Virulent Newcastle disease virus genotypes V.3, VII.2, and XIII.1.1 and their coinfections with infectious bronchitis viruses and other avian pathogens in backyard chickens in Tanzania.

Oropharyngeal (OP) and cloacal (CL) swabs from 2049 adult backyard chickens collected at 12 live bird markets, two each in Arusha, Dar es Salaam, Iringa, Mbeya, Morogoro and Tanga regions of Tanzania were screened for Newcastle disease virus (NDV) using reverse transcription real-time PCR (rRT-PCR). The virus was confirmed in 25.23% of the birds (n = 517; rRT-PCR CT ≤ 30), with the highest positivity rates observed in birds from Dar es Salaam region with higher prevalence during the dry season (September-November 2018) compared to the rainy season (January and April-May 2019). Next-generation sequencing of OP/CL samples of 20 out of 32 birds that had high amounts of viral RNAs (CT ≤ 25) resulted in the assembly of 18 complete and two partial genome sequences (15,192 bp and 15,045-15,190 bp in length, respectively) of NDV sub-genotypes V.3, VII.2 and XIII.1.1 (n = 1, 13 and 4 strains, respectively). Two birds had mixed NDV infections (V.3/VII.2 and VII.2/XIII.1.1), and nine were coinfected with viruses of families Astroviridae, Coronaviridae, Orthomyxoviridae, Picornaviridae, Pneumoviridae, and Reoviridae. Of the coinfecting viruses, complete genome sequences of two avastroviruses (a recombinant chicken astrovirus antigenic group-Aii and avian nephritis virus genogroup-5) and two infectious bronchitis viruses (a turkey coronavirus-like recombinant and a GI-19 virus) were determined. The fusion (F) protein F1/F2 cleavage sites of the Tanzanian NDVs have the consensus motifs 112 RRRKR↓F 117 (VII.2 strains) and 112 RRQKR↓F 117 (V.3 and XIII.1.1 strains) consistent with virulent virus; virulence was confirmed by intracerebral pathogenicity index scores of 1.66-1.88 in 1-day-old chicks using nine of the 20 isolates. Phylogenetically, the complete F-gene and full genome sequences regionally cluster the Tanzanian NDVs with, but distinctly from, other strains previously reported in eastern and southern African countries. These data contribute to the understanding of NDV epidemiology in Tanzania and the region.

Molecular Characterization of Complete Genome Sequence of an Avian Coronavirus Identified in a Backyard Chicken from Tanzania.

A complete genome sequence of an avian coronavirus (AvCoV; 27,663 bp excluding 3' poly(A) tail) was determined using nontargeted next-generation sequencing (NGS) of an oropharyngeal swab from a backyard chicken in a live bird market in Arusha, Tanzania. The open reading frames (ORFs) of the Tanzanian strain TZ/CA127/19 are organized as typical of gammaCoVs (Coronaviridae family): 5'UTR-[ORFs 1a/1b encoding replicase complex (Rep1ab) non-structural peptides nsp2-16]-[spike (S) protein]-[ORFs 3a/3b]-[small envelop (E) protein]-[membrane (M) protein]-[ORFs 4a/4c]-[ORFs 5a/5b]-[nucleocapsid (N) protein]-[ORF6b]-3'UTR. The structural (S, E, M and N) and Rep1ab proteins of TZ/CA127/19 contain features typically conserved in AvCoVs, including the cleavage sites and functional motifs in Rep1ab and S. Its genome backbone (non-spike region) is closest to Asian GI-7 and GI-19 infectious bronchitis viruses (IBVs) with 87.2-89.7% nucleotide (nt) identities, but it has a S gene closest (98.9% nt identity) to the recombinant strain ck/CN/ahysx-1/16. Its 3a, 3b E and 4c sequences are closest to the duck CoV strain DK/GD/27/14 at 99.43%, 100%, 99.65% and 99.38% nt identities, respectively. Whereas its S gene phylogenetically cluster with North American TCoVs and French guineafowl COVs, all other viral genes group monophyletically with Eurasian GI-7/GI-19 IBVs and Chinese recombinant AvCoVs. Detection of a 4445 nt-long recombinant fragment with breakpoints at positions 19,961 and 24,405 (C- and N-terminus of nsp16 and E, respectively) strongly suggested that TZ/CA127/19 acquired its genome backbone from an LX4-type (GI-19) field strain via recombination with an unknown AvCoV. This is the first report of AvCoV in Tanzania and leaves unanswered the questions of its emergence and the biological significance.

