Luca Borrelli, Ludovico Dipineto, Laura Rinaldi, Violante Romano, Emilio Noviello, Lucia

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1 JCM Accepted Manuscript Posted Online 19 August 2015 J. Clin. Microbiol. doi: /jcm Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 New diagnostic insights for Macrorhabdus ornithogaster infection Luca Borrelli, Ludovico Dipineto, Laura Rinaldi, Violante Romano, Emilio Noviello, Lucia Francesca Menna, Giuseppe Cringoli, Alessandro Fioretti Department of Veterinary Medicine and Animal Productions, Università di Napoli Federico II, via della Veterinaria 1, Napoli, Italy Corresponding authors: Ludovico Dipineto, ludovico.dipineto@unina.it; Luca Borrelli, e- mail: luca.borrelli@unina.it 9 10 ABSTRACT This study was aimed at proposing the use of a new rapid and user friendly diagnostic tool for the detection of Macrorhabdus ornithogaster in birds. The current report focuses on the diagnostic feasibility of different methods, paying particular emphasis on the application of the Mini-FLOTAC technique for diagnosis of M. ornithogaster. Mini-FLOTAC is particularly tailored for epidemiological monitoring and surveillance, where large numbers of faecal samples must be rapidly, yet reliably examined. Gram stain as standard method was used to validate the reliability of Mini-FLOTAC. This tool has not yet been used in avian species nor even in the diagnosis of yeast infections. In our study M. ornithogaster showed an excellent flotation performance never proved. Our results suggest that Mini-FLOTAC is a valid, sensitive and potentially low-cost alternative technique to use in the diagnosis of this yeast infections in birds. 21 INTRODUCTION Macrorhabdus ornithogaster (from the Greek words macrorhabdus long rod, and ornithogaster stomach of bird) is an anamorphic Ascomycota yeast which can infect many species of birds (1). Its phylogenetic analysis as well as growth and metabolic characteristics were recently described (1-3). 1

2 It colonizes the narrow junction (isthmus) of the glandular stomach (proventriculus) and grinding stomach (ventriculus) of birds and has not been identified elsewhere in the body or in the environment. Vegetative cells are elongated (2 20 μm) and divided by fission. Cells are single or in short chains of two to four. Ascospores are not formed. It is gram positive, but only the cytoplasm stains with the Gram stain (2). In mucosal scrapings and in the feces of infected birds, the organism is a stiff, straight rod μm long and 2-3 μm wide with rounded ends. In some circumstances, the long rods may bend slightly in a gentle curve. Viewed directly in a wet mount, small oblong refractile structures, the nuclei, found at regular intervals, are readily seen. Infection by M. ornithogaster has been described worldwide in a wide range of bird species including chickens, turkeys, ostriches, several species of parrots, passerines species, captive-bred and wild finches (3). There are different opinion whether M. ornithogaster can cause disease. It was detected, in fact, in its host both with and without obvious clinical signs. The majority of M. ornithogaster infections are benign or cause little detectable disease. Healthy birds can shed yeast cells while appearing normal on physical examination whereas sick birds may not shed them continuously (4). In vivo diagnosis of M. ornithogaster is based on the evaluation of clinical signs and/or on the microscopic examination of simple direct smears of fresh faeces possibly stained with a quick stain or Gram stain. M. ornithogaster in the faeces can be also detected by polymerase chain reaction (PCR) (3) and by cultural methods using cloacal cotton-tipped swabs (4). In the present paper the use of Mini- FLOTAC (5) is proposed as a new rapid tool for the detection of M. ornithogaster in live birds MATERIALS AND METHODS Sampling. The study was carried out from September 2013 to July 2014 in the Campania region (southern Italy). Sixty cages housing a total of 156 captive birds were examined. The cage was used as epidemiological unit and each cage hosted 2-3 birds. Animals were apparently healthy except a 2

