Effects of swab pool size and transport medium on the detection and isolation of avian influenza viruses in ostriches.

BACKGROUND: Rigorous testing is a prerequisite to prove freedom of notifiable influenza A virus infections in commercially farmed ostriches, as is the isolation and identification of circulating strains. Pooling 5 ostrich tracheal swabs in a 50 % v/v phosphate-buffered saline (PBS): glycerol transport medium (without antibiotics) is the current standard practice to increase reverse transcription real time PCR (RT-rtPCR) testing throughput and simultaneously reduce the test costs. In this study we investigated whether doubling ostrich tracheal swabs to 10 per pool would affect the sensitivity of detection of H5N8 high pathogenicity avian influenza virus (HPAIV) and H7N1 low pathogenicity avian influenza virus (LPAIV) by quantitative RT-rtPCR, and we also compared the effect of a protein-rich, brain heart infusion broth (BHI) virus transport media containing broad spectrum antimicrobials (VTM) on the efficacy of isolating the H5N8 and H7N1 viruses from ostrich tracheas, since the historical isolation success rate from these birds has been poor. RESULTS: Increasing the ostrich swabs from 5 to 10 per pool in 3 mls of transport medium had no detrimental effect on the sensitivity of the RT-rtPCR assay in detecting H5N8 HPAIV or H7N1 LPAIV; and doubling of the swab pool size even seemed to improve the sensitivity of virus detection at levels that were statistically significant (p less than or equal to 0.05) in medium and low doses of spiked H5N8 HPAIV and at high levels of spiked H7N1 LPAIV. On virus isolation, more samples were positive when swabs were stored in a protein-rich viral transport medium supplemented with antimicrobials in PBS: glycerol (10/18 vs. 7/18 for H5N8 HPAI); although the differences were not statistically significant, overall higher virus titres were detected (106.7 - 103.0 vs. 106.6 - 103.1 EID50 for H5N8 HPAIV and 105.5 - 101.4 vs. 105.1 - 101.3 EID50 for H7N1 LPAIV); and fewer passages were required with less filtration for both H5N8 HPAI and H7N1 LPAI strains. CONCLUSION: Ostrich tracheal swab pool size could be increased from 5 to 10 in 3mls of VTM with no loss in sensitivity of the RT-rtPCR assay in detecting HPAI or LPAI viruses, and HPAI virus could be isolated from a greater proportion of swabs stored in VTM compared to PBS: glycerol without antibiotics.

The spatiotemporal epidemiology of high pathogenicity avian influenza outbreaks in key ostrich producing areas of South Africa.

High pathogenicity avian influenza (HPAI) has become a major focus point worldwide due to its zoonotic potential and economic effects resulting from trade restrictions and high mortality rates in poultry. Key ostrich producing provinces of South Africa have experienced three H5N2 HPAI outbreaks (2004, 2006 and 2011) and one H5N8 HPAI (2017) outbreak over the past two decades. The Klein Karoo region in the Western Cape Province, a province with a largely Mediterranean climate, is the predominant ostrich producing region in the country. Understanding the epidemiology of HPAI in ostrich producing areas is an essential first step in developing effective and efficient control measures. This study investigated the spatiotemporal patterns associated with the 2011 (H5N2) and 2017 (H5N8) HPAI outbreaks in the key ostrich producing areas of South Africa. Six hundred and nine and 340 active ostrich farms/holdings were subjected to surveillance during 2011 and 2017 respectively, with over 70 % of these farms located within five local municipalities of the study area. Forty-two and fifty-one farms were affected in the 2011 and 2017 outbreaks respectively. Both HPAI outbreaks occurred predominantly in areas of high ostrich farm density. However, the temporal occurrence, spatial and directional distributions of the outbreaks were different. The 2011 outbreak occurred earlier in the South African autumn months with a predominantly contiguous and stationary distribution, whilst the 2017 outbreak onset was during the winter with a more expansive multidirectional spatial distribution. Results suggest potential dissimilarities in the important risk factors for introduction and possible mode of spread. The 2011 outbreak pattern resembled an outbreak characterised by point introductions with the risk of introduction possibly being linked to high ostrich farm density and common management and husbandry practices in the ostrich industry. In contrast, the 2017 outbreak appeared to have a more propagating mode of transmission. The findings highlight epidemiological features of HPAI outbreak occurrence within ostrich populations that could be used to inform surveillance and control measures including targeted surveillance within high-risk spatial clusters. The study emphasizes the importance of both; implementation of a multi-pronged approach to HPAI control and the need for constant evaluation of the interaction of the host, environment and agent with each outbreak, in order to strengthen disease control.

