Outbreaks Of African Horse Sickness Research Articles

Modelling bluetongue and African horse sickness vector (Culicoides spp.) distribution in the Western Cape in South Africa using random forest machine learning.

Culicoides biting midges exhibit a global spatial distribution and are the main vectors of several viruses of veterinary importance, including bluetongue (BT) and African horse sickness (AHS). Many environmental and anthropological factors contribute to their ability to live in a variety of habitats, which have the potential to change over the years as the climate changes. Therefore, as new habitats emerge, the risk for new introductions of these diseases of interest to occur increases. The aim of this study was to model distributions for two primary vectors for BT and AHS (Culicoides imicola and Culicoides bolitinos) using random forest (RF) machine learning and explore the relative importance of environmental and anthropological factors in a region of South Africa with frequent AHS and BT outbreaks. Culicoides capture data were collected between 1996 and 2022 across 171 different capture locations in the Western Cape. Predictor variables included climate-related variables (temperature, precipitation, humidity), environment-related variables (normalised difference vegetation index-NDVI, soil moisture) and farm-related variables (livestock densities). Random forest (RF) models were developed to explore the spatial distributions of C. imicola, C. bolitinos and a merged species map, where both competent vectors were combined. The maps were then compared to interpolation maps using the same capture data as well as historical locations of BT and AHS outbreaks. Overall, the RF models performed well with 75.02%, 61.6% and 74.01% variance explained for C. imicola, C. bolitinos and merged species models respectively. Cattle density was the most important predictor for C. imicola and water vapour pressure the most important for C. bolitinos. Compared to interpolation maps, the RF models had higher predictive power throughout most of the year when species were modelled individually; however, when merged, the interpolation maps performed better in all seasons except winter. Finally, midge densities did not show any conclusive correlation with BT or AHS outbreaks. This study yielded novel insight into the spatial abundance and drivers of abundance of competent vectors of BT and AHS. It also provided valuable data to inform mathematical models exploring disease outbreaks so that Culicoides-transmitted diseases in South Africa can be further analysed.

Molecular Identification of Culicoides Species and Host Preference Blood Meal in the African Horse Sickness Outbreak-Affected Area in Hua Hin District, Prachuap Khiri Khan Province, Thailand.

African horse sickness (AHS) was reported as an outbreak in Thailand in 2020. Hematophagous insects from the genus Culicoides are the suspected vector responsible for AHS transmission. Horses in Hua Hin district, Prachuab Khiri Khan province, Thailand, were affected and died from AHS in 2020. However, the potential Culicoides species and its host preference blood meal in the affected areas are unknown. To investigate the potential vectors of AHS, Culicoides were collected using ultraviolet light traps placed near horse stables. Six horse farms, including five farms with AHS history and one farm without AHS history, were included in this study. Morphological and molecular identification of the Culicoides species was performed. Polymerase chain reaction (PCR) targeting the cytochrome b oxidase I (COXI) gene for confirmation of the Culicoides species, identification of the prepronociceptin (PNOC) gene for host preference blood meal, and bidirectional sequencing were conducted. Consequently, 1008 female Culicoides were collected, consisting of 708 and 300 samples captured at positions A and B at a distance of <2 and >5 m from the horse, respectively. Twelve Culicoides species identified by morphology were noted, including C. oxystoma (71.92%), C. imicola (20.44%), C. actoni (2.28%), C. flavipunctatus (1.98%), C. asiana (0.99%), C. peregrinus (0.60%), C. huffi (0.60%), C. brevitarsis (0.40%), C. innoxius (0.30%), C. histrio (0.30%), C. minimus (0.10%), and C. geminus (0.10%). The PCR detection of the Culicoides COXI gene confirmed Culicoides species in 23 DNA samples. PCR targeting the PNOC gene revealed that the Culicoides collected in this study fed on Equus caballus (86.25%), Canis lupus familiaris (6.25%), Sus scrofa (3.75%), and Homo sapiens (3.75%) for their blood meal. Human blood was identified from two samples of C. oxystoma and a sample of C. imicola. Three dominant species including C. oxystoma, C. imicola, and C. actoni that were reported in the Hua Hin area prefer to feed on horse blood. Moreover, C. oxystoma, C. imicola, and C. bravatarsis also feed on canine blood. This study revealed the species of Culicoides in Hua Hin district, Thailand, after the AHS outbreak.

