Investigation of persistence of infectious Toxoplasma gondii in raw sausages using in-house developed and validated real time-PCR.

The question, if and to what extent raw-sausage-products represent a possible source of infection for the globally distributed and potentially health-threatening toxoplasmosis gave reason for this study. For this, the survival capability of Toxoplasma gondii in relation to the raw-sausage-manufacturing-process including different ripening-processes was investigated. To enable a fast and reliable parasite-detection, a real time-PCR-system based on a specific 529-bp-fragment of T. gondii and an internal amplification control (IAC) was developed and established. The applicability was tested in various experiments where T. gondii-tachyzoites were mixed into different types of raw-sausages and then investigated by using the real time-PCR-system. The latter was also used to investigate the possible infection-risk of raw-sausages. For this, two pigs were intravenously infected with T. gondii-tachyzoites and after having reached the typical slaughtering age, their meat was manufactured to different raw-sausage-products ("Mett"- and "Teewurst" as well as "Salami"). In order to prove the potential infectivity of these products under conditions close to reality, sausages in different ripening stages were fed to laboratory mice. The animals' organs (brain, heart and spleen) were examined employing the real time-PCR. T. gondii-DNA was detected in four out of 288 (1.4%) mice indicating that marketable raw-sausage-products generally bear a risk for consumers. However, the probability of an infection seems to be quite marginal.

Genetic resistance to Sarcocystis miescheriana in pigs following experimental infection.

Clinical and parasitological traits of Sarcocystis miescheriana differ in Pietrain and Meishan pigs. For further description and characterization of the genetic basis of this variation a F(2) family based on Pietrain boars and Meishan sows as founders was generated. One hundred and thirty-nine F(2) pigs were challenged orally at an age of 100 days with 50,000 sporozysts to produce the typical clinical picture of a moderate dose Sarcocystis infection. Heritabilities were estimated for clinical and clinical-chemical traits, for specific antibody responses to the infection and for bradyzoite numbers found in skeletal (Musculus longissimus dorsi: M.l.d.) and heart muscles at necropsy 70 days post-infection (p.i.) Apart from several low to moderate heritabilities, high heritabilities were observed for bradyzoite numbers in the M.l.d. (0.68), IgM antibody levels (0.74) during the acute (14 days p.i.) and titres of specific IgG antibodies (0.42) in the early stage of cyst formation (42 days p.i.). Marked heritabilities of these traits, which are basic for acute phase of the disease (14 days p.i.) or chronic Sarcocystosis presume genes that explain sufficient shares of variance (QTL). The model is considered valuable for screening of gene variants associated with resistance/susceptibility to Sarcocystis infection. Such gene variants could then be used in susceptibility-scoring or selection programs in the future.

Ultrasonography in filaria-infected rodents: detection of adult Litomosoides sigmodontis and Brugia malayi filariae.

OBJECTIVE: To evaluate the usefulness of ultrasonography (USG) in the detection of adult filariae in rodents. Wuchereria bancrofti are frequently detected using USG in humans, whereas adult Brugia malayi have not been so far. METHODS: A Meriones unguiculatus with Litomosoides sigmodontis infection was examined to visualize adult filariae of a similar length as W. bancrofti. Similarly, three Mastomys coucha, infected with B. malayi, were examined using USG to verify whether the adult worms, which are far smaller than W. bancrofti and L. sigmodontis, can be located using USG in the animals. RESULTS: Adult L. sigmodontis were detected using USG in the pleural cavity of M. unguiculatus, and in M. coucha adult B. malayi were visualized in the hearts, lungs, axillary lymph nodes and scrotum. Ultrasound findings were verified by dissection of the rodents. CONCLUSIONS: Although adult B. malayi are far smaller than L. sigmodontis and W. bancrofti, they can be detected using USG in rodents. USG may serve as an adjunctive tool to support parasitological examinations and can add information on filarial infections at any time point of an observation period, particularly in cryptic infections and without the need for invasive measures or killing of the rodent. Thus, USG can support the early detection of macrofilaricidal activities of new compounds and can be used to determine the location of adult worms in the animals. It is possible to give a rough estimate of the number of adult worms, but determination of the exact numbers of adult filariae in various locations is impossible with USG.