Whole Genome Characterization and Genetic Evolution Analysis of a New Ostrich Parvovirus.

Ostrich diseases characterized by paralysis have been breaking out in broad areas of China since 2015, causing major damage to the ostrich breeding industry in China. This report describes a parvovirus detected in ostriches from four different regions. The entire genomes of four parvovirus strains were sequenced following amplification by PCR, and we conducted comprehensive analysis of the ostrich parvovirus genome. Results showed that the length genomes of the parvovirus contained two open reading frames. Ostrich parvovirus (OsPV) is a branch of goose parvovirus (GPV). Genetic distance analysis revealed a close relationship between the parvovirus and goose parvovirus strains from China, with the closest being the 2016 goose parvovirus RC16 strain from Chongqing. This is the first report of a parvovirus in ostriches. However, whether OsPV is the pathogen of ostrich paralysis remains uncertain. This study contributes new information about the evolution and epidemiology of parvovirus in China, which provides a new way for the study of paralysis in ostriches.

Human surveillance and phylogeny of highly pathogenic avian influenza A(H5N8) during an outbreak in poultry in South Africa, 2017.

BACKGROUND: In June 2017, an outbreak of the highly pathogenic avian influenza A(H5N8) was detected in commercial poultry farms in South Africa, which rapidly spread to all nine South African provinces. OBJECTIVES: We conducted active surveillance for the transmission of influenza A(H5N8) to humans working with infected birds during the South African outbreak. METHODS: Influenza A(H5N8)-positive veterinary specimens were used to evaluate the ability of real-time PCR-based assays to detect contemporary avian influenza A(H5N8) strains. Whole genome sequences were generated from these specimens by next-generation sequencing for phylogenetic characterization and screening for mammalian-adaptive mutations. RESULTS: Human respiratory samples from 74 individuals meeting our case definition, all tested negative for avian influenza A(H5) by real-time PCR, but 2 (3%) were positive for human influenza A(H3N2). 54% (40/74) reported wearing personal protective equipment including overalls, boots, gloves, masks, and goggles. 94% (59/63) of veterinary specimens positive for H5N8 were detected on an influenza A(H5) assay for human diagnostics. A commercial H5N8 assay detected H5 in only 6% (3/48) and N8 in 92% (44/48). Thirteen (13/25; 52%) A(H5N8) genomes generated from veterinary specimens clustered in a single monophyletic clade. These sequences contained the NS (P42S) and PB2 (L89V) mutations noted as markers of mammalian adaptation. CONCLUSIONS: Diagnostic assays were able to detect and characterize influenza A(H5N8) viruses, but poor performance is reported for a commercial assay. Absence of influenza A(H5N8) in humans with occupational exposure and no clear impression of molecular adaptation for mammalian infection suggest that this avian pathogen continues to be low-risk human pathogen.

Risk of Human Infections With Highly Pathogenic H5N2 and Low Pathogenic H7N1 Avian Influenza Strains During Outbreaks in Ostriches in South Africa.

Background: Risk factors for human infection with highly pathogenic (HP) and low-pathogenic (LP) avian influenza (AI) H5N2 and H7N1 were investigated during outbreaks in ostriches in the Western Cape province, South Africa. Methods: Serum surveys were conducted for veterinarians, farmworkers, and laboratory and abattoir workers involved in 2 AI outbreaks in the Western Cape province: (1) controlling and culling of 42000 ostriches during (HPAI)H5N2 outbreaks in ostriches (2011) (n = 207); (2) movement control during (LPAI)H7N1 outbreaks in 2012 (n = 66). A third serosurvey was conducted on state veterinarians from across the country in 2012 tasked with disease control in general (n = 37). Antibodies to H5 and H7 were measured by means of hemagglutination inhibition and microneutralization assays, with microneutralization assay titers >40 considered positive. Results: Two of 207 (1%) participants were seropositive for H5 and 4 of 207 (2%) for H7 in 2011, compared with 1 of 66 (1.5%) and 8 of 66 (13%) in 2012. Although individuals in all professions tested seropositive, abattoir workers (10 of 97; 10.3%) were significantly more at risk of influenza A(H7N1) infection (P = .001) than those in other professions (2 of 171;1.2%). Among state veterinarians, 4 of 37(11%) were seropositive for H7 and 1 of 37 (2.7%) for H5. Investigations of (LP)H7N1-associated fatalities in wild birds and quarantined exotic birds in Gauteng, AI outbreaks in poultry in KwaZulu-Natal, and ostriches in Western Cape province provide possible exposure events. Conclusion: (LPAI)H7N1 strains pose a greater infection-risk than (HPAI)H5N2 strains to persons involved in control of outbreaks in infected birds, with ostrich abattoir workers at highest risk.

