Feline serum antibody responses to Toxoplasma gondii and characterization of target antigens.

The Toxoplasma gondii-specific target antigens for feline immunoglobulin M (IgM) and immunoglobulin G (IgG) immune responses were studied longitudinally using western blot immunoassay in 8 cats experimentally inoculated with T. gondii strain ME49. Multiple antigens were recognized by IgM and IgG during the course of infection. Dense bands were associated with 12 antigens in the IgM western blot immunoassay and 30 antigens in the IgG western blot immunoassay. Immunoglobulin M responses were maximal on week 4 postinoculation (PI) and were greatly diminished by week 20 PI. Immunoglobulin G responses were maximal on week 12 PI. On week 20 PI, the 19-kDa (6/8 samples), 26-kDa (8/8 samples), 28-kDa (8/8 samples), 31-kDa (7/8 samples), 35-kDa (6/8 samples), 51-kDa (6/8 samples), 55-kDa (7/8 samples), and 65-kDa (7/8 samples) antigens were recognized most commonly in the IgG western blot immunoassay. When the western blot immunoassay results were compared to enzyme-linked immunosorbent assay (ELISA) results, there was no clear advantage to the development of IgM-ELISA, IgG-ELISA, IgM western blot immunoassay, or IgG western blot immunoassay using a single antigen instead of multiple antigens as the detection system for the diagnosis of recent infection.

Surface epitope localization and gene structure of a Babesia bovis 44-kilodalton variable merozoite surface antigen.

Variation of Babesia bovis merozoite surface antigens occurs among geographic strains of the parasite. In this and a concurrent report, we investigate this variation at the gene and protein level. Using a monoclonal antibody (mAb 23/70.174), B. bovis gene sequences were identified that encoded a surface epitope of a 44-kDa merozoite surface antigen (MSA-2). This epitope is variably expressed among geographic isolates of B. bovis. Here, we describe the MSA-2 protein gene sequence, localize this surface epitope to a repeated amino acid sequence, and investigate the genomic organization of the gene in B. bovis strains from Mexico and Australia. The predicted protein sequence had hydrophobic regions at its amino and carboxy termini consistent with a signal peptide and a membrane anchor via glycosylphosphatidyl inositol, respectively. The surface epitope recognized by mAb 23/70.174 was localized within a 24-amino acid sequence which is repeated twice in tandem. Six different EcoRI bands hybridized to the MSA-2 gene sequence with varying intensities in genomic Southern blots of the homologous strain. Two of these appear to be alleles of the MSA-2 gene. Whereas 5' and 3' sequences of the MSA-2 gene sequence were detected in an Australia strain of B. bovis, internal gene sequences encoding the surface epitope were not. The 3' sequences of the MSA-2 gene also had significant sequence similarity with the MSA-1 gene of the Mexico strain B. bovis and a gene from the previously described BabR locus. These data indicate that the MSA-2 protein gene belongs to the BabR locus which encodes variable merozoite surface antigens.

A Babesia bovis 225-kilodalton protein located on the cytoplasmic side of the erythrocyte membrane has sequence similarity with a region of glycogen phosphorylase.

A Babesia bovis gene sequence is described which encodes a geographically conserved epitope (recognized by monoclonal antibody (mAb) 23.8.34) of a 225-kDa protein located on the surface of merozoites and associated with the infected erythrocyte membrane. The gene sequence, derived from both genomic and cDNA copies, is 2044 bp long and has one long open reading frame encoding about one third of the 225-kDa protein. The open reading frame is expressed in an approximately 6,400 nucleotide RNA transcript. A 73-amino acid sequence occurs as 4 complete and 1 partial tandem repeats at the carboxy terminus of the partial protein sequence. The epitope recognized by mAb 23.8.34 was localized to the repeat region. Based on epitope localization with mAb 23.8.34, the repeat was exposed on the surface of merozoites and located near the cytoplasmic face of the erythrocyte membrane. The amino terminus of the protein was non-repetitive and had 21% identity (60% similarity) to glycogen phosphorylase over a region of 151 amino acids. In addition, the corresponding 5' DNA sequence hybridized to as many as 8 restriction fragments on Southern blots of genomic DNA. In contrast, the DNA sequence of the repeat hybridized to a single fragment. Both the repeat and multiple non-repeat DNA sequences were detected in a different geographic strain of B. bovis. These results indicate that the 5' end of the 225-kDa protein gene is related to a larger gene family, independent of the 3' end of the gene encoding the repeat.

DNA probes distinguish geographical isolates and identify a novel DNA molecule of Babesia bovis.