Lethal LPS-independent side effects after microfilaricidal treatment in Acanthocheilonema viteae-infected rodents.

Mastomys coucha and jirds infected with Acanthocheilonema viteae, a filarial species free of endosymbiontic bacteria of the genus Wolbachia, suffer lethal side effects after effective microfilaricidal therapy with diethylcarbamazine and levamisole, whereas, M. coucha infected with the Wolbachia-infested species Brugia malayi or Litomosoides carinii tolerate corresponding treatment. Mortality in A. viteae infected, treated animals varied with microfilariae density in the blood. It was up to 100% in highly microfilaraemic M. coucha and jirds, but low or absent in animals with low microfilariae counts. Deaths occurred in most cases 5-24 h after treatment. Characteristic symptoms in animals, which died subsequently were a rapid drop in body temperature by 4-7 degrees C, an increase in hematokrit values by up to 10% and a moderate blood acidosis. Lethal effects in A. viteae infections did not depend on a particular status of hypersensitivity of the animals since desensitization procedures, which protected infected M. coucha against an otherwise lethal intravenous challenge with A. viteae homogenate did not protect against adverse reactions to a subsequent microfilaricidal treatment. The animals were protected from treatment induced death by injection of N-LMMA. Thus the final morbific agent seems NO. The data show that adverse effects after effective microfilaricidal therapy may be caused by microfilariae derived components different from Wolbachia-released LPS.

Detection of specific antibodies to Neospora caninum and Toxoplasma gondii in naturally infected alpacas (Lama pacos), llamas (Lama glama) and vicunas (Lama vicugna) from Peru and Germany.

Sera of an experimentally Neospora caninum infected llama and a non-infected control llama were used to establish an immunoblot, an ELISA and an IFAT to detect antibodies against N. caninum tachyzoites. Subsequently, serum samples collected from a total of 871 South American Camelids (SAC: Lama glama, Lama pacos, Lama vicugna) of two farms in Peru and from 32 SAC of a farm in central Germany were examined for antibodies against N. caninum and Toxoplasma gondii. Based on the recognition of specific bands in the immunoblot, sera of SAC from Peru were differentiated into N. caninum-positive (n = 18) and T. gondii-positive (n = 30) samples and into samples negative or inconclusive for both parasites. Using the immunoblot results as the reference, a modified version of the p38-ELISA and the IFAT were evaluated for detecting N. caninum antibodies in SAC sera. Applying a cut-off as determined by two graph-receiver operating characteristic analysis both, the ELISA and the IFAT, exhibited a sensitivity and specificity of about 95% in the SAC sera from Peru. Serological testing confirmed that SAC may become infected with N. caninum under field conditions in Peru. In addition to alpacas and llamas also 114 wild living vicunas had been examined for antibodies against N. caninum. However, only the alpacas and llamas but no vicunas were found N. caninum-positive. In contrast, T. gondii-seropositive animals were detected in all three SAC species. The lack of N. caninum-seropositive vicunas indicates that in the study area in Peru wild canids might not serve as definitive hosts of N. caninum while for T. gondii a life cycle including wild felids is likely. On the German farm no N. caninum- but only T. gondii-seropositive SAC (n = 14) were detected. The seroprevalence of T. gondii infection was significantly higher in adult SAC (alpacas in Peru, llamas in Germany) than in crias (i.e. < 12 months old foals) indicating that the predominant route of infection is post natal. Since the present study was restricted to a few farms, the seroprevalences determined are not representative. However, our results confirm natural infections with N. caninum and T. gondii in SAC. Whether these infections are linked to any disease, e.g. reproductive losses, has to be clarified in further studies.

Cross-sectional survey in pig breeding farms in Hesse, Germany: seroprevalence and risk factors of infections with Toxoplasma gondii, Sarcocystis spp. and Neospora caninum in sows.

