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The use of molecular methods for rotavirus characterisation provides increased sensitivity for typing and allows the identification of putative reassortant strains. Reagents and methods for genotyping the virus need constant modification because of the reassortant nature of the virus. This study was aimed at designing and evaluating new oligonucleotide degenerate primer pairs that provide increased sensitivity and specificity for detecting avian rotavirus. Gene-specific primer pairs were designed by analysing different rotavirus strains isolated during the last decade by downloading them from the GenBank. The alignments were generated using clustal analysis from the BioEdit program. Degenerate nucleotides were included due to the reassortant nature of rotavirus. The consensus sequences were aligned using the BioEdit program and then treated with the Fast PCR software to derive the primers. The derived primer sequences were submitted for a BLAST search to ensure alignment was exclusive to the desired target genes. The designed primers had specific bands and were efficient in detecting rotavirus in faecal samples than previously published primers. Thus, a successful surveillance of rotaviruses requires that primer pairs be updated regularly in order to detect the emergence of novel or "unusual types", which have occurred by genetic drift causing nucleotide changes at the primer binding sites that result in typing failures. We recommend the use of the proposed primers in molecular surveillance studies for efficient detection of avian rotavirus.

Aim: This study was designed to optimize and apply the use of loop-mediated isothermal amplification (LAMP) as an alternative to conventional polymerase chain reaction (PCR) for the detection of herpesvirus of turkeys (HVT) (FC 126 strain) in vaccinated and non-vaccinated poultry in Nigeria., Materials and Methods: HVT positive control (vaccine) was used for optimization of LAMP using six primers that target the HVT070 gene sequence of the virus. These primers can differentiate HVT, a Marek's disease virus (MDV) serotype 3 from MDV serotypes 1 and 2. Samples were collected from clinical cases of Marek's disease (MD) in chickens, processed and subjected to LAMP and PCR., Results: LAMP assay for HVT was optimized. HVT was detected in 60% (3/5) and 100% (5/5) of the samples analyzed by PCR and LAMP, respectively. HVT was detected in the feathers, liver, skin, and spleen with average DNA purity of 3.05-4.52 μg DNA/mg (A260/A280) using LAMP. Conventional PCR detected HVT in two vaccinated and one unvaccinated chicken samples, while LAMP detected HVT in two vaccinated and three unvaccinated corresponding chicken samples. However, LAMP was a faster and simpler technique to carry out than PCR., Conclusion: LAMP assay for the detection of HVT was optimized. LAMP and PCR detected HVT in clinical samples collected. LAMP assay can be a very good alternative to PCR for detection of HVT and other viruses. This is the first report of the use of LAMP for the detection of viruses of veterinary importance in Nigeria. LAMP should be optimized as a diagnostic and research tool for investigation of poultry diseases such as MD in Nigeria.

Background: Avian rotaviruses (RVs) cause gastrointestinal diseases of birds worldwide. However, prevalence, diversity, epidemiology and phylogeny of RVs remain largely under-investigated in Africa., Methods: Fecal samples from 349 birds (158 symptomatic, 107 asymptomatic and 84 birds without recorded health status) were screened by reverse transcription PCR to detect RV groups A and D (RVA and RVD). Partial gene sequences of VP4, VP6, VP7 and NSP4 for RVA, and of VP6 and VP7 for RVD were obtained and analyzed to infer phylogenetic relationship. Fisher's exact test and logistic regression were applied to identify factors potentially influencing virus shedding in chickens., Results: A high prevalence of RVA (36.1%; 126/349) and RVD (31.8%; 111/349) shedding was revealed in birds. In chickens, RV shedding was age-dependent and highest RVD shedding rates were found in commercial farms. No negative health effect could be shown, and RVA and RVD shedding was significantly more likely in asymptomatic chickens: RVA/RVD were detected in 51.9/48.1% of the asymptomatic chickens, compared to 18.9/29.7% of the symptomatic chickens (p < 0.001/p = 0.01). First RVA sequences were obtained from mallard ducks (Anas platyrhynchos) and guinea fowls (Numida meleagris). Phylogenetic analyses illustrated the high genetic diversity of RVA and RVD in Nigerian birds and suggested cross-species transmission of RVA, especially at live bird markets. Indeed, RVA strains highly similar to a recently published fox rotavirus (RVA/Fox-tc/ITA/288356/2011/G18P[17]) and distantly related to other avian RVs were detected in different bird species, including pigeons, ducks, guinea fowls, quails and chickens., Conclusion: This study provides new insights into epidemiology, diversity and classification of avian RVA and RVD in Nigeria. We show that cross-species transmission of host permissive RV strains occurs when different bird species are mixed.

