Acute toxoplasmosis in squirrel monkeys (Saimiri sciureus) in Mexico

Toxoplasma gondii causes fatal multisystemic disease in New World primates, with respiratory failure and multifocal necrotic lesions. Although cases and outbreaks of toxoplasmosis have been described, there are few genotyping studies and none has included parasite load quantification. In this article, we describe two cases of lethal acute toxoplasmosis in squirrel monkeys (Saimiri sciureus) of Mexico city. The main pathological findings included pulmonary edema, interstitial pneumonia, hepatitis and necrotizing lymphadenitis, and structures similar to T. gondii tachyzoites observed by histopathology in these organs. Diagnosis was confirmed by immunohistochemistry, transmission electron microscopy and both end point and real time PCR. The load was between <14 and 23 parasites/mg tissue. Digestion of the SAG3 gene amplicon showed similar bands to type I reference strains. These are the first cases of toxoplasmosis in primates studied in Mexico, with clinical features similar to others reported in Israel and French Guiana, although apparently caused by a different T. gondii variant.     

Toxoplasmosis in golden-headed lion tamarins (Leontopithecus chrysomelas) and emperor marmosets (Saguinus imperator) in captivity.

From 1991 to 1995, eight New World nonhuman primates of the family Callitrichidae belonging to the collection of Fundac?o Parque Zoologico de S?o Paulo died of toxoplasmosis. Of the eight affected nonhuman primates, four were Leontopithecus chrysomelas (one male, three females) and four were Saguinus imperator (two males, two females). The most commonly affected organs were the lungs, liver, and lymph nodes, with hemorrhagic and necrotic lesions. Histopathologic examination revealed protozoa that were morphologically consistent with Toxoplasma gondii. Immunohistochemical assays were strongly positive for T. gondii.     

Serodiagnosis of acute toxoplasmosis in macropods

The sera of 34 Australian macropods, the brains of which had been bioassayed for Toxoplasma gondii, were used to establish that a titre greater than 1/32 was significant for a direct agglutination test against toxoplasmosis. In addition, the concentration of 2-mercaptoethanol required to destroy the IgM fraction of macropod serum was confirmed in a modified direct agglutination test. To further validate the tests, the serological responses of three eastern grey kangaroos (Macropus giganteus) dosed orally with T. gondii oocysts and one M. giganteus injected with T. gondii cysts were studied. The tests were then used to investigate a diagnosis of acute toxoplasmosis in four Tasmanian pademelons (Thylogale billardierii) clinically suspected of acquiring toxoplasmosis naturally. One hundred and fifty-one Bennett's wallabies (Macropus rufogriseus rufogriseus) and 85 T. billardierii were also tested to determine the prevalence of acute toxoplasmosis of macropods in the wild. Four percent of M. r. rufogriseus and 1.2% of T. billardierii possessed T. gondii-specific IgM in their sera.     

Use of a multiplex polymerase chain reaction assay in the antemortem diagnosis of toxoplasmosis and neosporosis in the central nervous system of cats and dogs

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the detection of Toxoplasma gondii and Neospora caninum DNA in canine and feline biological samples. SAMPLE POPULATION; Biological samples from 7 cats with systemic (n = 4) or CNS (3) toxoplasmosis, 6 dogs with neospora- or toxoplasma-associated encephalitis, and 11 animals with nonprotozoal disease. PROCEDURE: Primers for T gondii, N caninum, and the canine ferritin gene (dogs) or feline histone 3.3 gene (cats) were combined in a single PCR assay. The DNA was extracted from paraffin-embedded brain tissue, CSF, or skeletal muscle. The PCR products with positive results were cloned, and sequence identity was confirmed. RESULTS: Of 7 cats and 4 dogs with immunohistochemical or serologic evidence of toxoplasmosis, PCR results were positive for all cats and 3 dogs for T gondii, and positive for T gondii and N caninum for 1 dog. Another dog had negative PCR results for both parasites. Of 2 dogs with immunohistochemical or serologic evidence of neosporosis, PCR results were positive for 1 for N caninum and positive for the other for T gondii. All negative-control samples yielded negative results for T gondii and N caninum on the PCR assay. CONCLUSIONS AND CLINICAL RELEVANCE: Standard tests for toxoplasmosis or neosporosis associated with the CNS rely on serologic, histologic, or immunohistochemical analysis and can be difficult to interpret. The multiplex PCR assay with built-in control reactions could be a complementary clinical tool for the antemortem diagnosis of toxoplasmosis or neosporosis associated with the CNS.     

