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 Newcastle disease virus obtained from Mawenzi live bird market in Morogoro, Tanzania in 2020-2021.

Newcastle disease (ND) is among the most important poultry diseases worldwide. It is the major threat to poultry production in Africa and causes major economic losses for both local and commercial chickens. To date, half of ND class II genotypes have been reported in Africa (I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII, and XXI). The information on the circulating NDV genotypes is still scarce despite the endemic nature of ND in most countries on the African continent.A total of 659 oro-cloacal swabs were collected from local chickens in Mawenzi live bird market located in Morogoro, Tanzania, between June 2020 and May 2021. Newcastle disease virus was detected by using reverse transcription real-time polymerase chain reaction (RT-qPCR) and conventional PCR followed by sequencing of PCR products. The prevalence of NDV in the surveilled live bird markets was 23.5%. Sequencing and phylogenetic analysis revealed the presence of sub-genotype VII.2. The detected sub-genotype VII.2 has phylogenetic links to Zambian NDV strains implying a Southeast dissemination of the virus, considering that it was first detected in Mozambique. This study underscores the need of active NDV surveillance to determine the distribution of this NDV genotype in the country and monitor its spread and contribution to the emergence of new ND viruses.

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.

Genetic Analyses of Response of Local Ghanaian Tanzanian Chicken Ecotypes to a Natural Challenge with Velogenic Newcastle Disease Virus.

Newcastle disease is a devastating poultry disease that often causes significant economic losses in poultry in the developing countries of Africa, Asia, as well as South and Central America. Velogenic Newcastle disease virus (NDV) outbreaks are associated with high mortalities, which can threaten household livelihoods, especially in the rural areas, and lead to loss of high-quality proteins in the form of meat and eggs, as well as household purchasing power. In this study, we exposed unvaccinated Ghanaian and Tanzanian chickens of six local ecotypes to velogenic NDV strains, measured NDV response traits, sequenced their DNA on a genotyping-by-sequencing platform, and performed variance component analyses. The collected phenotypes included: growth rates (pre- and post-exposure); lesion scores (gross lesion severity) in the trachea, proventriculus, intestine, and cecal tonsils; natural antibody levels; anti-NDV antibody levels at 7 days post exposure (dpe); tear and cloacal viral load at 2, 4, and 6 dpe; and survival time. Heritability estimates were low to moderate, ranging from 0.11 for average lesion scores to 0.36 for pre-exposure growth rate. Heritability estimates for survival time were 0.23 and 0.27 for the Tanzanian and Ghanaian ecotypes, respectively. Similar heritability estimates were observed when data were analyzed either separately or combined for the two countries. Survival time was genetically negatively correlated with lesion scores and with viral load. Results suggested that response to mesogenic or velogenic NDV of these local chicken ecotypes could be improved by selective breeding. Chickens that are more resilient to velogenic NDV can improve household livelihoods in developing countries.

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.

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.

Transmission of antibiotic-resistant Escherichia coli between cattle, humans and the environment in peri-urban livestock keeping communities in Morogoro, Tanzania.

Urban and peri-urban livestock farming is expanding world-widely because of increased urbanization and demands for food of animal origin. Such farming practices pose a public health risk as livestock are reservoirs of several zoonotic pathogens. In an attempt to determine the fecal transmission between livestock and people, 100 household clusters keeping cattle in close proximity of humans were selected in urban and peri-urban areas of Morogoro in Tanzania. One hundred eighteen ampicillin and tetracycline resistant Escherichia coli (40 from human stool, 50 from cattle feces, 21 from soil and seven from water samples) were isolated from 44 different clusters. Pulsed-field gel electrophoresis (PFGE) of XbaI digested chromosomal DNA was used to compare the genetic relatedness of the ampicillin- and tetracycline-resistant E. coli isolates. Indistinguishable PFGE band patterns of the ampicillin- and tetracycline-resistant E. coli isolates were found in samples from 23 (52%) clusters. This suggests that transfer of fecal microorganisms between cattle, humans, water and soils within the farms and from livestock farms to the neighborhood occurred commonly. Logistic regression showed that animal housing infrastructures (Odd Ratio=11.2, 95% CI=1.1-119.3) were associated with E. coli showing identical PFGE types within and between clusters. There is a need to improve animal husbandry and manure management practices to reduce risks of transmission of enteropathogens between livestock and humans in urban and peri-urban farming.