3 couple of cages housing two Carduelis carduelis each which showed signs of diarrhea. All birds did not receive any antifungal treatment during the sampling period. Before collecting the fecal samples, a sheet of sterile aluminum foil was placed under the grid of each cage for 24 hours. A total of 60 fecal samples were collected. Each faecal sample was placed in a sterile 10 ml tube and transported to the laboratory as soon as possible. The number of cages and birds for all sampled species are showed in Table Laboratory analysis. Each faecal sample was analyzed by Gram stain and Mini-FLOTAC technique to detect M. ornitogaster. For the examination by Gram stain, approximately half gram of fecal sample was homogenized, filtered and diluted (1:10) in physiologic saline solution in order to concentrate and separate M. ornithogaster from the other solid matter of the feces. Then, a drop of mixture was placed on a slide, heat fixed by a bunsen burner and Gram stained. Finally, the smear was observed under the light microscopic (Leitz Aristoplan LM, Wetzlar, Germany) using 40X and 100X magnification (Fig. 1). For the examination by the Mini-FLOTAC, approximately 1 gram of fresh fecal sample was homogenized, filtered and one to ten diluted in physiologic saline solution. The sample was then centrifuged for 5 minutes at 1500 rpm to concentrate and separate M. ornithogaster from the other solid matter of the feces. The pellet obtained was one to ten diluted in floating ZnSO4 (Zinc sulphate) solution and 1 ml of this product was placed in both chambers of Mini-FLOTAC and read after 10 min (Fig. 2 a and b). 67 RESULTS Gram stain identification. Twenty out of sixty cages (33.3%) were positive to M. ornithogaster by Gram stain (Table 2). In particular, 18 cages contained birds belonging to Fringillidae family and 2 cages to Estrildidae family Mini-FLOTAC detection. Twenty-two out of sixty cages (36.6%) were positive to M. ornithogaster (Table 2). In particular, 20 cages contained birds belonging to Fringillidae family and 3

4 cages to Estrildidae family. In addition, 2 cage samples housing Carduelis carduelis species which showed diarrhea were also positive to Isospora spp. (Fig. 3). 75 DISCUSSION The class Aves has one of the largest number of species of the terrestrial vertebrates. A statistical analysis shows that pet birds in Europe reach about 54 million of individuals (6) and in US about 21 million in (7). Recent studies pertaining to drug efficacy evaluation and detection of low-intensity gastro-intestinal infections in animals and humans are pointing to the urge for lowcost, sensitive, accurate and easy-to-perform quantitative tests to be used in veterinary and public health. Mini-FLOTAC has been already validated in veterinary parasitology for the diagnosis of helminths (e.g. ascarids, hookworms, trichurids, gastro-intestinal nematodes and liver flukes) in pets and livestock. More recently, Mini-FLOTAC has been extended to human parasitology and broadscale validation is underway for the diagnosis of major nematodes (e.g. ascarids, trichurids, hookworms, gastrointestinal strongyles) and trematodes (e.g. Schistosoma) infecting animals and humans in different parts of the world (5, 8, 9). Mini-FLOTAC has not yet been used in avian parasitology nor even in the diagnosis of yeast infections. In our study M. ornithogaster showed an excellent flotation capacity by using Mini-FLOTAC; the phenomenon responsible for flotation was described in yeasts by Palmieri et al. (10) where a fast flotation assay was used to select new floating yeast strains. Therefore, we used this special tool to detect accurately the infection of this ascomycetes in birds using flotation. Noteworthy, there are no epidemiological studies on the M. ornithogaster prevalence in cage birds available worldwide. Only one study in Spain by Lanzarot et al. (2013) reported 2/39 birds (5.13%) positive to M. ornithogaster. The prevalence reported in our study by Mini-FLOTAC was 36.6%. However, it should be noted that Lanzarot et al. (2013) used individual birds to calculate prevalence, whereas in our study the prevalence was calculated on the groups (cages).we present the first experience with the Mini-FLOTAC for the diagnosis of M. 97 ornithogaster. Our results, thus, suggest that it is a valid, sensitive and potentially low-cost 4

5 alternative technique to use in the diagnosis of this ascomycetes yeast. This study compared the Mini-FLOTAC with the standard technique (i.e. Gram stain) on qualitative diagnosis of M. ornithogaster infections in birds with similar results. However, Mini-FLOTAC reduces the stress from birds handling and allows to count the yeast cells in a microscopic field much clearer than the wet mount or Gram stain. Quantitative analysis by yeast counting may provide a good approach to evaluate the treatment choice and related efficacy/effectiveness. In addition, since Mini-FLOTAC is widely used for the study of parasitic infections such as protozoa (11), this method allows now to investigate mixed infections which are often concomitant in birds (i.e. coccidia infection). In this study we have found two goldfinches (Carduelis carduelis) fecal samples positive for both M. ornithogaster and Isospora sp., (Fig. 3) a protozoal infection frequently recovered in Fringillidae (12, 13). In a study conducted by Ozmen et al. (14), a co-infection with M. ornithogaster and Eimeria dunsingi (a coccidial species that cause a protozoan infection with high mortality in avian species) was found in death budgerigars (Melopsittacus undulatus), after a necropsy analysis. We demonstrated by using of Mini-FLOTAC the possibility to investigate infections caused by M. ornithogaster and other parasites in live birds, simultaneously, rapidly and with an unique stool sample. Mini-FLOTAC, in fact, has resulted a novelty in the microbiological approach of avian species since it is a valid, sensitive and potentially low-cost technique. Therefore, we advice the use of Mini-FLOTAC in the management of bird infectious diseases, treating birds when necessary in order to improve the animal welfare

