Sterilization of blow fly eggs, Chrysomya megacephala and Lucilia cuprina, (Diptera: Calliphoridae) for maggot debridement therapy application.

Maggot debridement therapy (MDT) is an application of sterile laboratory-reared blow fly larvae to remove necrotic tissue and disinfect wounds for medical conditions. For effective application, the blow fly larvae used in the wound treatment are required to be in aseptic condition. Here, we report the results of a detailed assessment of bacteria and fungi isolated from the eggs of two blow fly species, Chrysomya megacephala (F.) and Lucilia cuprina (Wiedemann) before and after sterilization by disinfectants Chlorhex-C, povidone-iodine, and sodium hypochlorite. We also assess the survival ability of larvae and their sterility after the cleansing process. The results indicate that the isolated microorganisms from the control group of both the species consisted of 10 species of gram-positive bacteria, 21 species of gram-negative bacteria, and 4 species of yeast. As for sterility testing, the eggs and the larvae of C. megacephala were found to have been completely sterilized after being subjected to thioglycollate medium for 5 days, leading to aseptic larvae. By contrast, some microorganisms from the bacterial culture were still detected in the L. cuprina larvae treated with Chlorhex-C and povidone-iodine. The survival ability of the larvae in both the species was not significantly different between the treated and the control groups. Due to its high disinfection efficacy in destroying microorganisms in both the blow fly eggs, sodium hypochlorite is recommended for preparing sterile larvae before using MDT.

Isolation, Identification, and Evaluation of Novel Probiotic Strains Isolated from Feces of Breast-Fed Infants.

OBJECTIVE: To isolate, identify, and evaluate the probiotic properties of lactic acid bacteria (LAB) isolated from the feces of breast-fed infants. MATERIAL AND METHOD: The probiotic tests included investigation of hemolysis activity, survival in simulated gastrointestinal tract conditions (acid and bile salt tolerance), susceptibility to antibiotics, and ability to inhibit selected bacterial pathogens (Escherichia coli O157:H7, Vibrio cholerae and Salmonella enterica subsp enterica serovar Typhimurium). The bacterial species identification was performed by both carbohydrate utilization and partial 16S ribosomal RNA sequencing. RESULTS: Five of fifty LAB isolates (UBU-03, UBU-06, UBU-09, UBU-34, and UBU-37) showed good probiotic properties. These five isolates showed non-hemolysis type (gamma-hemolysis), susceptibility to all antibiotics tested except for vancomycin, ability to survive in the simulated gastrointestinal conditions of both acid and bile salt solution, and ability to inhibit growth of E. coli O157: H7 and V. cholerae. Bacterial species identification revealed that all five isolates were firmly identified as Lactobacillus rhamnosus species. CONCLUSION: The L. rhamnosus strains that were isolated and characterized in this study could be considered as probiotic strains, and then used for further probiotic characterization in human cell cultures or animal models.

Antibacterial Activity of Excretions-Secretions from Chrysomya megacephala Against Escherichia coli.

BACKGROUND: The blowfly, Chrysomya megacephala, is distributed worldwide. Previous studies found maggot excretions-secretions from other blowfly species inhibited pro-inflammatory response and antimicrobial activity. OBJECTIVE: This study aimed to test the bactericidal activity of excretions-secretions from C. megacephala larvae. MATERIAL AND METHOD: A total of 1,500 3-day-old larvae were used to collect excretions-secretions (ES) modified by the Barnes method. The bactericidal activity ofthe excretions-secretions was test by Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli using suitable liquid culture assay. Scanning electron microscope (SEM) was used to investigate the morphological change ofthe bacteria. RESULTS: E. coli were significantly inhibited by excretions-secretions from C. megacephala larvae. P. aeruginosa and S. aureus were not found to inhibit growth. CONCLUSION: The excretions-secretions from C. megacephala larvae may have a medical property for the inhibition of bacterial growth.

