Long Time No Hear, Magnificent Wohlfahrtia! Morphological and Molecular Evidence of Almost Forgotten Flesh Fly in Serbia and Western Balkans.

The "beautiful viviparous fly", Wohlfahrtia magnifica, may have a magnificent appearance due to its striking morphology; however, it is a potentially deadly agent of obligate traumatic myiasis in humans and animals, with a serious impact on welfare and economics. The fly is found across the Palearctic realm, including the Western Balkan region, with reports from former Yugoslavian countries from the first half of the 20th century. In this paper, a recent case of wohlfahrtiosis recorded in Northern Serbia is evidenced using morphological and molecular techniques. Larvae were collected from two adult sheep with severe hoof myiasis and two young sheep with genital and interdigital myiasis. Morphological identification was performed for adults bred from the infested vulva and third-stage larvae (L3) collected from the hoof wounds, supported with barcoding sequences of the COI gene obtained from larval pairs from the hoof wounds of older and the genitalia of younger sheep. W. magnifica was identified according to the appearance of male fly terminalia and the morphology of L3, which was confirmed after the comparison of representative sequences of the COI gene (deposited in GenBank™ under accession numbers MT027108-MT027114) to those available in GenBank™. This finding represents the first reported case of wohlfahrtiosis in the Western Balkans in 80 years, highlighting the need to re-inform relevant stakeholders to achieve adequate disease control.

Transcriptomic profiling of different developmental stages reveals parasitic strategies of Wohlfahrtia magnifica, a myiasis-causing flesh fly.

BACKGROUND: Wohlfahrtia magnifica is an obligatory parasite that causes myiasis in several warm-blooded vertebrates. Adult females deposit the first-stage larvae directly onto wounds or natural body orifices (e.g., genitalia) of the host, from where they quickly colonize the host tissue and feed on it for development. The infestation of W. magnifica can lead to health issues, welfare concerns, and substantial economic losses. To date, little is known about the molecular mechanisms of the W. magnifica-causing myiasis. RESULTS: In this study, we collected parasitic-stage larvae of W. magnifica from wounds of naturally infested Bactrian camels, as well as pupae and adult flies reared in vitro from the wound-collected larvae, for investigating the gene expression profiles of the different developmental stages of W. magnifica, with a particular focus on examining gene families closely related to the parasitism of the wound-collected larvae. As key proteins related to the parasite-host interaction, 2049 excretory/secretory (ES) proteins were identified in W. magnifica through the integration of multiple bioinformatics approaches. Functional analysis indicates that these ES proteins are primarily involved in cuticle development, peptidase activity, immune response, and metabolic processes. The global investigation of gene expression at different developmental stages using pairwise comparisons and weighted correlation network analysis (WGCNA) showed that the upregulated genes during second-stage larvae were related to cuticle development, peptidase activity, and RNA transcription and translation; during third-stage larvae to peptidase inhibitor activity and nutrient reservoir activity; during pupae to cell and tissue morphogenesis and cell and tissue development; and during adult flies to signal perception, many of them involved in light perception, and adult behavior, e.g., feeding, mating, and locomotion. Specifically, the expression level analysis of the likely parasitism-related genes in parasitic wound-collected larvae revealed a significant upregulation of 88 peptidase genes (including 47 serine peptidase genes), 110 cuticle protein genes, and 21 heat shock protein (hsp) genes. Interestingly, the expression of 2 antimicrobial peptide (AMP) genes, including 1 defensin and 1 diptericin, was also upregulated in the parasitic larvae. CONCLUSIONS: We identified ES proteins in W. magnifica and investigated their functional distribution. In addition, gene expression profiles at different developmental stages of W. magnifica were examined. Specifically, we focused on gene families closely related to parasitism of wound-collected larvae. These findings shed light on the molecular mechanisms underlying the life cycle of the myiasis-causing fly, especially during the parasitic larval stages, and provide guidance for the development of control measures against W. magnifica.

Genome and Transcriptome Analyses Facilitate Genetic Control of Wohlfahrtia magnifica, a Myiasis-Causing Flesh Fly.

