Biologic and molecular characteristics of Toxoplasma gondii isolates from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), black-winged lory (Eos cyanogenia), and cats (Felis catus)

Toxoplasma gondii isolates can be grouped into 3 genetic lineages. Type I isolates are considered virulent to outbred mice, whereas Type II and III isolates are not. In the present report, viable T. gondii was isolated for the first time from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), and black-winged lory (Eos cyanogenia). For the isolation of T. gondii, tissues were bioassayed in mice, and genotyping was based on the SAG2 locus. Toxoplasma gondii was isolated from 3 of 6 skunks, 1 of 4 Canada geese, and 2 of 2 feral cats (Felis catus) from Mississippi. All donor animals were asymptomatic. Viable T. gondii was also isolated from 5 of 5 lories that had died of acute toxoplasmosis in an aviary in South Carolina. Genotypes of T. gondii isolates were Type III (all skunks, lories, and the goose) and Type II (both cats). All 5 Type III isolates from birds and 2 of the 3 isolates from skunks were mouse virulent.     

Toxoplasma gondii infections in captive black-footed ferrets (Mustela nigripes), 1992-1998: clinical signs, serology, pathology, and prevention

An epizootic of toxoplasmosis occurred among 22 adult and 30 kit black-footed ferrets (Mustela nigripes) maintained under quarantine conditions at the Louisville Zoological Garden (Louisville, Kentucky, USA) in June, 1992. Black-footed ferrets appear to be highly susceptible to acute and chronic toxoplasmosis. Clinical signs were observed in 19 adults and six kits and included anorexia, lethargy, corneal edema, and ataxia. Two adults and six kits died with acute disease. High antibody titers to Toxoplasma gondii were detected by latex agglutination and modified agglutination assay in 10 black-footed ferrets. One adult and six kits that died with acute clinical signs were necropsied and T. gondii-like organisms were found microscopically in multiple organs. Diagnosis of toxoplasmosis was confirmed by immunohistochemical staining with anti-T. gondii antibodies and by ultrastructural examination. Although the source of T. gondii for black-footed ferrets was not identified, frozen uncooked rabbit was the most likely source. Chronic toxoplasmosis resulted in the death of at additional 13 black-footed ferrets that were adults during the epizootic. Affected animals developed chronic progressive posterior weakness and posterior ataxia 6-69 mo after the epizootic began. Meningoencephalitis or meningoencephalomyelitis associated with chronic toxoplasmosis were identified at necropsy in all 13 ferrets. Precautions to prevent introduction of pathogens into the colony were insufficient to exclude T. gondii. Although toxoplasmosis may cause significant mortality in mustelids, the high mortality of black-footed ferrets in this epizootic was of concern due to their endangered status. This is the first detailed report of toxoplasmosis in black-footed ferrets.     

A new set of primers for the detection of Toxoplasma gondii in amniotic fluid using polymerase chain reaction

Abstract A new PCR system including a pair of primers, a probe and an internal control were designed from the B1 gene of Toxoplasma gondii . The system described allowed the detection of less than 10 tachyzoites of the RH strain of T. gondii . Among 21 amniotic fluid samples, this system diagnosed the cases of congenital toxoplasmosis which were simultaneously diagnosed using mice inoculation, in vitro culture, and serology from both amniotic fluid and fetal blood. These results show that these new primers allow for a highly sensitive detection of T. gondii DNA.     

Acute visceral toxoplasmosis in captive dik-dik (Madoqua guentheri smithi)

Acute toxoplasmosis was diagnosed in 2 captive dik-dik (Madoqua guentheri smithi) in the Houston Zoo. Both animals became ill suddenly and died in spite of supportive therapy. Toxoplasma gondii was identified in tissues of both animals immunohistochemically, and antibodies to T. gondii were found in titers of 1:800 or more in both animals upon examination by the modified agglutination rest. The cause of death was considered to be toxoplasmic pneumonia. This is the first report of toxoplasmosis in M. g. smithi.     