Addressing the recent outbreak of African horse sickness in Lagos, Nigeria.

Addressing the recent outbreak of African horse sickness in Lagos, Nigeria.

The impact of strategic ventilation adjustments on stress responses in horses housed full-time in a vector-protected barn during the African horse sickness outbreak in Thailand.

The severe outbreak of African horse sickness (AHS) in Thailand has forced horses to reside full-time inside barns that are covered by a small mesh net to prevent minuscule AHS insect vectors from gaining access. However, housing in the net-covered barn induces stress in horses, which compromises their welfare. Implementing strategic airflow adjustment while retaining the vector-protection characteristics has been proposed to help alleviate this problem. The present study aimed to investigate the effect of strategic ventilation adjustment on blood cortisol levels, heart rate and behaviour in horses in a vector-protected barn. Nine horses underwent two sequential stabling conditions: vector-protected barn housing and housing in a barn in which the air ventilation was explicitly adjusted. Heart rate was higher in the afternoon in horses housed in the barn without ventilation adjustment, whereas no change was observed in the barn with ventilation adjustment. The vector-protected housing increased the horses' behavioural scores. Blood cortisol level declined over time, and an earlier decrease was detected at 1400h in the barn with ventilation adjustment. Although airflow adjustment did not appear to statistically alter the stress response in horses during housing in the vector-protected barn, an earlier decline in cortisol level alongside an unchanged heart rate in horses during the day may indicate the positive impact of ventilation adjustment within the vector-protected barn. With limited options to reduce stress or discomfort in horses, this strategic protocol could, at least in part, be applied to managing horses' welfare during the AHS outbreak.

Sensitivity and specificity for African horse sickness antibodies detection using monovalent and polyvalent vaccine antigen-based dot blotting.

The immune responses of animals infected with African horse sickness (AHS) virus are determined by enzyme-linked immunosorbent assay (ELISA), complement fixation, and virus neutralization test. During the outbreaks of AHS in Thailand, the immune response after vaccination has been monitored using commercial test kits such as blocking ELISA, which are expensive imported products unavailable commercially in Thailand. This study aimed to assess the sensitivity and specificity of anti-AHS virus antibodies using dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine. A total of 186 horse sera, namely, 93 AHS-unvaccinated samples and 93 AHS-vaccinated samples, were used in this study. All sera underwent antibodies detection using commercial blocking ELISA and in-house dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine. The numbers of true positive, false positive, true negative, and false negative results in the dot blotting were compared with those in blocking ELISA and the sensitivity and specificity of dot blotting were assessed. For the monovalent antigen, there were 78, 19, 74, and 15 true positive, false positive, true negative, and false negative results, respectively, while for the polyvalent antigen, the corresponding numbers were 84, 34, 58, and 9. Meanwhile, the diagnostic sensitivity and specificity for monovalent antigen were 83.87% and 79.57%, respectively, but 90.32% and 62.37% for polyvalent antigen. Dot blotting for AHS antibodies detection using vaccine antigen showed high sensitivity and rather a high specificity compared with the findings with the commercial ELISA test kit. In countries where commercial ELISA test kits are not available and when the size of a serum sample is small, dot blotting could become a good alternative test given its advantages, including its simplicity, rapidity, and convenience. To the best of our knowledge, these findings are the first report on the use of dot blotting for detecting AHS antibodies in horses. In conclusion, monovalent antigen-based dot blotting could be used as a reliable alternative serodiagnostic test for monitoring AHS humoral immune response, especially in vaccinated horses.

Development of Differentiating Infected from Vaccinated Animals (DIVA) Real-Time PCR for African Horse Sickness Virus Serotype 1.

African horse sickness (AHS) is a highly infectious and often fatal disease caused by 9 serotypes of the orbivirus African horse sickness virus (AHSV). In March 2020, an AHS outbreak was reported in Thailand in which AHSV serotype 1 was identified as the causative agent. Trivalent live attenuated vaccines serotype 1, 3, and 4 were used in a targeted vaccination campaign within a 50-km radius surrounding the infected cases, which promptly controlled the spread of the disease. However, AHS-like symptoms in vaccinated horses required laboratory diagnostic methods to differentiate infected horses from vaccinated horses, especially for postvaccination surveillance. We describe a real-time reverse transcription PCR-based assay for rapid characterization of the affecting field strain. The development and validation of this assay should imbue confidence in differentiating AHS-vaccinated horses from nonvaccinated horses. This method should be applied to determining the epidemiology of AHSV in future outbreaks.

