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Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic phlebovirus-causing disease in domestic ruminants and humans in Africa, the Arabian Peninsula and some Indian Ocean islands. Outbreaks, characterized by abortion storms and a high morbidity rate in newborn animals, occur after heavy and prolonged rainfalls favouring the breeding of mosquitoes. However, the identity of the important mosquito vectors of RVFV is poorly known in most areas. Mosquitoes collected in the Ndumo area of tropical north-eastern KwaZulu-Natal (KZN), South Africa, were tested for RVFV nucleic acid using RT-PCR. The virus was detected in a single pool of unfed Aedes ( Aedimorphus ) durbanensis , indicating that this seasonally abundant mosquito species could serve as a vector in this area of endemic RVFV circulation. Phylogenetic analysis indicated the identified virus is closely related to two isolates from the earliest outbreaks, which occurred in central South Africa more than 60 years ago, indicating long-term endemicity in the region. Further research is required to understand the eco-epidemiology of RVFV and the vectors responsible for its circulation in the eastern tropical coastal region of southern Africa.

Rift Valley fever phlebovirus (RVFV) infects humans and a wide range of ungulates and historically has caused devastating epidemics in Africa and the Arabian Peninsula. Lesions of naturally infected cases of Rift Valley fever (RVF) have only been described in detail in sheep with a few reports concerning cattle and humans. The most frequently observed lesion in both ruminants and humans is randomly distributed necrosis, particularly in the liver. Lesions supportive of vascular endothelial injury are also present and include mild hydropericardium, hydrothorax and ascites; marked pulmonary congestion and oedema; lymph node congestion and oedema; and haemorrhages in many tissues. Although a complete understanding of RVF pathogenesis is still lacking, antigen-presenting cells in the skin are likely the early targets of the virus. Following suppression of type I IFN production and necrosis of dermal cells, RVFV spreads systemically, resulting in infection and necrosis of other cells in a variety of organs. Failure of both the innate and adaptive immune responses to control infection is exacerbated by apoptosis of lymphocytes. An excessive pro-inflammatory cytokine and chemokine response leads to microcirculatory dysfunction. Additionally, impairment of the coagulation system results in widespread haemorrhages. Fatal outcomes result from multiorgan failure, oedema in many organs (including the lungs and brain), hypotension, and circulatory shock. Here, we summarize current understanding of RVF cellular tropism as informed by lesions caused by natural infections. We specifically examine how extant knowledge informs current understanding regarding pathogenesis of the haemorrhagic fever form of RVF, identifying opportunities for future research.

Bluetongue is a serious non-contagious vector-borne viral disease in ruminants, causing poor animal welfare and economic consequences globally. Concern has been raised about the development of novel bluetongue virus (BTV) strains and their possibly altered virulence through the process of viral reassortment. Virulence is traditionally estimated in lethal dose 50 (LD 50 ) studies in murine models, but agreement with both in vitro and virulence in ruminants is questionable, and a refined experimental design is needed. Specific reassortants between wild-type and vaccine strains of BTV-1, -6 and -8 have previously been developed by reverse genetics. The aim of the present study was to rank the in vivo virulence of these parental and reassortant BTV strains by calculating LD 50 in a murine model by using an experimental design that is new to virology: a between-patient optimised three-level response surface pathway design. The inoculation procedure was intracranial. Fifteen suckling mice were used to establish LD 50 for each strain. Three parental and five reassortant virus strains were included. The LD 50 s varied from of 0.1 (95% confidence interval (CI) 0-0.20) to 3.3 (95% CI 2.96-3.72) tissue culture infectious dose 50/ml. The results support the hypothesis that reassortment in BTV may lead to increased virulence in mice with potential negative consequences for the natural ruminant host. The ranking showed low agreement with in vitro properties and virulence in ruminants according to existing literature. Refined design such as response surface pathway design was found suitable for use in virology, and it introduces significant ethical and scientific improvements.

This is a report of the complete genome sequences of plaque-selected isolates of five virus strains included in bottle A of the South African Onderstepoort Biological Products commercial live attenuated bluetongue virus vaccine., (Copyright © 2020 Coetzee et al.)

The Palyam serogroup orbiviruses are associated with abortion and teratogenesis in cattle and other ruminants. Of the 13 different serotypes that have been identified, the full genome sequence of only one, Kasba, has been published. We undertook to perform Next Generation Sequencing (NGS) and phylogenetic analysis on 12 Palyam serotypes plus field isolates of the African serotypes in our possession. The Palyam serogroup was found to be most closely related to the African horse sickness virus group and showed the most distant evolutionary relationship to the equine encephalosis viruses (EEV). Amino acid sequence analysis revealed that the gene encoding VP7 was the most conserved within serotypes and VP2 and VP5 showed the highest degree of variation. A high degree of sequence identity was found for isolates from the same geographical region. The phylogenetic analysis revealed two clades where the African serotypes were all very closely related in one clade and the other clade contained the Australian and Asian serotypes and one African serotype, Petevo. It was evident from the sequence data that the geographical origin of Palyam serogroup viruses played an important role in the development of the different serotypes.

Background: Rift Valley fever (RVF) is a mosquito-borne zoonotic disease characterized in South Africa by large epidemics amongst ruminant livestock at very long, irregular intervals, mainly in the central interior. However, the presence and patterns of occurrence of the virus in the eastern parts of the country are poorly known. This study aimed to detect the presence of RVF virus (RVFV) in cattle and goats in far northern KwaZulu-Natal province and to estimate the prevalence of antibodies to the virus and the incidence rate of seroconversion., Methodology: Cross-sectional studies were performed in communally farmed cattle (n = 423) and goats (n = 104), followed by longitudinal follow-up of seronegative livestock (n = 253) 14 times over 24 months, representing 160.3 animal-years at risk. Exposure to RVFV was assessed using an IgG sandwich ELISA and a serum neutralization test (SNT) and seroconversion was assessed using SNT. Incidence density was estimated and compared using multivariable Poisson models and hazard of seroconversion was estimated over time., Principal Findings: Initial overall seroprevalence was 34.0% (95%CI: 29.5-38.8%) in cattle and 31.7% (95%CI: 22.9-41.6%) in goats, varying by locality from 18-54%. Seroconversions to RVFV based on SNT were detected throughout the year, with the incidence rate peaking during the high rainfall months of January to March, and differed considerably between years. Overall seroconversion rate in cattle was 0.59 per animal-year (95% CI: 0.46-0.75) and in goats it was 0.41 per animal-year (95% CI: 0.25-0.64), varying significantly over short distances., Conclusions/significance: The high seroprevalence in all age groups and evidence of year-round viral circulation provide evidence for a hyperendemic situation in the study area. This is the first study to directly estimate infection rate of RVFV in livestock in an endemic area in the absence of reported outbreaks and provides the basis for further investigation of factors affecting viral circulation and mechanisms for virus survival during interepidemic periods., Competing Interests: The authors have declared that no competing interests exist.