6 120 REFERENCES Tomaszewski EK, Loiwn KS, Snowden KE, Kurtzrnan CR, Phalen DN Phylogenetic analysis identifies the megabacterium of birds as a novel anamorphic ascomycetous yeast, Macrorhabdus ornithogaster gen. nov., sp. nov. Int. J. Syst. Evol. Microb. 53: Hannafusa Y, Bradley A, Tomaszewski EE, Libal MC, Phalen DN Growth and metabolic characterization of Macrorhabdus ornithogaster. J. Vet. Diagn. Invest. 19: Phalen DN Update on the diagnosis and management of Macrorhabdus ornithogaster (formerly megabacteria) in avian patients. Vet Clin North Am Exot Anim Pract. 17(2): Lanzarot P, Blanco JL, Alvarez-Perez S, Abad C, Cutuli MT, Garcia ME Prolonged fecal shedding of megabacteria (Macrorhabdus ornithogaster) by clinically healthy canaries (Serinus canaria). Med. Mycol. 51, Cringoli G, Rinaldi L, Albonico M, Bergquist R, Utzinger J Geospatial (s)tools: integration of advanced epidemiological sampling and novel diagnostics. Geosp. Health 7(2): FEDIAF Facts & Figures Kelly D, McCarthy E, Menzel K, Engebretson M Avian Welfare Issues: An Overview Rinaldi L, Cringoli G Exploring the interface between diagnostics and maps of neglected parasitic diseases. Parasitology 141:

7 Maurelli MP, Rinaldi L, Alfano S, Pepe P, Coles GC, Cringoli G Mini-FLOTAC, a new tool for copromicroscopic diagnosis of common intestinal nematodes in dogs. Parasit Vectors 7: Palmieri MC, Greenhalf W, Laluce C Efficient flotation of yeast cells grown in batch culture. Biotechnol. Bioeng. 50(3): Silva LMR, Vila-Viçosa MJM, Maurelli MP, Morgoglione ME,. Cortes HCE, Cringoli G, Rinaldi L Mini-FLOTAC for the diagnosis of Eimeria infection in goats: An alternative to McMaster. Small Rumin. Res. 114, Ball SJ, Brown MA, Snow KR A new species of Isospora (Apicomplexa: Eimeriidae) from the greenfinch Carduelis chloris (Passeriformes: Fringillidae). Parasitol. Res. 111(4) Borrelli L, Dipineto L, Mirabile M, Pisano S, Gargiulo A, De Luca Bossa L, Russo T, Calabria M, Sensale M, Santaniello A, Rinaldi L, Cringoli G, Menna LF, Fioretti A Atoxoplasmosis in Carduelis carduelis, p Proceedings of the XLIX National Congress. Italian Society of Avian Pathology (SIPA), Forlì, Italy Ozmen O, Aydogan, A, Haligur M, Adanir R, Kose O, Sahinduran S The Pathology of Macrorhabdus ornithogaster and Eimeria dunsingi (Farr, 1960) Infections in Budgerigars (Melopsittacus undulatus). Israel J. Vet. Med. 68 (4):

8 FIG. 1. Gram stained Macrorhabdus ornithogaster (arrow) from faecal sample under light microscope (100X)

9 FIG. 2. Macrorhabdus ornithogaster (a) (arrow) observed under light microscope at 40X by using of Mini-FLOTAC tool (b)

10 FIG. 3. Mini-FLOTAC 100x magnification at light microscope in a Carduelis carduelis fecal sample: concomitant infection of Isospora sp. (white arrow) and Macrorhabdus ornithogaster (black arrow).

11 TABLE 1 Number of cages/fecal samples and bird species sampled. Family Species No. of cages and faecal samples Total no. of birds in the cages Estrildidae Erythrura gouldiae (Gould's finch) Taeniopygia guttata (zebra finch) 5 19 Lonchura striata domestica (society finch) 5 10 Fringillidae Carduelis carduelis (goldfinch) Serinus canaria (canary) TOTAL

12 TABLE 2 M. ornithogaster detected in faecal samples from birds using Gram stain and Mini- FLOTAC techniques. No. positive/no. cages tested by: Family Bird Species Gram stain Mini-FLOTAC Erythrura gouldiae 0/15 0/15 Estrildidae Taeniopygia guttata 2/5 2/5 Lonchura striata domestica 0/5 0/5 Fringillidae Carduelis carduelis 13/25 14/25 Serinus canaria 5/10 6/10