Establishment of a mouse allergy model for Culicoides (Diptera: Ceratopogonidae).

BACKGROUND: Culicoides is a genus of ubiquitous biting midges (Ceratopogonidae). Female midges have blood-sucking habit. They not only bite and harass humans and animals but also may be an important vector of disease transmission. Therefore, building an animal allergy model caused by Culicoides biting is very beneficial for studying its pathogenesis and exploring the therapeutic methods. MATERIAL AND METHOD: Kunming mice were used in this study to build the model and sensitised by two-step injection of midge extracts. Scratching behaviour and histological examination were used to check the immediate and delayed responses. Immunoglobulin E (IgE) and Immunoglobulin G (IgG) were detected using indirect enzyme-linked immunosorbent assay (ELISA) assay. Splenic cell proliferation and cytokine production were determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and ELISA assays. The response of cytokine gene expression to midge stimulation was analysed through quantitative real-time polymerase chain reaction (qPCR). RESULTS: Behavioural results revealed a significant increase in scratching frequency among the midge-sensitised animals (p < 0.05). Histological examination showed more inflammatory cytokine infiltration at the injection site of midge-sensitised mice comparing to the ones in the control group. The serum levels of IgE and IgG1 antibodies in the midge-sensitised group were significantly elevated (p < 0.05). After splenocytes were stimulated in vitro with midge extracts, the midge-sensitised group's splenocyte count significantly increased in comparison to the control group. The midge-sensitised group's qPCR data revealed a down-regulation of tumor necrosis factor alpha (TNF-α) expression and an increase in the expression of interleukin (IL)-4, IL-5, IL-10 and IL-13 but not in the control group (p < 0.05). CONCLUSIONS: In this study, an animal model of Culicoides-mouse sensitisation was successfully constructed using a two-step method. The mode of administration of the model was in good agreement with the natural immune pathway, and the immune response induced by the sensitisation of the model was similar to that produced by the bite of a midge.

Visceral Haemoproteus minutus Infection in a Major Mitchell's Cockatoo (Lophochroa leadbeateri).

A 1-year-old major Mitchell's cockatoo (Lophochroa leadbeateri) was presented for evaluation of weakness, diarrhea with undigested seeds in the droppings, and weight loss. Leukocytosis with severe heterophilia, monocytosis, and lymphocytosis was noted on the complete blood count. Altered plasma biochemical parameters included a slight increase in creatine kinase and mild hypoproteinemia. Two blood smears before and after 2 days of treatment revealed mild polychromasia and anisocytosis but no blood parasites. Radiographic and computed tomographic imaging of the cockatoo were helpful in identifying airsacculitis, pneumonia, and gastrointestinal motility disorders. The patient died 5 days after treatment for the presenting clinical problems. On the gross postmortem examination, dark red foci in the ventricular muscle layers and 1-3-mm white foci in the myocardium, opaque air sacs, and dark lungs were identified. Histopathologic examination of submitted tissue samples found severe granulomatous ventriculitis and myocarditis with intralesional Haemoproteus species megalomeronts. Qualitative polymerase chain reaction testing for the cytochrome b (cyt b) gene performed on pooled heart, liver, kidney, and intestinal tissues identified 99.5% homology to Haemoproteus minutus. This case report demonstrates the expansion of the geographic range of H minutus to France and potentially to Belgium, which may compromise breeding and conservation of Australian parrots living outdoors. Challenging diagnosis, rapid disease progression, and the absence of validated treatment protocols for psittacine patients suggest that the use of preventive measures to reduce the presence of insect vectors such as hippoboscid flies and biting midges (Culicoides) should be considered. Haemoproteus minutus should be considered and potentially screened by polymerase chain reaction testing on blood samples, especially in the case of highly susceptible avian species (eg, Australian parrots in Europe) that present with sudden weakness, heterophilic leukocytosis, and monocytosis associated with mild anemia.

Investigating the Role of African Horse Sickness Virus VP7 Protein Crystalline Particles on Virus Replication and Release.

African horse sickness is a deadly and highly infectious disease of equids, caused by African horse sickness virus (AHSV). AHSV is one of the most economically important members of the Orbivirus genus. AHSV is transmitted by the biting midge, Culicoides, and therefore replicates in both insect and mammalian cell types. Structural protein VP7 is a highly conserved major core protein of orbiviruses. Unlike any other orbivirus VP7, AHSV VP7 is highly insoluble and forms flat hexagonal crystalline particles of unknown function in AHSV-infected cells and when expressed in mammalian or insect cells. To examine the role of AHSV VP7 in virus replication, a plasmid-based reverse genetics system was used to generate a recombinant AHSV that does not form crystalline particles. We characterised the role of VP7 crystalline particle formation in AHSV replication in vitro and found that soluble VP7 interacted with viral proteins VP2 and NS2 similarly to wild-type VP7 during infection. Interestingly, soluble VP7 was found to form uncharacteristic tubule-like structures in infected cells which were confirmed to be as a result of unique VP7-NS1 colocalisation. Furthermore, it was found that VP7 crystalline particles play a role in AHSV release and yield. This work provides insight into the role of VP7 aggregation in AHSV cellular pathogenesis and contributes toward the understanding of the possible effects of viral protein aggregation in other human virus-borne diseases.

