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Introduction: Hypodermosis is a subcutaneous infestation in cattle that is caused by larvae of Hypoderma spp. and it is an economically important disease in the cattle industry. This study aimed to find the prevalence rate of hypodermosis and identify variations in the COX1 gene among isolates present in Sulaymaniyah, in the Kurdistan region of Iraq., Methodology: The study was conducted in a Sulaymaniyah slaughterhouse from March to July 2021. The carcasses of 867 cattle were carefully checked before and after skinning them to record the presence of boils containing the larvae of Hypoderma spp. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using TaqI enzyme, and sequencing of the COX1 gene were used for diagnosis and molecular characterization of Hypoderma spp., Results: The rate of infestation with Hypoderma bovis was 1.61% and the highest rate (3.57%) was detected in April. The disease was significantly (p < 0.05) higher in local breeds at 2.79%. PCR-RFLP confirmed that all recorded species were H. bovis. The result was further confirmed by Sanger sequencing of the COX1 gene of the isolated species. Comparison of the sequences of the conserved COX1 gene of the parasite led to identification of six different haplotypes in the research area. Two of the haplotypes were previously recorded internationally, while four new haplotypes associated with four novel mutations were recorded for the first time in the study region., Conclusions: Based on these results we can conclude that H. bovis is a widespread species in the research region., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Aram A Mohammed, Taib A Hama Soor, Mardin O Abdulqadir.)

The host switching of Hypoderma actaeon (Diptera: Oestridae), a specific parasite of red deer (Cervus elaphus), towards roe deer (Capreolus capreolus) has been recently reported in Spain. To provide information about the temporal and spatial spreading of H. actaeon infection in roe deer, 244 serum samples from animals hunted in Spain between 2013 and 2018 were analysed by an indirect enzyme-linked immunosorbent assay. The overall seropositivity was 13.9%. Seropositivity was higher in continental (27.7%) and mountainous (12%) areas from central Spain, followed by southern-Mediterranean (11.2%) and northern-oceanic regions (3.5%). Differences were significant between central-continental and northern-oceanic regions (P = 0.003). No differences were found according to the sex and age of roe deer (P > 0.05). In 2013, all seropositive animals were concentrated in two distant areas in central and southern Spain, suggesting that the host switch could have occurred independently in both regions. Changes in the pattern of distribution of red deer and roe deer could have favoured the spreading of this myiasis towards roe deer, indicating that roe deer may become infested by H. actaeon in areas where both cervids coexist at high densities., (© 2019 The Royal Entomological Society.)

Background: Despite intensive control measures including governmental campaigns using highly-efficacious systemic insecticides, there is evidence for persisting or recurring bovine Hypoderma species populations in parts of Europe, the USA and Canada. The present study evaluated the efficacy of LONGRANGE® (eprinomectin 5% w/v extended-release injection) against the infestation of cattle with Hypoderma lineatum, which is considered to be the predominant bovine warble fly in southern Europe and in North America., Methods: Thirty-six local breed cattle sourced in an endemic area in southern Italy and confirmed positive for Hypoderma exposure by ELISA were randomly assigned to three groups of 12 animals each. Cattle of one group served as control and received saline injectable solution, whereas those in the two other groups received LONGRANGE® by subcutaneous injection. LONGRANGE® was administered once, either when Hypoderma larvae were expected to be first-instars (L1) or after warbles development, with Hypoderma larvae moulting to the second-(L2) and third-(L3) instars. Cattle were checked at intervals for warbles and Hypoderma larvae were collected, examined for their viability and morphologically identified. The detection of Hypoderma on cattle was terminated when warbles were no longer emerging., Results: All intact larvae collected were identified as H. lineatum. No live larvae were collected from any animal treated with LONGRANGE® while live specimens were sampled from nine of the 12 control cattle (1 to 9 larvae per animal) (P = 0.0001 at α = 0.05). LONGRANGE® treatment was well accepted and no adverse events related to treatment or other health problems were observed., Conclusions: This study confirmed the continued 'preventive' (efficacy against migrating L1) and 'therapeutic' (efficacy against L2 and L3 in warbles) efficacy of LONGRANGE® against H. lineatum infestation of cattle under contemporary field conditions.

Fifteen apparently healthy Kandari cross-bred cattle aged about 4 years were purchased from Rurki, Patiala district of Punjab by a private dairy farmer in Erode, Tamil Nadu. Four animals showed eruptions on the lateral thoracic and dorsal abdominal regions of the body after 15-day period of quarantine. Manual palpation of the eruptions resulted in the emergence of larval stages of dipteran flies, identified by their morphology as Hypoderma from these animals. Molecular identification based on mitochondrial cytochrome oxidase-1 (COX-1) gene confirmed it to be Hypoderma lineatum. Treatment with oral ivermectin did not have any curative effects, with exacerbation of disease being noticed, as more than 500 eruptions subsequently emerged in each animal, which had to be culled. Consequences of long distance migration of host on parasite epidemiology are discussed. Awareness must be created among livestock farmers to prevent their economic loss while purchasing cattle from different parts of the country.

Background: The studies reported here were conducted to assess the efficacy of ivermectin long-acting injection (IVM LAI; IVOMEC® GOLD, Merial; 3.15 % w/v ivermectin) for the treatment and control of natural infestations of cattle by Hypoderma bovis and Hypoderma lineatum, which are the most economically important oestrid flies of cattle in the northern hemisphere., Methods: Cattle selected from herds with a history of Hypoderma infestation were grouped into blocks of three (Italy, 33 cattle; Germany, 30 cattle) or two (USA, 16 cattle) animals each, on the basis of positivity at the pre-treatment anti-Hypoderma antibody titres. Within each block, animals were randomly allocated to one of the following treatment regimens: saline (control); IVM LAI, administered at the predicted time of occurrence of first-instar larvae (Italy, Germany, USA); IVM LAI, administered at the predicted time of occurrence of second- and/or third-instar larvae (Italy, Germany). All treatments were administered by subcutaneous injection in correspondence of the area anterior to the shoulder at 1 ml/50 kg body weight, which corresponds to 630 mcg IVM/kg for IVM LAI., Results: No Hypoderma larvae emerged from animals treated with IVM LAI, whereas live H. lineatum (Italy) or H. bovis (Germany, USA) larvae were collected from saline-treated animals (P < 0.01). No adverse reactions to treatments were in any of the animals enrolled in the study., Conclusions: The results from this study demonstrate that ivermectin in a long-acting formulation is 100 % efficacious in the treatment of cattle naturally infested by H. bovis and H. lineatum larvae at all stages of development. IVM LAI can, therefore, be used as 'prophylactic' treatment for Hypoderma spp. infestations in absence of external evidence of their presence and thus prior to skin and carcass damage, and as 'therapeutic' treatment, when warbles are already present.