Comparison of Latex Agglutination, Indirect Hemagglutination, and ELISA Techniques for the Detection of Toxoplasma gondii-specific Antibodies in the Serum of Cats

The primary purpose of this study was to determine whether commercially available latex agglutination and indirect hemagglutination kits for the detection of Toxoplasma gondii-specific antibodies were capable of detecting T. gondii-specific immunoglobulin M (IgM) in the serum of cats. Serum samples from 35 cats containing either T. gondii-specific IgM, T. gondii-specific immunoglobulin G (IgG), or both were collected. Each serum sample was assayed using a latex agglutination kit, an indirect hemagglutination kit, an enzyme-linked immunosorbent assay (ELISA) for the detection of T. gondii-specific IgG, and an ELISA for the detection of T. gondii-specific IgM. When serum samples containing only T. gondii-specific IgM as determined by ELISA were assayed, the latex agglutination kit and the indirect hemagglutination kit detected antibodies in 33.3% and 13.3%, respectively. When T. gondii-specific IgG was present in a serum sample, the results from the latex agglutination kit, the indirect hemagglutination kit, and the IgG-ELISA were similar; however, there was a wide variation in titer magnitude results between the three assays. It was concluded that the latex agglutination kit and the indirect hemagglutination kit did not adequately detect T. gondii-specific IgM in feline serum.     

Toxoplasma gondii in an African crested porcupine (Hystrix cristata).

An adult female crested porcupine (Hystrix cristata) was evaluated for acute onset of neurologic signs including head tilt, circling, and ataxia. She was found dead in her holding area 2 days after initially exhibiting clinical signs. Necropsy was unremarkable. Histopathology of brain tissue revealed the presence of protozoal cysts associated with inflammation as the underlying cause of clinical signs and death. Immunohistochemical staining of brain tissue for Toxoplasma gondii was strongly positive. PCR on fresh brain confirmed T. gondii as the causative organism. An adult male in the same enclosure has demonstrated similar neurologic signs over the past 3 years and has failed to respond to various medical treatments. Clinical disease associated with T. gondii has not been previously reported in this porcupine species or any other Old World porcupines, although there are several reports of clinical toxoplasmosis involving New World porcupine species.     

Isolation and identification of mycobacteria in New World primates maintained in captivity

The presence of several Mycobacterium species was determined in 68 New World monkeys kept captive in the Cali Zoo. One hundred and thirty-three gastric lavage and blood samples were evaluated for mycobacterial presence by Ziehl-Neelsen (ZN) staining, culture and PCR amplification of the Mycobacterium tuberculosis Mtp40 species-specific gene. Mycobacteria other than tuberculosis (MOTT) were identified by PCR restriction fragment length polymorphism (RFLP). Different species of mycobacteria were detected in 65% of the primate population studied by Alpha Antigen PCR. Eleven percent were positive for Mtp40 PCR amplification, being diagnosed as having M. tuberculosis, and acid-fast bacilli were observed in 23% by ZN staining. MOTT were isolated from samples taken from 37 primates by culturing; according to the RFLP analysis, three strains were classified as belonging to the MAISS complex (Mycobacterium avium-intracellulare-scrofulaceum-simiae) and eight more, isolated from soil inside the cages, were categorized as environmental contaminants. Mycobacterium spp. were detected in 13 different New World primate species showing that PCR amplification of the Mtp40 gene is a better tool than culture for M. tuberculosis detection in captive animals and that RFLP is a useful technique for MOTT identification.     

