Fatal toxoplasmosis in a captive squirrel monkey (Saimiri boliviensis) in Portugal.

New World monkeys are especially vulnerable to develop severe clinical manifestations and succumb to acute toxoplasmosis. This study aimed to describe the histopathological findings and genotypic characterization of the Toxoplasma gondii strain involved in a lethal case occurring in a zoo-housed black-capped squirrel monkey (Saimiri boliviensis) in Portugal. Cyst-like structures suggestive of Sarcocystidae parasites and acute injuries in liver and brain were observed by light microscopy examination. By immunohistochemistry, calprotectin, T. gondii antigen and Iba1 antigen had a positive signaling in lung, liver and brain tissues. Toxoplasma gondii B1, ITS1 and 529 repetitive element fragments amplifications together with the genotyping of 13 microsatellite markers confirmed a systemic T. gondii infection linked to a non-clonal type II strain. This description is consistent to the majority T. gondii strains circulating in Europe.

Vaccination of squirrel monkeys (Saimiri spp.) with nanoparticle-based Toxoplasma gondii antigens: new hope for captive susceptible species.

Squirrel monkeys (Saimiri spp.), new world primates from South America, are very susceptible to toxoplasmosis. Numerous outbreaks of fatal toxoplasmosis in zoos have been identified around the world, resulting in acute respiratory distress and sudden death. To date, preventive hygiene measures or available treatments are not able to significantly reduce this mortality in zoos. Therefore, vaccination seems to be the best long-term solution to control acute toxoplasmosis. Recently, we developed a nasal vaccine composed of total extract of soluble proteins of Toxoplasma gondii associated with muco-adhesive maltodextrin-nanoparticles. The vaccine, which generated specific cellular immune responses, demonstrated efficacy against toxoplasmosis in murine and ovine experimental models. In collaboration with six French zoos, our vaccine was used as a last resort in 48 squirrel monkeys to prevent toxoplasmosis. The full protocol of vaccination includes two intranasal sprays followed by combined intranasal and s.c. administration. No local or systemic side-effects were observed irrespective of the route of administration. Blood samples were collected to study systemic humoral and cellular immune responses up to 1 year after the last vaccination. Vaccination induced a strong and lasting systemic cellular immune response mediated by specific IFN-γ secretion by peripheral blood mononuclear cells. Since the introduction of vaccination, no deaths of squirrel monkeys due to T. gondii has been observed for more than 4 years suggesting the promising usage of our vaccine. Moreover, to explain the high susceptibility of naive squirrel monkeys to toxoplasmosis, their innate immune sensors were investigated. It was observed that Toll-like and Nod-like receptors appear to be functional following T. gondii recognition suggesting that the extreme susceptibility to toxoplasmosis may not be linked to innate detection of the parasite.

Outbreak of toxoplasmosis in four squirrel monkeys (Saimiri sciureus) in Japan.

Toxoplasma gondii is a protozoan parasite that causes fatal disease in New World monkeys. Several reports have described outbreaks of toxoplasmosis in squirrel monkeys. Here, we report the death of four squirrel monkeys in a captive colony from acute toxoplasmosis, one of which developed toxoplasmosis about 1 year after the initial outbreak. Serum anti-T. gondii antibody was detected by a latex agglutination test in the animals, and one presented seropositive before clinical signs were observed. Macroscopically, the lungs were severely affected and three animals showed pulmonary edema. Microscopically, interstitial pneumonia was observed in all animals. In the liver and heart, multifocal mononuclear cell infiltration with necrosis was detected. Parasite loading tended to be higher in the lungs, liver and heart than in the spleen, kidney and brain. The parasite was isolated from the brain of one animal and this isolate showed type II restriction patterns in the SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2 and PK1 genes of T. gondii and type I restriction patterns in the L358 and Apico genes by PCR-Restriction Fragment Length Polymorphism analysis. The clinical signs were reduced in mice infected with this isolate compared with those infected with reference type II strain PLK in a bioassay. To our knowledge, this is the first report of isolation of the parasite from squirrel monkeys in Japan and offers the opportunity for genomic and pathogenic analyses to aid our understanding of acute toxoplasmosis.

