Alan John Guthrie

Re-emergence of bluetongue, African horse sickness, and other Orbivirus diseases

Arthropod-transmitted viruses (Arboviruses) are important causes of disease in humans and animals, and it is proposed that climate change will increase the distribution and severity of arboviral diseases. Orbiviruses are the cause of important and apparently emerging arboviral diseases of livestock, including bluetongue virus (BTV), African horse sickness virus (AHSV), equine encephalosis virus (EEV), and epizootic hemorrhagic disease virus (EHDV) that are all transmitted by haematophagous Culicoides insects. Recent changes in the global distribution and nature of BTV infection have been especially dramatic, with spread of multiple serotypes of the virus throughout extensive portions of Europe and invasion of the south-eastern USA with previously exotic virus serotypes. Although climate change has been incriminated in the emergence of BTV infection of ungulates, the precise role of anthropogenic factors and the like is less certain. Similarly, although there have been somewhat less dramatic recent alterations in the distribution of EHDV, AHSV, and EEV, it is not yet clear what the future holds in terms of these diseases, nor of other potentially important but poorly characterized Orbiviruses such as Peruvian horse sickness virus.

Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa

A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was conducted using samples collected from horses and zebra from different geographical locations in South Africa. A total of 488 samples were tested for the presence of Theileria equi and/or Babesia caballi using the reverse line blot hybridization assay. Ten percent of the samples hybridized to the Theileria/Babesia genus-specific probe and not to the B. caballi or T. equi species-specific probes, suggesting the presence of a novel species or genotype. The small subunit of rRNA gene (18S; approximately 1600bp) was amplified and sequenced from 33 of these 488 samples. Sequences were compared with published sequences from the public sequence databases. Twelve distinct T. equi and six B. caballi 18S rRNA sequences were identified. Alignments demonstrated extensive sequence variation in the V4 hypervariable region of the 18S rRNA gene within T. equi. Sequence variation was also found in B. caballi 18S rRNA genes, although there was less variation than observed for T. equi. Phylogenetic analysis based on 18S rRNA gene sequences revealed three T. equi clades and two B. caballi clades in South Africa. The extent of sequence heterogeneity detected within T. equi and B. caballi 18S rRNA genes was unexpected since concerted evolution is thought to maintain homogeneity within repeated gene families, including rRNA genes, in eukaryotes. The findings reported here show that careful examination of variants of the 18S rRNA gene of T. equi and B. caballi is required prior to the development of molecular diagnostic tests to detect these parasites in horses. Species-specific probes must be in designed in regions of the gene that are both conserved within and unique to each species.

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.

Lateral transmission of equine arteritis virus among Lipizzaner stallions in South Africa

REASONS FOR PERFORMING STUDY A serological study conducted in 1995 revealed that 7 stallions at the Lipizzaner Centre, Gauteng, South Africa, were seropositive for antibody to equine arteritis virus (EAV). A Lipizzaner stallion imported into South Africa from Yugoslavia in 1981 had previously (1988) been confirmed to be an EAV carrier. Despite being placed under life-long breeding quarantine, EAV had been transmitted between stallions at the Lipizzaner Centre. OBJECTIVES To investigate the phylogenetic relationships between the strain of EAV shed in the semen of the original carrier stallion and strains recovered from the semen of 5 other stallions; and to investigate the means whereby lateral transmission of EAV occurred among 7 in-contact, nonbreeding stallions at the Centre. METHODS EAV was isolated from semen collected from the seropositive stallions using RK-13 cells. Viral RNA was reverse transcribed and amplified by polymerase chain reaction using ORF 5-specific primers, subjected to sequence and phylogenetic analysis. RESULTS Phylogenetic analysis of strains of EAV recovered from the semen of 6 persistently infected stallions confirmed that all viruses were closely related and probably derived from a common ancestor, i.e. the stallion imported from Yugoslavia. Lateral transmission subsequently occurred among 7 in-contact, nonbreeding stallions at the Centre. It is speculated that these stallions may have been exposed to virus from bedding or fomites contaminated with semen. CONCLUSIONS These data confirm that lateral transmission of EAV can occur from shedding stallions to susceptible, in-contact horses, including other stallions, which may become persistently infected with the virus. POTENTIAL RELEVANCE The findings are consistent with lateral spread of a single, unique strain of EAV among a group; and suggest that transmission of EAV may be initiated by infection of one or more stallions with virus on bedding or other fomites contaminated with EAV- infected semen.

