Adriaan Olivier | University of Pretoria (original) (raw)

Papers by Adriaan Olivier

Avian Diseases, 2010

Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ost... more Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ostrich industry. In 2004 and again in 2006, low pathogenicity avian influenza H5N2 strains introduced from a wild bird reservoir mutated in ostriches to high pathogenicity avian influenza (HPAI), with serious economic consequences and export bans imposed by the European Union. Although no outbreaks of notifiable avian influenza have occurred in South Africa since 2006, the H9N2 virus caused a localized outbreak where ostriches displayed symptoms of green urine, depression, and mild morbidity. Most recently, an outbreak of H10N7 in farmed Pekin ducks (Anas platyrhynchos domestica) caused increased mortalities, but this was exacerbated by a secondary Escherichia coli infection, because an intravenous pathogenicity index of 0.00 was recorded. Each of the eight gene segments of the five strains isolated from 2007 to 2009 from farmed ostriches in the Oudtshoorn region (H6N8, H9N2), Pekin ducks (H10N7, Joostenburgvlakte region), and wild Egyptian geese (Alopochen aegypticus; H1N8, Baberspan wetlands; H4N2, Oudtshoorn region) were sequenced, genetically analyzed, and compared to previous South African isolates and viruses in the public data banks. An H5N8 strain was also detected by reverse-transcription PCR in cloacal swabs from swift terns (Sterna bergii) in the Mosselbaai region during 2007, although a virus could not be isolated. Initial phylogenetic results indicate that H6N8 and H9N2 ostrich and H10N7 Pekin duck viruses originated in the wild bird population that is geographically dispersed throughout southern Africa, based on the reassortment of viral genes from birds sampled outside of the ostrich farming areas. No evidence of internal genes associated with Asian HPAI H5N1 strains were detected in the South African isolates.

Avian Diseases Digest, 2010

Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ost... more Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ostrich industry. In 2004 and again in 2006, low pathogenicity avian influenza H5N2 strains introduced from a wild bird reservoir mutated in ostriches to high pathogenicity avian influenza (HPAI), with serious economic consequences and export bans imposed by the European Union. Although no outbreaks of notifiable avian influenza have occurred in South Africa since 2006, the H9N2 virus caused a localized outbreak where ostriches displayed symptoms of green urine, depression, and mild morbidity. Most recently, an outbreak of H10N7 in farmed Pekin ducks (Anas platyrhynchos domestica) caused increased mortalities, but this was exacerbated by a secondary Escherichia coli infection, because an intravenous pathogenicity index of 0.00 was recorded. Each of the eight gene segments of the five strains isolated from 2007 to 2009 from farmed ostriches in the Oudtshoorn region (H6N8, H9N2), Pekin ducks (H10N7, Joostenburgvlakte region), and wild Egyptian geese (Alopochen aegypticus; H1N8, Baberspan wetlands; H4N2, Oudtshoorn region) were sequenced, genetically analyzed, and compared to previous South African isolates and viruses in the public data banks. An H5N8 strain was also detected by reverse-transcription PCR in cloacal swabs from swift terns (Sterna bergii) in the Mosselbaai region during 2007, although a virus could not be isolated. Initial phylogenetic results indicate that H6N8 and H9N2 ostrich and H10N7 Pekin duck viruses originated in the wild bird population that is geographically dispersed throughout southern Africa, based on the reassortment of viral genes from birds sampled outside of the ostrich farming areas. No evidence of internal genes associated with Asian HPAI H5N1 strains were detected in the South African isolates.

Avian Diseases, 2016

The extensive nature of ostrich farming production systems bears the continual risk of point intr... more 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.

Anatomical record (Hoboken, N.J. : 2007), Jan 21, 2016

In ostrich husbandry, economic losses have mainly been attributed to low hatchability of eggs, wh... more In ostrich husbandry, economic losses have mainly been attributed to low hatchability of eggs, which has mostly been attributed to the structure of the eggshell. The main aim of this study was to investigate the morphology and the morphometry of the ostrich eggshell using micro-focus X-ray computer tomography and scanning electron microscopy. The mean weight and volume of the eggs were 1,312±56SE g and 1,333±44SE cm(3) , respectively. The mean thickness and the mean surface area of the eggshell was 1.83±0.10SE mm and 619±15SE cm(2) respectively and the mean total number of pores in the shell was 40,596±1832SE. No significant correlations were found between the thickness of the shell and the weight of the eggs, the volume of the egg and the thickness of the shell, the diameter of the pores and the number of pores, the volume of the pores and the number of pores or the surface area of the pores and the number of pores. The mean diameters of the pores on the blunt (air cell)- (0.02±0.0...

