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Abstract: We report the pathological, immunohistochemical, and molecular features of fatal acute systemic toxoplasmosis in an adult, female, free‐living southern muriqui (Brachyteles arachnoides) from São Paulo state, Brazil. PCR‐RFLP genotyping analysis identified the #21 genotype of Toxoplasma gondii. This represents the first report of acute toxoplasmosis involving this genotype in humans and animals. [ABSTRACT FROM AUTHOR]

To evaluate transplacental transmission of Toxoplasma gondii in naturally infected ewes, blood samples were collected from 55 pregnant ewes and their offspring, before ingestion of colostrum. From 16 offspring of positive ewes and nine offspring from negative ewes, blood samples were obtained after 48 h and 7, 14, 21, 28, 35, 42, 49 and 56 days after birth. T. gondii antibodies were detected in serum samples using the indirect fluorescence antibody test (IFAT ≥ 64). Four of the 30 positive ewes (13.3 %) had offspring positive for T. gondii before ingesting colostrum (vertical transmission). The colostrum antibody titers decreased every week, and only 20 % (2/10) of the lambs in continued to present detectable antibody titers until day 56 after birth. Therefore, vertical transmission of T. gondii in lambs was indication of occur and is an important route for transferring and maintaining the agent in sheep herds in the Brazilian semiarid region., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study was conducted to determine the seroprevalence of anti-Toxoplasma gondii antibodies in 152 free-living small wild mammals from distinct regions in the Caatinga biome, a semi-arid region in the Northeast of Brazil: the National Park of Serra das Confusões (NPSC), which is a preserved area in the state of Piauí, and the municipalities of Petrolina and Lagoa Grande, two non-preserved areas in the state of Pernambuco. Using the modified agglutination test (MAT), we found that 5.3% (4/75) and 3.3% (2/60) of small wild mammals were positive for IgG anti-T. gondii antibodies in the NPSC and Petrolina, respectively. All mammals from Lagoa Grande (0/17) tested negative on the MAT. Indirect infection of T. gondii was determined by MAT in Galea spixii, Monodelphis domestica and Thrichomys laurentius (from NPSC) and in Didelphis albiventris (from Petrolina). Seropositive animals were observed in both preserved and non-preserved areas within the Caatinga biome. Low seroprevalences observed can be related to the extreme temperature and humidity in this particular biome., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The study was conducted to determine the seroprevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in goats, sheep, dogs and cats from two distinct regions in the semi-arid region of Northeastern Brazil: Serra das Confusões National Park (SCNP), a preserved area; and municipality of Petrolina, a non-preserved area. Overall, by the indirect fluorescent antibody test (IFAT), the prevalence for IgG anti-T. gondii antibodies in goats was 5.1% (19/376) and 2.4% (9/376); in sheep, it was 10.2% (34/332) and 14.2% (47/332); in dogs, it was 19.7% (25/127) and 3.9% (5/127) and, in cats, it was 25.7% (9/35) and 5.7% (5/35), for T. gondii and N. caninum, respectively. For T. gondii infection, the risk factors associated with the seroprevalence was gender (female) and study area (Petrolina) for goats and only the study area (Petrolina) for dogs; and for N. caninum infection the risk factors were the age (six months to one year) and the study area (Petrolina) for sheep. The circulation of T. gondii and N. caninum was observed in both areas, with significative risk factors demonstrated of a degraded environment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Although few species of Sarcocystis are known to use marsupials of the genus Didelphis as definitive host, an extensive diversity of alleles of surface antigen genes (sag2, sag3, and sag4) has been described in samples of didelphid opossums in Brazil. In this work, we studied 25 samples of Sarcocystis derived from gastrointestinal tract of opossums of the genus Didelphis by accessing the variability of sag2, sag3, sag4, gene encoding cytochrome b (cytB) and first internal transcribed spacer (ITS1). Reference samples of Sarcocystis neurona (SN138) and Sarcocystis falcatula (SF1) maintained in cell culture were also analyzed. We found four allele variants of cytB, seven allele variants of ITS1, 10 allele variants of sag2, 13 allele variants of sag3, and 6 allele variants of sag4. None of the sporocyst-derived sequences obtained from Brazilian opossums revealed 100% identity to SN138 at cytB gene, nor to SN138 or SF1 at ITS1 locus. In addition, none of the sag alleles were found identical to either SF1 or SN138 homologous sequences, and a high number of new sag allele types were found other than those previously described in Brazil. Out of ten sag2 alleles, four are novel, while eight out of 13 sag3 alleles are novel and one out of six sag4 alleles is novel. Further studies are needed to clarify if such a vast repertoire of allele variants of Sarcocystis is the consequence of re-assortments driven by sexual exchange, in order to form individuals with highly diverse characteristics, such as pathogenicity, host spectrum, among others or if it only represents allele variants of different species with different biological traits., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The protozoan parasite Toxoplasma gondii is one of the most successful known eukaryotic pathogens on Earth. Virulence of T. gondii strains varies greatly in mice, and mounting evidence suggests that such variations may be relevant to the manifestation of human toxoplasmosis. Polymorphic rhoptry-secreted kinases and pseudokinases (ROP) have been demonstrated to account for murine virulence among the archetypal clonal parasite lineages that dominate the populations of North America and Europe. However, the distribution of virulence gene alleles in natural populations and the broad influence of these allele combinations on T. gondii virulence have not been examined in depth. In the present study, we performed PCR-RFLP genotyping analysis on a diverse array of globally distributed T. gondii strains at four ROP gene loci including ROP18, ROP5, ROP16 and ROP17 that were previously implicated in influencing T. gondii virulence and pathogenesis. We demonstrated through correlation with published virulence data that the combination of ROP18 and ROP5 allele types is highly predictive of T. gondii virulence across a broad range of global T. gondii isolates. These findings indicate that the importance of ROP18 and ROP5 in determining strain virulence is not limited to the North American/European archetypal lineages most commonly used in molecular studies, but also appears to apply to diverse isolates from South/central America and Asia. Restriction fragment length polymorphism analysis of these loci may thus serve as a valuable tool in determining the potential virulence of uncharacterized T. gondii strains in future studies., (Published by Elsevier Ltd.)

