Early immune responses and parasite tissue distribution in mice experimentally infected with oocysts of either archetypal or non-archetypal genotypes of Toxoplasma gondii.

In most of the world Toxoplasma gondii is comprised of archetypal types (types I, II and III); however, South America displays several non-archetypal strains. This study used an experimental mouse model to characterize the immune response and parasite kinetics following infection with different parasite genotypes. An oral inoculation of 50 oocysts per mouse from T. gondii M4 type II (archetypal, avirulent), BrI or BrIII (non-archetypal, virulent and intermediate virulent, respectively) for groups (G)2, G3 and G4, respectively was used. The levels of mRNA expression of cytokines, immune compounds, cell surface markers and receptor adapters [interferon gamma (IFNγ), interleukin (IL)-12, CD8, CD4, CD25, CXCR3 and MyD88] were quantified by SYBR green reverse transcription-quantitative polymerase chain reaction. Lesions were characterized by histology and detection by immunohistochemistry established distribution of parasites. Infection in G2 mice was mild and characterized by an early MyD88-dependent pathway. In G3, there were high levels of expression of pro-inflammatory cytokines IFNγ and IL-12 in the mice showing severe clinical symptoms at 8­11 days post infection (dpi), combined with the upregulation of CD25, abundant tachyzoites and tissue lesions in livers, lungs and intestines. Significant longer expression of IFNγ and IL-12 genes, with other Th1-balanced immune responses, such as increased levels of CXCR3 and MyD88 in G4, resulted in survival of mice and chronic toxoplasmosis, with the occurrence of tissue cysts in brain and lungs, at 14 and 21 dpi. Different immune responses and kinetics of gene expression appear to be elicited by the different strains and non-archetypal parasites demonstrated higher virulence.

Vertical transmission of Toxoplasma gondii in naturally infected ewes in the semiarid region of Brazil.

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.

Genetic Diversity of Toxoplasma gondii Isolates From Free-Range Chickens In Bahia, Brazil.

The genotyping of 25 isolates of Toxoplasma gondii from free-range chickens in the state of Bahia, Brazil, was performed by PCR-restriction fragment length polymorphism using 11 genetic markers: SAG1, 5'+3'SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3. The analysis revealed 8 genotypes, 3 of which had not been previously reported. Four genotypes were represented by single isolates, whereas the other genotypes were represented by 2 or more isolates. Five isolates showed mixed infections, and 2 of them were identical. None of the clonal types I, II, or III were found, but 2 isolates corresponded to the Brazilian clonal lineage BrIII. There was a single allele for the c22-8 marker. The CS3 marker demonstrated efficiency in the evaluation of virulence in mice. This study reaffirms the diverse genetic variability of T. gondii in Brazil.

Fatal toxoplasmosis in a southern muriqui (Brachyteles arachnoides) from São Paulo state, Brazil: Pathological, immunohistochemical, and molecular characterization.

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.

Detection of anti-Toxoplasma gondii antibodies in small wild mammals from preserved and non-preserved areas in the Caatinga biome, a semi-arid region of Northeast Brazil.

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.

Fatal toxoplasmosis in a southern muriqui (Brachyteles arachnoides) from São Paulo state, Brazil: Pathological, immunohistochemical, and molecular characterization

We report the pathological, immunohistochemical, and molecular features of fatal acute systemic toxoplasmosis in an adult, female, free­living southern muriqui (Brachyteles arachnoides) from São Paulo state, Brazil. PCR­RFLP genotyping analysis identified the #21 genotype of Toxoplasma gondii. This represents the first report of acute toxoplasmosis involving this genotype in humans and animals.

Relatamos as características patológicas, imuno-histoquímicas e moleculares da toxoplasmose sistêmica aguda fatal em um muriqui do sul adulto (feminino) de vida livre (Brachyteles arachnoides) do estado de São Paulo, Brasil. A análise de genotipagem por PCR ­ RFLP identificou o genótipo # 21 de Toxoplasma gondii. Isso representa o primeiro relato de toxoplasmose aguda envolvendo esse genótipo em humanos e animais

Report of the first clinical case of intestinal trichomoniasis caused by Tritrichomonas foetus in a cat with chronic diarrhoea in Brazil.

BACKGROUND: Tritrichomonas foetus is an emergent and important enteric pathogen of cats, which causes prolonged diarrhoea in cats. CASE PRESENTATION: This study describes a T. foetus infection in a seven-month-old, entire male domestic shorthair kitten with a six-month history of persistent large intestinal diarrhoea, faecal incontinence, prostration, apathy and weight loss. Parasites were microscopically observed and confirmed by PCR and DNA sequencing. Molecular analyses were carried out comparing the sequence obtained in this study with T. foetus and T. suis. Retrieved from GenBank. After treatment with ronidazole, the cat showed resolution of clinical signs. CONCLUSIONS: This is the first clinical case of T. foetus infection in a chronic diarrheic cat in Brazil and South America, confirming the presence of this pathogen in this part of the world and highlighting the importance of this protozoa being considered in the differential diagnosis of cats presenting diarrhoea of the large intestine. Our case report enriches our knowledge on the geographical distribution of T. foetus in cats in Brazil and provides further understanding of the clinical significance of feline intestinal trichomoniasis in this country.