Polymerase chain reaction in diagnosis of toxoplasmosis of the central nervous system: effect of methods for isolating DNA from samples of cerebrospinal fluid on results of the reaction

We examined influence of the method of isolation of DNA from cerebrospinal fluid samples on results of PCR in the diagnosis of toxoplasmosis of the central nervous system. Three different protocols of DNA isolation were used for DNA extraction from 360 samples made of cerebrospinal fluid spiked with tachyzoites of Toxoplasma gondii: thermic, enzymatic and enzymatic-filtering. Purified DNA samples were tested by PCR with primers T15 and T16 designed for the B1 gene of the parasite. Enzymatic method of DNA isolation appeared most effective allowing detection of T. gondii DNA in 50% of samples containing single parasite cell.     

Isolation and genotyping of Toxoplasma gondii from free-ranging chickens from Mexico

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the presence of T. gondii oocysts in the environment because chickens feed from the soil. In the present study, prevalence of T. gondii in 208 free-range chickens (Gallus domesticus) from Mexico was investigated. Blood, heart, and brain from each animal were obtained to test for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (1:10 or higher), were found in 13 (6.2%) chickens. Hearts and brains of 13 seropositive chickens were bioassayed in mice, and T. gondii was isolated from 6 chickens. All 6 isolates were avirulent for mice. Genotyping of chicken isolates of T. gondii using the SAG2 locus indicated that 5 were type III and 1 was type I. This is the first report of isolation of T. gondii from chickens from Mexico.     

Isolation and genotyping of Toxoplasma gondii from free-ranging chickens from Argentina

The prevalence of Toxoplasma gondii in free-ranging chickens can be considered a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 29 free-range chickens (Gallus domesticus) from Argentina was investigated. Blood, heart, and brain from each chicken were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT), were found in 19 of 29 (65.5%) chickens. Hearts and brains of seropositive (MAT > or = 1:5) chickens were bioassayed in mice. Toxoplasma gondii was isolated from 9 of 19 seropositive chickens. Genotyping of chicken isolates of T. gondii using the SAG2 locus indicated that 1 was type I, 1 was type II, and 7 were type III. This is the first report of isolation of T. gondii from chickens from Argentina.     

Seroprevalence and risk factors for Toxoplasma gondii infection on pig farms in central China

Toxoplasma gondii is a protozoan parasite that causes severe diseases in mammals, including humans, around the world. In China, pork is the main meat source; accordingly, T. gondii in pigs is considered an important source for human toxoplasmosis. Understanding the epidemiology of toxoplasmosis in pig farms is thus important for control of the disease in humans. The purpose of the present study was to investigate the epizootiology of T. gondii infections in pig farms in central China by assessing the seroprevalence and risk factors of this disease. In the present study, 3,558 sera samples were collected from pigs in 37 large-scale pig farms in this region and tested by AG-ELISA. The total seroprevalence was 24.5%, with the greatest prevalence in breeding pigs. The risk factors for toxoplasmosis suggest that high frequency of the contact of pigs with cats (P ≤ 0.01; IC 95%), high density of pig breeding (P ≤ 0.01; IC 95%), the presence of mosquitoes and flies (P ≤ 0.01; IC 95%), semi-patency pens (P ≤ 0.05; IC 95%), and low frequency of scavenging (P ≤ 0.01; IC 95%) were all associated with seroprevalence. In addition, the use of sulfonamides (P ≤ 0.01; IC 95%) significantly decreased seroprevalence. This is the first report of anti- T. gondii antibodies in pigs on large-scale pig farms in central China. The findings will provide useful information for designing control strategies of toxoplasmasis in pig farms.     

A novel Toxoplasma gondii calcium-dependent protein kinase

Toxoplasma gondii is an obligate intracellular parasite that infects all types of cells in humans. A family of calcium-dependent protein kinases (CDPKs), previously identified as important in the development of plants and protists, was recently shown to play a role in the infectivity of apicomplexans, and in motility and host cell invasion in particular. We report here the isolation of a new calcium-dependent protein kinase gene from the human toxoplasmosis parasite, Toxoplasma gondii. The gene consists of 12 exons. The encoded protein, TgCDPK4, consists of the four characteristic domains of members of the CDPK family and is most similar to PfCDPK2 from Plasmodium falciparum. We measured TgCDPK4 activity, induced by calcium influx, using a kinase assay. A calcium chelator (EGTA) inhibited this activity. These findings provide evidence of signal transduction involving members of the CDPK family in T. gondii.     

Biological and genetic characterisation of Toxoplasma gondii isolates from chickens (Gallus domesticus) from S?o Paulo, Brazil: unexpected findings.

