Diagnosis of visceral form of Isospora infection (atoxoplasma) in black spot syndrome in canaries, in Iran

The gastrointestinal form of Isospora is well described in Passeriformes, but there are few reports of the visceral form of Isospora. Therefore, to evaluate the visceral form of Isospora in canaries with "black spot" syndrome, gastrointestinal contents were prepared from 50 canaries that were lost and appeared with black spot under the skin of the abdomen. At the same time, tissue samples were collected from visceral tissues. After preparing the samples, the oocysts were counted in the digestive contents. A total of 7 canaries out of 50 canaries showed oocysts in their feces. Following the identification of infected birds, histopathological sections were prepared from their visceral tissues. Visceral tissues included the heart, liver, and intestine. Inflammation and hyperemia were seen in the microscopic view of the heart, but no developing stage of parasites was seen. The liver showed inflammation as well as the asexual reproductive stage of the parasite. The asexual reproductive stage of the parasite was also observed in the intestine. Therefore, Isospora seems to be involved in the syndrome of black spot in canaries by causing gastrointestinal and visceral lesions.


Introduction
Parasitic infection is one of the most common complications in pet birds, e.g. canaries. Protozoa can induce infection in the blood or gastrointestinal tract and Eimeria and Isospora are the more important gastrointestinal protozoa in Passeriformes (McDougald 2003). Sporulated Eimeria oocytes have 4 sporocytes, with two sporozoites, while Isospora oocytes have two sporocytes, with four sporozoites. Eimeria and Isospora have direct life cycles, and the infection spreads through the gastrointestinal tract (Marhoon and Al-maeahi 2020).
Isospora is very common in sparrows and parrots, while Eimeria is common in poultry and pigeons (Sanchez-Cordon et al. 2016). Infection with Isospora and Eimeria is expressed in the species mentioned as coccidiosis (Greiner 2008). Infected birds may be asymptomatic (subclinical infection), or may have clinical signs of dysentery, depression, anorexia, and eventually death in infected birds (McDougald 2003).
Few studies have been performed on Isospora infection in pet birds in Iran. Tavasoli and Alizadeh-Dastjerd (2004) studied the gastrointestinal parasites of pet birds in Urmia (Northwest of Iran) and reported that about 47% of the studied canaries showed Isospora infection (15). In a case report, Arabkhazaeli and Madani (2014) described visceral Isospora in a Myna (1). Although, the relatively high prevalence of this infection in the canary, the visceral form or atoxoplasma of this infection has not been investigated in Iran.
Atoxoplasma is an Isospora infection in Passeriformes that has a long lifespan that affects the reticuloendothelial system and intestinal epithelium. Clinical diagnosis of atoxoplasma is also difficult due to nonspecific symptoms, so the diagnosis of atoxoplasmosis should be based on detection of Isospora by light microscopy (Norton et al. 2006).
In Iran, there is no report on the prevalence of atoxoplasma in canaries, but according to clinical observations and field microscopic diagnoses, it seems that there is visceral infection in canaries. Recently, many cases of canary infections with clinical manifestations of black spot have been observed. The study aimed to evaluate the role of Isospora in causing black spot syndrome in dead canaries.

Materials and methods
Accidently, 50 canaries; referred to private veterinary clinics in Isfahan, central of Iran, with signs of a black spot under the abdomen were examined and necropsied. These birds had dark feces before mortality and counting the number of oocytes in the feces indicated canary coccidiosis (Isosporiasis). After the autopsy, hyperemia and dehydration symptoms were observed in the carcass. After the autopsy and sampling of visceral organs such as the intestine, heart, kidney, and liver, a microscopic section was prepared. The oocyte was also counted in the digestive contents.
To determine the status of bird infection with Isospora, it was necessary to convert oocysts in the feces into sprouted form for better diagnosis. The diagnosis of Isospora performed based on appearance characteristics including the number of sporocysts, the number of sporozoites, oocyte shape, presence of polar granule, sporocyst residuum, oocyte residuum and micropyle. This examination was performed to determining the infection and counting them in the next step. For this purpose, all samples were suspended in a solution of 2.5% potassium dichromate and incubated at a temperature of 25-27 °C for one week (Deger et al. 2003).
First, the presence and approximate number of oocysts of Isospora in gastrointestinal samples were qualitatively investigated. For this purpose, 3 samples were prepared in the form of leachate on the slide and examined by smearing. Then flotation method was used to count the number of oocytes (Hendrix 1998).
To prepare histopathology sections, tissue samples were collected in formalin 10% and after paraffinization, they were cut to a thickness of 0.5 microns with a microtome and stained with H&E stain (Bancroft and Stevens 1996).

