Apicomplexa : Eimeriidae )

Diurnal periodicity is a phenomenon that has been observed in coccidian of Isospora parasites of passerines, which have been eliminated great number of oocysts at dusk. The objective of this study was to evaluate the occurrence of periodicity of oocysts presence in the green-winged-saltator Saltator similis, and its use in the diagnosis of coccidiosis in wild birds in captivity. A total of 220 fecal samples were collected from birds, apprehended from illegal trading and kept in quarantine in CETAS/IBAMA, in the morning and late afternoon, from May to November 2010. It was observed that 1.82% of the samples collected in the morning were positive, while 31.36% of samples were positive in the late afternoon. In addition, the number of oocysts shed was greater in the afternoon. Therefore, it was concluded that the sampling in the late afternoon provided greater reliability for the diagnosis of coccidiosis in green-winged-saltators. Moreover, in this study a new isosporoid coccidian parasite from the green-winged-saltator S. similis was observed and is herein described. Isospora similisi n. sp. oocysts are spheroidal to sub-spheroidal, 27.5 × 25.9 μm, with a smooth and bi-layered wall, ~1.2 mm. Micropyle and oocyst residuum are absent, but splinter-like or comma-like granules are present. Sporocysts are ellipsoidal or slightly ovoidal, 17.4 × 12.2 mm. A stieda body and substieda body are present. The sporocyst residuum is composed of granules of different sizes. Sporozoites are vermiform with a single refractile body and a nucleus. This is the fourth description of an isosporoid coccidium infecting S. similis and the sixth description from Cardinalidae.


Introduction
The green-winged saltator Saltator similis Lafresnaye d'Orbigny, 1837 is a cardinalid bird resident in Brazil.Its natural populations have been decimated by illegal mining and trade, because of its beauty and vocal repertoire.Among the information available on this species, it is known that it does not present sexual dimorphism and its locomotion is mainly by jumping (SICK, 1997, IUCN, 2011).
However, in species of Brazilian passerines, there are no reports regarding diurnal periodicity in studies of prevalence or for diagnosis of coccidiosis in free-living wild birds or birds kept in captivity.Silva et al. (2010) reported that during periods of reproduction and moulting, wild birds kept in captivity were more prone to infection by Isospora spp.
This study aimed to determine the periodicity of oocysts shedding and identify new species of Isospora, not yet described in the scientific literature, from green-winged saltators S. similis recovered from the illegal trade of wild animals, which were kept under quarantine in preparation for release.

Materials and Methods
In a total of 220 fecal samples were obtained from 164 greenwinged saltators S. similis recovered from the trafficking of wild animals and kept in quarantine in CETAS ( Two hundred and twenty samples were collected according to Dolnik (2006) and Dolnik et al. (2009); fresh samples of feces, shed by a bird, were collected individually from a sheet of paper towel placed on the bottom of each cage, after cleaning in the morning (9-12h) and at late afternoon (15-17h).The collected samples were placed into plastic vials containing a 2.5% (w/v) solution of K 2 Cr 2 O 7 at a ratio of 1:6 (v/v).Samples were sent to the Coccidia and Coccidiosis Laboratory at Universidade Federal Rural do Rio de Janeiro (UFRRJ) for analysis.Upon receipt they were placed in a thin layer (~5 mm) of K 2 Cr 2 O 7 2.5% solution in Petri plates, with incubation at 23-28 °C for 10 days or until 70% of oocysts had sporulated.Oocysts were recovered by flotation in Sheather's sugar solution (S.G. 1.20) and were examined microscopically using the technique described by Duszynski and Wilber (1997).
The quantification of oocysts was conducted according by Dolnik (2006) and Dolnik et al. (2010), where the whole surface of the coverslip was observed to avoid counting errors which may be caused by the agglomeration of oocysts in some unobserved field.The results were expressed as oocysts per droplet (OoPD).
An additional 108 samples were used to determine each the periodicity of shedding of each Isospora species.54 samples were collected in the morning, between 9:00-12:00 PM, and 54 samples was collected in the late afternoon (3:00-5:00 PM).In order to describe the fourth (novel) species, sporulated oocysts were obtained from samples collected in late afternoon.
Statistical analysis by Fisher's test and Wilcoxon's test were performed using Excel XP (Microsoft Co., Redmond, WA, USA), as proposed by Sampaio (2002).