In spite of a wide host range and a world wide distribution, Toxoplasma gondii has a low genetic diversity. Most isolates of T. gondii can be grouped into two to three lineages. Type I strains are considered highly virulent in outbred laboratory mice, and have been isolated predominantly from clinical cases of human toxoplasmosis whereas types II and III strains are considered avirulent for mice. In the present study, 17 of 25 of the T. gondii isolates obtained from asymptomatic chickens from rural areas surrounding S?o Paulo, Brazil were type I. Antibodies to T. gondii were measured in 82 chicken sera by the modified agglutination test using whole formalin-preserved tachyzoites and mercaptoethanol and titres of 1:10 or more were found in 32 chickens. Twenty-two isolates of T. gondii were obtained by bioassay in mice inoculated with brains and hearts of 29 seropositive (> or =1:40) chickens and three isolates were obtained from the faeces of cats fed tissues from 52 chickens with no or low levels (<1:40) of antibodies. In total, 25 isolates of T. gondii were obtained by bioassay of 82 chicken tissues into mice and cats. All type I isolates killed all infected mice within 4 weeks whereas type III isolates were less virulent to mice. There were no type II strains. Tissue cysts were found in mice infected with all 25 isolates and all nine type I isolates produced oocysts. Infected chickens were from localities that were 18-200 km apart, indicating no common source for T. gondii isolates. This is the first report of isolation of predominantly type I strains of T. gondii from a food animal. Epidemiological implications of these findings are discussed.     

宠物弓形虫的感染及其防控

弓形虫病是一种世界性分布的人兽共患寄生虫病,对人类,尤其是妇女、儿童危害很大,估计全世界有1/3人受到该病的威胁。孕妇感染弓形虫后导致早产、流产、胎儿发育畸形;弓形虫是免疫功能低下患者的主要死亡原因之一。犬、猫是弓形虫的中间宿主和终末宿主,是人类感染弓形虫的主要来源。随着我国经济的迅速发展和人民生活水平的不断提高,城市中饲养犬、猫作为宠物的人越来越多,人、宠物间的亲密接触增加了弓形虫病传播给人的机会。加强对宠物犬、猫弓形虫病的研究及防控势在必行。本文就弓形虫的危害、宠物犬猫弓形虫感染及其防控措施作以综述。     

Toxoplasma gondii infections in cats from Paraná, Brazil: seroprevalence, tissue distribution, and biologic and genetic characterization of isolates

Cats are important in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete environmentally resistant oocysts. The prevalence of T. gondii was determined in 58 domestic cats from 51 homes from Santa Isabel do Ivai, Parana State, Brazil where a water-associated outbreak of acute toxoplasmosis had occurred in humans. Antibodies to T. gondii were found with the modified agglutination test in 49 of 58 (84.4%) cats at a serum dilution of 1:20. Tissues (brain, heart, and skeletal muscle) of 54 of these cats were bioassayed in T. gondii-free, laboratory-reared cats; T. gondii oocysts were excreted by 33 cats that were fed feline tissues. Brains from these 54 cats were bioassayed in mice; T. gondii was isolated from 7. Skeletal muscles and hearts of 15 cats were also bioassayed in mice; T. gondii was isolated from skeletal muscles of 9 and hearts of 13. The results indicate that T. gondii localizes in muscle tissue more than the brains of cats. In total there were 37 T. gondii isolates from 54 cats. Most isolates of T. gondii were virulent for mice. Genotyping of the 37 isolates of T. gondii, using the SAG2 locus, revealed that 15 isolates were type I and 22 were type III. The absence of type II genotype in cats in this study is consistent with the previous studies on T. gondii isolates from Brazil and is noteworthy because most T. gondii isolates from the United States are type II. These findings support the view that Brazilian and North American T. gondii isolates are genetically distinct. This is the first report of genotyping of T. gondii isolates from the domestic cat.     

