Newcastle disease vaccine virus I-2 fails to acquire virulence during repeated passage in vivo.

Background This study determined whether the naturally attenuated, thermotolerant Newcastle disease vaccine virus I-2 could acquire virulence after five in vivo passages through SPF chickens. Methods Study design was to international requirements including European Pharmacopoeia, Ph. Eur., v9.0 04/2013:0450, 2013. I-2 Working Seed (WS) was compared with five-times-passaged I-2 WS (5XP WS) in intracerebral pathogenicity index (ICPI), F o cleavage site sequencing and Safety tests. Results The first passage series used a 50% brain: 50% tracheal tissue challenge homogenate and was unsuccessful as I-2 was not detected after the fourth passage. A second passage series used 10% brain: 90% tracheal tissue homogenates. I-2 was isolated from tracheal tissue in each passage. However harvested titres were below the minimum challenge level (10 7 EID 50) specified for the ICPI and Safety tests, possibly reflecting I-2's inherently low pathogenicity (interestingly caecal tonsils yielded significant titres). Given this the WS and 5XP WS comparisons proceeded. ICPI values were 0.104 and 0.073 for the WS group and the 5XP WS group respectively confirming that I-2, whether passaged or not, expressed low pathogenicity. F 0 amino-acid sequences for both WS and 5XP WS were identified as 112R-K-Q-G-R-↓-L-I-G 119 and so compatible with those of avirulent ND viruses. In safety, no abnormal clinical signs were observed in both groups except for two chicks in the 5XP WS group, where one bird was withdrawn due to a vent prolapse, and another bird died with inconclusive necropsy results. Conclusions: These data, the issue of low passage titres with little or no virus isolation from brain tissues and the genomic copy approach suggest a need to amend Ph. Eur. v9.0 04/2013:0450, 2013 for naturally attenuated, low pathogenicity vaccine viruses such as I-2. From an international regulatory perspective, the study provides further definitive data demonstrating that Newcastle disease vaccine virus I-2 is safe for use.

Enterococcal-related vertebral osteoarthritis in South African broiler breeders: A case report.

Infections in broilers and broiler breeders by Enterococcus cecorum, causing clinical disease, have increasingly been described in various countries in the Northern Hemisphere over the past decade. This case report describes an outbreak of enterococcal-associated vertebral osteoarthritis (EVOA) in male broiler breeders in several flocks in South Africa. Male birds aged 4 and 9 weeks displayed the common presentation of lameness, paresis or complete paralysis. Autopsies of culled birds revealed masses on caudal thoracic vertebrae T5-T7, with vertebral osteomyelitis and spondylitis. Microbiological assays identified E. cecorum cultured from spondylitic lesions. Affected flocks were treated with amoxycillin at 25 mg/kg in the drinking water for 5 days, resulting in decreased numbers of lame birds and culls. The origin and pathogenesis of EVOA are poorly understood, which limits prevention to environmental factors that may inhibit systemic access by the enteric bacteria. Skeletal growth trends of male birds are thought to increase their susceptibility to bacterial colonisation at sites of skeletal strain, resulting in abscesses and lesions. Evidence points to the emergence of E. cecorum strains with increased pathogenicity; this highlights the need for greater understanding of the origins, treatment and prevention of EVOA to minimise its economic impact on poultry operations.

Serological and molecular investigation of Newcastle disease in household chicken flocks and associated markets in Eastern Shewa zone, Ethiopia.

Cross-sectional survey for Newcastle disease (ND) were conducted in nonvaccinated household flocks of village chickens to assess serological and virological ND status in households and associated live bird markets. In total, 1,899 sera and 460 pools of cloacal and tracheal swabs were sampled and tested using a commercial enzyme-linked immunosorbent assay (ELISA) and real-time reverse transcriptase polymerase chain reaction (rRT-PCR), respectively. Additionally, paired cloacal and tracheal swabs from 1,269 individual chickens were collected from markets and tested using RT-PCR. The prevalence of households with at least one seropositive chicken was higher during the dry season (27.4 %) than during the wet season (17.4 %) (P = 0.003). Viral genome was detected in 14.2 % of households during the wet season using a fusion (F) gene assay and in 24.2 % of households during the dry season using a polymerase (L) gene assay that targets both class I and class II viruses. At the markets sampled, overall bird level prevalence was 4.9 % for period 1 (F gene assay), and 38.2 % and 27.6 % for periods 2 and 3, respectively (L gene assay). Partial sequencing of the F gene (239 bp) cleavage site indicated that the majority of the circulating strains exhibited motifs specific to virulent strains. Seroepidemiology coupled with molecular analysis can be a useful tool to assess the status of NDV infection. The village chicken population in Ethiopia is endemically infected with virulent NDV that pose a significant threat to emerging small- and medium-scale commercial poultry production.

