Monoclonal antibodies against diagnostic Anisakis simplex antigens

Five monoclonal antibodies (UA2, UA3, UA5, UA6, and UA8) specific for Anisakis simplex are described. All are IgG1/κ monoclonal antibodies, except for UA2, which is an antibody IgM/κ. The molecular weights of the major components recognized in immunoblotting are 48 and 67 kDa (UA2); 139 kDa (UA3 and UA5; same epitope); 35, 38, and 139 kDa (UA6); and 205 kDa (UA8). UA2 was the only monoclonal antibody to recognize both components of an excretion-secretion antigen preparation and antigens in the excretory cell and esophageal glands of third-stage A.␣simplex larvae; antigens in the excretory cell were also recognized by UA3 and UA6. Cross-reactivity studies using a hyperimmune polyclonal rabbit serum reacting with various ascaridoid nematodes indicated that the antigens captured by our monoclonal antibodies were specific for A. simplex. Finally, comparative studies of our monoclonal antibodies and An2 (the only monoclonal antibody currently available for serodiagnosis of human anisakiasis), based on the calculation of multiples of normal activity for human anisakiasis sera, indicated that our monoclonal antibodies (and particularly UA3) recognized antigens that are good candidates for serodiagnostic purposes. Received: 13 February 1997 / Accepted: 16 March 1997     

A cDNA encoding the highly immunodominant antigen of Strongyloides stercoralis: γ-subunit of isocitrate dehydrogenase (NAD+)

A full length cDNA encoding the highly immunodominant 41 kDa antigen of Strongyloides stercoralis (P5), recognized by 83% of human patients [Siddiqui et al. (1997) Parasitol Res 83:655–658], is obtained. A clone containing a 1371 bp insert was selected following screening of the S. stercoralis cDNA library with antibodies specific to antigen P5. The nucleotide sequence of this insert identified a cDNA coding for the γ-subunit of isocitrate dehydrogenase (NAD⁺), GenBank Accession Number AF176568. The conceptually translated amino acid sequence of the open reading frame for the γ-subunit of S. stercoralis isocitrate dehydrogenase (NAD⁺) encodes a 388 amino acid residue protein with an apparent molecular weight of 43 kDa and a predicted pI of 7.15. The sequence is 71% A/T, reflecting the characteristic A/T codon bias of S. stercoralis. The amino acid sequence of the S. stercoralisγ-subunit of isocitrate dehydrogenase (NAD⁺) is compared with those of Caenorhabditis elegans, rat and human NAD⁺-ICDH. The diagnostic potential of the S. stercoralisγ-subunit of isocitrate dehydrogenase (NAD⁺) is also discussed. Received: 23 September 1999 / Accepted: 22 October 1999     

Seroprevalence of and risk factors for Toxoplasma gondii antibodies among asymptomatic blood donors in Egypt

A cross-sectional study was conducted to evaluate the seroprevalence of and risk factors for Toxoplasma gondii antibodies in 260 blood donors seen at blood banks in Mansoura University Hospital, Egypt. Blood donors were interviewed about sociodemographic characteristics and risk factors for T. gondii infection. A blood sample was taken to document their T. gondii antibody status using enzyme-linked immunosorbent assay. Overall, 155 (59.6%) of 260 blood donors were positive for anti-T. gondii IgG antibodies. Multivariate logistic regression analysis showed a significant association between T. gondii seropositivity and eating meat by-products (luncheon/shawerma) (adjusted odds ratio [OR] 80.82 [95% CI 18.62–350.81], P < 0.0001) or being non-educated (adjusted OR 32.25 [95% CI 7.46–139.44], P < 0.0001). These findings highlight that T. gondii is prevalent among blood donors in Egypt.     

Occurrence of antibodies anti-Neospora caninum, anti-Toxoplasma gondii, and anti-Leishmania chagasi in serum of dogs from Pará State, Amazon, Brazil

