Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro-CT.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro-computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a 'glandular band' by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio-opaque concretions. Moreover, the anatomy of O. ovis third-instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a 'band' and anterior Malpighian tubules narrowly uniform throughout their entire length.

Internal morphological changes during metamorphosis in the sheep nasal bot fly, Oestrus ovis.

During the larval stage, oestrid flies (Diptera: Oestridae) are obligate parasites, whereas during the adult stage they are free-living and do not feed. Like other cyclorrhaphous flies, oestrids undergo metamorphosis inside an opaque puparium, formed by the contracted and hardened cuticle of the third-instar larva. The present study documents the internal morphological changes taking place during metamorphosis of the sheep nasal bot fly, Oestrus ovis L., using non-invasive, micro-CT-based virtual histology and provides quantitative data of volumetric changes in specific organs. Virtual histological sections allowed visualisation of the progression and completion of the apolyses, which delimit the different intra-puparial stages, and the connection to the tracheal system of a large gas bubble, which plays an essential role during early metamorphosis. Overall, our results show that the sequence of morphological and volumetric changes in tissues and organs is similar to those found in other cyclorrhaphous flies, but they also reveal developmental differences that result in an adult vestigial digestive tract. Future studies could develop non-invasive, reliable methods for aging the intra-puparial forms of different oestrid species of veterinary importance, based on both qualitative and quantitative markers, thus improving our knowledge of their development and the efficiency of control strategies.

Micro-computed tomography visualization of the vestigial alimentary canal in adult oestrid flies.

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage as they acquire all necessary nutrients during the parasitic larval stage. The adult mouthparts and digestive tract are therefore frequently vestigial; however, morphological data on the alimentary canal in adult oestrid flies are scarce and a proper visualization of this organ system within the adult body is lacking. The present work visualizes the morphology of the alimentary canal in adults of two oestrid species, Oestrus ovis L. and Hypoderma lineatum (de Villiers), with the use of non-invasive micro-computed tomography (micro-CT) and compares it with the highly developed alimentary canal of the blow fly Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae). Both O. ovis and H. lineatum adults showed significant reductions of the cardia and the diameter of the digestive tract, an absence of the helicoidal portion of the midgut typical of other cyclorrhaphous flies, and a lack of crop and salivary glands. Given the current interest in the alimentary canal in adult dipterans in biomedical and developmental biology studies, further understanding of the morphology and development of this organ system in adult oestrids may provide valuable new insights in several areas of research.

A look into the Medical and Veterinary Entomology crystal ball.

Medical and Veterinary Entomology (MVE) represents a leading periodical in its field and covers many aspects of the biology and control of insects, ticks, mites and other arthropods of medical and veterinary importance. Since the first issue of the journal, researchers working in both developed and developing countries have published in MVE, with direct impact on current knowledge in the field. An increasing number of articles dealing with the epidemiology and transmission of vector-borne pathogens have been published in MVE, reflecting rapid changes in vector distribution, pathogen transmission and host-arthropod interactions. This article represents a gaze into the crystal ball in which we identify areas of increasing interest, discuss the main changes that have occurred in the epidemiology of parasitic arthropods since the first issue of MVE, and predict the principal scientific topics that might arise in the next 25 years for scientists working in medical and veterinary entomology.

The immunobiology of myiasis infections--whatever happened to vaccination?

The current state of myiasis vaccine technologies are reviewed mainly in the primary research genera of Lucilia and Hypoderma. The importance of myiasis flies as primary causes of morbidity and mortality in agricultural species and man has not diminished despite the existence of good control strategies. However, the development of vaccines against myiasis infections has been relatively quiescent for more than 10 years despite the rapid development of genomic and proteomic analysis and of skills in data interpretation. The value of vaccine research in an era of chemical primacy is analysed. In fact, recent findings of drug resistance and the impact of animal welfare concerns should mean a renewed interest in alternative controls. The reasons that this has not been true to date are explored and new possibilities discussed.

