Integrative taxonomy for the traditional coccidians (Chromista: Miozoa: Eimeriidae) from island canaries (Aves: Passeriformes: Fringillidae): Worldwide distribution, morphological and molecular characterization, revaluations and establishment of junior synonyms.

Island canaries Serinus canaria (Linnaeus) are finches native to the North Atlantic Islands, however, they have a worldwide distribution in captivity due to their relevance as a pet bird. Coccidians are the most reported parasites of passerines worldwide, both in the wild and in captivity, being frequently associated with disease in passerines kept in rehabilitation centers and commercial breeders. This study aimed to identify coccidians from island canaries kept in captivity in Brazil. Three hundred and fifteen genomic DNA extracted from fecal samples of island canaries from different breeders from Southern and Southeastern Brazil were used to perform a nested PCR assay to amplify a partial fragment of the 28S small subunit ribosomal RNA gene (28S) of Isospora spp. Microscopic screening and morphological identification of Isospora oocysts was performed in fecal samples corresponding to PCR positive DNA samples. Fecal samples have been formalin-stored for approximately four years. Positivity rate for both microscopy and PCR was 10.5% (33/315). Posteriorly, Isospora serini (Aragão, 1933) Box, 1975 and Isospora canaria Box, 1975 were morphologically identified from fresh fecal samples of island canaries maintained by a breeder in the State of São Paulo, Southeastern Brazil, providing a genotypic characterization via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) and 28S genes. The 28S and COI sequences referring to the morphological identification of I. canaria was, respectively, 100% and 99% similar to sequences deposited as Isospora serinuse Yang, Brice, Elliot & Ryan, 2015 from island canaries kept in a rehabilitation center in Australia. The COI sequence referring to the morphological identification of I. serini was 100% similar to a sequence of an extraintestinal Isospora, corroborating this identification/sequencing since I. serini is the first isosporan with an extra-intestinal cycle demonstrated. The comparison of morphological and molecular data from I. canaria and I. serini from this study with published data of Isospora spp. from canaries worldwide, allowed the specific identification from preliminary generic identifications, correction of misidentifications, as well as the establishment of junior synonyms. Finally, this study provides morphological and molecular data that ensure the correct identification of the two Isospora spp. from island canaries in future studies worldwide.

Cryptosporidium spp. in caged exotic psittacines from Brazil: Evaluation of diagnostic methods and molecular characterization.

The aim of this study was to evaluate the prevalence of and diagnostic methods for Cryptosporidium spp. in caged adult exotic parrots from Southern and Southeastern Brazil. Oocysts were purified from fecal samples from 463 psittacines by centrifugal-flotation in Sheather's sugar solution. Cryptosporidium spp. were detected by malachite green negative staining and nested PCR targeting the 18S rRNA gene. Cryptosporidium species were identified by sequencing nested PCR amplicons. Samples were also tested by duplex real-time PCR targeting the 18S rRNA gene of Cryptosporidium galli and Cryptosporidium avian genotype III. The prevalence rates of Cryptosporidium spp. determined by microscopy and nested PCR were 3.0% (14/463) and 5.0% (23/463), respectively. The nested PCR/sequencing identified avian genotype III (1.7%; 8/463), Cryptosporidium parvum (0.9%; 4/463) and Cryptosporidium canis (0.2%; 1/463). Duplex real-time PCR was positive for gastric Cryptosporidium in 9.5% (44/463) of the samples. Among them, 1.9% (9/463) were positive for C. galli, 5.8% (27/463) were positive for avian genotype III and 1.7% (8/463) showed mixed infections with C. galli and avian genotype III. With regards to the positive detection of Cryptosporidium spp., there was no statistically significant difference between nested PCR and microscopic analysis (p = .1237), and a fair agreement existed between them (Kappa = 0.242). A statistically significant difference (p < .0001) and fair agreement (Kappa = 0.317) were obtained between nested PCR/sequencing and duplex real-time PCR for the detection of gastric Cryptosporidium. We determined that nested PCR and duplex real-time PCR are the best options for the detection of Cryptosporidium spp. and gastric Cryptosporidium, respectively, and that avian genotype III is the most common Cryptosporidium genotype/species in psittacines.

