Evaluation of positive Rift Valley fever virus formalin-fixed paraffin embedded samples as a source of sequence data for retrospective phylogenetic analysis.

Rift Valley fever (RVF), caused by an arthropod borne Phlebovirus in the family Bunyaviridae, is a haemorrhagic disease that affects ruminants and humans. Due to the zoonotic nature of the virus, a biosafety level 3 laboratory is required for isolation of the virus. Fresh and frozen samples are the preferred sample type for isolation and acquisition of sequence data. However, these samples are scarce in addition to posing a health risk to laboratory personnel. Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are safe and readily available, however FFPE derived RNA is in most cases degraded and cross-linked in peptide bonds and it is unknown whether the sample type would be suitable as reference material for retrospective phylogenetic studies. A RT-PCR assay targeting a 490 nt portion of the structural GN glycoprotein encoding gene of the RVFV M-segment was applied to total RNA extracted from archived RVFV positive FFPE samples. Several attempts to obtain target amplicons were unsuccessful. FFPE samples were then analysed using next generation sequencing (NGS), i.e. Truseq® (Illumina) and sequenced on the Miseq® genome analyser (Illumina). Using reference mapping, gapped virus sequence data of varying degrees of shallow depth was aligned to a reference sequence. However, the NGS did not yield long enough contigs that consistently covered the same genome regions in all samples to allow phylogenetic analysis.

Review: Capripoxvirus Diseases: Current Status and Opportunities for Control.

Lumpy skin disease, sheeppox and goatpox are high-impact diseases of domestic ruminants with a devastating effect on cattle, sheep and goat farming industries in endemic regions. In this article, we review the current geographical distribution, economic impact of an outbreak, epidemiology, transmission and immunity of capripoxvirus. The special focus of the article is to scrutinize the use of currently available vaccines to investigate the resource needs and challenges that will have to be overcome to improve disease control and eradication, and progress on the development of safer and more effective vaccines. In addition, field evaluation of the efficacy of the vaccines and the genomic database available for poxviruses are discussed.

Bacterial enteritis in ostrich (Struthio Camelus) chicks in the Western Cape Province, South Africa.

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.

Lumpy skin disease: attempted propagation in tick cell lines and presence of viral DNA in field ticks collected from naturally-infected cattle.

Lumpy skin disease (LSD) is of substantial economic importance for the cattle industry in Africa and the Near and Middle East. Several insect species are thought to transmit the disease mechanically. Recent transmission studies have demonstrated the first evidence for a role of hard (ixodid) ticks as vectors of lumpy skin disease virus (LSDV). The aim of this study was to attempt in vitro growth of the virus in Rhipicephalus spp. tick cell lines and investigate in vivo the presence of the virus in ticks collected from cattle during LSD outbreaks in Egypt and South Africa. No evidence was obtained for replication of LSDV in tick cell lines although the virus was remarkably stable, remaining viable for 35 days at 28°C in tick cell cultures, in growth medium used for tick cells and in phosphate buffered saline. Viral DNA was detected in two-thirds of the 56 field ticks, making this the first report of the presence of potentially virulent LSDV in ticks collected from naturally infected animals.

Evidence of transstadial and mechanical transmission of lumpy skin disease virus by Amblyomma hebraeum ticks.

Lumpy skin disease (LSD) is an economically important disease caused by LSD virus (LSDV), a Capripoxvirus, characterized by fever and circumscribed skin lesions. It is suspected to be transmitted mechanically by biting flies. To assess the vector potential of Amblyomma hebraeum in transmission of LSDV, mechanical/intrastadial and transstadial modes of transmission of the virus by this tick species were investigated. Two cattle were artificially infected as sources (donors) of infection to ticks. Ticks were infected as either nymphs or adults. Male A. hebraeum ticks were partially fed on donor animals and transferred to recipient animals to test for mechanical/intrastadial transmission. Nymphal A. hebraeum were fed to repletion on donor animals. The emergent adult ticks were placed on recipient animals to test for transstadial transmission of the virus. Successful transmission of LSDV infection was determined in recipient animals by monitoring development of clinical signs, testing of blood for the presence of LSDV by real-time PCR, virus isolation and the serum neutralization test. This report provides further evidence of mechanical/intrastadial and, for the first time, transstadial transmission of LSDV by A. hebraeum. These findings implicate A. hebraeum as a possible maintenance host in the epidemiology of the disease.

An improved method for determining virucidal efficacy of a chemical disinfectant using an electrical impedance assay.

