Molecular and phylogenetic characterization of Cryptosporidium species in the saffron finch Sicalis flaveola.

BACKGROUND: Cryptosporidium is the most common protozoan that can infect a wide variety of animals, including mammals and birds. Fecal samples of six saffron finches, Sicalis flaveola, from a commercial establishment were screened for the presence of Cryptosporidium by the modified Ziehl-Neelsen technique and nested PCR of the 18S rRNA gene followed by sequencing of the amplified fragments. RESULTS: The species Cryptosporidium galli was identified in all six saffron fiches, in addition to Cryptosporidium andersoni in one of the birds, indicating a mixed infection. Only two birds had feathers that were ruffled and dirty with feces. Concomitant infection with Isospora spp. was observed in all birds. CONCLUSIONS: Saffron finches are a possible host of C. andersoni and this is the first report of this species in a captive bird and the third report of parasitism by C. galli in Sicalis flaveola.

A New Species of Eimeria Schneider, 1875 from the Saffron Finch, Sicalis flaveola (Aves: Passeriformes) in Brazil.

PURPOSE: The present study describes a new species of Eimeria found in free-living S. flaveola that inhabit the city of Eugenópolis in the state of Minas Gerais, Brazil. METHODS: Fecal samples from all captured birds were filtered through double gauze, mixed with 2.5% potassium dichromate (K2Cr2O7), placed in a Petri dish and incubated at 23-28 °C until 70% of the oocysts were sporulated. RESULTS: Eimeria flaveola n. sp. from the saffron finch, Sicalis flaveola (Aves: Passeriformes), was described in Brazil. Sporulated oocysts are subspherical; 22.67 ± 1.57 (18.47-26.48) × 21.19 ± 1.42 (16.69-24.91) µm; with a shape-index of 1.07 ± 0.05 (1.00-1.25) µm and a smooth, bilayered wall, ~ 1.09 µm. A micropyle and an oocyst residuum are absent, but one polar granule is present. Sporocysts are ovoidal, 14.24 ± 1.71 (9.02-19.33) × 9.98 ± 1.02 (6.80-12.87) µm; with a shape-index of 1.43 ± 0.16 (0.74-2.11). The Stieda body is button-shaped; sub-Stieda body and para-Stieda absent. The sporocyst residuum is formed by a compact rounded mass of granules. The sporozoite is claviform, 11.53 µm in length and 2.84 µm in width, with a refractile body and a nucleus. CONCLUSIONS: Based on its morphological and morphometric characteristics, we consider E. flaveola a newly identified species.

Lesions caused by Sarcocystis spp., parasites of opossums (Didelphis aurita), in acute and chronic infections in birds (Melopsittacus undulatus).

Protozoa of the genus Sarcocystis are obligatory heterogenous parasites with both definitive and intermediate hosts. Opossums (Didelphis aurita) can shed multiple species of Sarcocystis with birds as the intermediate host. The pathologies of Sarcocystis species in birds have not been thoroughly elucidated. Therefore, the aim of the present study to determine the main lesions that can occur in acute and chronic infections in intermediate hosts, when they ingest infective sporocysts that are shed in the opossum's feces, using budgerigars as a model. To this end, 12 budgerigars, Melopsittacus undulatus, were divided into two groups that received an inoculum with 60 and 120 sporocysts. Birds that died or were euthanized were necropsied, and the lung, tongue, liver, brain, heart, and skeletal striated muscles were collected and fixed in 10% formalin for histopathological analysis. The infectivity varied according to the sample and infective dose. Acute histopathological lesions were characterized by evidence of slightly degenerated hepatocyte cords that permeated the region of the blood vessel and hepatic sinusoids. Pulmonary tissue lesions were also observed in the parabronchial region with the presence of inflammatory infiltrates associated with areas of edema and atelectasis. In chronic infections, few mature cysts were observed in the chest, and many mature cysts in the thigh and tongue muscles. Thus, it was possible to conclude that lesions are highly characteristic in acute infection and, in chronic infections, cysts were present but without major lesions. In this case, the preferred organs of parasitism were the thigh and the tongue.