Sero-epidemiological survey for toxoplasmosis in wild New World monkeys (Cebus spp.; Alouatta caraya) at the Paraná river basin, Paraná State, Brazil

In this study, we captured 60 wild New World monkeys (Cebus spp.; Alouatta caraya) at the Paraná river basin, Paraná State, Brazil, and modified agglutination test (MAT) was performed to evaluate anti-Toxoplasma gondii antibodies. Prevalence was 30.2% (13/43) in Cebus spp. (capuchin monkeys) and 17.6% (3/17) for A. caraya (black and golden howler monkeys). MAT showed antibody titers of 16 (15/16) and 64 (1/16). Herein, we have observed an odds ratio (OR)=4.67 (1.060.05). The present work is the first report on serum occurrence of anti-T. gondii antibodies in wild capuchin monkeys and in wild black and golden howler monkeys.     

Serodiagnosis of Toxoplasma gondii infection in cats by enzyme-linked immunosorbent assay using recombinant SAG1

The gene encoding surface antigen 1 (SAG1, P30) of Toxoplasma gondii (T. gondii) was cloned into the plasmid pGEX-4T-3 and subsequently expressed in Escherichia coli (E. coli) as a glutathione-S-transferase (GST) fusion protein. The recombinant SAG1 (rSAG1) was refolded using 8M urea solution followed by dialysis and thereafter evaluated in an enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of toxoplasmosis. The test sera were adsorbed with GST to block non-specific reactivity to the GST-SAG1 fusion protein. The ELISA with rSAG1 was able to differentiate very clearly between sera from cats or mice experimentally infected with T. gondii and sera from normal cats or mice. The ELISA detected no cross-reactivity with sera from mice experimentally infected with the closely related parasite Neospora caninum (N. caninum). Some 193 cat sera were tested for antibodies to T. gondii, out of which 40 (20.7%) reacted positively by ELISA with the rSAG1 while another 79.3% cats reacted negative to the assay. Both positive and negative sera were confirmed by Western blot analysis. The results of ELISA were in agreement with those of a commercially available latex agglutination test (LAT) kit, although the former had higher titers than the latter.     

Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis

Loop-mediated isothermal amplification (LAMP) is a simple method that can amplify DNA with high specificity, sensitivity, and rapidity. In this study, we compared the performance of LAMP and real-time PCR assays for diagnosis of toxoplasmosis. We designed a real-time PCR assay targeting a 529 bp element repeated 200-300 times in the Toxoplasma gondii genome. The detection limits of the LAMP and real-time PCR assays were 10 fg/μL and 1 fg/μL of T. gondii DNA, respectively. Conventional PCR, LAMP, and real-time PCR methods were applied to detect T. gondii DNA in blood samples from 284 pigs and 292 sheep. Positive results were obtained with 0.4%, 3.2%, and 4.2% of the pig samples and 3.8%, 17.1%, and 17.8% of the sheep samples with conventional PCR, LAMP, and real-time PCR analyses, respectively. The real-time PCR assay provided the most sensitive diagnosis of toxoplasmosis, but the LAMP assay has potential as an alternative tool for detection of T. gondii in the field.     

Toxoplasmosis in black-faced kangaroos (Macropus fuliginosus melanops)

An epizootic of toxoplasmosis among captive black-faced kangaroos (Macropus fuliginosus melanops) is reported. Eight of 25 adult kangaroos had antibodies to Toxoplasma gondii. Serologic data indicated recent exposure to T. gondii in six kangaroos. Two kangaroos had high T. gondii antibody titers (greater than or equal to 16,384) in the modified agglutination test and their infants died when less than 7 months old. Toxoplasma gondii tachyzoites were found in several organs of one infant kangaroo (joey) that died at about 82 days of age and numerous cysts were seen in skeletal muscles of the other joey that died at about 7 months of age. Adult kangaroos had subclinical infections. The modified agglutination test and the dye test were more sensitive than the indirect hemagglutination and latex agglutination tests for the detection of T. gondii antibodies in kangaroo sera.     

