Larvicidal activity in vitro of essential oils against Cochliomyia hominivorax.

Cochliomyia hominivorax, from the family Calliphoridae, is responsible for causing most myiases in domesticated animals in Brazil. As alternatives to conventional treatments, essential oils (EOs) could possibly be used as natural control of ectoparasites. The aim of this study was to evaluate the in vitro activity of EOs from Salvia sclarea L., Rosmarinus officinalis L., Lavandula hybrida Reverchon, Citrus bergamia Risso, Citrus paradisi L., Juniperus virginiana L., Copaifera reticulata Ducke, Illicium verum Hook f., Pelargonium roseum, Cymbopogon winterianus Jowitt, Cymbopogon flexuosus Steud, Eugenia caryophyllus Spreng, Cinnamomum cassia Presl, Thymus vulgaris L. and Origanum vulgare L. against third-instar larvae of C. hominivorax. To evaluate the mortality rate, filter paper tests were performed at different concentrations and verified after 24 h and 48 h. Essential oils of T. vulgaris, O. vulgare and I. verum induced 100% mortality in the screening test, and their main active components (thymol, carvacrol and anethol, respectively) were also tested alone. In this new test, lethal concentrations (LC50) after 24 h and 48 h, respectively, were 407.1 and 314.2 µg.cm-² for T. vulgaris, 540.9 and 253.8 µg.cm-² for O. vulgare, 314.2 µg.cm-² (after 48 h) for I. verum, 255.6 and 102.3 µg.cm-² for thymol, 970.5 and 931.1 µg.cm-2 for carvacrol and 559.4 µg.cm-2 (after 48 h) for anethol. These results showed the potential of these EOs and their main components for development of ectoparasiticides for veterinary use, especially T. vulgaris EO and thymol.

Biological behavior of Chrysomya putoria (Wiedemann, 1819) (Diptera: Calliphoridae) after refrigeration: Logistics for use in Biotherapy.

The influence of refrigeration on the post-embryonic development of Chrysomya putoria larvae was evaluated, regarding its resistance in the logistics of storage and distribution in biotherapy. Previously sterilized larvae were submitted to four periods of storage under refrigeration (T1=12 h, T2=24 h, T3=48 h and T4=72 h) and control (without sterilization and refrigeration). Newly hatched larvae (0.200 g) were stored between 3 and 9ºC. After refrigeration, 40 neo-larvae (in triplicate) were transferred to 50 g of protein diet and incubated in an acclimatized chamber. There was a significant difference in the larval body mass (T1 and T2) and in the duration of larval, pupal and total development (T3 and T4). The sex ratios found in the four treatments did not differ from what was expected. Normality rates were 100% for all treatments. There was no significant difference between the Control, T1 and T2 treatments for larval, pupal and total viability. There was a significant difference between control (C) and T4 (larval viability), between C, T3 and T4 (pupa) and between C and T4 (total). C. putoria has resistance under refrigeration and storage of up to 56 h, presenting viability above 70% for use in biotherapy.

Identification, molecular characterization, and in silico structural analysis of larval salivary glands Netrin-A as a potent biomarker from Lucilia sericata (Diptera: Calliphoridae).

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6 kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.

Safety and efficacy of Lucilia cuprina maggots on treating an induced infected wound in Wistar rats.

Infection is one of the main complications that hinder wound healing. Currently, antibiotic-resistant bacteria, such as Methicilin-resistant Staphylococcus aureus (MRSA), are a concern worldwide for both humans and animals. Maggot therapy is re-emerging as an alternative to intractable wounds and may be an option to the traditional antibiotic treatment. Although the species of choice is Lucilia sericata, reports of clinical use have led us to evaluate the efficacy and safety of using Lucilia cuprina larvae on induced infected wounds in Wistar rats. In short, 32 male Wistar rats were divided into 4 groups: Group I - saline solution treated; Group II - antibiotic-treated; Group III - treated with larval debridement, and Group IV - without wound and treatment. Skin wounds were induced in groups I, II and III. All treatments were performed once and held for 48 h. Clinical, microbiological, histopathological, hematological, and biochemical analyses were done. Significant wound area contraction was found (>95%) in group III on day 9 compared to day 15 in group II. Complete elimination (0.0 ± 0.0 CFU/mL) of bioburden was achieved after the second treatment (day 6) in both the II and III groups, compared to an increase in Group I (6.51 ± 0.37 CFU/mL). A cleaner wound was also observed in the histopathological evaluation of group III, with adequate collagen formation and re-epithelialization on day 15. Furthermore, larvae increased blood platelet levels after the first treatment. L. cuprina larvae have proven safe and effective in accelerating wound treatment and eliminating MRSA.