A cross-sectional survey was performed to estimate the prevalences of antibodies to Toxoplasma gondii (ELISA, IFAT), Sarcocystis spp. (ELISA, using S. miescheriana as antigen) and Neospora caninum (ELISA, immunoblotting) in sows from breeding farms in southern Hesse, Germany. A total of 2041 plasma samples of sows from 94 randomly selected farms was examined. Data on farm profiles, husbandry management and sows were collected by a questionnaire and exploratively analysed. For T. gondii the ELISA results agreed well with the results obtained by IFAT (kappa=0.71). Antibodies to T. gondii were detected by ELISA in 19% of the sows. Sixty-nine percent of the farms had at least one seropositive sow, and a within-farm seroprevalence of >or=50% was observed in 14% of all farms. The prevalence of anti-T. gondii antibodies was positively correlated with the age of sows. The within-herd seroprevalence was significantly higher in farms with reproductive disorders than in those without such problems. On the farm level, the farm type 'piglet production' (versus 'pedigree breeding' or 'farrow-to-finish') was the only risk factor associated with the presence of T. gondii-seropositive sows. Antibodies to Sarcocystis spp. were found in 29% of the sows. Seventy-two percent of the farms harboured at least one seropositive sow, and a within-farm seroprevalence of >or=50% was detected in 23% of all farms. The seroprevalence increased significantly with the age of sows. On the farm level, only the farm type 'piglet production' (versus 'pedigree breeding') and the replacement of sows by purchasing (versus raising on the own farm) were identified as risk factors for seropositivity. Antibodies to N. caninum were detected in one sow using both the screening ELISA and the confirmatory immunoblotting technique. This may indicate the first natural N. caninum infection in pigs.

Alternative mechanism of Eimeria bovis sporozoites to invade cells in vitro by breaching the plasma membrane.

In vitro Eimeria bovis sporozoites invade a wide range of cell types, and in the case of bovine cells, they may develop to first-generation schizonts. Often, however, they subsequently leave their host cell to invade a new one, which seems contrary to the classical way of infecting a cell by forming a parasitophorous vacuole. Using a standard, "cell wound assay," we show that E. bovis can invade bovine endothelial cells by breaching the plasma membrane and may again leave the surviving cell. Eimeria bovis sporozoites also infected VERO and HT29 cells but obviously without damaging the plasma membrane. The same held true when bovine endothelial cells were exposed to tachyzoites of Toxoplasma gondii and Neospora caninum. According to a literature report dealing with Plasmodium yoelii sporozoites, breaching the membrane of certain host cells may be a common phenomenon for coccidian sporozoites but may not be for merozoites.

Toxoplasma gondii: changes of transepithelial ion transport in infected HT29/B6 cell monolayers.

An in vitro system was established to study the effect of coccidian parasites on ion transport systems in epithelial tissues using HT29/B6, a human colon carcinoma cell line, and Toxoplasma gondii as a model parasite. Ion transport was measured in perfusion chambers 5, 10 and 15 h post-infection using monolayers in which approximately 30% of the cells were parasitized. The infection had rapid effects on the conductance and unidirectional chloride fluxes of infected cell monolayers, which were two to three times higher than those of uninfected HT29/B6 cell monolayers throughout the observation period. However, the chloride net fluxes and short-circuit current were unaffected by the parasites, while the decrease of chloride seromucosal fluxes and conductance after addition of bumetanide were affected by the infection. The unidirectional mannitol fluxes, which correspond with water motion through paracellular pathways, were increased in infected HT29/B6 cell monolayers.

Prevalence, risk factors and economic importance of infestations with Sarcoptes scabiei and Haematopinus suis in sows of pig breeding farms in Hesse, Germany.

