Phylogenetic Characterisation of the Full Genome of a Bagaza Virus Isolate from Bird Fatalities in South Africa.

Bagaza virus (BAGV), a member of the Ntaya serogroup in the Flavivirus genus of the Flaviviridae, was isolated from the brain tissue of a Himalayan monal pheasant that died following neurological signs in Pretoria, South Africa in 2016. Next-generation sequencing was carried out on this isolate resulting in a genome sequence of 10980nt. The full genome sequence of this isolate, designated ZRU96-16, shared 98% nucleotide identity with a BAGV isolate found in Culex univitattus mosquitoes from Namibia and 97% nucleotide identity with a Spanish BAGV sequence isolated from an infected partridge. In total, seven amino acid variations were unique to ZRU96-16 after alignment with other BAGV and Israel turkey meningoencephalomyelitis (ITV) genomes. The 3'UTR sequence of ZRU96-16 was resolved with sufficient detail to be able to annotate the variable and conserved sequence elements within this region. Multiple sequence alignment of the 3'UTR suggested that it could be useful in lineage designation as more similar viruses carried similar mutations across this region, while also retaining certain unique sites. Maximum likelihood phylogenetic analysis revealed two clusters containing both BAGV and ITVs from Europe, the Middle East and Africa. Broadly, temporal clustering separated isolates into two groups, with one cluster representing viruses from the 1960-2000's and the other from 2010 onwards. This suggests that there is consistent exchange of BAGV and ITV between Europe and Africa. This investigation provides more information on the phylogenetics of an under-represented member of the Flaviviridae and provides an avenue for more extensive research on its pathogenesis and geographic expansion.

IDEAL, the Infectious Diseases of East African Livestock project open access database and biobank.

The Infectious Diseases of East African Livestock (IDEAL) project was a longitudinal cohort study of calf health which was conducted in Western Kenya between 2007-2010. A total of 548 East African shorthorn zebu calves were recruited at birth and followed at least every 5 weeks during the first year of life. Comprehensive clinical and epidemiological data, blood and tissue samples were collected at every visit. These samples were screened for over 100 different pathogens or infectious exposures, using a range of diagnostic methods. This manuscript describes this comprehensive dataset and bio-repository, and how to access it through a single online site ( http://data.ctlgh.org/ideal/ ). This provides extensive filtering and searching capabilities. These data are useful to illustrate outcomes of multiple infections on health, investigate patterns of morbidity and mortality due to parasite infections, and to study genotypic determinants of immunity and disease.

Bagaza Virus in Himalayan Monal Pheasants, South Africa, 2016-2017.

Bagaza virus (BAGV) has not been reported in birds in South Africa since 1978. We used phylogenetic analysis and electron microscopy to identify BAGV as the likely etiology in neurologic disease and death in Himalayan monal pheasants in Pretoria, South Africa. Our results suggest circulation of BAGV in South Africa.

The impact of co-infections on the haematological profile of East African Short-horn Zebu calves.

The cumulative effect of co-infections between pathogen pairs on the haematological response of East African Short-horn Zebu calves is described. Using a longitudinal study design a stratified clustered random sample of newborn calves were recruited into the Infectious Diseases of East African Livestock (IDEAL) study and monitored at 5-weekly intervals until 51 weeks of age. At each visit samples were collected and analysed to determine the infection status of each calf as well as their haematological response. The haematological parameters investigated included packed cell volume (PCV), white blood cell count (WBC) and platelet count (Plt). The pathogens of interest included tick-borne protozoa and rickettsias, trypanosomes and intestinal parasites. Generalized additive mixed-effect models were used to model the infectious status of pathogens against each haematological parameter, including significant interactions between pathogens. These models were further used to predict the cumulative effect of co-infecting pathogen pairs on each haematological parameter. The most significant decrease in PCV was found with co-infections of trypanosomes and strongyles. Strongyle infections also resulted in a significant decrease in WBC at a high infectious load. Trypanosomes were the major cause of thrombocytopenia. Platelet counts were also affected by interactions between tick-borne pathogens. Interactions between concomitant pathogens were found to complicate the prognosis and clinical presentation of infected calves and should be taken into consideration in any study that investigates disease under field conditions.