The mitochondrial genome of Dipetalonema gracile from a squirrel monkey in China.

Dipetalonema gracile is a common parasite in squirrel monkeys (Saimiri sciureus), which can cause malnutrition and progressive wasting of the host, and lead to death in the case of massive infection. This study aimed to identify a suspected D. gracile worm from a dead squirrel monkey by means of molecular biology, and to amplify its complete mitochondrial genome by polymerase chain reaction (PCR) and sequence analysis. The results identified the worm as D. gracile, and the full length of its complete mitochondrial genome was 13,584 bp, which contained 22 tRNA genes, 12 protein-coding genes, two rRNA genes, one AT-rich region and one small non-coding region. The nucleotide composition included A (16.89%), G (20.19%), T (56.22%) and C (6.70%), among which A + T = 73.11%. The 12 protein-coding genes used TTG and ATT as start codons, and TAG and TAA as stop codons. Among the 22 tRNA genes, only trnS1AGN and trnS2UCN exhibited the TΨC-loop structure, while the other 20 tRNAs showed the TV-loop structure. The rrnL (986 bp) and rrnS (685 bp) genes were single-stranded and conserved in secondary structure. This study has enriched the mitochondrial gene database of Dipetalonema and laid a scientific basis for further study on classification, and genetic and evolutionary relationships of Dipetalonema nematodes.

An Alternative Treatment Against Acanthocephala ( Prosthenorchis elegans) in Captive Squirrel Monkeys ( Saimiri sciureus) in Mexico.

We provide the first report of Acanthocephala ( Prosthenorchis elegans) in Mexican non-human primates. There has been no known treatment against this parasite except for surgical removal, and this has been relatively ineffective because of the small juveniles. We report the presence of P. elegans in a captive breeding colony of squirrel monkeys ( Saimiri sciureus) in Mexico, and we describe a successful treatment protocol. Treatment involved 2 steps: oral administration of the drugs loperamide chlorhydrate (0.5 mg/0.9 kg/3 days) and niclosamide (0.2 mg/0.9 kg/3 days) followed by surgical removal of adult worms from the intestine. Fecal examination during treatment revealed live adults but no living juveniles and no eggs. Surgery after 1 wk of treatment revealed the presence of adults and an absence of juvenile parasites. All adults were physically extracted during the surgery. All subjects recovered from surgery within 1 wk.

Adaptation of the Plasmodium falciparum FCB strain for in vitro and in vivo analysis in squirrel monkeys (Saimiri sciureus).

The asexual blood stages of the Plasmodium falciparum parasite are responsible for inducing the clinical symptoms and the most severe presentations of malaria infection that causes frequent mortality and morbidity in tropical and subtropical areas of the world, making the blood stages of infection a key target of new malaria treatment and prevention strategies. Progress towards the development of more effective treatment and prevention strategies has been hindered by the limited availability of infection models that permit the sequential analysis of blood stage parasites in vitro followed by in vivo analysis to confirm therapeutic benefits. To advance a model for in vitro and in vivo analysis of blood stage parasites, we examined nine laboratory strains of P. falciparum to determine which strains could adapt to growth in vivo in splenectomized squirrel monkeys (Saimiri sciureus). Only one of the nine laboratory strains tested, the FCB strain, adapted to in vivo growth. Morphological analysis show that the adapted ring-stage parasites have a different morphology from original parasites cultured in vitro, and more often they were found to localize at the edge of the infected red blood cell. No remarkable differences were observed for both trophozoites and schizonts. The adapted strain can be cultured back in vitro similar to the original parasite, indicating that the adapted parasite can develop both in vitro and in vivo. This squirrel monkey-adapted P. falciparum parasite is expected to be suitable and is advantageous for the research and development of vaccines and antimalarial drugs.

Long-term in vitro culture of Plasmodium vivax isolates from Madagascar maintained in Saimiri boliviensis blood.