A cross-sectional survey was performed in 110 randomly selected pig-breeding farms of southern Hesse, Germany to estimate the prevalence of ectoparasite infestations and to find possible risk factors. Ear scrapings of, if available, 10 sows per farm were examined for Sarcoptes scabiei var. suis (De Geer) (Acaridida: Sarcoptidae) by the potassium hydroxide digestion method, and a total of 2754 sows was inspected for skin lesions and infestations with Haematopinus suis (L.) (Anoplurida: Haematopinidae). Data on farm profiles and sows were collected by a questionnaire. In total, 19.1% and 2.5% of the sows were found to be infested with S. scabiei or H. suis, respectively. The percentage of mite or louse infestation was significantly higher in sows showing pruritus than in those without skin lesions. Both ectoparasite infestations were related neither to the age of sows nor their reproduction status, nor to the time interval to last ectoparasite treatment. Using farms as the unit of analysis, the estimated prevalence of mange mite and louse infestations was 45.4% and 14.5%, respectively. There was no significant association between the presence of S. scabiei and H. suis in the farms. Risk factors for S. scabiei infestation were mixed housing of dry and nursing sows in the same unit (vs. separate housing) and straw bedding (vs. strawless). For louse infestation, only mechanical cleaning of stable units (vs. additional use of disinfection methods) and pasturing of gilts and dry sows were identified as risk factors. The economic loss by S. scabiei infestation in the study population was assessed at euro 4200 per affected farm and year on average.

Development of Eimeria bovis in vitro: suitability of several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal, Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells.

Several bovine, human and porcine endothelial cell lines, bovine fetal gastrointestinal cells (BFGC), Madin-Darby bovine kidney (MDBK) and African green monkey kidney (VERO) cells were exposed in vitro to sporozoites of Eimeria bovis. Parasites invaded all cells used and changed their shape to more stumpy forms within 12 h. Sporozoites left their host cells and invaded new ones frequently within the first 12 h post-infection. Further development took place only in bovine cells, although parasites survived in the other cells for at least 3 weeks. Within the non-bovine cells, conspicuously enlarged parasitophorous vacuoles developed in VERO cells and reached a diameter of 15-20 microm. The best development to first generation schizonts with regard both to time required to mature, to schizont size and to merozoite yields was observed in BFGC, followed by bovine umbilical vein and bovine spleen lymphatic endothelial cells. MDBK cells were less suitable. The life cycle was completed (development of oocysts) only occasionally in BFGC. Results are considered under several aspects. Thus, infected VERO cells may represent a suitable tool for studying the parasitophorous vacuole, while infected endothelial cells represent a system quite narrow to the in vivo situation and should allow basic studies on parasite/host cell interactions and BFGC can be used for the mass production of E. bovis first generation merozoites.

Filaricidal efficacy of anthelmintically active cyclodepsipeptides.

PF 1022A, a novel anthelmintically active cyclodepsipeptide, and Bay 44-4400, a semisynthetic derivative of PF 1022A were tested for filaricidal efficacy in Mastomys coucha infected with Litomosoides sigmodontis, Acanthocheilonema viteae and Brugia malayi. The parent compound PF 1022A showed limited anti-filarial efficacy in L. sigmodontis and B. malayi infected animals. Oral doses of 5 x 100 mg/kg on consecutive days caused only a temporary decrease of microfilariaemia levels. By contrast, Bay 44-4400 was highly effective against microfilariae of all three species in single oral, subcutaneous and cutaneously applied (spot on) doses. Minimum effective doses (MED, reducing parasitaemia density by > or =95%) determined 3 and 7 days after treatment were 3.125-6.25 and 6.25-12.5mg/kg, respectively. Using the spot on formulation, doses of 6.25mg/kg (L. sigmodontis), 12.5mg/kg (A. viteae) and 25mg/kg (B. malayi) were required to cause reductions of microfilaraemia levels by > or =95% until day 56. Adulticidal effects, determined as minimum curative doses (MCD, eliminating adult parasites within 56 days by >95%) after single dose treatment were limited to A. viteae (MCD, 100mg/kg independent of the route of administration). Repeated oral treatment (100mg/kg on 5 consecutive days) killed all adult L. sigmodontis but did not affect B. malayi. However, single doses of 6.25 and 25mg/kg resulted in severe pathological alterations of intrauterine stages of L. sigmodontis and B. malayi, respectively. These alterations may be responsible for long-lasting reductions of microfilaraemia even when curative effects could not be achieved.