Objective: The aim of present study was to investigate the chronobiology and identification of Hypoderma species in cattle slaughtered in the Diyarbakir Province of Turkey., Methods: In total, 736 hides and subcutaneous tissue of slaughtered cattle were examined for the presence of second- and third-instar larvae in the slaughterhouse between November 2012 and May 2013. Third-instar larvae were collected from the slaughterhouses, and gDNA isolates were examined by PCR-RFLP analysis of the cytochrome c oxidase I (COI) gene of mt-DNA using TaqI enzyme., Results: In total, 62 out of 736 cattle (8.42%) were found to be positive for Hypoderma larvae. A total of 328 (90 second- and 238 third-instar) Hypoderma larvae were detected in the hide and subcutaneous tissue of the back of infested cattle. All the 238 third-instar larvae (100%) were identified as H. bovis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The mean number of Hypoderma larvae in each cow was 5.29% (62/736). In the examined cattle, second-instar larvae were observed starting from the second week of January and subcutaneous nodules were found until the last week of April., Conclusion: H. bovis was the dominant species detected in the Diyarbakir Province.

The occurrence of Hypoderma diana (Diptera: Hypodermatidae) in a herd of 15 horses was observed during a 4-year period (2010-2013) in northwest Bohemia. During this period, infestation by the warble fly H. diana increased from 12.5% in 2010 to 53.3% in 2013. Nodules were observed on the neck, back, stomach, legs, and head. The incidence of H. diana was probably influenced by the presence of a specific host mouflon (Ovis orientalis musimon).

For many years, hypodermosis control - which started in the 1920's - was unsatisfactory, partly due to a lack of efficacy or a too high toxicity of the available products. An improved control became possible with the introduction (1957) of the organophosphate Metrifonate (Neguvon®, Bayer AG). Subsequent large-scale campaigns according to the Federal Ordinance on epizootic diseases (1967) quite reduced hypodermosis in Switzerland. However, due to a limited use of the product (only young and alpine-grazing cattle were allowed to be treated, administratively indicated maximum dose, side-effects) and its incomplete efficacy, a full control was not possible. The decision to maintain hypodermosis as a "notifiable" disease then allowed further epidemiological studies. In 2000/2001 a concerted action in close cooperation with the cantonal veterinarians allowed a consequent and systematic treatment of all cattle in all endemic areas by the large-scale use of Eprinomectin (Eprinex®, Merial Inc.) for dairy cattle and of Ivermectin (Merial Inc.) as microdose "off-label" for young cattle has led to a rapid and sustainable success.

Systemic humoral and cellular immune responses were studied during natural infestations by Hypoderma lineatum in cattle at their first (G-1) and second exposure (G-2). Four out of seven animals in G-1 were palpation positive, with a mean intensity of 11.2 (12.81SD) warbles; the same proportion (4/7) presented warbles in G-2 but the intensity was 3.7 (2.21SD). The evolution of total IgG levels was characterized by a noticeable increment coinciding with the presence of warbles on the back, especially in G-2. The IgG1 isotype displayed a parallel evolution in both groups, with peak values prior to the appearance of first warbles. The IgG2 subclass followed an irregular pattern in both groups and IgM maintained low and constant levels throughout the study, mainly in G-1. CD4/CD8 ratios showed a predominance of CD4(+) throughout the infestation, principally in G-2 during the warble season. The evolution of IFN-γ in G-2 was constant, whereas in G-1 there was a gradual descent until warble emergence. The dynamics of the IL-10 differed between G-1 and G-2, although both groups showed a significant drop after the exit of the larvae that could be implicated in the termination of the inflammatory response. IL-4 and TNF-α levels did not show differences between groups. Our results suggest that the resistance mechanisms would become more apparent at the latest stages of the infestation by Hypoderma, supporting the hypothesis that considerable larval destruction in sensitized animals might take place after their arrival to the back., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The efficacy of eprinomectin in an extended-release injection (ERI) formulation was determined in cattle harboring naturally acquired infestations of first- or second- and third-stage larvae of Hypoderma spp. in three studies conducted according to the same protocol in the USA (two studies) and Germany (one study). Thirty cattle sourced from herds with a history of Hypoderma infestation were included in each study. Cattle were formed into replicates of three animals each on the basis of pre-treatment anti-Hypoderma antibody titers. Within replicates each animal was randomly allocated to one of the following treatments: ERI vehicle (control) at 1 mL/50 kg bodyweight, administered once on Day 0; Eprinomectin 5% ERI at 1 mL/50 kg bodyweight (1.0 mg eprinomectin/kg), administered once on Day 0 (when larvae were expected to be first instars); or Eprinomectin 5% ERI at 1 mL/50 kg bodyweight (1.0 mg eprinomectin/kg), administered once when larvae were second or third instars (study dependent, Day 73, 119, or 140). Treatments were administered by subcutaneous injection in front of the shoulder. In all studies, emerging and/or expressed Hypoderma larvae were recovered, speciated, and counted and viability was determined. Eprinomectin LAI treatment was 100% (p<0.05) efficacious against first- and second- or third-stage larvae of Hypoderma bovis (two studies) and Hypoderma lineatum (one study). All animals accepted the treatment well. No adverse reaction to treatments was observed in any animal in any study., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: The importance for the health of cattle Hypodermosis (Nokra) species that cause disease by Polymerase Chain Reaction- Restriction Fragment Length Polymorphism (PCR-RFLP) method was investigated., Methods: The molecular analysis of Hypoderma species specific sequences located on the Cytochrome C Oxidase I (COI) (mtDNA) gene by PCR-RFLP method was successfully carried out., Results: DNAs were obtained using the extraction kit and Polymerase chain reaction (PCR) foe amplification of the mitochondrial DNA gene yielded for 688bp COI gene on agarose gel. Using the PCR amplification and HinfI, RsaI and TaqI restriction enzymes, mitochondrial DNA gene regions were amplified with the RFLP technique., Conclusion: The mitochondrial COI gene region of the genus Hypoderma for the molecular identification of potential answers questions, such as the level, which indicates that it may be very useful. In this study, in the city of Van, it is also important that the Hypoderma species be defined in terms of the first molecular study.