Characterization of anti-microbial peptides and proteins from maggots of Calliphoridae and Sarcophagidae fly species (Diptera).

Removal of infected wounds using maggots has been known for centuries. Early research has shown that the maggot exosecretion, whole body, and fecal waste products of Calliphoridae and Sarcophagidae species contain a variety of alkaline peptides capable of inhibiting bacterial growth. Since the wide application of antibiotics such as penicillin, a number of bacterial infections have become insensitive to antibiotic treatment. In many of these instances, maggot therapy has been successfully applied for the treatment of chronic wounds. To identify and compare the expression patterns of anti-microbial peptides (AMPs) from some dipteran species, transcriptome analyses were conducted for the maggots of 11 Calliphoridae and Sarcophagidae species. Species of the subfamily Calliphorinae showed relatively higher expression levels of AMPs and anti-microbial proteins compared with those of Luciliinae and Sarcophagidae species. Furthermore, among all of the dipteran species examined, Lucilia illustris exhibited the highest transcription levels of AMPs. Cecropin A2 and defensin, whose expression levels were the highest among the anti-microbial peptides, were synthesized to test their biological activity. The synthesized peptides showed anti-microbial activities without hemolytic activities. In particular, cecropin A2 of L. illustris exhibited the highest anti-microbial activity against all of the bacteria and fungi examined, thereby possessing the potential to be developed as a new alternative to antibiotics. This comparative transcriptomic study may provide new insights into anti-microbial compositions of some dipteran species.

CRISPR/Cas9 Genome Editing in the New World Screwworm and Australian Sheep Blowfly.

Blowflies are of interest for medical applications (maggot therapy), forensic investigations, and for evolutionary developmental studies such as the evolution of parasitism. It is because of the latter that some blowflies such as the New World screwworm and the Australian sheep blowfly are considered major economic pests of livestock. Due to their importance, annotated assembled genomes for several species are now available. Here, we present a detailed guide for using the Streptococcus pyogenes Cas9 RNA-guided nuclease to efficiently generate both knockout and knock-in mutations in screwworm and sheep blowfly. These methods should accelerate genetic investigations in these and other closely related species and lead to a better understanding of the roles of selected genes in blowfly development and behavior.

Effect of Ointments of Sulphur or Pyrogenium on Larvae of Cochliomyia hominivorax (Diptera: Calliphoridae).

BACKGROUND: Myiasis by Cochliomyia hominivorax (Diptera: Calliphoridae) is a serious problem in animal health in tropical and sub-tropical regions. Ointment-type preparations are a good option of formulation in cases of myiasis in farm and pet animals. Sulphur and Pyrogenium have already shown in-vitro efficacy on C. hominivorax. This article describes an in-vitro experiment to test the inhibition of development from exposing larvae of C. hominivorax to two homeopathic ointments (prepared individually with Sulphur or Pyrogenium). METHODS: The homeopathic ointments were produced by mixing sterile lanolin, tocopherol and homeopathic medicine on a hydroalcoholic basis according to the Brazilian Homeopathic Pharmacopoeia. Larvae were obtained from naturally occurring myiases in sheep (wild larvae) or from a laboratory colony. The test consisted of exposing a group of 10 third-stage C. hominivorax wild larvae in contact with Sulphur or Pyrogenium ointment, or a group of 15 laboratory-propagated larvae in contact with the alcoholic vehicle of the ointment or homeopathic medicines prepared in sterile water (Sulphur or Pyrogenium), and observing the effect on the development, longevity and fertility of the blow-fly specimens. RESULTS: The C. hominivorax larval inhibition rate was 90.0% for the Sulphur ointment group and was 86.0% for the Pyrogenium ointment group. The non-alcoholic vehicle and the alcoholic vehicle inhibited the development of 24.0% and 22.08% of the larvae respectively. Sulphur prepared in sterile water inhibited the development of 74.67% and Pyrogenium in sterile water inhibited 73.33% of larvae. Specimens that survived contact with homeopathic ointments had their longevity decreased and did not reproduce. CONCLUSION: Ointments of Sulphur or Pyrogenium were able to inhibit the development of C. hominivorax larvae. The ointment vehicle was harmless.