Effects of Bay 44-4400, a new cyclodepsipeptide, on developing stages of filariae (Acanthocheilonema viteae, Brugia malayi, Litomosoides sigmodontis) in the rodent Mastomys coucha.

Bay 44-4400 was used as a spot on formulation and administered in single doses of 25 and 100 mg/kg to Acanthocheilonema viteae, Brugia malayi, and Litomosoides sigmodontis infected Mastomys coucha on various dates during prepatency, aiming to affect third stage larvae, fourth stage larvae or preadult worms. Microfilaraemia levels were controlled in comparison to untreated controls until necropsies were performed 100 days p.i. (A. viteae, L. sigmodontis) and 150 days p.i. (B. malayi) to determine the numbers of surviving worms and the condition of intrauterine developing stages. A significant proportion (86-100%) of larval and preadult stages of A. viteae were killed by Bay 44-4400 at a dose of 100 mg/kg. A dose of 25 mg/kg had only insignificant effects on the developing parasites, however, it strongly reduced microfilaraemia levels caused by surviving worms in the early phase of patency. Larval and preadult B. malayi and L. sigmodontis were not killed by Bay 44-4400 to a significant degree. Microfilaraemia developing by surviving parasites was generally and significantly reduced throughout the observation period when treatment was performed to affect the preadult parasites. In the other cases variable results were obtained. Intrauterine early embryonic stages were found to be pathologically altered in worms which had been treated at a preadult stage.

Cellular immune responses of filaria (Litomosoides sigmodontis) infected BALB/c mice detected on the level of cytokine transcription.

Cellular immune responses of BALB/c mice infected with 80 or 160 L3 of Litomosoides sigmodontis were studied over a period of 200 days postinfection (p.i.) by stimulating spleen cells with specific microfilariae and adult antigens and Concanavalin A (Con A). Effects were determined as the level of transcription of cytokine genes [interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, IL-10, IL-13] employing a semiquantitative reverse transcriptase-polymerase chain reaction technique. Con A stimulation resulted in generally enhanced transcription levels in infected animals. Exposure to filarial antigens stimulated T cells of infected animals dependent on time p.i. There was a general strong response in the early prepatency (24 days p.i.), a temporary almost complete downregulation of cytokine gene transcription except IL-10 towards the end of prepatency (45 days p.i.), and subsequently strong reactions particularly concerning IFN-gamma and IL-13 during patency and postpatency. The dose of infection as well as the mode of antigenic stimulation had generally only small effects on the cytokine gene transcription: following the same type of kinetics, infection with 160 L3 as well as the use of microfilarial antigen generally induced lower levels of cytokine gene transcription compared with infection with 80 L3 and stimulation with female antigen, respectively.

In vivo expression profiles of cytokine and iNOS mRNAs in rats infected with Eimeria separata.

Studies on cytokine (IFN-gamma, IL-2, IL-4, IL-5, IL-10) and inducible NO-synthase (iNOS) gene transcription in mesenteric lymph nodes (MLN) and the caecum wall were performed 0, 48, and 72h after primary and challenge infections of rats with Eimeria separata using RT-PCR. The amount of IFN-gamma mRNA was elevated in MLN and caeca 72h after primary and 48-72h after challenge infection when compared with uninfected controls. Increased amounts of IL-2 mRNA were only found in MLN of infected rats 72h post-infection (p.i.). In case of IL-10, infections did not affect the amount of mRNA in MLN, but led to markedly increased levels in the caecum wall of both infected groups 48 and 72h p.i. Levels of IL-4 mRNA remained unchanged after infections and IL-5 gene transcripts were undetectable. Amounts of iNOS mRNA (not investigated in MLN) were found strongly enhanced 48 and 72h p.i. in the caecum walls of all infected animals when compared with naive controls. The data are discussed in regard of the cellular source of the cytokines and their immunological role.