Natural infection by Procyrnea uncinipenis (Nematoda, Habronematidae), a parasite from rheas, an autoctone bird from South America, in emus Dromaius novaehollandiae, a ratite from New Zealand.

The present study reports a natural infection of emus, Dromaius novaehollandiae, by the nematode Procyrnea uncinipenis. Five adult emus from a scientific breeding farm at North Fluminense State University located in the city of Campos dos Goytacazes, Rio de Janeiro state, Brazil were necropsied, and their gastrointestinal tract were collected and examined for the presence of parasites from October 2013 to November 2015. Two of the five (40%) emus necropsied were infected with nematodes, and a portion of the nematodes were processed for light microscopy. In addition, two other nematodes (a male and a female) were prepared for scanning electron microscopy. In a female bird, one nematode was collected in the proventriculus and two nematodes in the gizzard and in the male bird four nematodes were collected in the gizzard. The morphological and morphometric analyzes allowed to identify the nematodes as P. uncinipenis, this being the first report of an infection by P. uncinipenis in emus. Therefore, we infer that these emus were naturally infected by nematodes that were considered specific to rheas.

The lungworm, Halocercus brasiliensis (Nematoda: Pseudaliidae), from Guiana Dolphins Sotalia guianensis from Brazil with Pathological Findings.

The Guiana dolphin, Sotalia guianensis, is a small cetacean species found on the Atlantic coast of Central and South America from Honduras to Santa Catarina State, Brazil. The nematode Halocercus brasiliensis infects this cetacean, resulting in lung pathologies and death. The present study aimed to conduct a morphological and morphometric study of specimens of H. brasiliensis collected from S. guianensis from the coast of the state of Espírito Santo, Brazil. For this study, 7 dolphins were collected and examined for the presence of lung parasites. Nematodes were collected and processed for light microscopy, and lung fragments were processed for histological analysis. The nematodes were identified as H. brasiliensis according to their morphology and morphometry. The histopathological analysis revealed mineralization of the bronchiolar cartilage and inflammatory process. The parasitic infections by H. brasiliensis in S. guianensis can contribute to the debilitating status of these cetaceans, resulting in their stranding and accidental capture in fishing nets.

Correction to: Description of a new species Physaloptera goytaca n. sp. (Nematoda, Physalopteridae) from Cerradomys goytaca Tavares, Pessôa & Gonçalves, 2011 (Rodentia, Cricetidae) from Brazil.

The name of Luanna Castro Oliveira was incorrectly spelled as Luana Castro Oliveira in the original version of this article.

Description of a new species Physaloptera goytaca n. sp. (Nematoda, Physalopteridae) from Cerradomys goytaca Tavares, Pessôa & Gonçalves, 2011 (Rodentia, Cricetidae) from Brazil.

Nematodes of the genus Physaloptera are common in rodents, including in species of the Family Cricetidae. There is no report of nematodes parasitizing Cerradomys goytaca, so this is the first one. For this study, 16 rodents were captured in the city of Quissamã, in the northern of Rio de Janeiro State. The rodents were necropsied, and the digestive tracts were analyzed under a stereomicroscope for the presence of parasites. The nematodes were fixed in hot AFA, clarified in Amann's lactophenol, mounted on slides with coverslip, and observed under an optical microscope. Part of the nematodes was fixed in Karnovisk solution for scanning electron microscopy. Nematodes presented evident sexual dimorphism. Oral openings had two semicircular pseudolabia, with an external lateral tooth and an internal lateral tripartite tooth on each pseudolabium. Males had a ventral spiral curved posterior ends with the presence of a caudal alae with 21 papillae with four pairs of pedunculated papillae arranged laterally, three pre-cloacal sessile papillae arranged rectilinearly and five pairs of post-cloacal sessile papillae. There was also a pair of phasmids located between the fourth and fifth pairs of post-cloacal papillae as well as two spicules that were sub-equal in size but of distinct shapes. The females have five uterine branches. The morphological and morphometrical analyses of the nematodes collected from C. goytaca were compared with other species, and the results indicated that this is a new species of the genus Physaloptera, Physaloptera goytaca n. sp.