Myiasis caused by Wohlfahrtia magnifica is a widespread parasitic infestation in mammals. The infested host suffers from damage as the developing larvae feed on its tissues. For the control of myiasis infestation, genetic methods have been shown to be effective and promising as an alternative to insecticides. Combining genome, isoform sequencing (Iso-Seq), and RNA sequencing (RNA-seq) data, we isolated and characterized two sex-determination genes, W. magnifica transformer (Wmtra) and W. magnifica transformer2 (Wmtra2), whose orthologs in a number of insect pests have been utilized to develop genetic control approaches. Wmtra transcripts are sex-specifically spliced; only the female transcript encodes a full-length functional protein, while the male transcript encodes a truncated and non-functional polypeptide due to the presence of the male-specific exon containing multiple in-frame stop codons. The existence of five predicted TRA/TRA2 binding sites in the male-specific exon and the surrounding intron of Wmtra, as well as the presence of an RNA-recognition motif in WmTRA2 may suggest the auto-regulation of Wmtra by its own protein interacting with WmTRA2. This results in the skipping of the male-specific exon and translation of the full-length functional protein only in females. Our comparative study in dipteran species showed that both the WmTRA and WmTRA2 proteins exhibit a high degree of similarity to their orthologs in the myiasis-causing blow flies. Additionally, transcriptome profiling performed between adult females and adult males reported 657 upregulated and 365 downregulated genes. Functional analysis showed that among upregulated genes those related to meiosis and mitosis Gene Ontology (GO) terms were enriched, while, among downregulated genes, those related to muscle cell development and aerobic metabolic processes were enriched. Among the female-biased gene set, we detected five candidate genes, vasa (vas), nanos (nanos), bicoid (bcd), Bicaudal C (BicC), and innexin5 (inx5). The promoters of these genes may be able to upregulate Cas9 expression in the germline in Cas9-based homing gene drive systems as established in some flies and mosquitoes. The isolation and characterization of these genes is an important step toward the development of genetic control programs against W. magnifica infestation.

Traumatic myiasis by Wohlfahrtia magnifica in sheep flocks from southeastern Spain: prevalence and risk factors.

Wohlfahrtia magnifica (Diptera: Sarcophagidae) is an obligate myiasis-causing fly of livestock that is widely distributed throughout the Mediterranean basin. A total of 73 683 sheep from 122 flocks in Albacete Province (southeastern [SE] Spain) were examined to detect active traumatic myiasis. The influence of several individual and flock factors on the prevalence was also assessed. The overall flock prevalence of traumatic myiasis was 95.9%, with an individual prevalence of 7.1%. Wohlfahrtia magnifica was identified in all of the positive animals except one that was infested by Lucilia sericata (Diptera: Calliphoridae). Adults of both genders showed a higher risk of infestation than juveniles, whereas males of all ages displayed an increased risk of infestation than females of all ages; the existence of a lambing building on the farm, poor husbandry facilities and the location area were also identified as risk factors. Prevalence was highest in the summer, and the fly season lasted from March to November. Genitalia were the main location of infestation in males (94.4%) and females (76.3%). Our results reveal that traumatic myiasis by W. magnifica is widespread in southeastern Spain, hence preventive measures minimizing the attractiveness of sheep to gravid flies are necessary to avoid health and welfare problems and significant production losses in sheep flocks.

Furuncular myiasis by Wohlfahrtia magnifica (Diptera: Sarcophagidae) in a healthy child

Rationale: Human myiasis is the invasion of tissue or organs by fly larvae. This could be obligatory, facultative, or accidental. Patient concerns: A 4-year-old Saudi boy complained of fever over the past three days with multiple inflamed painful dermal furuncles and worms-like discharge. Diagnosis: Furuncular obligatory myiasis caused by Wohlfahrtia magnifica. Interventions: Maggots were removed for identification. The wounds were cleaned with antiseptic dressings. Topical and oral antibiotics were applied. Outcomes: Seven days later, the wounds completely healed. Lessons: Although several reports correlated human myiasis with old age, low health status, mental retardation, and low socioeconomic status, but the patient in our case was a healthy child from a family with good socioeconomic status, good hygiene, no history of diseases or mental disability, but traveled to a village where the climate is suitable for fly breeding.