Results
Seventy-three of 220 samples examined in this study for evaluation of the incidence of oocysts shedding, were positive for Isospora spp.Only 1.82% of the samples collected in the morning had positive results.In the late afternoon, 31.36% of the samples were positive (Table 1).Furthermore, oocyst shedding was quantitatively greater in the late afternoon, with a mean value of 98 OoPD, in comparison with the mean value of <1 OoPD recorded for the morning period (Table 2).These results were highly significant (p = 0.0001).
Type material: One-half of the oocysts are kept in 10% aqueous buffered formalin (v/v) and the other half in 70% ethanol according Duszynski and Gardner (1991) Comments: Isospora similisi differs from other Isospora spp.from the same host family (Table 3).Only I. trincaferri had similar dimensions with I. similisi; however, it can be distinguished by the unique PG and bubble-shaped SB (BERTO et al., 2008(BERTO et al., , 2011b)).
The periodicity associated with Isospora spp. is presented in the Table 4. Firstly, a larger number of oocysts were recovered in the late afternoon.Secondly, in the late afternoon, four Isospora

Discussion
This study determined the most probable period of oocyst shedding, which should result in more reliable diagnosis of coccidian infection during quarantine at a center for wild animals, where the passerines had suffered constant stress since their capture until their seizure by IBAMA.During winter in Rio de Janeiro, Brazil, the sunset happens earlier, between 5:00-6:00 PM and the samples used were collected in autumn, winter and spring.The period of sampling occurred in the morning, between 9:00-12:00 PM, and in late afternoon, between 3:00-5:00 PM, which covered the main times for treatment and management of animal sheld at CETAS.According to Brawner III and Hill (1999), and Villanúa et al.Sampling in the period of the morning, between 9:00-12:00 AM, and late afternoon, between 3:00-5:00 PM. b Highly significant by Fisher's test.
Table 2. Distribution of the mean values of number of oocysts in the fecal samples of green-winged saltators, Saltator similis, by period.(2006), the time of day is a crucial factor that must be taken into consideration when undertaking the sampling of bird droppings.The majority of species of coccidia that infect passerines belong to the genus Isospora and during their developmental cycle, which includes oocyst shedding, there is a diurnal periodicity (BOUGHTON, 1937;DOLNIK, 2006).
In the present study only 1.82% of the samples collected in the morning were positive for coccidia but in the late afternoon, 31.36% of the samples were positive.Thus, these results are similar to those of Boughton (1933Boughton ( , 1937Boughton ( , 1988)), Dolnik (1999Dolnik ( , 2006)) 2011) and Pap et al. (2011), where, in spite of different latitudes and longitudes, it was reported that the late afternoon was the most reliable period for sampling because it is the period during which oocyst shedding is greatest.In birds kept in captivity, the oocysts shedding begin earlier and ended later, when compared with the free-living passerines which have peak of oocysts shedding between 1:00-9:00 PM.Moreover, the number of oocysts shed during the first days of captivity was reported to be significantly higher than during longer times post capture (DOLNIK, 1999).Based on that report, the sampling in the current study was performed, whenever possible, on the first day of arrival of passerines in quarantine, with the objective of ensuring a more reliable diagnosis.In a study with house sparrows P. domesticus was reported that the periodicity of oocysts shedding was not associated with feeding; however, it was associated with photoperiod, because after the reversal of light and darkness, there is a reversal in the time of oocyst shedding.On other hand, it is known that oocyst shedding is controlled by physiology of the host and physiological differences between dietary habits are probably responsible for these differences (LÓPEZ et al., 2007).In the wild, birds show two peaks of feeding activity, one in the morning and another in the late afternoon.During this period, the birds feed in the same areas.In the breeding season, they feed on the same territory all day and, during the migration season, many species gather in flocks to feed.Clearly, the presence of parasites in feces during this period would increase the chances of infecting new hosts (DOLNIK, 1999;MARTINAUD et al., 2009).It is of value to note, that, although the oocysts are relatively resistant to environmental factors, such as temperature and relative humidity, it has been reported that desiccation can reduce the infectivity of oocysts of Eimeria spp. in poultry production; this lack of infectivity was confirmed in an experiment with Isospora infection in Eurasian blackbirds T. merula.It was observed that the release of oocysts in the late afternoon represented an adaptation to provide resistance to desiccation and ultraviolet radiation, because if oocysts were released throughout the day, most would be quickly destroyed by the action of sunlight (MARTINAUD et al., 2009).In some species of birds which begin to migrate, the feeding peak disappears in the morning, but the feeding peak at late afternoon never disappears.Thus, these birds gather with others, at least in a period of day, increasing the chances of infection of new hosts (DOLNIK, 1999).
