This study investigated the prevalence, risk factors and species composition of ticks, fleas and lice infesting dogs and cats in and around Hawassa in southern Ethiopia. In total, 200 dogs and 100 cats were examined from November 2008 to April 2009. Of the dogs and cats examined, 99.5% and 91.5%, respectively, were infested with one or more species of ticks, fleas or lice. The overall prevalence was higher in dogs than in cats. A total of six different species of ectoparasites were collected and identified from dogs, but only three species from cats. Ctenocephalides felis was the predominant species amongst the animals, with a prevalence of 82.9% on dogs and 67% on cats. Other prevalent species on dogs included Ctenocephalides canis (73.8%), Heterodoxus spiniger (4%), nymphs of Amblyomma spp. (3.5%), Pulex irritans (2.5%) and Haemaphysalis leachi (0.5%). C. canis (18%) and P. irritans (6%) were also found on cats. More female than male fleas and lice were observed. The study revealed that the prevalence of fleas, ticks and lice on dogs was not significantly different between male and female animals or between young and adult dogs. However, the prevalence of these ectoparasites was significantly higher in female than in male and in adult than in young cats. The study showed that the prevalence of ectoparasites on both dogs and cats was significantly higher on animals with flea allergy dermatitis (FAD) than those without FAD, and on animals with lesions on their skin compared with those without lesions.

This study investigated the prevalence, risk factors and species composition of ticks, fleas and lice infesting dogs and cats in and around Hawassa in southern Ethiopia. In total, 200 dogs and 100 cats were examined from November 2008 to April 2009. Of the dogs and cats examined, 99.5% and 91.5%, respectively, were infested with one or more species of ticks, fleas or lice. The overall prevalence was higher in dogs than in cats. A total of six different species of ectoparasites were collected and identified from dogs, but only three species from cats. Ctenocephalides felis was the predominant species amongst the animals, with a prevalence of 82.9% on dogs and 67% on cats. Other prevalent species on dogs included Ctenocephalides canis (73.8%), Heterodoxus spiniger (4%), nymphs of Amblyomma spp. (3.5%), Pulex irritans (2.5%) and Haemaphysalis leachi (0.5%). C. canis (18%) and P. irritans (6%) were also found on cats. More female than male fleas and lice were observed. The study revealed that the prevalence of fleas, ticks and lice on dogs was not significantly different between male and female animals or between young and adult dogs. However, the prevalence of these ectoparasites was significantly higher in female than in male and in adult than in young cats. The study showed that the prevalence of ectoparasites on both dogs and cats was significantly higher on animals with flea allergy dermatitis (FAD) than those without FAD, and on animals with lesions on their skin compared with those without lesions.

Injury due to burning is known to impact on coagulation and haemostasis by disturbing the coagulation cascade and is also associated with impaired fibrinolysis. Also, venous thrombosis, pulmonary embolism and hypercoagulability are common during thermal injury. Using a Wistar albino rat model, we investigated in this study whether burn injury affects the ultrastructure of the fibrin networks. A typical fibrin network will contain mostly major, thick fibres with minor, thin fibres distributed amongst them. We found that the clot architecture changes after burn injury, showing more prominent minor, thin fibres in a netted appearance. Also, the clot showed areas of matted fibrin. We suggest that the thrombotic events associated with burn injury are due to the thickened and netlike areas formed when thrombin activates the coagulation cascade. This is due to impaired fibrinolysis activities, causing the resulting fibrin clots not to be successfully disseminated. Small fragments of these netted, clumped areas may therefore break loose and lead to thrombotic events after burn injuries. The current study therefore provided morphological evidence for thrombotic events associated with burn injury.

Injury due to burning is known to impact on coagulation and haemostasis by disturbing the coagulation cascade and is also associated with impaired fibrinolysis. Also, venous thrombosis, pulmonary embolism and hypercoagulability are common during thermal injury. Using a Wistar albino rat model, we investigated in this study whether burn injury affects the ultrastructure of the fibrin networks. A typical fibrin network will contain mostly major, thick fibres with minor, thin fibres distributed amongst them. We found that the clot architecture changes after burn injury, showing more prominent minor, thin fibres in a netted appearance. Also, the clot showed areas of matted fibrin. We suggest that the thrombotic events associated with burn injury are due to the thickened and netlike areas formed when thrombin activates the coagulation cascade. This is due to impaired fibrinolysis activities, causing the resulting fibrin clots not to be successfully disseminated. Small fragments of these netted, clumped areas may therefore break loose and lead to thrombotic events after burn injuries. The current study therefore provided morphological evidence for thrombotic events associated with burn injury.