African horse sickness: an ancient disease for a current threat

African horse sickness (AHS) is a major arthropod-borne disease that causes significant losses in horses in sub-Saharan Africa. It is caused by the African horse sickness virus (AHSV), which is transmitted during a blood meal by Culicoides biting midges. The distribution of historical African culicoid vectors increases due to global warming. In addition, recent (Thailand, 2020) and earlier (Iberian Peninsula, 1965-66/1987-90) AHS outbreaks outside Africa demonstrate the adaptation of the virus to endogenous species in AHS-free regions, similar to what has been observed for bluetongue disease in recent decades. Therefore, many regions are considered at risk of introduction of AHS which could have important economic consequences for the equine industry. Overall, this prone the European Union to launch research programs to get better diagnostic and prophylactic tools.

The financial burden of African Horse Sickness: a case of the European Union trade ban on South Africa’s horse industry

ABSTRACT Globalisation and the increased movement of goods such as live animals and animal products across national borders can exacerbate the introduction and spread of diseases. This risk can be mitigated through adherence to trade control measures such as the Sanitary and Phytosanitary Measures (SPS) of the World Trade Organization (WTO). However, compliance with SPS measures usually results in additional production and trade costs. This paper applied cost–benefit analysis, using stochastic scenario analysis, to estimate the financial burden of SPS measures on exporting horses from South Africa to the European Union (EU). These measures were instituted following a ban on the direct export of horses from South Africa to the EU, triggered by outbreaks of African Horse Sickness (AHS) in the AHS Controlled Area in the Western Cape Province. Analysis revealed that compliance to existing SPS measures by exporting a horse via a third country is 1.67 times more costly than exporting directly to the EU. A strengthened public-private sector partnership is recommended to jointly identify the most efficient and effective ways to develop capacity for collaborative judicious investment in order to build a resilient horse industry thereby enabling employment creation and economic growth.

African Horse Sickness Virus Serotype 1 on Horse Farm, Thailand, 2020.

To investigate an outbreak of African horse sickness (AHS) on a horse farm in northeastern Thailand, we used whole-genome sequencing to detect and characterize the virus. The viruses belonged to serotype 1 and contained unique amino acids (95V,166S, 660I in virus capsid protein 2), suggesting a single virus introduction to Thailand.

Use of nanopore sequencing to characterize african horse sickness virus (AHSV) from the African horse sickness outbreak in thailand in 2020.

African horse sickness (AHS) is a highly infectious and deadly disease despite availability of vaccines. Molecular characterization of African horse sickness virus (AHSV) detected from the March 2020 Thailand outbreak was carried out by whole-genome sequencing using Nanopore with a Sequence-Independent Single Primer Amplification (SISPA) approach. Nucleotide sequence of the whole genome was compared with closest matching AHSV strains using phylogenetic analyses and the AHSV-1 virus shared high sequence identity with isolates from the same outbreak. Substitution analysis revealed non-synonymous and synonymous substitutions in the VP2 gene as compared to circulating South African strains. The use of sequencing technologies, such as Nanopore with SISPA, has enabled rapid detection, identification and detailed genetic characterization of the AHS virus for informed decision-making and implementation of disease control measures. Active genetic information sharing has also allowed emergence of AHSV to be better monitored on a global basis.

InInfluence of full-time housing in vector-protected facilities on equine cortisol levels, heart rate, and behavior during the African horse sickness outbreak in Thailand

ABSTRACT Horses were provided full-time housing in unfamiliar vector-protected facilities during the African horse sickness (AHS) outbreak in Thailand. This study aimed to investigate the impact of this housing arrangement on the equine stress response. Nine healthy horses were housed in both a traditional barn and a vector-protected barn. Equine behavior and stress response data were collected in association with the housing environment and time of day. The mean behavioral score of horses housed in the vector-protected barn was lower at night than during the day. In addition, the horses’ mean heart rate at night was lower than their heart rate during the day, irrespective of housing condition. Furthermore, although blood cortisol peaked at 6:00 AM and was lowest at 6:00 PM under both housing conditions, daily fluctuations in blood cortisol levels were correlated with changes in humidity and temperature in both environments. Finally, horses housed in the traditional barn exhibited earlier decreases in cortisol levels relative to the horses in the vector-protected barn. This result indicates that housing horses in vector-protected facilities may impose stress.

