Gondii B1 Gene Research Articles

Reactivated and clinical Toxoplasma gondii infection in young lambs: Clinical, serological and pathological evidences

In this study, fatal toxoplasmosis with severe neurological clinical signs was evaluated in 20 lambs of a sheep flock containing 90 Akkaraman sheep, 60 lambs and 3 rams. The clinical signs in infected lambs (n=20) included incoordination, head shaking, tremors, shaking up, difficulty walking and subsequent death. No incidence of abortion observed in pregnant ewes. Two lambs with severe clinical signs were euthanized and necropsied following clinical and hematological examinations. Blood samples were also collected from the mothers of dead lambs and rams for Sabin-Feldman dye test to detect Toxoplasma gondii-specific antibodies. T. gondii infection was further confirmed by PCR analysis using T. gondii B1 gene specific primers. The histopathological findings included non-purulent myositis with T. gondii-like tissue cysts in the heart and skeletal musculature, severe necrotic vasculitis and multifocal necroses in the brain, liver and lungs. T. gondii immunoreactivity was present in the lungs, liver and spleen as well as tissue cyst-like structures. In differential diagnosis, Neospora caninum infection was excluded by immunohistochemical and PCR analyses. According to current literature, there has been no previous report on clinical toxoplasmosis in newborn lambs or goat kids, and the number of reports on clinical toxoplasmosis in small ruminants is limited to two adult goats with fatality following systemic toxoplasmosis. Our study indicated that toxoplasmosis occurs in lambs with severe neurological signs and subsequent death.

First Report of Genotyping of Toxoplasma gondii Isolates From Wild Birds in China

Toxoplasma gondii is an important cosmopolitan opportunistic protozoan parasite, which threatens the health of human beings and animals. Genetic characterization of isolates from South America has revealed high genetic diversity. In contrast, isolates from North America and Europe were highly clonal, with 3 major lineages known as the Types I, II, and III. However, limited information on T. gondii genotypes has been reported in The People's Republic of China. Here we conducted a survey to determine genetic diversity of this parasite in wild birds of China. In total, tissues from breast muscle of 178 wild birds, including 98 common pheasants ( Phasianus colchicus ), 35 tree sparrows ( Passer montanus ), 22 house sparrows ( Passer domesticus ), 20 saxaul sparrows ( Passer ammodendri ), and 1 cinnamon sparrow ( Passer rutilans ), were tested for T. gondii infection, 4 of which were found to be positive for the T. gondii B1 gene by PCR amplification. These positive DNA samples were typed at 10 genetic markers, including 9 nuclear loci, i.e., SAG1, 5'- and 3'-SAG2, alternative SAG2, SAG3, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico. Of these, 3 isolates were genotyped with complete data for all loci, and 2 genotypes (Type I and Type II variant) were identified. This is the first report of genetic typing of T. gondii isolates from wild birds from different regions in China. The results suggest that the Type I and II variant strains are circulating in wild birds in China, and these birds are potential reservoirs for T. gondii transmission.

Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time

To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1×10(2)-2.1×10(6)copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

Clinical, serological and histopathological signs of toxoplasmosis in broiler chickens (Gallus domesticus) after experimental infection

To identify the features of experimental toxoplasmosis in broiler chickens (Gallus domesticus), a total of 48 birds aged 25 days were randomly assigned to one of four groups of 12 birds each. Tachyzoites of Toxoplasma gondii were injected intraperitoneally at doses of 5 × 105 (group A), 1 × 106 (group B) and 1.5 × 106 (group C), and chickens in group D were treated with an injection of saline only (control). Before and after experimental infection, serum samples from all chickens were tested for antibodies against T. gondii with the Sabin–Feldman reaction. After infection, the clinical signs in all the chickens were recorded daily, and blood smears were prepared to determine parasitemia. Paraffin-embedded tissue sections were used for semi-nested polymerase chain reaction (PCR) to detect the T. gondii B1 gene. Half of the chickens in each group were killed 25 days and half were killed 35 days after infection. All serum samples from chickens in groups A, B and C contained titers of T. gondii antibodies. However, there were no clinical signs suggesting toxoplasmosis. On day 15, the protozoan was observed in blood smears in groups A, B, and C. Analysis by PCR was negative. Microscopic lesions were observed in the brain, heart, liver, pancreas, kidney, spleen, skeletal muscle, proventriculus and lungs, but not in the eyes. Although chickens in group A were exposed to the lowest dose of T. gondii tachyzoites, lesions in this group were relatively more severe than those observed in groups B and C, which were exposed to higher doses of tachyzoites. Group B showed acute signs of toxoplasmosis with few microscopic lesions, whereas group C showed no lesions. Although no stages of the parasite were found in histopathological sections of skeletal muscle, the potential risks of infected chicken meat for public health cannot be disregarded.

