Redescription of Hammondia hammondi and its differentiation from Toxoplasma gondii

Hammondia hammondi is a protozoan parasite that, until 1975, was misidentified as Toxoplasma gondii. Recently, the validity of H. hammondi has been questioned. In this article, the authors redescribe the parasite and its life cycle, provide accession numbers to its specimens deposited in a museum, and distinguish it structurally and biologically from T. gondii. Hammondia hammondi was found to be structurally, biologically, and molecularly different from T. gondii.     

Antigenic similarity between the coccidian parasites Toxoplasma gondii and Hammondia hammondi

The antigens that are present in the coccidian parasites Toxoplasma gondii and Hammondia hammondi were demonstrated and defined by using SDS-PAGE and immunoenzymatic techniques with 125I-labeled and unlabeled antigens of T. gondii and sera of mice infected orally or intraperitoneally with H. hammondi . All cell surface antigens of T. gondii that were labeled with 125I were recognized by antibodies in the sera of the mice infected with H. hammondi except the antigen of approximate molecular weight of 21.5 Kd. This suggests that this antigen is specific for T. gondii. Various antigens in the T. gondii-lysed antigen preparations were recognized by antibodies to H. hammondi . The number of recognized antigens increased as the infection of the mice with H. hammondi progressed. Oral infection with H. hammondi appeared to induce the formation of antibodies that recognized more T. gondii antigens than infection by intraperitoneal inoculation.     

Toxoplasma gondii and Hammondia hammondi: DNA comparison using cloned rRNA gene probes

A mung bean nuclease genomic library of purified DNA from tachyzoites of the RH strain of Toxoplasma gondii was prepared in the bacteriophage lambda gtll and recombinants containing rRNA gene fragments were detected by hybridization with radiolabeled total RNA from the closely related coccidian Eimeria acervulina. Ten recombinants were chosen at random, and five of these were investigated further using probes for the genes of the large and small rRNA of Plasmodium berghei. An insert (called TG4) that hybridized only to the 3' end of the large rRNA coding region of P. berghei and an insert (called TG18) that hybridized only to the small rRNA coding region of P. berghei were purified by electrophoresis in low melting point agarose. Radiolabeled E. acervulina total RNA, TG4, and TG18, were then used to compare the sizes of the large and small rRNA gene fragments after DNA extracted from three strains of T. gondii, and the type strain of the closely related coccidian Hammondia hammondi were cut by one of a series of 10 restriction endonucleases. The patterns obtained for the three T. gondii isolates were identical to those obtained for H. hammondi, for each enzyme tested. In addition, the guanine plus cytosine (G + C) content of H. hammondi DNA was found to be almost identical to that obtained previously for T. gondii DNA.     

Hammondia hammondi organelle proteins are recognized by monoclonal antibodies directed against organelles of Toxoplasma gondii.

Hammondia hammondi and Toxoplasma gondii, 2 closely related coccidia of cats, are known to share many antigenic molecules as shown by serologic cross reactivity. Monoclonal antibodies (MAbs) directed against the internal organelles of Toxoplasma gondii were tested by immunofluorescence assay and immunoelectron microscopy on the tachyzoites of H. hammondi. The MAbs anti-apex, anti-dense granules, anti-micronemes, and anti-rhoptries recognized, although weakly, the corresponding antigens on H. hammondi. This finding demonstrates that organelles of the 2 parasites are not only morphologically, but also antigenically, similar.     

Monoclonal antibodies to Hammondia hammondi allowing immunological differentiation from Toxoplasma gondii

Five murine monoclonal antibodies (MAbs) were developed against purified sporozoites of Hammondia hammondi. Despite a large antigenic similarity between the 2 closely related coccidia, H. hammondi and Toxoplasma gondii, these MAbs only reacted with H. hammondi. Three MAbs, ID3, 3F2, and 4C9-7, recognized antigens of 38 kDa localized in rhoptries (1D3), in rhoptries and in oocyst and cyst walls (3F2), and in rhoptries and the apical region (4C9-7). Another MAb, 4C9-10, reacted with a 27-kDa antigen in dense granules of sporozoites and tachyzoites, and MAB 11B3 labeled an antigen of >94 kDa located in the pellicular membrane of the 3 stages of the parasite. These MAbs could be used for a rapid discrimination of the 2 coccidia in epidemiological studies or for diagnostic purposes in tissues.     