A cross-sectional study was conducted to determine the occurrence of anti-Toxoplasma gondii, anti-Neospora caninum, and anti- Leishmania chagasi antibodies in dogs of the state of Pará, Brazil. For this purpose, 129 blood samples were collected from dogs of different ages and gender. Samples of 72 dogs were collected from 39 rural properties from 19 municipalities, and 57 samples were from stray dogs, collected after captivity by the Center of Zoonosis Control from the municipality of Santarém. The sera were analyzed for anti-T. gondii and anti-N. caninum antibodies by indirect fluorescent antibody tests with cutoff values of 1:16 and 1:50, respectively. For the presence of L. chagasi antibodies, enzyme-linked immunosorbent assay was used and positive results were confirmed by immunochromatographic method using the recombinant antigen K39. Of the total of 129 dogs, 90 (69.8%) were positive for T. gondii, 16 (12.4%) for N. caninum, and 30 (23.3%) for L. chagasi. Antibodies for all three parasites were found simultaneously in seven dogs (5.4%), mostly in urban dogs (six of seven). No association was observed related to gender and location (urban or rural) of dogs and occurrence of N. caninum and T. gondii antibodies although, regarding L. chagasi, higher prevalence was found in females (P < 0.02) and in dogs from urban location (P < 0.001). From the 39 farms, in 30 (76.9%) at least one dog was positive for T. gondii or N. caninum or both. Higher occurrence of Leishmania antibodies was observed in N. caninum-negative dogs (P < 0.05).     

Gerbils (Meriones unguiculatus) are highly susceptible to oral infection with Neospora caninum oocysts

Laboratory-reared gerbils (Meriones unguiculatus) were found to be highly susceptible to oral infection with Neospora caninum (NC-Liv strain) oocysts. Gerbils fed ∼1000 oocysts became sick or died at 6–13 days post feeding of oocysts (PFO). N. caninum was isolated in cell culture and from γ-interferon-knockout mice inoculated with homogenates of mesenteric lymph nodes of gerbils examined as early as 1 day PFO. Numerous N. caninum tachyzoites were found in ulcerative lesions in the intestines of gerbils examined at 7–9 days PFO. In a gerbil fed 10 oocysts, N. caninum tachyzoites were found in lesions in the brain. Gerbils fed 10 oocysts developed antibodies to N. caninum by 18 days PFO as determined by the Neospora agglutination test (titers ≥1:500). All gerbils remained negative for antibodies to Toxoplasma gondii as determined by the Toxoplasma agglutination test. Received: 10 September 1999 / Accepted: 10 September 1999     

Immune Response to Natural and Recombinant Antigens of Helicobacter pylori in Patients with Dyspeptic Complaints

 Sera of 223 dyspeptic patients with endoscopic findings of nonulcer dyspepsia (72%), gastric ulcer (15%) and duodenal ulcer (13%) were tested for antibodies against Helicobacter pylori with an enzyme immunoassay and an immunoblot technique using lysates of Helicobacter pylori cells as antigen source. One hundred and fifty-one (68%) sera were found to be positive for Helicobacter pylori IgG with both methods; 5% of the positive results in the enzyme immunoassay were false-positive due to cross-reactions mainly of proteins with a molecular mass of 43–66 kDa. Since cross-reactivity not only reduces the diagnostic value of the immunoassay but also complicates evaluation of the immunoblot results, an attempt was made to overcome these problems by using specific purified recombinant proteins instead of the crude cell preparations as antigens. Of the commonly recognised immunogens of Helicobacter pylori, antibodies against a cell surface protein of 26 kDa, the small urease subunit (29 kDa) and the cytotoxin-associated protein (130 kDa) were identified as highly sensitive serological markers for inclusion in a recombinant antigen mixture for Helicobacter pylori screening. Only the cytotoxinassociated protein was confirmed to be an indicator immunogen for ulcerogenic strains. To assess the reliability of recombinant fragments of this protein in serological screening, the reactivity of antibody to purified fragments of the cytotoxin-associated protein was compared with that to the natural protein. A C-terminal recombinant fragment of 58 kDa showed results identical to those obtained with the natural protein and was thus considered to be an appropriate component of an antigen mixture for serological detection of Helicobacter pylori.     

Characterization and in vitro translation analysis of pelargonium flower break virus

Summary.  Pelargonium flower break virus (PFBV) is one of the common viruses in the glasshouses of Western Europe and has been assigned to the genus Carmovirus. A Spanish isolate obtained from nursery-grown Pelargonium zonale plants (PFBV-m) has been characterized. The molecular weight of genomic RNA and coat protein of PFBV-m were determined to be 1.36 × 10⁶ (corresponding to approximately 4 kb) and 36,000, respectively. Only genomic-size RNA was encapsidated in PFBV virions; making necessary to purify double-stranded RNA from infected tissue in order to detect putative PFBV subgenomic RNAs. PFBV RNA directed the synthesis of a major polypeptide of 34 kDa and three other relevant polypeptides of estimated sizes 88–90 kDa, 42 kDa and 35–36 kDa. Antisera specific to PFBV immunoprecipitated the in vitro translated 35–36 kDa polypeptide indicating that this polypeptide is the PFBV coat protein. The PFBV in vitro translation pattern was very similar to that of CarMV although the relative levels of translated coat protein differed dramatically between the two viruses, most probably due to the lack of encapsidation of subgenomic PFBV. In vitro translation studies with a different biological clone obtained from the same PFBV-m isolate revealed a prominent additional polypeptide which is postulated to be a truncation of the 5′ proximal ORF. Received November 27, 1998 Accepted March 24, 1999     