BACKGROUND: Plasmodium vivax is the most prevalent human malaria parasite and is likely to increase proportionally as malaria control efforts more rapidly impact the prevalence of Plasmodium falciparum. Despite the prominence of P. vivax as a major human pathogen, vivax malaria qualifies as a neglected and under-studied tropical disease. Significant challenges bringing P. vivax into the laboratory, particularly the capacity for long-term propagation of well-characterized strains, have limited the study of this parasite's red blood cell (RBC) invasion mechanism, blood-stage development, gene expression, and genetic manipulation. METHODS AND RESULTS: Patient isolates of P. vivax have been collected and cryopreserved in the rural community of Ampasimpotsy, located in the Tsiroanomandidy Health District of Madagascar. Periodic, monthly overland transport of these cryopreserved isolates to the country's National Malaria Control Programme laboratory in Antananarivo preceded onward sample transfer to laboratories at Case Western Reserve University, USA. There, the P. vivax isolates have been cultured through propagation in the RBCs of Saimiri boliviensis. For the four patient isolates studied to-date, the median time interval between sample collection and in vitro culture has been 454 days (range 166-961 days). The median time in culture, continually documented by light microscopy, has been 159 days; isolate AMP2014.01 was continuously propagated for 233 days. Further studies show that the P. vivax parasites propagated in Saimiri RBCs retain their ability to invade human RBCs, and can be cryopreserved, thawed and successfully returned to productive in vitro culture. CONCLUSIONS/SIGNIFICANCE: Long-term culture of P. vivax is possible in the RBCs of Saimiri boliviensis. These studies provide an alternative to propagation of P. vivax in live animals that are becoming more restricted. In vitro culture of P. vivax in Saimiri RBCs provides an opening to stabilize patient isolates, which would serve as precious resources to apply new strategies for investigating the molecular and cellular biology of this important malaria parasite.

Isolation and molecular characterization of Toxoplasma gondii in a colony of captive black-capped squirrel monkeys (Saimiri boliviensis).

Toxoplasmosis is commonly asymptomatic; however, it can be a fatal multisystemic disease in some animal species, such as New World monkeys. An outbreak of acute fatal toxoplasmosis was reported in a colony of black-capped squirrel monkeys (Saimiri boliviensis) from the zoo of La Plata, Argentina. Post-mortem examination of two monkeys revealed macroscopical and microscopical lesions compatible with acute toxoplasmosis. The presence of Toxoplasma gondii was confirmed by immunohistochemistry on monkey tissues, bioassay in mice and PCR using the specific primers B22-B23. By PCR-RFLP analysis, T. gondii isolated in mice, deriving from both monkeys, showed the same restriction pattern, with most markers showing a type III restriction pattern, except for C22-8 (type II) and C29-2 (type I). To our knowledge this is the first report of fatal toxoplasmosis in S. boliviensis caused by a non-canonical or atypical genotype of T. gondii.

Editing the Plasmodium vivax genome, using zinc-finger nucleases.

Plasmodium vivax is a major cause of malaria morbidity worldwide yet has remained genetically intractable. To stably modify this organism, we used zinc-finger nucleases (ZFNs), which take advantage of homology-directed DNA repair mechanisms at the site of nuclease action. Using ZFNs specific to the gene encoding P. vivax dihydrofolate reductase (pvdhfr), we transfected blood specimens from Saimiri boliviensis monkeys infected with the pyrimethamine (Pyr)-susceptible Chesson strain with a ZFN plasmid carrying a Pyr-resistant mutant pvdhfr sequence. We obtained Pyr-resistant parasites in vivo that carried mutant pvdhfr and additional silent mutations designed to confirm editing. These results herald the era of stable P. vivax genetic modifications.

Transient transfection of Plasmodium vivax blood-stage parasites.

Here, we describe the methodology for transient transfection of Plasmodium vivax. The ability to genetically manipulate P. vivax has rendered this important human malaria parasite more amenable to molecular investigation. However, a systematic analysis of this parasite and its disease-mediating interactions with the human host still awaits further technological breakthroughs, foremost the establishment of a continuous in vitro culture system. Nevertheless, the first steps towards domesticating P. vivax for research purposes have been made. Transfection will eventually help to better understand the unique pathogenic features displayed by P. vivax, such as the host cell specificity for reticulocytes and the occurrence of relapses. Transfection will also be an invaluable tool for studies related to the emerging drug resistance, and it will help identify and validate novel targets for rational intervention.