Sterilization of Lucilia sericata (Diptera: Calliphoridae) Eggs for Maggot Debridement Therapy.

Maggot debridement therapy (MDT) is a therapy with the medical use of sterile fly larvae of certain species, particularly those within the Calliphoridae family including green bottle fly, Lucilia sericata (Meigan, Diptera: Calliphoridae), for treating chronically infected wounds and ulcers. Lucilia sericata flies were maintained under insectary conditions, and the eggs were sterilized using three treatments: hydrogen peroxide solutions, used as a hand disinfectant (Treatment 1-T1), hydrogen peroxide, surface disinfectant (Treatment 2-T2), and SaniHigene (Treatment 3-T3) and the control (without treatment). All three treatment caused the complete sterilization of eggs, and no bacterial colonies were found on the blood agar culture. The egg hatching rate after 72 h was much higher than after 24 h. Egg mortality in hydrogen peroxide solutions, T1 and T2, was 3-4% and less than in solution T3 (13%). Owing to less mortality and more sterility of the eggs, the aforementioned solutions are suggested to be appropriate for sterility in maggot therapy.

Biological Activity of Extract Solanum nigrum on some Biological Aspects of the Blue Fly Chrysomya albiceps (Diptera: Calliphoridae).

The current research evaluated the efficiency of alcoholic and alkaloid extracts of the leaves and fruits of the Solanum nigrum and investigated their effectiveness against the immature stages of the blue fly with 24-hour age at concentrations of 5, 10, 15, 20 mg/ml at the temperature of 30±1°C and relative humidity of 60±5%. It was revealed that the alcoholic extract of the fruits had the highest effect on killing the eggs of blue fly at all concentrations; accordingly, the death rates were estimated at 89.11% and 42.43% at the concentrations of 5 and 20 mg/ml, respectively, compared to that in the control, accounting for 9.27% mortality. It was also found that the alkaloid extract of the leaves of the plant outperformed in recording the highest rates of killing the eggs of blue fly, with death rates of 88.83% and 31.14% in the concentrations of 5 and 20 mg/ml, compared to the control group, amounting to 10.40%. Regarding the larval stages, the first stage was more sensitive than the other larval stages to all extracts of leaves and fruits of the plant. The highest mortality rates of the three larval stages were achieved by using the alcoholic extract of the fruits with the highest concentration of 20 mg/ml, compared to the alcoholic extract of the leaves; accordingly, the death rates of the third larval stage reached the highest rates of 57.11%, 68.20%, and 88.69% for the alcoholic extract of fruits and 53.19%, 68.64%, and 89.11% for that of leaves. The recorded data showed that the alkaloid extract of the S. nigrum leaves led to the highest mortality rates, compared to the alkaloid extract of the fruits, for all larval instars and at all concentrations. The mortality rates of the third larval stage with the highest concentration were 58.13% and 67.64%, respectively. The results of gas chromatography-mass spectrometry to investigate the chemical compounds in the alcoholic and alkaloid extracts of the leaves and fruits showed the presence of chemical compounds of varying numbers. The numbers of chemical compounds in the alcoholic extract of the leaves and fruits were 10 and 9, respectively, while it reached 17 and 23 for the alkaloid extract of the leaves and fruits of the plant, respectively.

Gene expression in Lucilia sericata (Diptera: Calliphoridae) larvae exposed to Pseudomonas aeruginosa and Acinetobacter baumannii identifies shared and microbe-specific induction of immune genes.

Antibiotic resistance is a continuing challenge in medicine. There are various strategies for expanding antibiotic therapeutic repertoires, including the use of blow flies. Their larvae exhibit strong antibiotic and antibiofilm properties that alter microbiome communities. One species, Lucilia sericata, is used to treat problematic wounds due to its debridement capabilities and its excretions and secretions that kill some pathogenic bacteria. There is much to be learned about how L. sericata interacts with microbiomes at the molecular level. To address this deficiency, gene expression was assessed after feeding exposure (1 h or 4 h) to two clinically problematic pathogens: Pseudomonas aeruginosa and Acinetobacter baumannii. The results identified immunity-related genes that were differentially expressed when exposed to these pathogens, as well as non-immune genes possibly involved in gut responses to bacterial infection. There was a greater response to P. aeruginosa that increased over time, while few genes responded to A. baumannii exposure, and expression was not time-dependent. The response to feeding on pathogens indicates a few common responses and features distinct to each pathogen, which is useful in improving the wound debridement therapy and helps to develop biomimetic alternatives.