Phylogenetic analysis of Newcastle disease viruses isolated from asymptomatic guinea fowls (Numida meleagris) and Muscovy ducks (Cariana moscata) in Nigeria.

Four Newcastle disease virus isolates were recovered from asymptomatic guinea fowl (Numida meleagris galeata) and Muscovy ducks (Cariana moscata). For the purpose of molecular identification and phylogeny, phylogenetic characterization was performed to identify the pathotypes. All four viruses had a cleavage motif (112)RRQKRF(117) which is characteristic of virulent strains. The isolates grouped with viruses previously reported as sub-lineage 5 g from chickens in Nigeria. This study report for the first time the identification of the virulent sub-lineage 5 g Newcastle disease virus from apparently healthy guinea fowl and domestic ducks in Nigeria, and since infections were sub-clinical, it suggest that these species could play a role in the spread and transmission of virulent Newcastle disease virus that can infect other poultry. The isolation and identification of virulent Newcastle disease virus in these unusual hosts and the high sequence similarity (99.3-100 %) between viruses in this study with strains reported for Niger and Cameroun gives insights into the ecology of virulent Newcastle disease viruses and suggests some cross-border movement and trade in live poultry.

Household-level risk factors for Newcastle disease seropositivity and incidence of Newcastle disease virus exposure in backyard chicken flocks in Eastern Shewa zone, Ethiopia.

A cross-sectional study with repeated sampling was conducted to investigate potential risk factors for Newcastle disease (ND) seropositivity and for incidence of ND virus (NDV) exposure in household flocks of backyard chickens in Eastern Shewa zone, Ethiopia. Data were collected from 260 randomly selected households in 52 villages in Adami Tulu Jido Kombolcha and Ada'a woredas (districts) using a structured questionnaire, and serum samples from chickens were tested for NDV antibodies using a blocking enzyme-linked immunosorbent assay (ELISA). Sampling took place during September 2009 and the same households were again sampled in May 2010. Household-level seroprevalence and incidence of NDV exposure were estimated in various ways using serological results from the two samplings, flock dynamics, and farmers' reports of ND in their flocks. The risk factors were assessed using multivariable mixed-effects logistic regression models. Household-level seroprevalence at the two sampling times was 17.4% and 27.4%, respectively, and the estimated incidence of household-level NDV exposure during the intervening period ranged between 19.7% and 25.5%. At the first sampling, reduced frequency of cleaning of poultry waste was associated with increased odds of seropositivity (OR=4.78; 95% CI: 1.42, 16.11; P=0.01) while hatching at home vs. other sources (buying in replacement birds, receiving as gift or buying fertile eggs) was associated with lower odds of seropositivity, both at the first sampling (OR=0.30; 95% CI: 0.11, 0.82; P=0.02) and the second sampling (OR=0.23; 95% CI: 0.10, 0.52; P<0.001). The risk of NDV exposure was shown to be higher with larger flock size at the beginning of the observation period (OR=3.6; 95% CI: 1.25, 10.39; P=0.02). Using an open water source (pond or river) for poultry compared to closed sources (tap or borehole) was associated with increased risk of NDV exposure (OR=3.14; 95% CI: 1.12, 8.8; P=0.03). The use of a grain supplement (OR=0.14; 95% CI: 0.03, 0.69; P=0.03) and hatching at home for flock replacement (OR=0.23; 95% CI: 0.10, 0.52; P=0.005) were associated with a lower risk of NDV exposure. Newcastle disease seroprevalence and incidence of NDV exposure were more heterogeneous between villages than between kebeles (aggregations of villages) and woredas in the study area. Further investigation of village-level risk factors would likely improve our understanding of ND epidemiology in backyard chickens.

Virulent Newcastle disease virus in Nigeria: identification of a new clade of sub-lineage 5f from livebird markets.

