Common techniques used to diagnose Cryptosporidium infection are microscopic analysis and n-PCR (Jex et al. 2008). Despite microscopy being an intensive procedure that demands time and experience, the extraction of DNA from fecal samples of S. flaveola was performed only by means of previous microscopy. As discussed by Nakamura et al. (2009), performing PCR on samples previously identified as positive by microscopy implies a lower cost, as the reagents are expensive. Microscopy is an affordable and quick technique; however, it does not identify Cryptosporidium species and is less sensitive and specific. Therefore, n-PCR was performed to allow the identification of the species after amplicon sequencing.
In the present study, we identified only 1 of the 4 species of Cryptosporidium commonly found in birds. The 100% positivity of saffron finches, family Emberizidae, was high, but the number of samples collected and analyzed was low, making it difficult to compare the prevalence with that reported in most studies of Cryptosporidium in captive and wild birds. One factor that may interfere with the rate of Cryptosporidium infection is a difference in the age of the animals (de Graaf et al., 1999; Nichols, 2008), although almost all reports of Cryptosporidium infections in captive or wild birds do not specify the age range of the animals examined.
Silva et al. (2010) carried out a study in which 480 samples of passerine feces were collected from Araçatuba, São Paulo. Of these samples, 105 were positive for Cryptosporidium, with n-PCR and sequencing revealing all infections to be of the species C. galli. Similarly, Antunes et al. (2008) detected the species C. galli in all samples studied through molecular analysis, with four canaries (Serinus canaria) and eight cockatiels (Nymphicus hollandicus) in captivity. These works corroborate the finding of the present study in which C. galli was detected in all PCR-positive samples.
The mean size of C. galli oocysts obtained in the present study was smaller than the mean size of C. galli reported by Ryan et al. (2003a) and by Qi et al. (2011). Since the sizes of the oocysts of different species of Cryptosporidium are very similar, oocyst morphometry alone is not sufficient to distinguish the species, making molecular studies necessary for accurate identification.
Among the species/genotypes of Cryptosporidium in birds, only two named species, C. baileyi and C. galli, were identified in the saffron finch, S. flaveola, in previous studies (Nakamura et al. 2009; Sevá et al. 2011; Nakamura et al. 2014). Nakamura et al. (2009) conducted a study of 966 stool samples from birds belonging to 18 families. These captive or wild birds came from three Brazilian states: Goiás, Paraná and São Paulo. In a specimen of S. flaveola, the species C. baileyi (GQ227475) was diagnosed through PCR and sequencing of the 18S rRNA gene. In 2012, Sevá and colleagues analyzed 242 fecal samples from wild birds seized by the environmental control agency of São Paulo State. Four S. flaveola were positive for Cryptosporidium, three birds harbored the species C. galli (GU816048, GU816069, HM126668), and one was positive for the species C. baileyi (GU816042). Nakamura et al. (2014) collected a total of 1027 fecal samples from birds of the orders Psittaciformes and Passeriformes. These birds were from captivity or the wild and came from Divisão Técnica de Medicina Veterinária e Manejo da Fauna Silvestre (DEPAVE-3) of São Paulo. Of the 108 positive samples, 40 were sequenced, one of which was from S. flaveola and was positive for C. galli according to n-PCR sequencing (accession number in GenBank not available). Even though our research represents the third diagnostic report of C. galli in S. flaveola, further studies are still needed on species or genotypes of Cryptosporidium that can infect this species of Passeriformes.
A coinfection of C. galli and C. andersoni occurred in one of the birds in the present study, although only monoinfections were previously found in S. flaveola (Nakamura et al. 2009; Sevá et al. 2011; Nakamura et al. 2014). According to Máca and Pavlásek (2016), the intensive rearing of birds in breeders can be problematic, as it is associated with a large number of birds in a relatively small area, increasing the possibility of bacterial, viral and parasitic diseases and their rapid spread compared to those in wild birds.
The birds in the present study lived in separate cages but were kept in the same environment and close to each other. As reported by Nakamura et al. (2014), this can result in the spread of infection through direct contact with feces or human transport of oocysts during routine management related to cleaning. In addition, the saffron finch cages were close to the cages of other bird species, which may have contributed to the interspecific spread of Cryptosporidium infections.
Specifically, C. galli infections are associated with other pathogens (Lindsay et al. 1991), and these associations can lead to weight loss, lameness, pelvic limb joint swelling and high mortality in captive birds (Antunes et al. al. 2008). Although the birds in the present study were infected with Isospora oocysts, they did not show any of these clinical symptoms. Due to the association of infections by C. galli and Isospora in the birds of the present study, it was not possible to determine which agent was responsible for the feathers ruffled and soiled with feces observed on two of the birds, since both infections can cause the observed characteristics. According to Cox et al. (2001), in mixed infections, the burden of one or both infectious agents may be increased, that of one or both may be suppressed, or that of one may be increased and that of the other suppressed.
Passerines infected with C. galli can shed oocysts intermittently for 12-13 months (Antunes et al. 2008; Silva et al. 2010). The determination of intermittent and prolonged shedding of C. galli oocysts in fecal samples, in addition to demonstrating that this species causes chronic infection in birds, also maintains the species between generations of birds through contact between parents and progeny. In view of this, it is necessary to adopt strict sanitary management measures to prevent the occurrence of infections in breeders, commercial establishments and nongovernmental organizations that receive apprehended wild birds.
The C. andersoni isolate from S. flaveola (Isolate 4b) clustered with the other isolates of the same species from previous studies (MT648437 and KT175411) with high (80%) bootstrap support (Figure 2). The branch of the C. andersoni species clustered with the isolates of C. galli, which is also a gastric parasite, suggesting that these two Cryptosporidium species are close relatives. Cryptosporidium andersoni is a species found mainly in cattle and humans (Chalmers and Katzer 2013; Ryan et al. 2014) but was previously reported in the bird Podargus strigoides in an Australian study (Ng et al., 2006) and in an ostrich, Struthio camelus, from a zoo in southwestern France (Osman et al. 2017). Similar to Ng et al. (2006), we were unable to determine whether the presence of C. andersoni oocysts in the fecal samples of birds analyzed in the present study was due to a real infection or accidental contamination by mechanical transport, since the birds in the present study had close contact with humans. In addition, animals can also be infected indirectly after drinking water contaminated with Cryptosporidium. In view of the above, studies are needed to discover whether birds are natural hosts or only carriers of C. andersoni, since studies have already reported that a species of Cryptosporidium may have a wider host range than originally assumed (Widmer and Sullivan 2012).