Efficacy of furosemide for prevention of exercise-induced pulmonary hemorrhage in Thoroughbred racehorses

OBJECTIVE To evaluate the efficacy of furosemide for prevention of exercise-induced pulmonary hemorrhage (EIPH) in Thoroughbred racehorses under typical racing conditions. DESIGN Randomized, placebo-controlled, blinded, crossover field trial. ANIMALS 167 Thoroughbred racehorses. PROCEDURES Horses were allocated to race fields of 9 to 16 horses each and raced twice, 1 week apart, with each of the 2 races consisting of the same race field and distance. Each horse received furosemide (500 mg, IV) before one race and a placebo (saline solution) before the other, with the order of treatments randomly determined. Severity of EIPH was scored on a scale from 0 to 4 after each race by means of tracheobronchoscopy. Data were analyzed by means of various methods of multivariable logistic regression. RESULTS Horses were substantially more likely to develop EIPH (severity score >or= 1; odds ratio, 3.3 to 4.4) or moderate to severe EIPH (severity score >or= 2; odds ratio, 6.9 to 11.0) following administration of saline solution than following administration of furosemide. In addition, 81 of the 120 (67.5%) horses that had EIPH after administration of saline solution had a reduction in EIPH severity score of at least 1 when treated with furosemide. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that prerace administration of furosemide decreased the incidence and severity of EIPH in Thoroughbreds racing under typical conditions in South Africa.

Development and evaluation of real-time PCR assays for the quantitative detection of Babesia caballi and Theileria equi infections in horses from South Africa

A quantitative real-time polymerase chain reaction (qPCR) assay using a TaqMan minor groove binder (MGB) probe was developed for the detection of Babesia caballi infection in equids from South Africa. Nine previously published sequences of the V4 hypervariable region of the B. caballi 18S rRNA gene were used to design primers and probes to target unique, conserved regions. The B. caballi TaqMan MGB qPCR assay was shown to be efficient and specific. The detection limit, defined as the concentration at which 95% of positive samples can be detected, was determined to be 0.000114% parasitized erythrocytes (PE). We further evaluated a previously reported Theileria equi-specific qPCR assay and showed that it was able to detect the 12 T. equi 18S rRNA sequence variants previously identified in South Africa. Both qPCR assays were tested on samples from two ponies experimentally infected with either T. equi or B. caballi. The qPCR assays were more sensitive than the indirect fluorescent antibody test (IFAT) and the reverse-line blot (RLB) during the early onset of the disease. The assays were subsequently tested on field samples collected from 41 horses, resident on three stud farms in the Northern Cape Province, South Africa. The IFAT detected circulating T. equi and B. caballi antibody in, respectively, 83% and 70% of the samples. The RLB detected T. equi parasite DNA in 73% of the samples, but none of the samples were positive for B. caballi, although 19 T. equi-positive samples also hybridized to the Babesia genus-specific probe. This could indicate a mixed T. equi and B. caballi infection in these samples, with either the B. caballi parasitaemia at a level below the detection limit of the B. caballi RLB probe, or the occurrence of a novel Babesia genotype or species. In contrast, the qPCR assays correlated fairly well with the IFAT. The B. caballi TaqMan MGB qPCR assay was able to detect B. caballi parasite DNA in 78% of the samples. The T. equi-specific qPCR assay could positively detect T. equi DNA in 80% of the samples. These results suggest that the qPCR assays are more sensitive than the RLB assay for the detection of T. equi and B. caballi infections in field samples.

An international parentage and identification panel for the domestic cat (Felis catus)

Seventeen commercial and research laboratories participated in two comparison tests under the auspices of the International Society for Animal Genetics to develop an internationally tested, microsatellite-based parentage and identification panel for the domestic cat (Felis catus). Genetic marker selection was based on the polymorphism information content and allele ranges from seven random-bred populations (n = 261) from the USA, Europe and Brazil and eight breeds (n = 200) from the USA. Nineteen microsatellite markers were included in the comparison test and genotyped across the samples. Based on robustness and efficiency, nine autosomal microsatellite markers were ultimately selected as a single multiplex ‘core’ panel for cat identification and parentage testing. Most markers contained dinucleotide repeats. In addition to the autosomal markers, the panel included two gender-specific markers, amelogenin and zinc-finger XY, which produced genotypes for both the X and Y chromosomes. This international cat parentage and identification panel has a power of exclusion comparable to panels used in other species, ranging from 90.08% to 99.79% across breeds and 99.47% to 99.87% in random-bred cat populations.

Helminth levels of working donkeys kept under different management systems in the Moretele 1 district of the North-West Province, South Africa.