Survey the Occurrence of Globocephalus urosubulatus (Nematoda: Ancylostomatidae) in Wild Boars (Sus scrofa) in the State of São Paulo, Brazil.

Although previous studies have characterized the helminth fauna of wild boars kept in captivity in Brazil, records on these helminths in free-ranging animals are still scarce. In view of this, we aimed in our work to investigate the occurrence and morphological and morphometric characteristics of gastrointestinal helminths in wild Sus scrofa from the northwest region of the State of São Paulo, Brazil. The digestive systems of 10 animals (5 males and 5 females of different ages) were used in this study. Each anatomical segment was washed and sieved under running water, and the helminths were separated and identified using light and scanning electron microscopy, according to their morphological characteristics. A total of 2750 (1152 males and 1598 females) nematode specimens were collected from the small intestine of these wild boars, and all of them presented the morphological characteristics of Globocephalus urosubulatus. However, one characteristic is of particular interest because it has not yet been reported in the literature: a marked asymmetry between the lobes and their respective rays of the copulatory bursa, with the left one being larger than the right one. In this research, we identified the presence of G. urosubulatus in all the examined free-ranging wild boars and reported for the first time in the literature the asymmetry in the copulatory bursa.

The Impact of Physical Effort on the Gut Microbiota of Long-Distance Fliers.

Flying pigeons (Columbia livia) are extensively studied for their physical endurance and superior sense of orientation. The extreme physical endurance of which these birds are capable creates a unique opportunity to investigate the possible impact of long-distance flying on the taxonomy and metabolic function of the gut microbiota. This project was enabled by access to two groups of pigeons raised by the same breeder in the same conditions, except that one group was trained in long-distance flying and participated in multiple races covering a total distance of over 2600 km over a 9-week period. In contrast, the second group did not fly. The fecal microbiota was analyzed using 16S amplicon sequencing, and the taxonomy and metabolic function were inferred from this sequence data. Based on phylogenetic distance and metabolic function, flying and non-flying pigeons were found to harbor distinct bacterial microbiota. The microbiota taxonomy varied extensively between the birds, whereas the inferred metabolic potential was relatively stable. Age was not a significant determinant of the fecal microbiota profile. In flying birds, the metabolic pathways annotated with biosynthesis were enriched, representing 60% of the 20 metabolic pathways that were most closely associated with flying.

Identification of Eimeria spp. in domestic chickens raised in alternative poultry production systems in the State of São Paulo, Brazil.

The objective of this study was to identify Eimeria spp. in alternative poultry production systems (APPS) in the State of São Paulo, Brazil. Fecal samples (168) and DNA extracted from fecal samples obtained in APPS located in different Municipalities in the State of São Paulo (93) were examined by microscopy or genera-specific PCR (ITS-1 locus). Samples positive for Eimeria spp. were examined using Eimeria lata, Eimeria nagambie, and Eimeria zaria species-specific PCR protocols (ITS-2 locus) and another E. lata-specific PCR (candidate IMP1 genomic locus) followed by molecular cloning (E. lata and E. zaria ITS-2 amplicons) and genetic sequencing. All positive DNA samples were also submitted to genera-specific nested PCR (18S rRNA gene) followed by next-generation sequencing to identify Eimeria spp. Eimeria nagambie, E. zaria, and Eimeria sp. were identified by ITS2-targeted species-specific PCRs and genetic sequencing. Next-generation sequencing identified, in order of prevalence: E. nagambie; Eimeria acervulina; Eimeria mivati; Eimeria praecox; Eimeria brunetti; Eimeria mitis; Eimeria sp.; Eimeria maxima; E. zaria, and Eimeria necatrix/tenella. Our results confirmed, for the first time in Brazil, the identification of E. nagambie, E. zaria, and Eimeria spp. ITS-2 and 18S rRNA gene sequences not yet described in Brazil.

