New Diagnostic Insights for Macrorhabdus ornithogaster Infection

Abstract

The aim of this study was to propose the use of a new rapid and user-friendly diagnostic tool for the detection of Macrorhabdus ornithogaster infection in birds. The current report focuses on the diagnostic feasibility of different methods, with particular emphasis on the application of the mini-Flotac technique for the diagnosis of M. ornithogaster infection. The mini-Flotac method is particularly tailored for epidemiological monitoring and surveillance, where large numbers of fecal samples must be rapidly, yet reliably, examined. Gram staining, as the standard method, was used to validate the reliability of the mini-Flotac method. This tool has not yet been used in avian species or in the diagnosis of yeast infections. In our study, M. ornithogaster showed excellent performance in a flotation assay, which had not been demonstrated previously. Our results suggest that the mini-Flotac method is a valid, sensitive, and potentially low-cost alternative technique for use in the diagnosis of this yeast infection in birds.

INTRODUCTION

Macrorhabdus ornithogaster (from the Greek words macrorhabdus for long rod and ornithogaster for stomach of bird) is an anamorphic Ascomycota yeast which can infect many species of birds (1). Its phylogenetic analysis and growth and metabolic characteristics were recently described (1–3). 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. The vegetative cells are elongated (2 to 20 μm) and divided by fission. The 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 Gram stain (2). In mucosal scrapings and in the feces of infected birds, the organism is a stiff, straight rod, 20 to 80 μm long and 2 to 3 μm wide, with rounded ends. In some circumstances, the long rods may bend slightly in a gentle curve. Viewed the organism is 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, passerine species, and captive-bred and wild finches (3). There are different opinions on whether M. ornithogaster can cause disease. In fact, it was detected in its hosts 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). The in vivo diagnosis of M. ornithogaster infection is based on the evaluation of clinical signs and/or on the microscopic examination of simple direct smears of fresh feces, possibly stained with a quick stain or Gram stain. M. ornithogaster cells in the feces can be also detected by PCR (3) and by culture methods using cloacal cotton-tipped swabs (4). In the present paper, the mini-Flotac technique (5) is proposed as a new rapid tool for the detection of M. ornithogaster infection 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 an epidemiological unit, and each cage hosted 2 to 3 birds. The animals were apparently healthy except for a two cages housing two Carduelis carduelis each, which showed signs of diarrhea. No birds received any antifungal treatment during the sampling period. Before collection of the fecal samples, a sheet of sterile aluminum foil was placed under the grid of each cage for 24 h. A total of 60 fecal samples were collected. Each fecal 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 1.

TABLE 1.

Number of cages/fecal samples and bird species sampled

Family	Species	No. of cages and fecal samples	Total no. of birds in the cages
Estrildidae	Erythrura gouldiae (Gould's finch)	15	43
	Taeniopygia guttata (zebra finch)	5	19
	Lonchura striata domestica (society finch)	5	10
Fringillidae	Carduelis carduelis (goldfinch)	25	60
	Serinus canaria (canary)	10	24
Total		60	156

Laboratory analysis.

To detect M. ornithogaster, each fecal sample was analyzed by Gram staining and by the mini-Flotac technique. For the examination by Gram staining, approximately 0.5 g of a fecal sample was homogenized, filtered, and diluted (1:10) in physiologic saline solution in order to concentrate and separate M. ornithogaster cells from the other solid matter of the feces. Then, a drop of the mixture was placed on a slide, heat fixed by a Bunsen burner, and Gram stained. Finally, the smear was observed under a light microscopic (Aristoplan LM; Leitz Wetzlar, Germany) using ×40 and ×100 magnification (Fig. 1). For the examination by the mini-Flotac technique, approximately 1 g of a fresh fecal sample was homogenized, filtered, and diluted (1:10) in physiologic saline solution. The sample was then centrifuged for 5 min at 1,500 rpm to concentrate and separate M. ornithogaster cells from the other solid matter of the feces. The pellet obtained was diluted (1:10) in floating ZnSO₄ (zinc sulfate) solution, and 1 ml of this product was placed in each of the two chambers of the mini-Flotac tool and read after 10 min (Fig. 2a and b).

FIG 1.

Gram stained Macrorhabdus ornithogaster (arrow) from a fecal sample under a light microscope. Bar = 50 μm.

FIG 2.

Macrorhabdus ornithogaster (a) (arrow) observed under a light microscope with use of the mini-Flotac tool (b). Bar = 50 μm.

RESULTS

Gram stain identification.