Newcastle disease (ND), caused by Avian Paramyxovirus Type 1, is a highly contagious and devastating viral disease of poultry of worldwide distribution with an enormous economic impact. Although ND is reported to be endemic in Nigeria, little information exists on the molecular epidemiology and the lineage distribution of the Newcastle disease viruses (NDVs) in the country, especially in the live bird markets (LBMs). Recent studies reported the identification of three unique sub-lineages. namely; 5f, 5g and 5h in West Africa, and sub-lineages 5f and 5g in particular in non-commercial farms in Nigeria. In this study, 33 NDV isolates, which included NDVs recovered from LBMs in Nigeria, during active surveillance from 2007 to 2008 and viruses recovered from outbreaks in backyard and commercial chicken farms within the same period were analysed. Based on determination of the F(0) cleavage site amino acid sequence and phylogenetic analysis, the isolates were classified as virulent; 16 strains were identified as sub-lineage 5g and 17 as sub-lineage 5f. Interestingly, 13 strains from the 5f group formed a distinct cluster that was not identified by other groups in similar studies. The close genetic similarities identified, provided evidence for the first time of the epidemiological link between the viruses circulating in the LBMs and those recovered from outbreaks in backyard and commercial chicken farms in Nigeria between 2007 and 2008. The emergence and identification of new sub-lineages provide an insight into the high rate of genetic drift occurring in NDV strains in Nigeria, and raises a lot of concerns about the efficacy of current ND control measures in the country.

Determination of the distribution of lentogenic vaccine and virulent Newcastle disease virus antigen in the oviduct of SPF and commercial hen using immunohistochemistry.

The control of Newcastle disease (ND) in South Africa has proved difficult since 2002 following the introduction of lineage 5d/VIId Newcastle disease virus (NDV) strain ("goose paramyxovirus" - GPMV) to which commercially available ND vaccines appeared less effective. Most of the ND infections, even in fully vaccinated hens were characterized consistently by a drop in egg production. In this study, commercial and SPF hens-in-lay were vaccinated with La Sota vaccine and challenged with a GPMV isolate. Immunohistochemical labeling was used to determine the distribution of viral antigen in the oviduct of the hens. Following reports that cloacal vaccination offered better protection against egg production losses than the oro-nasal route, the efficacy of cloacal and ocular routes of vaccination against challenge were compared. Results showed that La Sota vaccine offered birds 100% protection against the virulent ND (GPMV) virus challenge from clinical disease and death, but not against infection and replication of the GPMV, as birds showed varying degrees of macropathology. Histopathology of the oviduct of infected birds revealed multifocal lymphocytic inflammation in the interstitium as well as mild glandular ectasia and mild edema. Finely granular NDV-specific immunolabeling was demonstrated in the cytoplasm of epithelial cells and mononuclear (lymphohistiocytic) cells in the interstitium of the oviduct. Both vaccine and virulent GPMV showed greatest tropism for the uterus (versus the magnum and isthmus). There was no clear difference in the protection of the oviduct and in the distribution of oviductal GPMV antigens between the two routes of vaccination.

A survey of antimicrobial residues in table eggs in Khartoum State, Sudan, 2007-2008.

The risk to consumers of antimicrobial residues in table eggs produced in Khartoum State, Sudan, was studied. All producing layer farms (n = 175) in the state were sampled in April, June and August 2008. A total of 933 eggs from 335 layer houses were screened for antimicrobial residues by using the growth inhibition of Geobacillus stearothermophilus var. calidolactis in-house test. A high proportion of layer farms (72% in April, 61% in June and 66% in August) and layer houses (63% April, 59% in June and 61% in August) were found to have antimicrobial residues, with no significant difference in prevalence (p = 0.57) between study periods. The study showed that the consumer was at constant risk of exposure to antimicrobial residues in table eggs. The paper discusses reasons for the high prevalence of antimicrobial residues in Sudanese eggs and its implications, and makes recommendations to address this important public health problem.

A questionnaire survey of poultry layer farmers in Khartoum State, Sudan, to study their antimicrobial awareness and usage patterns.

An initial census of layer farms in Khartoum State, Sudan, was carried out in late 2007 and early 2008 and found that there were 252 layer farms with a total population of 2 221 800 birds. This paper reports the findings of the census. Based on this information, a structured questionnaire survey of 92 farms was then conducted in the state in April 2008 to collect data on antibiotic usage, demographic data and public health awareness. Ninety-eight per cent of participating farms comprised open-sided houses. It was found that 49% of the farms surveyed were on antibiotic treatment when the survey was conducted, whilst 59% of the farms had used antibiotics within the last 3 months. The study found that farmers and producers had a lack of knowledge about antimicrobial residues, their withdrawal periods and the risk posed by the consumption of these residues. The study also concluded that traditional farming systems in Sudan relied heavily on antimicrobial medication to control disease and almost half of the farms surveyed were treating their flocks with antimicrobials. In addition to this, there was a lack of disease control programmes which probably resulted in a massive use of antibiotics to control endemic diseases. This was further compounded by the absence of governmental supervision and control on the use of drugs.