Sensitivity and specificity of real-time reverse transcription polymerase chain reaction, histopathology, and immunohistochemical labeling for the detection of Rift Valley fever virus in naturally infected cattle and sheep.

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), histopathology, and immunohistochemical labeling (IHC) were performed on liver specimens from 380 naturally infected cattle and sheep necropsied during the 2010 Rift Valley fever (RVF) epidemic in South Africa. Sensitivity (Se) and specificity (Sp) of real-time RT-PCR, histopathology, and IHC were estimated in a latent-class model using a Bayesian framework. The Se and Sp of real-time RT-PCR were estimated as 97.4% (95% confidence interval [CI] = 95.2-98.8%) and 71.7% (95% CI = 65-77.9%) respectively. The Se and Sp of histopathology were estimated as 94.6% (95% CI = 91-97.2%) and 92.3% (95% CI = 87.6-95.8%), respectively. The Se and Sp of IHC were estimated as 97.6% (95% CI = 93.9-99.8%) and 99.4% (95% CI = 96.9-100%), respectively. Decreased Sp of real-time RT-PCR was ascribed to cross-contamination of samples. Stratified analysis of the data suggested variations in test accuracy with fetuses and severely autolyzed specimens. The Sp of histopathology in fetuses (83%) was 9.3% lower than the sample population (92.3%). The Se of IHC decreased from 97.6% to 81.5% in the presence of severe autolysis. The diagnostic Se and Sp of histopathology was higher than expected, confirming the value of routine postmortem examinations and histopathology of liver specimens. Aborted fetuses, however, should be screened using a variety of tests in areas endemic for RVF, and results from severely autolyzed specimens should be interpreted with caution. The most feasible testing option for countries lacking suitably equipped laboratories seems to be routine histology in combination with IHC.

Seroprevalence of Rift Valley fever and lumpy skin disease in African buffalo (Syncerus caffer) in the Kruger National Park and Hluhluwe-iMfolozi Park, South Africa.

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.

Demonstration of lumpy skin disease virus infection in Amblyomma hebraeum and Rhipicephalus appendiculatus ticks using immunohistochemistry.

Lumpy skin disease (LSD) is caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus. Transmission of the virus has been associated with haematophagous insects such as Stomoxys calcitrans as well as Aedes and Culex species of mosquitoes. Recent studies have reported the transmission of the virus by Amblyomma hebraeum, Rhipicephalus appendiculatus, and Rhipicephalus decoloratus ticks and the presence of LSDV in saliva of A. hebraeum and R. appendiculatus ticks. The aim of this study was to determine which tick organs become infected by LSDV following intrastadial infection and transstadial persistence of the virus in A. hebraeum and R. appendiculatus ticks. Nymphal and adult ticks were orally infected by feeding them on LSDV-infected cattle. Partially fed adult ticks were processed for testing while nymphs were fed to repletion and allowed to moult to adults before being processed for testing. The infection in tick organs was determined by testing for the presence of the viral antigen using monoclonal antibodies with immunohistochemical staining. The viral antigen was detected in salivary glands, haemocytes, synganglia, ovaries, testes, fat bodies, and midgut. Since the virus was shown to be able to cross the midgut wall and infect various tick organs, this may indicate potential for biological development and transmission of LSDV in ticks. This study strengthens the previously reported evidence of the occurrence of LSDV in tick saliva.

Transovarial passage and transmission of LSDV by Amblyomma hebraeum, Rhipicephalus appendiculatus and Rhipicephalus decoloratus.