Antibiotic resistance patterns of Enterococcus spp. isolated from Musca domestica and Chrysomya megacephala in ubon Ratchathani.

BACKGROUND: The housefly Musca domestica and the blowfly Chrysomya megacephala are found worldwide and are medically significant as mechanical vectors of various pathogens from unsanitary locations to food, resulting in diseases in humans. OBJECTIVES: This study aimed to test the antimicrobial activity against Enterococcus spp. isolated from M. domestica and C. megacephala by standard disk diffusion and minimal inhibitory concentration (MIC), and to study the potential of M. domestica and C. megacephala to transfer multi-drug resistant enterococcus to humans. MATERIAL AND METHOD: Seven hundred adult flies were collected from fresh-food markets, garbage piles, restaurants, school cafeterias, and rice paddy fields in Muang Ubon Ratchathani and Warinchamrap in Ubon Ratchathani Province. Antimicrobial susceptibility for Enterococcus spp. isolated from adult flies was performed by disk diffusion test and minimum inhibitory concentration (MIC) determination. RESULTS: One hundred and twenty isolates of Enterococcus spp. were taken from 67 M. domestica and 53 C. megacephala. Standard disk diffusion showed the Enterococcus spp. isolates exhibited susceptibility to ampilcillin (99.2%), chloramphenicol (74.20%), tetracycline (75.0%), vancomycin (50.8%), and erythromycin (42.5%). The MICs of antimicrobial agents for all isolates were < or = 0.25-8 microg/mL for vancomycin, 1- > 16 microg/mL for tetracycline, 4- > 16 microg/mL for chloramphenicol, and 0.5-8 microg/mL for ciprofloxacin. CONCLUSION: The study demonstrated the potential of M. domestica and C. megacephala to carry Enterococcus spp. Nine antimicrobial susceptibility patterns were obtained among the 120 enterococci isolates.

Isolation of Toxocara eggs from flies in Northeast Thailand.

BACKGROUND: Flies are natural carriers of pathogens and play a considerable role in the mechanical transmission of many pathogens, such as viruses, fungi, bacteria, and parasites, in various regions of the world. OBJECTIVE: To investigate the existence of Toxocara spp. eggs on two types of flies, the Oriental latrine fly, Chrysomya megacephala, and the housefly, Musca domestica, in Ubon Ratchathani, Northeast Thailand. MATERIAL AND METHOD: Flies were collected by the use of sterilized insect sweeping nets at five types of sites, fresh-food markets, garbage piles, restaurants, school cafeterias, and paddy fields from September 2010 to October 2011. After centrifugation, sediments from each fly species were examined for parasites under a light microscope. RESULTS: From the analysis of 555 C. megacephala and 439 M. domestica adult samples, Toxocara eggs were isolated from only the former species in all sites except for the restaurants in the Warinchamrap. No other helminthes and protozoa were found on the body surfaces of both fly species. CONCLUSION: The data suggest that C. megacephala is more likely to be a potential carrier of Toxocara eggs than M. domestica in areas frequented by humans in this region of Thailand.

Multi-epitope protein production and its application in the diagnosis of opisthorchiasis.