Avian Diseases, Apr 1, 2007

Low-pathogenicity (LPAI) and high-pathogenicity (HPAI) avian influenza viruses are periodically i... more 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.

Avian Diseases Digest, 2007

Low-pathogenicity (LPAI) and high-pathogenicity (HPAI) avian influenza viruses are periodically i... more 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.

Developments in biologicals, 2006

Wild waterfowl and shorebirds are known to be the natural reservoir for influenza A viruses. Surv... more Wild waterfowl and shorebirds are known to be the natural reservoir for influenza A viruses. Surveillance studies in waterfowl and shorebirds in North America show that influenza A viruses are repeatedly recovered from these birds. However, the virus recovery is influenced by geography, season, age and species of birds. In addition to the natural reservoir, the live-bird marketing system (LBMS) in certain regions of the United States has been recognized as a man-made reservoir of influenza viruses and has been linked to several outbreaks of low pathogenicity avian influenza (LPAI) in poultry. Outbreaks of LPAI in commercial poultry is attributed to movement of infected birds, dirty or improperly cleaned crates, and contaminated vehicles from the LBMS to poultry farms. However, in the majority of outbreaks in poultry, the source of infection is suspected to be wild aquatic birds or the source is unknown. Since 2002, three outbreaks of highly pathogenic avian influenza (HPAI) have occ...

Virus Genes, 2007

The first recorded outbreak of avian influenza (AI) in South African chickens (low pathogenicity ... more 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) /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 Nglycosylation, 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.

Avian Pathology, 2010

In the present study we collected 177 serum samples from ostriches (Struthio camelus) infected ex... more 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.

Avian Pathology, 2013

An ostrich farm of 929 birds that tested polymerase chain reaction-positive for highly pathogenic... more An ostrich farm of 929 birds that tested polymerase chain reaction-positive for highly pathogenic avian influenza H5N2 in a single sample was designated for culling, despite no evidence of sero-conversion as assessed by haemagglutination inhibition (HI) tests. A month later and immediately prior to culling, all birds were bled and tested with an IDEXX avian influenza virus (AIV) nucleoprotein (NP)-specific enzyme-linked immunosorbent assay (ELISA) and a high sero-prevalence was detected. To address the question of whether the NP-specific antibodies detected indicated exposure to H5 or non-H5 subtypes (H6N2 and H1N2 strains were also circulating regionally at the time), we developed two H5-specific ELISAs, both based on a recombinant H5 HA1 antigen. The H5 indirect ELISA used a horseradish peroxidase ostrich IgY conjugate that we produced in chicken eggs. The single-chain variable fragment (scFv) competitive ELISA (H5 scFv cELISA) used a scFv derived from an H5-immune chicken scFv library. By comparing IDEXX AIV ELISA results with those of the two H5-specific ELISAs and HI tests, we determined that up to 89% of the flock had been exposed to H5N2 AIV. We also detected evidence of suspected vaccination, since 17% of sera contained antibodies against the H5 glycoprotein but not the NP protein. Comparative analytical sensitivity indicated that HI tests are likely to miss up to 35% of H5-positive samples, and thus we consider that H5/H7-specific ELISAs should replace HI tests for ostrich testing in future.

Avian Diseases, 1999

The presence of virulent Newcastle disease virus (NDV) since the 1993-94 epidemic in southern Afr... more The presence of virulent Newcastle disease virus (NDV) since the 1993-94 epidemic in southern Africa holds major implications for the export of ostrich products from this region. A challenge experiment with this field strain was conducted in open-air feedlot facilities under strict biosecurity measures. The experiment was designed to follow vaccination and preslaughter quarantine regulations currently enforced in South African export ostrich facilities in order to determine the viremia period and immune response under these specific circumstances. One hundred forty-three slaughter ostriches were allocated into three test groups, according to the time period between pretrial vaccination and challenge (1-2 mo, 2-4 mo, 4-6 mo), and an unchallenged control group. All birds in the test groups were challenged by oral, tracheal, and ocular routes with a field isolate of NDV. They were slaughtered over the next 4 wk on nine separate occasions and bled on 12 occasions. Virus isolation was attempted from seven sets of pooled samples from each bird to determine the viremia period and the serum antibody concentrations were measured by hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) methods to establish an immune response curve. NDV could be back-isolated only up to day 9 postinfection and from only six ostriches with poor immune response titers and corresponding to a rise in antibody levels above an indirect ELISA optical density reading of 0.33. Virus could be recovered only from brain and respiratory tract tissue. The HI test was less sensitive than the ELISA. Immune response curves did not differ significantly between the groups and peaked on day 14 post-infection. From these data, ELISA titers would appear to be a good indicator of the probability that an ostrich will be clinically infected after velogenic NDV challenge. These results also suggest that the current vaccination schedule enforced by the South African Veterinary Authorities results in protective immunity in up to 95% of slaughter ostriches from export approved facilities. The standard 30-day preslaughter quarantine period introduced as part of Crimean-Congo hemorrhagic fever virus control measures also appears sufficient to encompass the determined NDV viremia period of 9-11 days in slaughter ostriches.