Effect of hyperprolactinaemia on <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> prevalence in humans

Recent studies have shown that hormones could induce anti-parasitic functions of the host immune system; thus, the aim of the present study was to estimate the seroprevalence of Toxoplasma gondii antibodies by an enzyme-linked immunosorbent assay in a Polish population of women and men with hyperprolactinaemia (n = 234) and hypoprolactinaemia (n = 41) and in a control group (n = 281) with the physiological level of prolactin (PRL). Women with hyperprolactinaemia revealed lower seroprevalence than those with normal PRL level (33.90% and 45.58%, respectively; p = 0.025). Detailed analysis of the results showed that twofold, threefold, fourfold and fivefold increase of the PRL concentration above the normal was correlated to the decrease of the T. gondii seroprevalence, but only in the group of women with a very high PRL level (>86 ng/ml) seroprevalence (12.50%) was significantly lower (p = 0.0004) than in the control subjects. These results confirm previously described suggestions on the relationship between hyperprolactinaemia and parasitic infection frequency. We postulate that a high level of PRL may be one of the important factors preventing T. gondii infection in women.     

Toxoplasma gondii in Capybara (Hydrochaeris hydrochaeris) antibodies and DNA detected by IFAT and PCR

Toxoplasmosis is considered nowadays as one of the most important foodborne diseases in the world. One of the emerging risks in acquiring infection with Toxoplasma gondii is the increasing popularity of wild animals and game meat. Capybara (Hydrochaeris hydrochaeris) is the world’s largest extant rodent and is used for human consumption in many areas of South America, and in case it carries T. gondii cysts, it may act as a source of infection. In the present study, we detected infection with T. gondii in capybaras from the south of Brazil. Antibodies to T. gondii were assayed in the serum of capybaras using the indirect fluorescent antibody test (IFAT ≥ 1:16). Blood, liver, heart, lymph nodes, and spleen tissues were collected and tested by polymerase chain reaction (PCR) for B1 gene and ITS1 region. The results showed that 61.5% (16/26) capybaras were seropositive to T. gondii. Titers of specific antibodies to T. gondii ranged from 1:16 to 1:512. Among the feral rodents studied, 7.7% (2/26) were PCR positive for B1 gene assay and 11.5% (3/26) were positive for ITS1 PCR assay; for both test, the prevalence was 15.4%. Liver, heart, and blood tissues were those which tested positive for the apicomplexan. Our findings show a high percentage of infection with T. gondii in asymptomatic capybaras. Based on those data, we hypothesize that the consumption of raw or undercooked capybara meat could be a source of infection for humans.     

The nucleotide sequence and genome organization of Japanese iris necrotic ring virus, a new species in the genus Carmovirus

Summary.  The genome of Japanese iris necrotic ring virus (JINRV) consists of a positive-sense ssRNA of 4014 nucleotides with six major open reading frames (ORFs). A 5′-non-coding region of 31 nucleotides precedes the first initiation codon. Like Carnation mottle virus (CarMV), the 5′-proximal three ORFs encode a 26 kDa protein (p26) and two readthrough proteins, i.e. an 85 kDa putative RNA replicase (p85) and a 99 kDa protein (p99). The central ORF encodes a small 8 kDa protein (p8). The 3′-proximal ORF encodes a 38 kDa capsid protein (p38). Another ORF encoding a 12 kDa protein (p12) overlaps the p99 ORF. JINRV RNA treated with bacterial alkaline phosphatase and tobacco acid pyrophosphatase could not be ligated to an oligoribonucleotide using T4 RNA ligase, indicating that the 5′ end of the viral RNA is uncapped. The 3′ end is not polyadenylated. Comparison of the genomic organization and the predicted amino acid sequences with those of other viruses confirmed that JINRV should be classified as a member of the genus Carmovirus, family Tombusviridae. Accepted September 23, 1999     

Nucleotide sequence and genome organisation of cherry mottle leaf virus and its relationship to members of the Trichovirus genus