Essential oil spray reduces clinical signs of insect bite hypersensitivity in horses.

OBJECTIVE: To assess the efficacy of an herbal spray combining various essential oils, with a claim of mast cell stabilisation, antipruritic, anti-inflammatory, and insect repellent effects on the clinical presentation of insect bite hypersensitivity (IBH) in horses. DESIGN: Double-blinded, placebo-controlled, randomised, cross-over clinical trial. METHODS: Twenty adult horses with clinical IBH were treated with a daily application of herbal spray or placebo for 28 days in a randomised, cross-over fashion, separated by a>28-day washout period. Horses were examined and scored prior to and after the completion of each treatment. Histopathology was performed on four horses. Owners kept daily diaries of observations. RESULTS: The herbal spray significantly reduced the severity of all assessed parameters (pruritus, excoriations, lichenification and alopecia; P < 0.05) compared with baseline values (pretreatment) and with placebo. Owners reported improvement of pruritus in 19/20 horses (95%) with complete resolution in 17 horses (85%) following treatment. Skin biopsies showed resolution of orthokeratosis in 4/4 horses, reduced thickness of the stratum spinosum in 2/4 horses and complete resolution of histopathological abnormalities in 1/4 horses after treatment, compared with either no change or deterioration of histopathologic lesions after placebo. No side effects were observed. CONCLUSIONS: The tested herbal spray may be an effective treatment for the management of equine IBH.

Detection of genotype 1 bovine leukemia virus from a C.schultzei pool: Do Culicoides spp. have a role on the transmission of bovine leukemia virus?

Bovine leukemia virus (BLV) is known as the etiological agent of Enzootic bovine leukosis (EBL), which is the most common neoplastic disease of cattle. While the major route of virus transmission is believed to be iatrogenic, BLV proviral DNA has been identified in biological materials, including nasal secretions, saliva, milk, colostrum, and semen, and in several insect species, including horses flies. However, insects' role in the natural transmission of BLV has not been clearly demonstrated. This study assessed the possible role of midges - Culicoides spp. - in BLV transmission. BLVs were genetically characterized and BLV infection seroprevelance was determined in 224 cattle sampled from 27 different small family herds in five different districts in Hatay province, southern Turkey. Out of the 25 Culicoides spp. pools, one (4.0%; 1/25) was a C.schultzei pool while 2.67% (6/224) of the sampled cattle were positive for BLV nucleic acid. The seroprevalance rates for the sampled herds and all sampled cattle were 7.40% (2/27) and 1.33% (3/224), respectively. According to the phylogenetic analysis, the sequences of the BLVs from the cattle (n = 6) and the one BLV-positive C.schultzei pool clustered on genotype 1 (G1) BLVs. Although these results do not reveal the exact role of Culicoides spp. or other midges flies in BLV transmission, the simultaneous presence of same substitions in BLVs from both cattle and a C.schultzei pool is noteworthy. Further studies on the env gene and other BLV gene regions detected from cattle and C.schultzei pools are ongoing to understand the possible epidemiological relationship between cattle and flies.

Diversity of biting midges Culicoides (Diptera: Ceratopogonidae), potential vectors of disease, in different environments in an Amazonian rural settlement, Brazil

Abstract INTRODUCTION: The Culicoides transmit a variety of pathogens. Our aim was to survey the Culicoides species occurring in an Amazonian rural settlement, comparing abundance, richness, and diversity in different environments. METHODS: Culicoides were captured using CDC light traps. The Shannon-Wiener (H') and Rényi indices were used to compare species diversity and evenness between environments, the equitability (J') index was used to calculate the uniformity of distribution among species, and similarity was estimated using the Jaccard similarity index. A permutational multivariate analysis of variance was applied to assess the influence of environment on species composition. A non-metric dimensional scale was used to represent the diversity profiles of each environment in a multidimensional space. RESULTS: 6.078 Culicoides were captured, representing 84 species (45 valid species/39 morphotypes). H' values showed the following gradient: forest > capoeira > peridomicile > forest edge. The equitability J' was greater in capoeira and forests compared to peridomiciles and the forest edge. The population compositions of each environment differed statistically, but rarefaction estimates indicate that environments of the same type possessed similar levels of richness. Species of medical and veterinary importance were found primarily in peridomiciles: C. paraensis, vector of Oropouche virus; C. insignis and C. pusillus, vectors of Bluetongue virus; C. filariferus, C. flavivenula, C. foxi, and C. ignacioi, found carrying Leishmania DNA. CONCLUSIONS: This study indicates that diversity was higher in natural environments than in anthropized environments, while abundance and richness were highest in the most anthropized environment. These findings suggest that strictly wild Culicoides can adapt to anthropized environments.