Many studies on the population dynamics of questing ticks on pastures and of parasitic ticks on cattle have been conducted. Few, however, have attempted to link the two in a single study. This study aimed to assess the population dynamics of questing ixodid ticks on pastures and of adult ticks on two breeds of cattle with different levels of susceptibility to tick infestation on the same pastures. Between January 2005 and December 2009 questing ixodid ticks were collected monthly from natural pastures at the Döhne Agricultural Development Institute and at the adjacent Campagna Production System in the Amahlathi District, Eastern Cape province, South Africa. Between February 2007 and January 2010 adult ticks were collected monthly from Bonsmara and Nguni cattle grazing these pastures. Ten tick species were collected from the pastures and 12 from the cattle. Significantly more questing larvae of Rhipicephalus appendiculatus, Rhipicephalus decoloratus, Rhipicephalus evertsi evertsi and Rhipicephalus microplus were recovered from the pastures grazed by Bonsmara cattle than from those grazed by Nguni cattle (p ≤ 0.05). Significantly more adult Hyalomma rufipes, Rhipicephalus follis, R. appendiculatus, R. decoloratus, R. evertsi evertsi and R. microplus were collected from the Bonsmara cattle than from the Nguni cattle (p ≤ 0.05). The study showed that Nguni cattle are less susceptible to tick infestation than are Bonsmara cattle and fewer questing ticks are collected from pastures grazed by Nguni cattle than by Bonsmara cattle.

Many studies on the population dynamics of questing ticks on pastures and of parasitic ticks on cattle have been conducted. Few, however, have attempted to link the two in a single study. This study aimed to assess the population dynamics of questing ixodid ticks on pastures and of adult ticks on two breeds of cattle with different levels of susceptibility to tick infestation on the same pastures. Between January 2005 and December 2009 questing ixodid ticks were collected monthly from natural pastures at the Döhne Agricultural Development Institute and at the adjacent Campagna Production System in the Amahlathi District, Eastern Cape province, South Africa. Between February 2007 and January 2010 adult ticks were collected monthly from Bonsmara and Nguni cattle grazing these pastures. Ten tick species were collected from the pastures and 12 from the cattle. Significantly more questing larvae of Rhipicephalus appendiculatus, Rhipicephalus decoloratus, Rhipicephalus evertsi evertsi and Rhipicephalus microplus were recovered from the pastures grazed by Bonsmara cattle than from those grazed by Nguni cattle (p ≤ 0.05). Significantly more adult Hyalomma rufipes, Rhipicephalus follis, R. appendiculatus, R. decoloratus, R. evertsi evertsi and R. microplus were collected from the Bonsmara cattle than from the Nguni cattle (p ≤ 0.05). The study showed that Nguni cattle are less susceptible to tick infestation than are Bonsmara cattle and fewer questing ticks are collected from pastures grazed by Nguni cattle than by Bonsmara cattle.

Bovine viral diarrhoea virus (BVDV) infection is an important viral infection affecting the cattle industry today. The prevalence of this infection in South African feedlots is unknown. Ear notch biopsies were collected from chronic poor doers and animals that appeared unthrifty upon entering feedlots, as well as animals entering the hospital pen with respiratory disease for the first time. A total of 1690 samples were collected: 1074 from the former category and 616 from the latter. A routine immunohistochemistry staining protocol showed that 49 animals tested positive, of which 43 (4%) came from the feedlot entry group and six (1%) from the hospitalised group. The prevalence of persistently infected cattle from this selected, nonrandom sample entering six large South African feedlots was found to be 2.9%, which is higher than the international rule of thumb that 0.5% of all cattle entering feedlots are persistently infected. There was no clear correlation between persistent infection and respiratory disease. Serum samples were also collected when possible and 10 positive cases were found. Results from enzyme-linked immunosorbent assays for antigen and antibody performed on these sera correlated well with those from the immunohistochemistry staining method in six cases, but in four cases the animals tested falsely positive owing to nonspecific staining. Immunohistochemistry staining on ear notch biopsies is thus a reliable diagnostic method to identify persistently infected animals with BVDV, but the pathologist should be aware of nonspecific positive staining.