Summary.  The nucleotide sequence of cherry mottle leaf virus (CMLV) was determined and compared to sequences of a number of plant viruses including the type member of the Trichovirus genus (apple chlorotic leafspot virus, ACLSV), and members of the Vitivirus genus including grapevine virus B, (GVB). The CMLV genome was determined to consist of 8003 nt excluding the poly(A) tail at the 3′ end of the genome. The overall A+U content of CMLV genomic RNA was 59. 1%, which is similar to ACLSV, but significantly different from GVB. Four putative open reading frames were identified (ORFs 1, 2, 3, and 4) encoding proteins of M_r 215. 8 kDa, 47 kDa, 21.6 kDa, and 15. 3 kDa, respectively. This differs from ACLSV which has 3 ORFS, and GVB which has 5 ORFs. Protein database searches showed no matches of CMLV ORF4 with ACLSV sequences, but found similarities between ORF4 of CMLV and ORF5 of GVB, suggesting that this may be a nucleic acid-binding protein. CMLV and ACLSV formed a common virus clade in phylogenetic analysis of the coat protein amino acid sequence and except for CMLV’s ORF4, these viruses show high levels of similarity throughout the genome. CMLV appears to be a member of the Trichovirus genus. Accepted November 19, 1999/Received August 12, 1999     

Calomys callosus (Rodentia: Cricetidae) trophoblast cells as host cells to Toxoplasma gondii in early pregnancy

The potential of the RH strain of Toxoplasma gondii to invade trophoblast cells of the cricetid rodent Calomys callosus in a congenital infection in the initial third of pregnancy was investigated in this study using morphological and immunocytochemical approaches. The animals were intraperitoneally inoculated on the 1st day of pregnancy and the infection was observed on day 7. Various numbers of parasites could be observed inside the parasitophorous vacuoles in trophoblastic cells under light and electron microscopy. The trophoblast cells showed characteristics of healthy cells, and no alteration other than parasite vacuoles in their cytoplasm could be detected. Polyclonal or monoclonal anti-T. gondii antibodies (respectively, anti-T. gondii components and the major surface parasite antigen p30) labeled both the parasite surface and parasitophorous vacuole membranes, regardless of the number of parasites inside the compartment. In addition, p30-containing trails were detected in the extracellular matrix surrounding trophoblastic cells similar to those found with other parasites during locomotion and the invasion process. Our results show the ability of T. gondii to infect trophoblast cells during the early blastocyst-endometrial relationship and open new possibilities for more accurate study of the invasion process of this parasite and the role of the trophoblast as an embryo defense barrier. Received: 30 December 1998 / Accepted: 7 February 1999     

Immunological characterization of Toscana virus proteins

Summary.  The genome of Toscana virus (Bunyaviridae family, Phlebovirus genus) consists of three single stranded RNA segments (L, M, S), with negative polarity. The L and M segments contain a single ORF in viral complementary sense and the S segment contains two ORFs in “ambisense” orientation. The M segment codes for three proteins in 3′–5′ genomic orientation: a 30 kDa non structural protein and two 65 kDa glycoproteins, GN, and GC. In this paper we report the expression in E. coli of the S segment ORFs and of three regions of the L ORF. The expressed proteins were used to produce monospecific polyclonal antibodies in mice. By using these antibodies the N and the NSs proteins were unequivocally assigned to the S viral-complementary and viral-sense ORFs, respectively, and the L protein to the L ORF. We have found that like N and L proteins, NSs protein is associated with the viral nucleocapsids in mature virions, suggesting its possible involvement in early events of viral replication. NSs protein was also found associated with cellular polysomes. In virus-infected cells the anti-L antibodies recognized proteins shorter than the full-length L protein, possibly products of L subgenomic segments. Interestingly these defective products were not found in mature virions, suggesting specific mechanisms in virion assembly. Accepted April 19, 1999 Received August 19, 1998     

Indian citrus ringspot virus: a proposed new species with some affinities to potex-, carla-, fovea- and allexiviruses