Safety and efficacy of inactivated African horse sickness (AHS) vaccine formulated with different adjuvants

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae causing African Horse Sickness (AHS) in equids with a mortality of about 95% in naïve horses. AHS causes serious losses in developing countries where horses play a central role in draft power and transportation. There are nine AHSV serotypes inducing no or low cross-neutralizing antibodies. AHSV is spread by biting Culicoides midges. AHS is endemic in sub-Saharan Africa, and a serious threat outside Africa, since Culicoides species in moderate climate conditions are spreading the closely related bluetongue virus. AHS outbreaks will be devastating for the equestrian industry in developed countries. Live-attenuated vaccines (LAVs) are licensed, marketed and in use in Africa. Their application is controversial with regard to safety issues. LAVs are not allowed in AHS-free countries. We here studied inactivated AHSV with different adjuvants in guinea pigs and horses. Subcutaneous and intramuscular vaccination were studied in horses. Local reactions were observed after prime and boost vaccination. In general, neutralizing antibodies (nAbs) titres were very low after prime vaccination, whereas boost vaccination resulted in high nAb titres for some adjuvants. Vaccinated horses were selected based on local reactions and nAb titres to study efficacy. Unfortunately, not all vaccinated horses survived virulent AHSV infection. Further, most survivors temporarily developed clinical signs and viremia. Further, the current prototype inactivated AHS vaccine is not suitable as emergency vaccine, because onset of protection is slow and requires boost vaccinations. On the other hand, inactivated AHS vaccine is completely safe with respect to virus spread, and incorporation of the DIVA principle based on NS3/NS3a serology and exploring a vaccine production platform for other serotypes is feasible. A superior adjuvant increasing the protective response without causing local reactions will be required to develop payable and acceptable inactivated AHS vaccines.

Outbreak of African horse sickness in Thailand, 2020.

African horse sickness was confirmed in horses in Thailand during March 2020. The virus was determined to belong to serotype 1 and is phylogenetically closely related to isolates from South Africa. This is the first incidence of African horse sickness occurring in South East Asia and of serotype 1 outside of Africa.

Update of the species checklist of Culicoides Latreille, 1809 biting midges (Diptera: Ceratopogonidae) of Morocco

BackgroundInvestigations of Culicoides fauna, including inventories, were carried out in Morocco at different periods after the country had faced major bluetongue and African horse sickness outbreaks. However, no comprehensive reference publication has provided a clear overview of the Culicoides species diversity. This study reviewed available data on Culicoides biting midge species in Morocco from 1968 to 2015 (published and grey literature in French and English) in order to revise the current checklist, in light of state of the art taxonomic and systematic knowledge, and confirmed the checklist with morphological and molecular identifications of specimens collected from the region of Rabat.MethodsLiterature related to Culicoides collections in Morocco was collated. Authors were contacted to obtain raw data and additional information for the collections. Fresh Culicoides material was collected and examined from two sites around Rabat, the capital of Morocco. Each collected individual was examined and morphologically identified, if possible, to the species level. In addition, molecular identification was performed to separate closely related species, to confirm difficult morphological identifications and to confirm new species records.ResultsA total of 6121 individuals of Culicoides spp. were collected and analyzed and at least 17 species were identified: C. cataneii/C. gejgelensis, C. circumscriptus, C. fagineus, C. festivipennis, C. imicola, C. jumineri, C. kingi, C. longipennis, C. montanus, C. newsteadi, C. obsoletus, C. paolae, C. parotti, C. puncticollis, C. sahariensis, C. scoticus and C. subfagineus. Seven species were confirmed using phylogenetic analyses. Two new species records for Morocco are reported: C. paolae and C. subfagineus.ConclusionsThe Moroccan fauna of Culicoides now includes 54 valid species. Further work would certainly increase this total, as one of the clades we identified was not affiliated to any described and valid species.

A field investigation of an African horse sickness outbreak in the controlled area of South Africa in 2016.