A role for host-parasite interactions in the horizontal transfer of transposons across phyla.

Horizontal transfer (HT), or the passage of genetic material between non-mating species, is increasingly recognized as an important force in the evolution of eukaryotic genomes. Transposons, with their inherent ability to mobilize and amplify within genomes, may be especially prone to HT. However, the means by which transposons can spread across widely diverged species remain elusive. Here we present evidence that host-parasite interactions have promoted the HT of four transposon families between invertebrates and vertebrates. We found that Rhodnius prolixus, a triatomine bug feeding on the blood of various tetrapods and vector of Chagas' disease in humans, carries in its genome four distinct transposon families that also invaded the genomes of a diverse, but overlapping, set of tetrapods. The bug transposons are approximately 98% identical and cluster phylogenetically with those of the opossum and squirrel monkey, two of its preferred mammalian hosts in South America. We also identified one of these transposon families in the pond snail Lymnaea stagnalis, a cosmopolitan vector of trematodes infecting diverse vertebrates, whose ancestral sequence is nearly identical and clusters with those found in Old World mammals. Together these data provide evidence for a previously hypothesized role of host-parasite interactions in facilitating HT among animals. Furthermore, the large amount of DNA generated by the amplification of the horizontally transferred transposons supports the idea that the exchange of genetic material between hosts and parasites influences their genomic evolution.

Observations on the Uganda I strain of Plasmodium malariae and Plasmodium brasilianum in Aotus and Saimiri monkeys and Anopheles mosquitoes.

Splenectomized Aotus lemurinus griseimembra, A. azarae boliviensis, A. nancymaae, A. vociferans, and Saimiri boliviensis monkeys were infected with the Uganda I/CDC strain of Plasmodium malariae. The maximum parasite counts were lower if the animals had been previously infected with Plasmodium vivax. Mosquito infection was concentrated in the 12 days following the rise in count above 1,000/microl. Mosquito infection and parasite counts were highest with A. l. griseimembra. Anopheles freeborni was more readily infected than An. gambiae, which was more readily infected than An. stephensi. Parasite counts and mosquito infection with P. brasilianum were much higher in S. boliviensis monkeys than with the Uganda I strain of P. malariae in this host, suggesting marked differences between the host-parasite-vector relationships and indicating that P. brasilianum in S. boliviensis monkeys may be a better reflection of the relationship of P. malariae in the human host.

Outbreaks of toxoplasmosis in a captive breeding colony of squirrel monkeys.

Toxoplasma gondii is highly virulent in New World monkeys, but despite numerous outbreaks observed in captive populations there are few reports of molecular characterization of strains. In this article, we describe two outbreaks of toxoplasmosis that occurred in 2001 and 2006 in an outdoor captive breeding colony of squirrel monkeys (Saimiri sciureus) kept by the Institut Pasteur in French Guiana. A microsatellite DNA analysis of the biological samples collected in the 2001 and 2006 outbreaks showed that two different Toxoplasma strains were involved. The 2001 strain exhibited a type II genotype whereas the 2006 strain showed a combination of type I, type III and atypical alleles. Infection could be related to oocysts contaminating water or food, or to ingestion of rats by monkeys. In 2006, a second episode was observed 3 weeks after the first, and was believed to be related to direct contamination by tachyzoites of bronchopulmonary origin from dying monkeys of the first event. During both outbreaks, a total of 50 monkeys died and none recovered spontaneously, confirming the virulence of both type II and non-type II Toxoplasma strains in New World monkeys.

Studies on the Salvador I strain of Plasmodium vivax in non-human primates and anopheline mosquitoes.