Lumpy skin disease (LSD), an acute, sub-acute or inapparent disease of cattle, is caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus in the family Poxviridae. LSD is characterised by high fever, formation of circumscribed skin lesions and ulcerative lesions on the mucous membranes of the mouth, respiratory and digestive tracts. It is an economically important disease due to the permanent damage to hides, the reduction in productivity and trade restrictions imposed on affected areas. Transmission has been associated with blood-feeding insects such as stable flies (Stomoxysis calcitrans) and mosquitoes (Aedes aegypti). Mechanical (intrastadial) and transstadial transmission by Amblyomma hebraeum and Rhipicephalus appendiculatus as well as transovarial transmission by R. decoloratus have been reported. In this study transovarial passage of LSDV to larvae and subsequent transmission to recipient animals were demonstrated. The finding of transovarial passage of LSDV in female ticks shows the potential for A. hebraeum, R. appendiculatus and R. decoloratus to be reservoir hosts for LSDV.

Evidence of vertical transmission of lumpy skin disease virus in Rhipicephalus decoloratus ticks.

Lumpy skin disease (LSD) is an economically important acute or sub-acute disease of cattle that occurs across Africa and in the Middle East. The aim of this study was to assess whether Rhipicephalus decoloratus ticks were able to transmit lumpy skin disease virus (LSDV) transovarially. Uninfected, laboratory-bred R. decoloratus larvae were placed to feed on experimentally infected "donor" cattle. After completion of the life cycle on donor animals, fully engorged adult female ticks were harvested and allowed to lay eggs. Larvae that hatched from these eggs were then transferred to feed on uninfected "recipient" cattle. The latter became viraemic and showed mild clinical disease with characteristic skin lesions and markedly enlarged precrural and subscapular lymph nodes. This is the first report of transovarial transmission of poxviruses by R. decoloratus ticks, and the importance of this mode of transmission in the spread of LSDV in endemic settings requires further investigation.

An investigation into an outbreak of Rift Valley fever on a cattle farm in Bela-Bela, South Africa, in 2008.

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.

Analyses of genes encoding Theileria parva p104 and polymorphic immunodominant molecule (PIM) reveal evidence of the presence of cattle-type alleles in the South African T. parva population.

Restriction fragment length polymorphism analysis of PCR products (PCR-RFLP) and sequencing of the variable region of the p104 and PIM genes was performed on samples obtained from South African T. parva parasites originating from cattle on farms with suspected theileriosis and from buffalo. p104 and PIM PCR-RFLP profiles similar to those of the T. parva Muguga stock, an isolate that causes ECF in Kenya, were obtained from three of seven cattle samples collected on a farm near Ladysmith in KwaZulu-Natal Province. Amino acid sequences of the p104 and PIM genes from two of these samples were almost identical to the T. parva Muguga p104 and PIM sequences. This result supports findings from a recent p67 study in which p67 alleles similar to those of the T. parva Muguga stock were identified from the same samples. While these results suggest the presence of a cattle-derived T. parva parasite, reports of cattle-to-cattle transmission could not be substantiated and ECF was not diagnosed on this farm. Although extensive diversity of p104 and PIM gene sequences from South African T. parva isolates was demonstrated, no sequences identical to known cattle-type p104 and PIM alleles were identified from any of the buffalo T. parva samples analyzed. 'Mixed' PIM alleles containing both cattle- and buffalo-type amino acid motifs were identified for the first time, and there appeared to be selection of cattle-type and 'mixed'-type PIM sequences in the cattle samples examined.

Four p67 alleles identified in South African Theileria parva field samples.