An adult, captive raised male Valley quail (Callipepla californica) acquired by a southern Brazilian aviary suddenly showed severe apathy, dyspnea and diarrhea, and died 18 hours after the onset of illness. At necropsy, pale muscles and whitish areas in the heart, splenomegaly, hepatomegaly, and consolidated red lungs were observed. Histological findings were mainly mononuclear inflammation with necrosis of liver, heart, spleen, bone marrow and lung. There were large numbers of Toxoplasma gondii tachyzoitesorganisms in the liver, heart, spleen, bone marrow, lungs, trachea, kidneys, adrenal glands, testes, intestines, and pancreas. These organisms were seen free in the organs' stroma or within macrophages and stained positively with polyclonal antiserum to T. gondii. Genomic DNA was extracted from the tissues and PCR was used to target the B1 gene of T. gondii. The genotypic characterization by PCR-RFLP with 11 markers (SAG1, SAG2 and alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3) revealed the ToxoDB-PCR-RFLP #87 genotype, the same as previously identified in a backyard chicken (TgCkBr156) in Rio Grande do Sul, Brazil.

The occurrence of antibodies against Toxoplasma gondii was determined in donkeys, mules, and horses from different regions of Brazil. Serum samples from 304 donkeys (67.11%), 118 horses (26.05%), and 31 mules (6.84%) were analyzed by means of the indirect fluorescent antibody test (cutoff=64). Antibodies against T. gondii were detected in 129 equids (28.47%) (82 donkeys, 32 horses, and 15 mules). Tissue samples from 19 seropositive and 50 seronegative animals were obtained in order to isolate the parasite by means of mouse bioassay, and T. gondii was isolated from a donkey. Through genotypic characterization of the isolate, by means of polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) using 11 genotypic markers, the genotype #163 (TgCkBr220), which has already been described in chickens in Brazil, was identified., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Toxoplasmosis is a parasitic zoonosis caused by Toxoplasma gondii protozoan with a worldwide distribution. The aim of this study was to determine the frequency of IgG anti-T. gondii antibodies in bats from São Paulo city, Brazil. A total of 616 serum samples were collected from 22 species of bats. Anti-T. gondii antibodies were searched using the indirect fluorescent antibody test (IFAT ≥ 1:16) and IgG anti-bat antibodies produced in sheep on samples collected during 2006-2011; 32.62% (201/616) of bats had T. gondii antibodies. The modified agglutination test (MAT ≥ 1:25) was performed on samples collected during 2010-2011; 18.61% (35/188) were seropositive. The concordance between IFAT and MAT (serum samples from 2010 to 2011) by Kappa (95% CI) was 0.144, resulting in a low agreement between the techniques. The specificity and sensitivity of MAT and IFAT have not been evaluated for the diagnosis of toxoplasmosis in bats. Thus, it was verified that bats are exposed to T. gondii during their lifetime and they are also part of the toxoplasmosis epidemiology., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Neospora caninum is widely distributed in the world and this parasite is one of the major causes of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum and several species of domestic and wild animals are intermediate hosts. Dogs can become infected by the ingestion of tissues containing cysts and then excrete oocysts. It is not yet known whether sporulated oocysts are