Megabacteriosis Occurrence in Budgerigars, Canaries and Lovebirds in Ribeirao Preto region - Sao Paulo State - Brazil

Ocorrência de Megabacteriose em Periquitos Autralianos, Canários e Agapornis na Região de Ribeirão Preto- Estado de São Paulo - Brasil

Ocorrência de Megabacteriose em Periquitos Autralianos, Canários e Agapornis na Região de Ribeirão Preto- Estado de São Paulo - Brasil

Megabacteriosis Occurrence in Budgerigars, Canaries and Lovebirds in Ribeirao Preto region - Sao Paulo State - Brazil

Autor(es) / Author(s)

Werther K1

Schocken-Iturrino R P1

Verona CES2

Barros LSS³

1- Docentes do Depto. de Patologia Veterinária, UNESP/ FCAV Campus de Jaboticabal - SP

2- Médico Veterinário

3- Médico Veterinário

Correspondência / Mail Address

Karin Werther

Depto. de Patologia Veterinária / FCAV - UNESP

Via de Acesso Prof. Paulo Donato Castellane s/n

14870-000 - Jaboticabal - SP - Brasil

E-mail: [e-mail]

Unitermos / Keywords

megabacteriose, síndrome do peito seco, doença do facão, proventriculite

megabacterium, proventriculitis, Going Light Syndrom

RESUMO

Este trabalho tem como objetivo relatar a ocorrência de um agente etiológico, denominado na Europa, Australia e EUA como "megabactéria", observado em estômago de pequenas aves (canários belgas, agapornis e periquitos australianos), provenientes da região de Ribeirão Preto, Estado de São Paulo/SP.

As necropsias de 64 aves silvestres (4 periquitos australianos, 12 agapornis e 48 canários), realizadas no perído de 1994 a 1997, foram analisadas, constatando-se em 56% dos casos a presença de estruturas filiformes, acidofílicas sob coloração Giemsa, gram positivas, existentes no muco do proventrículo, descritas na literatura como "megabactérias".

Foram testados diversos tipos de meios de cultura para reprodução in vitro deste microrganismo. Foram ainda comparadas as dimensões (comprimento e largura) dessa bactéria obtida apartir do esfregaço fresco de muco proventricular e da "megabactéria" proveniente de cultivo in vitro. Também foram listados os principais achados macroscópicos do animais portadores desta bactéria.

ABSTRACT

This paper reports the occurrence of an megabacterium-like organism at small birds from the Northeast area of São Paulo State - Brazil.

The results presented herein were obtained from 64 necropsy along four years (1994-1997). Sixty four birds (4 budgerigars Melopsittacus undulatus, 12 lovebirds Agapornis spp and 48 canaries Serinus canaria) were studied.

At 56% of the examined birds showed at the proventricular mucus smear a presence of rod-shaped (similar to a toothpick) organisms, Gram positive and acidophilic in Giemsa staining, called megabacteria.

Different kind of culture media was testes to replicated these organism in vitro. Also the dimension (large and width) of the fresh microorganism from the proventricular mucus and from the first culture was compared.

The macroscopic alterations observed at the necropsies was also listed.

INTRODUCTION

Megabacteriosis, also called "Going Light Syndrome" is a rather common disease in birds, especially in budgerigars (Melopsittacus undulatus) and canaries (Serinus canaria). Many authors have already reported the occurrence of this disease in the United States, Europe and Australia Schweighardt et al. (1984), Van Herck (1984), Baker (1985, 1987, 1992), Gylstorff & Grimm (1987), Scalam & Graham (1990), Ungerechts (1990), Uyttebroek & Ducatelle (1990), Anderson (1993), Filippich et al. (1993), Gerlach (1993), Filippich & Parker (1994) and Ritchie et al. (1994).

In other birds, such as Lesser Sulphur-crested cockatoo, (Cacatua sulphurea sulphurea) Anderson (1993), cockatiels (Nymphicus hollandicus) Tsai et al. (1992) and lovebirds (Agapornis spp.) Filippich et al. (1993), megabacteriosis have also been reported.

According to Gylstorff & Grimm (1987), the disease is characterised by a high morbidity and low mortality. The gastrointestinal symptoms are complex and the birds show progressive weight loss, which lasts for months, despite which they continued to feed. Other symptoms reported by Baker (1987) are diarrhoea that dirties the feathers around the cloaca and presence of not totally digested grains in faeces and mucous vomit Baker (1992). Faeces with blood were described in more severe cases by Gylstorff & Grimm (1987). Anatomopathological findings included caquexy, pale and thickened mucosae in proventriculus and a thin layer of white mucus between proventriculus and ventriculus Gylstorff & Grimm (1987), Baker (1992), Anderson (1993), Ritchie et al. (1994). Haemorrhagic ulcers in proventriculus were described, and koilin layer was loose or very thin, with grinding plates not much visible.

