Newcastle disease and avian influenza A virus in wild waterfowl in South Africa.

In an intensive ostrich farming area in South Africa with a history of ostrich influenza outbreaks, we conducted a survey of avian influenza virus (AIV) and Newcastle disease virus (NDV) in wild aquatic birds. During late autumn and winter 1998, the time of year when outbreaks in ostriches typically start to occur, 262 aquatic birds comprising 14 species were sampled and tested for both virus infections. From eight samples, AIV, serotype H10N9, could be isolated. All isolates were apathogenic as determined by the intravenous pathogenicity index (0.00). Conversely, none of 33 sera of these wild birds showed antibodies against H10. However, one bird was found serologically positive for H6 AIV. This AIV serotype was later isolated from ostriches during an avian influenza outbreak in this area. No NDV was isolated although 34 of 46 serum samples contained NDV-specific antibodies. This is the first H10N9 isolate to be reported from Africa. In addition, our data support the notion that wild aquatic birds may function as a reservoir for AIV and NDV in South Africa.

Ostrich diseases.

Scientific knowledge of ostrich diseases is incomplete and very fragmented, with specific details on technical aspects of diagnostic and/or screening tests completely absent in most cases. Salmonella Typhimurium is common in multispecies collections and causes mortality in chicks younger than three months on commercial farms, but is rarely found in chicks older than six months, or slaughter birds of twelve to fourteen months in southern Africa. Campylobacter jejuni and Chlamydia psittaci are occasionally reported, mainly in young ostriches, but both remain a diagnostic challenge. Crimean-Congo haemorrhagic fever is transmitted to domestic animals including ostriches, principally by ticks of the genus Hyalomma. In the ostrich, the disease causes no clinical symptoms during a viraemia of approximately four days. Spongiform encephalopathy has not been reliably reported in ostriches, while anthrax has occurred rarely in modern times but was reportedly an important cause of death approximately 100 years ago in South Africa. Salmonella Gallinarum and S. Pullorum are unknown in ostriches. Pasteurella multocida occurs but is easily contained with antibiotics. Mycoplasma spp. are regularly found in an upper respiratory disease syndrome complicated by opportunistic bacterial pathogens. Ostriches of all ages are susceptible to challenge by velogenic Newcastle disease virus (NDV), but standard inactivated La Sota poultry vaccines can stimulate protective immunity lasting over six months. The viraemic period in vaccinated slaughter ostriches is between nine and eleven days and there are no indications of a carrier state or presence of the virus in the meat or any other tissues after this period, with peak immunoglobulin G response reached on day fourteen post infection. Haemagglutination inhibition tests are significantly less sensitive and less specific than enzyme-linked immunosorbent assays. Cloacal and choanal swabs used for direct virological screening in clinically affected cases (field and experimental) could not detect NDV. All avian influenza isolates reported from ostriches have been non-pathogenic to poultry, even the H5 and H7 subtypes. Some of the latter have been associated with mortality of ostrich chicks in localised outbreaks during periods of inclement weather and with significant wild bird (waterfowl) contact. Borna disease causes a nervous syndrome in ostrich chicks, but to date, has only been reported in Israel. Eastern and Western equine encephalomyelitides cause fatal disease in ostriches and other ratites, with mortality ranging from less than 20% to over 80% in affected flocks. These diseases are present in North, Central and South America where the associated ornithophilic mosquito vectors occur. Equine and human vaccines are apparently safe and efficacious in ratites. Wesselsbron disease, infectious bursal disease (type 2), adenovirus and coronavirus infections have been reported from ostriches but the significance of these diseases is unclear. Due to the paucity of data regarding ostrich diseases and the unvalidated state of most poultry tests in this unique group of birds, strict observation of a pre-slaughter quarantine of thirty days is strongly advised, whilst live exports and fertile eggs should be screened through the additional use of sentinel chickens and/or young ostriches.

Enterocin 012, a bacteriocin produced by Enterococcus gallinarum isolated from the intestinal tract of ostrich.