Besides birds, pigs are another important reservoir of influenza A viruses that can be transmitted to human, as highlighted by the emergence and spread of the pandemic (H1N1) virus (pdm/09) in 2009. Surveillance in pigs is therefore necessary for public health and influenza pandemic preparedness. Nevertheless, there is a serious lack of data on influenza in Africa, especially in swine. We therefore collected serum samples from pigs in Nigeria (2009, 2012) and Cameroon (2011) in which the presence of anti-influenza A neutralizing antibodies was investigated. Our serological survey suggests that, before the 2009 pandemic, only rare swine and human H3N2 or human H1N1 infections occurred in Nigeria in swine. However, in 2011-2012, 27.4% of pigs in Nigeria and 5.6% in Cameroon had antibodies against H1N1 viruses. Higher antibody titres against pdm/09 suggested that pigs were exposed to this or a similar virus, either by multiple introductions or sustained circulation, and that reactivity against American and European swine H1N1 viruses resulted from cross-reaction., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In West and Central Africa, virulent Newcastle disease virus (NDV) strains of the recently identified genotypes XIV, XVII, and XVIII are enzootic in poultry, representing a considerable threat to the sector. The increasing number of reports of virulent strains in wild birds at least in other parts of the world raised the question of a potential role of wild birds in the spread of virulent NDV in sub-Saharan Africa as well. We investigated 1,723 asymptomatic birds sampled at live-bird markets and sites important for wild-bird conservation in Nigeria and 19 sick or dead wild birds in Côte d'Ivoire for NDV class I and II. Typical avirulent wild-type genotype I strains were found in wild waterfowl in wetlands in northeastern Nigeria. They were unrelated to vaccine strains, and the involvement of inter- or intracontinental migratory birds in their circulation in the region is suggested. Phylogenetic analyses also revealed that genotype VI strains found in pigeons, including some putative new subgenotype VIh and VIi strains, were introduced on multiple separate occasions in Nigeria. A single virulent genotype XVIII strain was found in a dead wild bird in Côte d'Ivoire, probably as a result of spillover from sick poultry. In conclusion, screening of wild birds and pigeons for NDV revealed the presence a variety of virulent and avirulent strains in West Africa but did not provide strong evidence that wild birds play an important role in the spread of virulent strains in the region.

Despite rampant Newcastle disease virus (NDV) outbreaks in Africa for decades, the information about the genetic characteristics of the virulent strains circulating in West and Central Africa is still scarce. In this study, 96 complete NDV fusion gene sequences were obtained from poultry sampled in Cameroon, Central African Republic, Côte d'Ivoire, and Nigeria between 2006 and 2011. Based on rational criteria recently proposed for the classification of NDV strains into classes, genotypes, and subgenotypes, we revisited the classification of virulent strains, in particular those from West and Central Africa, leading to their grouping into genotype XIV and newly defined genotypes XVII and XVIII, each with two subgenotypes. Phylogenetic analyses revealed that several (sub)genotypes are found in almost every country. In Cameroon, most strains were related to vaccine strains, but a single genotype XVII strain was also found. Only three highly similar genotype XVII strains were detected in Central African Republic. Subgenotypes XVIIa, XVIIIa, and XVIIIb cocirculated in Côte d'Ivoire, while subgenotypes XIVa, XIVb, XVIIa, XVIIb, and XVIIIb were found in Nigeria. While these genotypes are so far geographically restricted, local and international trade of domestic and exotic birds may lead to their spread beyond West and Central Africa. A high genetic diversity, mutations in important neutralizing epitopes paired with suboptimal vaccination, various levels of clinical responses of poultry and wild birds to virulent strains, strains with new cleavage sites, and other genetic modifications found in these genotypes tend to undermine and complicate NDV management in Africa.