Acute toxoplasmosis in pigs in Brazil caused by Toxoplasma gondii genotype Chinese 1.

This study reports the clinicopathological, immunohistochemical, and molecular findings from two cases of systemic toxoplasmosis in pigs showing apathy and dyspnea. In the post-mortem examination, severe diffuse necrotizing bronchointerstitial pneumonia with numerous intralesional tachyzoites of Toxoplasma gondii was observed. The lungs had not collapsed but were diffusely reddened, and the parenchyma showed friable whitish subpleural nodules with multifocal to coalescent distribution and diameters of 0.5-1.0 cm. The histopathological findings comprised mononuclear inflammation and multifocal areas of necrosis in alveolar septa (cases 1 and 2). In addition, esophagitis and ulcerations in the mucosa of the stomach and the small and large intestines were observed (case 1). Immunohistochemical analysis using anti-T. gondii antibodies on lung tissue in both cases revealed strong immunolabeling of free tachyzoites and tachyzoites in the cytoplasm of histiocytes and in cysts. Nested PCR targeting a 155-bp fragment of the B1 gene of T. gondii was positive for the DNA extracted from lung fragments from the two pigs. Genotyping of the samples by means of PCR-RFLP (10 markers) and by means of microsatellites (15 of them) revealed that these animals were infected with T. gondii that was molecularly characterized as the non-archetypal genotype Chinese 1. This presents worldwide circulation, but it had not previously been described in Brazil. The microsatellite analysis showed that the animals were infected with the same T. gondii isolate circulating in the environment.

Diversity of Sarcocystis spp shed by opossums in Brazil inferred with phylogenetic analysis of DNA coding ITS1, cytochrome B, and surface antigens.

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.

The ROP18 and ROP5 gene allele types are highly predictive of virulence in mice across globally distributed strains of Toxoplasma gondii.

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.

Seroprevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in domestic mammals from two distinct regions in the semi-arid region of Northeastern Brazil.

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.

Fatal systemic toxoplasmosis in Valley quail (Callipepla californica).

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.

Occurrence of antibodies against Toxoplasma gondii and its isolation and genotyping in donkeys, mules, and horses in 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.

Seroepidemiology of Toxoplasma gondii infection in bats from São Paulo city, Brazil.

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.

Isolation and biological and molecular characterization of Toxoplasma gondii from canine cutaneous toxoplasmosis in Brazil.

Cutaneous toxoplasmosis is a rare manifestation. This study represents a case report of an immunosuppressed dog that developed nodular dermal lesions caused by Toxoplasma gondii. The isolate (TgDgBr20) was characterized as mouse virulent and was genotyped as type BrI (ToxoDB genotype 6) using PCR-restriction fragment length polymorphism (RFLP) and as Africa 1 through microsatellite analysis.

Geographical patterns of Toxoplasma gondii genetic diversity revealed by multilocus PCR-RFLP genotyping.

In recent years, an extensive collection of Toxoplasma gondii samples have been typed using a set of 10 PCR-RFLP genetic markers. Here we summarize the data reported until the end of 2012. A total of 1457 samples were typed into 189 genotypes. Overall, only a few genotypes dominate in the northern hemisphere, which is in stark contrast to the southern hemisphere where hundreds of genotypes coexist with none being notably dominant. PCR-RFLP genotype #1 (Type II clonal), #2 (Type III), #3 (Type II variant) and #10 (Type I) are identified globally. Genotypes #2 and #3 dominate in Africa, genotypes #9 (Chinese 1) and #10 are prevalent in Asia, genotypes #1, #2 and #3 are prevalent in Europe, genotypes #1, #2, #3, #4 and #5 dominate in North America (#4 and #5 are collectively known as Type 12). In Central and South America, there is no clear dominance of any genotype even though a few have relatively higher frequencies. Statistical analysis indicates significant differences among populations in Africa, Asia, Europe, North America, and Central and South America, with only Europe and North America exhibiting similar diversity. Collectively, the results revealed distinct population structures and geographical patterns of diversity in T. gondii.

Experimentally induced clinical Cystoisospora canis coccidiosis in dogs with prior natural patent Cystoisospora ohioensis-like or C. canis infections.