Summary.  An isolate of Indian citrus ringspot virus from Kinnow mandarin in northern India had flexuous particles with evident cross-banding and a modal length of 650 nm. It was mechanically transmitted to five herbaceous hosts including Phaseolus vulgaris cv Saxa, in which it became systemic. In thin sections, virus particles were observed in the cytoplasm of parenchyma cells but no specific inclusions were seen. The virus was purified from infected Saxa bean leaves and an antiserum prepared. There was no serological cross-reaction with representative allexi-, capillo-, potex- and trichoviruses, except a faint one-way reaction with Potato virus X. Purified virus yielded a major band, the presumed coat protein (CP), of about 34 kDa, and a single ssRNA of about 7.5 kb, which was infectious. Two ORFs encoding putative proteins of 34 kDa and 23 kDa were located in the 3′ part of the RNA. The product of the 34 kDa ORF was confirmed as the CP by expression in E. coli. The derived amino acid sequence of the CP contained some short motifs similar to those of potex-, fovea-, carla- and allexiviruses but otherwise there was no strong similarity to any of these. The 23 kDa ORF contained a zinc finger-like sequence, as in similar ORFs in carla- and allexiviruses but overall amino acid homology with these was low. The virus does not appear to fall into any known genus. A new species is proposed. Serological and molecular diagnostic reagents were prepared. Received July 14, 1999/Accepted February 10, 2000     

Antigenicity of Trichomonas vaginalis heat-shock proteins in human infections

Patients infected with Trichomonas vaginalis mount humoral and cellular immune responses that often do not protect against reinfection. The oxidative stressors produced by leukocytes may trigger a heat-shock-like response in T. vaginalis trophozoites, helping the parasite to survive host immune defenses. The antigenicity of T. vaginalis heat-shock proteins (HSPs) was examined by immunoprecipitation of T. vaginalis heat-induced proteins with sera from infected patients and controls. When T. vaginalis was heat-shocked, HSPs of 169–167 and 140–137 kDa were specifically recognized by sera from infected male and female patients. However, the majority of T. vaginalis HSPs were also immunoprecipitated by control sera; all sera recognized 72- to 71-kDa, 47- to 45-kDa, 38- to 37-kDa, 35-kDa, and 31-kDa heat-induced proteins. At least 15 proteins from non-heat-shocked T. vaginalis were immunoprecipitated by sera from infected patients and controls, indicating that natural or cross-reacting antibodies could participate in host responses to trichomoniasis. Molecules of 158, 135, 89, and 74–72 kDa were immunoprecipitated from some non-heat-shocked parasites only by patients' sera. A 38-kDa T. vaginalis protein was immunoprecipitated only by sera from infected females and may be specific for infection in women. Received: 26 August 1999 / Accepted: 15 September 1999     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> antibody profile in HIV-infected pregnant women and the risk of congenital toxoplasmosis

The purpose of this study was to investigate the antibodies to Toxoplasma gondii in human immunodeficiency virus (HIV)-infected pregnant women and to determine the association between serological profile and the risk of congenital toxoplasmosis. The study, conducted in a public maternity ward from May 2002 to April 2005, included all HIV-infected women who delivered live infants during the 36 months, and, as a control group, all HIV-negative women that delivered live infants in the first 12 months of the study. Antibodies to T. gondii were detected in 1,624 of 2,421 HIV-negative women (67%; 95% confidence interval [CI] 65–69%) and in 121 of 168 HIV-infected patients (72%; 95% CI 65–79%). A total of 547 HIV-negative and 103 HIV-infected patients were tested at delivery and had positive T. gondii-specific IgG. In HIV-negative women, the median of the specific IgG concentration was 79 (interquartile range 38–160), and in HIV-infected patients, it was 283 (interquartile range 94–704) (P < 0.001). In the group of co-infected women, the only infant with congenital toxoplasmosis was born to a mother with acute toxoplasmosis infection acquired during pregnancy who did not have a high specific IgG concentration or a positive result for specific IgM. We concluded that high T. gondii-specific IgG values were much more frequent among HIV-infected pregnant women, but it did not translate into an increased risk of maternal–fetal transmission of toxoplasmosis.     

Coinfection is an important factor in epidemiological studies: the first serosurvey of the aoudad (<Emphasis Type="Italic">Ammotragus lervia</Emphasis>)