BACKGROUND: Opisthorchiasis and cholangiocarcinoma (CCA) continue to be public health concerns in many Southeast Asian countries. Although the prevalence of opisthorchiasis is declining, reported cases tend to have a light-intensity infection. Therefore, early detection by using sensitive methods is necessary. Several sensitive methods have been developed to detect opisthorchiasis. The immunological detection of antigenic proteins has been proposed as a sensitive method for examining opisthorchiasis. METHODS: The Opisthorchis viverrini antigenic proteins, including cathepsin B (OvCB), asparaginyl endopeptidase (OvAEP), and cathepsin F (OvCF), were used to construct multi-antigenic proteins. The protein sequences of OvCB, OvAEP, and OvCF, with a high probability of B cell epitopes, were selected using BepiPred 1.0 and the IEDB Analysis Resource. These protein fragments were combined to form OvCB_OvAEP_OvCF recombinant DNA, which was then used to produce a recombinant protein in Escherichia coli strain BL21(DE3). The potency of the recombinant protein as a diagnostic target for opisthorchiasis was assessed using immunoblotting and compared with that of the gold standard method, the modified formalin-ether concentration technique. RESULTS: The recombinant OvCB_OvAEP_OvCF protein showed strong reactivity with total immunoglobulin G (IgG) antibodies against light-intensity O. viverrini infections in the endemic areas. Consequently, a high sensitivity (100%) for diagnosing opisthorchiasis was reported. However, cross-reactivity with sera from other helminth and protozoan infections (including taeniasis, strongyloidiasis, giardiasis, E. coli infection, enterobiasis, and mixed infection of Echinostome spp. and Taenia spp.) and no reactivity with sera from patients with non-parasitic infections led to a reduced specificity of 78.4%. In addition, the false negative rate (FNR), false positive rate (FPR), positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy were 0%, 21.6%, 81.4%, 100%, and 88.9%, respectively. CONCLUSIONS: The high sensitivity of the recombinant OvCB_OvAEP_OvCF protein in detecting opisthorchiasis demonstrates its potential as an opisthorchiasis screening target. Nonetheless, research on reducing cross-reactivity should be undertaken by detecting other antibodies in other sample types, such as saliva, urine, and feces.

Molecular identification and genetic variation of forensically important fly species (Order: Diptera) in Thailand using DNA barcoding.

Forensic entomology plays a crucial role in criminal investigations by providing vital insights into minimum postmortem interval (PMImin) and corpse relocation by identifying insect species that colonize in decomposing remains. This study aimed to identify and analyze the genetic variation of forensically significant fly species in Thailand, using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I COI gene. A total of 3,220 fly specimens were collected from 18 provinces across six regions of Thailand from October 2017 to September 2022. These specimens were classified by morphological identification into 21 species among three Dipteran families: Calliphoridae, Muscidae, and Sarcophagidae, with Chrysomya megacephala Diptera: Calliphoridae being the most abundant species. DNA barcoding confirmed the morphological identifications with 100 % accuracy, showing low intraspecific K2P distances0.0 to 1.1 %) and significant interspecific K2P distances 2.5 % to 17.2 %. A Neighbour-Joining (NJ) analysis was conducted to assess the molecular identification capabilities of the barcoding region. This analysis successfully recovered nearly all species as distinct monophyletic groups. The species groupings obtained were generally consistent with both morphological and molecular identifications. These findings underscore the effectiveness of DNA barcoding for precise species identification and contribute to a comprehensive database of forensically important flies in Thailand, thus facilitating improved forensic investigations and biodiversity studies.

Forensically important flesh fly species in Thailand: morphology and developmental rate.

Forensically important flesh fly species in Thailand have been investigated for their larval morphology and developmental rate to increase the forensic entomology database in Thailand and nearby countries. Sarcophaga (Liosarcophaga) dux Thomson, Sarcophaga (Liopygia) ruficornis (Fabricius), and Sarcophaga (Boettcherisca) peregrina (Robineau-Desvoidy) are the species of greatest forensic importance. The third instars of these species are similar in morphological appearance, making it difficult or impossible to differentiate between them based on external morphological characteristics. This study compares these important characteristics and reveals that the most distinctive is the posterior spiracle, followed by the number of papillae on the anterior spiracle. For confirmation of these species, the unique characteristics of adult males are also shown for accurate identification. Both the third instar and adult male characteristics are keys to identifying these species. In addition, the developmental rate of S. dux was examined in northern Thailand during 2002-2003 under natural ambient temperature (approximately 24-28 degrees C) and a natural light/dark photoperiod (approximately 12:12 h), indicating relatively rapid larval development in summer (March-June), pre-pupae (stop moving) initiated at 72 h. In the rainy season (July-October), pre-pupae initiated at 72 or 96 h, whereas pre-pupae initiated at 96 h in winter.