Vector competence is strongly affected by a small deletion or point mutations in bluetongue virus.

BACKGROUND: Transmission of vector-borne virus by insects is a complex mechanism consisting of many different processes; viremia in the host, uptake, infection and dissemination in the vector, and delivery of virus during blood-feeding leading to infection of the susceptible host. Bluetongue virus (BTV) is the prototype vector-borne orbivirus (family Reoviridae). BTV serotypes 1-24 (typical BTVs) are transmitted by competent biting Culicoides midges and replicate in mammalian (BSR) and midge (KC) cells. Previously, we showed that genome segment 10 (S10) encoding NS3/NS3a protein is required for virus propagation in midges. BTV serotypes 25-27 (atypical BTVs) do not replicate in KC cells. Several distinct BTV26 genome segments cause this so-called 'differential virus replication' in vitro. METHODS: Virus strains were generated using reverse genetics and their growth was examined in vitro. The midge feeding model has been developed to study infection, replication and disseminations of virus in vivo. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV variants and propagation in the midge was examined using PCR testing. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies. RESULTS: A 100 nl blood meal containing ±105.3 TCID50/ml of BTV11 which corresponds to ±20 TCID50 infected 50% of fully engorged midges, and is named one Midge Alimentary Infective Dose (MAID50). BTV11 with a small in-frame deletion in S10 infected blood-fed midge midguts but virus release from the midgut into the haemolymph was blocked. BTV11 with S1[VP1] of BTV26 could be adapted to virus growth in KC cells, and contained mutations subdivided into 'corrections' of the chimeric genome constellation and mutations associated with adaptation to KC cells. In particular one amino acid mutation in outer shell protein VP2 overcomes differential virus replication in vitro and in vivo. CONCLUSION: Small changes in NS3/NS3a or in the outer shell protein VP2 strongly affect virus propagation in midges and thus vector competence. Therefore, spread of disease by competent Culicoides midges can strongly differ for very closely related viruses.

Bluetongue disease in sheep: a review / O vírus da língua azul em ovinos: uma revisão

The present review aims to show the main aspects related to bluetongue virus (BTV) infection in sheep. The bluetongue (BT) is a viral, infectious, and non-contagious disease caused by a virus (BTV) of the Orbivirus genus, transmited by a hematophagous vector of the Culicoides genus, to domestic and wild ruminants, mainly to sheep, the most susceptible species. It is caused by the association of endemic with climate conditions, with high temperatures and humidity. Economic loss is directly linked to death, abortion, weight loss, loss of milk, and meat production, and, indirectly, to the restriction on the export of animals and their by-products. The study concludes that the BTV is worldwidely spread, and probably persists due to the warm and humid climate that leads to the proliferation of Culicoides sp., being necessary to adopt measures that reduce the risk factors associated to the BTV infection.(AU)

A presente revisão objetivou apresentar os principais aspectos relacionados à infecção causada pelo vírus da língua azul em ovinos. A língua azul é uma doença viral, infecciosa e não contagiosa, causada por um vírus (BTV) do gênero Orbivírus, transmitida por meio de vetores hematófagos do gênero Culicoides a ruminantes domésticos e selvagens, principalmente aos ovinos, a espécie mais susceptível. A infecção ocorre de forma endêmica, associada a condições climáticas com elevada temperatura e umidade. As perdas econômicas estão ligadas diretamente à morte, ao abortamento, à perda de peso, à perda na produção de leite e carne, e, indiretamente, devido à restrição na exportação de animais e seus subprodutos. O estudo conclui que a língua azul está disseminada mundialmente e persiste, provavelmente, devido ao clima quente e úmido que propicia a proliferação de Culicoides sp., sendo necessário adotar medidas que diminuam os fatores de risco associados à infecção pelo vírus.(AU)

Culicoides species composition and abundance on Irish cattle farms: implications for arboviral disease transmission.