A major problem with the testing of virucidal efficacy using current protocols is that scoring of virus-induced cytopathic effect (CPE) is dependent on subjective visual interpretation using light microscopy. The current report details the use of an electrical impedance assay (xCELLigence, ACEA Biosciences) for its utility in virucidal efficacy testing. In this study, the xCELLigence system was used in a procedure developed from guidelines given by the Deutsche Vereiniging zur Bekämpfung der Viruskrankheiten (DVV) (German Association for the Control of Virus Diseases) in order to demonstrate the inactivation of infectious bursal disease virus using a commercial virucide. Although the modified DVV assay using the xCELLigence system yielded identical results (i.e. a 5-log10 reduction in viral infectivity) as the traditional DVV assay, the system allows virucidal efficacy and cytotoxicity to be measured in a more precise and reproducible fashion.

Seminal transmission of lumpy skin disease virus in heifers.

It is known that lumpy skin disease virus (LSDV) can be shed in bull semen following infection and also that artificial insemination (AI) poses a biosecurity risk. However, it is not known whether the use of LSDV infected semen in AI poses a biosecurity risk. The aim of this study was to investigate whether LSDV, transmitted through semen, can infect cows and their embryos. Two controlled trials were performed simultaneously. Eleven young beef heifers, naïve to LSDV, were synchronized using an OvSynch protocol and inseminated on Day 0 with fresh semen spiked with a field strain of LSDV on day 0. Six of the heifers were superovulated on Day 1 using pregnant mare serum gonadotropin, and embryos were flushed from these heifers on Day 6. Blood and serum samples were collected from Day 4 until Day 27 to determine the presence of LSDV by PCR and virus isolation, and the presence of antibodies against LSDV by SNT. The first clinical signs of LSD were noticed on Day 10, followed by severe generalized LSD in three heifers and mild LSD in two more heifers. Two heifers were humanely euthanized due to severe unresponsive stranguria. LSDV was detected by PCR, virus isolation or electron microscopy in blood, embryos and organs of experimentally infected animals; and eight heifers had seroconverted by Day 27. Two control animals were not affected. This is the first report of experimental seminal transmission of LSDV in cattle.

Evidence of lumpy skin disease virus over-wintering by transstadial persistence in Amblyomma hebraeum and transovarial persistence in Rhipicephalus decoloratus ticks.

Lumpy skin disease is a debilitating cattle disease caused by the lumpy skin disease virus (LSDV), belonging to the genus Capripoxvirus. Epidemics of the disease usually occur in summer, when insect activity is high. Limited information is available on how LSDV persists during inter-epidemic periods. Transmission of LSDV by mosquitoes such as Aedes aegypti has been shown to be mechanical, there is no carrier state in cattle and the role of wildlife in the epidemiology of the disease seems to be of minor importance. Recent studies in ticks have shown transstadial persistence of LSDV in Rhipicephalus appendiculatus and Amblyomma hebraeum as well as transovarial persistence of the virus in Rhipicephalus decoloratus, R. appendiculatus and A. hebraeum. The over-wintering of ticks off the host as part of their life cycles is well known: A. hebraeum and R. appendiculatus over-winter, for example, on the ground as engorged nymphs/unfed (emergent) adults while R. decoloratus over-winters on the ground as engorged females. In this study, transstadial and transovarial persistence of LSDV from experimentally infected A. hebraeum nymphs and R. decoloratus females after exposure to cold temperatures of 5 °C at night and 20 °C during the day for 2 months was reported. This observation suggests possible over-wintering of the virus in these tick species.

Detection of lumpy skin disease virus in saliva of ticks fed on lumpy skin disease virus-infected cattle.

Lumpy skin disease is an economically important disease of cattle that is caused by the lumpy skin disease virus (LSDV), which belongs to the genus Capripoxvirus. It is endemic in Africa and outbreaks have also been reported in the Middle-East. Transmission has mostly been associated with blood-feeding insects but recently, the authors have demonstrated mechanical transmission by Rhipicephalus appendiculatus as well as mechanical/intrastadial and transstadial transmission by Amblyomma hebraeum. Saliva is the medium of transmission of pathogens transmitted by biting arthropods and, simultaneously, it potentiates infection in the vertebrate host. This study aimed to detect LSDV in saliva of A. hebraeum and R. appendiculatus adult ticks fed, as nymphs or as adults, on LSDV-infected animals, thereby also demonstrating transstadial or mechanical/intrastadial passage of the virus in these ticks. Saliva samples were tested for LSDV by real-time PCR and virus isolation. Supernatants obtained from virus isolation were further tested by real-time PCR to confirm that the cytopathic effects observed were due to LSDV. Lumpy skin disease virus was detected, for the first time, in saliva samples of both A. hebraeum and R. appendiculatus ticks. At the same time, mechanical/intrastadial and transstadial passage of the virus was demonstrated and confirmed in R. appendiculatus and A. hebraeum.