Cryptosporidium proventriculi in Captive Cockatiels (Nymphicus hollandicus).

Cockatiels (Nymphicus hollandicus) are among the most commonly sold psittacines pets. The aim of this study was to evaluate the occurrence of Cryptosporidium spp. in domestic N. hollandicus and identify risk factors for this infection. We collected fecal samples from 100 domestic cockatiels in the city of Araçatuba, São Paulo, Brazil. Feces from birds of both genders and older than two months were collected. Owners were asked to complete a questionnaire to identify how they handle and care for their birds. Based on nested PCR targeting the 18S rRNA gene, the prevalence of Cryptosporidium spp. in the cockatiels sampled was 9.00%, 6.00% based on Malachite green staining, 5.00% based on modified Kinyoun straining, and 7.00% when the Malachite green was combined with Kinyoun. Applying multivariate logistic regression to test the association between Cryptosporidium proventriculi positivity and potential predictors showed that gastrointestinal alterations was a significant predictor (p < 0.01). Amplicons from five samples were sequenced successfully and showed 100% similarity with C. proventriculi. In summary, this study demonstrates the occurrence of C. proventriculi in captive cockatiels.

Evidence of the Zoonotic Transmission of Cryptosporidium among Children and Pets.

We investigated the zoonotic transmission of Cryptosporidium among the children (n = 188), dogs (n = 133), and cats (n = 55) living in 188 households. Fecal samples were examined using ELISA and confirmed via nested PCR. Coproantigens oocysts were detected in 3.7% of children, 8.3% of dogs, and 5.5% of cats. We found strong evidence of two cases of the zoonotic transmission of Cryptosporidium canis between children and dogs. Furthermore, four children and their respective pets (one dog and three cats) were infected with Cryptosporidium parvum, but we cannot exclude the hypotheses that the oocysts were transmitted from children to animals or that both hosts were infected by a shared source, such as contaminated water or food. The presence of an infected animal elevated the risk of zoonotic transmission by 129.7-fold (95% CI: 13.92-1209.68). Furthermore, sharing a bed with pets was identified as a risk factor for infection in children (OR: 9.9, 95% CI: 1.37-71.2). In conclusion, the zoonotic transmission of Cryptosporidium among children and pets cohabiting in the same household may be quite common, especially when infected animals lie or sleep on children's beds. These findings unequivocally highlight the public health concern surrounding C. canis.

Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples.

The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples.

Efficacy of imidacloprid/flumethrin collar in preventing canine leishmaniosis in Brazil.

The Leishmania infantum (synonym, Leishmania chagasi) causes life-threatening infection, namely canine leishmaniosis (CanL), which is a chronic zoonosis prevalent in various countries and spread by the bite of the infected Lutzomyia female sandfly in South America. The objective of the study was to assess the effectiveness of a polymer matrix collar containing made up of 10% imidacloprid and 4.5% flumethrin for the prevention of canine leishmaniosis from the hyperendemic region falling under Araçatuba municipality (Brazil). The research included a total of 146 dogs chosen from 75 households. Test were initiated via physical examination; weighing and biological sample collection (blood, popliteal lymph node and conjunctival swab) of these dogs were done in March 2018 (Day 0; GA, control = 69, GB, treated = 77) to initiate laboratory tests. Post-inclusion, the animals were monitored on the 120th, 240th, 360th and 480th days, respectively. The usage of collars continued between 0 and 480 days before being substituted in second (D240) and fourth (D480) follow-up visits. On the whole, 25 dogs in GA (36.2%) and three in GB (3.9%) were found positive for L. infantum infection in a minimum of one diagnostic test used in the research. Therefore, the average collar effectiveness for protection from L. infantum infection was 89.2% (p < .01). In the last follow-up, the average incidence density rate for GA was 30.7%, whereas for GB, it was 2.9%. The imidacloprid/flumethrin collars evaluated in the research were found to be safe and extremely efficient for the prevention of L. infantum infection through Lutzomyia species among the large population of dogs in highly prone endemic regions. This is a dependable and efficient technique aimed at reducing the occurrence and propagation of this illness among the population of canines, which would eventually reduce the human-health-related hazards. In Brazil, Lutzomyia spp. is a leading vector of the infection; thus, the collar can be used to limit infection in dogs and humans.