BACKGROUND: Following the emergence of Schmallenberg virus (SBV) in Ireland in 2012, a sentinel herd surveillance program was established in the south of Ireland with the primary aim of investigating the species composition and abundance of Culicoides on livestock farms in the region. METHODS: Ultraviolet-light trapping for Culicoides was carried out on 10 sentinel farms. Each site was sampled fortnightly over 16 weeks (21st July to 5th November 2014). One Onderstepoort Veterinary Institute UV light trap was run overnight at each site and catches were transferred immediately into 70% ethanol. Culicoides were morphologically identified to species level. Collection site habitats were characterised using the Phase 1 habitat survey technique (Joint Nature Conservation Committee). RESULTS: A total of 23,929 individual Culicoides from 20 species was identified, including one species identified in Ireland for the first time, Culicoides cameroni. The most abundant species identified were Culicoides obsoletus/Culicoides scoticus (38%), Culicoides dewulfi (36%), Culicoides pulicaris (9%), Culicoides chiopterus (5%) and Culicoides punctatus (5%), comprising 93% of all Culicoides specimens identified. Collection site habitats were dominated by improved grassland and a combination of broadleaf woodland and native woodland species. CONCLUSIONS: The most abundant species of Culicoides identified were the putative vectors of bluetongue virus (BTV) and SBV in northern Europe. Their presence and abundance demonstrates the potential for future transmission of arboviruses among livestock in this region.

Serosurvey of bluetongue, caprine arthritis-encephalitis (CAE) and Maedi-Visna in Barbary sheep (Ammotragus lervia) of a southern Brazilian zoo / Estudo sorológico dos vírus da língua azul, da artrite-encefalite caprina e Maedi-Visna em aoudads (Ammotragus lervia) em um zoológico do Sul do Brasil

Bluetongue (BT) is an infectious and non-contagious disease of compulsory notification which may affect domestic and wild ruminants, transmitted by Culicoides spp. midges. Despite the high morbidity and mortality in sheep, role of wild animals in the BT cycle remains unclear. Caprine arthritis-encephalitis (CAE) and Maedi-Visna virus (MVV) have been reportedly found in goats and sheep, but not described in wildlife species. Accordingly, serum samples from 17 captive Barbary sheep (Ammotragus lervia) from Curitiba zoo, southern Brazil, were tested for bluetongue, caprine arthritis-encephalitis (CAE) and Maedi-Visna viruses by agar gel immunodiffusion (AGID) and enzyme linked immunosorbent assay (ELISA). Antibodies for bluetongue were observed in 6/17 (35.3%) Barbary sheep by AGID test and in 7/17 (41.2%) by ELISA. All samples were negative for the presence of antibodies against caprine arthritis-encephalitis (CAE) and Maedi-Visna viruses. These findings indicate that Barbary sheep may be infected by bluetongue virus and act as wildlife reservoir in both captive and free-range environments.(AU)

A língua azul é uma doença infecciosa e não contagiosa, de notificação obrigatória, que pode afetar ruminantes domésticos e silvestres, transmitida por mosquitos do gênero Culicoides spp. Apesar da alta morbidade e mortalidade em ovelhas, o papel de animais silvestres no ciclo do vírus da língua azul é desconhecido. A artrite encefalite caprina (CAE) e Maedi-visna vírus (MVV) tem sido encontrados em cabras e ovelhas, porém não há descrição em espécies selvagens. Amostras de soro de 17 aoudads (Ammotragus lervia), mantidos em cativeiro no Zoológico de Curitiba, Sul do Brasil, foram testadas para os vírus da língua azul, da artrite encefalite caprina (CAE) e Maedi-visna, utilizando imunodifusão em gel de ágar e o teste de ELISA (enzyme linked immunosorbent assay). Foram observados anticorpos para o vírus da língua azul em 35,3% (6/17) aoudads utilizando a imunodifusão em gel de ágar e 41,2% (7/17) no ELISA. Todas as amostras foram negativas para a presença de anticorpos contra os vírus da artrite encefalite caprina e Maedi-visna. Esses resultados indicam que os aoudads podem ser infectados pelo vírus da língua azul e atuar como um reservatório silvestre tanto em cativeiro quanto em vida livre.(AU)

Yungahelea, a new genus of predaceous midge from northwestern Argentina (Culicomorpha: Ceratopogonidae)

ABSTRACT A new monotypic genus and species of predaceous midge from the southernmost area of the Argentinean Yungas, Yungahelea australis Spinelli and Ronderos, is described and illustrated from male and female adults. It belongs to a group containing the Ceratopogonini genera Parabezzia Malloch, Diaphanobezzia Ingram and Macfie, Spinellihelea Borkent, Grogan and Picado, Leptohelea Wirth and Blanton, and Fittkauhelea Wirth and Blanton. Phylogenetic interpretation indicates that Yungahelea is the sister group of Spinellihelea or the clade composed by Parabezzia and Diaphanobezzia.