Two new species of Sarcophaga s. lat. from Thailand with a key to species (Diptera: Sarcophagidae).

During a distribution survey of flies of medical importance around northern Thailand, we discovered two new species of flesh fly Sarcophaga (s. lat.) (Diptera: Sarcophagidae) that also are new to science. The species are Sarcophaga nanensis sp. nov. and Sarcophaga kiyokoae sp. nov. Adults of the former species was recovered from Pua River of Nan province (19 degrees 12'29" N, 100 degrees 56' 36" E) 442 m in northern Thailand, whereas the latter species was collected around Kuang River (19 degrees 0'3" N, 99 degrees 17'16" E) 551 m of the lowland forests of Chiang Mai province, northern Thailand. Although these two sarcophagid flies showed similarity in their male terminalia in having large aedeagi and rounded juxta, illustrations of the sternite 5, cercus, surstylus, and anterolateral margin of the aedeagus allow differentiation between them. A key is provided to the adult males of the flesh flies of medical importance in Thailand.

Sensilla of the antenna and palp of Hydrotaea chalcogaster (Diptera: Muscidae).

Hydrotaea chalcogaster is a fly species of medical and forensic importance in many parts of the world. In this study, we investigated the sensilla of the antenna and palp of the adult female fly using scanning electron microscopy. The antennal scape has one type of sensillum, the sharp-tipped sensillum trichodeum; whereas, the antennal pedicel also possessed this type of sensillum in addition to an unidentified type. Three types of sensilla were found on the flagellum: (1) sensilla basiconica, with both large and small sensilla basiconica showing wall pores, (2) sensilla coeloconica, with a smooth surface, and (3) sensory pits, with wall pores of pegs. The arista is located dorso-laterally on the flagellum and has three segments. Short microtrichia are located around the distal end of its second segment and on the proximal half of the third segment. Both large sharp-tipped sensilla chaetica and small sensilla basiconica with wall pores were observed on the palps. Results of this study contribute to our overall understanding of the ultrastructural morphology of sensilla on the antenna and palp of H. chalcogaster.

Fine structure of the eggshell of the blow fly, Lucilia cuprina.

The fine structure of the eggshell of blow fly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae), was examined using scanning and transmission electron microscopy. Eggs, 1.09+/-0.07 mm in length and 0.25+/-0.05 mm in width, bore a relatively wide plastron that extending along almost the entire length. The polygonal pattern of chorionic sculpture was indistinct. The ultrathin section indicated a multi-layered eggshell having an exochorion, outer endochorion, pillars, an inner endochorion, innermost chorionic layer, and a wax layer. This study provides new information about the fine morphology of blow flies eggs. A key to differentiate the eggs of forensically important flies in Thailand is given.

Male genitalia of flesh fly Parasarcophaga (Liosarcophaga) dux (Diptera: Sarcophagidae) revealed by scanning electron microscopy.

The male genitalia of flesh fly Parasarcophaga (Liosarcophaga) dux (Diptera: Sarcophagidae) was observed using scanning electron microscopy (SEM). The phallus is a short, broad structure that is formed by a tubular base connected to a trumpet-shaped, anteroventrally expanded ventralia. The juxta projects forward from a broad base, which is connected to an upright long structure, the harpes. The juxta is apically bifurcated. The lateral styli bear three small spines curved inward. The pregonite and postgonite are slightly curved upward apically. The cerci are pointed and curved apically. Sensillae were observed on the surfaces of postgonite, surstylus, epandrium, and cerci.

Morphological Characteristics of Terminalia of the Wasp-Mimicking Fly, Stomorhina discolor (Fabricius).