Bovine viral diarrhoea virus (BVDV) infection is an important viral infection affecting the cattle industry today. The prevalence of this infection in South African feedlots is unknown. Ear notch biopsies were collected from chronic poor doers and animals that appeared unthrifty upon entering feedlots, as well as animals entering the hospital pen with respiratory disease for the first time. A total of 1690 samples were collected: 1074 from the former category and 616 from the latter. A routine immunohistochemistry staining protocol showed that 49 animals tested positive, of which 43 (4%) came from the feedlot entry group and six (1%) from the hospitalised group. The prevalence of persistently infected cattle from this selected, nonrandom sample entering six large South African feedlots was found to be 2.9%, which is higher than the international rule of thumb that 0.5% of all cattle entering feedlots are persistently infected. There was no clear correlation between persistent infection and respiratory disease. Serum samples were also collected when possible and 10 positive cases were found. Results from enzyme-linked immunosorbent assays for antigen and antibody performed on these sera correlated well with those from the immunohistochemistry staining method in six cases, but in four cases the animals tested falsely positive owing to nonspecific staining. Immunohistochemistry staining on ear notch biopsies is thus a reliable diagnostic method to identify persistently infected animals with BVDV, but the pathologist should be aware of nonspecific positive staining.

Despite many studies regarding tick ecology, limited information on long-term changes in tick populations exist. This study assessed the long-term population dynamics of the less frequently collected questing ixodid ticks in the Kruger National Park (KNP). From 1988 to 2002, monthly dragging of the vegetation was performed in three habitats (grassland, woodland and gully) at two sites in the KNP (Nhlowa Road, Landscape Zone 17, and Skukuza, Landscape Zone 4). Amblyomma marmoreum and Rhipicephalus evertsi evertsi were collected as larvae most commonly. Most A. marmoreum larvae were collected at Skukuza and numbers peaked from March to July. More R. evertsi evertsi larvae were collected at Nhlowa Road and numbers peaked in summer and in winter, while at Skukuza there was a single peak in spring. Haemaphysalis elliptica, Rhipicephalus simus and Rhipicephalus turanicus were collected as adults most commonly. More Ha. elliptica and R. turanicus were collected at Nhlowa Road than at Skukuza, while R. simus numbers from the two sites were approximately equal. Ha. elliptica were collected most often between February and June, and R. simus and R. turanicus during February and March. All three species were collected more frequently in gullies than in grassland or woodland. Their numbers increased in 1994/1995 following an eruption of rodents, the preferred hosts of the immature stages. The different host-seeking strategies of ticks largely determine the development stage at which they are likely to be collected during vegetation dragging and reflect a complex interaction between ticks, their hosts and the environment.

Despite many studies regarding tick ecology, limited information on long-term changes in tick populations exist. This study assessed the long-term population dynamics of the less frequently collected questing ixodid ticks in the Kruger National Park (KNP). From 1988 to 2002, monthly dragging of the vegetation was performed in three habitats (grassland, woodland and gully) at two sites in the KNP (Nhlowa Road, Landscape Zone 17, and Skukuza, Landscape Zone 4). Amblyomma marmoreum and Rhipicephalus evertsi evertsi were collected as larvae most commonly. Most A. marmoreum larvae were collected at Skukuza and numbers peaked from March to July. More R. evertsi evertsi larvae were collected at Nhlowa Road and numbers peaked in summer and in winter, while at Skukuza there was a single peak in spring. Haemaphysalis elliptica, Rhipicephalus simus and Rhipicephalus turanicus were collected as adults most commonly. More Ha. elliptica and R. turanicus were collected at Nhlowa Road than at Skukuza, while R. simus numbers from the two sites were approximately equal. Ha. elliptica were collected most often between February and June, and R. simus and R. turanicus during February and March. All three species were collected more frequently in gullies than in grassland or woodland. Their numbers increased in 1994/1995 following an eruption of rodents, the preferred hosts of the immature stages. The different host-seeking strategies of ticks largely determine the development stage at which they are likely to be collected during vegetation dragging and reflect a complex interaction between ticks, their hosts and the environment.