Mechanical transmission of lumpy skin disease virus by Rhipicephalus appendiculatus male ticks.

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.

Genetic characterization of bovine viral diarrhoea (BVD) viruses: confirmation of the presence of BVD genotype 2 in Africa.

Bovine viral diarrhoea virus (BVDV) has emerged as one of the economically important pathogens in cattle populations, with a worldwide distribution and causing a complex of disease syndromes. Two genotypes, BVDV 1 and 2, exist and are discriminated on the basis of the sequence of the 5' non-coding region (5' NCR) using real-time PCR. Real-time PCR is more sensitive, specific, and less time-consuming than conventional PCR, and it has less risk of cross-contamination of samples. Limited information exists on BVDV genetic subtypes in South Africa. The aim of this study was to determine the genotypes of BVDV currently circulating in South African feedlots. A total of 279 specimens (219 tissue samples, 59 trans-tracheal aspirates and 1 blood sample) were collected from dead and living cattle with lesions or clinical signs compatible with BVDV infection. Pooled homogenates from the same animals were prepared, and total RNA was extracted. A screening test was performed on the pooled samples, and positive pools were investigated individually. A Cador BVDV Type 1/2 RT-PCR Kit (QIAGEN, Hilden, Germany) was used for the real-time PCR assay on a LightCycler(®) V2.0 real-time PCR machine (Roche Diagnostics, Mannheim, Germany). The results were read at 530 and 640 nm for BVDV 1 and 2, respectively. Bovine viral diarrhoea virus was detected in a total of 103 samples that included 91 tissue samples, 1 blood sample and 11 trans-tracheal aspirates. Eighty-five (82.5 %) of the strains were genotype 1 and 18 (17.5 %) were genotype 2. Comparing the sequencing data, genotypes 1 and 2 from the field strains did not cluster with vaccine strains currently used in feedlots in South Africa. The present study revealed the presence of BVDV genotype 2 in cattle in South Africa based on the high sequence similarity between genotype 2 field strains and strain 890 from North America. The presence of genotype 2 viruses that phylogenetically belong to different clusters and coexist in feedlots is consistent with the possibility of multiple virus introductions. These results represent the first documented evidence for the presence of BVDV genotype 2 in African cattle.

Detection and characterisation of papillomavirus in skin lesions of giraffe and sable antelope in South Africa.

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.

Molecular epidemiology of human and animal tuberculosis in Ibadan, Southwestern Nigeria.

From 2005 to 2007, Mycobacterium tuberculosis complex (MTC) strains were isolated from cattle, goats and pigs samples collected at the Bodija abattoir and from human samples from tuberculosis patients and livestock traders at the Akinyele cattle market in Ibadan, Southwestern Nigeria. Seventy four isolates obtained from humans (24) and livestock (50) were identified as MTC strains. Thirty two isolates were spoligotyped. Nineteen of these 32 isolates were identified as M. tuberculosis whilst 13 were identified as Mycobacterium bovis. M. bovis was isolated from two humans, whereas M. tuberculosis was isolated from a bovine, a pig and a goat. All the M. bovis isolates identified in this study belonged to the Africa 1 clonal complex. Multiple locus VNTR [variable number of tandem repeats] analysis (MLVA) was carried out on the 74 isolates. Three major clusters were defined. Group A consisted of 24 M. tuberculosis isolates (MLVA genotypes 1-18). One strain was isolated from a bovine and one from a pig. Group B consisted of 49 M. bovis strains (MLVA genotypes 19-48), mainly of cattle origin but also included four goat, nine pig and two human isolates. Group C consisted of a single M. tuberculosis isolate (MLVA genotype 49) obtained from a goat. Spoligotyping and MLVA confirmed it as clustering with the East Africa Indian clade found in humans in Sudan and the Republic of Djibouti. The isolation of three M. tuberculosis strains from livestock raises the question of their epidemiological importance as a source of infection for humans.

A potential role for ixodid (hard) tick vectors in the transmission of lumpy skin disease virus in cattle.