In Southern Africa, where 150,000 working donkeys provide an important alternative to mechanisation in resource-poor communities, very little is known about their helminth status, or about the impact of helminths on their work output. The aim of this study was to investigate the helminth status of working donkeys under different management systems. Donkey owners in three different areas (one rural and two semi-rural) of the Moretele 1 district of North-West Province, South Africa, were visited and structured interviews were used to assess the management systems under which the donkeys were kept. Faecal samples were collected from 93 donkeys in the study once a month for 14 months. Faecal samples were analysed for nematode and trematode eggs and cultured to produce third-stage larvae for the identification of the nematode species. Final comparisons between management system subgroups, as well as between areas, age groups and sexes were made. Four management systems were identified. (1) The first system identified consisted of donkeys which were kept in a small yard at all times. They were fed hay but no supplementary food. (2) The second system consisted of donkeys which were allowed to roam freely around the village most of the time and rounded up and held in an enclosure when needed for work. (3) The third system was identical to the second management system except that the donkeys were given supplementary food during winter. (4) The fourth system was only found in the one area where each owner owned 10 ha of land and here the donkeys were allowed to roam freely on the owners land and brought into enclosures prior to working. Helminth species composition and faecal egg count numbers differed between these four systems. The main difference noted was that donkeys from management system one showed significantly higher numbers of strongyle eggs and higher percentages of some of the strongyle larvae. Management system two had a higher Strongyloides mean egg count and prevalence than the other areas. Parascaris and Gastrodiscus egg counts differed between all four systems. Since the results showed differences in the number and species of helminths in donkeys kept under the four management systems, suggestions are made as to which management system would facilitate reduction of helminth parasites in the animals. Although supplementary feeding in Moretele 1 is fairly rare, it would seem that donkeys which do have access to better food resources have lower egg counts than donkeys on limited grazing.

Comparison of two trapping methods for Culicoides biting midges and determination of African horse sickness virus prevalence in midge populations at Onderstepoort, South Africa

Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of a variety of pathogens including African horse sickness virus (AHSV), a member of the family Reoviridae, genus Orbivirus. AHSV causes African horse sickness (AHS), an endemic disease of equids with an extremely high mortality rate in horses in sub-Saharan Africa. Culicoides (Avaritia) imicola Kieffer is considered to be the principal vector of AHSV and is the dominant Culicoides species in South Africa. Due to the global distribution of Culicoides vectors, there is a potential risk of AHS spreading from endemic areas to areas traditionally free of the disease, which could have a severe economical impact on the affected equine industry. As part of any risk assessment it is essential to monitor known vectors as well as potential vector species. In the present study, sampling of Culicoides insects was compared using overnight collections in the conventional Onderstepoort light trap and mechanical aspiration of midges at sunset from bait horses. Culicoides imicola was confirmed as the predominant species using both trapping methods. Other species, mainly Culicoides (Avaritia) bolitinos Meiswinkel and Culicoides (Avaritia) gulbenkiani Caeiro, were highly underrepresented in the light trap collections, but made a significant contribution to the mechanical aspiration catches. The time for optimal collection differed between the trapping methods, leading to the conclusion that mechanical aspiration is a useful addition to conventional light trap collection and possibly the better choice when investigating insect vectors. An infection rate of 1.14% was calculated for the midge population based on real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays of collected Culicoides midges, which exceeds previous estimates. This is probably due to the increased sensitivity of the RT-qPCR assay used in this study as compared to the virus isolation assays used in previous studies. RT-qPCR-positive midges were present in midge pools obtained from both light trap and mechanical aspiration. Seven of the positive pools consisted of C. imicola only, four contained mixed species and one pool contained no C. imicola, suggesting the presence of AHSV in midges of other species.

West Nile virus infection of Thoroughbred horses in South Africa (2000-2001)

REASONS FOR PERFORMING STUDY West Nile virus (WNV) infection is endemic in southern Africa. With the recent emergence of WNV infection of horses in Europe and the USA the present study was performed to estimate the risk of seroconversion to WNV in a cohort of 488 young Thoroughbred (TB) horses. OBJECTIVES To estimate the risk of seroconversion to WNV among a cohort of South African TB yearlings sold at the 2001 National Yearling Sales (NYS) and to determine whether the risk varied geographically. Two horses were also infected with a recent South African isolate of WNV to evaluate its virulence in horses. METHODS Serum samples were collected from the cohort of 488 TB yearlings at the 2001 NYS. Serum samples that were collected from the same horses at the time that they were identified were sourced from our serum bank. Sera from 243 of the dams that were collected at the time that the foals were identified were also sourced from our serum bank. These sera were subjected to serum neutralisation (SN) tests for antibody to WNV. RESULTS Approximately 11% of yearlings seroconverted to WNV on paired serum samples collected from each animal approximately 12 months apart. Studfarms with WNV-seropositive yearlings were widely distributed throughout South Africa and SN tests on sera from their dams indicated that exposure to WNV was even more prevalent (75%) in this population. Neurological disease was not described in any of the horses included in this study and 2 horses inoculated with a recent lineage 2 South African isolate of WNV showed no clinical signs of disease after infection and virus was not detected in their blood. CONCLUSIONS Infection of horses with WNV is common in South Africa, but infection is not associated with neurological disease. POTENTIAL RELEVANCE In contrast to recent reports from Europe, North Africa, Asia and North America, the results of our field and experimental studies indicated that exposure of horses to the endemic southern African strains of WNV was not associated with neurological disease.