Life history parameters of the cattle long-nosed sucking louse, Linognathus vituli.

Cattle sucking lice, Linognathus vituli (L.) (Phthiraptera: Linognathidae), were obtained from naturally infected cattle and maintained within 'arenas' affixed to the backs of cattle confined in controlled environment chambers maintained at a constant temperature of 15 °C. Temperatures measured within the arenas at an ambient temperature of 15 °C were constant at about 34 °C and only slightly above the temperature on nearby skin. The effect of temperature on egg development was determined using a gradient of temperatures between 25 °C and 41 °C. Eggs did not develop at temperatures of < 26 °C or > 39 °C. Survival of eggs was highest at temperatures of 30 °C and 35 °C. The earliest hatch was observed at 5 days post-oviposition (at 33-35 °C). Development was extended to as long as 13 days at the lower temperatures. Kaplan-Meier survival probabilities were compared for lice kept at two densities in the arenas and showed there to be no effect of density on louse survival. Similarly, the mean number of eggs/louse/day over an 8-day period was not influenced by louse density.

Immunohistochemical characterization of inflammatory cells in the skin of cattle undergoing repeated infestations with Hypoderma lineatum (Diptera: Oestridae) larvae.

Cutaneous T- and B-cell responses were studied in three groups of cattle (n=4) after a primary (G1), secondary (G2) and tertiary (G3) experimental infestation with Hypoderma lineatum first instars (L1). Cattle were each infested with 25 L1 deposited onto the skin. Skin biopsies were taken at 0, 6, 12, 48, 96 and 144 h post infestation (hpi). The kinetics of infiltration of CD3(+), CD4(+) and CD8(+) T lymphocytes and B cells at the infestation site was evaluated immunohistochemically. The number and pattern of infiltration of CD3(+) T cells were similar in the different groups of animals, showing a progressive increase until 96 hpi. The number of CD4(+) T helper cells increased significantly at 96, 6 and 48 hpi in G1, G2 and G3, respectively. CD8(+) cytotoxic T-cell infiltration was similar in the different groups, except at 48 hpi when there was a significant difference between G1 and G3. The CD4:CD8 ratio showed predominance of CD4(+) cells throughout the response. The number of B cells increased with the number of previous infestations, with significant differences between G1 and G3 at 12 hpi. All of the cell types concentrated around the superficial and deep dermal vessels. During primary larval infestations there was a predominance of CD4(+) cells, whereas in cattle sensitized by previous infestations, B cells were most abundant in the infiltrate. These findings suggest that humoral immunity may play a significant role in bovine immunity to H. lineatum.

Evaluation of diagnostic techniques for Anoplocephala perfoliata in horses from Alberta, Canada.

Accurate diagnosis of parasites within individual hosts remains a difficult task. Incorrect and/or inaccurate diagnosis restricts the potential for targeted treatment of individuals and limits our understanding of key epidemiological characteristics of potential pathogens of domestic stock. In this study, we compared the specificity and sensitivity of four diagnostic methods for determination of the presence and intensity of the cestode Anoplocephala perfoliata in horses. Over 400 horses from an abattoir in south-central Alberta were evaluated for the presence and number of cestodes. Thirty one horses were infected with 1 to >1000 worms. Diagnosis based upon faecal egg counts of horses with known numbers of worms was least accurate in detecting worm presence. Detection of circulating antibodies to the cestode was most sensitive using Western blot analysis (100%), but had lower specificity (87%). A serum-based ELISA had a lower sensitivity (70%) for detection of antibodies. A coproantigen ELISA had 74% sensitivity and 92% specificity, and there was a positive correlation between antigen concentration and tapeworm intensity. The coproantigen ELISA may have the potential as a diagnostic tool for determining the presence and intensity of this potentially pathogenic cestode.

The mitochondrial genome of the common cattle grub, Hypoderma lineatum.