Genome and transcriptome sequencing of the green bottle fly, Lucilia sericata, reveals underlying factors of sheep flystrike and maggot debridement therapy.

The common green bottle blow fly Lucilia sericata (family, Calliphoridae) is widely used for maggot debridement therapy, which involves the application of sterile maggots to wounds. The larval excretions and secretions are important for consuming necrotic tissue and inhibiting bacterial growth in wounds of patients. Lucilia sericata is also of importance as a pest of sheep and in forensic studies to estimate a postmortem interval. Here we report the assembly of a 565.3 Mb genome from long read PacBio DNA sequencing of genomic DNA. The genome contains 14,704 predicted protein coding genes and 1709 non-coding genes. Targeted annotation and transcriptional analyses identified genes that are highly expressed in the larval salivary glands (secretions) and Malpighian tubules (excretions) under normal growth conditions and following heat stress. The genomic resources will underpin future genetic studies and in development of engineered strains for genetic control of L. sericata and for biotechnology-enhanced maggot therapy.

Therapeutic Targeting of Nrf2 Signaling by Maggot Extracts Ameliorates Inflammation-Associated Intestinal Fibrosis in Chronic DSS-Induced Colitis.

Intestinal fibrosis is induced by excessive myofibroblast proliferation and collagen deposition, which has been regarded as a general pathological feature in inflammatory bowel disease (IBD). Therefore, identifying clinical markers and targets to treat and prevent intestinal fibrosis is urgently needed. The traditional Chinese medicine maggot, commonly known as "wu gu chong", has been shown to reduce oxidative stress and alleviate inflammation in chronic colitis. This study investigated the mechanisms underlying the effects of maggot extract (ME) on inflammation-associated intestinal fibrosis in TGF-ß1-stimulated human intestinal fibroblasts (CCD-18Co cells) and dextran sodium sulphate (DSS)-induced chronic colitis murine model. To assess the severity of inflammation and fibrosis, histological and macroscopic evaluation were carried out. The results showed that ME was a significant inhibitor of body weight loss and colon length shortening in mice with chronic colitis. In addition, ME suppressed the intestinal fibrosis by downregulating TGF-ß1/SMADs pathway via upregulation of Nrf2 expression at both protein and mRNA levels. ME markedly increased the expression of Nrf2, thus resulting in a higher level of HO-1. After treatment with Nrf2 inhibitor (ML385) or siRNA-Nrf2 for deactivating Nrf2 pathway, the protective effects of ME were abolished both in vitro and in vivo. Moreover, the histopathological results for the major organs of DSS mice treated with ME showed no signs of clinically important abnormalities. Treatment with ME had no effect on the viability of CCD-18Co cells, suggesting its low in vitro cytotoxicity. Furthermore, ME could mediate intestine health by keeping the balance of the gut microbes through the enhancement of beneficial microbes and suppression of pathogenic microbes. In conclusion, this is the first ever report demonstrating that ME ameliorates inflammation-associated intestinal fibrosis by suppressing TGF-ß1/SMAD pathway via upregulation of Nrf2 expression. Our findings highlight the potential of Nrf2 as an effective therapeutic target for alleviating intestinal fibrosis.

Larval excretion/secretion of dipters of Lucilia cuprina species induces death in promastigote and amastigote forms of Leishmania amazonensis.