This paper presents data on the prevalence, biology and control of warble fly infestation (WFI) in cattle and goats in Pakistan. A questionnaire for obtaining information on biology and prevalence was circulated amongst field veterinary staff and livestock farmers in all five provinces (Punjab, Sindh, Balochistan, Khyber Pakhtunkhwa [KPK] and Gilgit-Baltistan) and in the Federally Administered Tribal Areas of the country. A total of 1,019 questionnaires were received (Punjab = 296, Sindh = 246, KPK = 318, Balochistan = 151, Gilgit-Baltistan = 8). Warble fly infestation was reported from each province and from the federally administered tribal areas, particularly from hilly, semi-hilly and sandy desert areas (the Cholistan desert, which adjoins the Rehim Yar Khan, Bahawalpur and Bahawalnagar districts, and the Nara area of Sanghar district). Warbles (nodules) started appearing on the backs of the infested animals from September through December and disappeared from October through March. The prevalence of WFI varied from 5% to 75%. It was highest in hilly areas and gradually decreased towards the plains. A map was developed of warble fly-infested areas. Four field trials were conducted to study the efficacy of different drugs indicated for the control of warble fly infestation. A total of 2,094 cattle and 3,876 goats were given five different injectables (avermectins); namely, Ivomec, Endectin, Euvectin, Dectomax and Promectin (ivermectin) during the first three weeks of September. A control group was given normal saline. All the medicines were found to be effective in controlling infestation.

Soluble fractions of Hypoderma lineatum third instar fat body, haemocytes and haemolymph were formulated with Quil A and used to immunize four groups of calves while a fifth group remained untreated. Calves received two subcutaneous injections of the soluble fractions, or adjuvant only delivered two weeks apart. Two weeks after the last injection the calves were exposed to 50 newly hatched larvae of H. lineatum which were placed on the skin and allowed to penetrate. Survival of larval stages was monitored by weekly palpation and collection of emergent third instars. Antibody responses to the immunogens were evaluated by immunoblots and following infestation antibody responses to first instar antigens were evaluated by an ELISA. Non-immunized calves and calves injected with adjuvant were all palpation positive for cattle grubs. In groups immunized with fat body, haemocyte and haemolymph components 100%, 33% and 33% were palpation positive for grubs respectively. First instar mortality, as reflected in palpable grubs, was high in the groups receiving injections with tissue components (99.3%, 95.1%, 95.8%, 83.9 and 80.4% mortality for those groups receiving fat body, haemocyte, haemolymph, adjuvant or control respectively). Second and third instar mortality was also higher in the immunized groups (100.0%, 91.7%, 91.7% for fat body, haemocyte, and haemolymph respectively) in comparison to the adjuvant only (14.0%) and unvaccinated (33.3%) groups. No viable flies emerged from pupae originating from larvae emergent from any of the immunized groups. Calves receiving the tissue extracts developed antibodies to several protein components following the second immunization which were still present 13 weeks post-infestation. Several proteins appeared to be common among the three tissue extracts and were recognized by antibodies from the immunized calves. All groups of calves became positive for antibodies to first instar antigens, although in some immunized calves the antibodies were transient., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

This study was carried out to investigate the prevalence and economic losses due to in cattle of hypodermosis between May 2008 and April 2009 in Afyonkarahisar province of Turkey. A total of 6,598 cattle-3,498 in pasture and 3,100 in slaughterhouse-were examined for hypodermosis. One hundred ninety-five out of 6,598 cattle (2.96%) were found positive. Concerning this infestation rate, the prevalence of hypodermosis in field was 5.3%, whereas the prevalence was 0.3% in slaughter house. Hypoderma bovis and Hypoderma lineatum were recorded in slaughter house. First-stage Hypoderma spp. larvae weren't found in cattle organs in slaughterhouse. The annual economic loss on skins is estimated to be about 18,288 TL (currency exchange rate is 1 USD:1.499 TL).

During the 2003-2005 hunting seasons, a total of 120 Cervidae, including 39 red deer (Cervus elaphus hispanicus) and 81 fallow deer (Dama dama), were examined for subcutaneous myiasis. Animals were shot from January to June in southern Spain. Specific antibodies against Hypodermatinae (Diptera: Oestridae) were detected by indirect enzyme-linked immunosorbent assay (iELISA) using a crude larval extract (CLE) and a purified antigen [hypodermin C (HC)] obtained from first instars of Hypoderma lineatum (De Villers) (Diptera: Oestridae). Hypoderma actaeon Brauer was the only species detected in this study, which represents the first confirmation of this species in fallow deer from Spain. The overall prevalence of animals presenting subcutaneous larvae (14.2%) was considerably lower than the prevalences determined by iELISA with CLE (43.3%) and HC (40.0%). Red deer showed a higher prevalence of Hypoderma than fallow deer. The concordance between larval examination during the hunting season and iELISA using both antigens was low, whereas the concordance between the CLE and HC ELISAs was good. Larval antigens obtained from H. lineatum constitute a good tool for the diagnosis of H. actaeon in Cervidae, especially when the hunting season does not coincide with the maximum presence of larvae on the back., (© 2010 The Authors. Medical and Veterinary Entomology © 2010 The Royal Entomological Society.)