Prevalence of enteropathogens in suckling and weaned piglets with diarrhoea in southern Germany.

Faecal samples from suckling (n = 205) and weaned piglets (n = 82) with diarrhoea from 24 farms in Southern Germany were examined for shedding of important metazoic parasitic, viral and bacterial pathogens using culture, microscopic and electronmicroscopic methods. Escherichia coli isolates were tested further for the enterotoxin genes est-Ia and elt-I by colony blot hybridization. Isospora suis was diagnosed in 26.9% and Cryptosporidium parvum in 1.4% of the piglets investigated. The proportion of coronavirus-positive animals was 13.4% and 4% were positive for rotavirus. It was found that 17.6% of the animals were infected with enterotoxigenic E. coli (ETEC; 10.1% ETEC-ST-Ia and 8.6% ETEC-LT-I, respectively). The occurrence of the pathogens was significantly associated with the age of the animals examined (P < 0.001). Isospora suis was predominantly isolated from suckling piglets (in the second and third week of life), while in weaned piglets (fourth week of life) rotavirus and ETEC were most prevalent. On 22 of the 24 piglet production farms examined at least one of the investigated pathogens was detected. Coronavirus was diagnosed in 66.7%, I. suis in 62.5%, rotavirus in 20.8% and C. parvum in 8.3% of the farms. These results underline the fact that despite the hygienic, technical and immune preventive efforts during the last years, enteropathogens are still common in German piglet production units.

Lymphocyte subpopulations in the caecum mucosa of rats after infections with Eimeria separata: early responses in naive and immune animals to primary and challenge infections.

This study aimed to characterise the local (intestinal) immune response of rats after primary and challenge infections with Eimeria separata. Naive rats and rats which had been immunised by two moderate infections were exposed to a heavy infection with 100000 oocysts per animal. Necropsies were performed 0, 24 and 48 h after infection and lymphocyte subpopulations were microscopically quantified in the caecum mucosa after marking by immunohistological techniques. There was no difference between naive and immune rats concerning the number of CD45R(+) (B) cells, whereas significantly more CD3(+) (T) cells were found in the caecum wall of the immune rats. CD4(+) T cells predominated in animals after primary infection, whereas CD8(+) T cells represented the major T-cell subset in challenged rats. The proportion of TCRgammadelta(+) T cells did not differ in the mucosa between the groups examined, whereas challenged rats showed significantly increased numbers of TCRalphabeta(+) T cells in the caecum wall when compared with animals after a primary infection. Thus, CD4(+) T cells may be particularly involved in the immune response to a primary infection of rats with E. separata whereas immunity to a challenge infection seems to be mediated predominantly by CD8(+) and TCRalphabeta(+) T cells.

Immunity in rats against Eimeria separata: oocyst excretion, effects on endogenous stages and local tissue response after primary and challenge infections.

The aim of the study was the characterization of the local immune response of Lewis rats to Eimeria separata, a caecum-dwelling coccidium. Rats infected twice at 10-day intervals with 5,000 oocysts developed a high degree of immunity to a heavy challenge with 100,000 oocysts, reducing the oocyst production by > 98% when compared with naive recipients. Histopathological investigations performed over a period of 0-72 h post-infection (pi) showed that 1st generation schizonts, developed within 24 h pi, represented the major target stages, although later stages were also affected. Preinfected animals showed significantly more lymphocytes in the caecum wall than naive animals. An increase in lymphocyte numbers after challenge observed in both groups was enhanced in challenged animals up to 36 h pi. The number of lamina propria lymphocytes predominantly was increased after primary infection whereas in repeatedly infected animals the increase also concerned intraepithelial lymphocytes. In addition, the numbers of plasma cells were enhanced in the caecum wall of immune animals. Macrophage infiltration in the caecum wall followed a similar time course in both groups up to 36 h pi. A subsequent further rise up to 48 h pi was enhanced in naive rats. Tissue infiltrations with eosinophils and mast cells were observed predominantly in the repeatedly infected rats. No obvious changes occurred with intestinal neutrophils and goblet cells. In conclusion, caecum tissue alterations suggest an early local immune response, which is related to development and maturation of the parasite.