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 103.5 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.

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 103.5 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.

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.

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.

Free-range rural chickens (FRCs) dominate the poultry industry in developing countries and chickens are exposed to multi-host infections, including Newcastle disease virus (NDV). The knowledge about the characteristics of NDV from FRCs is limited. This study investigated the persistence, spread and risks of NDV from FRCs. NDV isolates (n = 21) from unvaccinated FRCs in Tanzania were characterised by conventional intracerebral pathogenicity index (ICPI) and sequence analysis of a partial region of the deduced fusion protein encompassing the cleavage site. Results showed that five isolates were screened as lentogenic, nine as mesogenic and six as velogenic. Phylogenetic analysis of the 21 isolates compared to reference sequences revealed three, four, nine and five isolates in genotypes 1, 2, 3c and 4a, respectively. Genotype 3c also included published sequences of Tanzanian isolates obtained from exotic birds and chicken isolates from Uganda. The analysis showed that NDV were persistently present among chicken populations and possibly spread through live chicken markets or migration of wild birds. Differences in amino acid sequences detected around the cleavage site separated the isolates in six types. However, cleavage site pattern could not fully differentiate mesogenic isolates from velogenic isolates.

Free-range rural chickens (FRCs) dominate the poultry industry in developing countries and chickens are exposed to multi-host infections, including Newcastle disease virus (NDV). The knowledge about the characteristics of NDV from FRCs is limited. This study investigated the persistence, spread and risks of NDV from FRCs. NDV isolates (n = 21) from unvaccinated FRCs in Tanzania were characterised by conventional intracerebral pathogenicity index (ICPI) and sequence analysis of a partial region of the deduced fusion protein encompassing the cleavage site. Results showed that five isolates were screened as lentogenic, nine as mesogenic and six as velogenic. Phylogenetic analysis of the 21 isolates compared to reference sequences revealed three, four, nine and five isolates in genotypes 1, 2, 3c and 4a, respectively. Genotype 3c also included published sequences of Tanzanian isolates obtained from exotic birds and chicken isolates from Uganda. The analysis showed that NDV were persistently present among chicken populations and possibly spread through live chicken markets or migration of wild birds. Differences in amino acid sequences detected around the cleavage site separated the isolates in six types. However, cleavage site pattern could not fully differentiate mesogenic isolates from velogenic isolates.

Several studies on ticks infesting small mammals, including elephant shrews, have been conducted in South Africa; however, these studies have included only a single four-toed elephant shrew and no hedgehogs. This study thus aimed to identify and quantify the ixodid ticks infesting four-toed elephant shrews and Southern African hedgehogs. Four-toed elephant shrews (Petrodromus tetradactylus) were trapped in dense shrub undergrowth in a nature reserve in north-eastern KwaZulu-Natal. They were separately housed, first in cages and later in glass terraria fitted with wire-mesh bases to allow detached ticks to fall through for collection. Southern African hedgehogs (Atelerix frontalis) were hand caught on a farm in the eastern region of the Northern Cape Province and all visible ticks were collected by means of tweezers while the animals were anaesthetised. The ticks from each animal were preserved separately in 70% ethanol for later identification and counting. The immature stages of five ixodid tick species were collected from the elephant shrews, of which Rhipicephalus muehlensi was the most common. It has not been recorded previously on any species of elephant shrew. Three ixodid tick species were collected from the hedgehogs. Large numbers of adult Haemaphysalis colesbergensis, which has not been encountered previously on hedgehogs, were collected from these animals. Four-toed elephant shrews are good hosts of the larvae and nymphs of R. muehlensi, and Southern African hedgehogs are good hosts of adult H. colesbergensis.