Susceptibility and Status of Avian Influenza in Ostriches.

The extensive nature of ostrich farming production systems bears the continual risk of point introductions of avian influenza virus (AIV) from wild birds, but immune status, management, population density, and other causes of stress in ostriches are the ultimate determinants of the severity of the disease in this species. From January 2012 to December 2014, more than 70 incidents of AIV in ostriches were reported in South Africa. These included H5N2 and H7N1 low pathogenicity avian influenza (LPAI) in 2012, H7N7 LPAI in 2013, and H5N2 LPAI in 2014. To resolve the molecular epidemiology in South Africa, the entire South African viral repository from ostriches and wild birds from 1991 to 2013 (n = 42) was resequenced by next-generation sequencing technology to obtain complete genomes for comparison. The phylogenetic results were supplemented with serological data for ostriches from 2012 to 2014, and AIV-detection data from surveillance of 17 762 wild birds sampled over the same period. Phylogenetic evidence pointed to wild birds, e.g., African sacred ibis (Threskiornis aethiopicus), in the dissemination of H7N1 LPAI to ostriches in the Eastern and Western Cape provinces during 2012, in separate incidents that could not be epidemiologically linked. In contrast, the H7N7 LPAI outbreaks in 2013 that were restricted to the Western Cape Province appear to have originated from a single-point introduction from wild birds. Two H5N2 viruses detected in ostriches in 2012 were determined to be LPAI strains that were new introductions, epidemiologically unrelated to the 2011 highly pathogenic avian influenza (HPAI) outbreaks. Seventeen of 27 (63%) ostrich viruses contained the polymerase basic 2 (PB2) E627K marker, and 2 of the ostrich isolates that lacked E627K contained the compensatory Q591K mutation, whereas a third virus had a D701N mutation. Ostriches maintain a low upper- to midtracheal temperature as part of their adaptive physiology for desert survival, which may explain the selection in ratites for E627K or its compensatory mutations-markers that facilitate AIV replication at lower temperatures. An AIV prevalence of 5.6% in wild birds was recorded between 2012 and 2014, considerably higher than AIV prevalence for the southern African region of 2.5%-3.6% reported in the period 2007-2009. Serological prevalence of AI in ostriches was 3.7%, 3.6%, and 6.1% for 2012, 2013, and 2014, respectively. An annual seasonal dip in incidence was evident around March/April (late summer/autumn), with peaks around July/August (mid to late winter). H5, H6, H7, and unidentified serotypes were present at varying levels over the 3-yr period.

Tracking socioeconomic vulnerability using network analysis: insights from an avian influenza outbreak in an ostrich production network.

BACKGROUND: The focus of management in many complex systems is shifting towards facilitation, adaptation, building resilience, and reducing vulnerability. Resilience management requires the development and application of general heuristics and methods for tracking changes in both resilience and vulnerability. We explored the emergence of vulnerability in the South African domestic ostrich industry, an animal production system which typically involves 3-4 movements of each bird during its lifetime. This system has experienced several disease outbreaks, and the aim of this study was to investigate whether these movements have contributed to the vulnerability of this system to large disease outbreaks. METHODOLOGY/PRINCIPAL FINDINGS: The ostrich production system requires numerous movements of birds between different farm types associated with growth (i.e. Hatchery to juvenile rearing farm to adult rearing farm). We used 5 years of movement records between 2005 and 2011 prior to an outbreak of Highly Pathogenic Avian Influenza (H5N2). These data were analyzed using a network analysis in which the farms were represented as nodes and the movements of birds as links. We tested the hypothesis that increasing economic efficiency in the domestic ostrich industry in South Africa made the system more vulnerable to outbreak of Highly Pathogenic Avian Influenza (H5N2). Our results indicated that as time progressed, the network became increasingly vulnerable to pathogen outbreaks. The farms that became infected during the outbreak displayed network qualities, such as significantly higher connectivity and centrality, which predisposed them to be more vulnerable to disease outbreak. CONCLUSIONS/SIGNIFICANCE: Taken in the context of previous research, our results provide strong support for the application of network analysis to track vulnerability, while also providing useful practical implications for system monitoring and management.