Biting midges (Diptera: Ceratopogonidae) from an urban forest fragment in Central Amazon (Brazil): Effects of opening areas on abundance, richness, and composition

ABSTRACT We assessed the immature stages of Ceratopogonidae (Diptera) in artificial containers in an urban forest fragment in Manaus (Brazil), including their behavioral, biological and ecological information. In addition, we evaluated the effects of deforestation in an open and forested area on Ceratopogonidae communities. Immatures were sampled between August 2012 and July 2014 in artificial containers installed in both habitat types. We collected 685 immatures of seven morpho-species of Bezzia Kieffer, Culicoides Latreille, Dasyhelea Kieffer, Forcipomyia Meigen, and Palpomyia Meigen. In the open area, we recorded higher temperature and electrical conductivity values than in the forested area; however, these variables did not differ between seasons. Water volume was higher in open area and in rainy season, while pH was similar in both areas and seasons. Species richness was higher in forested area, but did not differ between seasons. We did not record differences in abundance between areas or seasons. Community composition differed between areas, but not between seasons. We provide the first records of Culicoides (Hoffmania) insignis Lutz and C. (Haematomyidium) quasiparaensis Clastrier in artificial containers from the state of Amazonas. Our results suggest that the preservation of forested areas in Amazonas is fundamental for the maintenance of the life cycle of some species of Ceratopogonidae.

Dermatite alérgica à picada de Culicoides em muar: relato de caso / Allergic dermatitis due to bite of Culicoides in muar: case report

A dermatite alérgica à picada de ectoparasitos é uma enfermidade alergoparasitária bastante comum entre animais domésticos, sendo relatada principalmente em pequenos ruminantes e em animais de companhia. Contudo, a doença é pouco diagnosticada na clínica de equídeos devido a similaridades nosológicas com outras dermatopatias. Objetivou-se, com este relato de caso, descrever a síndrome clínica, o plano diagnóstico e a conduta terapêutica de um muar acometido por essa enfermidade. Atendeu-se, no Hospital Veterinário da Universidade Federal Rural do Pernambuco, uma mula de oito anos de idade, que apresentava lesões cutâneas pápulo-crostosas e pruriginosas com evolução clínica de dois anos. Em três situações anteriores, a doença havia sido tratada como dermatite fúngica por outros médicos veterinários. Para o diagnóstico, foram solicitados exame citopatológico e parasitológico de pele, cultivo bacteriológico e fúngico, análise histopatológica e hemograma. Os exames demonstraram uma dermatite superficial perivascular eosinofílica crônica, sendo indicada a terapia tópica com dimetilsufóxido, sulfadiazina, ureia e vitamina A. O protocolo terapêutico mostrou-se satisfatório, permitindo completa remissão do quadro clínico. Este trabalho relatou achados clínicos e patológicos da dermatite alérgica à picada de Culicoides spp. em muar, além de alertar sobre a importância de exames complementares para a realização do diagnóstico diferencial e para o direcionamento terapêutico adequado.(AU)

Allergic dermatitis to ectoparasite bites is a common parasitic disease among domestic animals, being reported mainly in small ruminants and companion animals. However, the disease is poorly diagnosed in equine clinics due to nosological similarities with other skin diseases. The aim of this case report was to describe the clinical syndrome, the diagnostic plan and the therapeutic management of a mule affected by this disease. An 8-year-old mule was observed at Universidade Federal Rural de Pernambuco, presenting papular-crusted and pruritic cutaneous lesions with clinical evolution of two years. In three previous situations, the disease had been treated as fungal dermatitis by other veterinarians. For the diagnosis, cytopathological and parasitological examination of the skin, bacteriological and fungal culture, histopathological analysis and blood count were performed. The exams showed a chronic eosinophilic perivascular superficial dermatitis. A topical therapy with dimethyl sulfoxide, sulfadiazine, urea, and vitamin A was indicated. The therapeutic protocol was satisfactory, allowing complete remission of the clinical condition. This work reported clinical and pathological findings of allergic dermatitis to the bites of Culicoides spp. in muar, in addition to alerting about the importance of complementary examinations for the accomplishment of the differential diagnosis and adequate therapeutic orientation.(AU)

Successful long-term maintenance of Mansonella perstans in an in vitro culture system.