Identification of opossums Didelphis aurita (Wied-Neuweid, 1826) as a definitive host of Sarcocystis falcatula-like sporocysts.

This study was conducted to identify the Sarcocystis species that infect the opossum Didelphis aurita in order to determine which sporocysts they are excreating in to the environment and help determine the role of D. aurita in the epidemiology of Sarcocystis. Sporocysts were obtained from intestinal tracts of 8 of 13 D. aurita trapped in Rio de Janeiro state, Brazil, and were orally inoculated into Melopsittacus undulatus and Balb/c nude Mus musculus. Portions of organs and muscles were processed for histology, immunohistochemistry, transmission electron microscopy (TEM), and PCR using primers JNB 33/54, and ITS. Amplification products were subjected to RFLP using DraI and HinfI. Some birds were euthanized 6, 7, 13, 16, and 24 days after inoculation (DAI). All other birds and all mice were euthanized 60 DAI. Schizonts were observed in the lungs using histology and immunostaining in birds examined prior to 60 DAI. Sarcocysts with a ~ 1.5-µm-thick wall were found in the breast, thigh, and tongue of some birds. Sarcocystis asexual stages were isolated in cell cultures inoculated with sporozoites. Parasite DNA isolated from bird tissues and cell cultures demonstrated that S. falcatula-like parasites were present in all samples derived from positive opossums. Asexual stages molecularly characterized as S. lindsayi-like were isolated in cell culture from one opossum with an apparent multiple infection. This study demonstrated that D. aurita is a definitive host for S. falcatula-like parasites and indicates that S. lindsayi-like parasites can be found in coinfections of this opossum species.

Short communication: first report of nematodes parasitizing the four-eyed-fish, Anableps anableps (Pisces, Cyprinodontiformes).

The genus Anableps is composed of species Anableps anableps, Anableps dowi, and Anableps microlepis. These fishes are tropical and usually live on the surface of brackish water, being popularly known as four-eyed-fishes due to the presence of prominent eyes and a pupil split horizontally. A. anableps and A. microlepis are considered as sister species that live in sympatry in South America. A. dowi, however, is restricted to the Pacific Ocean (Central America) and is considered the most primitive species of this genus. The aims of this study were to investigate the presence of endoparasites in A. anableps from the Parnaíba's Delta and characterize them morphologically. During the necropsy, larvae of Contracaecum sp. in the third larval stage (L3) were collected from the pancreas of A. anableps, but no endoparasites were observed in other organs. The worms had a cuticular tooth and excretory pore located at the anterior end, a thread like body, whitish color, and without distinction of sex. The length of the ventricular appendix of the larvae was much greater than in other studies. This is the first report of endoparasitism in A. anableps and the first report of nematodes in four-eyed-fishes.

Zoonotic Enterocytozoon bieneusi genotypes found in brazilian sheep.

The presence of Enterocytozoon bieneusi in sheep has been reported in only three countries worldwide. The present study has found E. bieneusi in Brazilian sheep for the first time; in 24/125 (19.2%) fecal samples by PCR and on 8/10 (80%) farms from three diverse locations. A significantly greater number of lambs (34.1%) were found infected than older sheep (11.1%) (P=0.0036); most of the lambs were less than 6months of age. Farms with an intensive production system had a lower infection rate (10.5%) of infection than semi-intensive farms (23%), but this difference was not statistically significant. Sequencing analysis of the internal transcribed spacer (ITS) region of the rRNA gene revealed four known E. bieneusi genotypes (BEB6, BEB7, I, and LW1) and two novel genotypes (BEB18 and BEB19). Genotypes LW1 and BEB19 clustered within designated zoonotic Group 1 while genotypes BEB6, BEB7, I, and BEB18, and clustered within Group 2. BEB6 was the most prevalent (45.8%), followed by BEB7 (33.3%). Genotypes BEB6, I, and LW1 are zoonotic and can pose a risk to human health for immunocompromised individuals.