Molecular characterization of Newcastle disease viruses in Ostriches (Struthio camelus L.): further evidences of recombination within avian paramyxovirus type 1.

Newcastle disease virus (NDV) strains isolated from ostriches have been genotyped for the first time by partial sequencing of the F gene to determine the epidemiologic role that this species can play within ND outbreaks. Fifteen additional NDV strains, mostly isolated from chickens but also from pigeons and penguins, were also included in the study to determine genetic relationships with ostriches NDV isolates. High genetic diversity was demonstrated in ostrich NDV isolates, as the 10 isolates were grouped in four distinct NDV genotypes. In agreement with the results obtained when chicken isolates have been molecularly characterized, the predominant genotype in ostriches was the genotype VII. More interestingly, evidences of recombination between genotype II and VII were observed in one ostrich isolate and in two further chicken isolates. Therefore, it seems that ostriches may play a relevant role in the ecology and epidemiology of ND particularly in those regions where they have an increasing farming importance as minor poultry species.

Characterization of a highly pathogenic avian influenza H5N1 virus isolated from an ostrich.

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among poultry and wild birds has posed a potential threat to human public health. An influenza pandemic happens, when a new subtype that has not previously circulated in humans emerges. Almost all of the influenza pandemics in history have originated from avian influenza viruses (AIV). Birds are significant reservoirs of influenza viruses. In the present study, we performed a survey of avian influenza virus in ostriches and H5N1 virus (A/Ostrich/SuZhou/097/03, China097) was isolated. This H5N1 virus is highly pathogenic to both chickens and mice. It is also able to replicate in the lungs of, and to cause death in, BALB/c mice following intranasal administration. It forms plaques in chicken embryo fibroblast (CEF) cells in the absence of trypsin. The hemagglutinin (HA) gene of the virus is genetically similar to A/Goose/Guangdong/1/96(H5N1) and belongs to clade 0. The HA sequence contains multiple basic amino acids adjacent to the cleavage site, a motif associated with HPAI viruses. More importantly, the existence of H5N1 isolates in ostriches highlights the potential threat of wild bird infections to veterinary and public health.

Evaluation of different serological tests for the detection of antibodies against highly pathogenic avian influenza in experimentally infected ostriches (Struthio camelus).

In the present study we collected 177 serum samples from ostriches (Struthio camelus) infected experimentally with A/ostrich/South Africa/Middleton/2004 (H5N2) highly pathogenic avian influenza virus. We tested these samples using the haemagglutination inhibition (HI) test, the agar gel immunodiffusion test and three enzyme-linked immunosorbent assay kits. We considered the HI test, with homologous antigen and including pre-treatment of sera with 10% chicken red blood cells, as the gold standard. Detectable specific antibodies appeared on day 7 post-infection and persisted until the termination of the experiment. The relative sensitivity and specificity of the tests under evaluation and Cohen's K value were calculated. The results reported herein could be of assistance to decision-makers in drafting guidelines for the definition of the health status of ostriches and for trade purposes.

Ostrich involvement in the selection of H5N1 influenza virus possessing mammalian-type amino acids in the PB2 protein.

Amino acids at positions 627 and 701 in the PB2 protein (PB2-627 and PB2-701, respectively) of avian influenza A viruses affect virus replication in some mammalian cells. Highly pathogenic H5N1 influenza viruses possessing mammalian-type PB2-627 were detected during the Qinghai Lake outbreak in 2005 and spread to Europe and Africa. Via a database search, we found a high rate of viral isolates from Ratitae, including ostrich, possessing mammalian-type PB2-627 or -701. Here, we report that H5N1 avian influenza viruses possessing mammalian-type amino acids in PB2-627 or -701 are selected during replication in ostrich cells in vitro and in vivo.

Characterisation of a highly pathogenic influenza A virus of subtype H5N2 isolated from ostriches in South Africa in 2004.