A genomic DNA library of Babesia bovis was screened to identify DNA probe candidates for direct detection of the parasite. Two sequences, Bo6 and Bo25, had the highest sensitivity and further analysis revealed unique characteristics of each of these. Neither sequence hybridized detectably to bovine DNA. Bo6 detected 100 pg of both a Mexican and an Australian isolate of B. bovis, but Bo6 also detected 1.0 ng of Babesia bigemina DNA under identical conditions. A unique characteristic of Bo6 is that it hybridizes to an apparent 7.4-kilobase DNA in undigested genomic DNA of both B. bovis and B. bigemina. The sequence is well conserved between the 2 geographic isolates of B. bovis, but it is apparently divergent in B. bigemina. Bo25 did not hybridize detectably to bovine or B. bigemina DNA. This sequence detected 100 pg of homologous B. bovis Mexican isolate DNA, but the sensitivity was reduced to 1 ng for the Australian isolate DNA. The restriction enzyme profile of the Bo25 sequence in genomic DNA differed markedly in the number, size, and intensity of bands between the 2 B. bovis geographic isolates tested. Thus, the Bo25 sequence can distinguish geographic isolates of B. bovis.

A recombinant surface protein of Babesia bovis elicits bovine antibodies that react with live merozoites.

Ten monoclonal antibodies (MoAbs) were generated against five surface-exposed proteins (16 kDa, 42 kDa, 44 kDa, 60 kDa, 225 kDa) on merozoites of Babesia bovis. A genomic library constructed in the lambda gt11 expression vector was screened with MoAbs in a plaque immunoassay for identification of clones expressing recombinant surface proteins. Two recombinant clones were identified (lambda Bo44-15 and lambda Bo44-16) that encoded a protein recognized by a MoAb specific for an epitope on the native 44-kDa surface protein. Southern blot analysis using radiolabeled Bo44-15 DNA (1.25 kb) against merozoite DNA and bovine leukocyte DNA confirmed the parasite-specificity of the cloned insert and revealed multiple bands of hybridization with merozoite DNA. Western blot analyses of lambda Bo44-15 lysogen preparations demonstrated that recombinant protein production in this clone was IPTG-induced and that the recombinant molecule was a beta-galactosidase fusion protein. Additionally, recombinant 44-kDa protein, purified by immunoaffinity chromatography, reacted with specific MoAb in Western blot assay indicating that the integrity of the epitope was retained during purification. Immune sera from calves immunized with purified recombinant Bo44-15 protein immunoprecipitated metabolically radiolabeled merozoite protein of 44 kDa indicating that antibody induced by recombinant Bo44-15 protein recognized native 44-kDa protein. Also, these sera reacted with the surface of live merozoites as evidenced by indirect immunofluorescence assay. Serum antibody titers determined by this assay had a wide range.

Identification of antigens of Sarcocystis cruzi sporozoites, merozoites, and bradyzoites with monoclonal antibodies.

Sporozoites and culture-derived merozoites of Sarcocystis cruzi were used to elicit monoclonal antibodies (MAb's) in mice. Some of these antibodies reacted with the surface of live sporozoites and merozoites as determined by immunofluorescence. An array of similar antigens was identified in Western blots of sporozoites by both anti-merozoite MAb's and an anti-sporozoite MAb. At least 1 antigen in blots of bradyzoites was identified by anti-merozoite MAb's and a cluster of antigens was identified by an anti-sporozoite antibody. These results indicate that several surface epitopes of sporozoites and merozoites are shared with molecules of bradyzoites and that antigen patterns of molecules bearing these epitopes in 3 stages of Sarcocystis may be either distinct or similar.

Eimeria from bats of the world. II. A new species in Tadarida femorosacca from Sonora, Mexico.

Between 1979 and 1980, 104 bats representing 13 species in 4 families were collected in California and New Mexico, U.S.A., and Baja California and Sonora, Mexico, and were examined for coccidia; only 3 (3%) had oocysts in their feces. Bats examined and their infection rates were: Molossidae: 0 of 12 Tadarida brasiliensis, 1 of 18 (6%) T. femorosacca; Natalidae: 0 of 1 Natalus stramineus; Phyllostomatidae: 0 of 1 Choeronycteris mexicana, 0 of 2 Leptonycteris sanborni, 0 of 1 Macrotus californicus; Vespertilionidae: 0 of 9 Antrozous pallidus, 0 of 28 Eptesicus fuscus, 0 of 1 Lasionycteris noctivagans, 0 of 3 Lasiurus borealis, 2 of 22 (9%) L. cinereus, 0 of 1 L. ega, 0 of 5 Pipistrellus hesperus. Sporulated oocysts were only found in T. femorosacca and these represent a new species, Eimeria tadarida n. sp. They are subspheroidal to ellipsoidal, 19 x 25 (16-23 x 20-30) microns; a micropyle is absent, and fragments within the oocyst may be oocyst residuum or multiple polar bodies. The oocyst wall, approximately 1.5 microns, is composed of a mammillated outer layer and smooth inner layer. Sporocysts are ovoidal, 8 x 12 (6-9 x 10-14) microns, and have a small Stieda body and a wide substieda body. This is only the 14th eimerian to be described from bats worldwide. Only unsporulated or partially sporulated oocysts of an eimerian were seen in 2 L. cinereus. These measured 28 x 25 (27-29 x 24-26) microns and had a mammillated outer oocyst wall.