Molecular characterization of a Litomosoides sigmodontis protein involved in the development of the microfilarial sheath during embryogenesis.

A cDNA clone Ls110 was isolated from a female Litomosoides sigmodontis expression library using an antiserum raised against the microfilarial sheath. The complete cDNA encodes a protein (Ls110) of 382 amino acids. Southern and PCR analyses revealed the presence of Ls110 in L. sigmodontis as a single copy gene. The transcription of the Ls110 gene was limited to female worms. In these worms the transcription was confined to the epithelial cells of the uterus. The protein Ls110 was detected not only in the epithelial layer of the uterus but also secreted in the lumen of the uterus. All the intra-uterine embryonic stages showed the protein bound to their egg shell/sheath, except the early multicellular embryonic stages and fully developed microfilariae. The transient occurrence of Ls110 on these structures of intra-uterine stages besides the presence of a cysteine-rich N-terminal region (SXC-like domain) suggest that the protein may play a role in the formation of the microfilarial sheath during embryogenesis.

Intracellular calcium and pH conditions of cultured cells infected with Eimeria bovis or E. separata.

Loading of Eimeria bovis-infected Vero cells with membrane-permeant acetoxymethyl esters (AM-esters) of ion-sensitive dyes provided us with a noninvasive method for investigation of the permeability of the parasitophorous vacuole membrane (PVM) and simultaneous measurement of Ca2+ and H+ concentrations in different compartments of the infected cells. The distribution patterns of the cleaved membrane-impermeant dyes argue against the existence of nonselective pores in the PVM. There is also no indication of a parasitophorous duct connecting the vacuolar space with extracellular media. The pH inside the parasitophorous vacuole (PV) was lower than that in the cytoplasm of the host cell or the parasite, whereas the [Ca2+] in these compartments did not differ significantly. In HT29 cells infected with E. separata for 24 h the Ca2+ response to extracellular adenosine triphosphate (ATP) was significantly reduced, indicating influences on the host cell's intracellular signaling.

Immunomodulatory effects in Litomosoides sigmodontis-infected Mastomys coucha.

The levels of parasite-specific IgG1 and IgG2 antibodies and mitogen-induced and parasite-specific proliferative T-cell responses were determined in Litomosoides sigmodontis-infected Mastomys coucha throughout an observation period of 400 days post infection (p.i.). These were compared with the respective reactions in animals that had been immunized with intrauterine stages/microfilariae of the parasite and in animals that had been challenged after immunization as determined at up to 60 days after challenge. IgG1 antibodies to adult antigen developed early and reached a plateau at 120 days p.i., whereas IgG2 antibodies were not found before day 60 p.i., increased with rising parasitemia, reached a plateau at 200 days p.i., and, in some animals, even became the predominant IgG subclass. Proliferative responses of spleen lymphocytes to concanavalin A (Con A) and lipopolysaccharides (LPS), but not Con-A-induced interleukin 2 (IL-2) production, were found to be suppressed in infected animals during patency as compared with uninfected controls. Spleen cells of infected animals showed a weak proliferative reaction to male antigen but were unresponsive to female and microfilarial antigen during prepatency and early patency. Subsequently, when microfilaremia decreased (200 days p.i.), continuously increasing responses to all antigens were observed. Immunized M. coucha developed specific IgG1 and IgG2 antibodies, and their spleen cells showed strong proliferative responses to the three L. sigmodontis antigens. Challenge infections down-regulated the proliferative responses of spleen cells to filarial antigens as early as during the prepatent phase of the challenge infection but supported existing IgG1 and IgG2 responses.