The mitochondrial DNA of the cattle grub Hypoderma lineatum (de Villers) (Diptera: Oestridae) was completely sequenced. The entire molecule was 16,354 bp long and presented a heavy bias towards A + T, which accounted for 77.8% of the whole genome. Hypoderma lineatum genes were organized in the same order and orientation as in the mitochondrial genomes available for other species belonging to the Oestroidea superfamily and compared in this study [Chrysomya putoria (Wiedemann), Cochliomyia hominivorax (Coquerel), Lucilia sericata (Meigen) and Dermatobia hominis (L.)], except for the occurrence of a 102-bp non-coding region partially present in other species. The complete sequence of H. lineatum will represent a useful dataset to evaluate the evolutionary pattern of mtDNA within Oestroidea by using molecular information in diagnostic, taxonomic and evolutionary studies.

Local and systemic cytokine responses during larval penetration in cattle experimentally infested with Hypoderma lineatum (Diptera: Oestridae).

Local and systemic cytokine responses were studied in 3 groups of cattle, with 4 animals each, experimentally infested with Hypoderma lineatum (De Villers) first instars (L1). The first group was undergoing a primary infestation (G-1), the second group was undergoing a secondary infestation (G-2) and the third group was infested for their third consecutive year (G-3). Cattle were infested with 25 L1 deposited on the skin. Blood and skin samples were taken at 0, 6, 12, 48, 96 and 144h post-infestation (h.p.i.). Interleukin 10 (IL-10), IL-4 and interferon gamma (IFN-gamma) production was studied by immunohistochemistry and sandwich ELISAs. IL-4(+) cells showed a significant increase at 6h.p.i. in both reinfested groups (G-2 and G-3) when compared with G-1. In all groups the number of IL-4(+) cells decreased significantly at 48h.p.i. IL-10(+) cells increased in G-1 at 6 and 48h.p.i., whereas in both reinfested groups increased at 12h.p.i. with a peak at 48h.p.i. IFN-gamma(+) cells showed a significant increment at 6h.p.i. in all groups, followed by a rapid descent at 12 (G-1 and G-2) and 48h.p.i. (G-3). Penetration of the skin by H. lineatum did not have any significant effect on IFN-gamma serum concentrations and, except for IL-10 there were no correlation between local production and serum concentrations of cytokines. The increase of both Th1 (IFN-gamma) and Th2-type cytokines (IL-4 and IL-10) indicates that bovine T-cell response during the first phases of the infestation by H. lineatum is apparently a Th0 response.

Biodiversity and extinction versus control of oestrid causing myiasis in Mediterranean area.

Oestrid larvae causing myiasis display a wide degree of biodiversity, in terms of species of domestic and wild mammals infected and anatomical sites. The presence in some regions of southern Europe of a high number of different species of oestrids in domestic animals stimulated interest in exploring the basis of such degree of parasitic biodiversity in the Mediterranean region. However, broad spectrum anti-parasitic treatments (e.g. macrocyclic lactones) constitute a critical factor for the selection of species of Oestrids and for the maintenance of their biodiversity in a given area. The dynamic equilibrium that oestrid larvae have established with the host and the environment as well as the span of biodiversity they represent may be considered to be at odds with maintaining animal welfare and reducing animal production losses.

Comparative scanning electron microscopy of Gasterophilus third instars.

Comparison of cuticular features, including spine distribution and shape, structure of the maxillae and mandibles, the cephalic sensillae and the terminal abdominal segments of third instar Gasterophilus haemorrhoidalis, Gasterophilus inermis, Gasterophilus intestinalis, Gasterophilus meridionalis, Gasterophilus nasalis and Gasterophilus pecorum was conducted using scanning electron microscopy. One or more features distinguished among the species, with the exception of G. haemorrhoidalis and G. intestinalis, which shared all morphological characteristics. The features presented in this study complement and extend those presented in Zumpt's compendium. The function of some features described by Zumpt (e.g. the 'warts' on the rim of the respiratory chamber) is clarified and the presence of previously described sensory structures in the grooves on the maxillae of G. intestinalis is called into question.

Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions.

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.

First description of the endogenous life cycle of Hypoderma sinense affecting yaks and cattle in China.

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.

Control of the cattle louse Bovicola bovis with the fungal pathogen Metarhizium anisopliae.

The effects of the entomopathogenic fungus Metarhizium anisopliae (Metschnikoff) were evaluated against the common louse parasite of cattle, Bovicola bovis (Piaget) (Trichodectidae: Ischnocera). Two different concentrations and formulations of conidial suspensions were applied to contained populations of adult female lice. In vitro, lice immersed in suspensions of M. anisopliae formulated in 0.03% Tween 80 developed infections; at the highest concentration (1x10(8)conidia ml-1) a mean of 71% (+/-11.52%, 95% C.I.) of lice became infected. Lice exposed to the Tween 80 only in vitro, showed high levels of survival and zero infection. In vivo, fungal conidia were applied to louse populations contained in 7 cm diameter circular arenas glued to the backs of Holstein cattle, maintained in controlled climate conditions. Conidia were formulated in either Tween 80 or silicone oil. The treatment with M. anisopliae resulted in high levels of infection and there was no overall difference between the two formulations in the number of infections observed. At the highest concentration (1x10(8)conidia ml-1) a mean of 73% (+/-15.57%, 95% C.I.) lice became infected. It is concluded that the strategic seasonal use of a fungal pathogen on cattle, applied in early winter, may be of value in suppressing the winter increase in abundance, preventing the population increasing to clinically significant levels.

Hypoderma sinense: solving a century-old enigma.

Among the species of Hypoderma (Diptera: Oestridae) that have been described and named over the last three centuries, Hypoderma sinense Pleske has been the subject of several scientific discussions. Hypoderma sinense was described by T. Pleske in 1926 on the basis of only three females collected by the Russian explorer P. K. Kozlov nearly 25 years earlier during a scientific expedition to China (1900-1901). This species was examined by the foremost oestrid authorities and synonomized with H. lineatum. Recently a unique, unidentified species of Hypoderma was observed to infect cattle and yaks in China. Molecular and morphological observations confirmed the unique nature of the third-stage larvae. This data initiated a debate within the scientific community regarding the proper name of this species, in particular with reference to previous taxonomical discussion on the validity of H. sinense. The present work provides a historical overview of the Russian scientific expeditions that collected the specimens and of the explorers and the entomologists who contributed to the description of H. sinense. The morphological examination of the original type material of H. sinense and the comparison with females of H. lineatum indicated that the H. sinense lectotype, deposited at the Zoological Institute of the Russian Academy of Sciences, St Petersburg, was within the range of variation of H. lineatum. Comparisons of the cox1 (688 bp) sequence obtained from the leg of a paralectotype of H. sinense with those of H. bovis (Linneaus), H. lineatum (De Villers) and of a sixth valid species of Hypoderma identified as "H. sinense" available in GenBank revealed differences of 9.7%, 7.2% and 0.3%, respectively. On the basis of these results, we concluded that the nominal species H. sinense should be treated as valid.

Skin immune responses in cattle after primary and secondary infections with Hypoderma lineatum (Diptera: Oestridae) larvae.

Cellular immune responses were examined in the skin of cattle after primary and secondary experimental infections with Hypoderma lineatum larvae. Skin biopsies were taken at 0, 6, 12, 48 and 96 h post-infection (h.p.i.). In primary infected animals the penetration of Hypoderma larvae was characterized by moderate inflammatory responses. The pattern of cellular changes in previously infected animals suggested an allergic or rheumatic process, probably as a consequence of the development of a type III hypersensitivity reaction against the second or successive reinfections. Perivascular infiltration with CD3(+) T lymphocytes was marked in infected groups, but especially in previously infected animals, with a significant increase with respect to uninfected controls at 48 h.p.i. B cells remained close to control values during primary infection, while they increased significantly 12 h.p.i. in reinfected animals. IgG(+) plasma cells were also very abundant during secondary infections, with significant differences from primary infected and uninfected animals from 6 h.p.i. onwards. These results suggest that secondary infections allow cellular responses which may be effective in killing some of the entering larvae, resulting in a degree of resistance.