BACKGROUND: Approximately 114 million people are infected with Mansonella perstans in large proportions of Africa. In contrast to other filariae that infect humans, M. perstans-infected individuals show no distinct pathology or specific clinical picture, indicating a well-tuned adaptation to the host. In addition, since M. perstans adult worms reside in serous cavities which are difficult to access, research has been hindered and there is a paucity of knowledge about the biology of M. perstans, especially the development of the different life stages as well as M. perstans-driven immune responses. Thus in this study, an in vitro culture system was developed which allows an in-depth analysis of M. perstans. RESULTS: Culicoides species were caught in Ediki (Kumba), Southwest Region within Cameroon following a blood meal on a microfilaremic donor that had 1500 microfilariae/ml of peripheral blood and kept in captivity for 12 days at 23 °C. In a pilot experiment, 15 infective larvae were obtained from the midges and co-cultured with a confluent monolayer of monkey kidney epithelial cells (LLC-MK2) in DMEM medium supplemented with 10% FBS for up to 77 days. The resulting survival rates of 33% revealed that the cell-conditioned medium was suitable for long-term maintenance of M. perstans worms. To confirm these preliminary observations, 249 infective larvae were cultured for 50 days and their development was monitored daily and microscopically graded for motility. In total, 170 (68.3%) filariae survived and 124 (49.8%) larvae moulted between days 21-30 to become L5 stage larvae which were motile and showed continuous vigorous movement. CONCLUSION: We have established an in vitro culture system for the generation and long-term maintenance of viable M. perstans worms. This technique will be an important tool to study parasite biology and development, the role in host immunity, and might be helpful to discover novel treatment strategies against this filariae.

Allergic dermatitis caused by Culicoides in Texel sheep in the state of Pará, Brazil / Dermatite alérgica causada por Culicoides em ovinos Texel no estado do Pará

Allergic dermatitis was studied at a farm located in the municipality of Castanhal in the state of Pará, Brazil, from December 2009 to December 2012. Clinical and epidemiological data were obtained during monthly visits to the farm. Skin biopsies were performed for histopathological examinations, and insects were captured with Centers for Disease Control and Prevention (CDC) traps and live-bait entomological vacuum devices. The property housed a herd of both genders, with 45 Santa Inês sheep of different ages and 72 Texel adults. Of the 72 Texel sheep, 56 were affected. Of these, 24 exhibited alopecia, erythema, edema, and crust in the ears, on top of the head, and less often, around the eyes. In addition to the lesions described above, 14 animals exhibited hyperpigmentation of the ears and/or around the eyes and crust in the nose. Another 18 animals exhibited chronic lesions characterized by deformed and thickened ears, alopecia with hyperpigmentation, and in most cases, secondary bacterial infections with abscesses and/or myiasis. Some of these animals had lost part of their ears. Histologically, the lesions were characterized as orthokeratotic hyperkeratosis, acanthosis, vacuolization, and necrosis of epidermal cells. An inflammatory infiltrate was present in the dermis, which was composed mainly of eosinophils and macrophages, some lymphocytes and mast cells, and few neutrophils, in addition to edema and collagen deposition. The Santa Inês sheep were not affected. Among the captured insects, 294 specimens of Culicoides (Hoffmania) plaumanni Spinelli, 57 specimens of C. (Hoffmania) insignis Lutz, and 27 specimens of other Culicoides species were identified. We conclude that C. plaumanni, C. insignis, and possibly other Culicoides species are involved in the etiology of allergic dermatitis in Texel sheep in the state of Pará. Santa Inês sheep in the same region and under the same conditions were not affected, which suggests lower susceptibility of these animals.(AU)

A doença foi estudada em uma propriedade localizada no município de Castanhal, Pará, no período de dezembro de 2009 a dezembro de 2012. A propriedade possuía um plantel de 117 ovinos, 45 da raça Santa Inês e 72 Texel. Dos 72 ovinos da raça Texel, 56 estavam afetados. Os principais sinais clínicos observados foram alopecia, eritema, edema, hiperpigmentação e crostas nas orelhas, parte superior da cabeça e ao redor dos olhos. Em alguns animais as orelhas estavam deformadas, espessadas e com abscessos e/ou miíase. Histologicamente, as lesões caracterizavam-se por hiperqueratose ortoqueratótica, acantose, vacuolização e necrose das células da epiderme. Na derme havia infiltrado inflamatório composto principalmente por eosinófilos e macrófagos, edema e deposição de colágeno. Entre os insetos capturados foram identificados 294 exemplares de Culicoides (Hoffmania) plaumanni Spinelli, 57 de Culicoides (Hoffmania) insignis Lutz e 27 exemplares pertencentes a outras espécies de Culicoides. Conclui-se que C. plaumanni, C. insignis e, possivelmente, outras espécies de Culicoides estejam envolvidos na etiologia da dermatite alérgica nos ovinos da raça Texel no estado do Pará. Ovinos da raça Santa Inês que se encontravam na mesma área e nas mesmas condições não foram afetados, sugerindo menor susceptibilidade.(AU)

Description of fourth instar larva and pupa of Atrichopogon delpontei Cavalieri and Chiossone (Diptera: Ceratopogonidae) from Brazilian Amazonia

ABSTRACT The fourth instar larva and pupa of Atrichopogon delpontei Cavalieri and Chiossone are described for the first time. The immatures were collected from stream margins in the northern Brazilian states Rondônia and Piauí, and subsequently reared to adults. Larvae and pupae are illustrated and photomicrographed. Details on the rearing process and feeding behavior in laboratory, bionomics and notes on habitats are also provided.