The aim of this study was to determine the seroprevalence of hypodermosis in cattle in East-Turkey and to identify Hypoderma species using morphological and molecular methods. For this purpose, a total of 778 serum samples of cattle were collected from Erzurum provinces of east-Turkey from December 2008 to February 2009. The sera were analyzed using a Hypodermin C antigen by means of indirect ELISA. In addition, 10 Hypoderma spp. larvae were collected from slaughtered animals in the abattoir for morphological identification and molecular characterization on the basis of mitochondrial CO1 gene sequence analysis and PCR-RFLP. Two hundred and twenty three (28.6%) out of 778 cattle were seropositive for hypoderma antibodies. All positive cattle were female of a local breed. Seven out of 10 Hypoderma larvae were morphologically classified as third instar larvae (L3) of H. bovis and 3 were classified as L3 of H. lineatum. The TaqI restriction enzyme was used to differentiate the Hypoderma species on the basis of the 438 and 250 bp bands for H. bovis and the 488 and 200 bp bands for H. lineatum resulting from PCR-RFLP. According to the alignment of the mitochondrial CO1 sequences of the Hypoderma species and the PCR-RFLP results, all examined larva samples were classified as H. bovis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Bovine hypodermosis affecting livestock performance and the leather industry was still widespread in France up to the nineties despite successive directives from the Ministry of Agriculture since 1941, encouraging livestock owners to treat, on a voluntary basis. In 1969 the French Ministry of Agriculture, asked the national Farmers' Animal Health Organisation (FAHO), to plan a durable hypodermosis control programme and a working group including all the partners in cattle production was set up, under the coordination of the national FAHO. Improved systems of hypodermosis control, including new treatment and surveillance methods were developed. Among the main benefits obtained from this original work, were (i) the identification of scientific data which allowed inexpensive and accurate immuno-surveillance procedures, and a highly effective low-cost treatment, Ivomec, administered at the micro dose rate (2 microg/kg), which is environmentally non-threatening, and (ii) the development of a new strategy to manage the control programmes progressively, on a regional basis, in two to three concentric zones over three successive years at a maximum. The current programme, coordinated at the National level since 1998, has been implemented in each region of France. The compulsory systemic winter treatments directed against the endo-parasitic stage, carried out by technicians and veterinarians involved the entire bovine population in controlled zones. As each zone reached a hypodermosis herd prevalence of under 5%, usually after two years, the treatments were suspended. However treatments of the infected farms and contiguous farms were maintained. An immuno-survey was carried out, each winter, to evaluate the prevalence of the disease and detect any residual foci or re-infestations. Since 2002, bovine hypodermosis in France is under control with immuno-surveillance maintained at a very low cost. In 2006 hypodermosis became a notifiable disease.

This study was carried out to investigate the prevalence of hypodermosis in cattle between January and June 2005 in Nigde province, which is located in the middle of Turkey. A total of 1336 cattle, which were slaughtered in Nigde, were investigated for Hypoderma larvae. The 68 out of 1336 cattle (%5.08) were found positive for Hypoderma larvae. A total of 536 Hypoderma larvae were found in the skin and subcutaneous tissue of the back of infested cattle. The 489 out of 536 larvae (%91.23) were identified as Hypoderma bovis and 47 out of 536 (%8.77) as H. lineatum. Number of Hypoderma larvae counted on single infested cattle varied between 1-45 and the mean number of Hypoderma larvae per cattle was 7.88 (536/68). Hypodermosis was recorded for the first time in cattle from Nigde.

The aim of this study was to determine the seroprevalence of hypodermosis in cattle in the east and southeast of Turkey. For this purpose, a total of 634 sera samples of cattle were collected from Malatya, Elazig and Diyarbakir provinces of east and southeast of Turkey from November 2005 to February 2006. The sera were analyzed using a Hypodermin C antigen by means of indirect ELISA. One hundred and forty eight (23.3%) out of 634 cattle were seropositive for hypoderma antibodies. The highest percentage of seropositivity were detected at Elazig province (26.3%) followed by Malatya (22.3%) and Diyarbakir provinces (22.1%). The seropositivity rate was higher in female (31%) than male (14.1%). When the mean is considered by animal breed, the highest seropositivity was detected at local breed (27.7%) following crossbreed (26.8%) and purebreed (19.7%). There was a positive relation between age and seropositivity. Seropositivity rate was 15.9% in 2 and under ages while these rates were 38.1% and 30.4% in 3-4 ages and 5 and up ages, respectively.

The potential for cross-transmission of Hypoderma lineatum from cattle to domestic goats (Capra hircus) was examined using artificial infestation techniques. Two routes of infestation, subcutaneous injection and dermal penetration, were used to expose goats to newly hatched first instars. Presence of antibodies and appearance of circulating antigen (hypodermin C) were evaluated at selected intervals for up to 40 weeks post-infestation. In addition, immunoblots against H. lineatum first-instar proteins were conducted using sera taken at 10 weeks post-infestation. Goats were palpated for the presence of developing larvae at sub-dermal sites beginning at week 30 pi. No developing larvae were palpated at any time, regardless of the route of infestation nor was circulating antigen detected in any infested goats. Antibodies were present at weeks 6 and 10 and week 27 pi in both infested groups. Immunoblots indicated all infested goats produced antibodies to first instar H. lineatum antigens. H. lineatum appears to be incapable of completing development in domestic goats although the transient appearance of ELISA detectable antibodies and the presence of bands on immunoblots suggests that at least some larvae survive long-enough to engender a humoural response. The host specificity of H. lineatum is discussed in light of the general concepts of host-parasite relationships of oestrids.