Nested PCR-based detection of Toxoplasma gondii in German shepherd dogs and stray cats in South Korea

The prevalence of Toxoplasma gondii was surveyed by using a nested polymerase chain reaction (PCR) that was targeted to T. gondii B1 gene in German shepherd dogs and stray cats. Sixty-four (46.3%) out of 138 German shepherd dogs and 50 (47.2%) out of 106 stray cats were tested positive by the nested PCR assay, respectively. There was no significant difference in gender or age in German shepherd dogs and stray cats. In the five positive dogs and five positive cats, the nucleotide partial sequence of the T. gondii B1 gene was identified by direct sequence analysis. All the sequences were identical to each other and the corresponding sequence, T. gondii B1 gene (Accession No. AF179871). The results suggest that the prevalence of T. gondii is high, and the nested PCR assay is useful for early detection of T. gondii for asymptomatic dogs and cats.

Detection of Toxoplasma gondii by Polymerase Chain Reaction in Cerebrospinal Fluid from Human Immunodeficiency Virus-1-infected Japanese Patients with Focal Neurological Signs

We aimed to determine the effectiveness of using the polymerase chain reaction (PCR) to detect Toxoplasma gondii in cerebrospinal fluid (CSF) specimens from Japanese patients infected with human immunodeficiency virus (HIV)-1. Twenty-six HIV-positive individuals presenting with focal neurological signs and a possible diagnosis of T. gondii encephalitis (TE) were enrolled in the study between April 1997 and March 2003. Eight patients were diagnosed as having TE using the accepted diagnostic criteria; PCR amplified the T. gondii B1 gene in CSF samples from five of these eight patients. CSF samples from the 18 patients without TE were negative for T. gondii DNA. The sensitivity, specificity and positive and negative predictive values for detecting T. gondii in CSF using PCR were 62.5%, 100%, 100% and 85.7%, respectively. These results suggest that PCR might be a clinically useful technique for detecting T. gondii DNA in patients infected with HIV showing focal neurological signs. Improvements in sensitivity are needed, however.

Rapid and sensitive diagnosis of Toxoplasma gondii infections by PCR

DNA was extracted with a modified Qiagen DNA Mini Kit method from 20 clinical samples and was amplified by PCR using specific primers for the T. gondii B1 gene. T. gondii was detected correctly in 18 of the 20 clinical samples in < 5 h, with a detection limit of two parasites/sample. The results were in good agreement with those obtained by a more complicated and time-consuming procedure involving two-step nested PCR and either liquid hybridisation or colorimetric detection using internal probes.