Toxoplasmosis in Tammar wallabies (Macropus eugenii) in the Budapest Zoo and Botanical Garden (2006-2010)

Smaller macropodid species (commonly referred to as wallabies) are extremely susceptible to toxoplasmosis: in most cases, infection with Toxoplasma gondii leads to death within a short time. Between June 2006 and July 2010, T. gondii was detected by immunohistochemical examination in six Tammar wallabies (Macropus eugenii) that died in the Budapest Zoo and Botanical Garden; in another four specimens histopathology revealed T. gondii-like organisms (which could not be differentiated from Neospora caninum solely by morphology), and in another 11 animals toxoplasmosis as the possible cause of death could not be excluded. The current zoo population of 12 Tammar wallabies was tested for T. gondii IgG antibodies by the modified agglutination test (MAT), with negative results. We suppose that most of the deaths were due to acute toxoplasmosis resulting from a recent infection.     

A review of toxoplasmosis in cattle

Worldwide reports of natural and experimentally-induced Toxoplasma gondii infections in cattle are reviewed and tabulated. Serologic tests employed in most studies in the past are of suboptimal sensitivity for the diagnosis of T. gondii infection in cattle; therefore data need to be interpreted with caution. From the evidence available it is concluded that: T. gondii is probably not important in causing abortion or clinical illness in cattle but further experimental studies are desirable; T. gondii is eliminated quickly from bovine tissues; Whether beef plays a part in the epidemiology of toxoplasmosis is unresolved; Milk from T. gondii-infected cows is of negligible importance in the epidemiology of toxoplasmosis.     

Flow cytometric analysis on cross-reactivity of human-specific CD monoclonal antibodies with splenocytes of Aotus nancymaae, a non-human primate model for biomedical research

The reactivity of 204 monoclonal antibodies (mAb) out of 377 commercially available antibodies collected by the animal homologue group of the HLAD8 was analyzed by single colour flow cytometry. Most of these mAb were originally developed against human cell surface molecules. Fifty-eight mAb (28%) showed reactivity with spleen cells of Aotus nancymaae, a non-human primate animal model in biomedical research. Out of these 58 mAb, 22 also showed reactivity with mononuclear cells derived from rhesus macaques and cynomolgus monkeys indicating that the epitopes recognized are evolutionary conserved between human, Old and New World monkeys. This novel panel of A. nancymaae reactive mAb will increase the potential to explore complex host-pathogen interactions in non-human primate animal models, particularly in malaria vaccine research.     

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G1 (infected group, n=10) and G2 (uninfected group, n=8). Infection was performed with 4 x 10(4) VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T. gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T. gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T. gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) x IFA (ah) (r=0.62, P=0.05), MAT(s) x MAT (ah) (r=0.97, P<0.0001); however, there was no significant difference between r-ELISA(s) x r-ELISA (ah) (r= 0.14, P=0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis.     

Serologic diagnosis of toxoplasmosis in experimentally infected pregnant goats and transplacentally infected kids

Eight pregnant goats were inoculated orally with 10 to 1,000 oocysts of Toxoplasma gondii at 83 to 102 days of gestation. Serum samples from the goats and from the kids born to them were analyzed, using the Sabin-Feldman dye test (DT), a commercially available modified agglutination test (MAT), and a latex agglutination test. Six of the does were observed for greater than 1 year; during this time, they delivered twice. All does developed DT and MAT antibody titers of greater than or equal to 1:2,048 within 29 days after inoculation, and the high titers persisted through the 2nd pregnancy; therefore, serologic results alone should not be relied on for the diagnosis of T gondii-induced abortion in does. On the other hand, all transplacentally infected kids had DT or MAT antibody titers of 1:2,048 before ingesting colostrum, indicating the usefulness of serologic evaluation of the fetus or stillborn kid in the diagnosis of abortion. Antibody was not found in the sera of noninfected kids born to Toxoplasma-infected does. The passively acquired colostral antibody declined by 5 months. Therefore, specific antibody found in adult goats is probably actively acquired. The commercially available MAT was simple, sensitive, and reliable for the diagnosis of caprine toxoplasmosis. The latex agglutination test needs further improvement, as titers rarely exceeded 1:256.     