Avian Diseases, 2007

Low-pathogenicity (LPAI) and high-pathogenicity (HPAI) avian influenza viruses are periodically i... more 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.

Avian Diseases, 2010

Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ost... more Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ostrich industry. In 2004 and again in 2006, low pathogenicity avian influenza H5N2 strains introduced from a wild bird reservoir mutated in ostriches to high pathogenicity avian influenza (HPAI), with serious economic consequences and export bans imposed by the European Union. Although no outbreaks of notifiable avian influenza have occurred in South Africa since 2006, the H9N2 virus caused a localized outbreak where ostriches displayed symptoms of green urine, depression, and mild morbidity. Most recently, an outbreak of H10N7 in farmed Pekin ducks (Anas platyrhynchos domestica) caused increased mortalities, but this was exacerbated by a secondary Escherichia coli infection, because an intravenous pathogenicity index of 0.00 was recorded. Each of the eight gene segments of the five strains isolated from 2007 to 2009 from farmed ostriches in the Oudtshoorn region (H6N8, H9N2), Pekin ducks (H10N7, Joostenburgvlakte region), and wild Egyptian geese (Alopochen aegypticus; H1N8, Baberspan wetlands; H4N2, Oudtshoorn region) were sequenced, genetically analyzed, and compared to previous South African isolates and viruses in the public data banks. An H5N8 strain was also detected by reverse-transcription PCR in cloacal swabs from swift terns (Sterna bergii) in the Mosselbaai region during 2007, although a virus could not be isolated. Initial phylogenetic results indicate that H6N8 and H9N2 ostrich and H10N7 Pekin duck viruses originated in the wild bird population that is geographically dispersed throughout southern Africa, based on the reassortment of viral genes from birds sampled outside of the ostrich farming areas. No evidence of internal genes associated with Asian HPAI H5N1 strains were detected in the South African isolates.

Avian Diseases, 2012

The third outbreak of highly pathogenic avian influenza (HPAI) H5N2 in less than seven years affe... more The third outbreak of highly pathogenic avian influenza (HPAI) H5N2 in less than seven years affected ostriches of South Africa's Western Cape during 2011. Twenty farms tested PCR positive for the presence of HPAI H5N2 between March and November 2011. Three HPAI H5N2 (AI2114, AI2214, AI2512) and 1 H1N2 (AI2887) viruses were isolated during this period, but H6N2 and H1N2 infections of ostriches were also confirmed by PCR. HPAI H5N2 isolate AI2114 produced an intravenous pathogenicity index (IVPI) score of 1.37 in chickens whereas isolate AI2214 produced an IVPI score of 0.8. The former virus had an additional, predicted N-linked glycosylation site at position 88 of the hemagglutinin protein as well as an E627K mutation in the PB2 protein that was lacking from AI2214. Four variations at HA0 were detected in the PCR-positive cases. Phylogenetically, the branching order of outbreak strains indicated a lack of reassortment between outbreak strains that implied a single outbreak source and a wild duck origin for the progenitor outbreak strain. The 2011 outbreak strains had no genetic relationships to the previous 2004 and 2006 HPAI H5N2 outbreak viruses. Molecular clock analysis based on the N2 neuraminidase genes estimated a recent common ancestor for the outbreak tentatively dated at September 2010. Deep sequencing results of 16 clinical PCR-positive samples yielded data in the range of 573 to 12,590 base pairs (bp), with an average of 4468 bp of total genomic sequence recovered per sample. This data was used to confirm the lack ofreassortment and to assign samples into one of two epidemiologic groups to support epidemiologic tracing of the spread of the outbreak. One farm (no. 142), thought to have played a major epidemiologic role in the outbreak, was confirmed by deep sequencing to contain a mix of both epidemiologic virus groups.