An outbreak of African horse sickness (AHS) caused by AHS virus type 1 occurred within the South African AHS surveillance zone during April and May 2016. The index case was detected by a private veterinarian through passive surveillance. There were 21 cases in total, which is relatively low compared to case totals during prior AHS outbreaks in the same region (and of the same AHS virus type) in 2004, 2011 and 2014. The affected proportion of horses on affected properties was 0.07 (95% CI 0.04, 0.11). Weather conditions were conducive to high midge activity immediately prior to the outbreak but midge numbers decreased rapidly with the advent of winter. The outbreak was localized, with 18 of the 21 cases occurring within 8km of the index property and the three remaining cases on two properties within 21km of the index property, with direction of spread consistent with wind-borne dispersion of infected midges. Control measures included implementation of a containment zone with movement restrictions on equids. The outbreak was attributed to a reversion to virulence of a live attenuated vaccine used extensively in South Africa. Outbreaks in the AHS control zones have a major detrimental impact on the direct export of horses from South Africa, notably to the European Union.

African horse sickness virus (AHSV) with a deletion of 77 amino acids in NS3/NS3a protein is not virulent and a safe promising AHS Disabled Infectious Single Animal (DISA) vaccine platform

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. Currently, nine serotypes have been defined showing limited cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African Horse Sickness (AHS) in equids with a mortality up to 95% in naïve domestic horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are competent vectors of closely related bluetongue virus. AHS outbreaks cause huge economic losses in developing countries. In the developed world, outbreaks will result in losses in the equestrian industry and will have an enormous emotional impact on owners of pet horses. Live-attenuated vaccine viruses (LAVs) have been developed, however, safety of these LAVs are questionable due to residual virulence, reversion to virulence, and risk on virulent variants by reassortment between LAVs or with field AHSV. Research aims vaccines with improved profiles. Reverse genetics has recently being developed for AHSV and has opened endless possibilities including development of AHS vaccine candidates, such as Disabled Infectious Single Animal (DISA) vaccine. Here, virulent AHSV5 was recovered and its high virulence was confirmed by experimental infection of ponies. ‘Synthetically derived’ virulent AHSV5 with an in-frame deletion of 77 amino acids codons in genome segment 10 encoding NS3/NS3a protein resulted in similar in vitro characteristics as published NS3/NS3a knockout mutants of LAV strain AHSV4LP. In contrast to its highly virulent ancestor virus, this deletion AHSV5 mutant (DISA5) was completely safe for ponies. Two vaccinations with DISA5 as well as two vaccinations with DISA vaccine based on LAV strain AHSV4LP showed protection against lethal homologous AHSV. More research is needed to further improve efficacy, to explore the AHS DISA vaccine platform for all nine serotypes, and to study the vaccine profile in more detail.

Sellers' Revisited: A Big Data Reassessment of Historical Outbreaks of Bluetongue and African Horse Sickness due to the Long-Distance Wind Dispersion of Culicoides Midges.

The possibility that outbreaks of bluetongue (BT) and African horse sickness (AHS) might occur via long-distance wind dispersion (LDWD) of their insect vector (Culicoides spp.) was proposed by R. F. Sellers in a series of papers published between 1977 and 1991. These investigated the role of LDWD by means of visual examination of the wind direction of synoptic weather charts. Based on the hypothesis that simple wind direction analysis, which does not allow for wind speed, might have led to spurious conclusions, we reanalyzed six of the outbreak scenarios described in Sellers’ papers. For this reanalysis, we used a custom-built Big Data application (“TAPPAS”) which couples a user-friendly web-interface with an established atmospheric dispersal model (“HYSPLIT”), thus enabling more sophisticated modeling than was possible when Sellers undertook his analyzes. For the two AHS outbreaks, there was strong support from our reanalysis of the role of LDWD for that in Spain (1966), and to a lesser degree, for the outbreak in Cyprus (1960). However, for the BT outbreaks, the reassessments were more complex, and for one of these (western Turkey, 1977) we could discount LDWD as the means of direct introduction of the virus. By contrast, while the outbreak in Cyprus (1977) showed LDWD was a possible means of introduction, there is an apparent inconsistency in that the outbreaks were localized while the dispersion events covered much of the island. For Portugal (1956), LDWD from Morocco on the dates suggested by Sellers is very unlikely to have been the pathway for introduction, and for the detection of serotype 2 in Florida (1982), LDWD from Cuba would require an assumption of a lengthy survival time of the midges in the air column. Except for western Turkey, the BT reanalyses show the limitation of LDWD modeling when used by itself, and indicates the need to integrate susceptible host population distribution (and other covariate) data into the modeling process. A further refinement, which will become increasingly important to assess LDWD, will be the use of virus and vector genome sequence data collected from potential source and the incursion sites.