Autofluorescence of Toxoplasma gondii and related coccidian oocysts

This is the first report of blue autofluorescence as a useful characteristic in the microscopic detection of Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Neospora caninum, Besnoitia darlingi, and Sarcocystis neurona oocysts or sporocysts. This autofluorescence is of sufficient intensity and duration to allow identification of these oocysts from complex microscopic sample backgrounds. As with the autofluorescence of related coccidia, the oocysts glow pale blue when illuminated with an ultraviolet (UV) light source and viewed with the correct UV excitation and emission filter set.     

Tazonomy of Toxoplasma.

After reviewing reports of the hosts, structure and life cycle of Toxoplasma, the genus is placed in the apicomplexan family Eimeriidae and the folllowing 7 species are recognized: Toxoplasma gondii (Nicolle & Manceaux) (type species) from about 200 species of mammals and birds, with oocysts in felids; Toxoplasma alencari (Da Costa & Pereira) from the frog Leptodactylus ocellatus; Toxoplasma brumpti Coutelen from the iguana Iguana tuberculata; Toxoplasma colubri Tibaldi from the snakes Coluber melanoleucus and Coluber viridiflavus; Toxoplasma hammondi (Frenkel & Dubey) (a new combination for Hammondia hammondi) from the house mouse with oocysts in the domestic cat; Toxoplasma ranae Levine & Nye from the leopard frog Rana Pipiens; and Toxoplasma serpai Scorza, Dagert & Iturriza Arocha from the toad Bufo marinus.     

A structural study of the Neospora caninum oocyst

Oocysts of Neospora caninum were collected from the faeces of a dog fed mouse brains containing tissue cysts of the NC-beef strain of N. caninum. Sporulated oocysts were spherical to subspherical, and were 11.7x11.3 microm. The length/width ratio was 1.04. No micropyle or oocyst residuum was present. Polar granules were not present, although occasionally tiny refractile granules were seen among sporocysts. Sporocysts were ellipsoidal, did not contain a Stieda body, and were 8.4x6.1 microm. The length/width ratio for sporocysts was 1.37. A spherical or subspherical sporocyst residuum was present, and was composed of a cluster of small, compact granules of 4.3x3.9 microm, or was represented by many dispersed granules of similar size. Sporozoites were elongate and 7.0-8.0x2.0-3.0 microm in situ. No refractile bodies were present and the nucleus was centrally or slightly posteriorly located. The features of the oocyst of N. caninum are similar to those of Hammondia heydorni oocysts from dog faeces and Toxoplasma gondii and Hammondia hammondi oocysts from cat faeces.     

Specific detection of Neospora caninum oocysts in fecal samples from experimentally-infected dogs using the polymerase chain reaction

Neospora caninum oocysts, passed in the feces of a definitive host (dog), were isolated, and genomic DNA was extracted. A polymerase cahin reaction (PCR) targeting the N. caninum-specific Nc 5 genomic sequence was performed using the isolated DNA. A synthesized competitor molecule containing part of the Nc 5 sequence was included in the assay as a check against false-negative PCR results and to quantify N. caninum oocyst DNA in fecal samples. A standard curve of the ratio of fluorescence intensity of PCR-amplified competitor to that of oocyst DNA was constructed to compare oocyst equivalents from fecal samples containing unknown numbers of N. caninum oocysts and to assess the sensitivity of the assay. The specificity of the assay was determined using the Nc 5-specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine coccidians Hammondia heydorni, Cryptosporidium parvum, Sarcocystis cruzi, S. tenella, and Isospora ohioensis and the canine helminth parasites Strongyloides stercoralis, Toxocara canis, Dipylidium caninum, and Ancylostoma caninum were not amplified. In addition, genomic DNA sequences from oocysts of coccidian parasites that might contaminate dog feces, such as Hammondia hammondi, Toxoplasma gondii, or Eimeria tenella, were not amplified in the PCR assay. The assay should be useful in epidemiological surveys of both domestic and wild canine hosts and in investigations of oocyst biology in experimental infections.     