A Strange Gingival Orifice in an Infant: A Case of Oral Myiasis.

Myiasis is a parasitic infection of humans and animals caused by dipterous larvae and is often seen in rural areas. For the first time in the literature as a case of gingival myiasis in an 18-month-old healthy child with good oral hygiene is reported. This case presents the clinical and histopathologic features of oral myiasis. The patient had a swelling in the upper lip for three days and a perforation in the gingiva. After mechanical removal of larvae intraoral symptoms resolved within six days. The larva was identified as Wohlfahrtia magnifica according to the histopathologic evaluation. Dentists working in rural areas should be aware of this very rare condition and its treatment.

Emerging pathogens: A case of Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica bacteremia.

Wohlfahrtiimonas chitiniclastica and Ignatzschineria indica are rare causes of infection in humans and have been linked to infestation with fly larvae in open wounds. Both organisms are emerging causes of disease globally and co-infection resulting in bacteremia is rare. An 82-year-old male with bilateral lower extremity infections was hospitalized due to fall with associated right lower extremity pain. On exam, a maggot infested ulcer was identified on his right lower extremity. On day three of hospitalization, blood cultures grew gram-negative and gram-variable rods, and methicillin-resistant Staphylococcus aureus. Further analysis of the gram negative and gram variable rods revealed W. chitiniclastica and I. indica respectively. Both I. indica and W. chitiniclastica were pan sensitive to all antimicrobials tested with the exception of tetracyclines to which W. chitiniclastica was fully resistant and I. indica was intermediately sensitive. The patient was treated with two weeks of IV ceftriaxone and was discharged with plans to complete a six-week course of IV daptomycin due to MRSA bacteremia. All repeat blood cultures were negative. Until recently W. chitiniclastica and I. indica infections have been documented only in farm and feral animals. Major risk factors for infection include: poor hygiene, open wounds, peripheral vascular disease, and myiasis. Due to the rarity of infection, identification of both organisms can be difficult, therefore a high index of suspicion is required.

A formulation of neem and hypericum oily extract for the treatment of the wound myiasis by Wohlfahrtia magnifica in domestic animals.

Untreated, traumatic, or wound myiases can cause severe consequences to animal health and welfare as well as economic losses to livestock productions. For healing myiasis-caused wounds, disinfectant such as creolin is wrongly but currently used in association with insecticides. Though effective, creolin is highly toxic to the patients, is inadequate with respect to the repellent effect, and may delay the healing of treated wounds. In this uncontrolled study, the efficacy of the patented plant-derived formulation 1 Primary Wound Dressing© (1-PWD), composed of neem oil (Azadirachta indica A. Juss.) and the oily extract of Hypericum perforatum (L.) flowers, has been investigated. Forty-four domestic animals of different species suffering from wound myiasis lasting for up to 25 days, at different parts of the body, were enrolled in the study. No systemic or local antibiotic or disinfectants' treatment was administered. Larvae recovered on open wounds and adults reared from mature larvae were identified as Wohlfahrtia magnifica. All the treated wounds healed in a range of 10 to 32 days without further infestation. None of the recruited animals presented bacterial complications. Data herein presented indicate that the tested natural plant-derived formulation is able to manage the infestation caused by W. magnifica larvae and the healing process of traumatic infested wounds in several domestic animal species, without any side effect on the living tissue and without the need to use local or systemic chemical or other products.

Molecular identification and detection of Wohlfahrtia magnifica in ovine vulvar myiasis in Gansu, China.

Traumatic myiasis causes substantial economic losses to farmers worldwide. In the present study, six flocks of sheep (2261 sheep) were investigated in Gansu, China, and 207 of 552 larvae were genetically characterized based on three genes, including cyt b, EF-1α, and white gene, by polymerase chain reaction and sequence analysis. A survey of sheep in China revealed that the prevalence of vulvar myiasis of six sheep flocks was 5.00% (11/220, Flock1), 4.85% (10/206, Flock2), 4.50% (9/200, Flock3), 5.00% (15/300, Flock4), 4.68% (15/320, Flock5), 0% (0/1015, Flock6), respectively. The sequence and phylogenetic analysis showed that only Wohlfahrtia magnifica was detected in the field samples. This is the first report of ovine vulvar myiasis caused by W. magnifica in Gansu, China. Some prophylactic measures are strongly recommended to reduce the risk of sheep acquiring traumatic myiasis in Gansu, China.

