Identification and partial sequencing of a crocodile poxvirus associated with deeply penetrating skin lesions in farmed Nile crocodiles, Crocodylus niloticus.

When large numbers of crocodile skins were downgraded because of the presence of small pin prick-like holes, collapsed epidermal cysts were found deep in the dermis of juvenile crocodiles while forming cysts were observed in hatchlings. Histopathology of these forming cysts showed the presence of intracytoplasmic inclusions in proliferating and ballooning epidermal cells. Pox virions were seen in electron microscope preparations made from the scabs of such early lesions. The partial sequencing of virus material from scrapings of these lesions and comparison of it with the published sequence of crocodile poxvirus showed the virus associated with the deep lesions to be closely related, but different. To differentiate between the two forms of crocodile pox infection it is suggested that the previously known form should be called "classical crocodile pox" and the newly discovered form "atypical crocodile pox". The application of strict hygiene measures brought about a decline in the percentage of downgraded skins.

Characterization of pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa from 2001 to 2006.

Pigeon paramyxovirus type 1 (PPMV-1), a variant of Newcastle disease virus that primarily affects doves and pigeons has been isolated in South Africa since the mid-1980s. Phylogenetic evidence indicates that pigeon paramyxovirus type 1 viruses were introduced into South Africa on multiple occasions, based on the presence of two separate lineages, 4bi and 4bii, that have been circulating in Europe and the Far East since the early 1990s. During 2006, a PPMV-1 virus was isolated from an African ground hornbill (Bucorvus leadbeateri) which became acutely infected with PPMV-1 and died, probably after scavenging off infected dove carcasses in the region, since a closely-related PPMV-1 strain was also isolated from doves collected nearby. The hornbill isolate had ICPI and MDT values characteristic of PPMV-1 strains. The threat of PPMV-1 to poultry production and biodiversity in southern Africa highlights the importance of monitoring the spread of this strain.

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.

Rift Valley fever.

Rift Valley fever (RVF) is an arthropod-borne viral disease of ruminants, camels and humans. It is also a significant zoonosis which may be encountered as an uncomplicated influenza-like illness, but may also present as a haemorrhagic disease with liver involvement; there may also be ocular or neurological lesions. In animals, RVF may be inapparent in non-pregnant adults, but outbreaks are characterised by the onset of abortions and high neonatal mortality. Jaundice hepatitis and death are seen in older animals. Outbreaks of RVF are associated with persistent heavy rainfall with sustained flooding and the appearance of large numbers of mosquitoes, the main vector. Localised heavy rainfall is seldom sufficient to create conditions for an outbreak; the simultaneous emergence of large numbers of first generation transovarially infected mosquitoes is also required. After virus amplification in vertebrates, mosquitoes act as secondary vectors to sustain the epidemic.

A South African overview of the virus, vectors, surveillance and unique features of bluetongue.

The origin of bluetongue (BT) is probably African and the disease was first recognised in South Africa in Merino sheep in the late 18th Century. Diagnostic and research findings for a number of years have been summarised to obtain data relevant to the distribution of BT and its serotypes in the country. The role of ruminant game and cattle as maintenance hosts for BT virus (BTV) is mentioned although cattle appear to have largely replaced antelope in this role. Only about 30% of over 1,000 game animals tested for export were found to be BT-antibody positive. An outbreak of a bluetongue-like disease in cattle is mentioned as are the BT and epizootic haemorrhagic disease of deer (EHD) isolates in the outbreak. A summary by serotype and province of sheep isolates is given and it is pointed out that the sheep population in a province does not reflect the number of isolates made and the province with the largest sheep population has almost the smallest number of BTV isolates and vice-versa. South Africa currently has 21 of the 24 BTV serotypes with 17, 20 and 21 being exotic to the country. The recent retrospective typing of serotype 17 in South Africa is being investigated, as type 17 crosses strongly with type 20, which is absent and also with type 4 which is present. 1, 3, 4 and 2 were the most common serotypes while 18, 19, 22 and 23 were not found among the isolates. Mention is made of BTV isolates made from Culicoides bolitinos catches during two devastating outbreaks of African horse sickness in an unvaccinated population. A six-year Culicoides monitoring project is mentioned and the many BTV isolates made of a variety of serotypes. BTV is endemic in Africa and in South Africa unvaccinated indigenous breeds appear to have achieved a balance with the virus. Indeed, it is possible to find virus, antibody and lesions in asymptomatic animals in different situations. Bluetongue creates a significant trade barrier but the virus remains interesting among a number of other uniquely African viruses.

Transmission potential of South African Culicoides species for live-attenuated bluetongue virus.

