostasis under heat stress. Differential gene expression showed that Leghorns mainly performed metabolic regulation in response to heat stress and NDV infection, while Fayoumis regulated both immune and metabolic functions. This study provides novel insights and enhances our understandings of liver response to heat stress of heat resilient and susceptible inbred chicken lines.

As a major infectious disease in chickens, Newcastle disease virus (NDV) causes considerable economic losses in the poultry industry, especially in developing countries where there is limited access to effective vaccination. Therefore, enhancing resistance to the virus in commercial chickens through breeding is a promising way to promote poultry production. In this study, we investigated gene expression changes at 2 and 6 days post inoculation (dpi) at day 21 with a lentogenic NDV in a commercial egg-laying chicken hybrid using RNA sequencing analysis. By comparing NDV-challenged and non-challenged groups, 526 differentially expressed genes (DEGs) (false discovery rate (FDR) &lt, 0.05) were identified at 2 dpi, and only 36 at 6 dpi. For the DEGs at 2 dpi, Ingenuity Pathway Analysis predicted inhibition of multiple signaling pathways in response to NDV that regulate immune cell development and activity, neurogenesis, and angiogenesis. Up-regulation of interferon induced protein with tetratricopeptide repeats 5 (IFIT5) in response to NDV was consistent between the current and most previous studies. Sprouty RTK signaling antagonist 1 (SPRY1), a DEG in the current study, is in a significant quantitative trait locus associated with virus load at 6 dpi in the same population. These identified pathways and DEGs provide potential targets to further study breeding strategy to enhance NDV resistance in chickens.

High ambient temperature is one of the most important environmental factors negatively impacting poultry production and health. Genetics is an important contributor in mitigating the stress response to heat. Two genetically distinct highly inbred lines of similar body size (Leghorn and Fayoumi) were characterized for phenotypic differences in response to heat. At 14 days of age, birds were exposed to 38°C with 50% humidity for 4 hours, then 35°C until the conclusion of the experiment. Non-treated individuals were kept at 29.4°C for the first week and then 25°C throughout the experiment. Birds in the heat-stress group were inoculated at day (d) 21 with Newcastle disease virus (NDV) La Sota strain to investigate the effects of heat stress and NDV infection. Thirteen blood parameters were measured using the iSTAT blood analyzer at three stages: 4 h, 6 d, and 9 d post heat-stress treatment, representing acute heat (AH) exposure, chronic heat (CH1) exposure, and chronic heat exposure after virus infection (CH2), respectively. Most blood parameters were significantly changed with heat stress in Leghorns at AH and in Fayoumis at CH1 and CH2. The Leghorn line had significant acute responses with disrupted acid-base balance and metabolic disorders. The heat-resilient Fayoumis maintained a relatively well-balanced acid-base balance. The current study provides the comprehensive profile of biomarker signatures in blood associated with heat tolerance and suggests that PO2, TCO2, HCO3, and base excess can be served as potential biomarkers that can be used to genetically improve heat tolerance in poultry.

Newcastle Disease (ND) is a continuing global threat to domestic poultry, especially in developing countries, where severe outbreaks of velogenic ND virus (NDV) often cause major economic losses to households. Local chickens are of great importance to rural family livelihoods through provision of high-quality protein. To investigate the genetic basis of host response to NDV, three popular Tanzanian chicken ecotypes (regional populations) were challenged with a lentogenic (vaccine) strain of NDV at 28 days of age. Various host response phenotypes, including anti-NDV antibody levels (pre-infection and 10 days post-infection, dpi), and viral load (2 and 6 dpi) were measured, in addition to growth rate. We estimated genetic parameters and conducted genome-wide association study analyses by genotyping 1399 chickens using the Affymetrix 600K chicken SNP chip. Estimates of heritability of the evaluated traits were moderate (0.18&ndash, 0.35). Five quantitative trait loci (QTL) associated with growth and/or response to NDV were identified by single-SNP analyses, with some regions explaining &ge, 1% of genetic variance based on the Bayes-B method. Immune related genes, such as ETS1, TIRAP, and KIRREL3, were located in regions associated with viral load at 6 dpi. The moderate estimates of heritability and identified QTL indicate that NDV response traits may be improved through selective breeding of chickens to enhance increased NDV resistance and vaccine efficacy in Tanzanian local ecotypes.

Newcastle disease virus (NDV) is a highly contagious avian pathogen that poses a tremendous threat to poultry producers in endemic zones due to its epidemic potential. To investigate host genetic resistance to NDV while under the effects of heat stress, a genome-wide association study (GWAS) was performed on Hy-Line Brown layer chickens that were challenged with NDV while under high ambient temperature to identify regions associated with host viral titer, circulating anti-NDV antibody titer, and body weight change. A single nucleotide polymorphism (SNP) on chromosome 1 was associated with viral titer at two days post-infection (dpi), while 30 SNPs spanning a quantitative trait loci (QTL) on chromosome 24 were associated with viral titer at 6 dpi. Immune related genes, such as CAMK1d and CCDC3 on chromosome 1, associated with viral titer at 2 dpi, and TIRAP, ETS1, and KIRREL3, associated with viral titer at 6 dpi, were located in two QTL regions for viral titer that were identified in this study. This study identified genomic regions and candidate genes that are associated with response to NDV during heat stress in Hy-Line Brown layer chickens. Regions identified for viral titer on chromosome 1 and 24, at 2 and 6 dpi, respectively, included several genes that have key roles in regulating the immune response.