First reports of nasal and traumatic myiasis infection in endangered Przewalski's horses (Equus ferus przewalskii).

Myiasis has great economic and medical importance. However, myiasis in wildlife that is caused by oestroid flies is relatively rarely recorded compared with that in humans and domestic animals. Recently, during our research on the conservation of Przewalski's horse (PH), we observed two new records of oestroid flies parasitizing wildlife in China. The first is the horse nasal bot fly, Rhinoestrus sp. (Diptera: Oestridae), found in a dead PH from Kalamaili Nature Reserve. One morphotype (R. purpureus-like) was identified. The second is the Wohlfahrt's wound myiasis fly, Wohlfahrtia magnifica (Schiner, 1862) (Diptera: Sarcophagidae), which was collected from an open wound of a PH in the Wild Horse Breeding Research Centre. These observations extend the records of known hosts of these two oestroid myiasis agents. To the knowledge of the authors, infestation by Rhinoestrus and Wohlfahrtia larvae causing myiasis in wildlife has not been reported in China previously.

Dataset of traumatic myiasis observed for three dominant screw worm species in North West Pakistan with first report of Wohlfahrtia magnifica (Schiner).

Regional surveys were carried out in different parts of North West Pakistan among domestic animals (N=57,921) including pets and livestock identifying cases of traumatic myiasis (n=1037). A total of four surveys focused general livestock population during Eid ul Adha (Eid surveys; incidence=1.21%) while another four surveys (Miscellaneous surveys; incidence=7.34%) targeted animal population brought to veterinary hospitals and dispensaries. Timeframe spanned four years from 2012 to 2015. Maggots were sampled and location of the wound was recorded for each host. Taxonomic identification used light and electron microscopic techniques. Our dataset shows three species as principle agents of myiasis (n=882) including Chrysomya bezziana Villeneuve (n=394), Wohlfahrtia magnifica (n=244) and Lucilia cuprina Wiedemann (n=244). Others (n=155) including Chrysomya megacephala (Fabricius), Chrysomya rufifacies (Macquart), Lucilia sericata (Meigen), Lucilia illustris (Meigen), Lucilia porphyrina (Walker), Hemipyrellia ligguriens (Wiedemann), Calliphora vicina (Robineau-Desvoidy), Sarcophaga crassipalpalis (Macquart) and Sarcophaga species were identified as species of minor importance. The obligatory screwworm species W. magnifica is a first report from Pakistan. The results based on this dataset are presented in a recent publication "Distribution Modeling of three screwworm species in the ecologically diverse landscape of North West Pakistan" (Zaidi et al., 2016) [1].

Periungual myiasis caused by wohlfahrtia magnifica mimicking an ingrown toenail.

Myiasis is the infestation of organs and tissues of human being or other vertebrate animals with dipterous larvae. Myiasis is generally rare in humans, especially in children. We present the first case of periungual myiasis by Wohlfahrtia magnifica in a 5-year-old female. Wohlfahrtia magnifica is an obligatory parasite, which has been described as cause of ophthalmomyiasis, otomyiasis, oral myiasis, vulvar myiasis and wound myiasis. Treatment of myiasis and ingrown toenail is generally non-operative although both entities can be serious and/or non-responsive to conservative therapy.

Canine Myiasis and Its Causal Agents in Northeastern Iran.

BACKGROUND: Myiasis is defined as the infestation of live human and vertebrate animals with dipterous larvae for a certain period. There are reports indicating that dogs are the most common species affected by myiasis. This study was conducted to identify myiasis-causing flies in owned and stray dogs in Mashhad (Northeastern Iran). METHODS: A total of 435 owned dogs and 800 stray dogs were examined for myiasis. Myiasis cases were cured and fly larvae were identified by microscopy using the relevant standard identification keys. RESULTS: Ten out of 435 owned dogs (2.29 %) and 18 out of 800 stray dogs (2.25 %) had myiasis. The causative agents of myiasis in dogs based on their frequencies were as follows: Wohlfahrtia magnifica (50%), Lucilia sericata (28.57%) and Chrysomya albiceps (21.42%). CONCLUSION: W. magnifica was the most important myiasis-causing fly among the dogs sampled here, sometimes causing very serious damages. However, when treatment was given early enough, the larvae removed and the wound disinfected, the animals usually made a full recovery.