Prevalence and economic losses of warble fly infestation (WFI) in cattle and buffaloes were recorded in Dera Ghazi Khan and Rajan Pur districts of southern Punjab (Pakistan). A total of 18,000 cattle and 9000 buffaloes were examined in the field and slaughter house. Only one species of Hypoderma, i.e. Hypoderma lineatum was recorded from this area. The prevalence of WFI was higher in slaughter house versus field, cattle versus buffaloes, males versus females, and young versus old animals in both the districts. The highest month-wise prevalence was recorded in December and the lowest in July. Organ-wise prevalence of WFI was highest in oesophagus followed in order by rumen and spleen. The economic losses based on the value of warbled and warble-free hides were estimated as Rs. 22.8 million per annum (currency exchange rate is Rs. 57.5=US 1 dollar).

Larvae belonging to five species of Hypoderma spp. (Diptera, Oestridae) cause myiasis in wild and domestic ruminants that is characterized by migrations within deep tissues. In China hypodermosis is one of the most important arthropod diseases affecting ruminants and, moreover, represents a significant zoonosis, with numerous reports of Hypoderma spp. affecting farmers. Recently, a sixth species, Hypoderma sinense Pleske, has been rediscovered but the endogenous migration pathway within the host body is completely unknown and it represents a major constraint for the control of larval infection. In December 2003 a total of 165 larval stages of Hypoderma spp. were collected from different anatomical sites of 40 yaks slaughtered at an abattoir in the province of Gansu, China. The morphological characters and size of the recovered larvae were used to infer migratory routes and 45 specimens were also subjected to a polymerase chain reaction (PCR) assay of cox1 mtDNA and amplicons sequenced. All the larvae molecularly processed were identified as H. sinense and sequence identity was confirmed by a PCR-restriction fragment length polymorphism (PCR-RFLP) tool carried out using BfaI and HinfI endonucleases. The finding of H. sinense larvae only in the oesophagus or both in oesophagus and subcutaneous tissue of 12 and 15 animals, respectively, indicates that H. sinense larvae migrate through the oesophagus similarly to Hypoderma lineatum (De Villers). The description of the endogenous life cycle of H. sinense will help to determine the timing of specific treatment programmes to guarantee the improvement of animal welfare and health, thus resulting in an increase in livestock production in China.

A chemoprophylactic field trial was conducted to assess the efficacy of pour-on eprinomectin applied at the approximate dose of 50 mcg/kg to dairy cattle with naturally occurring hypodermosis. Two-hundred-eleven cattle, selected from two herds with a high prevalence of Hypoderma spp. infestation, were divided in three groups: Group A (N = 71) was treated with pour-on eprinomectin at the recommended dosage of 500 mcg/kg, Group B (N = 64) at the lower dose of 50 mcg/kg, a third group (Group C, N = 76) served as untreated control group. Treatments were performed in November-December 2002 and the animals were examined for the presence of warbles in the following April and June. No larvae emerged in the treated groups, whereas a variable number of warbles (ranging from 1 to 28) were found in control animals. Adverse reactions were not observed in any animal, and only minor side effects were observed. A larger field trial carried out in the following year (1064 treated and 131 untreated control cattle) confirmed the chemoprophylactic efficacy of minidosed eprinomectin against Hypoderma spp. Administration of eprinomectin minidoses in dairy cattle is interesting because of the low costs involved and no need for milk withdrawal.

A total of 1276 cattle from 76 farms in 8 villages were examined by palpation of the skin for evidence of hypodermosis. The effects of age, sex, breed, and colour of cattle on hypodermosis were investigated. The overall prevalence was 31.9%. The mean number of warble fly larvae on infested animals was 8.46. Hypodermosis infestations were encountered in all the villages visited. Within a herd, the intensity of infestion decreased with the age of cattle. Infestation rates of warble fly larvae were lower in native cattle, in females, and in piebald animals.

A large-scale serological surveillance was conducted for hypodermosis in the north of China. A total of 4175 sera samples of yaks and cattle were collected from five provinces in north China that included Xinjiang, Inner Mongolia, Heilongjiang, Jiling and Gansu provinces, between 2001 and 2002, and were examined for anti-hypoderma antibody by ELISA. The results indicate that the naturally infested positive rates of Hypoderma spp. in the above provinces were 51.77%, 27.02%, 13.00%, 6.03% and 44.41%, respectively. These results suggest a significant warble fly population in the north of China. Positive sera were collected from infested cattle in Pingliang county of the Gansu province monthly between August 2001 and July 2002. These sera were used to evaluate the seasonal kinetics of anti-hypoderma antibody. The kinetics demonstrated that anti-hypoderma antibody was elevated from October to December. Thus, serological surveillances associated with low infestation rates and chemical therapy would best be performed from October to December in the region.

In order to estimate the presence and the seroprevalence of hypodermosis in Albania, 625 head of cattle were bled during two sampling seasons (i.e. from February to March 2003 and from November to December 2003). The cattle came from three collection sites in Northern (site A), Central (site B) and Southern (site C) Albania. Milk samples were collected monthly from four animals from October 2002 to May 2003 during the lactating period. The animals were also clinically examined for the presence of warbles by manual palpation from April to July 2003 and 2004 and third instar larvae were collected and morphologically identified. Serum and milk samples were processed by ELISA. One hundred and thirty-three (38.6%) out of 344 and 116 (41.3%) out of 281 animals were found to be seropositive for Hypoderma during the first and the second sampling season. In particular, the animals from site C presented the highest percentage of seropositive results (i.e. 72.8% and 97.8% in the first and in second year, respectively) followed by the animals from sites A (i.e. 35.8% and 23.8% in the first and in second year, respectively) and B (i.e. 17.8% and 3.4% in the first and in second year, respectively). The kinetics of anti-Hypoderma antibodies in milk samples showed the highest antibody titres from October to February 2003. All the seropositive animals in both the sampling periods showed the presence of one or more warbles under the skin during April and May 2003 and 2004 and the third-stage larvae collected were morphologically identified as Hypoderma bovis. The results of this survey indicate that hypodermosis is widespread in Albania and that early prophylactic treatments must be carried out accordingly. The hope is not only to reduce the parasitic intensity with obvious benefits for livestock production, but also to avoid the risk of spreading this parasitic disease to neighboring countries.