Despite being considered an invasive ungulate outside its native range (North Africa), little information exists regarding the role of the aoudad (also called Barbary sheep, Ammotragus lervia) as a pathogen reservoir. Furthermore, in most epidemiological surveys the potential role of coinfections (e.g. a first infection may make the host more immuno-competent or susceptible against a second pathogen) as a risk factor is often neglected. In this study we first performed a serological survey for selected pathogens (Mycobacterium bovis, M. avium subsp. paratuberculosis, Chlamydophila abortus, bovine viral diarrhoea/border disease viruses (BVDV-BDV), Salmonella spp., Brucella melitensis and Toxoplasma gondii) on free (n = 66) and captive (n = 25) aoudad from south-east Spain. Then, by using Akaike’s information criterion, we evaluated the importance of coinfection in two statistical models that included the effects of population, age, and sex. Our results show that neither free nor captive aoudad had antibodies against Brucella melitensis, Chlamydophila abortus, or BVDV-BDV. However, compared to other wild ungulates in Spain, aoudads have high prevalence of antibodies against M. bovis (free = 49.5%; captive = 8%), very high prevalence of antibodies against M. avium subsp. paratuberculosis (free = 19.4%; captive = 56%), and intermediate prevalence of antibodies against Salmonella spp. (free = 13.4%; captive = 0%) or T. gondii (free = 1.5%; captive = 24%). Although the additive effects of population and age were included in our set of selected models, coinfection was the most influential factor to detect antibodies response against mycobacterials and salmonella infections. The direction of this influence could be exclusion of disease between tuberculosis and paratuberculosis seroreactor animals, or enhanced susceptibility to disease between tuberculosis and salmonella seroreactor animals. In conclusion, we believe that wildlife managers must pay more attention to the potential risk posed by aoudads as hosts (and probably reservoirs) of paratuberculosis and tuberculosis mycobacterials, while epidemiologists should be more aware of coinfection as an important factor in epidemiological surveys, especially in wildlife populations where multiple infections are common.     

Schisantherin A Exhibits Anti-inflammatory Properties by Down-Regulating NF<Emphasis Type="Italic">-</Emphasis>κB and MAPK Signaling Pathways in Lipopolysaccharide-Treated RAW 264.7 Cells

Schisantherin A, a dibenzocyclooctadiene lignan isolated from the fruit of Schisandra sphenanthera, has been used as an antitussive, tonic, and sedative agent under the name of Wuweizi in Chinese traditional medicine. In the present study, we carry out a screening program to identify the anti-inflammatory potentials of schisantherin A. We found that schisantherin A reduced lipopolysaccharide (LPS (1 mg/L))-induced levels of TNF-α, IL-6, NO, and PGE2 (p < 0.01 or p < 0.05), and also reduced levels of iNOS and COX-2 in RAW 264.7 macrophages in a concentration-dependent manner. We further investigated signal transduction mechanisms to determine how schisantherin A affects. RAW264.7 cells were pretreated with 0.5, 2.5, or 25 mg/L of schisantherin A 1 h prior to treatment with 1 mg/L of LPS. Thirty minutes later, cells were harvested and mitogen activated protein kinases (MAPKs) activation and IκBα was measured by Western blot. Alternatively, cells were fixed and nuclear factor-κB (NF-κB) activation was measured using immunocytochemical analysis. Signal transduction studies showed that schisantherin A significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH2-terminal kinase (JNK) phosphorylation protein expression. Schisantherin A also inhibited p65-NF-κB translocation into the nucleus by IκBα degradation. By using specific inhibitors of ERK, JNK and p38, we found that schisantherin A may inhibit TNF-α mostly through ERK pathway. Therefore, schisantherin A may inhibit LPS-induced production of inflammatory cytokines by blocking NF-κB and MAPKs signaling in RAW264.7 cells.     

Immunogenic characterization of the chimeric surface antigen 1 and 2 (SAG1/2) of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> expressed in the yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. These results showed that the P. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. gondii. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. gondii. Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Specific production of IFN-γ, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice. Finally, vaccinated mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.005), and their survival time increased significantly compared to the negative control.     

Nitric oxide is not involved in the killing of Trypanosoma cruzi by chicken macrophages

It is known that chicken macrophages derived in vitro from blood monocytes have the capacity to destroy Trypanosoma cruzi, but Toxoplasma gondii can survive within these cells. This study was performed to determine the involvement of nitric oxide (NO) in the killing of T. cruzi by chicken macrophages. Activated (by interferon-γ and lipopolysaccharide) mouse peritoneal macrophages were used as controls. Macrophages were infected with T. cruzi and T. gondii; after 2, 24, and 48 h, NO was assayed using the Griess reagent. Respiratory-burst involvement, revealed by the reduction of nitroblue tetrazolium (NBT), was determined in chicken macrophages. Chicken macrophages did not produce NO; mouse macrophages were capable of producing NO with no multiplication of parasites. Reduction of NBT could be detected in chicken macrophages that interacted with T. cruzi but was absent in those that interacted with T. gondii. These results demonstrate that chicken macrophages do not use NO as a microbicidal agent when infected with T. cruzi or T. gondii. Received: 1 June 1999 / Accepted: 18 August 1999