Occurrence and characterization of Shiga toxin-producing Escherichia coli O157:H7 and other non-sorbitol-fermenting E. coli in cattle and humans in urban areas of Morogoro, Tanzania.

Escherichia coli strains such as Shiga toxin-producing E. coli (STEC), enteropathogenic E. coli, enterotoxigenic, attaching, and effacing E. coli, and enteroinvasive E. coli cause diarrhea in humans. Although other serotypes exist, the most commonly reported STEC in outbreaks is O157:H7. A cross-sectional study was conducted to isolate and characterize non-sorbitol-fermenting (NSF) E. coli O157:H7 from urban and periurban livestock settings of Morogoro, Tanzania. Human stool, cattle feces, and soil and water samples were collected. Observations and questionnaire interview studies were used to gather information about cattle and manure management practices in the study area. E. coli were isolated on sorbitol MacConkey agar and characterized by conventional biochemical tests. Out of 1049 samples, 143 (13.7%) yielded NSF E. coli. Serological and antimicrobial tests and molecular typing were performed to NSF E. coli isolates. These procedures detected 10 (7%) pathogenic E. coli including STEC (n=7), enteropathogenic E. coli (EPEC) (n=2), and attaching and effacing E. coli (A/EEC) (n=1) strains. The STEC strains had the ability to produce VT1 and different VT2 toxin subtypes that caused cytopathic effects on Vero cells. The prevalence of STEC in cattle was 1.6%, out of which 0.9% was serotype O157:H7 and the overall prevalence of diarrheagenic E. coli in cattle was 2.2%. The serotypes O157:H7, O142:H34, O113:H21, O+:H-, O+:H16, and O25:H4 were identified. One ESBL-producing isolate showed the MLST type ST131. To our knowledge, this is the first finding in Tanzania of this recently emerged worldwide pandemic clonal group, causing widespread antimicrobial-resistant infections, and adds knowledge of the geographical distribution of ST131. Cattle manure was indiscriminately deposited within residential areas, and there was direct contact between humans and cattle feces during manure handling. Cattle and manure management practices expose humans, animals, and the environment to pathogenic E. coli and other manure-borne pathogens. Therefore, there is a need to improve manure management practices in urban and periurban areas to prevent pathogen spread and associated human health risks.

Human and animal Campylobacteriosis in Tanzania: A review.

The thermotolerant species of Campylobacter have become very important in public health, particularly as agents of infectious diarrhoea in human beings. Though the mechanism by which they cause disease is yet to be fully explained, they have been recognized as the leading cause of bacterial enteritis in both developed and developing countries. The organisms colonize different animal species without causing any symptoms of disease; and humans acquire infections through contact with or consumption of contaminated meat especially raw/undercooked poultry meat. The growing trend of antibiotic resistant Campylobacter isolates continues to pose significant public health challenges. In this review we present the available information generated in Tanzania about Campylobacter infections in humans and animals. We conducted a structured literature search of PUBMED and ScienceDirect electronic databases and identified 15 articles. Studies on humans reported Campylobacter infections in both symptomatic and asymptomatic subjects; with higher prevalence in children under the age of five years. Studies on animals found colonization of both domestic and wild species. Among isolates, some demonstrated antimicrobial resistance. The available information for both human and animal Campylobacteriosis in the country is sparse. It however provides an insight of the bacteriological and epidemiological aspects of Campylobacter infections in the country and eventually creates more awareness on the need to develop control strategies. Since the organism is zoonotic its control strategies should adopt the "One Health" approach involving collaborative efforts from veterinary and human medicine.

Teacher-pupil model: improving poultry health and management through children.

The perception of risks of exposure to avian influenza and other poultry diseases among adults in Tanzania is influenced by their previous experiences, beliefs, and values, which can stand in the way of learning new approaches. We tested a novel disease risk communication approach centered on elementary school pupils, involving their teachers and parents. Age-appropriate training modules were developed and taught to teachers who then taught their pupils through extracurricular activities. The pupils practiced what they learned through club projects and subsequently transmitted what they learned to their parents. In 2009 we developed a poultry health and production curriculum as part of efforts to prevent and control poultry diseases, including avian influenza, in Tanzania. The curriculum developed for veterinarians and veterinary paraprofessionals was adapted for use with elementary school children and translated into Kiswahili. Twenty teachers from 10 primary schools in Mzumbe ward, Morogoro, were trained by poultry veterinary extension experts on teaching the curriculum to standard 5 pupils (ages 11-12 yr). Pupils and teachers practiced the curriculum in four demonstration chicken coops established on the grounds of the Changarawe, Lubungo, Masanze, and Mzumbe primary schools. By October 2011, at the conclusion of the funded project, a total of 202 girls and 193 boys had been trained. Additionally, 34 adults from surrounding villages made official learning tours to the schools and received training from their children and teachers involved in the projects. With at least 75% of the 395 pupils coming from different households, it can be safely assumed that over 250 households have heard about poultry disease risks and how to manage poultry to prevent those risks.