New findings of Enterocytozoon bieneusi in beef and dairy cattle in Brazil.

Microsporidia are widely recognized as important human pathogens with Enterocytozoon bieneusi as the most common species infecting humans and animals, including cattle. Although Brazil has the second largest cattle herd in the world and it is the largest exporter of beef there are no data on the presence or impact of E. bieneusi on this important population. To fill this knowledge gap, fecal specimens were collected from 452 cattle from pre-weaned calves to adult cattle in the state of Rio de Janeiro. Host factors including age, gender, dairy/beef, body composition, and fecal consistency were included in the study. Using molecular methods, E. bieneusi was found in 79/452 (17.5%) fecal specimens. This represents the first report of this parasite in Brazilian cattle. A significantly higher prevalence was found in calves less than 2 months of age (27.6%) and those 3-8 months of age (28.8%) versus heifers (14.1%) and adults (1.4%) (P<0.05). Dairy cattle (26.2%) had a higher prevalence than beef cattle (9.7%) (P<0.001). No correlation was found between infection and gender, body composition, and fecal consistency. Molecular characterization of the internal transcribed spacer (ITS) revealed 12 genotypes; five previously reported in cattle (BEB4, BEB8, D, EbpA and I), and seven novel genotypes (BEB11-BEB17). A phylogenetic analysis showed that 6 genotypes (D, EbpA, BEB12, BEB13, BEB15, and BEB16) identified in 18 animals clustered within the designated zoonotic Group 1 while the other 6 genotypes (I, BEB4, BEB8, BEB11, BEB14, BEB17) identified in 61 animals clustered within Group 2. The identification of genotypes in Brazilian cattle that have previously been reported in humans highlights the potential risk of zoonotic transmission and suggests that the role of cattle in transmission of human infections requires further study.

Gastrointestinal nematodes in ostriches, Struthio camelus, in different regions of the state of Rio de Janeiro, Brazil.

The ratite group is composed of ostriches, rheas, emus, cassowaries and kiwis. Little research has been done on parasitism in these birds. The aim of this study was to determine the distribution of infections by gastrointestinal nematodes in ostriches in the state of Rio de Janeiro. For this, fecal samples were collected from 192 on 13 farms. From each sample, four grams of feces were used to determine the eggs per gram of feces (EPG) count, by means of the McMaster technique. Part of the feces sample was used for fecal cultures, to identify 100 larvae per sample. The results were subjected to descriptive central trend and dispersion analysis, using confidence intervals at the 5% error probability level in accordance with the Student t distribution, and Tukey's test with a 95% confidence interval. The mean EPG in the state was 1,557, and the municipality of Três Rios had the lowest average (62). The city of Campos dos Goytacazes presented the highest mean EPG of all the municipalities analyzed. The northern region presented the highest mean EPG, followed by the southern, metropolitan, coastal lowland and central regions. Libyostrongylus species were observed on all the farms: L. douglassii predominated, followed by L. dentatus and Codiostomum struthionis.

Isospora dromaii n. sp. (Apicomplexa, Eimeriidae) isolated from emus, Dromaius novaehollandiae (Casuariiformes, Casuariidae).

A new species of Coccidia (Protozoa: Apicomplexa: Eimeriidae) isolated from emus, Dromaius novaehollandiae, which was observed in Brazil is described and named. Oocysts of Isospora dromaii n. sp. are subspheroidal to ovoid in shape, measure 21.6 × 19.8 µm, and have a double and smooth wall thickness of approximately 1.4 µm. In this species, micropyle, oocyst residuum, and polar granules are absent. The sporocysts are slightly ovoid in shape and measure 13.7 × 10.0 µm. Nipple-like Stieda body and prominent sub-Stieda body are present. The sporocyst residuum is composed of small dispersed granules of varying sizes. The sporozoites are characterized by an oblong refractile body and one centrally located nucleus. This is the first description of isosporid coccidia infecting birds of the family Dromaiidae.