able to induce a patent infection in dogs, i.e. a shedding of N. caninum oocysts in feces. The objective of this study was to experimentally examine the infection of dogs by sporulated oocysts. The oocysts used in the experiment were obtained by feeding dogs with brain of buffaloes (Bubalus bubalis) positive for anti-N. caninum antibodies by indirect fluorescent antibody test (IFAT ≥200). Oocysts shed by these dogs were confirmed to be N. caninum by molecular methods and by bioassay in gerbils, and sporulated N. caninum oocysts were used for the oral infection of four dogs. The dogs were 8 weeks old and negative for antibodies to N. caninum and Toxoplasma gondii. Dogs 1 and 4 received an inoculum of 10,000 sporulated oocysts each; dog 2 an inoculum of 5000 sporulated oocysts and dog 3 received 1000 sporulated oocysts of N. caninum. The total feces excreted by these dogs were collected and examined daily for a period of 30 days. No oocysts were found in their feces. The dogs were monitored monthly for a 6-month period to observe a possible seroconversion and when this occurred the animals were eliminated from the experiment. Dogs 1 and 4 seroconverted 1 month after the infection with titer, in the IFAT, of 1600 and 800, respectively; the other two dogs presented no seroconvertion during the 6-month period. Dogs 1 and 2 were euthanized 180 days after infection and were examined for the detection of N. caninum in tissues (brain, muscle, lymph node, liver, lung, heart and bone marrow) by immunohistochemistry and PCR with negative results in both techniques. Bioassay in gerbils with brain of these dogs was also performed and again the results were negative. In conclusion, dogs infected with sporulated oocysts of N. caninum were not able to shed oocysts in feces. However, a higher dose of infection stimulated the production of antibodies against N. caninum in the dogs., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Recent studies indicated that Toxoplasma gondii isolates of many domestic animal hosts from Brazil are genetically and biologically different from those in USA and Europe. Despite of high pathogenicity of this parasite to small ruminants, the epidemiology and genetic diversity of T. gondii in these animals are not well understood in Brazil. In this study, a total of 28 T. gondii samples (16 isolates from sheep in São Paulo state, and 12 isolates from goats in the states of São Paulo and Rio Grande do Norte) were genotyped using genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. Eleven genotypes were identified from these T. gondii isolates. Eight isolates (4 from sheep and 4 from goats) were grouped into the common clonal type BrI lineage. One sheep isolate was grouped to the type BrIII lineage. Five isolates grouped to three previously identified genotypes in Brazil, and 13 isolates grouped to six novel genotypes. Mixed genotype was found in one isolate from goat in São Paulo. No classical clonal Type I, II or III isolates were found, confirming previous reports that these clonal lineages are rare in Brazil. The allele types at the CS3 locus are strongly linked to mouse virulence of the parasite. The results of this study indicate that even though a large number of T. gondii genotypes have been identified from a variety of animal hosts in Brazil, high percentage of new genotypes are continuously identified from different animal species, suggesting extremely high diversity of T. gondii in the population., ((c) 2010 Elsevier B.V. All rights reserved.)