A review is presented on studies conducted in New World monkeys and chimpanzees with the Salvador I strain of Plasmodium vivax. This isolate has been adapted to Aotus and Saimiri (squirrel) monkeys and developed as a model for the testing of antimalarial vaccines. After the injection of 10,000 sporozoites, the median prepatent period in S. boliviensis monkeys was 21.5 days. In 103 sporozoite-induced infections in splenectomized monkeys, the median maximum parasite count ranged from 2,139 to 202,368/microL, with a median maximum parasite count of 48,174/microL. Median maximum parasite counts in Aotus lemurinus griseimembra, A. nancymaae, A. azarae boliviensis, and A. vociferans monkeys were 19,902, 18,390, 21,420, and 18,210/microL, respectively and ranged from 124 to 156,000/microL. Mosquito infections were readily obtained in different species of Anopheles mosquitoes. The S. boliviensis monkey and Salvador I strain seems suitable for the testing of sporozoite and liver stage vaccines but not for blood-stage vaccines against P. vivax unless adapted further in spleen-intact Saimiri boliviensis monkeys.

Plasmodium inui shortii: studies in old world and new world monkeys.

Plasmodium inui shortti was studied in monkeys (66 Macaca mulatta, 2 M. fascicularis, 12 Saimiri boliviensis, 4 Aotus lemurinus griseimembra, and 1 A. nancymaae). Prepatent periods for 30 sporozoite transmissions by Anopheles stephensi, An. dirus, and An. maculatus mosquitoes ranged from 10 to 48 days with a median of 15.5 days. In rhesus monkeys, mean maximum parasite counts for intact animals were 181,970/muL; for splenectomized animals, the mean maximum parasite count was 1,167,890/muL.

An outbreak of Toxoplasmosis amongst squirrel monkeys in an Israeli monkey colony.

An outbreak of Toxoplasmosis in a colony of squirrel monkeys (Saimiri sciureus) in Israel is described. Serological, pathological, and molecular findings of monkeys, as well as rodents and pigeons from the vicinity are summarized. Seventy-nine percent (19/24) of monkeys were T. gondii seropositive at titer 1:16 whilst 4% (1/24) were also seropositive at titer 1:64 using the Modified Agglutination Test (MAT). Eighty four percent (21/25) of rats were positive at titer 1:16 and 8% (2/25) of rats were positive at titer 1:32. DNA amplification of a 529bp repeated sequence of T. gondii was detected in the liver and lungs of all monkeys tested, 6/7 in myocardial extractions and 5/6 in brain extractions. Sequence analysis of the SAG2 locus disclosed that T. gondii detected was of Type III genotype. The source of disease was thought to be contamination of feed with infective feline oocysts. As a result of this study, the implementation of a program to capture and remove resident feral cats, to discontinue the feeding of stray cats, and to control rodent populations in the park was introduced.

Prevalence and molecular phylogenetic characterization of Trypanosoma (Megatrypanum) minasense in the peripheral blood of small neotropical primates after a quarantine period.

Neotropical primates of the Cebidae and Callitrichidae, in their natural habitats, are frequently infected with a variety of trypanosomes including Trypanosoma cruzi, which causes a serious zoonosis, Chagas' disease. The state of trypanosome infection after a 30-day quarantine period was assessed in 85 squirrel monkeys (Saimiri sciureus) and 15 red-handed tamarins (Saguinus midas), that were wild-caught and exported to Japan as companion animals or laboratory animals, for biomedical research, respectively. In addition to many microfilariae of Mansonella (Tetrapetalonema) mariae at a prevalence of 25.9%, and Dipetalonema caudispina at a prevalence of 3.5%, a few trypomastigotes of Trypanosoma (Megatrypanum) minasense were detected in Giemsa-stained thin films of blood from 20 squirrel monkeys at a prevalence of 23.5%. Although few T. minasense trypomastigotes were found in Giemsa-stained blood films from tamarins, a buffy-coat examination detected trypanosomes in 12 red-handed tamarins (80.0%), and PCR amplification of a highly variable region of the small subunit ribosomal RNA genes (SSU rDNA) for Trypanosoma spp. detected the infection in 14 of the 15 tamarins (93.3%). Nucleotide sequences of the amplicons were identical for trypanosomes from tamarins and squirrel monkeys, indicating a high prevalence but low parasitemia of T. minasense in imported Neotropical nonhuman primates. Based on the SSU rDNA and 5.8S rDNA, the molecular phylogenetic characterization of T. minasense indicated that T. minasense is closely related to trypanosomes with Trypanosoma theileri-like morphology and is distinct from Trypanosoma (Tejeraia) rangeli, as well as from T. cruzi. Using some blood samples from these monkeys, amplification and subsequent sequencing of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene fragments detected 4 trypanosome genotypes, including 2 types of T. cruzi clade, 1 type of T. rangeli clade, and 1 T. rangeli-related type, but failed to indicate its phylogenetic position based on the gGAPDH gene. Furthermore, species ordinarily classified in the Megatrypanum by morphological criteria do not form a clade in any molecular phylogenetic trees based on rDNA or gGAPDH genes.