Results
The OPG results in the early stage of identification of infected canaries to Isospora demonstrated that 7 canaries from 50 sampled canaries with black spot lesions were infected with Isospora. The oocyte number in 7 infected canaries was varied from 47 to 120 with an average of 75 ± 28 per gram of intestinal content. In most infected canaries, the most clinical finding before death was loose-dark feces, anorexia, and weight loss. Furthermore, all infected canaries showed a variety of symptoms comprising diarrhea, anorexia, weight loss, and lethargy. These birds have the appearance of puffy with cluttered and irregular feathers. Before death, in one canary neurological symptoms in the form of tremors were observed.
Seven canaries had intestinal infections as well as black spot lesions, but the two infected canaries showed the development stage of parasites in the livers. The canaries with parasitic lesions in the liver had most OPG count than other infected canaries. Although, other tissues such as the heart and kidney were found pathological changes, we did not find any development stage of the parasite in the heart and kidney of infected canaries.
At autopsy, the liver was congested and edematous. The intestines also had hyperemia and hemorrhage. The heart was also congested and dark. Histopathological examination showed the development of the parasite (asexual reproduction) in the liver and intestines. Stages of parasite life were observed in the intestines in the middle parts of the cell (near the glands) and above the nucleus (Fig. 1). Pathological lesions in the liver included severe hyperemia and hepatocyte necrosis (Fig. 2). Cell swelling was also seen to some extent. The heart also suffered from severe hyperemia and infiltration of mononuclear inflammatory cells, as well as signs of necrosis of the myocytes (Fig. 3).

Discussion
The most common gastrointestinal protozoa infections in birds belong to the Cryptosporidia, Eimeria, and Isospora is more prevalent in Passeriformes (McDougald 2003). There are two common species of Isospora in canary; Is. serini and Is. canaria. In addition to these two species, other species have been reported but are less common. The Isospora infection is caused by the ingestion of sporulated oocytes Fig. 1 Microscopic view of intestine in a canary with visceral coccidiosis (Schizogenic stage illustrated by arrows) (H&E staining, X400) (Barreto et al. 2020). The difference between these two species in the life cycle, is the length of the latent period, the length of the oocyte excretion period, and the place of oocyte growth in both sexual and asexual stages. Is. serini grows asexual stages in mononuclear phagocytes of liver, lung, and spleen and sexual stage in epithelial cells of intestinal tissue while Is. canaria spends sexual and asexual stage in the epithelial cells of the intestinal (Norton et al. 2006;Saki and Ozer 2012).
There are several reports of Isospora infection in canaries. Saki and Ozer (2012) studied the infection of 64 canaries with diarrhea in Turkey from 2002 to 2006 and found that 18 canaries (28.1%) had Isospora oocytes in their feces. De Freitas et al. (2003) examined the incidence of Isospora infection in 327 canaries in Brazil and found that 167 canaries (50.5%) were infected with the Isospora. In Iran, Tavassoli and Alizadeh-Dastjerdi (15) studied the parasitic infection of the gastrointestinal tract of pet birds in Urmia. The results showed that infection with Isospora was observed in 100 canaries (46.6%) which, in 75 birds, the number of oocytes was less than 20 and in infected birds, it reached 650 oocytes per gram of feces. In a recent study, the Isospora infection was studied in 50 canaries with black spot symptoms. Results showed 14% Isospora infection according to oocyte counting in digestive contents. Therefore, it seems Isospora infection can be one of the most causes of black spot syndrome in Iranian canaries. This finding is confirmed by observing visceral lesions at autopsy as well as pathology results that demonstrated visceral form or atoxoplasma form of Isospora infection. Box's (1997) research has shown that Isospora can live outside the gut and inside monocytes for several months. Box (2009) described the extraintestinal form of Isospora and found that oocyst production in visceral organs such as the liver had a chronic trend. Box (2009) also showed that the number of parasites in mononuclear macrophages was proportional to the number of oocysts excreted. In our study, the examined canaries had the symptom of enteric for a relatively long time and eventually died. The study also found that birds that showed visceral form had the highest oocyst excretion, which is confirmed by Box (2009).
One naturally infected parrot reported that merozoites were present in lymphocytes, monocytes, and macrophages, and were numerous in the gut, but eventually spread by blood to the liver, lungs, brain, and kidneys. Histologically, the lesions included necrosis and infiltration of mononuclear cells into the liver, spleen, and lamina properia of the small intestine. A large number of mononuclear cells were observed in the capillaries, and protozoan-like were observed within liver and dilated blood vessels (Greiner 2008). In our study, histological lesions due to the asexual reproductive stages of Isospora were observed in the liver. Asexual reproductive stages of the parasite were also observed in intestinal tissue. The parasite was not observed in the heart tissue, but lesions such as severe hyperemia and dilation of blood vessels and necrosis of myocytes, as well as inflammation in the heart, were observed.
Although in the recent study, pathological lesions were shown in the kidney and heart, most pathological lesions and parasites were observed in the liver. Previously, Norton et al. (2006) stated that the liver shows the most lesions in the form of atoxoplasma of isospora infection. So, in addition to hemorrhagic lesions in the intestine, lesions in the liver appear to be a special symptom, in the form of atoxoplasmosis.
In conclusion, the gastrointestinal and visceral forms of Isospora in canary can be involved in the occurrence of black spot syndrome.