Outbreaks of infectious bursal disease in vaccinated chicken flocks are frequent in Nigeria. For the control of infectious bursal disease, live vaccines based on foreign infectious bursal disease virus (IBDV) strains are used. The present study investigated the phylogenetic relationship between field and vaccine IBDV strains from northwestern Nigeria. Thirty field IBDV strains and three commercial vaccines strains were characterized through sequencing the VP2 hypervariable region. In addition, the complete genome segment A coding region for two vaccines and two field strains was sequenced. The deduced amino acid sequences (position 212 to 331) of IBDV strains from Nigeria and other regions of the world were aligned and possible regional and virulence markers were identified associated with VP2 minor hydrophilic peaks. Reversion to virulence of a vaccine strain with a Q to L mutation at position 253 was observed. Phylogenetic analyses revealed a unique cluster of northwest Nigerian field IBDV strains alone or related to imported characterized classical and very virulent IBDV vaccines. The results suggest that when IBDV strains spread from their region of origin to a different region they mutate alongside indigenous field strains but may retain their identity on the VP2 region.

The efficacy of Stresroak--an Ayurvedic product (India) was tested in cockerel chicks. Eighty day-old chicks were randomly allocated into four groups with 20 birds per group. Group 1 received Stresroak at the recommended dosage of 1 ml/20 birds for 5 days prior to the NDV vaccinations (i/o, LaSota and Komarov). Group 2 received Stresroak at double the recommended dosage prior to vaccinations. Group 3 received no treatment but had all the afore mentioned vaccinations while Group 4 received neither treatments nor vaccinations and therefore served as the control group. Sera samples were analyzed by Haemagglutination Inhibition (HI) tests. There was no significant immunostimulation by Stresroak except on days 42 and 63 of life during the period of this study. It is therefore concluded that the manufacturer's claim of immunomodulatory property of Stresroak could not be validated in the humoral immunity studied. It is therefore suggested that further works be conducted using immunosuppressed hosts like birds with subclinical IBD and coccidiosis. In addition, research can be extended to the cell-mediated immunity.

Shiga toxin-producing Escherichia coli (STEC) are major food-borne pathogens associated with gastroenteritis and sometimes fatal haemolytic uraemic syndrome complication. Farm animals are asymptomatic carriers of STEC and contaminated meat is an important vehicle for zoonotic transmission from animals to humans. This study investigated the presence, virulence traits and antimicrobial susceptibility of seven potentially human pathogenic STEC serogroups (O157, O26, O91, O103, O111, O128 and O145) in the faeces and meat of food-producing animals in Ibadan, Nigeria. One hundred and fifty-four (7.3%) of 2133 samples were positive for STEC serogroups. The pathogens were detected in the faeces of cattle (15.2%), sheep (10.7%), goats (7.5%) and pigs (5.6%) as well as in beef (3.8%), goat-meat (1.7%) and pork (4.0%). All seven investigated STEC serogroups were found in cattle, all except O145 were found in sheep, three serogroups (O157, O26 and O111) were found in goats and three (O157, O111 and O128) in pigs. The rate of detection of each of the serogroups in all 2133 samples was: O157 (5.0%), O26 (0.2%), O91 (0.3%), O103 (0.3%), O111 (1.0%), O128 (0.2%) and O145 (0.1%). Of all 154 isolates, 11.0% had shiga toxin type 1 gene (stx(1)), 25.3% had stx(2) and 41.6% had stx(1)/stx(2); intimin gene (eaeA) was detected in 56.5% and enterohaemolysin gene (hlyA) in 75.3%. Among the O157 isolates, 24.5% were negative for stx genes but positive for eaeA and/or hlyA while 7.6% were negative for all four virulence genes. Fourteen different combinations of virulence genes were encountered but stx(1)/stx(2)/eaeA/hlyA combination was the most predominant. The percentage resistance of the isolates to the tested antimicrobial agents was: ampicillin (82.5%), chloramphenicol (42.9%), ciprofloxacin (22.1%), enrofloxacin (25.3%), nalidixic acid (37.7%), neomycin (24.0%), norfloxacin (20.8%), streptomycin (50.7%) and tetracycline (75.3%). One hundred and forty-eight (96.1%) of all 154 isolates were resistant to at least one of the tested antimicrobial agents while 69.5% were categorised as multi-drug resistant. Potentially pathogenic multi-drug resistant STEC isolates were recovered from the meat production chain in Nigeria. Unhygienic practices that predominate during slaughter and processing were observed to have contributed to faecal contamination and presence of STEC in meat., (Copyright 2010 Elsevier B.V. All rights reserved.)

