Abstract
We report the incidence of bacteraemia associated withIgnatzschineria spp. for the first time from animal clinical case presumably, as a post complication of maggot wound in a White Yorkshire pig. We described a clinical history of a febrile adult white Yorkshire pig and the isolation of Ignatzschineria spp. from blood sample. The isolate was characterized phenotypically and further identified by 16S rRNA sequence analysis. Its occurrence may be misdiagnosed in veterinary hospitals especially in low-resource settings, often leading to the underreporting of such emerging infections, since the diagnostic facilities are still in very primitive phase in developing countries. More information on speciation is needed with much about the epidemiology and pathogenesis of this emerging pathogen in order to explore its role in the lives of animals and humans. Novel pathogens continue to emerge in human, domestic animal, wildlife and plant populations, yet the population dynamics of this kind of biological invasion remain poorly understood. This rapid communication may redirect the scientific community working for animal and human health worldwide to unveil such rare emerging infections.
Keywords: Bacteraemia, Maggot infestation, Ignatzschineria spp., Myiasis in animals, Open wound, Emerging pathogen.
Introduction
Myiasis is an infestation of animals and humans by various dipteran larvae, it was defined by Zumpt (1965) as “the infestation of live vertebrate animals with dipterous larvae, which at least for a certain period, feed on host’s dead or living tissues, liquid body substances, or ingested food”. Such invasions may result in mild to severe illness or even death. Flies of several families are obligatory parasites on domestic animals, the most important family causing wound myiasis isCalliphoridae and included genera of flies are Calliphora, Lucilia, Chrysomyia  and Cochliomyia   (Nadrah et al., 2021) but flies belonging to family Calliphoridae and Sarcophagidaemostly cause traumatic wound and subsequent myiasis (Roy and Dasgupta, 1982) which is one of the commonest and widespread clinical problems in veterinary practice in India (Chhabra & Pathak, 2009). Exposed lesions attract and stimulate females to oviposit (Francesconi & Lupi, 2012). Because flies and their larvae are the hosts to several bacterial species, there are possibilities of secondary bacterial infections of the wound and also bacteraemia sets in if invaded to the bloodstream. The bacteria which are most frequently associated with myiasis-induced bacteraemia are Wohlfahrtiimonas spp. and Ignatzschineriaspp. (Ahmad et al., 2022; Lysaght et al., 2018). Both the genera are well known inhabitants of the salivary glands of larvae of many fly species (Barker et al., 2014). The presence of Ignatzschineriaspp. has been reported from diverse environmental samples and animal excreta (Juteau et al., 2004; De Luna et al., 2009). Here we describe the very first incidence of myiasis with subsequentIgnatzschineria spp. bacteraemia from animal clinical case, presumably as a post complication of maggot wound in an adult male of White Yorkshire pigs.
Materials and Methods
In September 2021, a febrile male pig aging about 2 years of breed White Yorkshire was presented with a history of pyrexia, cachexia and partial anorexia from village Nardaha of Chhattisgarh state, India (Latitude: 21.2918203ON Longitude: 81.4313321OE). On physical examination it was found that pig was wounded at the base of pinna and as per the history the animal was suffered with maggot infested wound and treated with a subcutaneous dose of Ivermectin @ 200 mcg/kilogram of body weight. The animal belonged to a herd of about 250 animals, inclusive of piglets and adult animals which were kept under semi-intensive production system. Peripheral blood smear was prepared from febrile pig, air dried and blood samples were collected aseptically. The collected samples were transported to the laboratory on ice and processed for isolation of the pathogens.
Sheep Blood agar (SBA) and MacConkey agar (MLA) were used as primary culture media for preliminary isolation of organisms according to methods described by Quinn et al. (2013). Briefly, blood samples were streaked on SBA and MLA plates and incubated at 37oC for 24 hours. Single colony of was picked up from primary culture and re-streaked on fresh SBA plate and incubated at 37oC for 24 hours to obtain pure culture. The organism was characterized by Gram’s reaction, biochemical reactions and further identified by 16S rRNA sequence analysis. The isolate was subjected to antibiotic sensitivity testing by determination of minimum inhibitory concentration (MIC). The EZY MIC™ Strips were obtained from HiMedia Laboratories Pvt. Ltd., Mumbai, India. The isolates were tested against most commonly used antibiotics and the results were interpreted as per CLSI 2020 guidelines with breakpoints for other
non-Enterobacteriaceae applied. The antibiotics used for testing were amoxycillin clavulanic acid, oxytetracycline, tetracycline, doxycycline, kanamycin, gentamicin, vancomycin, rifampicin, cotrimoxazole, ciprofloxacin, ofloxacin, pefloxacin, levofloxacin, cefotaxime, cefotaxime-clavulanic acid, ceftriaxone, streptomycin and colistin.
Results and Discussions
Bacteriological cultures revealed as dominant growth of non-haemolytic, smooth, greyish- white colonies of about 1 mm diameter size on sheep blood agar (Fig. 1) and non-lactose-fermenting colonies on MacConkey agar at 37˚C for 24 hours of incubation. Phenotypically, the organism was Gram-negative bacilli (Fig. 2), positive for oxidase, catalase, nitrate reduction and phenyl alanine deamination while negative for urease, H2S production and did not utilize citrate, lysine, and ornithine. The isolate could not able to ferment adonitol, arabinose, cellobiose, glucose, dulcitol, galactose, inositol, inulin, lactose, mannitol, mannose, raffinose, rhamnose, salicin, sorbitol and sucrose and trehalose. Antibiotic Sensitivity Testing showed resistance towards tetracycline group of antibiotics while showed susceptibility to all other tested antimicrobials. According to the test results the animals were treated with Enrofloxacin @ 5mg/Kg body weight per day for 5 days, the animal was afebrile from the 2nd day onward. The 16S rRNA gene was amplified by PCR, and the amplicon underwent Sanger sequencing (GCC Biotech Pvt. Ltd. Kolkata, India). The nucleotide sequence data were analysed by BLAST (https://blast.ncbi.nlm.nih.gov), which yielded 98.5% sequence homology to both Ignatzschineriaspp., assembled the genome sequences and submitted to GenBank under accession numbers OP021690 and OP021691.This sequence was analyzed on the nucleotide BLAST (BLASTn) suite of tools available on the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Default parameters were employed to search the highly similar sequences among 16S rRNA genes in the database.