Leishmaniasis is a neglected tropical disease that affects millions of people around the world. Larval excretion/secretion (ES) of the larvae of flies of the Calliphoridae family has microbicidal activity against Gram-positive and Gram-negative bacteria, in addition to some species of Leishmania. Our study aimed at assessing the in vitro efficacy of Lucilia cuprina larval ES against the promastigote and amastigote forms of Leishmania amazonensis, elucidating possible microbicidal mechanisms and routes of death involved. Larval ES was able to inhibit the viability of L. amazonensis at all concentrations, induce morphological and ultrastructural changes in the parasite, retraction of the cell body, roughness of the cytoplasmic membrane, leakage of intracellular content, ROS production increase, induction of membrane depolarization and mitochondrial swelling, the formation of cytoplasmic lipid droplets and phosphatidylserine exposure, thus indicating the possibility of apoptosis-like death. To verify the efficacy of larval ES on amastigote forms, we performed a phagocytic assay, measurement of total ROS and NO. Treatment using larval ES reduced the percentage of infection and the number of amastigotes per macrophage of lineage J774A.1 at all concentrations, increasing the production of ROS and TNF-α, thus indicating possible pro-inflammatory immunomodulation and oxidative damage. Therefore, treatment using larval ES is effective at inducing the death of promastigotes and amastigotes of L. amazonensis even at low concentrations.

Wound healing potential: evaluation of molecular profiling and amplification of Lucilia sericata angiopoietin-1 mRNA mid-part.

OBJECTIVE: High prevalence of chronic ulcers and the burden of disease necessitate the increasingly significant production of new recombinant proteins in the world. The angiopoietin-1 enzyme is a part of the growth factors group which is secreted by Lucilia sericata (Diptera: Calliphoridae) larvae when they meet lesions to ensure maggot therapy. It is one of the most potent proteins in wound healing. Given its essential role, the angiopoietin-1 gene of L. sericata was characterized, which provided some necessary information on its identity. RESULTS: The mid-part of the angiopoietin-1 mRNA sequence was thus characterized based on the design of different primers such as exon-exon junction, conserved regions, and specific region primers via conventional polymerase chain reaction (PCR). Its structural features were configured by in silico method. The sequence of mid-part (390 bp) of angiopoietin-1 was determined empirically, and BLAST analysis unraveled its high identity (85%) with the sequence of angiopoietin-1 mRNA of the larval housefly, Musca domestica. The homology of this enzyme also exhibited that its nucleic acid sequence was very similar to the domains of angiopoietin-1 in Lucilia cuprina. The current data are instructive and critical to evaluate the action of this enzyme in recombinant protein production in future molecular studies on wound healing.

Clinical study of Maggot therapy for Fournier's gangrene.

Fournier's Gangrene is a fulminating necrotizing fasciitis of the perineum and genitalia. Standard treatment involves immediate excision of all necrotic tissue, aggressive antibiotic coverage, and supportive medical care. Still, the infection is commonly fatal or disfiguring. Wound treatment with disinfected blowfly larvae (maggot debridement therapy or MDT) has been shown to be highly effective, with multiple studies demonstrating effective debridement, disinfection, and promotion of granulation tissue. MDT also has been associated with preservation of viable tissue and minimised blood loss. This report describes a prospective clinical study of MDT for Fournier's gangrene aimed to test the hypothesis that early use of maggots could decrease the number of surgical treatments required to treat Fournier's gangrene. Subjects were provided with one initial surgical excision, followed by debridement using only medical grade Lucilia sericata larvae. Only two subjects were enrolled, both diabetic men. Intensive care and culture-directed antimicrobial coverage were administered as usual. Maggot debridement was associated with the disappearance of necrotic tissue, control of infection and granulation tissue growth. In both subjects, wounds healed without requiring further surgical resection or anatomical reconstruction. Maggot therapy decreased the number of surgical procedures that otherwise would have been necessary, and led to favourable outcomes.

Phantom pain as an adverse effect after maggot (Lucilia sericata) debridement therapy: a case study.

Maggot debridement therapy, also known as larval therapy, is a well known method of treatment for hard-to-heal and intractable wounds. This case study describes severe phantom pain as a rare adverse event of maggot therapy in patients after post-traumatic amputation of the leg. We also hypothesise and discuss the possibility that the phantom pain may be a result of maggot activity, not only through tissue debridement but also through nerve nourishment and nerve regeneration.

Evaluation of Chilean Boldo Essential Oil as a Natural Insecticide Against Chrysomya megacephala (Diptera: Calliphoridae).