Between 2002 and 2007, more than 1000 chickens from commercial farms, live bird markets and backyard farms in Nigeria and Niger were tested for the presence of the infectious bronchitis virus (IBV) genome. Phylogenetic analysis of full-length sequences of the spike 1 (S1) gene revealed a new genotype of IBV that we refer to as 'IBADAN'. The minimum genetic distance to the closest 'non-IBADAN' strains (UK/7/93 at the nucleotide level; H120 and M41 at the amino acid level) reached 24 and 32 % at the nucleotide and amino acid levels, respectively. The full genome of the IBADAN reference strain (NGA/A116E7/2006) had a genetic distance of 9.7-16.4 % at the nucleotide level with all available fully sequenced strains. As IBV S1 plays a major role in antigenicity, the antigenic relatedness of NGA/A116E7/2006 was compared with strains of other serotypes. NGA/A116E7/2006 did not cross-react with antisera against IT02, M41, D274, Connecticut or 793/B strains in virus neutralization assays. NGA/A116E7/2006 cross-reacted with the QX-like strain ITA/90254/2005 but only to a low level (antigenic relatedness of 33 %), suggesting that IBADAN also represents a new serotype. A comparison of S1 sequences identified several amino acids that may play a role in IBV antigenicity. Despite the absence of obvious clinical signs in poultry infected by IBADAN strains, it is important to test the cross-protection of current vaccine strains.

Forty-four Newcastle disease virus (NDV) strains, obtained between 2002 and 2007 from different poultry species in Nigeria, Niger, Burkina Faso and Cameroon, were phylogenetically analysed based on partial F sequences. Lineage 2 viruses were genetically identical or similar to the locally used LaSota vaccine strain and were mostly detected in commercial farms. Lineage 1, 3 and 4 strains were only sporadically found, and their origin was less clear. Twenty-one strains from backyard farms and live bird markets formed three new clusters within lineage 5, tentatively named 5f, 5g and 5h. All of these strains were predicted to be virulent based on their F protein cleavage site sequence. Minimal genetic distances between new and previously established sublineages ranged from 9.4 to 15.9%, and minimal distances between the new sublineages were 11.5 to 17.3%. Their high genetic diversity and their presence in three different Sub-Saharan countries suggest that these new sublineages represent the NDV variants indigenous to West Africa.

Eight new full-length sequences from highly pathogenic avian influenza viruses (H5N1) from 4 states in southwest Nigeria were analyzed. All gene sequences were more closely related to the first strains found in Nigeria in 2006 than to any strain found outside the country. Six viruses had evolved by at least 3 reassortment events (AC HA/NS, AC NS) from previously identified sublineages A (EMA 2) and C (EMA 1). Our results suggest that highly pathogenic avian influenza viruses (H5N1) initially imported into Nigeria in 2006 have been gradually replaced by various reassortments. In all reassortants, nonstructural genes were derived from sublineage C with 2 characteristic amino acids (compared with sublineage A). If the high prevalence of reassortants was typical for West Africa in 2007, the absence of such reassortants anywhere else suggests that reintroductions of influenza A (H5N1) from Africa into Eurasia must be rare.