In histopathological examination of the proventriculus, it was observed epithelial proliferation and degeneration Gylstorff & Grimm (1987), mucosae necrosis Anderson (1993), Ritchie et al. (1994) and a thin koilin layer Baker (1992). Inflammatory cells were infiltrated in basal layer and there was mucosae fibrosis in chronic cases Anderson (1993). Baker (1992) reported the presence of mononuclear cells, such as lymphocytes and macrophages and occasionally heterophils, infiltrated in the proventriculus mucosae. The presence of few inflammatory cells was described by Ritchie et al. (1994). Megabacteria were also found in large quantities in the proventriculus Gylstorff & Grimm (1987), in the tubular glands of the proventriculus Anderson (1993) and in high density in the lesion focus Ritchie et al. (1994).

Initially, Uyttebroek & Ducatelle (1980) described structures similar to fungus hyphaes in proventriculus from canaries and budgerigars. The authors concluded that the aetiological agent, responsible for the clinical disease, was a fungus.

According to Ungerechts (1990), it has already been confirmed that megabacterium, a large rod-like or toothpick-like bacterium (1 x 90 mm), is the aethiological agent. Microbiologically, megabacteria are characterized as Gram-positive, PAS positive and acidophilic in Giemsa stain bacteria.

Other authors Scanlan & Graham (1990) also reported that the responsible agent for the disease are Gram-positive bacteria measuring between 1.5 to 3.0 mm wide and 20 to 50 mm long. They also suggested that megabacterium is a component of the normal gastrointestinal flora of the budgerigar, since they isolated these bacteria from healthy birds.

These bacteria are found in faeces and in the mucus of the isthmus between proventriculus and ventriculus Gylstorff & Grimm (1987), Filippich et al. (1993), Ritchie et al. (1994).

It has been reported that in vitro culture of these bacteria is difficult, but it is possible if Lactobacillus MRS Broth culture medium in a moist chamber it is used Gylstorff & Grimm (1987). According to Ritchie et al. (1994), in vitro culture of these organisms, in order to describe biochemical characteristics, which are necessary for a taxonomical classification, demonstrated circular colonies with irregular edge board and diameters between 3 and 4 mm. In general, megabacteria need a 48-hour incubation to grow. The sub-cultures were more difficult to be obtained, since the microrganisms stopped their growth in successive sub-culturing.

A successful megabacteria culture in blood agar plate submitted to three different incubation conditions was reported by Scanlan & Graham (1990). One plate was aerobically incubated, the second one was incubated aerobically plus 10% CO₂ and the third one anaerobically (80% N, 10% H and 10% CO₂), all of them at 37ºC. The best growth obtained was at 10% CO₂ in air. They also reported the culture of megabacteria by using sodium azide agar plate, although there was no growth when MacConkey agar and tryptose agar plates were used. The megabacteria cultured in blood agar produced colonies that increased in size from 1-2 mm to 6-8 mm when they were subcultured. The bacteria morphology themselves decreased by half. Gylstorff & Grimm (1987) observed that in vitro megabacteria were resistant to routinely used antibiotics, although the drugs had not been specified.

Diagnosis of megabacteriosis may be done post-mortem, using faeces smears and/or proventricular mucus smears Gylstorff & Grimm (1987). Megabacteriosis cause an increase in the pH of the proventricular mucus from 2.7 to 7.0-7.3 Ritchie et al. (1984), therefore, pH measurement can also help in diagnosis. In vivo diagnosis have been done through proventriculus washing, direct smears of faeces Gram-stained, and contrast radiography, which showed a narrowing between proventriculus and ventriculus.

Due to the large loose of birds by breeders with characteristics symptoms of weight loss and sudden death, the aim of this wok was to isolate from necropsies an pathological agent of this disease.

MATERIAL AND METHODS

Necropsy of sixty-four birds was done at the Department of Veterinary Pathology of the Faculty of Agricultural and Veterinary Sciences - Campus of Jaboticabal - UNESP, being 4 budgerigars (M. undulatus), 12 lovebirds (Agapornis sp) and 48 canaries (S. canaria). The animals were brought by private bird breeders of the Northeast region of Sao Paulo State.

During the necropsy the nutritional status was observed by decreased of weight and pectoral muscle mass. The macroscopic alterations of the organs was also observed. Faecal and proventricular mucus smears were stained with Gram and Giemsa. These samples were observed by light microscopy magnified 10x, 20x and 40x. Fragments collected from proventriculus were fixed in 10% phosphate-buffered formol solution, pH 7.0 and processed afterwards using the histopathology routine methods, hematoxylin-eosin stained and examined by light microscopy with up to 100x magnification.

Three animals (two budgerigars and one lovebirds) were submitted to contrast radiography with orally barium sulfate. The proventriculus pH was measured in one lovebirds.