Electrocardiographic alterations in persons with a positive serology for toxoplasmosis]

To each of 11,161 randomly taken people from urban and peri-urban localities of the first seven regions of Chile (geographic area of distribution of Chagas' disease in the country), with negative serology for Chagas' disease, an indirect hemagglutination test (IHAT) for toxoplasmosis and an EKG were practiced. The IHAT for toxoplasmosis resulted positive in 3,519 individuals (31.5%). The EKG showed alterations in 10.9% of the IHAT positive individuals and in 7.9% of the IHAT negative ones. This difference between the proportion of altered EKG in IHAT positive people and altered EKG in those with negative IHAT is statistically significative with p < 0.001. These results suggest the convenience of considering toxoplasmosis as a cause of chronic myocardiopathy in epidemiological studies on Chagas' disease, since not discard the presence of Toxoplasma gondii infection should overvaluete the magnitude of the impact of Trypanosoma cruzi in the genesis of such a myocardiopathy.     

Seroprevalence of Toxoplasma gondii in swine from slaughterhouses in Lima, Peru, and Georgia, U.S.A

Toxoplasma gondii is an important pathogen transmitted by food, with raw or undercooked meat as the main foodborne source of toxoplasmosis. In Peru, 2-4 million people have antibodies to T. gondii. It is believed that more than 60 million people in the United States are infected with T. gondii. In this study, the prevalence of T. gondii in pigs from Peru and the United States was determined by Western blot. The presence of IgG antibodies to T. gondii from serum samples was determined. Blood samples were collected from 137 pigs at a slaughterhouse in Lima, Peru, and 152 pigs at a slaughterhouse in Georgia. Of the serum samples collected from swine, 27.7% (n = 38) from Peru and 16.4% (n = 25) from the United States were positive for T. gondii. Swine represent a significant source of human infection with T. gondii in Peru and the United States.     

Natural toxoplasma gondii infections in European brown hares and mountain hares in Finland: proportional mortality rate, antibody prevalence, and genetic characterization

In material examined postmortem in Finland from May 2006 to April 2009, acute generalized toxoplasmosis was the immunohistochemically confirmed cause of death in 14 (8.1%) of 173 European brown hares (Lepus europaeus) and four (2.7%) of 148 mountain hares (Lepus timidus). Sera from 116 of the European brown hares and 99 of the mountain hares were screened with a commercial direct agglutination test for Toxoplasma gondii-specific IgG antibodies at a dilution of 1:40. All sera from cases of fatal toxoplasmosis had high titers of antibodies reactive to T. gondii. In contrast, none of 107 European brown hares and four (4%) of 96 mountain hares that died of other causes were antibody-positive. The proportional mortality rates and the T. gondii antibody prevalences among noncases differed significantly between the two host species (P<0.05). Direct genetic characterization of the causative agent was performed on DNA extracted from formalin-fixed, paraffin-embedded tissue of the hares with fatal toxoplasmosis. Based on the results with six microsatellite markers (B18, TUB2, TgM-A, W35, B17, and M33; all six in 15 cases and four in three cases), all the cases were caused by T. gondii genotype II; the size of the PCR product at the seventh marker (M48) varied (213-229 base pairs). The presence of T. gondii genotype II, which is endemic in Europe, is now confirmed in Finnish wildlife: Natural infections with T. gondii parasites belonging to this widespread genotype caused fatal generalized toxoplasmosis in the two species of wild hares.     

Ocular toxoplasmosis: evaluation of lacrimal-specific secretory IgA levels in both patients with active and inactive phases of the disease

Ocular toxoplasmosis can result in recurrent uveitis. Studies have shown that a correlation between active ocular toxoplasmosis and the presence of anti-Toxoplasma gondii secretory IgA (SIgA) in tears. This study compares anti-T. gondii SIgA levels in patients' tears during the acute and inactive phases of toxoplasmic uveitis. Twenty-nine positive tear specific SIgA for T. gondii patients with acute toxoplasmic uveitis were selected and were followed-up for at least two years, when the anti-T. gondii SIgA tears levels were determined. Specific SIgA for T. gondii was negative in 22 patients (75.86%) and positive in seven patients (24.13%) of whom six (85.7%) were followed over three years. Average SIgA levels during the acute phase are 1.54 and decrease significantly to 0.72 (p = 0.0001) during the inactive phase of disease. Because anti-T. gondii SIgA in the tear is negative in 75.86% of patients after the acute phase of infection, T. gondii SIgA levels may be used as a complementary diagnostic marker for active ocular toxoplasmosis.     