Diarrhea caused by intestinal coccidia (Cystoisospora species) is a common problem in pet dogs and in dogs in animal shelters. Cystoisospora canis has the largest oocysts of the 4 named species of coccidia infecting dogs. The present study examined an isolate of C. canis obtained from a dog from São Paulo, SP, Brazil. Oocysts sporulated within 2 days at room temperature, and 20 sporulated oocysts were measured at 37.6 by 28.6 µm (range 35-42 by 26-31 µm). Most sporulated oocysts contained 2 sporocysts, each with 4 sporozoites, although a few (<1%) were Caryospora-like and contained 1 sporocyst with 8 sporozoites. Two experiments using a total of 11 female 6-wk-old beagles were conducted to determine the pathogenicity of oral infection with 5 × 10(4) sporulated oocysts of this isolate of C. canis. Five of the 11 dogs had natural infections with Cystoisospora ohioensis-like (n = 4) or C. canis (n = 1) species prior to the predicted patent period of 9-10 days. Ten of the dogs developed diarrhea with occasional blood, and 3 dogs were affected to the extent that clinical treatment for coccidiosis using sulfadimethoxine was recommended. Dog CRU had a natural C. canis infection and did not develop clinical disease after oral infection with C. canis oocysts. This dog had a prepatent period of 9 days and a patent period of 3 days, corresponding to experimental infection with the new isolate of C. canis. It excreted fewer C. canis oocysts than did the other dogs. The 4 dogs with natural C. ohioensis-like infection all developed clinical disease, and 1 required treatment. The prepatent period was 9-10 days, and the patent period was 10-11 days in these dogs. All 6 dogs not naturally infected with Cystoisospora developed clinical disease, and 2 required treatment. The prepatent period was 9-10 days, and the patent period was 8-12 days. The present study confirms that C. canis is a primary pathogen for young dogs. It demonstrates that prior infection with C. canis but not C. ohioensis-like coccidia confers some resistance to clinical disases and a decrease in oocyst production in dogs challenged with C. canis.

Felid herpesvirus 1 as a causative agent of severe nonsuppurative meningoencephalitis in a domestic cat.

Felid herpesvirus 1 is an important respiratory pathogen of domestic cats. This report presents the first case of severe nonsuppurative meningoencephalitis caused by this virus in a cat.

Occurrence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies in dogs with visceral leishmaniasis / Ocorrência de anticorpos anti-Neospora caninum e anti-Toxoplasma gondii em cães com leishmaniose visceral

Uninfected dogs and those naturally infected with Leishmania chagasi exhibiting different clinical forms of disease were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies. Blood samples were collected from 110 mongrel dogs. Sera were tested using the indirect fluorescent antibody test (IFAT), and the animals with visceral leishmaniasis (VL) (n=60) were classified clinically. Out of the 110 sera investigated, 5 (4.5 percent) were positive for N. caninum (IFAT>50) and 36 (32.7 percent) for T. gondii (IFAT>16). Anti-L. chagasi antibody titers in asymptomatic dogs (n=10) were found to be significantly lower (P<0.05) than those in oligosymptomatic ones (n=22), which were in turn significantly lower (P<0.05) than those in symptomatic ones (n=28). No association between Leishmania and N. caninum infections was observed. Among dogs infected with L. chagasi, a tendency (P=0.053) towards an association between the infection with T. gondii and the appearance of VL symptoms was observed, suggesting that the clinical manifestation of VL in dogs may enhance their susceptibility to T. gondii. The possible influence of the immunosuppressive status of canine leishmaniasis in the different clinical forms of the disease is discussed.

A presença de anticorpos anti-Neospora caninum e anti-Toxoplasma gondii foi avaliada em cães não infectados e naturalmente infectados com Leishmania chagasi manifestando diferentes formas clínicas da enfermidade. Amostras de sangue foram coletadas de 110 cães sem raça definida. Os soros foram avaliados por meio da reação de imunofluorescência indireta (RIFI) e os animais com leishmaniose visceral (LV) (n=60) foram classificados clinicamente. Dos 110 soros analisados, 5 (4,5 por cento) foram reativos para N. caninum (RIFI>50) e 36 (32,7 por cento) para T. gondii (RIFI>16). Os títulos de anticorpos anti-L. chagasi em cães assintomáticos (n=10) foram significativamente (P<0,05) mais baixos que aqueles verificados em oligossintomáticos (n=22), que por sua vez foram significativamente menores (P<0,05) que em cães sintomáticos (n=28). Não foi observada associação entre infecções por Leishmania e N. caninum. Entre os cães infectados com L. chagasi, verificou-se uma tendência de associação (P=0.053) entre infecção com T. gondii e aparecimento de sinais clínicos da LV, o que sugere que a manifestação clínica da LV em cães pode aumentar sua susceptibilidade ao T. gondii. A provável influência do quadro de imunossupressão em diferentes formas clínicas da leishmaniose canina é abordada.