Origins of Wohlfahrtia magnifica in Italy based on the identification of mitochondrial cytochrome b gene haplotypes.

To identify the geographical origins of larvae of Wohlfahrtia magnifica (Diptera: Sarcophagidae) causing myiasis of sheep in Italy, comparative DNA sequence analysis of the mitochondrial cytochrome b gene was performed, based on gene fragments amplified by PCR from genomic DNA isolated from individual specimens. DNA extractions of 19 larvae from Lazio, Molise, Puglia, and Sicilia generated 17 readable sequences homologous to 2 haplotypes, either CB_magn01 or CB_magn02; DNA extracts from 4 adult flies from Calabria (reared from larvae) produced 4 readable sequences belonging to the haplotype CB_magn01. The two haplotypes found represent both the East and West phylogenetic lineages of W. magnifica, which is consistent with the species' arrival from central/southeast Europe (East lineage) and/or from southwest Europe/northwest Africa (West lineage). This is the first report of the sympatric occurrence of the two lineages, which could have resulted from natural or human-assisted dispersal. Polymorphic nuclear loci will have to be characterized in order to explain the origins and lack of mitochondrial haplotype diversity of this pest in Italy, where it poses increasing veterinary problems.

Traumatic myiasis agents in Iran with introducing of new dominant species, Wohlfahrtia magnifica (Diptera: Sarcophagidae).

OBJECTIVE: To study agents of animal wound myiasis in various geographical districts of Fars province. METHODS: This study has been done in Fars province, located in the southern part of Iran. Sums of 10 358 domestic animals have been visited from April 2011 to March 2012. The infected wounds in any parts of animal body were sampled by means of forceps. RESULTS: About 61% of all animal wound myiasis were caused by larvae of Wohlfahrtia magnifica. The most wound myiasis cases due to this species occurred in central part of Fars province. There wasn't any significant difference between sheep and goat in infestation with myiasis (P>0.05). The infestation rate of myiasis in cattle community was 0.86%. CONCLUSIONS: The infestation rate of livestock was lower than other works in Iran and some other countries like Saudi Arabia. Chrysomya bezziana has been mentioned as main myiasis agent in Iran. But in this study it cleared that similarly to some European countries, the common animal myiasis agent in Iran is Wohlfahrtia magnifica. Introducing new species as principal agent for myiasis can help public health and animal husbandry policy makers to prepare sufficient and effective control and/or preventive measures for this disease.

Improved method for screening mitochondrial cytochrome b markers to identify regional populations of the Old World screwworm fly and other myiasis agents.

A new protocol was developed to overcome obstacles to the high-throughput sequence analysis of the 716-717 nucleotides at the carboxyl terminal of the mitochondrial gene cytochrome b (cyt b) of the myiasis flies Chrysomya bezziana and Wohlfahrtia magnifica. For both of these obligate parasites, cyt b haplotypes provide diagnostic markers for phylogeographic populations, markers that identify the origins of emerging populations causing economically important myiasis in livestock and, in the case of C. bezziana (Old World screwworm fly), could help select reproductively-compatible populations for use in the Sterile insect technique as part of area wide integrated pest management. High sequence quality is important for unambiguously detecting the few mutations that are diagnostic for regional cyt b haplotypes and their lineages. A key innovation is the design of a new forward primer for the specific PCR amplification and high-quality sequencing of cyt b. The improved protocol will facilitate the use of this established comparative cyt b sequence analysis, not only by teams lacking the resources for whole genome sequencing (WGS) but also by those requiring reference sequences for developing comparative mitogenomics based on WGS.

A retrospective and geographical epidemiological survey of traumatic myiasis in southern Italy.