Avian Diseases, 2010

Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ost... more Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ostrich industry. In 2004 and again in 2006, low pathogenicity avian influenza H5N2 strains introduced from a wild bird reservoir mutated in ostriches to high pathogenicity avian influenza (HPAI), with serious economic consequences and export bans imposed by the European Union. Although no outbreaks of notifiable avian influenza have occurred in South Africa since 2006, the H9N2 virus caused a localized outbreak where ostriches displayed symptoms of green urine, depression, and mild morbidity. Most recently, an outbreak of H10N7 in farmed Pekin ducks (Anas platyrhynchos domestica) caused increased mortalities, but this was exacerbated by a secondary Escherichia coli infection, because an intravenous pathogenicity index of 0.00 was recorded. Each of the eight gene segments of the five strains isolated from 2007 to 2009 from farmed ostriches in the Oudtshoorn region (H6N8, H9N2), Pekin ducks (H10N7, Joostenburgvlakte region), and wild Egyptian geese (Alopochen aegypticus; H1N8, Baberspan wetlands; H4N2, Oudtshoorn region) were sequenced, genetically analyzed, and compared to previous South African isolates and viruses in the public data banks. An H5N8 strain was also detected by reverse-transcription PCR in cloacal swabs from swift terns (Sterna bergii) in the Mosselbaai region during 2007, although a virus could not be isolated. Initial phylogenetic results indicate that H6N8 and H9N2 ostrich and H10N7 Pekin duck viruses originated in the wild bird population that is geographically dispersed throughout southern Africa, based on the reassortment of viral genes from birds sampled outside of the ostrich farming areas. No evidence of internal genes associated with Asian HPAI H5N1 strains were detected in the South African isolates.

Avian Diseases Digest, 2010

Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ost... more Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ostrich industry. In 2004 and again in 2006, low pathogenicity avian influenza H5N2 strains introduced from a wild bird reservoir mutated in ostriches to high pathogenicity avian influenza (HPAI), with serious economic consequences and export bans imposed by the European Union. Although no outbreaks of notifiable avian influenza have occurred in South Africa since 2006, the H9N2 virus caused a localized outbreak where ostriches displayed symptoms of green urine, depression, and mild morbidity. Most recently, an outbreak of H10N7 in farmed Pekin ducks (Anas platyrhynchos domestica) caused increased mortalities, but this was exacerbated by a secondary Escherichia coli infection, because an intravenous pathogenicity index of 0.00 was recorded. Each of the eight gene segments of the five strains isolated from 2007 to 2009 from farmed ostriches in the Oudtshoorn region (H6N8, H9N2), Pekin ducks (H10N7, Joostenburgvlakte region), and wild Egyptian geese (Alopochen aegypticus; H1N8, Baberspan wetlands; H4N2, Oudtshoorn region) were sequenced, genetically analyzed, and compared to previous South African isolates and viruses in the public data banks. An H5N8 strain was also detected by reverse-transcription PCR in cloacal swabs from swift terns (Sterna bergii) in the Mosselbaai region during 2007, although a virus could not be isolated. Initial phylogenetic results indicate that H6N8 and H9N2 ostrich and H10N7 Pekin duck viruses originated in the wild bird population that is geographically dispersed throughout southern Africa, based on the reassortment of viral genes from birds sampled outside of the ostrich farming areas. No evidence of internal genes associated with Asian HPAI H5N1 strains were detected in the South African isolates.

Avian Diseases, 2016

The extensive nature of ostrich farming production systems bears the continual risk of point intr... more 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.

Anatomical record (Hoboken, N.J. : 2007), Jan 21, 2016

In ostrich husbandry, economic losses have mainly been attributed to low hatchability of eggs, wh... more In ostrich husbandry, economic losses have mainly been attributed to low hatchability of eggs, which has mostly been attributed to the structure of the eggshell. The main aim of this study was to investigate the morphology and the morphometry of the ostrich eggshell using micro-focus X-ray computer tomography and scanning electron microscopy. The mean weight and volume of the eggs were 1,312±56SE g and 1,333±44SE cm(3) , respectively. The mean thickness and the mean surface area of the eggshell was 1.83±0.10SE mm and 619±15SE cm(2) respectively and the mean total number of pores in the shell was 40,596±1832SE. No significant correlations were found between the thickness of the shell and the weight of the eggs, the volume of the egg and the thickness of the shell, the diameter of the pores and the number of pores, the volume of the pores and the number of pores or the surface area of the pores and the number of pores. The mean diameters of the pores on the blunt (air cell)- (0.02±0.0...