Detection and molecular characterization of Giardia spp. in captive Psittaciformes in Brazil.

This study aimed to perform the detection and molecular characterization of Giardia spp. in Psittaciformes from the Southern and Southeastern regions of Brazil. Fecal samples were obtained from 359 adult exotic captive Psittaciformes belonging to 13 genera, randomly selected from 33 aviaries located in the Southern and Southeastern regions of Brazil during a bird exhibition at the 2015 Ornithological Championship of the Ornithological Federation of Brazil (FOB). Nested polymerase chain reaction targeting the small subunit rRNA gene identified Giardia spp. in 93/359 (25.9%) fecal samples and 25/33 (75.8%) aviaries. Genetic sequencing identified G. psittaci in 12 birds from six genera. Zoonotic Giardia species was not detected in fecal samples from Psittaciformes.

Detection and characterization of Cryptosporidium species and genotypes in three chicken production systems in Brazil using different molecular diagnosis protocols.

The objective of this study was to determine the occurrence of Cryptosporidium spp. in domestic chickens raised in different chicken production systems in Brazil using three nested PCR protocols. The purification and concentration of oocysts present in 190 fecal samples from chickens raised in extensive, semi-intensive and intensive production systems were accomplished by centrifugal flotation in Sheather's solution and were followed by the extraction of genomic DNA. The detection and molecular characterization of Cryptosporidium species and genotypes were performed using three nested polymerase chain reaction (nested PCR) protocols targeting the 18S rRNA gene followed by sequencing of the amplified fragments. Subgenotyping of C. meleagridis was performed using a nested PCR reaction targeting the gp60 gene. Sample identified as Cryptosporidium sp. genetically similar to Cryptosporidium xiaoi and Cryptosporidium bovis by 18S rRNA gene sequencing were further analyzed by nested PCR targeting the actin gene and subsequent sequencing of the amplified fragment. Positive amplification for Cryptosporidium spp. was observed in 12.6% (24/190) of the samples, including C. baileyi (9.8%; 18/190), C. meleagridis (0.5%, 1/190), C. parvum (2.1%; 4/190) and Cryptosporidium sp. (0.5%; 1/190). Subgenotyping of C. meleagridis revealed the presence of the zoonotic subtype IIIgA23G3R1. Sequencing of the 18S rRNA gene and the actin gene fragments revealed a Cryptosporidium genotype in an extensive poultry system genetically related to C. xiaoi and C. bovis. There was no significant difference in the frequency of positive results obtained by the three nested PCR protocols (p > 0.05); additionally, the agreement obtained by Kappa index ranged from substantial (0.70) to almost perfect (0.9).

Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods.

This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples / Validação da nested PCR em tempo real em um tubo para detecção de Cryptosporidium spp. em amostras fecais de aves

Abstract The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10/36 (27.8%) of positive samples.

Resumo O objetivo deste trabalho foi validar um protocolo de nested PCR em tempo real em um tubo (nPCR-TR-1T) seguida de sequenciamento genético para detectar e caracterizar as espécies e genótipos de Cryptosporidium em aves. Um total de 443 amostras de DNA genômico, extraído de amostras fecais de aves, foi analisado pela nPCR-TR-1T e pela nested PCR convencional. Pela nPCR-TR-1T, foi observada positividade para Cryptosporidium spp. de 20,3% (90/443), em contraste com a nested PCR convencional, que apresentou positividade de 8,1% (36/443). O teste de sensibilidade analítica mostrou que a nPCR-TR-1T detecta aproximadamente 0,5 oocisto (2 esporozoítos) por reação. A avaliação da especificidade analítica não revelou amplificação de microrganismos que comumente apresentam amplificação inespecífica com primers utilizados para o diagnóstico de Cryptosporidium spp. O cálculo da repetibilidade evidenciou o mesmo resultado em 27 de 30 amostras (90%). Em relação à reprodutibilidade da nPCR-TR-1T, foi observado o mesmo resultado em 80% (24/30) das amostras examinadas. Foi possível realizar o sequenciamento em todas as 90 amostras amplificadas pela nPCR-TR-1T, com identificação de C. baileyi, C. galli, C. meleagridis, C. proventriculi e Cryptosporidium genótipo I em aves. O sequenciamento dos fragmentos amplificados pela nested PCR convencional foi possível em 10/36 (27,8%) das amostras positivas.

Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes.

In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.

First description of Cryptosporidium parvum in carrier pigeons (Columba livia).

The carrier pigeon and the domestic pigeon are different breeds of the species Columba livia. Carrier pigeons are used for recreational activities such as bird contests and exhibitions. Due to the close contact with humans, these birds may potentially represent a public health risk, since they can host and disseminate zoonotic parasites, such as those belonging to the genus Cryptosporidium (phylum Apicomplexa). The purpose of this work was the detection by microscopic and molecular techniques of Cryptosporidium spp. oocysts in fecal samples of carrier pigeons, and subsequently to sequence the 18S ribosomal RNA marker of positive samples to identify the species. A total of 100 fecal samples were collected individually in two pigeon breeding facilities from Formiga and Araçatuba, cities located in Minas Gerais state and São Paulo state, Brazil, respectively. The age of the birds ranged from one to 12 years; 56 were females and 44 males. Fecal smears were stained with negative malachite green, whereas the molecular characterization was based on the sequence of a ∼800bp fragment of the 18S rRNA gene. Microscopic examination of fecal smears revealed 4% (4/100) oocyst positivity. On the other hand, 7% (7/100) of positivity were found using nested PCR. Three samples were 99% to 100% similar to Cryptosporidium parvum 18S rDNA type A (Genbank AH006572) and the other three samples had 99% to 100% similarity to C. parvum 18S rDNA type B (Genbank AF308600). To our knowledge, this is the first report of C. parvum oocysts in carrier pigeons.

First description of Cryptosporidium hominis GP60 genotype IkA20G1 and Cryptosporidium parvum GP60 genotypes IIaA18G3R1 and IIaA15G2R1 in foals in Brazil.

The present study focuses on Cryptosporidium infections of foals in Brazil. A total of 92 animals of different breeds from 11 farms in the vicinity of Araçatuba in the state of São Paulo, were examined. According to PCR targeting the 18S rRNA gene, Cryptosporidium sp. DNA was detected in 21.7% (20/92) of foals. Good quality 18S rRNA, actin, HSP70 and gp60 genes nPCR amplicons were obtained from five fecal samples. PCR amplification and sequencing of a fragment of the GP60 sporozoite surface glycoprotein gene revealed C. parvum genotypes IIaA18G3R1, IIaA15G2R1. Interestingly, we also detected in two foals a GP60 genotype related to the human parasite C. hominis.

Natural canine infection by Leishmania infantum and Leishmania amazonensis and their implications for disease control.

Leishmaniasis is a major public health problem worldwide. Because Leishmania can adapt to new hosts or vectors, knowledge concerning the current etiological agent in dogs is important in endemic areas. This study aimed to identify the Leishmania species detected in 103 samples of peripheral blood from dogs that were naturally infected with these protozoa. The diagnosis of leishmaniasis was determined through parasitological examination, the indirect enzyme-linked immunosorbent assay (ELISA) and the polymerase chain reaction (PCR). The Leishmania species were identified by means of PCR-restriction fragment length polymorphism (PCR-RFLP). The samples were subjected to PCR using oligonucleotide primers that amplify the intergenic region ITS1 of the rRNA gene in order to identify the species. The amplified DNA was digested using the restriction enzyme HaeIII. A restriction profile identical to L. amazonensis was shown in 77/103 samples and the profile was similar to L. infantum in 17/103. However, a mixed profile was shown in 9/103 samples, which impeded species identification. In conclusion, the infection in these dogs was predominantly due to L. amazonensis, thus indicating that diagnosing of cases of canine leishmaniasis needs to be reexamined, since the causative agent identified is not restricted to L. infantum.