Proteins and antigens of merozoites and sporozoites of Eimeria bovis (Apicomplexa).

Proteins and antigens of first-generation merozoites and sporozoites of Eimeria bovis were examined using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and lactoperoxidase iodination procedures. SDS-PAGE gels revealed both common and unique protein bands in merozoite and sporozoite extracts, ranging in molecular weight (Mr) from 15,000 to 215,000. Nitrocellulose immunoblots of separated proteins, when probed with sera obtained from immunized calves, revealed numerous IgG-binding antigens of Mr 18,000 to 180,000 in merozoites and Mr 28,000 to approximately 118,000 in sporozoites. Although merozoite and sporozoite preparations each contained antigens of different molecular weights, 4 antigens had the same migratory distance in both preparations (Mr 58,000, 70,000, 83,000, 98,000). Of 3 types of immune sera used to probe immunoblots, serum taken from a calf that had been inoculated with oocysts of E. bovis and boosted 10 wk later by subcutaneous injection with 2 X 10(7) live merozoites emulsified in Freund's complete adjuvant consistently identified and reacted more intensely with more antigens of merozoites and sporozoites than the other immune sera tested. Autoradiographic analysis of radioiodinated parasites revealed major surface proteins on merozoites of between 15,000 and 18,000 Mr and 3 surface proteins on sporozoites of Mr 28,000, 77,000, and 183,000. All but the 183,000 protein elicited an IgG antibody response in the host.

In vitro excystation of Sarcocystis capracanis, Sarcocystis cruzi and Sarcocystis tenella (Apicomplexa).

Improved rates of in vitro excystation of sporozoites from sporocysts of Sarcocystis capracanis, Sarcocystis cruzi, and Sarcocystis tenella were obtained by pretreating sporocysts with an aqueous sodium hypochlorite (NaOCl) solution followed by incubation in excysting fluid (EF). After pretreatment with NaOCl, sporocysts were washed 4 times in Hanks' balanced salt solution and then incubated in various EF (pH 7.4) at 38.5 C in 5% CO2-95% air. Maximum rates of excystation (free sporozoites/(sporozoites in sporocysts + free sporozoites) X 100) for all 3 species of Sarcocystis occurred at 4 hr after incubation in EF. These rates were 17% for S. capracanis after incubation in EF containing 2% trypsin + 10% caprine bile; 90% for S. cruzi in 2% trypsin + 10% bovine bile; and 20% for S. tenella in 2% trypsin + 10% caprine bile. Only a 40% excystation rate occurred in sporocysts of S. cruzi that had been stored previously for 14 days in aqueous potassium dichromate. Excysted sporozoites of S. capracanis, S. cruzi, and S. tenella penetrated and developed to mature meronts in bovine pulmonary artery endothelial cells or bovine monocytes.

In vitro cultivation of meronts of Sarcocystis cruzi.

Several established cell lines were tested for their ability to support in vitro development of meronts of Sarcocystis cruzi. Sporozoites penetrated bovine monocytes (BM), bovine pulmonary artery endothelial cells (CPA), Madin-Darby bovine kidney cells and mouse macrophages, but developed to meronts in BM and CPA only. Sporozoites developed to large meronts that contained approximately 180-350 merozoites, whereas merozoites formed small meronts with 50-100 merozoites. Mature large meronts were present at 18-86 days after inoculation (DAI) in BM and at 16-72 DAI in CPA. Small meronts were present at 23-115 and 23-91 DAI in BM and CPA. Considerably more merozoites developed in CPA than in BM. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that merozoites harvested at 36 and 48 DAI each had 1 unique protein as well as numerous common proteins.

Lymphokine-induced inhibition of growth of Eimeria bovis and Eimeria papillata (Apicomplexa) in cultured bovine monocytes.