Avian Diseases, Apr 1, 2007

Low-pathogenicity (LPAI) and high-pathogenicity (HPAI) avian influenza viruses are periodically i... more 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.

Avian Diseases Digest, 2007

Low-pathogenicity (LPAI) and high-pathogenicity (HPAI) avian influenza viruses are periodically i... more 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.

Developments in biologicals, 2006

Wild waterfowl and shorebirds are known to be the natural reservoir for influenza A viruses. Surv... more Wild waterfowl and shorebirds are known to be the natural reservoir for influenza A viruses. Surveillance studies in waterfowl and shorebirds in North America show that influenza A viruses are repeatedly recovered from these birds. However, the virus recovery is influenced by geography, season, age and species of birds. In addition to the natural reservoir, the live-bird marketing system (LBMS) in certain regions of the United States has been recognized as a man-made reservoir of influenza viruses and has been linked to several outbreaks of low pathogenicity avian influenza (LPAI) in poultry. Outbreaks of LPAI in commercial poultry is attributed to movement of infected birds, dirty or improperly cleaned crates, and contaminated vehicles from the LBMS to poultry farms. However, in the majority of outbreaks in poultry, the source of infection is suspected to be wild aquatic birds or the source is unknown. Since 2002, three outbreaks of highly pathogenic avian influenza (HPAI) have occ...

Virus Genes, 2007

The first recorded outbreak of avian influenza (AI) in South African chickens (low pathogenicity ... more 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) /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 Nglycosylation, 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.

Avian Pathology, 2010

In the present study we collected 177 serum samples from ostriches (Struthio camelus) infected ex... more 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.

Avian Pathology, 2013

An ostrich farm of 929 birds that tested polymerase chain reaction-positive for highly pathogenic... more An ostrich farm of 929 birds that tested polymerase chain reaction-positive for highly pathogenic avian influenza H5N2 in a single sample was designated for culling, despite no evidence of sero-conversion as assessed by haemagglutination inhibition (HI) tests. A month later and immediately prior to culling, all birds were bled and tested with an IDEXX avian influenza virus (AIV) nucleoprotein (NP)-specific enzyme-linked immunosorbent assay (ELISA) and a high sero-prevalence was detected. To address the question of whether the NP-specific antibodies detected indicated exposure to H5 or non-H5 subtypes (H6N2 and H1N2 strains were also circulating regionally at the time), we developed two H5-specific ELISAs, both based on a recombinant H5 HA1 antigen. The H5 indirect ELISA used a horseradish peroxidase ostrich IgY conjugate that we produced in chicken eggs. The single-chain variable fragment (scFv) competitive ELISA (H5 scFv cELISA) used a scFv derived from an H5-immune chicken scFv library. By comparing IDEXX AIV ELISA results with those of the two H5-specific ELISAs and HI tests, we determined that up to 89% of the flock had been exposed to H5N2 AIV. We also detected evidence of suspected vaccination, since 17% of sera contained antibodies against the H5 glycoprotein but not the NP protein. Comparative analytical sensitivity indicated that HI tests are likely to miss up to 35% of H5-positive samples, and thus we consider that H5/H7-specific ELISAs should replace HI tests for ostrich testing in future.

Avian Diseases, 1999

The presence of virulent Newcastle disease virus (NDV) since the 1993-94 epidemic in southern Afr... more The presence of virulent Newcastle disease virus (NDV) since the 1993-94 epidemic in southern Africa holds major implications for the export of ostrich products from this region. A challenge experiment with this field strain was conducted in open-air feedlot facilities under strict biosecurity measures. The experiment was designed to follow vaccination and preslaughter quarantine regulations currently enforced in South African export ostrich facilities in order to determine the viremia period and immune response under these specific circumstances. One hundred forty-three slaughter ostriches were allocated into three test groups, according to the time period between pretrial vaccination and challenge (1-2 mo, 2-4 mo, 4-6 mo), and an unchallenged control group. All birds in the test groups were challenged by oral, tracheal, and ocular routes with a field isolate of NDV. They were slaughtered over the next 4 wk on nine separate occasions and bled on 12 occasions. Virus isolation was attempted from seven sets of pooled samples from each bird to determine the viremia period and the serum antibody concentrations were measured by hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) methods to establish an immune response curve. NDV could be back-isolated only up to day 9 postinfection and from only six ostriches with poor immune response titers and corresponding to a rise in antibody levels above an indirect ELISA optical density reading of 0.33. Virus could be recovered only from brain and respiratory tract tissue. The HI test was less sensitive than the ELISA. Immune response curves did not differ significantly between the groups and peaked on day 14 post-infection. From these data, ELISA titers would appear to be a good indicator of the probability that an ostrich will be clinically infected after velogenic NDV challenge. These results also suggest that the current vaccination schedule enforced by the South African Veterinary Authorities results in protective immunity in up to 95% of slaughter ostriches from export approved facilities. The standard 30-day preslaughter quarantine period introduced as part of Crimean-Congo hemorrhagic fever virus control measures also appears sufficient to encompass the determined NDV viremia period of 9-11 days in slaughter ostriches.