Several studies on ticks infesting small mammals, including elephant shrews, have been conducted in South Africa; however, these studies have included only a single four-toed elephant shrew and no hedgehogs. This study thus aimed to identify and quantify the ixodid ticks infesting four-toed elephant shrews and Southern African hedgehogs. Four-toed elephant shrews (Petrodromus tetradactylus) were trapped in dense shrub undergrowth in a nature reserve in north-eastern KwaZulu-Natal. They were separately housed, first in cages and later in glass terraria fitted with wire-mesh bases to allow detached ticks to fall through for collection. Southern African hedgehogs (Atelerix frontalis) were hand caught on a farm in the eastern region of the Northern Cape Province and all visible ticks were collected by means of tweezers while the animals were anaesthetised. The ticks from each animal were preserved separately in 70% ethanol for later identification and counting. The immature stages of five ixodid tick species were collected from the elephant shrews, of which Rhipicephalus muehlensi was the most common. It has not been recorded previously on any species of elephant shrew. Three ixodid tick species were collected from the hedgehogs. Large numbers of adult Haemaphysalis colesbergensis, which has not been encountered previously on hedgehogs, were collected from these animals. Four-toed elephant shrews are good hosts of the larvae and nymphs of R. muehlensi, and Southern African hedgehogs are good hosts of adult H. colesbergensis.

Despite its important role as vector for African horse sickness virus (AHSV), very little information is available on the dissemination of this virus in Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae). This study reports on the applicability of a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect AHSV in dissected midges. A total of 96 midges were fed on AHSV-infected blood, after which one test group was dissected into head/thorax and abdomen segments immediately after feeding and the other only after 10 days of incubation. The majority of the midges (96%) ingested the virus successfully and there was no significant difference between the virus concentration in the heads/thoraxes and the abdomens immediately after feeding. After incubation, virus was detected in 51% of the midges and it was confined to the abdomen in the majority of these. The fact that virus was detected only in the heads/thoraxes of four Culicoides midges after incubation suggests the presence of a mesenteronal escape barrier. Replication in the salivary glands was not shown. An increase of the mean virus concentration in the abdomen after incubation indicates localised viral replication. The real-time RT-qPCR is recommended for further studies investigating the replication and dissemination of AHSV in Culicoides midges.

Despite its important role as vector for African horse sickness virus (AHSV), very little information is available on the dissemination of this virus in Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae). This study reports on the applicability of a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect AHSV in dissected midges. A total of 96 midges were fed on AHSV-infected blood, after which one test group was dissected into head/thorax and abdomen segments immediately after feeding and the other only after 10 days of incubation. The majority of the midges (96%) ingested the virus successfully and there was no significant difference between the virus concentration in the heads/thoraxes and the abdomens immediately after feeding. After incubation, virus was detected in 51% of the midges and it was confined to the abdomen in the majority of these. The fact that virus was detected only in the heads/thoraxes of four Culicoides midges after incubation suggests the presence of a mesenteronal escape barrier. Replication in the salivary glands was not shown. An increase of the mean virus concentration in the abdomen after incubation indicates localised viral replication. The real-time RT-qPCR is recommended for further studies investigating the replication and dissemination of AHSV in Culicoides midges.

Despite its important role as vector for African horse sickness virus (AHSV), very little information is available on the dissemination of this virus in Culicoides (Avaritia) imicola Kieffer (Diptera: Ceratopogonidae). This study reports on the applicability of a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) to detect AHSV in dissected midges. A total of 96 midges were fed on AHSV-infected blood, after which one test group was dissected into head/thorax and abdomen segments immediately after feeding and the other only after 10 days of incubation. The majority of the midges (96%) ingested the virus successfully and there was no significant difference between the virus concentration in the heads/thoraxes and the abdomens immediately after feeding. After incubation, virus was detected in 51% of the midges and it was confined to the abdomen in the majority of these. The fact that virus was detected only in the heads/thoraxes of four Culicoides midges after incubation suggests the presence of a mesenteronal escape barrier. Replication in the salivary glands was not shown. An increase of the mean virus concentration in the abdomen after incubation indicates localised viral replication. The real-time RT-qPCR is recommended for further studies investigating the replication and dissemination of AHSV in Culicoides midges.