Newcastle disease (ND) is one of the most challenging infectious diseases affecting poultry production in Africa, causing major economic losses. To date, Newcastle disease virus isolates from several African countries have been grouped into class II NDV genotypes I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII and XXI. Although ND is endemic in many African countries, information on circulating genotypes is still scarce. In Tanzania, outbreaks with genotypes V and XIII have been reported. In West and Central Africa, genotypes XIV, XVII, and XVIII are the most predominant. To investigate other genotypes circulating in Tanzania and Ghana, we performed molecular genotyping on isolates from Tanzania and Ghana using the MinION, a third-generation portable sequencing device from Oxford Nanopore Technologies. Using the MinION, we successfully sequenced the NDV F gene hypervariable region of 24 isolates from Tanzania and four samples from Ghana. In Tanzania, genotypes V, VII and XIII were detected. All isolates from Ghana belonged to genotype XVIII. The data obtained in this study reflect the genetic diversity of NDV in Africa and highlight the importance of surveillance for monitoring the distribution of NDV genotypes and viral evolution.

Background Newcastle disease virus, in its most pathogenic form, threatens the livelihood of rural poultry farmers where there is a limited infrastructure and service for vaccinations to prevent outbreaks of the virus. Previously reported studies on the host response to Newcastle disease in chickens have not examined the disease under abiotic stressors, such as heat, which commonly experienced by chickens in regions such as Africa. The objective of this study was to elucidate the underlying biological mechanisms that contribute to disease resistance in chickens to the Newcastle disease virus while under the effects of heat stress. Results Differential gene expression analysis identified genes differentially expressed between treated and non-treated birds across three time points (2, 6, and 10 days post-infection) in Fayoumi and Leghorn birds. Across the three time points, Fayoumi had very few genes differentially expressed between treated and non-treated groups at 2 and 6 days post-infection. However, 202 genes were differentially expressed at 10 days post-infection. Alternatively, Leghorn had very few genes differentially expressed at 2 and 10 days post-infection but had 167 differentially expressed genes at 6 days post-infection. Very few differentially expressed genes were shared between the two genetic lines, and pathway analysis found unique signaling pathways specific to each genetic line. Fayoumi had significantly lower viral load, higher viral clearance, higher anti-NDV antibody levels, and fewer viral transcripts detected compared to Leghorns. Fayoumis activated immune related pathways including SAPK/JNK and p38 MAPK signaling pathways at earlier time points, while Leghorn would activate these same pathways at a later time. Further analysis revealed activation of the GP6 signaling pathway that may be responsible for the susceptible Leghorn response. Conclusions The findings in this study confirmed our hypothesis that the Fayoumi line was more resistant to Newcastle disease virus infection compared to the Leghorn line. Within line and interaction analysis demonstrated substantial differences in response patterns between the two genetic lines that was not observed from the within line contrasts. This study has provided novel insights into the transcriptome response of the Harderian gland tissue during Newcastle disease virus infection while under heat stress utilizing a unique resistant and susceptible model. Electronic supplementary material The online version of this article (10.1186/s12917-018-1583-0) contains supplementary material, which is available to authorized users.

Behind each eye of the chicken resides a unique lymph tissue, the Harderian gland, for which RNA sequencing (RNA-seq) analysis is novel. We characterized the response of this tissue to Newcastle disease virus (NDV) in two inbred lines with different susceptibility to NDV across three time points. Three-week-old relatively resistant (Fayoumi) and relatively susceptible (Leghorn) birds were inoculated with a high-titered (107EID50) La Sota strain of NDV via an oculonasal route. At 2, 6, and 10 days post infection (dpi) Harderian glands were collected and analyzed via RNA-seq. The Fayoumi had significantly more detectable viral transcripts in the Harderian gland at 2 dpi than the Leghorn, but cleared the virus by 6 dpi. At all three time points, few genes were declared differentially expressed (DE) between the challenged and nonchallenged birds, except for the Leghorns at 6 dpi, and these DE genes were predicted to activate an adaptive immune response. Relative to the Leghorn, the Fayoumi was predicted to activate more immune pathways in both challenged and nonchallenged birds suggesting a more elevated immune system in the Fayoumis under homeostatic conditions. Overall, this study helped characterize the function of this important tissue and its response to NDV.

A high prevalence and diversity of avian influenza (AI) viruses were detected in a population of wild mallards sampled during summer 2011 in California, providing an opportunity to compare results obtained before and after virus culture. We tested cloacal swab samples prior to culture by matrix real-time PCR, and by amplifying and sequencing a 640bp portion of the hemagglutinin (HA) gene. Each sample was also inoculated into embryonated chicken eggs, and full genome sequences were determined for cultured viruses. While low matrix Ct values were a good predictor of virus isolation from eggs, samples with high or undetectable Ct values also yielded isolates. Furthermore, a single passage in eggs altered the occurrence and detection of viral strains, and mixed infections (different HA subtypes) were detected less frequently after culture. There is no gold standard or perfect reference comparison for surveillance of unknown viruses, and true negatives are difficult to distinguish from false negatives. This study showed that sequencing samples prior to culture increases the detection of mixed infections and enhances the identification of viral strains and sequences that may have changed or even disappeared during culture.