Molecular characterisation of Newcastle disease virus isolates from different geographical regions in Mozambique in 2005.

Newcastle disease (ND) is regarded as a highly contagious and economically important disease in poultry and has a worldwide distribution. Viral determinants for Newcastle disease virus (NDV) virulence are not completely understood and viruses of different pathotypes can be found at live-bird markets in different geographical areas. The prevalence of Newcastle disease in village poultry in Mozambique is not well documented and strains of NDV involved in yearly outbreaks are unknown. The fusion (F) protein is an important determinant of pathogenicity of the virus and is used commonly for phylogenetic analysis. Newcastle disease viruses from various geographical regions of Mozambique were sequenced and compared genetically to published sequences obtained from GenBank. Samples were collected in three different areas of Mozambique and NDV was isolated by infection of embryonated chicken eggs. Sequence analysis of the F-protein encoding gene was used to classify 28 isolates from Mozambique into genotypes and compare these genotypes phylogenetically with existing genotypes found in GenBank. The isolates obtained from Mozambique grouped mainly into two clades. In the first clade, 12 isolates grouped together with sequences of isolates representing genotypes from Mozambique that were previously described. In the second clade, 16 isolates group together with sequences obtained from GenBank originating from Australia, China, South Africa and the USA. Eleven of these isolates showed a high similarity with sequences from South Africa. The number of samples sequenced (n = 28), as well as the relatively small geographical collection area used in this study, are too small to be a representation of the circulating viruses in Mozambique in 2005. Viruses characterised in this study belonged to lineage 5b, a similar finding of a previous study 10 years ago. From this data, it merely can be concluded that no new introduction of the virus occurred from 1995 to 2005 in Mozambique.

Identification of risk factors associated with highly pathogenic avian influenza H5N1 virus infection in poultry farms, in Nigeria during the epidemic of 2006-2007.

We conducted a matched case-control study to evaluate risk factors for infection with highly pathogenic avian influenza (HPAI) H5N1 virus in poultry farms during the epidemic of 2006-2007 in Nigeria. Epidemiologic data were collected through the use of a questionnaire from 32 case farms and 83 control farms. The frequency of investigated exposure factors was compared between case and control farms by using conditional logistic regression analysis. In the multivariable analysis, the variables for (i) receiving visitors on farm premises (odds ratio [OR]=8.32; 95% confidence interval [CI]=1.87, 36.97; P<0.01), (ii) purchased live poultry/products (OR=11.91; 95% CI=3.11-45.59; P<0.01), and (iii) farm workers live outside the premises (OR=8.98; 95% CI=1.97, 40.77; P<0.01) were identified as risk factors for HPAI in poultry farms. Improving farm hygiene and biosecurity should help reduce the risk for influenza (H5N1) infection in poultry farms in Nigeria.

Outbreaks of avian influenza H6N2 viruses in chickens arose by a reassortment of H6N8 and H9N2 ostrich viruses.

The first recorded outbreak of avian influenza (AI) in South African chickens (low pathogenicity H6N2) occurred at Camperdown, KwaZulu/Natal Province (KZN) in June 2002. To determine the source of the outbreak, we defined the phylogenetic relationships between various H6N2 isolates, and the previously unpublished gene sequences of an H6N8 virus isolated in 1998 from ostriches in the Leeu Gamka region (A/Ostrich/South Africa/KK98/98). We demonstrated that two distinct genetic H6N2 lineages (sub-lineages I and II) circulated in the Camperdown area, which later spread to other regions. Sub-lineages I and II shared a recent common H6N2 ancestor, which arose from a reassortment event between two South African ostrich isolates A/Ostrich/South Africa/9508103/95 and (H9N2) A/Ostrich/South Africa/KK98/98 (H6N8). Furthermore, the H6N2 sub-lineage I viruses had several molecular genetic markers including a 22-amino acid stalk deletion in the neuraminidase (NA) protein gene, a predicted increased N-glycosylation, and a D144 mutation of the HA protein gene, all of which are associated with the adaptation of AI viruses to chickens. The H6N2 NS1 and PB1 genes shared recent common ancestors with those of contemporary Asian HPAI H5N1 viruses. Our results suggest that ostriches are potential mixing vessels for avian influenza viruses (AIV) outbreak strains and support other reports that H6 viruses are capable of forming stable lineages in chickens.