Complete genome sequence of seven virulent Newcastle disease virus isolates of sub-genotype XIII.1.1 from Tanzania.

We report the complete genome sequences of seven virulent Newcastle disease viruses (NDVs) that were isolated from chickens from live bird markets in the Arusha, Iringa, Mbeya, and Tanga regions of Tanzania in 2012. Phylogenetic analysis revealed that all isolates belong to sub-genotype XIII.1.1.

Genome Sequences and Characterization of Chicken Astrovirus and Avian Nephritis Virus from Tanzanian Live Bird Markets.

The enteric chicken astrovirus (CAstV) and avian nephritis virus (ANV) are the type species of the genus Avastrovirus (AAstV; Astroviridae family), capable of causing considerable production losses in poultry. Using next-generation sequencing of a cloacal swab from a backyard chicken in Tanzania, we assembled genome sequences of ANV and CAstV (6918 nt and 7318 nt in length, respectively, excluding poly(A) tails, which have a typical AAstV genome architecture (5'-UTR-ORF1a-ORF1b-ORF2-'3-UTR). They are most similar to strains ck/ANV/BR/RS/6R/15 (82.72%) and ck/CAstV/PL/G059/14 (82.23%), respectively. Phylogenetic and sequence analyses of the genomes and the three open reading frames (ORFs) grouped the Tanzanian ANV and CAstV strains with Eurasian ANV-5 and CAstV-Aii viruses, respectively. Compared to other AAstVs, the Tanzanian strains have numerous amino acid variations (substitutions, insertions and deletions) in the spike region of the capsid protein. Furthermore, CAstV-A has a 4018 nt recombinant fragment in the ORF1a/1b genomic region, predicted to be from Eurasian CAstV-Bi and Bvi parental strains. These data should inform future epidemiological studies and options for AAstV diagnostics and vaccines.

Strategies to Upgrade Animal Health Delivery in Village Poultry Systems: Perspectives of Stakeholders From Northern Ghana and Central Zones in Tanzania.

Village chicken production holds much potential for the alleviation of malnutrition and poverty in rural communities in Africa. Owing to their subsistence nature, however, such systems are rife with infectious poultry diseases such as Newcastle disease (ND). Strategies common for the management of ND and other poultry diseases in intensive production systems, including vaccination and biosecurity measures, have seen limited success in the village production systems. New approaches are needed that can successfully deliver animal health inputs and services for the effective management of poultry health challenges in low-input systems. Our study utilized focus group discussions with men and women farmers as well as other poultry value chain actors such as input suppliers, live bird traders and processed poultry meat retailers, to investigate potential options for delivery of animal health care to village poultry systems in northern Ghana and central Tanzania. ND was commonly reported as a major disease constraint in the study sites of the two countries, with resulting fatalities particularly impactful on men and women producers and on traders. We therefore also conducted interviews that focused specifically on the gender component of village chicken production. The key health related challenges prioritized by women and men participants included limited access to, and poor quality of, vaccines and veterinary drugs, a shortage of veterinary officers, and insufficient knowledge and training of farmers on flock management practices. Women, more than men, emphasized the difficulties of accessing poultry health services. Our assessments suggest that for poultry health care delivery in the studied communities to be effective, there is need to improve the supply of good quality drugs and vaccines in rural areas, respond to the needs of both men and women, and recognize the different incentives for farmers, traders and other value chain actors. Community-based approaches and increased use of ICT technology such as mobile phones have much to offer in this regard.

Phenotypic variability and population structure analysis of Tanzanian free-range local chickens.

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.

Molecular Characterization of Newcastle Disease Viruses Isolated from Chickens in Tanzania and Ghana.

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.

Isolation and Characterization of Newcastle Disease Virus from Live Bird Markets in Tanzania.