Field-collected Culicoides were fed on sheep blood-virus mixtures, each containing one of four live-attenuated vaccine strains of bluetongue virus (BTV), namely: BTV-1, BTV-4, BTV-9, and BTV-16. A South African field isolate of BTV-1 was used as the non-attenuated control virus. Titres of vaccine strains in blood meals ranged from 5.1 to 6.1 log(10)TCID(50)/ml; the titre of the field isolate of BTV-1 was 7.1 log(10)TCID(50)/ml. Recovery rates of vaccine viruses from Culicoides assayed immediately after feeding varied from 0% to 10.6%. This indicates that virus concentrations in blood meals were too low to ensure that all individuals ingested detectable amounts of virus. Thus, the oral susceptibility of Culicoides to infection with BTV vaccine strains determined in this study might be an underestimation. Of a total of 6 540 Culicoides that survived a 10-day extrinsic incubation period at 23.5 degrees C, 124 tested positive for BTV; 65 individuals yielded vaccine strains, and the remaining 59, the field isolate of BTV-1. Infection prevalences with the vaccine viruses ranged from 11.0% in C. bolitinos fed on blood containing 6.1 log(10)TCID/ml of BTV-1 down to 0.3% in C. imicola fed on a blood containing 5.3 log(10)TCID/ml of BTV-4. The infection rate for C. imicola and C. bolitinos fed on the field isolate of BTV-1 was 9.5% and 36.0%, respectively. In most infected midges the replication levels of vaccine strains were below the postulated threshold for a systemic infection with an orbivirus as previously calculated in the larger American vector, C. sonorensis (>2.5 log(10)TCID(50)/midge) but some individuals replicated BTV vaccine strains to high titres. This carries an implication that if ruminants become viraemic after vaccination with live-attenuated BTV vaccines, they might act as a source for the infection of Culicoides vectors.

Indirect enzyme-linked immunosorbent assay for the detection of antibody against Rift Valley fever virus in domestic and wild ruminant sera.

An indirect enzyme-linked immunosorbent assay (I-ELISA) for the detection of specific IgG immunoglobulins against Rift Valley fever virus (RVFV) was validated in-house. A total of 3055 sera from sheep (n = 1159), goats (n = 636), cattle (n = 203), African buffalo (n = 928), and other wild ruminants (n = 129), including eland, kudu, and black wildebeest, was used. Sera from domestic ruminants were collected in West (n = 10), South (n = 1654) and East Africa (n = 334), and sera from wild ruminants (n = 1064) were collected in South Africa. In addition, 136 sera from eight experimentally RVFV-infected sheep, taken during a period of 28 days post infection (dpi), were used to study the kinetics of RVFV antibody production. Field sera were tested by the serum neutralization (VN) test and experimental sera by VN and haemagglutination-inhibition (HI) test. Based on VN test results, negative sera were regarded as reference controls from RVFV-free, and positive sera were regarded as reference controls from RVFV-infected subpopulations of animals. ELISA data were expressed as the percentage positivity (PP) of an internal high positive control. The two-graph receiver operating characteristics approach was used for the selection and optimization of I-ELISA cut-offs including the misclassification costs term and Youden index (J). In addition, cut-off values were determined as the mean plus two-fold standard deviation of the result observed with the RVFV-free subpopulations. Established optimal cut-offs were different for each of the data sets analyzed, and ranged from 1.65 PP (buffalo) to 9.1 PP (goats). At the cut-off giving the highest estimate of combined measure of diagnostic accuracy (highest J value), the I-ELISA test parameters were determined as follows: (1) Diagnostic sensitivity (%): cattle--84.31, buffalo--94.44, sheep--98.91, goats--99.18. (2) Diagnostic specificity (%): cattle--99.34, buffalo--98.28, sheep--99.16, goats--99.23 and other game ruminants--99.26. In the group of RVFV-experimentally infected sheep, seroconversion In all individuals was detected by VN on 4-6 dpi, by HI on 5-7 dpi, and by I-ELISA on 6-7 dpi. All tests showed the same kinetic pattern of immunological response. Antibody levels were low for a very short period before increasing to high titres, after which it was easily detectable by all tests. Compared to traditional tests, the lower sensitivity of I-ELISA in the detection of the earliest stage of immunological response may be practically insignificant, particularily when this assay is used in population-based, disease-surveillance programmes. The high sensitivity and specificity of I-ELISA established in this study, especially for the statistically more representative subpopulations of animals tested, seem to support this prediction. Test parameters determined in this study should, however, be regarded as in-house diagnostic decision limits, for which further updating is recommended, particularly for specimens from other countries, and preferably by applying a standardized method for sampling of new subpopulations of animals to be targeted by the assay.

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.

The prevalence of different African horsesickness virus serotypes in the Onderstepoort area near Pretoria, during an outbreak of African horsesickness in South Africa in 1995/1996.