In order to detect and characterize avian metapneumovirus, organs or swabs were collected from 697 chicken and 110 turkeys from commercial farms in Southwestern Nigeria and from 107 chickens from live bird markets in Southeastern China. In Nigeria, 15% and 6% of the chicken and turkey samples, respectively, and 39% of the chicken samples from China, were positive for aMPV genome by PCR. The sequence of a 400 nt fragment of the attachment protein gene (G gene) revealed the presence of aMPV subtype A in both Nigeria and Southeastern China. Essentially identical subtype A viruses were found in both countries and were also previously reported from Brazil and the United Kingdom, suggesting a link between these countries or a common source of this subtype. In Nigeria, subtype B was also found, which may be a reflection of chicken importations from most major poultry-producing countries in Europe and Asia. In order to justify countermeasures, further studies are warranted to better understand the metapneumoviruses and their impact on poultry production.

In Africa, highly pathogenic avian influenza H5N1 virus was first detected in northern Nigeria and later also in other regions of the country. Since then, seven other African countries have reported H5N1 infections. This study reports a comparison of full-length genomic sequences of H5N1 isolates from seven chicken farms in Nigeria and chicken and hooded vultures in Burkina Faso with earlier H5N1 outbreaks worldwide. In addition, the antigenicity of Nigerian H5N1 isolates was compared with earlier strains. All African strains clustered within three sublineages denominated A (south-west Nigeria, Niger), B (south-west Nigeria, Egypt, Djibouti) and C (northern Nigeria, Burkina Faso, Sudan, Côte d'Ivoire), with distinct nucleotide and amino acid signatures and distinct geographical distributions within Africa. Probable non-African ancestors within the west Asian/Russian/European lineage distinct from the south-east Asian lineages were identified for each sublineage. All reported human cases in Africa were caused by sublineage B. Substitution rates were calculated on the basis of sequences from 11 strains from a single farm in south-west Nigeria. As H5N1 emerged essentially at the same time in the north and south-west of Nigeria, the substitution rates confirmed that the virus probably did not spread from the north to the south, given the observed sequence diversity, but that it entered the country via three independent introductions. The strains from Burkina Faso seemed to originate from northern Nigeria. At least two of the sublineages also circulated in Europe in 2006 as seen in Germany, further suggesting that the sublineages had already emerged outside of Africa and seemed to have followed the east African/west Asian and Black Sea/Mediterranean flyways of migratory birds.

As the avian influenza virus H5N1 swept from Asia across Russia to Europe, Nigeria was the first country in Africa to report the emergence of this highly pathogenic virus. Here we analyse H5N1 sequences in poultry from two different farms in Lagos state and find that three H5N1 lineages were independently introduced through routes that coincide with the flight paths of migratory birds, although independent trade imports cannot be excluded.

Eight poultry farms in Nigeria, including chickens from nine breeder, 14 broiler, 28 pullet, 11 layer, and three cockerel flocks, were tested for antibody seroprevalence to the following poultry viruses of potential economic importance: infectious bronchitis virus (IBV), avian reovirus, avian pneumovirus (APV), infectious laryngotracheitis virus (ILTV), avian influenza virus (AIV), and avian leukosis virus (ALV). Serum samples were collected between 1999 and 2004 and were tested for antibodies using commercial enzyme-linked immunosorbent assay (ELISA) kits. Seroprevalence was very high for IBV (84%); intermediate for reovirus (41%), APV (40%), and ILTV (20%); and very low for ALV (<5%) antibodies. By commercial ELISA, the seroprevalence of antibodies against AIV was, in some flocks, up to 63%. However, more specific assays did not confirm AIV antibodies, indicating that all flocks tested were free of avian influenza antibodies. Birds seemed to be first infected by IBV (at about 7 wk of age), then by reovirus at 12 wk, before they became infected by APV (week 25) and ILTV (week 30). This is the first report of serological evidence of the above viruses in West Africa. Further studies are necessary to assess economic losses due to these avian viruses and the costs and benefits of countermeasures.

Between February 2002 and May 2004, chicken anemia virus (CAV) was detected by PCR in organ samples from 14 flocks of poultry farms in Lagos, Ogun and Oyo States in Southwestern Nigeria. The farms reported low (<5%) to high mortalities (up to 100%) with various lesions at necropsy. The complete VP1 gene of 30 of these positive strains was sequenced. Strains that diverged by up to 4.4% on a nucleotide level differed only by up to 2.5% at the amino acid level (7 aa) as a result of clustered silent mutations. No amino acid substitutions specific for Nigerian strains were observed. Some birds had a CAV mixed infection. Genetic clustering of the VP1 gene did not correlate with differences in flock mortality but the co-infection of CAV with IBDV may be particularly lethal. This first molecular epidemiological study of CAV in Africa shows that the Nigerian strains cluster with viruses from very diverse geographic origins and were almost as diverse (4.4%) as all other strains combined (5.8%).