Sarcocystis neurona n. sp. (Protozoa: Apicomplexa), the etiologic agent of equine protozoal myeloencephalitis

Sarcocystis neuronan n. sp. is proposed for the apicomplexan taxon associated with myeloencephalitis in horses. Only asexual stages of this parasite presently are known, and they are found within neuronal cells and leukocytes of the brain and spinal cord. The parasite is located in the host cell cytoplasm, does not have a parasitophorous vacuole, and divides by endopolygeny. Schizonts are 5-35 microns x 5-20 microns and contain 4-40 merozoites arranged in a rosette around a prominent residual body. Merozoites are approximately 4 x 1 micron, have a central nucleus, and lack rhoptries. Schizonts and merozoites react with Sarcocystis cruzi antiserum but not with Caryospora bigenetica. Toxoplasma gondii, Hammondia hammondi, or Neospora caninum antisera in an immunohistochemical test.     

Hepatic sarcocystosis in a horse

Hepatic sarcocystosis was diagnosed in a horse in association with refractory bacterial osteomyelitis and plasma cell tumor of the maxilla and hepatic salmonellosis. Gross lesions included pleural, pericardial, and peritoneal effusions, hepatomegaly, gastric ulceration, colonic edema, and proliferative tissues filling 2 maxillary dental alveoli. Histologically, liver was characterized by severe suppurative, necrotizing, periportal hepatitis, and severe periacinar necrosis. Hepatocytes frequently contained protozoal schizonts in various stages of development. In mature schizonts, merozoites were often arranged radially around a central residual body, consistent with asexual division by endopolygeny. Ultrastructural features of merozoites included an apical conoid and polar ring, anterior micronemes, central nuclei, and absence of rhoptries. These protozoa did not react to antisera raised against Neospora caninum, Sarcocystis neurona, Toxoplasma gondii, or Hammondia hammondi. The microscopic and ultrastructural characteristics and immunoreactivity of this organism are consistent with a Sarcocystis sp. other than S. neurona. This is the first report of Sarcocystis-associated hepatitis in a horse. The life cycle of this organism and source of infection are unknown.     

Intergenic spacer (IGS) polymorphism: a new genetic marker for differentiation of Toxoplasma gondii strains and Neospora caninum

The region between the 28S and 18S rRNA genes, including the intergenic spacer (IGS) region and the 5S rRNA gene, from 32 strains of Toxoplasma gondii and the NC1 strain of Neospora caninum was amplified and used for DNA sequencing and/or restriction fragment length polymorphism (RFLP) analysis. The 5S rDNA sequences from 20 strains of T. gondii were identical. The IGS region between the 5S and 18S rRNA genes (nontranscribed spacer 2 or NTS 2) showed 10 nucleotide variations. Six of the 10 variant positions correlated with the murine virulence of the strains. Intraspecific polymorphisms distinguished the virulent strains of zymodemes 5, 6, and 8 from other virulent strains (in zymodeme 1). RFLP methods (IGS-RFLP) were developed and used to characterize the virulent and avirulent patterns among 29 T. gondii strains. Sequence diversity of 19.8% was found between T. gondii and N. caninum when comparing a region of 919 bp at the 3' end of NTS 2. The sequence variation in ribosomal IGS could therefore be a useful marker for Toxoplasma strain identification and for distinguishing N. caninum from T. gondii.     

Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis

The utility of sequence polymorphisms in the dense granule antigen GRA6 gene as typing markers for Toxoplasma gondii was investigated. The coding region of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucleotide polymorphisms at 24 positions out of 690 bp, which correlated with murine-virulence. Types I, II, and III could be distinguished from each other on the basis of three, 10, and six variable positions, respectively. Two deletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substitutions, and the two gaps of 15 bp and 3 bp caused the deletion of six amino acids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolates of this apicomplexan. The large variety of amino acid changes supports the view that the GRA6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, II, III) of T. gondii.     

A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture

Tissue cysts of parasites of the genus Hammondia are rarely described in naturally or experimentally infected intermediate hosts. However, ultrastructural examinations on tissue cyst stages of Hammondia sp. are needed, e.g. to compare these stages with those of Neospora caninum and other related parasites. We describe a cell culture system employed to examine the in vitro development of tissue cysts of a Hammondia sp.-like parasite (isolate FOX 2000/1) which uses the European fox as a definitive host. Cells of a diploid finite cell line from embryonal bovine heart (KH-R; CCLV, RIE 090) were infected by inoculation of sporozoites und cultivated for up to 3 months. Transmission electron microscopic examination of 17 day old cell culture material revealed the presence of cyst walls. Infected cell cultures cultivated for 2 months were used to feed a fox. Six to 13 days post infection the fox shed large numbers (n=1.2 x 10(7)) of Hammondia-sp. like oocysts which could not be distinguished from those used to infect the cell culture as determined by DNA sequencing of the internal transcribed spacer 1 and the D2/D3 domain of the large subunit ribosomal DNA. To find out the proportion of parasitophorous vacuoles that had developed into tissue cysts, the expression of bradyzoite markers was examined by probing infected cell cultures with mouse polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (anti-BAG1) and rat monoclonal antibodies against a cyst wall protein (mAbCC2). Nineteen and 90 days post infection all parasitophorous vacuoles in the cell cultures were positive with anti-BAG1 and mAbCC2. This shows that biologically viable (i.e. infectious) tissue cysts of a fox-derived Hammondia sp. isolate (FOX 2000/1) can be efficiently produced in this cell culture system. Since in vitro cystogenesis of dog-derived Hammondia heydorni has not been observed yet, in vitro cyst formation might be one trait to separate fox-derived Hammondia sp. from H. heydorni on a species level.     

Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites

Sporozoites of avian Eimeria species differed markedly in their ability to invade cells in vitro. Invasion by E. tenella and E. adenoeides was significantly greater in baby hamster kidney (BHK) and chicken cecal cell (CC) cultures than in primary chicken (PCK) or turkey kidney (PTK) cell cultures. Moreover, invasion of BHK cell cultures by E. adenoeides was significantly greater than that of other Eimeria species, and invasion by E. acervulina sporozoites was significantly lower. Monoclonal antibody 1209-C2 (MAb 1209-C2) reacted by immunofluorescent labeling (IFA) with refractile bodies of sporozoites of 5 species of Eimeria and Caryospora bigenetica, but not with sporozoites of Toxoplasma gondii, Hammondia hammondi, or Cryptosporidium parvum, which have no refractile bodies. The MAb also cross-reacted with formalin-fixed BHK, CC, turkey cecal (TC) cells, and PTK. Pretreatment of BHK cells with MAb 1209-C2 significantly reduced invasion of the cells by sporozoites of E. tenella, E. acervulina, E. meleagrimitis, and C. bigenetica, but did not alter invasion by T. gondii, C. parvum, or H. hammondia. Apparently, reactivity of MAB 1209-C2 with the sporozoites was required for inhibition of invasion despite the fact that the inhibition resulted from pre-treatment of the host cell. Conversely, although MAb 1209-C2 also reacted moderately with PTK and TC cells, pre-treatment of these cell cultures with the MAb did not inhibit invasion by either MAB 1209-C2-reactive or -nonreactive parasites. Collectively, the data indicated that refractile body antigens of sporozoites of Eimeria and Caryospora, which are recognized by MAb 1209-C2, may function in cellular invasion, but also suggest that cellular invasion is probably not mediated by interactions between the conserved epitopes in sporozoites and cultured host cells that are recognized by the MAb.     

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.     

Inhibition of Toxoplasma gondii and Plasmodium falciparum infections in vitro by NSC3852, a redox active antiproliferative and tumor cell differentiation agent

We searched the National Cancer Institute (NCI) compound library for structures related to the antitumor quinoline NSC3852 (5-nitroso-8-quinolinol) and used a computer algorithm to predict the antiprotozoan activity for each of 13 structures. Half of these compounds inhibited Toxoplasma gondii tachyzoite propagation in human fibroblasts at 相似文献   

A comparison of isozymes of five axenic Giardia isolates

The relative mobilities of six enzymes from the trophozoites of five axenically-cultured isolates of Giardia from human, cat, and guinea pig hosts were compared by starch and polyacrylamide gel electrophoresis. The six enzymes compared were malate dehydrogenase (NAD+) (MDH) (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (ME) (EC 1.1.1.40), hexokinase (EC 2.7.1.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6P) (EC 1.1.1.49), and alpha-glycerophosphate dehydrogenase (EC 1.1.1.8). The latter three enzymes have not been previously reported in Giardia. On the basis of zymogram patterns, the five Giardia isolates were divided into three zymodemes. Zymodeme I comprised human-1/England, human-1/Bethesda, and cat-1/Portland, Zymodeme II the guinea pig-1/Portland isolate, and Zymodeme III the human-1/Portland isolate. These zymodemes were further substantiated when several physical and kinetic properties of three of the enzymes, MDH, ME, and G6P, were examined. Our results, in which Giardia isolated from different mammalian hosts share multiple isoenzymes, question the validity of the practice of assigning Giardia species names on the basis of the animal host from which the protozoan was obtained.