Stomorhina discolor (Fabricius), a species of blow fly that mimics wasps, is distributed worldwide, but detailed information about characteristics of its adult terminalia is incomplete. To help fill this gap in the information, the morphology of adult stages of S. discolor was investigated using light microscopy and scanning electron microscopy. Observations using the light microscope revealed unique characteristics of the male genitalia that are markedly different from other blow fly species. More morphological detail, including observation of several sensilla (e.g., sensilla trichoid and sensilla basiconica) along the male terminalia and female ovipositor, was seen under the scanning electron microscope. These details can be taxonomically valuable for identifying males and females of S. discolor and may help address matters concerning copulation in this species.

Effect of cold argon plasma on eggs of the blow fly, Lucilia cuprina (Diptera: Calliphoridae).

Non-thermal plasma has been used in many medical applications, including treatment of living cells, blood coagulation, wound healing, and sterilization. The process uses an environmentally friendly gas (e.g., argon, helium, oxygen, nitrogen, or hydrogen) to destroy bacteria cells with no serious adverse effect on humans or animals. However, information on the effect of argon plasma on blow fly eggs is lacking. In this study, we explored the ability of cold argon plasma to destroy the eggs of the Australian sheep blow fly, Lucilia cuprina (Wiedemann, 1830); its larvae are a myiasis-producing agent in both human and animals. We tested the effect of cold argon plasma exposure for 1, 2, 3 and 5min on L. cuprina eggs. Since the temperature of cold Ar plasma is around 30°C, to clarify the effect of temperature on the fly eggs, hot air from an electric dryer was tested for comparison. Cold argon plasma exposure in eggs significantly reduced the survival rates of second instar larvae at all exposures tested; the effects were time dependent, with a stronger effect at longer exposure (32% survival rate after a 1-min treatment; 20%, 2min; 20%, 3min; and 6%, 5min), compared to the control (86%). No significant differences were observed in larval survival rates from eggs treated with hot air (80-84%, after 1- to 5-min treatments) versus the control (86%). These results were supported by observing the treated eggshells under a scanning electron microscope (SEM), we found noticeable aberrations only in the plasma treated groups. The emission spectrum of the argon gas discharge revealed emission lines of hydroxyl radicals at 309.1nm; these may cause the deterioration of the treated L. cuprina eggs. Our results have shown the possibility of using cold argon plasma in medical applications, in particular treating myiasis wounds.

Morphological comparison between Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton (Diptera: Calliphoridae) puparia, forensically important blow flies.

In Thailand, the hairy maggots of the blow flies, Chrysomya rufifacies (Macquart) and Chrysomya villeneuvi Patton, are of forensic importance. Both flies are closely related species, not only in the morphological appearance of their larvae and puparia, but also on the aggressive feeding habit of the former. In our continuing studies of forensically important flies, identification of immature ones needs particular attention. In this study, we reported the morphological comparison between the puparia of these two blow fly species using scanning electron microscopy (SEM). Observation revealed that the cuticular sculpture of tubercles along the dorsal and lateral segments had markedly different features: with C. rufifacies having many sharp spines assembling only at the tip, while of C. villeneuvi bore stout spines throughout the tubercle. A larger number of globules at the bubble membrane on the dorsolateral border of the fifth segment was found in C. villeneuvi (average 225) than in C. rufifacies (average 35), and more papillae was observed on the anterior spiracle in C. villeneuvi (13-15) than in C. rufifacies (9-12). However, the morphology of distinct net-like patches of the integument and structure of the posterior spiracle of both species were almost identical. Morphological comparison in this study permitted identification of the puparia of both fly species, particularly in areas where they co-exist.

Morphological observation of puparia of Chrysomya nigripes (Diptera: Calliphoridae) from human corpse.