Our objective was to determine the seroprevalence of Hypoderma spp. and to develop a spatial model describing the risk surface of warble-fly infection in Belgian cattle herds (adjusting for herd size, herd type, local temperature, rainfall, relative air humidity and land-cover). This survey was carried out in 390 selected herds of all types (dairy, mixed and beef) from December 1997 to March 1998, which were included in a national infectious bovine rhinotracheitis and paratuberculosis (Johne's-disease) survey. All animals >24 months old were blood sampled and an ELISA was used on pooled serum samples (10 animals per pool). The herd seroprevalence was 48.7% (95% confidence interval: 43.6-53.8); positive herds were mainly in the south of the country and along the North Sea coast. The logistic multiple-regression model of herd-level seropositivity indicated that mixed-type and beef-cattle herds have more than four-fold and two-fold increases in the odds of being Hypoderma-positive, respectively, compared with dairy herds.

Sixty yaks were autopsied to determine the migration pattern of warble fly larvae. In August, first instars were observed in the body of yak for the first time. These larvae peaked in number in October. From November to February, second instars were detected and their number peaked in January. Third instars appeared in January and peaked in March. Forty-five yaks were administered with ivermectin: 15 animals in September, 15 in October and 15 in November. Between December and June, the number of warbles was checked by palpation. Although some warbles were observed in the September- and November-treated groups, no warbles were detected in the October-treated group. Treatment of yaks with ivermectin was most effective for warble fly in October.

A chemotherapy trial was conducted to determine the lowest dosage of injectable preparation of ivermectin against Hypoderma spp. infestation in yaks in Tibetan areas in Tianzhu county, Gansu province, in northwest of China. One hundred and sixty yaks were randomly divided into four groups of 40 yaks for the trial. The first three groups were treated by subcutaneous injection in the neck with 0.1% ivermectin (respectively, 1, 5, 10 microg/kg body weight). The fourth group was not treated and considered as control group. All the experiments were performed in November 2000 and the animals were examined for the presence of warbles in the next March and May. The results indicated that there was no warbles found on the back of treated animal while third stage larvae were palpated on back of some of the yaks in control group. It is concluded that dosage of 1 microg/kg ivermectin injectable was sufficient to kill or stop development of larvae of Hypoderma spp. in naturally infected yaks if administrated in November.

An antigen capture ELISA, using a murine monoclonal antibody recognising recombinant hypodermin C (rHyC), was used to evaluate the influence of early treatment with eprinomectin (Eprinex) or fenthion (Spotton) on the kinetics of circulating hypodermin C in calves naturally infested with Hypoderma lineatum. No viable larvae were collected from treated animals, whereas a variable number of warbles were found in control animals. Treatment provoked a decrease in circulating HyC levels that was significant 9 days post-treatment (p.t.). Circulating antigen levels in the treated cattle remained detectable for approximately 99 days p.t. In contrast, control animals had no detectable antigen at 64 days p.t., 42 days earlier than in the treated animals. These results suggest that larvae were either gradually killed, resulting in slow release of antigen or they were encapsulated, leading to the slow liberation of antigen. Kinetics of circulating HyC did not differ among the two insecticide treatments. Antibodies persisted, in all groups, throughout the 120-day study. These results suggest that the antigen capture ELISA will be useful as a technique for detecting successful treatment of cattle grub infestations and for the detection of new infestations in previously infested cattle.

Cattle hypodermosis, due to insect larvae, is widely spread over the northern hemisphere. Very efficient insecticides are available and their use in most countries are done on an individual level but never cover the whole cattle population of a country. Untreated animals remain the reservoir of the disease and annually re-infest the cattle population. The economic effects of this disease on animal production (meat, milk and the leather industry) but also on the general cattle health status, have led many European countries to launch organised control programs. The first example of definitive hypodermosis control goes back one hundred years ago when Danish farmers eradicated hypodermosis from the Danish islands by manual elimination of the warbles. Since then, more and more European countries have considered the feasibility and economic returns of such programs. The various factors which foster these programs are related to (i) biological factors, (parasite cattle specificity, synchronous biological cycles of both species of insects involved), (ii) the development of more and more efficient insecticides used only once a year by systemic application, with high efficiency at very low dosages against the first larval stage of Hypoderma spp., (iii) the development of acute techniques of detection of the disease for the monitoring of hypodermosis free countries and (iv) the durable successful results obtained in more and more European countries. Although the programs were imposed by different partners of the livestock channel production (farmers, dairy industry, leather industry) and have been engaged within the last 50 years in many European countries (Denmark, The Netherlands, Ireland, the United Kingdom, the Czech Republic, Germany, France and Switzerland) common features have emerged among these different eradication programs. They all need a preliminary statement of the economic impact of this pest and the farmers' awareness of the economic returns of such programs. The programs' efficacy depends: (i) on a good knowledge ofthe epidemiology of the parasites, (ii) on the simultaneous implementation of the control program on the whole national cattle population whatever the structure monitoring the treatments (veterinary services, farmers association), (iii) on a national Warble fly legislation making the treatments compulsory and (iv) on an acute epidemiological survey as soon as the status of a hypodermosis free country is reached and the treatments are suspended. The sanitary and financial returns of such programs are a benefit to all the partners of livestock production, to the quality of the environment and to the consumers.

An antigen capture assay for the detection of circulating hypodermin C was developed for diagnosis of hypodermosis. A murine monoclonal antibody to recombinant hypodermin C was raised using rapid immunization and a one-step hybridization-cloning technique. A highly reactive, specific monoclonal antibody was tested using sera spiked with known quantities of purified, native hypodermin C or with recombinant hypodermin C. Sensitivity of 96.4% and specificity of 95.6% for the antigen capture assay was assessed using a panel of sera from animals unexposed to cattle grubs and from cattle with palpation proven cattle grub infestations. Data from this panel of sera was used to establish the cut-off OD for further testing. The kinetics of circulating hypodermin C was assessed using the assay in three groups of cattle artificially infested with 50, 100 or 200 first instar Hypoderma lineatum. Antigen was first detected approximately 6 weeks after infestation. The amount of antigen detected increased in each group of animals reaching peaks at different times in each group. Levels of antigen fell quickly following arrival of grubs at the back and completion of the molt to second instar.