Chicken anemia virus (CAV) was isolated for the first time from the Nigerian chicken population. The virus was recovered from necropsied birds from broiler and pullet flocks that suffered disease outbreaks tentatively diagnosed as infectious bursal disease. A sensitive polymerase chain reaction (PCR) assay detected CAV DNA in tissues of necropsied birds. Restriction endonuclease analysis performed with the 733-bp PCR product and the Cfo I enzyme indicated at least two different CAVs were circulating among the Nigerian chicken population. Four isolates were obtained from pooled liver and thymus tissues using the MDCC-MSB1 cell line. These isolates were found to be antigenically closely related to the Cuxhaven-1 (Cux-1) reference strain of CAV when reacted with four monoclonal antibodies prepared against the Cux-1 virus. One of the isolates (isolate A) induced thymus atrophy, bone marrow aplasia, and low hematocrit values when inoculated into 1-day-old specific-pathogen-free chickens. These findings not only demonstrate that CAV is present in Nigeria, but they also likely represent the first cell culture isolation of the virus in Africa.

Fifty-eight outbreaks of Infectious bursal disease virus (IBDV) were observed in vaccinated chicken flocks in four Southwestern states of Nigeria between 1995 and 2000. Bursa samples from 40 flocks were found virus-positive in VP2-specific nested RT-PCR. Sequences of the hypervariable region of VP2 were compared to reference strains of the different IBDV variants including also 1988 isolates from Nigeria. Sequence analysis revealed that all 40 Nigerian isolates belonged to the very virulent (vv) variant. The maximum sequence diversity of 5.7% was higher than in all other vvIBDV sequences listed in Genbank (3.6%). Two clusters within Nigerian isolates are unique to this region. Serotype 1 IBDV was also detected in four symptomatic turkey flocks. The turkey isolates were found within 2 of the 3 VV-clusters of chicken isolates. Full length sequence of a turkey isolate (NIE009t) confirmed its close relation to vvIBDV strain D6948NET for both segment A (1.4% sequence diversity) and segment B (2.1%). Thus, turkeys should be considered susceptible to vvIBDV infection. The unusually high sequence diversity of vvIBDV may be an indication of a West-African origin of this virus, from where it spread to other continents.

Sera samples from seven poultry farms in southwest Nigeria consisting of 7 broiler, 10 pullet, 1 layer, 1 cockerel, and 1 broiler breeder flocks were tested for the presence of chicken infectious anemia virus (CIAV) antibodies using a commercial enzyme-linked immunosorbent assay kit. Eleven of the 20 flocks (55%) and six out of seven (86%) farms were positive for CIAV antibodies. The seroprevalence largely depended on the age of the flocks. Seroprevalence was higher within the older pullet and layer flocks (83%-100%) than in the younger broiler flocks (0%-83%). In essence, all flocks older than 6 to 8 wk became infected. This is the first report of serologic evidence of CIAV in Subsaharan Africa. Since Southwest Nigeria is the main port of entry of imported chicken and the hub of major poultry breeders, the disease can probably be found throughout the country and beyond. Further studies are necessary to assess economic losses due to CIAV and the cost benefit of countermeasures.

Infectious bursal disease virus antigen was detected in the bursa of Fabricius of infected birds by the use of the counter immunoelectroosmophoresis technique. Eight suspected outbreaks were confirmed in this way and 82 out of 89 bursal samples submitted for laboratory confirmation were positive. The test was also suitable for the detection of antibody to infectious bursal disease virus in sera of infected birds. Precipitin lines were visible within 30 min as compared with 18 to 24 h with the Ouchterlony agar gel precipitation test. Counter immunoelectroosmophoresis is recommended for rapid confirmation of infectious bursal disease of poultry.