Previous studies characterizing the Theileria parva p67 gene in East Africa revealed two alleles. Cattle-derived isolates associated with East Coast fever (ECF) have a 129bp deletion in the central region of the p67 gene (allele 1), compared to buffalo-derived isolates with no deletion (allele 2). In South Africa, Corridor disease outbreaks occur if there is contact between infected buffalo and susceptible cattle in the presence of vector ticks. Although ECF was introduced into South Africa in the early 20th century, it has been eradicated and it is thought that there has been no cattle to cattle transmission of T. parva since. The variable region of the p67 gene was amplified and the gene sequences analyzed to characterize South African T. parva parasites that occur in buffalo, in cattle from farms where Corridor disease outbreaks were diagnosed and in experimentally infected cattle. Four p67 alleles were identified, including alleles 1 and 2 previously detected in East African cattle and buffalo, respectively, as well as two novel alleles, one with a different 174bp deletion (allele 3), the other with a similar sequence to allele 3 but with no deletion (allele 4). Sequence variants of allele 1 were obtained from field samples originating from both cattle and buffalo. Allele 1 was also obtained from a bovine that tested T. parva positive from a farm near Ladysmith in the KwaZulu-Natal Province. East Coast fever was not diagnosed on this farm, but the p67 sequence was identical to that of T. parva Muguga, an isolate that causes ECF in Kenya. Variants of allele 2 were obtained from all T. parva samples from both buffalo and cattle, except Lad 10 and Zam 5. Phylogenetic analysis revealed that alleles 3 and 4 are monophyletic and diverged early from the other alleles. These novel alleles were not identified from South African field samples collected from cattle; however allele 3, with a p67 sequence identical to those obtained in South African field samples from buffalo, was obtained from a Zambian field isolate of a naturally infected bovine diagnosed with ECF. The p67 genetic profiles appear to be more complex than previously thought and cannot be used to distinguish between cattle- and buffalo-derived T. parva isolates in South Africa. The significance of the different p67 alleles, particularly the novel variants, in the epidemiology of theileriosis in South Africa still needs to be determined.

Low cross-reactivity of T-cell responses against lipids from Mycobacterium bovis and M. avium paratuberculosis during natural infection.

Although CD1 proteins are known to present mycobacterial lipid antigens to T cells, there is little understanding of the in vivo behavior of T cells restricted by CD1a, CD1b and CD1c, and the relative immunogenicity and immunodominance of individual lipids within the total array of lipids that comprise a bacterium. Because bovines express multiple CD1 proteins and are natural hosts of Mycobacterium bovis and Mycobacterium avium paratuberculosis (MAP), we used them as a new animal model of CD1 function. Here, we report the surprisingly divergent responses against lipids produced by these two pathogens during infection. Despite considerable overlap in lipid content, only three out of 69 animals cross-react with M. bovis and MAP total lipid preparations. The unidentified immunodominant compound of M. bovis is a hydrophilic compound, whereas the immunodominant lipid of MAP is presented by CD1b and was identified as glucose monomycolate (GMM). The preferential recognition of GMM antigen by MAP-infected cattle may be explained by the higher expression of GMM by MAP than by M. bovis. The bacterial species-specific nature of the CD1-restricted, adaptive T-cell response affects the approach to development of lipid based immunodiagnostic tests.

Development and evaluation of a real-time polymerase chain reaction test for the detection of Theileria parva infections in Cape buffalo (Syncerus caffer) and cattle.

Corridor disease, caused by the tick-borne protozoan parasite Theileria parva, is a controlled disease in South Africa. The Cape buffalo is the reservoir host and uninfected buffalo have become sought-after by the game industry in South Africa, particularly for introduction into Corridor disease-free areas. A real-time polymerase chain reaction (PCR) test for detection of T. parva DNA in buffalo and cattle was developed to improve the sensitivity and specificity of the official diagnostic test package in South Africa. Oligonucleotide primers and hybridization probes were designed based on the 18S ribosomal RNA (rRNA) gene. Amplification of control DNA using Theileria genus-specific primers resulted in detection of T. taurotragi and T. annulata, in addition to T. parva. A T. parva-specific forward primer was designed which eliminated amplification of all other Theileria species, except for Theileria sp. (buffalo); however only the T. parva product was detected by the T. parva-specific hybridization probe set. The real-time PCR assay requires less time to perform, is more sensitive than the other molecular assays previously used in T. parva diagnostics and can reliably detect the parasite in carrier animals with a piroplasm parasitaemia as low as 8.79 x 10(-4)%.