Application of loop-mediated isothermal amplification assay in the detection of herpesvirus of turkey (FC 126 strain) from chicken samples in Nigeria.

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.

Characterization of field and vaccine infectious bursal disease viruses from Nigeria revealing possible virulence and regional markers in the VP2 minor hydrophilic peaks.

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.

Potentially zoonotic shiga toxin-producing Escherichia coli serogroups in the faeces and meat of food-producing animals in Ibadan, Nigeria.

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.

Effects of stresroak as an immunomodulator on birds vaccinated with newcastle disease vaccine.

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.

Avian metapneumovirus subtype A in China and subtypes A and B in Nigeria.

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.

Molecular and antigenic evolution and geographical spread of H5N1 highly pathogenic avian influenza viruses in western Africa.

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.

Seroprevalence of avian influenza virus, infectious bronchitis virus, reovirus, avian pneumovirus, infectious laryngotracheitis virus, and avian leukosis virus in Nigerian poultry.

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.

Avian flu: multiple introductions of H5N1 in Nigeria.

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.

Molecular epidemiology of chicken anemia virus in Nigeria.

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%).

Isolation and preliminary characterization of chicken anemia virus from chickens in Nigeria.

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.

Serologic evidence of chicken infectious anemia in commercial chicken flocks in southwest Nigeria.

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.

High sequence diversity in infectious bursal disease virus serotype 1 in poultry and turkey suggests West-African origin of very virulent strains.

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.

Counter immunoelectroosmophoresis in the diagnosis of infectious bursal disease of poultry.

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.