Atypical mechanoreceptors in larvae of biting midges Forcipomyia nigra (Diptera: Ceratopogonidae).

The dorsolateral setae of Forcipomyia nigra have been investigated. They are mechanoreceptors, sensilla trichoidea, innerved by a single neuron. The tubular body is located at the base of the hair shaft. Although the fine structure of the dorsolateral setae is similar to dorsal setae that perform a double function: secretion of a hygroscopic viscous substance through the pores and mechanoreception dorsolateral setae, they do not secrete any fluid. In both types of setae, trichogen cells (hair forming cells) produce the hair shafts and thereafter do not retract from the cavity of the setae. They contain a large polyploid nucleus and expanded bundles of microtubules. In dorsolateral mechanoreceptors, the microtubules form a network around the nucleus of the trichogen cell and are especially numerous in the cytoplasm invading the interior of the seta, which is evidenced by immunofluorescence light microscopy. No tormogen cell, responsible for the production of the setal socket, was found. Our observations indicate that the dorsolateral setae are solely mechanoreceptors but their trichogen cells reveal some glandular activity.

The Biting Midge Culicoides sonorensis (Diptera: Ceratopogonidae) Is Capable of Developing Late Stage Infections of Leishmania enriettii.

BACKGROUND: Despite their importance in animal and human health, the epidemiology of species of the Leishmania enriettii complex remains poorly understood, including the identity of their biological vectors. Biting midges of the genus Forcipomyia (Lasiohelea) have been implicated in the transmission of a member of the L. enriettii complex in Australia, but the far larger and more widespread genus Culicoides has not been investigated for the potential to include vectors to date. METHODOLOGY/PRINCIPAL FINDINGS: Females from colonies of the midges Culicoides nubeculosus Meigen and C. sonorensis Wirth & Jones and the sand fly Lutzomyia longipalpis Lutz & Nevia (Diptera: Psychodidae) were experimentally infected with two different species of Leishmania, originating from Australia (Leishmania sp. AM-2004) and Brazil (Leishmania enriettii). In addition, the infectivity of L. enriettii infections generated in guinea pigs and golden hamsters for Lu. longipalpis and C. sonorensis was tested by xenodiagnosis. Development of L. enriettii in Lu. longipalpis was relatively poor compared to other Leishmania species in this permissive vector. Culicoides nubeculosus was not susceptible to infection by parasites from the L. enriettii complex. In contrast, C. sonorensis developed late stage infections with colonization of the thoracic midgut and the stomodeal valve. In hamsters, experimental infection with L. enriettii led only to mild symptoms, while in guinea pigs L. enriettii grew aggressively, producing large, ulcerated, tumour-like lesions. A high proportion of C. sonorensis (up to 80%) feeding on the ears and nose of these guinea pigs became infected. CONCLUSIONS/SIGNIFICANCE: We demonstrate that L. enriettii can develop late stage infections in the biting midge Culicoides sonorensis. This midge was found to be susceptible to L. enriettii to a similar degree as Lutzomyia longipalpis, the vector of Leishmania infantum in South America. Our results support the hypothesis that some biting midges could be natural vectors of the L. enriettii complex because of their vector competence, although not Culicoides sonorensis itself, which is not sympatric, and midges should be assessed in the field while searching for vectors of related Leishmania species including L. martiniquensis and "L. siamensis".

Cloning and expression of recombinant equine interleukin-3 and its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes.

Interleukin-3 is a growth and differentiation factor for various hematopoietic cells. IL-3 also enhances stimulus-dependent release of mediators and cytokine production by mature basophils. Function of IL-3 has not been studied in horses because of lack of horse-specific reagents. Our aim was to produce recombinant equine IL-3 and test its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes (PBL). Equine IL-3 was cloned, expressed in E. coli and purified. PBL of 19 healthy and 20 insect bite hypersensitivity (IBH)-affected horses were stimulated with Culicoides nubeculosus extract with or without IL-3. Sulfidoleukotriene (sLT) production was measured in supernatants by ELISA and mRNA expression of IL-4, IL-13 and thymic stromal lymphopoietin (TSLP) assessed in cell lysate by quantitative real-time PCR. Recombinant equine IL-3 (req-IL-3) had a dose dependent effect on sLT production by stimulated equine PBL and significantly increased IL-4, IL-13 and TSLP expression compared to non-primed cells. IL-3 priming significantly increased Culicoides-induced sLT production in IBH-affected but not in non-affected horses and was particularly effective in young IBH-affected horses (≤ 3 years). A functionally active recombinant equine IL-3 has been produced which will be useful for future immunological studies in horses. It will also allow improving the sensitivity of cellular in vitro tests for allergy diagnosis in horses.