A prospective study was undertaken during 2013 and 2014, to determine the prevalence of African horse sickness virus (AHSV) in Culicoides midges and the incidence of infection caused by the virus in 28 resident horses on two equine establishments on the East Rand, Gauteng Province, South Africa. Field caught Culicoides midges together with whole blood samples from participating horses were collected every two weeks at each establishment. Culicoides midges and blood samples were tested for the presence of AHSV RNA by real-time quantitative reverse transcription polymerase chain reaction. Nine immunised horses became infected with AHSV during the study period, although infections were subclinical. African horse sickness virus was also identified from a field-collected midge pool. The observations recapitulate previously published data in another setting, where further investigation is warranted to determine what role subclinical infection plays in the diseases epidemiology.

Bluetongue is primarily a disease of sheep in South Africa, while cattle and goats are mostly subclinically infected. The viraemia of bluetongue virus in cattle lasts much longer than in sheep and the role of cattle in the epidemiology of bluetongue in South Africa is poorly understood. Bluetongue virus has a segmented double-stranded ribonucleic acid genome and reassortment of genomes is a common feature. The aim of the study was to investigate whether reassortment occurs between vaccine and field strains when simultaneously administered to cattle. Six cattle between the ages of 6 and 12 months were infected with five strains of modified live vaccine bluetongue virus and a virulent field isolate of bluetongue virus 4. Blood samples were subsequently collected daily from these animals from day 1 to day 39 post-inoculation. Viruses were directly isolated during viraemia from the buffy coat on Vero cells using the plaque forming unit method. Analysis of plaques indicated that no reassortants between virulent field and vaccine strains occurred and the virulent bluetongue virus 4 was identified as the predominant virus strain. However, a reassortant virus between two bluetongue virus vaccine strains was isolated from the buffy coat. Whole genome sequences from the vaccine viruses were compared to the suspected reassortant and it was found that segment 8 exchanged between the bluetongue virus 8 and bluetongue virus 9 vaccine strains. The use of the live-attenuated bluetongue virus multivalent vaccine in South Africa causes circulation of different vaccine serotypes in Culicoides spp. and susceptible hosts and cattle might provide the ideal host for reassortment to occur.

Canine distemper virus (CDV) causes a severe contagious disease in a broad range of hosts. This is the first study to genetically characterise CDV strains from four different wildlife species in South Africa. The phylogenetic diversity of CDV is examined, using the haemagglutinin gene. The South African wildlife CDV isolates showed a high degree of similarity to CDV in South African domestic dogs. Phylogenetic analyses confirmed the presence of 12 geographical lineages with CDV strains from South African wildlife falling within the Southern African lineage. The study reveals two possible co-circulating sub-genotypes corresponding to the northern and southern regions of South Africa respectively. CDV strains from the non-canid species were distinct, but similar to CDV isolates from domestic dog and wild canids. Residues at amino acid sites of the SLAM binding region support the notion that CDV strains encoding 519I / 549H are better adapted to non-canid species than canid species. The amino acids present at site 530 are conserved regardless of host species. Strains from South African wild carnivores showed no difference between host species with all strains presenting 530N. All non-canid strains in this study presented the combination 519I/549H. No evidence of host adaptation or lineage grouping was observed for the Nectin-4 binding region. Further studies should include CDV strains isolated from various hosts from a wider geographical range in South Africa., Competing Interests: The authors have declared that no competing interests exist.

Canine distemper virus causes global multihost infectious disease. This report details complete genome sequences of three vaccine and two new wild-type strains. The wild-type strains belong to the South African lineage, and all three vaccine strains to the America 1 lineage. This constitutes the first genomic sequences of this virus from South Africa., (Copyright © 2017 Loots et al.)

Canine distemper virus (CDV) has emerged as a significant disease of wildlife, which is highly contagious and readily transmitted between susceptible hosts. Initially described as an infectious disease of domestic dogs, it is now recognized as a global multi-host pathogen, infecting and causing mass mortalities in a wide range of carnivore species. The last decade has seen the effect of numerous CDV outbreaks in various wildlife populations. Prevention of CDV requires a clear understanding of the potential hosts in danger of infection as well as the dynamic pathways CDV uses to gain entry to its host cells and its ability to initiate viral shedding and disease transmission. We review recent research conducted on CDV infections in wildlife, including the latest findings on the causes of host specificity and cellular receptors involved in distemper pathogenesis.

In early 2014, abortions and death of ruminants were reported on farms in Maputo and Gaza Provinces, Mozambique. Serologic analysis and quantitative and conventional reverse transcription PCR confirmed the presence of Rift Valley fever virus. The viruses belonged to lineage C, which is prevalent among Rift Valley fever viruses in southern Africa.

Several studies have demonstrated the ability of certain viruses to overwinter in arthropod vectors. The over-wintering mechanism of bluetongue virus (BTV) is unknown. One hypothesis is over-wintering within adult Culicoides midges (Diptera; Ceratopogonidae) that survive mild winters where temperatures seldom drop below 10 °C. The reduced activity of midges and the absence of outbreaks during winter may create the impression that the virus has disappeared from an area. Light traps were used in close association with horses to collect Culicoides midges from July 2010 to September 2011 in the Onderstepoort area, in Gauteng Province, South Africa. More than 500 000 Culicoides midges were collected from 88 collections and sorted to species level, revealing 26 different Culicoides species. Culicoides midges were present throughout the 15 month study. Nine Culicoides species potentially capable of transmitting BTV were present during the winter months. Midges were screened for the presence of BTV ribonucleic acid (RNA) with the aid of a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. In total 91.2% of midge pools tested positive for BTV RNA. PCR results were compared with previous virus isolation results (VI) that demonstrated the presence of viruses in summer and autumn months. The results indicate that BTV-infected Culicoides vectors are present throughout the year in the study area. Viral RNA-positive midges were also found throughout the year with VI positive midge pools only in summer and early autumn. Midges that survive mild winter temperatures could therefore harbour BTV but with a decreased vector capacity. When the population size, biting rate and viral replication decrease, it could stop BTV transmission. Over-wintering of BTV in the Onderstepoort region could therefore result in re-emergence because of increased vector activity rather than reintroduction from outside the region.