Cryptosporidium infections in birds--a review.

Cryptosporidiosis is one of the main protozoan infections in birds. It manifests as either a respiratory or a digestive illness, and it affects a very large number of avian species across several continents. The aim of this review is to report on the main results of studies on cryptosporidiosis among birds and the importance of these results to veterinary medicine and public health.

The detection of Cryptosporidium serpentis in snake fecal samples by real-time PCR.

Infection by Cryptosporidium serpentis occurs in reptiles, particularly in snakes. This disease is characterized by chronic infection with the presence of hypertrophic gastritis. The objectives of this study were to use real-time polymerase chain reaction (PCR) targeting the heat shock protein 70 (Hsp70) gene for the detection of C. serpentis in fecal samples from snakes and to determine the analytical and epidemiological specificity and sensitivity of this approach relative to the gold standard of nested PCR for the amplification of a fragment of the 18S subunit of the ribosomal RNA (18S rRNA) gene followed by the sequencing of amplified fragments (nPCR/S). Individual fecal samples were collected on a single occasion from 503 asymptomatic adult snakes housed in the serpentarium of the Butantan Institute in São Paulo, Brazil. The nested PCR revealed that 60 samples (11.98%) were positive for Cryptosporidium sp. The sequencing of amplified fragments, which was possible for 38 samples, resulted in the identification of Cryptosporidium tyzzeri (7), Cryptosporidium muris (4), Cryptosporidium varanii (12) and C. serpentis (15) in fecal samples from several snake species. The real-time PCR approach indicated that 17 samples (3.37%) were positive for C. serpentis, whereas the nPCR/S indicated that 15 samples (2.98%) were positive for C. serpentis. The epidemiological sensitivity and specificity of real-time PCR were 93.8% and 99.5%, respectively. Thus, we conclude that real-time PCR targeting the Hsp70 gene is a sensitive and specific method for the detection of C. serpentis in snake fecal samples.

Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods / Detecção e caracterização molecular de Cryptosporidium spp. em canários mantidos em cativeiro (Serinus canaria) por diferentes métodos de diagnóstico

Abstract This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

Resumo Este trabalho teve como objetivos determinar a ocorrência e realizar a caracterização molecular de Cryptosporidium spp. em 498 amostras fecais de canários (Serinus canaria) criados em cativeiro, utilizando três métodos de diagnóstico: análise microscópica pela coloração negativa com verde malaquita, nested PCR seguida de sequenciamento dos fragmentos amplificados e PCR duplex em tempo real específica para detecção de Cryptosporidium galli e Cryptosporidium genótipo III de aves. A positividade total para Cryptosporidium spp. (total de amostras positivas em pelo menos um método de diagnóstico) obtida pela análise microscópica, nested PCR e PCR duplex em tempo real foi de 13,3% (66/498). As taxas de positividade para Cryptosporidium spp. foram 2,0% (10/498) e 4,6% (23/498) por microscopia e nested PCR, respectivamente. O sequenciamento de 20 amostras amplificadas pela nested PCR identificou C. galli (3,0%; 15/498), Cryptosporidium genótipo I de aves (0,8%; 4/498) e Cryptosporidium avium (0,2%; 1/498). A PCR duplex em tempo real revelou positividade de 7,8% (39/498) para C. galli e 2,4% (12/498) para Cryptosporidium genótipo III de aves. A análise microscópica diferiu significativamente da nested PCR para detecção de Cryptosporidium spp. A PCR duplex em tempo real apresentou maior sensibilidade que a nested PCR/sequenciamento para detectar as espécies/genótipos gástricos de Cryptosporidium.