Sporozoites of Eimeria bovis penetrated and developed normally to first-generation meronts in bovine monocytes (BM) and Madin-Darby bovine kidney (MDBK) cells that had been pretreated with culture medium (CM) or supernatant (NS) from nonstimulated bovine T cells. At 240 h after sporozoite inoculation (ASI), the mean percent development (meronts/[sporozoites + meronts]) in CM- and NS-pretreated BM was 52 and 28%, respectively; values for MDBK cells were 36 and 35%, respectively. Pretreatment of BM and MDBK cells with supernatant (ConAS) from concanavalin A-stimulated bovine T cells had no effect on the ability of sporozoites to penetrate cells; however, at 240 h ASI, only 1% of the sporozoites in ConAS-pretreated BM cultures had developed to meronts. In contrast, ConAS had no adverse effect on the ability of E. bovis sporozoites to develop to first-generation meronts in MDBK cells. At 240 h ASI, E. bovis meronts in ConAS-pretreated BM were abnormal in appearance and retarded in development, whereas sporozoites appeared structurally normal by light microscopy. Pretreatment of BM with ConAS had no effect on the ability of sporozoites of Eimeria papillata (Apicomplexa) to penetrate cells. Sporozoites of E. papillata did not develop to meronts in ConAS-pretreated BM and, in contrast to E. bovis, most sporozoites were destroyed intracellularly.

Ultrastructure of Cryptosporidium parvum oocysts and excysting sporozoites as revealed by high resolution scanning electron microscopy.

Cryptosporidium parvum oocysts isolated from calf feces were examined by scanning electron microscopy during excystation. Intact C. parvum oocysts were spheroid to ellipsoid, approximately equal to 3.5 X 4.0 micron, with length : width ratio = 1.17. The oocyst wall had a single suture at one pole, which spanned 1/3 to 1/2 the circumference of the oocyst. During excystation the suture dissolved, resulting in a slit-like opening, which the sporozoites used to exit the oocyst. Sporozoites were 3.8 X 0.6 micron and had a rough outer surface.

Eimeria species (Apicomplexa: Eimeriidae) infecting Peromyscus rodents in the southwestern United States and northern Mexico with description of a new species.

Of 198 deermice (Peromyscus spp) collected from various localities in the southwestern United States and northern Mexico, 106 (54%) had eimerian oocysts in their feces when examined. These included 50 of 106 (47%) Peromyscus truei, 34 of 54 (63%) Peromyscus maniculatus, 4 of 17 (24%) Peromyscus leucopus, and 18 of 21 (86%) Peromyscus eremicus. The following Eimeria were identified from infected mice: Eimeria arizonensis and Eimeria langebarteli from P. truei; E. arizonensis, Eimeria peromysci, and Eimeria delicata from P. maniculatus; E. arizonensis and Eimeria lachrymalis n. sp. from P. eremicus; and E. langebarteli from P. leucopus. Of the 106 Peromyscus found positive for Eimeria, 97 (91.5%) harbored only a single eimerian species at the time of examination. Sporulated oocysts of E. lachrymalis n. sp. were ellipsoid, 27-35 X 17-21 (30.8 +/- 1.7 X 19.1-0.9) micron, possessed a smooth wall and one polar granule, but lacked a micropyle and an oocyst residuum. Sporocysts were teardrop-shaped, 9-13 X 6-10 (10.9 +/- 0.9 X 7.9 +/- 0.5) micron, and had a Stieda body and sporocyst residuum, but no substieda body. Prepatent periods in experimental infections were 3-6 days after inoculation (DAI) for E. arizonensis (hosts: P. eremicus, P. maniculatus, P. truei); 4-5 DAI for E. peromysci (host: P. maniculatus); 6-9 DAI for E. langebarteli (hosts: P. truei, P. leucopus); and 8-10 DAI for E. lachrymalis (host: P. eremicus). Patency in these infections lasted 6-11 days for E. arizonensis, 5-10 days for E. peromysci, 14-40+ days for E. langebarteli, and 19-50+ days for E. lachrymalis. Eimeria lachrymalis appears to produce occult infections in P. eremicus that can be reactivated upon inoculation of the host with E. arizonensis.

Eimeria ladronensis n. sp. and E. albigulae (Apicomplexa: Eimeriidae) from the woodrat, Neotoma albigula (Rodentia: Cricetidae).

Of 50 white-throated woodrats (Neotoma albigula) collected from Socorro Co., New Mexico, 21 (42%) had eimerian oocysts in their feces when examined. Of the 21 Neotoma found positive for Eimeria, 19 (90%) harbored a single eimerian species at time of examination. Eimeria albigulae Levine, Ivens & Kruidenier, 1957, was found in 18 (86%), and E. ladronensis n. sp. was found in five (24%) infected woodrats. Sporulated oocysts of E. ladronensis are ellipsoidal, 19-25 X 13-15 (21.4 +/- 1.3 X 14.1 +/- 1.1) micron, have a smooth wall and one or two polar granules, but lack a micropyle and an oocyst residuum. Sporocysts are tapered at one end, 7-10 X 6-7 (8.5 +/- 0.7 X 6.5 +/- 0.3) micron, and have a Stieda body and sporocyst residuum, but no substieda body. Prepatent periods for E. albigulae and E. ladronensis n. sp. are 5-6 and 8-9 days, respectively; patent periods are 7-18 and approximately 11 days, respectively.