In controlled experiments utilizing artificial infestations, a topical formulation of ivermectin (IVOMEC Pour-On for Cattle) was 100% effective against migrating first instar Hypoderma lineatum for 3 weeks following treatment. Larvae were apparently killed early in the infestation as no significant level of specific antibodies was found in the treated calves. At 4 weeks post-treatment the efficacy remained high at 96%; mortality of larvae in the one calf in which warbles were observed and from which mature third instars were collected, was higher than that seen in the untreated calves, indicating some level of treatment induced effect. No specific antibodies were detected in calves that did not develop palpable warbles. Antibody kinetics in those calves from which viable larvae emerged were typical. The length of activity of this product against early stages of the cattle grub makes it practical to apply treatment up to 3 weeks before the end of fly activity.

The investigation was carried out in Basilicata region (Southern Italy) from October 1997 to June 1998. Fifteen dairy cows bred in semiconfined conditions on a farm with a history of hypodermosis were sampled once a month for sera and milk; bulk milk from these animals was also collected monthly from the farm's tanker. Samples were tested for anti-Hypoderma spp. antibodies (Abs) with an ELISA technique and clinical parasitological examination was carried out monthly from January to July on all the animals in order to detect grubs. Blood and single and bulk milk samples yielded similar antibody kinetics and patterns in accordance with results obtained in previous immunological surveys in Italy. All animals were warbled in the spring time. November-January was confirmed to be the most suitable period for seroepidemiological survey for weather conditions in Southern Italy. The ELISA test proved once again to be very useful because it is simple to perform and cost effective. Either blood or milk samples may be used for epidemiological surveys; bulk milk may be very useful for the preliminary detection of hypodermosis on farms or in areas where there is no data available on the diffusion of the disease.

Stage specific survival of larvae and pupae of the common cattle grub, Hypoderma lineatum de Villers, was compared in cattle artificially infested with larvae delivered by subcutaneous injection as a single infestation of 100 larvae, in trickle infestations of either two doses of 50 larvae, separated by 15 days, or in four doses of 25 larvae, separated by 9, 6, and 6 days, respectively. The duration of the migratory and 'warble' phases were also compared between treatments. The kinetics of the antibody response was monitored throughout the larval development by ELISA. No difference was noted in the level of antibodies among the three treatments. Mortality of first-instars differed among the infestation groups with mortality in the single pulse infestation group

Pasteurised and ultra high temperature (UHT)-treated milk were tested over the period November 1997-February 1998 in order to evaluate the applicability of an hypodermosis ELISA test on commercial milk samples. Milk samples from six dairy companies were analysed and the development of anti-Hypoderma antibodies recorded for the period from September 1998 to June 1999. For one dairy (no. 3) bulk milk samples were also obtained from the tankers, transporting milk away from the farms, and analysed; the results were compared to the parasitological status of the cows whose milk had been collected on the farms. Out of 32 pasteurised samples tested, 23 were positive, exceeding the cut-off (22%), while 9 samples were negative. UHT milk was always negative. The antibody levels in milk samples from four of the six companies were highest during January and decreased gradually during February to May 1999. The bulk milk samples also had high antibody levels and 47.2% of milk producing cows had lesions of hypodermosis. It was concluded that testing commercial milk for antibodies is an efficient way of detecting the presence of hypodermosis in cattle, especially in those countries for which no data on this disease are available.

The cDNA encoding the entire mature hypodermin C (HC) of Hypoderma lineatum was cloned and expressed in Escherichia coli as a glutathione S-transferase fusion protein using pGEX vector. The recombinant HC protein (rHC) was tested by Western blotting to detect antibodies to H. lineatum in cattle. Western blotting with rHC as antigen clearly differentiated between H. lineatum-infested cattle sera and normal cattle sera. Forty-six out of forty-eight serum samples from cattle in Central Mongolia were positive, whereas all 30 serum samples from cows in Hokkaido, Japan, were negative by Western blotting. The result of Western blotting was identical to that of a previously developed enzyme-linked immunosorbent assay. These data demonstrated that Western blotting, with rHC expressed in E. coli, might be a useful method for the diagnosis of cattle hypodermosis.

An infestation with Hypoderma diana in roe deer in the Netherlands is described. The condition of the infested deer was poor. Hypoderma diana is no threat for cattle.

The dynamics of the anti-Hypoderma antibody response after a treatment with injectable and pour-on ivermectin against first instars of Hypoderma sp. were determined in naturally infested cows. Cattle were randomly assigned to three groups: group 1, which served as an untreated control; group 2, treated with pour-on ivermectin at a dose of 500 microg/kg of body weight, and group 3, which received 200 microg/kg of body weight of ivermectin by subcutaneous injection. Both ivermectins showed total efficacy against L-1 of Hypoderma sp. In untreated control animals, grubs became detectable in the 2 mo after treatment. Indirect enzyme-linked immunosorbent assay (ELISA) showed a significant increase in group 3 antibody levels 1 mo after treatment, whereas in group 2 the increase was not significant. Following the peak of anitibody activity in group 3, antibody levels declined at a faster rate than in those treated topically. In both treated groups, cattle remained serologically positive for 4 mo after ivermectin treatment. Thus, serological surveillance programs for cattle grubs conducted during this posttreatment period will not be influenced by treatment with ivermectin. The persistence of antibody levels after larval destruction makes the indirect ELISA unsuitable for the prediction of the efficacy of treatment.

Hypodermosis in red deer, Cervus elaphus Linnaeus (Artilodactyla: Cervidae), caused by the warble fly, Hypoderma actaeon Latreille (Diptera: Oestridae), was studied at Quintos de Mora (Toledo, Spain) over a one-year period between October 1994 and September 1995. One hundred and twenty-five red deer were examined for the presence of warble fly larvae. The prevalence of warbles was 44.8% and the intensity of infection was 38.29 (SD +/- 61.32) warbles/deer infected. Clear seasonal variations were observed in prevalence and intensity, with the highest values in the autumn and winter. Statistically significant differences were observed in prevalence and intensity among age groups. In contrast, no differences were found in prevalence and intensity between males and females.