This is a report of the complete genome sequences of plaque-selected isolates of each of the five virus strains included in a South African commercial trivalent bluetongue virus (BTV) attenuated live virus vaccine, a BTV-4 field strain isolated from Rustenburg, South Africa, in 2011, and a bluetongue reassortant (bluetongue virus 4 strain 4/O. aries-tc/ZAF/11/OBP-115) isolated from experimentally vaccinated cattle. Full-genome sequencing and phylogenetic analyses show that the bluetongue virus 9 strain 9/B. taurus-tc/ZAF/15/Onderstepoort_B02b is a reassortant virus containing segments from both BTV-9 and BTV-8., (Copyright © 2016 van den Bergh et al.)

This is a report of the complete genome sequences of plaque-selected isolates of each of the four virus strains included in a South African commercial tetravalent African horse sickness attenuated live virus vaccine., (Copyright © 2015 Guthrie et al.)

Bluetongue virus (BTV) has since 1998 extended its distribution further North than where it has previously been encountered. Changes in the epidemiology of Bluetongue (BT), as well as novel features of recent outbreaks of BTV in Europe, have stimulated research on BTV‑vector‑host interaction. The outbreak of BTV‑8 in Northern Europe from 2006‑2008 is particular noteworthy in this regard, as the European strain of BTV‑8 demonstrated novel properties, including high virulence - especially for cattle - and the capability to cross the ruminant placenta. The virus was in addition transmitted by indigenous European Culicoides species that had not previously been implicated in the widespread transmission of BTV. Recent advances in the scientific understanding of BTV‑vector‑host interaction include increased knowledge of the virus' replication cycle, the role of biotic factors in influencing viral infection of the insect vector, increased knowledge of BTV immunology and pathogenesis in the mammalian host, and increased knowledge of virulence and pathogenicity features of newly discovered serotypes/strains of the virus. New research on aspects of BTV‑vector‑host interaction has been driven in part by developments in molecular biology and experimental infection biology, of which next generation sequencing, the expression of individual viral proteins in cell culture, the establishment of a reverse genetics system for the virus, the development of novel in vitro and in vivo infection models, and refinement of existing BTV experimental infection methodologies have proven instrumental. Moreover, these developments have also provided the opportunity for the development of novel vaccine strategies. This article provides a synopsis of selected recent advances that have been made in the understanding of BTV‑vector‑host interaction, with a particular focus on research that has been conducted in Europe over the last 5 years.

This is a report of the complete genome sequences of plaque-selected isolates of each of the three virus strains included in a South African commercial trivalent African horse sickness attenuated live virus vaccine., (Copyright © 2015 Guthrie et al.)

Rift Valley fever (RVF) is an arthropod-borne viral disease of importance in livestock and humans. Epidemics occur periodically in domestic ruminants. People in contact with infected livestock may develop disease that varies from mild flu-like symptoms to fatal viraemia. Livestock vaccination may assist in disease control. Rift Valley fever virus (RVFV) Clone 13 is a relatively new vaccine against RVF, derived from an avirulent natural mutant strain of RVFV, and has been shown to confer protective immunity against experimental infection with RVFV. The hypothesis tested in the current trial was that rams vaccinated with RVFV Clone 13 vaccine would not experience a reduction in semen quality (measured by evaluating the percentage progressively motile and percentage morphologically normal spermatozoa in successive ejaculates) relative to unvaccinated control animals. Ram lambs were screened for antibodies to RVFV using a serum neutralisation test. Animals without detectable antibodies (n = 23) were randomly allocated to either a test group (n = 12) or a control group (n = 11). Animals in the test group were vaccinated with RVFV Clone 13 vaccine. Daily rectal temperature measurements and weekly semen and blood samples were taken from all animals. Seven animals were eliminated from the statistical analysis because of potential confounding factors. Logistic regression analysis was performed on data gathered from the remaining animals to determine whether an association existed between animal group, rectal temperature and semen quality parameters. No correlation existed between the treatment group and values obtained for the semen quality parameters measured. There was no statistically significant post-vaccination decline in the percentage of live morphologically normal spermatozoa, or the percentage of progressively motile spermatozoa, either when assessed amongst all animals or when assessed within individual groups. A repeat study with a larger sample size and a more comprehensive pre-screening process may be indicated to avoid the inclusion of unsuitable animals.

Ostrich (Struthio camelus) chicks less than 3 mo age are observed to experience a high mortality rate that is often associated with enteritis. This study was undertaken to investigate the infectious bacteria implicated in ostrich chick enteritis. Postmortems were performed on 122 ostrich chicks aged from 1 d to 3 mo and intestinal samples were subjected to bacterial culture. Bacterial isolates were typed by PCR and serotyping. Escherichia coli (E. coli; 49%) was the most frequently isolated from the samples followed by Clostridium perfringens (C. perfringens; 20%), Enterococcus spp. (16%), and Salmonella spp. (7%). Of the E. coli, 39% were categorized as enteropathogenic E. coli, 4% enterotoxigenic E. coli, and no enterohaemorrhagic E. coli were found. The majority (93%) of C. perfringens was Type A and only 7% was Type E. C. perfringens Types B through D were not present. The netB gene that encodes NetB toxin was identified from 16% of the C. perfringens isolated. All the C. perfringens Type E harbored the netB gene and just 10% of the C. perfringens Type A had this gene. Three Salmonella serotypes were identified: Salmonella Muenchen (S. Muenchen; 80%), S. Hayindongo (13%), and S. Othmarschen (7%). The indication is that the cause of enteritis in ostrich chicks is bacterial-involving: enteropathogenic E. coli and enterotoxigenic E. coli; C. perfringens Types A and E (with the possible influence of netB gene); and S. Muenchen, S. Hayindongo, and S. Othmarschen., (© 2015 Poultry Science Association Inc.)