The proventriculus mucus from five animals was cultured, Gram smears were made. The mucus cultures were incubated at 37ºC for 24 to 48 hours, using the following media:

• Brain heart infusion broth (Merck), incubated under aerobic and anaerobic conditions (80 % N₂, 10% H₂, 10% CO₂)

• Tryptose broth (Brucella) under anaerobic conditions (80 % N₂, 10% H₂, 10% CO₂)

• and Agar Brucella (Difco) under anaerobic conditions (80 % N₂, 10% H₂, 10% CO₂)

• Lactobacillus MRS broth (Difco), incubated under aerobic and anaerobic conditions (80 % N₂, 10% H₂, 10% CO₂)

• Rogosa SL Agar plate (Difco) under aerobic and anaerobic (80 % N₂, 10% H₂, 10% CO₂) conditions

• Blood agar plate, incubated under aerobic and anaerobic (30% CO₂, 70% N₂) conditions

Dimensions (length and width of the organism) were obtained using an image analyzer, it was analyzed one hundred megabacteria from culture (first culture) and one hundred from fresh material of the proventriculus mucus.

RESULTS

From the 64 lovebirds, budgerigars and canaries examined, megabacterium-like microorganism were found in proventriculus mucus smears of 36 birds (56%). Clinical and anatomopathological parameters of the positive animals were considered and are shown in graphic 01.

In 8 cases (22%), progressive weight loss and sudden death were reported, although there was normal feeding behaviour. Contrast radiography using barium sulphate was done in three cases, two of which had alterations that suggested megabacteriosis, i.e., the isthmus between proventriculus and ventriculus was narrower. The mucus smears confirmed this assumption.

In thirteen (36%) microscopic exams of faeces, it was possible to visualize megabacteria. The most important post-mortem findings were the poor nutritional status, ranging from reduced to caquetic in 17 cases (47%) and the enlargement of proventriculus, which was seen in 27 cases (75%). Mucoid proventricular contents were found in all 36 cases (100%). Smears of the mucus were observed by optic microscopy (40x magnification) and uniform elongated toothpicks-like structures were found in all of them (Figure 1). In one case, the pH of the proventricular mucus was 7.0.

On histopathological examination proventriculitis with the presence of toothpick-like structures near the mucus glands openings were found (Figure 2).

Microscopically the presence of megabacterium-like microorganisms was detected in:

• BHI medium (Merck) that was cultured in anaerobic conditions

• Tryptose broth (Brucella) under anaerobic conditions

• Lactobacillus MRS broth (Difco), incubated under aerobic

There was no growth on Rogosa SL Agar plate (Difco) under aerobic and anaerobic conditions nor on the blood agar plate incubated under aerobic and anaerobic conditions.

Using an image analyzer (Videoplan) the megabacteria (cultivated in BHI broth and fresh material) were measured and the results are at table 1.

DISCUSSION

The owners of small birds such as canaries, lovebirds and budgerigars frequently report a disease commonly known as "dry breast" or "knife disease", which presents a history of progressive weight loss, with sudden death. The common name is evidenced by prominent keel due to the loss of breast muscle mass. The owners report that the animals in the final phase of the disease showed difficulty in breaking seeds and feeding properly. Considering this and the existence of a similar pathology widely known as "Going light Syndrome" or "megabacteriosis" in Europe Gylstorff & Grimm (1987) and USA Ritchie et al. (1994), the animals described above were necropsied.

The microbiological characteristics described in literature and the characteristics of the agent found in this study are quite similar. There are great differences in the dimensions of megabacteria described here and in the literature, i.e., the mean width found here is the triple and the mean length is a third of the literature values Ungerechts (1990). These differences were observed in the fresh material and in the initial culture, which suggests that the morphology of the bacteria shows great variation. The fresh material showed bacteria narrower but longer than those found in the initial culture.

As reported by other authors Gylstorff & Grimm (1987), Ritchie et al. (1994), there was some difficulty in culturing this bacteria, since there was just discrete growth in anaerobic BHI medium, in Brucella broth and in MRS broth (incubated in anaerobic conditions). The isolation of a pure culture or subculture of megabacteria was not possible, which prevented further examination/tests. As reported by Gylstorff & Grimm (1987) and Ritchie et al. (1994), the material was cultured in MRS medium and in moist chamber, but no subcultures were obtained.

Data from symptoms, pH measurement of the mucus in one case and anatomopathological findings (enlargement of proventriculus and mucus presence), which were characteristic of megabacteriosis, were similar in these cases as those described by many other authors Gylstorff & Grimm (1987), Baker (1992), Anderson, (1993), Ritchie et al. (1994).

Direct smear of the proventricular mucus and faeces showed structures similar to the microorganism widely described in the literature as megabacterium (Gylstorff & Grimm, 1987, Ritchie et al. 1994).

In histopathology, it was observed few alterations in comparison to those described by Gylstorff & Grimm (1987), Baker (1992), Anderson (1993). Similar data such as few inflammatory cells and presence of megabacteria near the openings of the proventriculus glands were cited by Ritchie et al. (1994).

Radiographic technique was also used in in vivo diagnosis of megabacteriosis Ritchie et al. (1994), which was later confirmed by necropsy.

The presence of this pathology in São Paulo State and the lack of an efficient treatment reveals the importance of other studies about the disease and the aethiological agent, since it causes the death of many adult and young birds.

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Publication Dates

• Publication in this collection
  02 June 2003
• Date of issue
  Aug 2000