OBJECTIVES: The HPAI H5N2 strain that caused an outbreak in ostriches of the Eastern Cape Province, South Africa in 2004 was characterized. DESIGN: Haemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) were performed on sera from ostrich farms in the outbreak region, and intravenous pathogenicity (IVPI) tests, reverse-transcriptase-polymerase-chain reaction (RT-PCR), nucleic acid sequencing and phylogenetic comparisons were performed on the HPAI H5N2 virus isolated during the outbreak. RESULTS: The deduced amino acid sequence at the HA0 cleavage site determined by RT-PCR and nucleotide sequencing was PQREKRRKKRGLF and thus the virus fell within the definition of a highly pathogenic virus, but in an IVPI test in chickens on the virus isolated from the index case and a value of 0.63 was recorded, which is below the criterion for highly pathogenic viruses in this in vivo test. After a further passage in embryonated eggs a second IVPI was carried out and an elevated value of 1.19 was obtained. Cloacal swabs were taken from the initial IVPI birds, inoculated into embryonated chickens eggs and a third IVPI was then performed on the resulting haemagglutinating, infective allantoic fluid. An index of 2.73 was recorded. CONCLUSIONS: HI tests appeared to be the more sensitive test compared to AGID when testing for antibodies to avian influenza in sera. An ostrich-derived virus with a virulent HA0 cleavage site was not initially virulent in chickens but after passage in the latter the virulence increased. Phylogenetic analyses demonstrated the link between AI viruses carried by wild ducks and those infecting ostriches.

Novel reovirus isolation from an Ostrich (Struthio camelus) in Japan.

An orthoreovirus was isolated from an Ostrich (Struthio camelus) and rapidly identified as orthoreovirus by the rapid determination of viral RNA sequences (RDV) system and electron microscopy. Phylogenetic analysis of the sigma A protein indicated that the isolate belonged to avian species and was closely related to chicken orthoreovirus strain 138. The results of the present study indicated that an ostrich orthoreovirus is slight different from other chicken orthoreoviruses and provided evidence of diversity among avian orthoreoviruses. To our knowledge, this is the first genetic report of an orthoreovirus isolated from an ostrich.

Risk factors for seropositivity to H5 avian influenza virus in ostrich farms in the Western Cape Province, South Africa.

In a 2005 serological survey, carried out in response to an outbreak of H5N2 avian influenza (AI) in ostriches in the Eastern Cape Province, 16.3% of ostrich farms in the Western Cape Province of South Africa were found to be seropositive to H5 AI virus. We subsequently carried out a questionnaire-based census survey on all available registered Western Cape ostrich farms that still existed at the end of 2005 (367 farms, of which 82 were seropositive), in order to identify risk factors associated with farm-level seropositivity. A farm was classified as seropositive for H5 AI virus if one or more birds had tested positive (haemagglutination inhibition titre >1:16) in the 2005 survey, which had been designed to detect a minimum within-group seroprevalence of 10%. For each farm, risk factor information was collected using a questionnaire administered during a face-to-face interview with each farm owner or manager. Information was obtained on the ostrich population, movements of birds, environmental factors, management practices, and frequency of contact between ostriches and various wild bird species. Multiple logistic regression models were developed for the whole Western Cape Province and also for the two largest ostrich farming regions, "Klein Karoo" and "Southern Cape". Seroprevalence differed between regions, being highest in the Klein Karoo (31.6%). In all three models, increased risk of farm-level H5 AI virus seropositivity was associated with increasing numbers of ostriches, excluding chicks, present on the farm. Increased risk of seropositivity was associated with reduced frequency of cleaning of feed troughs (<1x/week vs. >1x/week), both overall (odds ratio (OR)=4.5; 95% confidence interval (CI): 1.5, 13.3) and in the Southern Cape (OR=53.6; 95% CI: 3.3, 864), and with failure to clean and disinfect transport vehicles, both overall (OR=2.3; 95% CI: 1.1, 4.8) and in the Klein Karoo (OR=2.6; 95% CI: 1.1, 6.5). Increased risk of seropositivity was also associated with increasing frequency of contact of ostriches with certain wild bird species: overall with white storks (Ciconia ciconia), in the Southern Cape with gulls (Larus spp.), and in the Klein Karoo with Egyptian geese (Alopochen aegyptiaca).

A rapid and sensitive real-time reverse transcription PCR for the pathotyping of South African H5N2 avian influenza viruses.

A Fluorescence resonance energy transfer (FRET) real-time reverse-transcription (rRT-PCR) assay was developed that distinguishes stains of South African and European highly pathogenic (HPAI) from low pathogenicity (LPAI) H5 avian influenza viruses in the absence of virus isolation, irrespective of the length of insertion at the hemagglutinin cleavage site (H0). The assay was used to pathotype H5-type viruses detected by rRT-PCR in ostrich tracheal swabs collected during the 2006 HPAI H5N2 outbreak in the Western Cape Province.