Inclusion body hepatitis is an acute disease of chickens ascribed to viruses of the genus Aviadenovirus and referred to as fowl adenovirus (FAdV). There are 12 FAdV types (FAdV1to FAdV8a and FAdV8b to FAdV11), classified into five species based on their genotype (designated FAdVA to FAdVE). A total of 218 000 chickens, 2-29 days of age, were affected over a 1-year period, all testing positive by microscopy, virus isolation and confirmation with polymerase chain reaction (PCR). Affected birds were depressed, lost body weight,were weak and had watery droppings. Pathological changes observed during necropsy indicated consistent changes in the liver, characterised by hepatomegaly, cholestasis and hepatitis. Lesions were also discernible in the spleen, kidney and gizzard wall and were characterised by splenomegaly, pinpoint haemorrhages, nephritis with haemorrhage,visceral gout and serosal ecchymosis of the gizzard wall. Histopathological lesions were most consistently observed in the liver but could also be seen in renal and splenic tissue. Virus isolation was achieved in embryonated eggs and most embryos revealed multifocalto diffuse hepatic necrosis, with a mixed cellular infiltrate of macrophages and heterophils(necro-granulomas), even in the absence of macroscopic pathology. Virus isolation results were verified by histopathology and PCR on embryonic material and further characterised by nucleotide sequence analysis. Two infectious bursal disease virus isolates were also made from the Klerksdorp flock. Nucleotide sequence analysis of the L1 hexon loop of all the FAdV isolates indicated homology (99%) with prototype strains P7-A for FAdV-2, as well as for FAdV-8b.

In South Africa, outbreaks of African horse sickness (AHS) occur in summer; no cases are reported in winter, from July to September. The AHS virus (AHSV) is transmitted almost exclusively by Culicoides midges (Diptera: Ceratopogonidae), of which Culicoides imicola is considered to be the most important vector. The over-wintering mechanism of AHSV is unknown. In this study, more than 500 000 Culicoides midges belonging to at least 26 species were collected in 88 light traps at weekly intervals between July 2010 and September 2011 near horses in the Onderstepoort area of South Africa. The dominant species was C. imicola. Despite relatively low temperatures and frost, at least 17 species, including C. imicola, were collected throughout winter (June-August). Although the mean number of midges per night fell from > 50 000 (March) to < 100 (July and August), no midge-free periods were found. This study, using virus isolation on cell cultures and a reverse transcriptase polymerase chain reaction (RT-PCR) assay, confirmed low infection prevalence in field midges and that the detection of virus correlated to high numbers. Although no virus was detected during this winter period, continuous adult activity indicated that transmission can potentially occur. The absence of AHSV in the midges during winter can be ascribed to the relatively low numbers collected coupled to low infection prevalence, low virus replication rates and low virus titres in the potentially infected midges. Cases of AHS in susceptible animals are likely to start as soon as Culicoides populations reach a critical level.

Fowl adenovirus (FAdV) is a member of the genus Aviadenovirus and causes a number of economically important poultry diseases. One of these diseases, inclusion body hepatitis (IBH), has a worldwide distribution and is characterised by acute mortality (5% - 20%) in production chickens. The disease was first described in the United States of America in 1963 and has also been reported in Canada, the United Kingdom, Australia, France and Ireland, but until now, not in South Africa. Adenoviruses isolated from the first outbreak of IBH in South Africa were able to reproduce the disease in chicken embryo livers. The aim of the present study was to characterise the viruses and determine the pathogenicity of the FAdV strains responsible for the first reported case of IBH in South Africa. Polymerase chain reaction (PCR) amplification of the L1 loop region of the fowl adenovirus hexon gene using degenerate primer pair hexon A/B was used to identify the viruses that were isolated. Restriction fragment length polymorphism (RFLP) of the amplification products was used for the differentiation of 14 isolates of fowl adenovirus. Sequencing of the PCR products followed by amino acid comparison and phylogenetic analysis using the L1 loop region of the hexon protein was done to determine the identity of the isolates. Amino acid sequences of the hexon genes of all the South African isolates were compared with those of reference strains representing FAdV species. Amino acid comparison of 12 South Africa field isolates to FAdV reference strains revealed a high sequence identity (> 93.33%) with reference strains T8-A and 764. Two of the isolates had high sequence identity (93.40%) with reference strains P7-A, C2B and SR48. Phylogenetic analysis of the L1 loop region of the hexon protein of all 14 South African isolates was consistent with their RFLP clusters. The mortality rates of embryos challenged with 106 egg infective doses (EID50) FAdV 2 were 80% - 87% and mortality rates for embryos challenged with 105.95 (EID50) FAdV 8b were 65% - 80%.

Bluetongue (BT) is a non-contagious disease of sheep and other domestic and wild ruminants caused by the bluetongue virus (BTV). Currently 26 serotypes of the virus have been identified. In South Africa, 22 serotypes have been identified and BT is controlled mainly by annual vaccinations using a freeze-dried live attenuated polyvalent BTV vaccine. The vaccine is constituted of 15 BTV serotypes divided into three separate bottles and the aim is to develop a vaccine using fewer serotypes without compromising the immunity against the disease. This study is based on previously reported cross-neutralisation of specific BTV serotypes in in vitro studies. Bluetongue virus serotype 4 was selected for this trial and was tested for cross-protection against serotype 4 (control), 1 (unrelated serotype), 9, 10 and 11 in sheep using the serum neutralisation test. The purpose of the study was to determine possible cross-protection of different serotypes in sheep. Of those vaccinated with BTV-4 and challenged with BTV-1, which is not directly related to BTV-4, 20% were completely protected and 80% showed clinical signs, but the reaction was not as severe as amongst the unvaccinated animals. In the group challenged with BTV-10, some showed good protection and some became very sick. Those challenged with BTV-9 and BTV-11 had good protection. The results showed that BTV-4 does not only elicit a specific immune response but can also protect against other serotypes.

Rift Valley fever and lumpy skin disease are transboundary viral diseases endemic in Africa and some parts of the Middle East, but with increasing potential for global emergence. Wild ruminants, such as the African buffalo (Syncerus caffer), are thought to play a role in the epidemiology of these diseases. This study sought to expand the understanding of the role of buffalo in the maintenance of Rift Valley fever virus (RVFV) and lumpy skin disease virus (LSDV) by determining seroprevalence to these viruses during an inter-epidemic period. Buffaloes from the Kruger National Park (n = 138) and Hluhluwe-iMfolozi Park (n = 110) in South Africa were sampled and tested for immunoglobulin G (IgG) and neutralising antibodies against LSDV and RVFV using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the serum neutralisation test (SNT). The I-ELISA for LSDV and RVFV detected IgG antibodies in 70 of 248 (28.2%) and 15 of 248 (6.1%) buffaloes, respectively. Using the SNT, LSDV and RVFV neutralising antibodies were found in 5 of 66 (7.6%) and 12 of 57 (21.1%), respectively, of samples tested. The RVFV I-ELISA and SNT results correlated well with previously reported results. Of the 12 SNT RVFV-positive sera, three (25.0%) had very high SNT titres of 1:640. Neutralising antibody titres of more than 1:80 were found in 80.0% of the positive sera tested. The LSDV SNT results did not correlate with results obtained by the I-ELISA and neutralising antibody titres detected were low, with the highest (1:20) recorded in only two buffaloes, whilst 11 buffaloes (4.4%) had evidence of co-infection with both viruses. Results obtained in this study complement other reports suggesting a role for buffaloes in the epidemiology of these diseases during inter-epidemic periods.