Chrysomya megacephala (Fabricius, 1794) is a vector of enteric bacteria, protozoa, helminths, and viruses. These Diptera can also be responsible for secondary myiasis in several animal species. Therefore, it is easy to understand the relevance of studies focusing on C. megacephala dissemination control. The employment of essential oils as natural insecticides must be considered as a promising alternative for the replacement of synthetic insecticides. In this context, the essential oil obtained from Chilean boldo leaves should be highlighted. The aim of the present work was to assess the insecticidal activity of Chilean boldo essential oil against C. megacephala blowflies in different life stages (larva, pupa, and adult). The essential oils were extracted from commercial samples of Chilean boldo leaves by hydrodistillation and were mixed to produce a pool that was employed in the study. Gas chromatographic techniques were used to enable the identification and quantification of the pool's components. Larvae, pupae, and adult insects of C. megacephala were exposed (topical application) to different concentrations of this essential oil pool. After that, the larvicidal, pupicidal, and insecticidal actions of the oil were tested. Its toxicity might be associated with compounds such as eucalyptol, linalool, α-pinene, limonene, and ascaridole, either acting alone or by synergic effects. Interestingly, the pupae appeared to be stronger than the larvae and adult insects, needing higher doses of essential oil to be killed. The oil's toxic effects could be useful to control C. megacephala dissemination in all of its development phases.

Changing attitudes toward maggot debridement therapy in wound treatment: a review and discussion.

Maggot debridement therapy is the real-time placement of maggots into a wound by health professionals for the treatment of diabetic ulcers or hard-to-heal wounds infected with antibiotic-resistant bacteria. Maggot debridement therapy shortens healing and disinfects wounds. This paper is a literature review of maggot debridement therapy in the clinical setting today and addresses the costs and benefits of this therapy. It includes recommendations to engage healthcare providers and increase awareness of this therapeutic treatment. A case study is presented on the use of maggot therapy for full debridement of a necrotic wound and clearing of a meticillin-resistant Staphylococcus aureus infection. There is also discussion on how to engage healthcare workers and reduce anxieties about the use of maggots as a treatment for hard-to-heal wounds. Education and awareness are the key factors in changing healthcare workers attitudes to maggot debridement therapy.

Histological and Immunohistochemical Study of Wounds in Sheep Skin in Maggot Therapy by Using Protophormia terraenovae (Diptera: Calliphoridae) Larvae.

A study was performed to evaluate the implication of Protophormia terraenovae larvae as a surgical therapy for wounded skin. Three groups of sheep (n = 25) were considered based on larval doses. Groups 1 and 2 were artificially infested with low and high concentrations of L1 stage P. terraenovae, respectively, and group 0 served as a control. Skin biopsies were taken at 4 and 14 d postinfestation (D.P.If). A histopathological study was carried out to evaluate the lesions with a score, numbers of eosinophils and mast cells, and an immunohistochemical analysis of CD3, CD79α, and CD68 as T lymphocytes, B lymphosytes, and macrophages, respectively. The results indicated that higher larval doses led to faster regeneration by 14 D.P.If. Furthermore, the higher larval doses showed a high number of the CD68 marker and eosinophils and a low number of CD3 and CD79α markers and mast cells. In addition, the number of mast cells, T lymphocytes, and macrophage markers increased when the lesion progressed; however, a low number of immunolabeled CD79α cells and eosinophils were observed. The results indicate a possible positive effect of larvae in the healing of certain wounds.

First Report on the Use of Larvae of Cochliomyia macellaria (Diptera: Calliphoridae) for Wound Treatment in Veterinary Practice.

Factors contributing to the delay or prevention of the natural wound healing process include infection and ineffective conventional treatment. Alternative therapies, such as the maggot debridement therapy (MDT), may be helpful for successful treatment in these cases. Aiming to disseminate information about the possibility of using other species of flies for wound treatment, besides the best known Lucilia sericata Meigen, 1826 (Diptera, Calliphoridae), we report here a successful MDT case with the application of Cochliomyia macellaria (Fabricius, 1775) larvae to treat an infected wound with extensive area of necrotic tissue in a dog. Five sterile larvae were applied to each square-cm of lesion and kept on the animal for only 48 h. The healing was successful, from both qualitative and quantitative points of view. The ratio of wound healing (RWH) reached almost 50% on the 5th day and 100% on the 14th day after MDT. Although the overall animal prognosis had been unfavorable, mainly due to the sepsis, the patient began to recover and had improved clinical condition from the fifth day after MDT. This study shows the importance and effectiveness of MDT in promoting faster and more complete healing of a complex wound.