Morphological variability and genetic identity in Rhinoestrus spp. causing horse nasal myiasis.

Larvae of Rhinoestrus purpureus (Brauer) and Rhinoestrus usbekistanicus Gan (Diptera: Oestridae) cause nasal myiases of equids. During a recent epidemiological survey in southern Italy some morphological and taxonomical doubts arose concerning the identification of Rhinoestrus third stage larvae on the basis of the features of the posterior spiracles and the distribution of dorsal spines on the third segment. Four different morphotypes were retrieved: R. usbekistanicus-like, R. purpureus-like and two morphotypes with shared features. The genes encoding for the mitochondrial cytochrome oxidase I (COI) and for the ribosomal subunits 16S and 28S of the four morphotypes of Rhinoestrus were investigated to determine whether they belonged to a single taxon or they displayed genetic differences indicative of more than one species. The three genes showed a very low level of sequence variation (COI 0-0.43%, 16S 0-1.45%, 28S 0-0.23%) falling within the intraspecific ranges previously described for Oestridae species. Finally, the peritreme features and the spinulation of the third segment of the four morphotypes examined could not be used to differentiate the two species.

On the ubiquity and phylogeny of Wolbachia in lice.

Wolbachia are intracellular bacteria that occur in an estimated 20% of arthropod species. They are of broad interest because they profoundly affect the reproductive fitness of diverse host taxa. Here we document the apparent ubiquity and diversity of Wolbachia in the insect orders Anoplura (sucking lice) and Mallophaga (chewing lice), by detecting single or multiple infections in each of 25 tested populations of lice, representing 19 species from 15 genera spanning eight taxonomic families. Phylogenetic analyses indicate a high diversity of Wolbachia in lice, as evidenced by the identification of 39 unique strains. Some of these strains are apparently unique to lice, whereas others are similar to strains that infect other insect taxa. Wolbachia are transmitted from infected females to their offspring via egg cytoplasm, such that similar species of lice are predicted to have similar strains of Wolbachia. This predicted pattern is not supported in the current study and may reflect multiple events of recent horizontal transmission between host species. At present, there is no known mechanism that would allow for this latter mode of transmission to and within species of lice.

A third species of Hypoderma (Diptera: Oestridae) affecting cattle and yaks in China: molecular and morphological evidence.

Cattle and yak hypodermosis in China is caused by Hypoderma bovis and H. lineatum, with a prevalence reaching up to 98-100% of the animals and maximum intensities exceeding 400 warbles for each animal. A third species, H. sinense, is also considered by Chinese researchers to affect livestock. The molecular characterization of the most variable region of the mitochondrial cytochrome oxidase I gene and of the ribosomal 28S gene has been performed for the third-stage larvae collected from cattle and yaks in China and identified (on the basis of the spinulation on the ventral side of the 10th segment) as H. bovis, H. lineatum, and H. sinense. Amplicons were digested with the HinfI and BfaI restriction enzymes, which provided diagnostic profiles to simultaneously differentiate the 3 Hypoderma species. Third-stage larvae of H. sinense were also examined by scanning electron microscopy, which revealed proper morphological characteristics different from those of H. bovis and H. lineatum. The molecular and morphological evidence herein reported support the existence of a third species of Hypoderma affecting cattle and yaks in China, and the results provide new tools for unequivocal identification of this species and present key components for the evaluation of its endogenous cycle and pathogenicity in animals and humans.