This article presents Chrysomya nigripes Aubertin as a blow fly species of forensic importance in Thailand, and morphological observation of fly puparia using scanning electron microscopy (SEM). Morphologically, we focused on the characteristics of puparia used to accurately identify fly species. Numerous puparia of C. nigripes were found aggregated, adhering side by side, on the tibia of a skeletonized corpse, which was recovered from a forested area of Chiang Mai, northern Thailand. In the triangular shape of the anterior end of the puparia, three thoracic segments and broad hairy patches beginning dorsolaterally at the sixth segments were distinguishing characteristics. This study, showing pupariation of the flies along the bone of a corpse as well as morphological features, provides important guidance in identifying C. nigripes puparia. A key to differentiate puparia of C. nigripes from the other flies of forensic importance in Thailand is given.

Susceptibility of Musca domestica and Chrysomya megacephala to Permethrin and deltamethrin in Thailand.

Susceptibility to permethrin and deltamethrin was assessed in the house fly, Musca domestica L., and blow fly Chrysomya megacephala (F.). The laboratory strain of M. domestica was captured in Muang District (downtown), Chiang Mai, northern Thailand, and the field strains were collected from Muang District, two agricultural areas of Hang Dong District and San Khampaeng District. These strains displayed susceptibility to both permethrin and deltamethrin insecticides, with LD50 values of 0.0049, 0.0019, 0.0003, and 0.0005 microg/fly and 0.1058, 0.0185, 0.1251, and 0.0981 microg/fly, respectively. Tests on the laboratory strain of C. megacephala and the field strains collected from four areas of Chiang Mai (Muang District [downtown], two agricultural areas of Hang Dong District, and San Khampaeng District) and the wooded area near the National Forest revealed slightly lower susceptibility compared with house fly, with LD50 values of permethrin equal to 0.0028,0.0027,0.0024, 0.0007, and 0.0034 microg/fly and of deltamethrin equal to 0.0461, 0.0267,0.1327,0.1151, and 0.0244 microg/fly. These results indicated that the adult populations of both fly species were susceptible to both insecticides.

Ultramorphology of eggshell of flesh fly Liosarcophaga dux (Diptera: Sarcophagidae).

Eggs of Liosarcophaga dux (Thomson) (Diptera: Sarcophagidae) are elongated and slightly bean-shaped, measuring approximately 1.5 mm in length. Each is covered externally by an eggshell comprised of polygonal patterns. In this study, scanning and transmission electron microscopy were used to examine the eggshell of this species of flesh fly for the first time. No plastron region or median area was detected. Ultrathin sectioning of the eggshell revealed multiple layers in the shell that could be observed using transmission electron microscopy.

Larval morphology of Chrysomya nigripes (Diptera: Calliphoridae), a fly species of forensic importance.

The morphology of all instars of Chrysomya nigripes Aubertin, a blow fly species of forensic importance, is presented with the aid of both light microscopy and scanning electron microscopy (SEM). Morphological features of the cephalopharyngeal skeleton, anterior spiracle, posterior spiracle, and dorsal spines between the prothorax and mesothorax are highlighted. No consistent features were found, even using SEM, for distinguishing the first instar of C. nigripes from that of Chrysomya megacephala (F.) or Chrysomya rufifacies (Macquart), two other commonly associated blow fly species in corpses in Thailand. Several features observed in second and third instars proved to be valuable characteristics for separating these species.

First report of human myiasis caused by Chrysomya megacephala and Chrysomya rufifacies (Diptera: Calliphoridae) in Thailand, and its implication in forensic entomology.

We report a forensic entomology case associated with human myiasis in Chiang Mai Province, northern Thailand. The remains of a 53-yr-old-male were concurrently infested with third instars of the two blow fly species, Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), near a severe tumor lesion presented on the lower right leg. The presence of third instars, approximately 5 d old, on the day following postmortem indicated that myiasis occurred before death. This is the first report of both fly species acting as a myiasis-producing agent in Thailand. Unsynchronized data between the age of fly larvae due to myiasis premortem and verified age/ condition of the corpse suggest a potential complication and error in the estimation of postmortem interval if other predisposition fly infestations are not considered.