Background: Although reported sporadically from various countries, feline babesiosis appears to be a significant clinical entity only in South Africa, where Babesia felis is usually incriminated as the causative agent. Babesia lengau, recently described from asymptomatic cheetahs, has now possibly been incriminated as the causative agent in two severe clinical cases in domestic cats., Findings: Both cats were euthanised in extremis. While typical feline babesiosis in South Africa is an afebrile disease with a chronic manifestation, there was acute onset of severe clinical signs in both cats and their body temperatures were above the normal range when they were presented for treatment. Haemolytic anaemia was confirmed in one case. To our knowledge, this is the first report of cerebral babesiosis in cats.On reverse line blot 18S rDNA PCR products obtained from both cats showed positive hybridization profiles with the B. lengau species-specific probe. The two partial parasite 18S rRNA gene sequences obtained, showed high sequence similarity (99.9%) to B. lengau. In a representative tree constructed by the neighbor-joining method using the two-parameter model of Kimura the two obtained partial 18S rDNA sequences and that of B. lengau formed a monophyletic group with B. conradae and sequences previously isolated from humans and wildlife in the western USA., Conclusion: All clinical cases of feline babesiosis in South Africa are not necessarily caused by B. felis. Other piroplasms, e.g. B. lengau, may be incriminated in clinical cases, especially those occurring outside the known endemic area.

Lumpy skin disease (LSD) is an economically important, acute or sub-acute, viral disease of cattle that occurs across Africa and in the Middle East. The aim of this study was to investigate if lumpy skin disease virus (LSDV) can be transmitted mechanically by African brown ear ticks (Rhipicephalus appendiculatus Neum.). Laboratory-bred R. appendiculatus males were fed on experimentally infected viraemic 'donor' cattle. Partially fed male ticks were then transferred to feed on an uninfected 'recipient' cow. The recipient animal became viraemic, showed mild clinical signs of LSD and seroconverted. Additionally, R. appendiculatus males were found to transmit LSDV through feeding on skin lacking visible lesions, demonstrating that viraemic animals without lesions at the feeding site of ticks may be a source of infection. This is the first time that transmission of poxviruses by a tick species has been demonstrated and the importance of this mode of transmission in the spread of LSDV in endemic settings is discussed.

In 2008, a suspected outbreak of Rift Valley fever (RVF) was reported on a farm in the Bela-Bela area, Limpopo Province, South Africa. Seven calves died on the affected dairy farm, where no RVF vaccination programme was practised. No apparent clinical disease was reported in the other 300 cattle (33 calves included) or 200 sheep on the farm. During the outbreak, blood samples from 77.7% (233/300) of the cattle and 36.5% (73/200) of the sheep were collected on the affected farm and 55 blood samples were taken from cattle on a neighbouring farm. Eight weeks later, 78% of the cattle (234/300) and 42.5% of the sheep (85/200) were bled on the affected farm only. All sera were tested by an Immunoglobulin M (IgM)-capture Enzymelinked immunosorbent assay (ELISA) and by an indirect Immunoglobulin G (IgG) ELISA. Selected IgM-positive (n = 14), IgG-positive (n = 23) and samples negative for both IgM and IgG-specific antibodies against RVF virus (n = 19) were tested using the serum neutralisation test (SNT). Sera from IgM-positive (n = 14) and negative (n = 20) animals were also tested by a TaqMan polymerase chain reaction (PCR). On the affected farm, 7% (16/233) of the cattle were IgM-positive and 13.7% (32/233) IgG-positive at the first bleed and 2% were IgM-positive at the second bleed, whilst the number of cattle positive for IgG-specific antibodies increased by 21.3% compared with the first bleed. Only 1.4% of sheep were positive for both IgM and IgG antibodies at the first collection; at the second bleed, IgM-positive cases decreased to 1.2%, whilst IgG-positive cases increased to 2.4%. Whilst no IgM-positive cattle were found on the neighbouring farm, 5.5% of cattle were IgG-positive. The SNT confirmed most of the ELISA results, whilst PCR results were all negative. Although serology results indicated virus circulation on both farms, the negative PCR results demonstrated that the animals were not viraemic at the time they were sampled. The movement of infected mosquito vectors by wind over long distances into a low-lying area that favoured their breeding on the Bela-Bela farm may have led to an outbreak of the disease there, but the reason for the low level of virus circulation amongst susceptible animals remains unclear.

Bluetongue (BT) is a non-contagious, infectious, arthropod transmitted viral disease of domestic and wild ruminants that is caused by the bluetongue virus (BTV), the prototype member of the Orbivirus genus in the family Reoviridae. Bluetongue was first described in South Africa, where it has probably been endemic in wild ruminants since antiquity. Since its discovery BT has had a major impact on sheep breeders in the country and has therefore been a key focus of research at the Onderstepoort Veterinary Research Institute in Pretoria, South Africa. Several key discoveries were made at this Institute, including the demonstration that the aetiological agent of BT was a dsRNA virus that is transmitted by Culicoides midges and that multiple BTV serotypes circulate in nature. It is currently recognized that BT is endemic throughout most of South Africa and 22 of the 26 known serotypes have been detected in the region. Multiple serotypes circulate each vector season with the occurrence of different serotypes depending largely on herd-immunity. Indigenous sheep breeds, cattle and wild ruminants are frequently infected but rarely demonstrate clinical signs, whereas improved European sheep breeds are most susceptible. The immunization of susceptible sheep remains the most effective and practical control measure against BT. In order to protect sheep against multiple circulating serotypes, three pentavalent attenuated vaccines have been developed. Despite the proven efficacy of these vaccines in protecting sheep against the disease, several disadvantages are associated with their use in the field.