Lumpy skin disease (LSD) is an economically important cattle disease. The disease is endemic in many African countries, but outbreaks have also been reported in Madagascar and the Middle East. The aim of this study was to investigate the potential role of ixodid (hard) ticks in the transmission of the disease. Cattle were infected with a virulent, South African field isolate of lumpy skin disease virus (LSDV). Three common African tick species (genera Rhipicephalus, Amblyomma and Rhipicephalus (Boophilus)) in different life cycle stages were fed on the infected animals during the viraemic stage and on skin lesions. Post-feeding, the partially fed male ticks were transferred to the skin of non-infected 'recipient' animals, while females were allowed to lay eggs that were then tested using the polymerase chain reaction (PCR) method and virus isolation. Nymphs were allowed to develop for 2-3 weeks after which time they were tested. The non-infected 'recipient' cattle were closely monitored, both skin and blood samples were tested using PCR and virus isolation, and serum samples were tested by the serum neutralization test. This is the first report showing molecular evidence of potential transmission of LSDV by ixodid ticks. The study showed evidence of transstadial and transovarial transmission of LSDV by R. (B.) decoloratus ticks and mechanical or intrastadial transmission by R. appendiculatus and A. hebraeum ticks.

Comparison of the capillary and agarose electrophoresis based multiple locus VNTR (variable number of tandem repeats) analysis (MLVA) on Mycobacterium bovis isolates.

Electrophoretic techniques that can be used for genotyping of bacterial pathogens ranges from manual, low-cost, agarose gels to high-throughput capillary electrophoresis sequencing machines. These two methods are currently employed in the electrophoresis of PCR products used in multiple locus VNTR (variable number of tandem repeats) analysis (MLVA), i.e. the agarose electrophoresis (AE) and the capillary electrophoresis (CE). Some authors have suggested that clusters generated by AE are less reliable than those generated by CE and that the latter is a more sensitive technique than the former when typing Mycobacterium tuberculosis complex (MTC) isolates. Because such a claim could have significant consequences for investigators in this field, a comparison was made on 19 Belgian Mycobacterium bovis strains which had previously been genotyped using CE VNTR analysis. The VNTR profiles of the CE VNTR analysis were compared with those obtained by AE VNTR analysis at 14 VNTR loci. Our results indicated that there were no differences in copy numbers at all loci tested when the copy numbers obtained by the AE VNTR analysis were compared with those obtained by CE VNTR analysis. The use of AE VNTR analysis in mycobacterial genotyping does not alter the sensitivity of the MLVA technique compared with the CE VNTR analysis. The AE VNTR can therefore be regarded as a viable alternative in moderately equipped laboratories that cannot afford the expensive equipment required for CE VNTR analysis and data obtained by AE VNTR analysis can be shared between laboratories which use the CE VNTR method.

Evaluation of PCR assays for the detection of Campylobacter fetus in bovine preputial scrapings and the identification of subspecies in South African field isolates.

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.

Sites of persistence of lumpy skin disease virus in the genital tract of experimentally infected bulls.

The objectives of this work were to determine the site of persistence of lumpy skin disease virus (LSDV) in bulls shedding the virus in semen for a period longer than 28 days, to determine if the virus is present in all fractions of semen and to study lesions that developed in the genital tract. Six serologically negative postpubertal bulls were experimentally infected with a virulent field isolate of LSDV. The polymerase chain reaction (PCR) was performed on sheath washes, vesicular fluid, supernatant and cell-rich fractions of semen from day 10 to day 26 postinfection (p.i.). Bulls that were positive by PCR on the whole semen sample collected on day 28 p.i. were slaughtered and tissue samples from their genital tracts submitted for histopathological evaluation, immunoperoxidase staining, virus isolation and PCR. Two of the bulls developed severe lumpy skin disease (LSD) and were found to be shedding viral DNA in their semen on day 28 p.i. Viral DNA was identified in all semen fractions from all bulls, but mostly from the cell-rich fraction and from the severely affected bulls. The PCR assay was positive on postmortem samples of testes and epididymides from the two severely affected bulls. Virus could be recovered from the testes of these two bulls and from the epididymis of one of them. Immunoperoxidase staining was positive for LSDV staining in sections of testes and epididymides exhibiting necrosis. This study suggests that the testis and epididymis are sites of persistence of LSDV in bulls shedding virus in semen for prolonged periods and revealed that viral DNA is present in all fractions of the ejaculate.

Detection of Anaplasma antibodies in wildlife and domestic species in wildlife-livestock interface areas of Kenya by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay.

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.

Experimental reproduction of severe bluetongue in sheep.

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.

Absence of lumpy skin disease virus in semen of vaccinated bulls following vaccination and subsequent experimental infection.

Twelve serologically negative bulls were used, six were vaccinated with a modified live LSD vaccine and six unvaccinated. All were then experimentally infected with a virulent field strain of LSDV. No clinical abnormality was detected following vaccination, and mild clinical signs were seen in four vaccinated bulls following challenge. Virus was not found in semen of vaccinated bulls. Two of the unvaccinated bulls developed severe LSD and four showed mild symptoms, all excreted the virus in the semen following challenge. This study confirmed the ability of LSD vaccination to prevent the excretion of LSDV in semen of vaccinated bulls.