The economic impact of Bluetongue and other orbiviruses in sub-Saharan Africa, with special reference to Southern Africa.

Bluetongue (BT) and African horse sickness (AHS) are considered the most important orbiviral diseases in Southern Africans countries. The general endemic status makes these diseases challenging to be quanti ed in terms of their economic impact. Using country reported data from BT and AHS outbreaks and cases, as well as international trade data, the economic impact of BT and AHS is evaluated on local, regional, and global scales. Local scale impact in the Southern African region is underestimated as shown by the underreporting of BT and AHS. Exceptions occur during epidemic cycles of the diseases and when the diseases impact regional animal movement and global trade, as in the case of AHS in South Africa. While BT is not directly implicated as a signi cant non-tari barrier for regional movement, there are unspeci ed clauses in import permits which refer to the 'OIE listed diseases' and the freedom thereof includes endemic diseases like BT. African horse sickness has a much more tangible regional and global economic impact because of movement restrictions within AHS control zones in South Africa and through international movement of horses from this country.

Foraging range of arthropods with veterinary interest: New insights for Afrotropical Culicoides biting midges (Diptera: Ceratopogonidae) using the ring method

The identification of blood meal source of arthropod vector species contributes to the understanding of host-vector-pathogen interactions. The aim of the current work was to identify blood meal source in Culicoides biting midge species, biological vectors of internationally important arboviruses of livestock and equids, using a new ecological approach. We examined the correlation between blood meal source identified in engorged Culicoides females collected in a suction light trap and the available vertebrate hosts along four rings (200, 500, 1000 and 2000m) centered at the trap site and described the foraging range of the three main vector species of veterinary interest present in the study area, Culicoides imicola, Culicoides kingi and Culicoides oxystoma. The study was performed in four sites localized in the Niayes region of Senegal (West Africa) where recent outbreaks of African horse sickness occurred. Blood meal source identification was carried out by species-specific multiplex PCRs with genomic DNA extracted from the abdomen of engorged females collected during nine night collections for twenty-six collections. The four most abundant hosts present in the studied area (horse, cattle, goat and sheep) were surveyed in each ring zone. The blood meal source varied according to Culicoides species and host availability in each site. C. oxystoma and C. imicola females mainly fed on horses readily available at 200m maximum from the trap location whereas females of C. kingi fed mainly on cattle, at variable distances from the traps (200 to 2000m). C. oxystoma may also feed on other vertebrates. We discuss the results in relation with the transmission of Culicoides-borne arboviruses and the species dispersion capacities.

Host preferences and circadian rhythm of Culicoides (Diptera: Ceratopogonidae), vectors of African horse sickness and bluetongue viruses in Senegal

African horse sickness- and bluetongue virus are orbiviruses transmitted by Culicoides biting midges (Diptera: Ceratopogonidae) to horses and to ruminants, respectively. Since the last epizootic outbreak of African horse sickness in 2007 in Senegal, extensive investigations have been undertaken to improve our knowledge on Culicoides species involved locally in the transmission of the virus. The purpose of this study was to compare and quantify the host preferences of potential vectors of these orbiviruses on horse and sheep and to study their circadian rhythm. We found that Culicoides oxystoma and species of the sub-genus Avaritia (Culicoides imicola, Culicoides bolitinos and Culicoides pseudopallidipennis) had a preference for horse when compared to sheep (the predicted ratio between horse and sheep was 80 for C. oxystoma and 26 for C. imicola), and were mostly crepuscular: C. oxystoma had continuous activity throughout the diel with peaks in numbers collected after sunrise and sunset, while C. imicola was mostly nocturnal with peak after sunset. Unexpectedly, species of the subgenus Lasiohelea was also collected during this study. This diurnal biting species was a nuisance pest for both animal species used as bait.