Diagnosis of toxoplasmic retinochoroiditis with atypical clinical features

PURPOSE: To determine the value of aqueous humor analysis for confirming the diagnosis of ocular toxoplasmosis in patients who present with atypical clinical features and to relate the results of local antibody production and polymerase chain reaction (PCR) with the extent of active retinitis and the immune status of the patient. DESIGN: Retrospective case series. METHODS: Sixty-seven consecutive patients with retinitis or retinochoroiditis that was clinically consistent with atypical ocular toxoplasmosis underwent diagnostic anterior chamber paracentesis and serological studies. The aqueous humor was analyzed both by PCR to detect Toxoplasma gondii B1 gene and by the Goldman-Witmer coefficient to determine levels of local anti- T. gondii antibody production. RESULTS: In nine of the 67 cases, PCR was positive for T. gondii; seven of these were negative for local antibody production. All nine patients had illnesses associated with immunosuppression or advanced old age and all had active retinitis with a mean area of 11.5 disk areas (DA). Twenty-five of the remaining 58 cases were positive for local antibody production. These 25 had a mean area of active retinitis measuring 2.6 DA, and 24 of these patients were immunocompetent. All 34 cases with laboratory evidence of ocular toxoplasmosis diagnosed by either method responded to anti- T. gondii agents. The remaining 33 were negative for T. gondii infection by these two methods; some had laboratory evidence of other infections. CONCLUSIONS: Although in the present study, the sensitivity and specificity of the aqueous humor PCR and Goldman-Witmer coefficient could not be ascertained in the laboratory diagnosis of toxoplasmic retinochoroiditis, the PCR method appears to confirm the diagnosis in immunocompromised individuals with large atypical foci of retinitis. Conversely, determination of local antibody production may be appropriate for proper diagnosis in immunocompetent individuals presenting with small foci of retinitis.

Detection of Toxoplasma gondii Parasitemia in Experimentally Inoculated Cats

Toxoplasma gondii B1 gene polymerase chain reaction (PCR) amplification utilizing a flanking and nesting reaction was compared to mouse bioassay on feline whole blood samples collected before and after experimental inoculation with T. gondii. Samples were collected from 5 cats prior to inoculation with T. gondii and on days 3, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 84, 112, 140, 143, 147, 150, 154, 161, 168, 175, and 182 after inoculation. Cats were challenged with T. gondii orally on day 140. Bioassay was found to be less effective for detection of parasitemia than B1 gene PCR. Parasitemia was detected in all 5 cats by PCR multiple times after primary and challenge inoculation. Detection of T. gondii parasitemia by PCR utilizing the flanking reaction described here may be useful in predicting the oocyst shedding period in individual cats. As none of the cats developed signs of systemic illness, yet were chronically parasitemic, T. gondii whole-blood PCR is not helpful as a diagnostic test for clinical feline toxoplasmosis.

Polymerase chain reaction for the detection of Toxoplasma gondii within aqueous humor of experimentally-inoculated cats

The purpose of this study was to determine the temporal appearance of T. gondii in aqueous humor of cats orally inoculated with T. gondii using polymerase chain reaction (PCR) for the detection of the B1 gene. Serum and aqueous humor were collected from five SPF cats prior to oral inoculation with T. gondii and days 7, 14, 21, 28, 42, 84, 140, 147, 154, 161, 168, and 182 after inoculation. Cats were inoculated orally with T. gondii tissue cysts on day 0 and day 140. T. gondii-specific IgM and IgG were measured in serum and aqueous humor from the cats at each sample date. T. gondii B1 gene PCR was performed on all the aqueous humor samples and the amplified DNA was detected by Southern blotting. Chorioretinitis developed in three out of the five cats, but anterior uveitis was not detected. All cats developed T. gondii-specific IgG titers in serum, and had T. gondii-specific IgG C-values >1 in both eyes at varying times during the study. T. gondii was detected by PCR and Southern blotting in aqueous humor in both eyes of all cats at times varying from days 14–84 after primary inoculation and days 14–42 after challenge inoculation.

Vertical transmission of Toxoplasma gondii from chronically infected house (Mus musculus) and field (Apodemus sylvaticus) mice determined by polymerase chain reaction.