Finally, we conclude that estimations of the prevalence and parasite load performed without considering the time of day are not reliable.A good method for accuracy would be to restrict the period of sampling.Collection of samples for quantification of parasite load for coccidia would be restricted to the second half of the late afternoon, while those intended for prevalence studies could be carried out between 1/2 and 4/5 of the late afternoon as suggested by López et al. (2007).The results of the current study were similar, because the sampling was restricted to the late afternoon, specifically between 3:00-5:00 PM.The oocysts' shedding was quantitatively greater in the late afternoon, with a mean value of 98 OoPD, in comparison with the mean value of <1 OoPD in the morning period, even in wild passerines kept in a quarantine regime.
The illegal animal trade had led to the reduction and extinction of some bird species worldwide.In addition, another consequence is the introduction of new parasites, as occurred with Isospora vanriperorum Levine, 1982, first described from the northern   LOPES et al., 2007;BERTO et al., 2011b).
The ease of transmission of Isospora among birds from the same family, the large number of cardinalid species in New World and the illegal bird trade may serve to ensure a wide distribution of these parasites in South, Central and North America (BERTO et al., 2011b).
In Brazil, the native birds are protected by law and supervised by the Brazilian Institute of the Environment and Natural Renewable Resources (IBAMA), therefore a symbiotype could not be collected (IBAMA, 2011).
According to Duszynski and Wilber (1997) and Berto et al. (2011b), a new coccidian species should be morphologically compared with other congeneric coccidian species, that share similar features and belong to the same host family.Thus, I. similisi sporulated oocysts was compared to all of described species from Cardinalidae.
As observed in the current study, a few variations in SB and SSB (Figure 1b,c) were also observed in sporocysts of I. similisi according to Grulet et al. (1982), Berto et al. (2009a, b, c, 2010, 2011a, c), Coelho et al. (2011a, b) and Pereira et al. (2011); however, these variations are not significant enough to separate and describe it as new species.These variations could possibly be the result of the sporulation process, the position of the SZ inside of the sporocyst, or the position of the oocyst and sporocyst under the coverslip.
Based upon its morphological features, I. similisi is considered as new to science, being the fourth description from S. similis and the sixth description from the host family Cardinalidae.Interestingly, based upon the OoPD values, this species was considered to be the most competitive among the four species of Isospora associated with S. similis.a Sampling in the period of the morning, between 9:00-12:00 AM. b Sampling in the late afternoon, between 3:00-5:00 PM. c Number of oocysts per droplet.
. Both samples were deposited in the Parasitology Collection, in the Department of Animal Parasitology, at UFRRJ, located in Seropédica, Rio de Janeiro, Brazil.Phototypes and line drawings are deposited as well.The repository number is P-41/2011.Type locality: Seropédica.(22° 43' 23.79"' S and 43° 42' 36.94"'W), Rio de Janeiro, Brazil.Sporulation time: seven days.Site of infection: Not investigated.Prevalence: 59% (32 of 54 birds examined).Etymology: The specific epithet is derived from the specific name of the type host.
spp. were indentified (I.trincaferri; I. vanriperorum; I. saltatori; and I. similisi), in contrast, in the morning only I. trincaferri and I. vanriperorum were indentified.Isospora similisi showed the highest mean of OoPD in the late afternoon; and I. trincaferri and I. vanriperorum have equal mean values of OoPD in the morning.

Table 1 .
Fecal samples of green-winged saltators, Saltator similis, used in the evaluation of the incidence of oocyst shedding. a

Table 4 .
Periodicity of shedding Isospora oocysts from green-winged saltators Saltator similis at CETAS/IBAMA, Seropédica in the State of Rio de Janeiro, Brazil.