Avian Diseases, 2007

Low-pathogenicity (LPAI) and high-pathogenicity (HPAI) avian influenza viruses are periodically i... more 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.

Avian Diseases, 2010

Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ost... more Influenza A strains emerging from wild birds are a constant threat to South Africa's valuable ostrich industry. In 2004 and again in 2006, low pathogenicity avian influenza H5N2 strains introduced from a wild bird reservoir mutated in ostriches to high pathogenicity avian influenza (HPAI), with serious economic consequences and export bans imposed by the European Union. Although no outbreaks of notifiable avian influenza have occurred in South Africa since 2006, the H9N2 virus caused a localized outbreak where ostriches displayed symptoms of green urine, depression, and mild morbidity. Most recently, an outbreak of H10N7 in farmed Pekin ducks (Anas platyrhynchos domestica) caused increased mortalities, but this was exacerbated by a secondary Escherichia coli infection, because an intravenous pathogenicity index of 0.00 was recorded. Each of the eight gene segments of the five strains isolated from 2007 to 2009 from farmed ostriches in the Oudtshoorn region (H6N8, H9N2), Pekin ducks (H10N7, Joostenburgvlakte region), and wild Egyptian geese (Alopochen aegypticus; H1N8, Baberspan wetlands; H4N2, Oudtshoorn region) were sequenced, genetically analyzed, and compared to previous South African isolates and viruses in the public data banks. An H5N8 strain was also detected by reverse-transcription PCR in cloacal swabs from swift terns (Sterna bergii) in the Mosselbaai region during 2007, although a virus could not be isolated. Initial phylogenetic results indicate that H6N8 and H9N2 ostrich and H10N7 Pekin duck viruses originated in the wild bird population that is geographically dispersed throughout southern Africa, based on the reassortment of viral genes from birds sampled outside of the ostrich farming areas. No evidence of internal genes associated with Asian HPAI H5N1 strains were detected in the South African isolates.

Avian Diseases, 2012

The third outbreak of highly pathogenic avian influenza (HPAI) H5N2 in less than seven years affe... more The third outbreak of highly pathogenic avian influenza (HPAI) H5N2 in less than seven years affected ostriches of South Africa's Western Cape during 2011. Twenty farms tested PCR positive for the presence of HPAI H5N2 between March and November 2011. Three HPAI H5N2 (AI2114, AI2214, AI2512) and 1 H1N2 (AI2887) viruses were isolated during this period, but H6N2 and H1N2 infections of ostriches were also confirmed by PCR. HPAI H5N2 isolate AI2114 produced an intravenous pathogenicity index (IVPI) score of 1.37 in chickens whereas isolate AI2214 produced an IVPI score of 0.8. The former virus had an additional, predicted N-linked glycosylation site at position 88 of the hemagglutinin protein as well as an E627K mutation in the PB2 protein that was lacking from AI2214. Four variations at HA0 were detected in the PCR-positive cases. Phylogenetically, the branching order of outbreak strains indicated a lack of reassortment between outbreak strains that implied a single outbreak source and a wild duck origin for the progenitor outbreak strain. The 2011 outbreak strains had no genetic relationships to the previous 2004 and 2006 HPAI H5N2 outbreak viruses. Molecular clock analysis based on the N2 neuraminidase genes estimated a recent common ancestor for the outbreak tentatively dated at September 2010. Deep sequencing results of 16 clinical PCR-positive samples yielded data in the range of 573 to 12,590 base pairs (bp), with an average of 4468 bp of total genomic sequence recovered per sample. This data was used to confirm the lack ofreassortment and to assign samples into one of two epidemiologic groups to support epidemiologic tracing of the spread of the outbreak. One farm (no. 142), thought to have played a major epidemiologic role in the outbreak, was confirmed by deep sequencing to contain a mix of both epidemiologic virus groups.