The aim of this study was to determine if Toxoplasma gondii are present in oysters (Crassostrea rhizophorae) and mussels (Mytella guyanensis) under natural conditions using a bioassay in mice and molecular detection methods. We first compared two standard protocols for DNA extraction, phenol-chloroform (PC) and guanidine-thiocyanate (GT), for both molluscs. A total of 300 oysters and 300 mussels were then acquired from the fish market in Santos city, São Paulo state, Brazil, between March and August of 2008 and divided into 60 groups of 5 oysters and 20 groups of 15 mussels. To isolate the parasite, five mice were orally inoculated with sieved tissue homogenates from each group of oysters or mussels. For molecular detection of T. gondii, DNA from mussels was extracted using the PC method and DNA from oysters was extracted using the GT method. A nested-PCR (Polymerase Chain Reaction) based on the amplification of a 155 bp fragment from the B1 gene of T. gondii was then performed. Eleven PCR-RFLP (Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, CS3 and Apico, were used to genotype positive samples. There was no isolation of the parasite by bioassay in mice. T. gondii was not detected in any of the groups of mussels by nested-PCR. DNA of T. gondii was apparently detected by nested-PCR in 2 groups of oysters (3.3%). Genotyping of these two positive samples was not successful. The results suggest that oysters of the species C. rhizophorae, the most common species from the coast of São Paulo, can filter and retain T. gondii oocysts from the marine environment. Ingestion of raw oysters as a potential transmission source of T. gondii to humans and marine mammals should be further investigated., ((c) 2010 Elsevier B.V. All rights reserved.)

Recent studies indicate that Toxoplasma gondii isolates of many domestic hosts from Brazil are genetically and biologically different from T. gondii isolates from USA and Europe. However, little is known about genetics of T. gondii isolates from wild mammals in Brazil. In this study, genotypes of 36 T. gondii isolates from capybaras (Hydrochaeris hydrochaeris) from six counties in São Paulo state, Brazil, were determined. Sixteen genotypes were identified using 11 genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. No classical clonal Type I and Type II isolates were found, confirming other findings that these lineages are rare in Brazil. Eight of these 36 isolates were grouped into the common clonal lineages in Brazil, previously designed as Types BrI, BrII and BrIII. Seven of the 16 genotypes were reported for the first time in this study. Three of the 36 isolates showed mixed infections. Analysis of mortality rates in infected mice indicated that Type BrI is highly virulent, Type BrII is intermediately virulent and Type BrIII is non-virulent, which is in agreement with previous report. The allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. These genotyping results support previous findings that the T. gondii population is highly diverse in Brazil.

Hammondia heydorni is a cyst forming coccidia closely related to other apicomplexans, such as Toxoplasma gondii, Neospora caninum and Hammondia hammondi with a two-host life cycle. Dogs and other canids as red foxes (Vulpes vulpes) and coyotes (Canis latrans) may serve as definitive hosts for H. heydorni. Sporulated oocysts are infective for cattle, sheep and goats, which may serve as intermediate hosts. Herein, we describe the ability of crab-eating fox (Cerdocyon thous), a wild carnivore that is commonly found from northern Argentina to northern South America, to serve as definitive host of H. heydorni. The whole masseter muscle and brain from two 2-year-old bovines were collected, minced and pooled together for the fox infection. The bovine pooled tissues were equally administered to four foxes, in two consecutive days. Two foxes shed subspherical unsporulated oocysts measuring 10-15microm, after 8 and 9 days post-infection, respectively. One of the foxes eliminated oocysts for 5 days, while the other fox shed oocysts for 9 days. A DNA sample of oocysts detected at each day of oocyst elimination was tested by two PCRs, one of them carried out employing primers directed to the common toxoplasmatiid 18S and 5.8S ribosomal RNA coding genes (PCR-ITS1) and the other based on heat-shock protein 70kDa coding gene (PCR-HSP70). These samples were also submitted to a N. caninum specific nested-PCR protocol based on a N. caninum specific gene (Nc5-nPCR). All of them were positive by PCR-ITS1 and PCR-HSP70 but negative by Nc5-nPCR. The PCR-ITS1 and PCR-HSP70 nucleotide sequences amplified from the oocysts shed by the foxes revealed 100% identity with homologous sequences of H. heydorni. In conclusion, it is clear that H. heydorni also uses the crab-eating fox as a definitive host. The crab-eating fox is usually reported to live in close contact with livestock in several regions of Brazil. Therefore, it is reasonable to infer that such carnivores may play an important role in the sylvatic and domestic cycles of H. heydorni infection.