Twenty out of 60 cages (33.3%) were positive for M. ornithogaster by Gram staining (Table 2). In particular, 18 of these cages contained birds belonging to the Fringillidae family and 2 cages contained birds of the Estrildidae family.

TABLE 2.

M. ornithogaster detected in fecal samples from birds using Gram staining and mini-Flotac techniques

Family	Bird species	No. positive/no. cages tested by:
		Gram staining	Mini-Flotac
Estrildidae	Erythrura gouldiae	0/15	0/15
	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

Mini-Flotac detection.

Twenty-two out of 60 cages (36.6%) were positive for M. ornithogaster (Table 2). In particular, 20 of these cages contained birds belonging to the Fringillidae family and 2 cages contained birds of the Estrildidae family. In addition, samples from 2 cages housing the Carduelis carduelis species, which showed diarrhea, were also positive for Isospora sp. (Fig. 3).

FIG 3.

Carduelis carduelis fecal sample examined under a light microscope with use of the mini-Flotac tool: concomitant infection with Isospora sp. (white arrow) and Macrorhabdus ornithogaster (black arrow). Bar = 20 μm.

DISCUSSION

With its many species, the class Aves is one of the largest classes of the terrestrial vertebrates. A statistical analysis showed that in 2013-2014 about 54 million individuals in Europe (6) and about 21 million individuals in the United States had contact with pet birds (7). Recent studies on the evaluation of drug efficacy and detection of low-intensity gastrointestinal infections in animals and humans point to the need for low-cost, sensitive, accurate, and easy-to-perform quantitative tests for use in veterinary and public health. The mini-Flotac technique has already been validated in veterinary parasitology for the detection of helminths (e.g., ascarids, hookworms, trichurids, gastrointestinal nematodes, and liver flukes) in pets and livestock. More recently, the mini-Flotac technique has been extended to human parasitology, and broad-scale validation is under way for the detection of major nematodes (e.g., ascarids, trichurids, hookworms, and gastrointestinal strongyles) and trematodes (e.g., Schistosoma) infecting animals and humans in different parts of the world (5, 8, 9). The mini-Flotac method has not yet been used in avian parasitology or in the diagnosis of yeast infections. In our study, M. ornithogaster showed excellent flotation performance with use of the mini-Flotac tool; the phenomenon responsible for flotation in yeasts was described by Palmieri et al. (10), who used a fast flotation assay to select new floating yeast strains. Therefore, we used this special tool to accurately detect infections with this ascomycete in birds using flotation. Of note, there are no epidemiological studies on the prevalence of M. ornithogaster in cage birds available worldwide. Only one study by Lanzarot et al. in Spain (4) reported 2/39 birds (5.13%) positive for M. ornithogaster. The prevalence reported in our study by using the mini-Flotac technique was 36.6%. However, it should be noted that Lanzarot et al. (4) used individual birds to calculate the prevalence, whereas in our study, the prevalence was calculated on the groups (cages).We present the first experience with the mini-Flotac technique for the diagnosis of M. ornithogaster. Our results suggest that it is a valid, sensitive, and potentially low-cost alternative technique to use in the diagnosis of infections with this ascomycete yeast. In this study, we compared the mini-Flotac technique with the standard technique (i.e., Gram staining) for the qualitative diagnosis of M. ornithogaster infections in birds and found similar results. However, the mini-Flotac technique reduces the stress to the birds associated with handling and allows the yeast cells to be counted in a microscopic field that is much clearer than the wet mount or Gram stain. Quantitative analysis by yeast counting may provide a good approach for evaluating the treatment choice and related efficacy/effectiveness. In addition, since the mini-Flotac technique is widely used for the study of parasitic infections such as those caused by protozoa (11), this method also allows investigation of mixed infections (e.g., fungi, protozoa, and helminths), which are often found in birds. In this study, we found two goldfinches (Carduelis carduelis) that had fecal samples positive for coinfection with M. ornithogaster and Isospora sp., which is protozoon frequently recovered in Fringillidae (Fig. 3) (12, 13). In a study conducted by Ozmen et al. (14), coinfection with M. ornithogaster and Eimeria dunsingi (a coccidial species that cause a protozoan infection with high mortality in avian species) was found in dead budgerigars (Melopsittacus undulatus), after a necropsy analysis. By using the mini-Flotac technique, we demonstrated the feasibility of investigating infections caused by M. ornithogaster and other parasites in live birds simultaneously and rapidly with a stool sample. The mini-Flotac method, in fact, is a novel microbiological approach in avian species since it is a valid, sensitive, and potentially low-cost technique. Therefore, we advise the use of the mini-Flotac method in the management of infectious diseases in birds, with treatment when necessary in order to improve the animal welfare.