Serological evidence of Toxoplasma gondii infection in captive marine mammals in Mexico

Toxoplasma gondii infection in marine mammals is important because they are considered as a sentinel for contamination of seas with T. gondii oocysts, and toxoplasmosis causes mortality in these animals, particularly sea otters. Serological evidence of T. gondii infection was determined in 75 captive marine mammals from four facilities in southern and central geographical regions in Mexico using the modified agglutination test (MAT). Antibodies (MAT, 1:25 or higher) to T. gondii were found in 55 (87.3%) of 63 Atlantic bottlenose dolphins (Tursiops truncatus truncatus), 3 of 3 Pacific bottlenose dolphins (Tursiops truncatus gillii), 2 of 4 California sea lions (Zalophus californianus), but not in 3 West Indian manatees (Trichechus manatus), and 2 Patagonian sea lions (Otaria flavescens). Seropositive marine mammals were found in all 4 (100%) facilities sampled. All marine mammals were healthy and there has not been any case of clinical toxoplasmosis in the facilities sampled for at least the last 15 years. The seroprevalence of T. gondii infection in marine mammals of the same species did not vary significantly with respect to sex and age. This is the first report on the detection of antibodies to T. gondii in marine mammals in Mexico.     

Antemortem diagnosis and treatment of toxoplasmosis in two cats on cyclosporin therapy

Clinical toxoplasmosis was diagnosed antemortem in two cats being treated with therapeutic doses of cyclosporin. The diagnosis was made by detecting tachyzoites on cytological examination of bronchoalveolar lavage fluid from one case and pleural effusion from the other. Despite early diagnosis and aggressive treatment in both cases, only one cat survived. Reactivation of latent Toxoplasma gondii infection secondary to cyclosporin-induced immunosuppression was considered likely in both cases. The presence of respiratory signs in cats treated with cyclosporin should alert clinicians to the possibility of clinical toxoplasmosis. Consideration should be given to determining the serostatus of cats to T gondii prior to use of drugs which are potent inhibitors of cell mediated immunity, such as cyclosporin. Two cases of feline toxoplasmosis are presented.     

Comparison of various primer sets for detection of Toxoplasma gondii by polymerase chain reaction in fetal tissues from naturally aborted foxes

Tissues from 4 aborted polar foxes (3 samples of brain and 4 samples of liver) were selected for Toxoplasma gondii PCR assay. Positive results of serological tests of mothers and immunofluorescence test (IFT) of fetal organ smears were the criteria of sample selection. Five sets of primers designed from B1 gene and ITS1 sequences of T. gondii were used for detection of the parasite in fetal fox tissues. All used primer sets successfully amplified T. gondii DNA in PCR from organs which were positive by IFT. Single tube nested PCR also showed positive result from a sample negative by IFT, but this product was not confirmed. The studies showed usefullness of PCR for routine diagnosis of toxoplasmosis in carnivores.     

Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections

In this study, the technical performance of culture, two commercially available polymerase chain reaction (PCR) tests, rapid plate agglutination (RPA) test, hemagglutination inhibition (HI) test, and eight commercially available enzyme-linked immunosorbent assays (ELISAs) were compared for the detection of avian mycoplasma infections from 3 days postinfection (d.p.i.) through 35 d.p.i. The tests were carried out on samples from specified pathogen-free layers that were infected at 66 wk of age with recent Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) field strains, MS and MG ATCC strains, and Mycoplasma imitans (MIM), respectively. Results showed a high percentage of positive samples in the homologous infected groups and a high percentage of negative samples (100%) in the uninfected and heterologous infected groups during 35 d.p.i. of both culture and PCR tests. For the group infected with the MG 15302 ATCC strain, serology was more sensitive than bacteriology. All MG and MS tests, with the exception of MG ELISA kit D showed a lower percentage of positive samples during 35 d.p.i. for the detection of the MG and MS ATCC strain infection compared with that of the field strains. Also, the number of cross-reactions (false positives) in the serologic tests was lower after infection with an ATCC strain than after an infection with the MG or MS field strain. Contradictory to other studies, the ELISAs and the RPA test using undiluted serum showed a relatively high number of false-positive results. The MG ELISAs (except ELISA kit D) showed more false-positive results (up to 37%) in the MIM-infected group than in the MS-infected groups. This was not unexpected, as MIM and MG have a close antigenic relationship. The results of the serologic tests in this study showed that a certain level of false-positive results can be expected in about any serologic test. Although the level of false-positive results varied between several serologic tests, this study showed that it is not advisable to rely completely on one test (system) only.