During 1995/1996 parts of South Africa experienced exceptionally high rainfall. Large numbers of Culicoides midges were seen and an outbreak of African horsesickness (AHS) followed. In the Onderstepoort area, near Pretoria in Gauteng, a number of horses died of suspected AHS. Virus isolation and typing was done from blood and/or organ samples of 21 suspected cases as well as from five zebra which were kept in the area. Virus was isolated from 14 of the 21 suspected cases but not from the zebra. The neutralizing antibody response of the zebra to the nine different African horsesickness virus (AHSV) serotypes was determined. Results indicated the highest prevalence of serotypes 2 and 4 followed by serotypes 1, 6 and 9. Reverse transcription polymerase chain reaction (RT-PCR) was performed on total RNA extracted from blood samples of the zebra. AHSV RNA was indicated in three of five zebra by agarose gel electrophoresis analysis of amplicons and in four of five zebra after Southern blot hybridization using a 32P-labelled probe. RT-PCR can be used together with serological techniques in studies of AHS to further clarify the epizootiology of the disease.

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.

Some epidemiological and economic aspects of a bluetongue-like disease in cattle in South Africa--1995/96 and 1997.

In December 1995 to March 1996 and the early summer of 1997 South Africa experienced above average rainfall which favoured the occurrence of Culicoides transmitted diseases. During this period several outbreaks of an uncommon disease of cattle occurred over a large part of the country. The clinical signs were similar to those of infection with the viruses of bluetongue (BT) and epizootic haemorrhagic disease of deer (EHD). Virus isolation from cattle and Culicoides yielded both viruses. Dual infections occurred on several farms. Typing of BT isolates yielded types 2, 3, 6 and 8. On at least two farms more than one BT virus serotype was involved. On one farm only EHD virus could be isolated from cattle and Culicoides. Serological tests confirmed that on this farm the disease was caused by EHD. In 1932/33, when a similar disease was reported conditions were vastly different. Rainfall figures show that the 1932/33 season was exceptionally dry. Techniques available at that time could not identify EHD and the cause was reported to be BT. The occurrence of BT in a dry season and over a much wider area than the distribution in South Africa of Culicoides imicola, the only proven vector for BT, is a clear indication that other species less dependent on high rainfall are involved. The present isolation of BT virus from three of five pools of parous C. bolitinos is evidence that this species, which breeds in cattle dung, may be an additional vector for BT.

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.

An outbreak of encephalomyocarditis-virus infection in free-ranging African elephants in the Kruger National Park.

A cluster of four deaths in late December 1993, marked the onset of an outbreak of disease of African elephants (Loxodonta africana) in the Kruger National Park (KNP) in South Africa, which has an estimated population of 7,500 elephants. Mortalities peaked in January 1994, with 32 deaths, and then declined steadily to reach pre-outbreak levels by September, but sporadic losses continued until November. During the outbreak altogether 64 elephants died, of which 53 (83%) were adult bulls. Archival records revealed that, in addition to the usual losses from known causes such as poaching and intraspecific fighting, sporadic deaths from unexplained causes had, in fact, occurred in widely scattered locations from at least 1987 onwards, and from that time until the perceived outbreak of disease there had been 48 such deaths involving 33 (69%) adult bulls. Carcases had frequently become decomposed or had been scavenged by the time they were found, but seven of eight elephants examined early in 1994 had lesions of cardiac failure suggestive of encephalomyocarditis (EMC)-virus infection, and the virus was isolated from the heart muscles of three fresh carcases. The results of tests for neutralizing antibody on 362 elephant sera collected for unrelated purposes from 1984 onwards and kept frozen, indicated that the virus had been present in the KNP since at least 1987. Antibody prevalences of 62 of 116 (53%) 18 of 139 (13%) and seven of 33 (21%) were found in elephants in three different regions of the KNP in 1993 and 1994. Studies had been conducted on myomorph rodents in the KNP for unrelated purposes since 1984, and trapping attempts were increased during the perceived outbreak of disease in elephants. There was a striking temporal correlation between the occurrence of a population explosion (as evidenced by markedly increased catch rates per trap-night) and a surge in prevalence of antibody to EM virus in rodents, and the occurrence of the outbreak of disease in elephants.

Hepatitis in farmed hatchling Nile crocodiles (Crocodylus niloticus) due to chlamydial infection.

An investigation into the cause of acute mortality in farmed hatchling crocodiles Crocodylus niloticus led to the isolation of chlamydia from the livers of affected animals. Prominent pathological finds were acute hepatitis with intracellular chlamydial colonies and generalized oedema. A chlamydia presumed to be C. psittaci was isolated from livers of affected hatchlings. Mortality subsided after treatment with oxytetracycline. This disease is now recognized as being a major problem on crocodile farms in Zimbabwe.