Epidemiological investigation into the introduction and factors for spread of Peste des Petits Ruminants, southern Tanzania.

A study was carried out to confirm and identify sources and elucidate factors associated with the introduction of Peste des Petits Ruminants (PPR) in southern Tanzania. This study was conducted in Tandahimba and Newala districts of Mtwara region following suspected outbreak of PPR in the area. Qualitative data were collected using semi-structured questionnaires and in-depth interviews of key informants who included goat and sheep owners with suspected cases of PPR and animal health service providers as well as local administrative authority. Additionally, 216 serum samples and 28 swabs were collected for serological and virological laboratory disease confirmation. The results show that PPR was first introduced in Likuna village of Newala district in February 2009 through newly purchased goats from the Pugu livestock market located about 700 km in the outskirts of Dar es Salaam city. Factors which contributed to spread of PPR included communal grazing and the cheap prices of sick animals bought by livestock keepers for slaughtering in other villages. Laboratory findings confirmed presence of PPR in the area by RT-PCR and serological analysis revealed that seroprevalence was 31%. These findings have confirmed, for the first time, introduction of PPR in southern Tanzania. The presence of PPR poses high risk of southward spread of the disease to other southern African countries in the SADC region thus calling for concerted and collaborative efforts in prevention and control of the disease to avoid losses. Further elaborate studies on the spread, prevalence and risk factors associated with the disease should urgently be investigated.

Preparation for the prevention and control of highly pathogenic avian influenza in rural Tanzanian village settings.

Free-ranging local chicken flocks are important for the livelihood of resource-poor rural farmers in Tanzania, as they provide a critical source of animal protein and a ready source of income through the sale of chickens and eggs. An occurrence of highly pathogenic avian influenza (HPAI) in the village setting of Tanzania would result in a disastrous loss of livelihood. This paper attempts to offer an alternative method for preventing and controlling HPAI in village settings of Tanzania through community-based approaches.

Implementing poultry vaccination and biosecurity at the village level in Tanzania: a social strategy to promote health in free-range poultry populations.

A social strategy was tested for implementing Newcastle disease (ND) vaccination and biosecurity improvements among free-ranging chicken at village level in Tanzania. In addition to training the local poultry vaccinators, data recorders and poultry-keepers, the strategy involved training and empowering leaders at the district, ward and village level. The trainings covered poultry health, management, and marketing of village chickens, with an emphasis on ND vaccination and improving biosecurity against avian influenza (AI), The study sites included villages in one ward in each of three each three districts (Iringa, Mtwara-Mikindani, and Mvomero) of mainland Tanzania. Ninety-six local leaders at district level and 101 leaders at ward levels were trained. In addition, 196 farmers (households) were trained, as well as 86 vaccinators and 26 data recorders. Data recorders were also trained as poultry first aid workers. ND vaccination was conducted by the vaccinators, supervised by their local leaders with technical assistance from veterinarians. A total of 158,343 village chickens were vaccinated in three rounds of vaccination three months apart. The training and empowerment of local leaders and local implementers was the key element for success as it fostered the feeling of local ownership of the program and prevented conflicts with other development activities within the villages. We conclude that most animal health programs will increase their odds of success by involving local leaders and by addressing the current challenges facing the farmers. Further assessment on the usefulness of this approach is needed.

The efficacy of crude extract of Aloe secundiflora on Candida albicans.

In-vitro studies on the efficacy of crude extracts of Aloe secundiflora on Candida albicans was conducted. Five mature leaves of Aloe secundiflora were collected and the crude extract was prepared, then autoclaved. The extract was then tested on Candida albicans grown on solid media. The results from these studies revealed complete inhibition of Candida albicans growth caused by Aloe secundiflora crude extract on solid media. The findings from this study suggest that the crude extract of Aloe secundiflora can inhibit the growth of Candida albicans. Further studies are required to establish the in-vivo activity of the crude extract, the active ingredient, dosage and safety of Aloe secundiflora, before recommending for clinical use.