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoró, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%) had at least one seropositive animal for T. gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent's antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region.

The prevalence and risk factors for anti-Toxoplasma gondii antibodies were investigated in goats of the Seridó Oriental microregion, Rio Grande do Norte state, Northeast region of Brazil. Three hundred and sixty-six blood samples from goats collected by jugular venopuncture were used. For the serologic diagnosis of Toxoplasma gondii infection, the indirect fluorescent-antibody test (IFAT) with cut-off value 1:64 was carried out. The prevalence of anti-T. gondii antibodies was 30.6% [95% CI=25.9-35.6%] with titers ranging from 1:64 to 1:16,384. The multivariate logistic regression analysis showed that the risk factors associated to anti-T. gondii antibodies were presence of cats in the herd, extensive/semi-intensive management systems and lack of mineral supplementation.

The objective of this study was to obtain information of epidemiological nature through genotypic characterization of Cryptosporidium isolates from dogs, cats and bovines from the state of São Paulo, Brazil. The extraction of DNA from oocysts was carried out and polymerase chain reaction was accomplished using specific primers to 18S rRNA gene. The amplicons were directed sequenced. Seven cat samples, nine dog samples and nine bovine samples were analysed. From the seven cat samples the genotypic analyses revealed Cryptosporidium felis in all. These were the first genotypic characterization of Cryptosporidium from domestic felines in Brazil. In nine sequenced samples from dogs, genotypic identities compatible with Cryptosporidium canis were revealed in all samples. The genotypic analyses in bovines revealed Cryptosporidium parvum in eight samples and Cryptosporidium bovis in another sample, the last one being a non-zoonotic species, not related to clinical symptoms and described for the first time in Brazil.

The nucleotide sequence of glutamate dehydrogenase (gdh) coding genes were obtained from cysts of Giardia duodenalis isolated from feces of naturally infected cats (n=19), dogs (n=27), humans (n=37) and cattle (n=5). The samples were from several municipalities within the state of São Paulo, Brazil and were collected from January 2004 to August 2006. Sequences analysis of the 37 specimens recovered from humans revealed 29 G. duodenalis assemblage AII and 8 G. duodenalis assemblage B. Among samples from cats, 11 were classified into assemblage F and 8 into assemblage AI. Only the host-adapted assemblages C and D were detected in samples from dogs (7 and 20, respectively). Among the samples from cattle, the genotype livestock was found in four samples and the assemblage AI was detected in one sample. The molecular identification of assemblages of G. duodenalis isolates from different hosts reveals that genetic diversity of this protozoon in Brazil is similar to that of Giardias from other parts of the world.

The prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies was investigated in goats slaughtered in the public slaughterhouse of Patos, State of Paraíba, Northeast region of Brazil, and possible associations between sex of the animals and antibody prevalence were verified. Three-hundred six blood samples from goats collected before slaughter by jugular venopuncture were used. For the serologic diagnosis of T. gondii and N. caninum, the indirect fluorescent-antibody test (IFAT) with cut-off values 64 and 50, respectively, was carried out. The prevalence of anti-T. gondii antibodies was 24.5% [95% CI=19.8-29.7%] with titers ranging from 64 to 4096, and anti-N. caninum antibodies was 3.3% (95% CI=1.6-5.9%) with titers ranging from 50 to 400. There were no associations between sex of animals and prevalence of anti-T. gondii and anti-N. caninum antibodies.