The pupae of the biting midges of the world (Diptera: Ceratopogonidae), with a generic key and analysis of the phylogenetic relationships between genera.

The pupae of species in each of the 45 genera of Ceratopogonidae known in this stage are diagnosed and described. A standard set of terms is provided, with a glossary, for all pertinent structures of the pupal stage within a context of other Culicomorpha. The variety of terms provided by previous authors are synonymized. Some pupal structures are directly related to developing adult structures and these are discussed. A key to the genera (and to the subgenera of Forcipomyia Meigen) allows for their identification. Pupation and pupal behaviour is summarized. A table of all previous descriptions of each stage of the immatures (egg, larva, pupa) is provided, showing that 13% of all validly named extant Ceratopogonidae are known as pupae. This study examined 45% of these species. All species known as fossil pupae are discussed. A phylogenetic analysis based primarily on pupal characters confirms the relationships between the subfamilies as well as the relationships between the genera in Leptoconopinae, Forcipomyiinae and Dasyheleinae. The question of the monophyly of the Culicoidini remains unresolved. Results confirm the paraphyly of the Ceratopogonini and, for the first time, the Sphaeromiini sensu lato, which is divided into Hebetulini (new tribe), Johannsenomyiini Crampton (new status) and Sphaeromiini sensu novum. Sphaeromiini sensu novum includes Sphaeromias Curtis, Leehelea Debenham, Homohelea Kieffer and Xenohelea Kieffer and forms the sister group of the Palpomyiini. Other genera in Sphaeromiini sensu lato not known as pupae are discussed. The genus Mallochohelea Wirth is shown to be polyphyletic and one group of species is therefore recognized as members of the new genus Anebomyia (type species = Mallochohelea atripes Wirth). A number of species previously placed in Stilobezzia Kieffer are shown to belong to Schizonyxhelea Clastrier. Study of the type species of the monotypic genus Nemoromyia Liu and Yu showed it to be a member of the Palpomyia distincta species group and the generic name is therefore a new junior synonym of Palpomyia. Examination of pupae showed that the following species were misplaced and are now in other genera as follows: Schizonyxhelea brevicostalis (Das Gupta & Wirth), 1968: 28 (Stilobezzia) new combination, Schizonyxhelea bulla (Thomsen), 1935: 289 (Stilobezzia) new combination, Schizonyxhelea caribe (Lane & Forattini), 1958: 208 (Stilobezzia) new combination, Schizonyxhelea diminuta (Lane & Forattini), 1958: 209 (Stilobezzia) new combination, Schizonyxhelea obscura (Lane & Forattini), 1958: 216 (Stilobezzia) new combination, Schizonyxhelea panamensis (Lane & Forattini), 1958: 218 (Stilobezzia) new combination, Schizonyxhelea thomsenae (Wirth), 1953: 83 (Stilobezzia) new combination, Schizonyxhelea scutata (Lane & Forattini), 1961: 92 (Stilobezzia) new combination, Pellucidomyia geari (de Meillon & Wirth), 1981: 547 (Macropeza) new combination, Dibezzia prominens (Johannsen) 1932: 435 (Johannsenomyia) new combination, Nilobezzia theileri (de Meillon & Wirth, 1981:552) (Sphaeromias) new combination, Anebomyia atripes (Wirth, 1962: 281) (Mallochohelea) new combination, Anebomyia fluminea (de Meillon & Wirth), 1981: 550 (Mallochohelea) new combination, Anebomyia siricis (de Meillon), 1961: 50 (Sphaeromias) new combination, Anebomyia texensis (Wirth), 1962: 283 (Mallochohelea) new combination, Anebomyia yunnana (Yu & Zou), in Yu et al. 2005: 1503 (Mallochohelea) new combination, Bezzia goezii (Schrank), 1803: 72 (Tipula) new combination (also a nomen nudum), Palpomyia nemorosa (Liu & Yu), 1991: 26 (Nemoromyia) new combination. Bezzia xanthogaster (Kieffer), 1919: 130 (Probezzia) is a junior homonym of Probezzia xanthogaster (Kieffer), 1917: 329 (Bezzia) and is here given the new name Bezzia gilvigaster Borkent. The fossil genus Paraculicoides Pierce, 1966: 94 and its type species Paraculicoides rouseae Pierce, 1966: 94 are removed from the Ceratopogonidae and likely belong in the Psychodidae.