Buprenorphine does not affect acute murine toxoplasmosis and is recommended as an analgesic in Toxoplasma gondii studies in mice

Groups of mice were infected with tachyzoites of the RH strain of Toxoplasma gondii, treated with the opioid analgesic buprenorphine, sodium sulfadiazine, a combination of buprenorphine and sodium sulfadiazine, or nothing in the drinking water, on days -1 to 12 postinfection. Mice in the T. gondii-infected buprenorphine-treated group did not live significantly longer (P > 0.05) than mice given T. gondii and not treated with buprenorphine. Clinical observations of mice indicated that buprenorphine treatment reduced distress and pain in mice with acute toxoplasmosis. Mice treated with sodium sulfadiazine alone or sodium sulfadiazine combined with buprenorphine survived the 28-day study. Mice treated with buprenorphine and not infected with T. gondii also survived the 28 days. This study demonstrates that buprenorphine does not adversely interfere with acute T. gondii infection and indicates that buprenorphine can be given to mice to alleviate pain and distress associated with a T. gondii infection, and not adversely influence the results of toxoplasmosis studies. Analgesic (buprenorphine) treatment should now be the standard of care for mice in acute toxoplasmosis studies.     

Infection and immunity with the RH strain of Toxoplasma gondii in rats and mice.

Infection and immunity to toxoplasmosis induced by the RH strain of Toxoplasma gondii was compared in Sprague-Dawley (SD) and Wistar rats and in outbred Swiss Webster mice. All rats injected with up to 1,000,000 RH-strain tachyzoites remained clinically normal, whereas mice injected with only 1 live tachyzoite died of acute toxoplasmosis. Rats could be infected with 1 tachyzoite of the RH strain as shown by antibody development and by bioassay in mice. However, after 8 days, RH-strain organisms were recovered only inconsistently from SD and Wistar rat brains. Contrary to a report of sterile immunity to T. gondii infection in rats after immunization with live RH tachyzoites, we found infection immunity after challenge with the VEG strain. Toxoplasma gondii tissue cysts of the VEG strain could be recovered from most SD and Wistar rats, first injected with live RH-strain tachyzoites and then challenged with oocysts of the VEG strain. Our RH strain, and probably many others, passed for 50+ yr as tachyzoites has lost not only the capacity to form oocysts, but also shows a marked reduction or absence of tissue cyst (bradyzoites) formation.     

Prevalence of Toxoplasma gondii infection in wild birds in Durango, Mexico

There is a lack of information concerning the prevalence of Toxoplasma gondii infection in wild birds in Mexico. In the present study, serum samples and tissues from 653 birds from Durango State, Mexico, were evaluated for T. gondii infection. Antibodies to T. gondii (modified agglutination test, titer 1∶25 or higher) were found in 17 (2.6%) of the 653 birds, including 1 of 2 curve-billed thrashers (Toxostoma curvirostre), 2 (1 Anas platyrhynchos, 1 Anas diazi) of 4 ducks, 1 of 2 eagles (Aquila sp.), 5 (27.8%) of 18 great-tailed grackles (Quiscalus mexicanus), 7 (1.3%) of 521 rock pigeons (Columba livia), and 1 (14.3%) of 7 quail (Coturnix coturnix). The seroprevalence of T. gondii infection in birds captured in a park outside the city zoo (11.6%, 8/69) was significantly higher than that found in birds from other regions (1.5%, 9/584, OR = 8.38; 95% CI: 2.82-24.77; P = 0.0001). Brains and hearts of 23 birds (17 seropositive, 6 seronegative) were bioassayed in mice for the isolation of T. gondii . Viable T. gondii was isolated from 1 of 7 seropositive pigeons. The DNA obtained from the T. gondii isolate from the pigeon was genotyped using the PCR-RFLP typing using 11 markers (B1, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) and revealed an atypical genotype. This is the first report of T. gondii infection in great-tailed grackles, the Mexican duck, and curved-billed thrashers and the first survey of wild birds in Mexico.     

High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli

This report describes a simple, highly efficient and reproducible method for obtaining large quantities of highly pure recombinant Toxoplasma gondii antigens, which can be used for diagnostic application. The obtained T. gondii SAG1, GRA1, and GRA7 antigens (as fusion proteins), expressed in Escherichia coli, contained polyhistidine tags at the N- and C-ends that allowed single-step isolation by metal-affinity chromatography on Ni(2+)-IDA-Sepharose columns. The immunoreactivity of the recombinant antigens was tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of T. gondii infection.