A survey on the prevalence and geographical distribution of traumatic myiasis in sheep, and the risk factors for the disease, was carried out in a region of southern Italy. A total of 138 sheep flocks were selected and visited to acquire data on the presence or absence of traumatic myiasis using both a questionnaire for retrospective analysis and animal inspection. Prevalences registered at the farm and animal levels, respectively, were 8.7% and 6.3% in 2010, and 5.8% and 5.0% in 2011. Records of the occurrence of the parasitic disease in this region are recent: a statistically significant (P < 0.01) progressive increase in the number of farms affected (from 0.7% to 8.7%) has been registered since 2007. Wohlfahrtiosis was found in 11 of 95 (11.6%) geographical units sampled and three significant (P < 0.05) clusters of spatial farm aggregation were identified in the southern part of the study area. A total of 158 presently uninfested farms were considered to be at high risk for transmission as a result of their proximity to infested farms. The spreading of Wohlfahrtia magnifica (Diptera: Sarcophagidae) in southern regions of Italy represents a warning that the risk for infestation may become more significant in other Italian regions, as well as other European countries.

A report on the genital myiasis by Wohlfahrtia magnifica in camel herds in southwest of Iran.

Myiasis is a disease of vertebrate animals caused by different fly larvae. Wohlfahrtia magnifica is responsible for serious losses in animal husbandry in Eurasia. Larvae of W. magnifica parasitize several warm-blooded vertebrates and are responsible for a severe traumatic myiasis of mucosal membranes or wounds. This myiasis has been reported in many European areas, but for the first time was reported from Iran. Larvae infestation was observed in five camels out of 35 (14.28%) around the perinea and vaginal region of camels in a herd in southwest of Iran. The larvae samples were collected and transferred to the laboratory of parasitology for identification. This is the first report of infestation of a camel herd (camelus dromedarius) in Iran. The treatment was performed and prevented the loss from camels and improved their physical condition. Although the myiasis is not a lethal disorder, however knowledge of the disease is necessary from preventive, diagnostic and curative standpoint.

Traumatic myiasis agents in Iran with introducing of new dominant species, Wohlfahrtia magnifica (Diptera: Sarcophagidae)

Objective: To study agents of animal wound myiasis in various geographical districts of Fars province. Methods: This study has been done in Fars province, located in the southern part of Iran. Sums of 10 358 domestic animals have been visited from April 2011 to March 2012. The infected wounds in any parts of animal body were sampled by means of forceps. Results: About 61% of all animal wound myiasis were caused by larvae of Wohlfahrtia magnifica. The most wound myiasis cases due to this species occurred in central part of Fars province. There wasn't any significant difference between sheep and goat in infestation with myiasis (P>0.05). The infestation rate of myiasis in cattle community was 0.86%. Conclusions: The infestation rate of livestock was lower than other works in Iran and some other countries like Saudi Arabia. Chrysomya bezziana has been mentioned as main myiasis agent in Iran. But in this study it cleared that similarly to some European countries, the common animal myiasis agent in Iran is Wohlfahrtia magnifica. Introducing new species as principal agent for myiasis can help public health and animal husbandry policy makers to prepare sufficient and effective control and/or preventive measures for this disease.

Traumatic myiasis agents in Iran with introducing of new dominant species, Wohlfahrtia magnifica (Diptera：Sarcophagidae)

Objective: To study agents of animal wound myiasis in various geographical districts of Fars province.Methods:of 10358 domestic animals have been visited from April 2011 to March 2012. The infected wounds in any parts of animal body were sampled by means of forceps.Results:This study has been done in Fars province, located in the southern part of Iran. Sums The most wound myiasis cases due to this species occurred in central part of Fars province. There wasn’t any significant difference between sheep and goat in infestation with myiasis (P>0.05). The infestation rate of myiasis in cattle community was 0.86%. About 61% of all animal wound myiasis were caused by larvae of Wohlfahrtia magnifica. Conclusions: The infestation rate of livestock was lower than other works in Iran and some other countries like Saudi Arabia. Chrysomya bezziana has been mentioned as main myiasis agent in Iran. But in this study it cleared that similarly to some European countries, the common animal myiasis agent in Iran is Wohlfahrtia magnifica. Introducing new species as principal agent for myiasis can help public health and animal husbandry policy makers to prepare sufficient and effective control and/or preventive measures for this disease.