The experiments were carried out on 285 cows from 10 herds from different regions of Poland. Extensity of the H. bovis invasion in each herd varied from 10 to 86%. The highest extensity appeared in herds from eastern and north-eastern Poland and the lowest in herds from southern and central regions of the country. Seroconversion of Hypoderma bovis antibodies was calculated for animals in which warbles were detected by clinical examination. The highest serum densities were observed in herds in March, April and May. The results of these investigations demonstrated that the best term for collecting blood samples to examine cattle for hypodermosis is the winter-spring season.

The aim of these investigations was to prepare the antigen and control sera for the ELISA. Positive control sera were obtained from 163 cows showing clinical symptoms of H. bovis infection and negative control sera from 60 cows free from the infection. The antigen for the ELISA was obtained from H. bovis L1 larvae. Three basic fraction were isolated from whole excreted/secreted antigen by elution from gel and by preparative isoelectrofocusing. Using the Western-Blotting method it was demonstrated that Hy A (22-24 kDa and pI 4-5) had the best antigenic property. As can be seen from these investigations the developed ELISA kit for the detection of H. bovis antibodies in cows is highly specific and sensitive.

The aim of investigations was to determine the stability of ELISA components preserved under different conditions. The following components for the ELISA were used: the secreted/excreted antigen prepared from H. bovis L1 larvae, its fraction containing hypodermine A, conjugat, positive and negative control sera. Freezing at -20 degrees C, lyophilization and preservation at 4 degrees C were applied. Seven kits for the ELISA containing components preserved using described methods, were prepared. The identical packets of sera were examined by the ELISA directly after preparation of the kits (day '0'), and next, at 3-4 week intervals for 9 months. The experiments showed that the best method of conservation of ELISA components for the detection of H. bovis antibodies was lyophilization. The kits prepared in this way did not lose their characteristics for 9 months and their specificity and sensitivity were close 100%.

In two test areas in Austria (Burgenland, Lower Austria) ivermectin has been used to control oestrinosis and hypodermosis in red- and roe deer since many years (1985 and 1987, respectively). The results until 1992, using the injectable solution Ivomec-injection 1% only, have been reported (Kutzer, 1994). In continuation of these tests Ivomec-Praemix 0.6% was examined and it turned out that the treatment was extremely successful. In practice Ivomec-Praemix 0.6% has to be given preference due to its stability. The most favourable dosage for red- and roe deer was 2 x 0.4 mg ivermectin/kg bodyweight administered at an interval of one week, although equally good results could be obtained with 1 x 0.3-0.4 mg ivermectin/kg bodyweight. With the above mentioned dosages against Pharyngomyia picta and Cephenemyia stimulator an up to 100% and against Hypoderma actaeon a 100% success of treatment could be achieved. The administration in two doses has the advantage to eradicate intestinal and lung nematodes at the same time if there are any. Finally, it was found that the controlling measures should not be interrupted for more than one year, if no treatment was done in the neighbouring hunting grounds.

An experimental survey was carried out in western Spain to investigate both the chronobiology of Hypoderma spp. and the immunoresponse of their bovine hosts. This study was initiated with a new system of obtaining Hypoderma spp. larvae directly from their host, including the eclosion of adults from their pupae, infestation under natural but controlled conditions, and confirmation of the resulting infection. This survey was carried out over 2 cattle grub seasons; it was possible to infest and reinfest the experimental animals and to monitor them by both parasitological methods and by an enzyme-linked immunosorbent assay. This method permitted the evaluation of the development of anti-Hypoderma antibodies during the experiment. The experimental design also enabled us to establish the period of detectable H. lineatum infection to be from December until the end of April with the largest number of warbles observed during March and April. After a pupal period of < 30 d, adults were seen in April and May. Hypoderma bovis (de Geer) showed a delay of 2 m.o. relative to H. lineatum (de Villiers). This study reports a completed biological life cycle of Hypoderma spp. under controlled conditions in both natural and experimental environments.

An indirect ELISA test using as antigen a recombinant parasite protein, hypodermin C, was developed to measure Hypoderma-specific antibodies in cattle sera and compared with natural hypodermin C. To evaluate the field efficacy of the ELISA test, 334 serum samples were collected from cows raised at farms in Galicia for a serological survey. Compared with an ELISA based on natural parasite antigen, the recombinant hypodermin C gave excellent results, with a sensitivity of 95.8% and a specificity of 95.7%. Considering the cut-off point, with the recombinant hypodermin C, 70.9% of the animals had positive levels of antibodies to Hypoderma and with natural hypodermin C, 73.6%. Recombinant hypodermin C appears to be a useful alternative to the natural parasite antigen for the serodiagnosis of Hypoderma sp in cattle.

Between 1989 and 1997 different factors related to warble fly infestation (prevalence, intensity, climate, breed) were studied in north-eastern Algeria. Mean prevalence during the period was 76%. This figure was obtained from a cattle market survey of four different regions (Jijel, Constantine, Guelma, and El Tarf). One-hundred percent of the herds were found to be infested in El Tarf and 61% of animals were infested. Within a herd, the intensity of infestation decreased with the age of the cattle. The mean number of warbles per animal decreased as reinfestations occurred. The peak of the emergence of warbles observed between January and March was progressively delayed in the older animals. Two species of Hypoderma were present in cattle in Algeria (H. bovis and H. lineatum) with a predominance of H. lineatum (63%) in the semi-arid area (Batna) and a majority of H. bovis (75%) in humid area (Jijel). Warbles appeared earlier in Batna (October) than in Jijel (November). The Prim'Holstein breed was more susceptible to the warble fly infestation than Montbeliard or local breeds. The implementation of a control programme for hypodermosis in Algeria is urgent. Control can be achieved through a mass treatment of cattle. This approach must take into account the factors described in this study which influence the epidemiology of the disease.