Recent outbreaks of bluetongue virus (BTV) serotypes 2 and 8 in many European countries provided an opportunity to investigate the possibility of improving the safety of the modified live vaccines administered mainly in South Africa. Modified live vaccines (MLV) released at a titre of 5 x 104 PFU/mL, raised concerns and prompted the need to determine the minimum titre which will still be protective and also safe. The BTV serotypes 2 and 8 vaccines were produced at the following titres: 102 PFU/mL, 103 PFU/mL and 104 PFU/mL, and were injected into 24 sheep which were then monitored. Blood was collected on days 0, 3, 6, 9, 12, 15, 18, 21, 25, 28 and 4 months post vaccination, for seroconversion and viraemia studies. These sheep were later challenged at 4 months post vaccination using BTV infected cell culture material, they were then observed and bled and again tested for viraemia. There was no viraemia post vaccination, however, a febrile reaction did occur and seroconversion was demonstrated at low titres for both BTV 2 and 8. Although viraemia was demonstrated post challenge, sheep vaccinated with the low titre BTV 2 vaccine showed more than a 90% protection index at a lower titre of 103 PFU/mL, compared with BTV 8 that showed a protection index above 90% at all the titres used. It is recommended that for BTV 2 vaccine, sheep should be vaccinated at a titre of 103 PFU/mL and at a titre of 102 PFU/mL with BTV 8 vaccine.

Bovine tuberculosis (BTB), a chronic disease of mammals caused by Mycobacterium bovis, is a threat to South African wildlife. It has been reported that African buffaloes (Syncerus caffer) are reservoir hosts of BTB in South African wildlife populations. This study reports on the molecular identification and typing of 31 M. bovis isolates collected between 1993 and 2008, mainly from buffaloes but also from two lions and a bush pig, in the Hluhluwe-iMfolozi Park (HiP) in KwaZulu-Natal. To study the dynamics of BTB in the buffalo populations, 28 M. bovis isolates from the HiP and epidemiologically related parks were characterised using regions of difference deletion analysis for species identification and spoligotyping, variable number of tandem repeats (VNTR), polymorphic G-C-rich sequences and IS6110 restriction fragment length polymorphism (RFLP) genotyping methods. At least three distinct M. bovis genotypes were found amongst HiP samples. The combination of VNTR typing (using a 16-loci panel) and IS6110 RFLP revealed the presence of three additional genetic profiles in individual buffaloes, demonstrating that the highest level of discrimination was achieved by these typing methods. One of the observed spoligotypes (SB0130) was dominant and represented 75% of isolates from buffaloes. A novel M. bovis spoligotype (SB1474), which is reported for the first time in this study, was observed in 14.3% of isolates from buffaloes. Based on the observed genetic relationships, the findings suggest independent introductions from at least three unrelated sources. These findings improve the knowledge regarding the diversity of circulating M. bovis strains in the HiP.

Papillomavirus was detected electron microscopically in cutaneous fibropapillomas of a giraffe (Giraffa camelopardalis) and a sable antelope (Hippotragus niger). The virus particles measured 45 nm in diameter. Histopathologically, the lesions showed histopathological features similar to those of equine sarcoid as well as positive immunoperoxidase-staining of tissue sections for papillomavirus antigen. Polymerase chain reaction (PCR) detected bovine papillomavirus (BPV) DNA. Bovine papillomavirus-1 was characterised by real-time PCR in the sable and giraffe, and cloning and sequencing of the PCR product revealed a similarity to BPV-1. As in the 1st giraffe, the lesions from a 2nd giraffe revealed locally malignant pleomorphism, possibly indicating the lesional end-point of papilloma infection. Neither virus particles nor positively staining papillomavirus antigen could be demonstrated in the 2nd giraffe but papillomavirus DNA was detected by real-time PCR which corresponded with BPV-1 and BPV-2.

Bluetongue (BT), a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV), can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS) protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 102.88 and 1 x 10(3.5) respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 103.5. No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo.

In a previous paper, we reported on a large number of cheetah blood specimens that gave positive signals only for Babesia and/or Theileria genus-specific probes on the reverse line blot (RLB) assay, indicating the presence of a novel species or variant of an existing species. Some of these specimens were investigated further by microscopic, serological, sequencing, and phylogenetic analyses. The near-full-length 18S rRNA genes of 13 samples, as well as the second internal transcribed spacer (ITS2) region, were amplified, cloned, and sequenced. A species-specific RLB probe, designed to target the hypervariable V4 region of the 18S rRNA gene for detection of the novel Babesia sp., was used to screen an additional 137 cheetah blood specimens for the presence of the species. The prevalence of infection was 28.5%. Here we describe the morphology and phylogenetic relationships of the novel species, which we have named Babesia lengau sp. nov.

As a result of the high lability and slow growth of Campylobacter fetus subspecies, the laboratory diagnosis of bovine genital campylobacteriosis has always been difficult. This is especially true under South African conditions, where farms are far apart, laboratories are only present in major centres and there are high ambient temperatures. In order to overcome the shortcomings associated with traditional diagnostic methods, the implementation of a molecular assay was sought. This work describes how a previously published PCR assay (MG3F/ MG4R primers) was adapted, optimised and applied in the diagnostic laboratory to test preputial samples directly for the presence of Campylobacter fetus. Field evaluation of the assay revealed an analytical sensitivity and specificity of 85.7% and 99%, respectively. Subsequent genotyping and phenotyping of a diverse collection of South African field isolates revealed that South Africa has an unexpected and previously unreported high incidence of Campylobacter fetus subsp. venerealis biovar intermedius strains. These strains were not identified correctly by the subspecies-specific primer set evaluated. Until such time that cost- effective genotyping methods are available to diagnostic laboratories in South Africa, and other countries with these atypical Campylobacter fetus subsp. venerealis strains, the need for bacterial culture will persist. Identification to subspecies level of isolates at present remains dependent upon a single phenotypic criterion, namely tolerance to 1% glycine.

The seroprevalence of Anaplasma antibodies in wildlife (eland, blue wildebeest, kongoni, impala, Thomson's gazelle, Grant's gazelle, giraffe and plains zebra) and domestic animal (cattle, sheep and goat) populations was studied in wildlife/livestock interface areas of Kenya. Serum samples were analyzed by competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA), using a recombinant antigen (MSP-5) from Anaplasma marginale surface membrane. A monoclonal antibody, FC-16, was used as the primary antibody, while anti-mouse conjugated to horseradish peroxidase was used as the secondary antibody. The results indicate a high seroprevalence in both wildlife and livestock populations, in contrast to earlier reports from Kenya, which indicated a low seroprevalence. The differences are attributed to the accurate analytical method used (CI-ELISA), as compared with agglutination techniques, clinical signs and microscopy employed by the earlier workers.