Occurrence and risk factors associated to Toxoplasma gondii infection in sheep from Rio de Janeiro, Brazil.

Toxoplasmosis is an important cause of abortion in sheep and a zoonotic risk to humans, leading to significant hazards to health and to economic losses. This study examined the seroprevalence and associated risk factors for infection with Toxoplasma gondii in 379 sheep from 12 flocks in Rio de Janeiro state, Brazil. Using the modified agglutination test (MAT), 202 (53.3 %) of 379 were seropositive with titers of 1:25 in 65, 1:50 in 40, 1:100 in 23, 1:200 in 11, 1:400 in 36, 1:800 in 7, 1:1,600 in 1, and 1:3,200 or higher in 19 sheep. The most significant factors associated with T. gondii seropositivity in sheep were age, gender, and veterinary care. Finding of T. gondii antibodies in 97 of the 202 sheep in titers of 1:100 or higher is indicative of persistently infected animals. This high level of seropositivity requires urgent control measures to reduce impact on animal productivity and public health.

Didelphis aurita (Marsupialia: Didelphidae): a new host for Sarcocystis lindsayi (Apicomplexa: Sarcocystidae).

Nine opossums, Didelphis aurita , were captured in the city of Seropédica, State of Rio de Janeiro, Brazil and examined for species of Sarcocystis. Sporocysts were observed in the mucosal scrapings of the small intestine from 3 opossums. Five budgerigars, Melopsittacus undulatus , were infected with sporocysts from each of these infected opossums and 5 budgerigars were used as controls. Of the 15 sporocyst-treated budgerigars, 5 birds that received sporocysts from 1 of the infected opossums developed tissue parasites. Meronts in the vascular endothelium of the lung venous capillaries and cysts in the skeletal and cardiac muscle cells were observed in histological sections stained with hematoxylin and eosin. The microscopic cysts, which were predominantly in the tongue and leg muscles, ranged from 65.3 to 118.1 µm in length and 14.0 to 29.4 µm in width and from 0.9 to 1.9 µm in thickness of the cystic wall. Sections examined by transmission electron microscopy revealed that the cyst wall contained numerous slender and jagged-shaped protrusions, each with a finger-like formation at the end. The morphology, especially of the cyst wall, and the morphometry of the tissue cysts indicate that the parasite is Sarcocystis lindsayi and, therefore, the opossum, D. aurita , is now considered a definitive host for this species in Brazil.

Compromised nutrition in gerbils infected by Cystoisospora felis detected through an animal performance analysis tool.

The impact of Cystoisospora felis infection on the nutritional efficiency of gerbils was studied. The variables weight gain and feed intake were measured during four weeks in 28 laboratory gerbils, of which 14 were inoculated with 3.5 × 10(5) sporulated oocysts of C. felis and the remaining 14 were controls. The animals from both groups were weighted, killed, eviscerated and had their carcasses and tissues weighted and compared. A modern tool designed for measuring nutritional performance of farm animals was applied. The results showed compromised nutritional efficiency of the infected animals within the first week after infection. The consequences of these results are discussed here, including the potential impact of infection on farm animals performance.

Molecular characterization of Cryptosporidium in Brazilian sheep.

Feces were collected from 125 sheep between January and December 2007, on ten farms in the State of Rio de Janeiro, Brazil, and examined for the presence of Cryptosporidium. Ninety samples were collected from lambs 2 to 6 months of age, and 35 were from sheep over 12 months of age. All samples were subjected to molecular analysis by polymerase chain reaction (nested PCR) in two steps of the SSU rRNA. Two samples (1.6%) from the lambs were positive, and after sequencing were identified as Cryptosporidium ubiquitum. This species has been reported worldwide and it is considered a zoonotic pathogen since it has been found and in several animal species and humans. However, because of the low frequency of C. ubiquitum found, the risk for public health in this region may not be high.