Enterococcus gallinarum strain 012, isolated from the duodenum of ostrich, produced enterocin 012 which is active against Ent. faecalis, Lactobacillus acidophilus, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp., Clostridium perfringens, Pseudomonas aeruginosa and Salmonella typhimurium. One of the four pathogenic strains of Escherichia coli isolated from the intestinal tract of ostrich was inhibited by enterocin 012. No antimicrobial activity was recorded against Bacillus cereus, Cl. sporogenes, Cl. tyrobutyricum, Leuconostoc cremoris, Pediococcus pentosaceus, Staphylococcus carnosus and Streptococcus thermophilus. Enterocin 012 was resistant to treatment with lysozyme, catalase, lipase and papain, but sensitive to Proteinase K, alpha-chymotrypsin, trypsin and pepsin. Treatment of enterocin 012 with gastric juice from the duodenum resulted in a 50% loss of antibacterial activity. Half of the activity was lost when incubated at 80 degrees C for 30 min, or when kept overnight at a pH of 1.0-5.0 and pH 11.0 and 12.0, respectively. Enterocin 012 production started in mid-logarithmic growth and reached a maximum of 800 AU ml-1, but increased further to 1600 AU ml-1 in the stationary growth phase. The peptide is approximately 3.4 kDa in size, as determined after partial purification with Amberlite XAD-1180 and ammonium sulphate precipitation, followed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mechanism of antimicrobial activity against Lact. sake LMG 13558 is bactericidal and caused cell lysis of active growing cells.

Experimental infection of vaccinated slaughter ostriches in a natural, open-air feedlot facility with virulent Newcastle disease virus.

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.

Experimental infection of ostriches with Crimean-Congo haemorrhagic fever virus.

Following the occurrence of an outbreak of Crimean-Congo haemorrhagic fever (CCHF) among workers at an ostrich abattoir in South Africa in 1996, 9 susceptible young ostriches were infected subcutaneously with the virus in order to study the nature of the infection which they undergo. The ostriches developed viraemia which was demonstrable on days 1-4 following infection, with a maximum intensity of 4.0 log10 mouse intracerebral LD50/ml being recorded on day 2 in 1 of the birds. Virus was detectable in visceral organs such as spleen, liver and kidney up to day 5 post-inoculation, 1 day after it could no longer be found in blood. No infective virus was detected in samples of muscle, but viral nucleic acid was detected by reverse transcription-polymerase chain reaction in muscle from a bird sacrificed on day 3 following infection. It was concluded that the occurrence of infection in ostriches at abattoirs could be prevented by keeping the birds free of ticks for 14 days before slaughter.

Pancreatic cryptosporidiosis in ostriches.

Four 4-month-old ostriches exhibiting poor growth were submitted for necropsy. Gross findings included marked emaciation and pancreatic atrophy. Histological examination revealed pancreatic cryptosporidiosis with large numbers of Crypto-sporidium sp. present in the ductal epithelium causing necrosis and lymphoplasma-cytic inflammation. The gross and histological findings in these birds and the transmission electron microscopic features of the parasites are described.

Experimental infection of vaccinated slaughter ostriches with virulent Newcastle disease virus.

A virulent Newcastle disease virus (NDV) isolate from an outbreak in commercial poultry, with virulence indices of MDT = 47-48 h; IVPI = 2,17 and ICPI = 1,8; was used to inoculate 10x vaccinated (standard poultry vaccines) as well as 10x unvaccinated slaughter ostriches via intratracheal, ocular and nasal routes, in a controlled environment. All unvaccinated ostriches developed clinical signs (mainly respiratory); two of them died while the other eight recovered. No vaccinated ostriches developed any clinical signs. All remaining (18) ostriches were slaughtered 14 d after the last mortality. Virulent NDV could be re-isolated from the dead birds, but not from organs, muscle (fresh), muscle (24 h chilled), gastro-intestinal tract, bone-marrow or respiratory system taken from the slaughtered ostriches. It is suggested that it would be extremely unlikely that the international trade in ostrich meat could act as a mechanism for spreading virulent NDV from endemic to non-endemic parts of the world.

Thermostability analysis of major histocompatibility complex class I molecules by temperature gradient gel electrophoresis.

Empty major histocompatibility complex (MHC) class I molecules present on the surface of RMA-S (26 degrees C) cells were loaded with the iodinated peptides APGNYPAL, FAPGNYPAL (SEV-9) and RGYVYQGL (VSV-8), respectively. The thermostability of these peptide-loaded MHC class I molecules was assessed using temperature gradient native polyacrylamide gel electrophoresis. A linear temperature gradient perpendicular to the direction of electrophoresis yielded a graphical representation of the melting of MHC class I molecules. The class I signal disappeared when the peptide melted out of the groove, and gave rise to a second signal due to released peptide. APGNYPAL-loaded class I molecules melted at 11 degrees C with considerable release even at 0 degrees C. VSV-8-loaded class I molecules melted first at 36 degrees C, whereas SEV-9-loaded molecules melted at about 22 degrees C. A discrimination between the binding of SEV-9 to Kb and Db molecules was seen in the melting patterns. Results are discussed in correlation with known crystallographic structures of class I molecules containing peptides in the binding groove.