Sheep inoculated with a virulent South African strain of bluetongue (BT) virus serotype 4 developed severe clinical signs and lesions characteristic of fulminant BT, including coronitis, hemorrhage and ulceration of the mucosal lining of the oral cavity and forestomaches, hemorrhage in the wall of the pulmonary artery, and focally extensive necrosis of skeletal muscle, especially of the neck. At necropsy, up to 14 days after infection, the infected sheep exhibited striking pulmonary edema, edema of the subcutaneous tissues and fascial planes of the head and neck, and pleural and pericardial effusion of varying severity. A reliable model for experimental reproduction of fulminant BT in sheep will facilitate future studies to better characterize the pathogenesis of this disease, particularly as it regards the mechanisms responsible for the increased vascular permeability that characterizes BT and related orbiviral diseases such as African horse sickness.

The epidemiology of serotype SAT-2 foot-and-mouth disease was investigated in sub-Saharan Africa by phylogenetic analysis using the 1D gene encoding the major antigenic determinant. Fourteen genotypes were identified of which three are novel and belong to East Africa, bringing the total number of genotypes for that region to eight. The genotypes clustered into three lineages that demonstrated surprising links between East, southern and south-western Africa. One lineage was unique to West Africa. These results established numerous incursions across country borders in East Africa and long term conservation of sequences for periods up to 41 years. Ethiopia, Kenya and Uganda have all experienced outbreaks from more than one unrelated strain, demonstrating the potential for new introductions. The amount of variation observed within this serotype nearly equalled that which was found between serotypes; this has severe implications for disease control using vaccination.

Lumpy skin disease virus (LSDV), a poxvirus of the genus Capripoxvirus, is shed in the semen of infected bulls. The screening of semen for infectious virus requires a sensitive diagnostic method. The isolation of the virus on cell cultures and/or the polymerase chain reaction (PCR) are sensitive diagnostic tests which may be used to screen semen for LSD viral DNA prior to artificial insemination. Although cell culture detects infectious virus and is a sensitive method, there are major difficulties in using this method due to the toxic effect of semen on the cells. The aim of this study was to find a method that decreases the toxic effect of semen and enhances the isolation of LSDV on cell culture. Semen samples from LSDV sero-negative bulls were collected and infected with a field isolate of LSDV, strain V248/93, with a titre of 6.5 log TCID50. The semen samples were treated with one of four different methods: centrifugation, serial dilution, filtration and chemical treatment with kaolin. The samples subjected to centrifugation, serial dilution and filtration were supplemented with gentamycin. Semen toxicity on cell cultures was eliminated when supernatants of semen samples centrifuged at 2000 rpm for 1, 3 and 5 min and serially diluted were used to inoculate confluent monolayer bovine dermis cells. The toxicity recorded when the pellet fractions of semen samples centrifuged for 5 min at 2000 rpm was comparable to results obtained from serially diluted samples supplemented with gentamycin. Filtration and kaolin treatment of semen samples did not remove the toxic effect.

To assess post-evisceration contamination of broiler carcasses, 300 samples were randomly selected during routine slaughter in the winter of 2004. The samples originated from 50 chicken carcasses, taken directly after evisceration, as well as 25 samples from ready-to-sell packages of fresh intestines (mala) and livers. The samples were taken in batches over a period of 4 weeks to allow randomised sampling from different farms of origin. Conventional culture-based detection methods of Campylobacter spp. usually need 4-6 days to produce a result. The polymerase chain reaction (PCR) used for this study took less than 32 hours. The average contamination rates with Campylobacter in both the skin and liver samples were 24%, and 28% for intestines. Chicken and chicken products, especially livers and intestines, form an integral part of the traditional diet of many Black South Africans, as they are cheap and readily available in bulk and un-chilled for direct distribution, mainly through street vending and other informal retail outlets. This sudy showed that Campylobacter spp. are prevalent in poultry in South Africa. The handling of poultry meat and products contaminated with this organism in households and the potential for cross-contamination of other foods presents a high risk of infection to consumers in South Africa. The study also emphasised the need for further research in this field.

Lumpy skin disease (LSD) is a disease of cattle, primarily in Africa and Madagascar and rarely in the Middle East. It is caused by a capripoxvirus that belongs to the family Poxviridae. The disease is of economic importance in endemic areas. Effective control of LSD requires accurate and rapid laboratory techniques to confirm a tentative clinical diagnosis. Comparative studies on different diagnostic tests used at different stages of the disease have not been done. The aim of this study was to compare several of these tests. Six seronegative bulls, between 11 and 20 months of age, were infected intravenously and kept in an insect-free facility. The course of the infection was monitored. During a 3-month period blood samples and skin biopsies were collected for virus isolation and polymerase chain reaction (PCR). Skin biopsies were also examined using transmission electron microscopy (TEM). The incubation period in infected animals varied from 4-5 days. The length of the viraemic period did not correlate with the severity of clinical disease. Viraemia was detected from 1-12 days using virus isolation and from 4-11 days using the PCR, which is longer than has previously been reported. Virus was isolated from skin biopsies until Day 39 post infection (p.i.) and PCR could demonstrate viral DNA until Day 92 p.i. Transmission electron microscopy of negatively stained skin biopsies detected LSD virus only in one of the four bulls that developed skin lesions until Day 33 p.i. The PCR was a fast and sensitive method to demonstrate viral DNA in blood and skin samples. It could detect viral nucleic acid in skin lesions 53 days longer than virus isolation. Virus isolation from blood and skin samples was sensitive and reliable, but as a single test it may be too time-consuming to use although this depends on how rapidly the diagnosis must be confirmed. In conclusion, this study showed the PCR to be superior in detecting LSD virus from blood and skin samples. However, virus isolation is still required when the infectivity of the LSD virus is to be determined.

Partial 1D gene characterization was used to study phylogenetic relationships between 17 serotype O foot-and-mouth disease (FMD) viruses in Ethiopia as well as with other O-type isolates from Eritrea, Kenya, South and West Africa, the Middle East, Asia and South America. A homologous region of 495 bp corresponding to the C-terminus end of the 1D gene was used for phylogenetic analysis. This study described three lineages, viz. African/Middle East-Asia, Cathay and South American. Within lineage I, three topotypes were defined, viz. East and West Africa and the Middle East-Asia together with the South African isolate. The Ethiopian isolates clustered as part of topotype I, the East African topotype. Two clades (based on < 12 % nucleotide difference) A and B were identified within the East African isolates, with clade A being further classified into three significant branches, A1 (80% bootstrap support), A2 (89% bootstrap support) and A3 (94% bootstrap support). Clade B consisted of two Kenyan isolates. Within topotype I, the 17 Ethiopian isolates showed genetic heterogeneity between themselves with sequence differences ranging from 4.6-14 %. Lineage 2 and 3 could be equated to two significant topotypes, viz. Cathay and South America. Comparison of amino acid variability at the immunodominant sites between the vaccine strain (ETH/19/77) and other Ethiopian outbreak isolates revealed variations within these sites. These results encourage further work towards the reassessment of the type O vaccine strain currently being used in Ethiopia to provide protection against field variants of the virus.