Chickens in live bird markets (LBMs) from six different regions of Tanzania were surveyed for Newcastle disease (ND) virus (NDV) and avian influenza virus in 2012. ELISA-based serology, virus isolation, and characterization, including pathotyping was conducted. Virulent NDV was isolated from almost 10% of the tested samples, with two distinct genotypes being detected. One genotype was similar to recent viruses circulating in Kenya and Uganda, which share a northern border with Tanzania. Several viruses of this genotype were also isolated from Tanzania in 1995, the last time surveillance for NDV was conducted in the country. The second genotype of virus from Tanzania was closely related to viruses from Mozambique, a southern neighbor, and more distantly to viruses from South Africa, Botswana, and several European countries. Partial fusion gene sequence from the isolated viruses showed identical fusion cleavage sites that were compatible with virulent viruses. Selected viruses were tested by the intracerebral pathogenicity index, and all viruses tested had scores of >1.78, indicating highly virulent viruses. Serology showed only a third of the chickens had detectable antibody to NDV, suggesting that vaccination is not being commonly used in the country, despite the availability of vaccines in agricultural-related markets. All samples were taken from clinically healthy birds, and it is believed that the birds were sold or slaughtered before showing ND clinical signs. LBMs remain a biosecurity risk for farmers through the return of live infected birds to the farm or village or the movement of virus on fomites, such as uncleaned wooden cages.

Aislamiento y caracterización de virus de la enfermedad de Newcastle de mercados de aves vivas en Tanzania. Se llevó a cabo un muestreo de pollos en mercados de aves vivas (LBM) de seis regiones diferentes de Tanzania para detectar al virus de la enfermedad de Newcastle (NDV) y el virus de la influenza aviar en el año 2012. Se llevaron a cabo la serología basada en la prueba de ELISA, el aislamiento viral y la caracterización, incluyendo la determinación del patotipo. Formas virulentas del virus de Newcastle se aislaron de casi el 10% de las muestras analizadas y se detectaron dos genotipos distintos. Un genotipo era similar a los virus recientes que circulan en Kenia y Uganda, países que comparten una frontera al norte de Tanzania. Varios virus de este genotipo también se aislaron de Tanzania en el año 1995, la última vez que se realizó la vigilancia del virus de Newcastle en el país. El segundo genotipo de virus de Tanzania estaba estrechamente relacionado con virus de Mozambique, país vecino al sur, y más distantemente con virus de Sudáfrica, Botswana y de varios países europeos. Una secuencia parcial del gene de fusión de los virus aislados mostró sitios de disociación en la proteína de fusión idénticos que eran compatibles con los virus virulentos. Los virus seleccionados fueron analizados mediante el índice de patogenicidad intracerebral y todos los virus analizados tuvieron puntajes mayores de 1.78, lo que indica que son virus altamente virulentos. La serología mostró que solo un tercio de los pollos tenían anticuerpos detectables contra el virus de Newcastle, lo que sugiere que la vacunación no se usa comúnmente en el país, a pesar de la disponibilidad de vacunas en los mercados agrícolas. Todas las muestras fueron recolectadas de aves clínicamente sanas y se cree que las aves fueron vendidas o sacrificadas antes de mostrar signos clínicos de la enfermedad de Newcastle. Los mercados de aves vivas siguen siendo un riesgo de bioseguridad para los agricultores mediante el regreso de aves vivas infectadas a la granja o a los pueblos o por el movimiento de virus en fómites, como las jaulas de madera sin limpiar.

Genetic Analyses of Tanzanian Local Chicken Ecotypes Challenged with Newcastle Disease Virus.

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 (0.18-0.35). Five quantitative trait loci (QTL) associated with growth and/or response to NDV were identified by single-SNP analyses, with some regions explaining ≥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.

First Complete Genome Sequence of a Subgenotype Vd Newcastle Disease Virus Isolate.

A Newcastle disease virus was isolated from a chicken from a live bird market in the Mbeya region of Tanzania. Complete genome characterization of the isolate identified it as a member of subgenotype Vd. This is the first complete genome sequence of this subgenotype.