Pathological findings in the caeca of naturally infected ostriches, Struthio camelus Linnaeus, 1758 (Aves, Struthionidae) parasitized by Codiostomum struthionis (Horst, 1885) Railliet and Henry, 1911 (Nematoda, Strongylidae).

In order to characterize lesions associated with Codiostomum struthionis in ostriches, 10 caeca were examined on both macro- and microscopic levels. Parasites were found in the distal third of the caecum and characterized as C. struthionis. Thickened mucosa was identified macroscopically where parasites were observed in high concentrations. Nodular areas were also observed in the distal third of the infected caeca, as well as hemorrhagic areas abutting small ulcers surrounded by edema. These findings were not observed in healthy controls. The concentration of C. struthionis found in infected animals was directly correlated with the severity of lesions observed in each caecum. These results allowed us to infer that C. struthionis is responsible for lesions in ostrich caeca.

Further study of Codiostomum struthionis (Horst, 1885) Railliet and Henry, 1911 (Nematoda, Strongylidae) parasite of ostriches (Struthio camelus Linnaeus, 1758) (Aves, Struthioniformes).

Codiostomum struthionis is a nematode parasite of the ostrich caecum. Little is known about its pathology, being considered by many authors as a non-pathogenic parasite. Infections by C. struthionis are sometimes overlooked because its eggs are indistinguishable from another ostrich nematode, Libyostrongylus spp. Fecal cultures and infective larvae identification are necessary for proper identification. The aim of this study is to provide improved morphological characterization of adults and infective larvae of C. struthionis. Ten caeca of adult ostriches were collected and washed in 0.09% saline solution. Male and female nematodes were collected and quantified separately. Nematodes were fixed in A.F.A. for optical microscopy or fixed in Karnovsky solution for scanning electron microscopy. To obtain infective larvae, fecal samples were collected at sites of high concentration of parasites in the caeca and fecal cultured. The resultant larvae were identified and measured with light microscope at 400x. Nine of the 10 slaughtered ostriches were parasitized by C. struthionis. All nematodes were found in the distal third of the caeca. A total of 566 parasites were recovered (234 males and 332 females). All the cultured larvae had characteristics of C. struthionis (rounded cephalic region with a flat extremity, an acute larvae tail termination and a long and filamentous sheath tail). All the adult parasites were characterized as C. struthionis. Through the analysis of the infective larvae it was determined that the morphology of the larvae tail was the best trait to use in the distinction of this species (live bird diagnosis).

Morphological diagnosis of infective larvae of Libyostrongylus douglassii (Cobbold, 1882) Lane, 1923 and L. dentatus Hoberg, Lloyd and Omar, 1995 (Nematoda: Trichostrongylidae) of ostriches.

The differentiation of the species of the Libyostrongylus genus is only possible with the obtainment of the adult parasites in the ostriches proventriculus and gizzard. The present work confirms that it is possible to differentiate the infective larvae of L. douglassii and L. dentatus allowing the differential diagnosis of these species by fecal culture. To show this, adult females from both species were collected from ten proventriculus from adult ostriches and separated by species. Both groups were macerated individually added to sterilized feces for standard fecal cultures. The infective larvae were recovered, identified, quantified and measured. All proventriculus analyzed were parasitized by Libyostrongylus spp. and a clear heterogeneous location for each species was observed. The infective larvae from the fecal cultures of macerated L. douglassii presented a mean total length of 874.3+/-33.80 microm, and a short sheath tail (29.5+/-4.11 microm) with acute termination. The infective larvae from the macerated L. dentatus presented mean total length of 856.0+/-43.63 microm, long sheath tail (61.2+/-9.52 microm) with filamentous termination. The mean measures of the tails of both species had a significant difference. The differentiation of the infective larvae of L. douglassii and L. dentatus by fecal cultures will facilitate the diagnosis of both species for further understanding the Libyostrongylus biology.