Thirty-one viruses causing SAT-2 outbreaks in seven West African countries between 1974 and 1991, and four viruses representative of East and Central Africa were genetically characterized in this study. Four major viral lineages (I-IV) were identified by phylogenetic analysis of an homologous 480 nucleotide region corresponding to the C-terminus end of VP1. Lineage I comprised two West African genotypes with viruses clustering according to year of isolation rather than geographical origin. Lineage II was represented by viruses isolated between 1979 and 1983 in two neighbouring West African countries, Senegal and The Gambia. Viruses from Nigeria and Eritrea, representative of West and East Africa respectively, constituted lineage III, whilst lineage IV, comprising viruses from Central and East Africa, was regionally and genetically distinct. This study revealed that unrestricted animal movement in West Africa is a major factor in disease dissemination and has also provided the first indication of trans-regional virus transmission.

Low cheetah (Acinonyx jubatus) birth rates were observed for a long time in a captive breeding facility in which Salmonella, which was possibly present in contaminated beef, was isolated from still-born lion (Panthera leo) cubs. Salmonella, including 14 isolates of Salmonella serovar typhimurium and 19 isolates of Salmonella serovar muenchen, was subsequently isolated 47 times from 378 meat samples at the facility during a 13-mo period. Salmonella, including 26 isolates of S. serovar typhimurium, 10 of S. serovar muenchen, and 11 other serovars, also was isolated 54 times from 119 fecal samples. Only three plasmid profiles were identified in 59 S. typhimurium isolates from both meat and fecal samples. Although random-amplified polymorphic DNA fingerprinting using different primers in the polymerase chain reaction was able to distinguish between S. typhimurium and S. muenchen and to demonstrate similar chromosomal DNA fingerprints in some of the isolates from meat and feces, the results were not consistent enough to prove that the Salmonella in the feces originated from contaminated meat. However, the predominance of only two serovars in the meat fed to carnivores and in the feces of these animals suggests that the meat was the source of the Salmonella organisms in the feces.

In order to detect the prevalence of Cowdria ruminantium in the vector tick, Amblyomma hebraeum, free-living, unfed adult ticks were collected with the aid of pheromone/CO2 traps. Ticks were collected at the Rietgat communal grazing area, as well as in the southwestern Kruger National Park and in the Songimvelo Game Reserve, all located in heartwater-endemic areas of South Africa. The presence of C. ruminantium in these ticks was determined by polymerase chain reaction (PCR) analysis. Ticks from the Rietgat communal grazing area were assayed in 2 batches and 4.7% of the one and 11.3% of the other were positive for infection, while 5.7% of the ticks collected in the Kruger National Park and 25% in the Songimvelo Game Reserve were positive. These results support the contention that a vector-wildlife cycle of transmission of C. ruminantium, the cause of heartwater in domestic ruminants, can be maintained in the absence of the latter animals.

Conventional methods of ensuring the safety and soundness of cows' milk for human consumption, such as pasteurisation, are not always practical in poor socioeconomic conditions or in rural communities that lack modern amenities. Activation of lactoperoxidase (LP) system and souring of milk were investigated as potential alternative methods to sustain the safety of milk by inhibiting certain microorganisms with known pathogenic potential. The activation of the LP-system inhibited the growth of Staphylococcus aureus and Escherichia coli by the order of 2 log values. The inhibition of Brucella abortus was negligible. The replication of Coxiella burnetti in milk was not disturbed even after 17 h of LP-system activation at 20 degrees C, but the outcome of the LP-system treatment on Mycobacterium bovis could not be determined as the conventional culturing technique used to grow this organism did not allow full recovery. Souring inhibited the growth of S. aureus and E. coli also by the order of 2 log values. From the results obtained in this investigation are concluded that the activation of the LP-system and souring can be used to inhibit the growth of S. aureus and E. coli in cows' milk, thereby increasing its safety.

Intra-oral carcinoma is the third most common malignancy among men in developing countries, and carries a high mortality rate, particularly in Africa, where patients often present initially with lesions at an advanced stage. The present study was undertaken to determine the prevalence of human papillomavirus (HPV) DNA in oral squamous carcinoma in the west of the Northern Transvaal, an area where a large number of new cases has been diagnosed over the past few years. Paraffin blocks from 66 cases (51 men, 15 women; mean age 58.7 years) of oral squamous carcinoma were randomly selected. Blocks contained samples of both tumour and adjacent normal epithelium. The presence of HPV antigen was established by means of immunocytochemistry and HPV DNA by in situ hybridisation with radiolabelled probes for HPV-6, 11, 16 and 18. Immunocytochemistry for viral antigen was negative in all the specimens. HPV-18 was detected in normal epithelium adjacent to the tumour in one case only. It appears from our study that HPV is of limited importance in oral squamous cell carcinogenesis in the population studied.

A series of 10 oesophageal and 10 laryngeal squamous carcinomas was examined by means of immunocytochemistry and in situ DNA hybridisation to demonstrate human papillomavirus (HPV) infection. Changes in the epithelium adjacent to the carcinoma were found in 5 of 10 oesophageal and 7 of 10 laryngeal carcinomas. Viral antigens could not be detected with immunocytochemistry in any of the specimens. HPV 6, 11 and 16 were detected in 3 oesophageal specimens. In one of these, HPV 16 was detected in normal as well as malignant cells. HPV 7 was detected for the first time in a laryngeal carcinoma. Our results confirm previous reports of possible HPV involvement in the pathogenesis of aerodigestive carcinomas.

Rotavirus infections in a nursery ward for newborn white infants in South Africa do not apparently differ from those encountered in other temperate countries. Neonates began to excrete rotavirus as early as 2 days after birth; a peak incidence of excretion occurred during the winter months. After a period of 4 years the incidence of excretion remained virtually unchanged. Rotaviruses also colonized and spread very rapidly in a newly commissioned nursery ward, possibly via respiratory infections. Passively acquired antibodies had no influence on the prevention of infections but may play a role in the suppression of clinical symptoms since none of the newborn population studied developed gastro-enteritis. The immunosorbent electron microscopy technique used to detect rotaviruses in this study yielded results that compared well with results obtained previously by other techniques.