Phylogenetic analysis of low-pathogenicity avian influenza H6N2 viruses from chicken outbreaks (2001-2005) suggest that they are reassortants of historic ostrich low-pathogenicity avian influenza H9N2 and H6N8 viruses.

Low-pathogenicity (LPAI) and high-pathogenicity (HPAI) avian influenza viruses are periodically isolated from South African ostriches, but during 2002 the first recorded outbreak of LPAI (H6N2) in South African chickens occurred on commercial farms in the Camperdown area of KwaZulu/Natal (KZN) Province. Sequence analysis of all eight genes were performed and phylogenetic analysis was done based on the hemagglutinin and neuraminidasc sequences. Results from phylogenetic analyses indicated that the H6N2 chicken viruses most likely arose from a reassortment between two South African LPAI ostrich isolates: an H9N2 virus isolated in 1995 and an H6N8 virus isolated in 1998. Two cocirculating sublineages of H6N2 viruses were detected, both sharing a recent common ancestor. One of these sublineages was restricted to the KZN province. The neuraminidase gene contained a 22-amino acid deletion in the NA-stalk region, which is associated with adaptation to growth in chickens, whereas the other group, although lacking the NA-stalk deletion, spread to commercial farms in other provinces. The persistence of particular H6N2 types in some regions for at least 2 yr supports reports from Asia and southern California suggesting that H6N2 viruses can form stable lineages in chickens. It is probable that the ostrich H6N8 and H9N2 progenitors of the chicken H6N2 viruses were introduced to ostriches by wild birds. Ostriches, in which AI infections are often subclinical, may serve as mixing vessels for LPAI strains that occasionally spill over into other poultry.

First finding of libyostrongylosis in farm-reared ostriches (Struthio camelus) in Croatia: unusual histopathological finding in the brain of two ostriches, naturally infected with Libyostrongylus douglasi.

In the present work, the very first finding of Libyostrongylus douglasi in farm-reared ostriches in Croatia, was described, not only as the main cause of the death, but also as the cause of persistent infection. The results of histopathological examination of almost all parenchymatous organs in two routinely necropsied ostriches were presented, including atypical histopathological finding in the brain and the result of the L. douglasi scanning electron microscopical (SEM) examination. In order to determine the parasite species to which the larval form found in the brain belonged, polymerase chain reaction (PCR) was performed. Total DNA was isolated from fresh L. douglasi, and from archival formalin-fixed and paraffin-embedded brain section. Additionally, the smears of the proventricular mucosal lining were cytologically examined. Virological examination for newcastle disease virus (NDV) was also performed. As there was very limited information concerned ostrich's health status in Croatian farms, a preliminary evaluation of the parasite infestation level in the Croatian ostrich population over the period 2001-2002 was also done, and an attempt at characterising individual parasite species, was made.

Molecular characterization of H5N2 avian influenza viruses isolated from South African ostriches in 2006.

Highly pathogenic avian influenza (HPAI) H5N2 reemerged in ostriches in South Africa during 2006, and a low-pathogenic AI H5N2 virus was also isolated. Molecular and phylogenetic characterization was performed to determine whether the outbreak strains were genetically derived from the supposedly eradicated Eastern Cape ostrich outbreak HPAI H5N2 strain of 2004. It was demonstrated that although the 2004 and 2006 South African H5N2 strains shared a common ancestor, the two outbreaks were not related. Not only were extensive reassortments with wild bird viruses involved in the evolution of the 2006 strains, but the precursor HA molecule HA0 cleavage site sequence of the 2006 HPAI H5N2 virus also contained fewer basic amino-acid insertions. Multiple transmission events occurred from wild birds to ostriches in 2006, and it appears that a reservoir of H5N2 with pathogenic potential for poultry is established in the South African wild duck population.

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.

Serological and virological studies of Newcastle disease and avian influenza in slaughter-age ostriches (Struthio camelus) in Japan.

Serum samples from 191 ostriches (Struthio camelus) in Japan were tested for antibodies to Newcastle disease virus (NDV) and avian influenza virus (AIV). Twenty-two (12%) contained NDV-specific neutralizing antibodies by a virus-neutralization (VN) test without vaccination. Antibodies to AIV were not detected in the any sera by an agar gel precipitation test. Seven serum samples that had vaccinated with live NDV by eye drop were all positive by the VN test at 1 month post vaccination. A haemagglutination inhibition (HI) test for NDV seemed not to be suitable for ostriches because of non-specific agglutination of chicken red blood cells. No haemagglutinating viruses were isolated. This is the first report on detection of antibodies against NDV in ostriches in Japan.