Observations on the sporozoite transmission of Plasmodium vivax to monkeys.

Saimiri boliviensis monkeys were infected via sporozoites with the Salvador I strain of Plasmodium vivax that had been stored frozen for periods ranging from 12 to 5,312 days. Prepatent periods ranged from 16 to 53 days.

Comparative phylogeography of Trypanosoma rangeli and Rhodnius (Hemiptera: Reduviidae) supports a long coexistence of parasite lineages and their sympatric vectors.

To make reliable interpretations about evolutionary relationships between Trypanosoma rangeli lineages and their insect vectors (triatomine bugs of the genus Rhodnius) and, thus, about the determinant factors of lineage segregation within T. rangeli, we compared phylogenies of parasite isolates and vector species. Sixty-one T. rangeli isolates from invertebrate and vertebrate hosts were initially evaluated in terms of polymorphism of the spliced-leader gene (SL). Further analysis based on SL and SSUrRNA sequences from 33 selected isolates, representative of the overall phylogenetic diversity and geographical range of T. rangeli, supported four phylogenetic lineages within this species. By comparing the phylogeny of Rhodnius species with that inferred for T. rangeli isolates and through analysis of the geographical range of the isolates, we showed that there is a very significant overlap in the distribution of Rhodnius species and T. rangeli lineages. Congruence between phylogeographical analysis of both T. rangeli lineages and complexes of Rhodnius species are consistent with the hypothesis of a long coexistence of parasites and their vectors, with lineage divergence associated with sympatric species of Rhodnius apparently without association with particular vertebrate hosts. Separation of T. rangeli isolates from vectors of distinct complexes living in sympatry favours the absence of gene flow between the lineages and suggests evolution of T. rangeli lineages in independent transmission cycles, probably associated to specific Rhodnius spp. ecotopes. A polymerase chain reaction assay based on SL intergenic sequences was developed for simultaneous identification and lineage genotyping of T. rangeli in epidemiological surveys.

Studies on sporozoite-induced and chronic infections with Plasmodium fragile in Macaca mulatta and New World monkeys.

Plasmodium fragile continues to be investigated because of its biologic similarities to the human malaria parasite, Plasmodium falciparum. Two strains of P. fragile are available for study; one strain is able to infect mosquitoes, whereas the other strain is transmissible only by blood inoculation. The Sri Lanka strain of P. fragile was transmitted to Macaca mulatta, Macaca fascicularis, Aotus lemurinus griseimembra, Aotus nancymaae, Aotus vociferans, and Saimiri boliviensis monkeys via sporozoites that developed to maturity only in Anopheles dirus mosquitoes. The prepatent periods ranged from 12 to 35 days for macaques and from 15 to 30 days for New World monkeys after intravenous injection of sporozoites. Eight rhesus monkeys were infected with the Nilgiri strain and followed for 482 days. Parasitemia in 6 animals persisted at relatively high density through the period of observation. Erythrocyte, hematocrit, and hemoglobin values reached their lowest levels 3 wk after infection and slowly recovered; however, the values did not approach preinfection levels as long as parasitemia persisted in the monkeys. The mean corpuscular volume and corpuscular hemoglobin concentration reached their peak and lowest values, respectively, at day 38 and then returned to the preinfection level. The mean corpuscular hemoglobin value decreased to its lowest level at day 87 and then returned to preinfection level.