Captive-bred Mus musculus (house mice) and Apodemus sylvaticus (field mice) were each infected with 50 oocysts of Toxoplasma gondii M1 strain per os and infection in them and their offspring was assessed by polymerase chain reaction (PCR) amplification of the T. gondii B1 gene in brain tissue and by serology, using the modified agglutination test (MAT). The chronically infected female A. sylvaticus (n = 10) and M. musculus (n = 23) were mated at least 6 weeks after infection (and subsequently to produce up to 6 litters) and their pups examined 3 weeks after weaning at 6 weeks of age. By PCR, in offspring of A. sylvaticus and M. musculus respectively, vertical transmission was demonstrated in 82.7% (n = 83) and 85.0% (n = 207) of all pups (N.S., P > 0.05), 95% (n = 21) and 100% (n = 30) of all litters (N.S., P > 0.05), with a mean (+/- S.E.) proportion of each litter infected of 0.87 (0.06) and 0.86 (0.04) (N.S., P > 0.05). There was no change in any of these variables between first and subsequent litters. By serology, whilst MAT suggested 100% vertical transmission in A. sylvaticus, it under-estimated rates of infection in offspring of M. musculus. A limited series of bioassays from M. musculus tissues confirmed the good correlation of PCR and the poor correlation of MAT with mouse inoculation. These results indicate that vertical transmission in A. sylvaticus and M. musculus is extremely efficient and probably endures for the life of the breeding female. This mechanism favours parasite transmission and dispersion by providing a potential reservoir of infection in hosts predated by the cat.

Polymerase chain reaction for the detection of Toxoplasma gondii in aqueous humor of cats

Abstract Objectives To develop Toxoplasma gondii B1 gene polymerase chain reaction (PCR) for use with aqueous humor of cats, and to report PCR and antibody detection results in naturally exposed cats with and without uveitis. Sample Population Serum and aqueous humor samples from client-owned, healthy cats (n = 23) and client-owned cats with uveitis (n = 43). Procedure T gondii-specific IgM and IgG were measured in serum and aqueous humor from all cats. The Goldman-Witmer coefficient for ocular antibody production was calculated for cats positive for T gondii-specific IgM or IgG in aqueous humor. Aqueous humor from all cats was assessed by the B1 gene PCR. Results T gondii was detected in aqueous humor by PCR from 2 of 23 (8.7%) healthy cats and 8 of 43 (18.6%) cats with uveitis. T gondii-specific IgM in either serum or aqueous humor was detected in 5 of 8 (62.5%) cats with uveitis and T gondii in aqueous humor. All cats with uveitis and T gondii in aqueous humor had anterior segment disease. In 5 of 8 (62.5%) cats with uveitis and T gondii in aqueous humor, ocular production of T gondii antibodies was not detected. T gondii was not detected in aqueous humor from 14 of 17 (82.4%) cats with ocular production of T gondii-specific antibody. Conclusions The presence of T gondii in aqueous humor may correlate to clinical disease in some, but not all, cats. Clinical Relevance T gondii-specific aqueous humor antibody tests and PCR should be used together to aid in the diagnosis of ocular toxoplasmosis in cats. (Am J Vet Res 1996;57:1589–1593)

Detection of Toxoplasma gondii parasitemia by gene amplification, cell culture, and mouse inoculation

Diagnosis of Toxoplasma gondii infection is difficult, especially in immunosuppressed people. The AIDS epidemic has increased the number of people at risk and increased the need for better diagnostic methods. We have compared three methods for detection of T. gondii parasitemia. Rabbits were infected subcutaneously with 10(4) T. gondii tachyzoites. Blood samples were obtained, and buffy coat or leukocyte fractions were prepared. We sought the T. gondii B1 gene by gene amplification by the polymerase chain reaction, and we sought viable T. gondii cells by inoculating fibroblast cell cultures and by mouse inoculation. Thirty-two blood samples were obtained from seven infected rabbits, and 18 were obtained from four control, uninfected rabbits. Parasitemia was detected in 20 of 32 samples (62%) from infected samples by mouse inoculation, 12 of 32 samples (37%) by gene amplification and detection, and 8 of 32 samples (25%) by cell culture. Mouse inoculation requires use of live animals and has a long turnaround time. Currently, cell culture is the least sensitive but most practical and widely available method for the detection of T. gondii parasitemia. Gene amplification and detection was more sensitive than cell culture and may become available in clinical laboratories as techniques are developed further and automated.