Cooperative role of macrophages and neutrophils in host Antiprotozoan resistance in mice acutely infected with Cryptosporidium parvum.

Severe experimental infections with Cryptosporidium parvum have been reported in immunocompromised animals such as SCID mice (mice without functional T cells and B cells). In a C. parvum infection with 1 x 10(6) oocysts/mouse in SCID beige (SCIDbg) mice (SCID mice lacking functional NK cells), oocyst shedding was first demonstrated 18 days after infection. However, shedding was shown as early as 3 days after the same infection in SCIDbgMN mice. All of the SCIDbgMN mice died within 16 days of C. parvum infection, while 100% of the SCIDbg mice exposed to the parasite survived. SCIDbgMN mice are SCIDbg mice depleted of functional macrophages (Mphi) and neutrophils (PMN), suggesting that the severity early after C. parvum infection is strongly influenced by the functions of Mphi and PMN. All SCIDbgMN mice orally infected with a lethal dose of C. parvum survived after they were inoculated with Mphi from SCIDbg mice exposed to C. parvum (CP-Mphi) or resident Mphi previously cultured with PMN from C. parvum-infected SCIDbg mice (CP-PMN). However, all SCIDbgMN mice inoculated with CP-PMN alone or resident Mphi alone died after C. parvum infection. CP-Mphi were identified as classically activated Mphi (M1Mphi), and CP-PMN were characterized as PMN-I. In in vitro studies, resident Mphi converted to M1Mphi after transwell cultivation with CP-PMN. These results indicate that the resistance of SCIDbg mice early after C. parvum infection is displayed through the function of M1Mphi which are converted from resident Mphi influenced by CP-PMN (PMN-I).

Hymenolepis microstoma: direct life cycle in immunodeficient mice.

The mouse bile duct tapeworm Hymenolepis microstoma requires beetles as the obligatory intermediate host. However, when congenitally athymic NMRI-nu mice were infected with the mature tapeworm and allowed to eat their own faeces with tapeworm eggs, the oncospheres penetrated the intestinal tissue and developed to cysticercoids. After excysting, growth to adult worms occurs in the lumen of the small intestine and bile duct. Furthermore, the same happened when NMRI-nu mice, non-obese diabetic severe combined immunodeficiency (NOD/Shi-scid) mice and NOD/Shi-scid, IL-2 Rgamma(null) (NOG) mice were orally inoculated with shell-free eggs of this parasite. Differences between the cysticercoids of H. microstoma and H. nana developed in the mouse intestinal tissues were: (i) the time course for the development of fully matured cysticercoids of H. microstoma in mice was about 11 days but only 4 days for H. nana; and (ii) cysticercoids of H. microstoma developed in mice had a tail while those of H. nana had none.

NOD-scid mouse as an experimental animal model for cysticercosis.

The major three species of human taeniid cestodes, Taenia solium, T. saginata and T. saginata asiatica (= T. asiatica) which require humans as the definitive host are still not rare in developing countries. Among these, T. solium is the most serious with medical and economic importance. Neurocysticercosis (NCC) in humans is now recognized as the major cause of neurologic disease in the world. As these human taeniid cestodes obligatory require domestic animals such as swine, cattle and swine as the major intermediate host animals respectively, it is not easy to analyze the basic research in these domestic animals. In this brief review, we introduce experimental animal model for these three species in order to obtain fully developed metacestode stage in severe combined immunodeficiency (scid) mice. Non-obese diabetic scid (NOD-scid) mice are expected to be a satisfactory animal model and to have advantages for analysis by several view points of developmental biology with gene expression throughout development, antigenic homology of cyst fluid of these three species, evaluation of drug efficacy or metacestocidal drug designs, confirmation of unknown taeniid gravid segments for identification based on the morphology and DNA analysis of metacestodes. The animal model is not only available for human Taenia spp but can also be applied to other taeniid cestodes of economic importance or in veterinary parasitology.

SCID mice as a tool for evaluation of heteroxenous life cycle pattern of Caryospora (Apicomplexa, Eimeriidae) species.

Adult severe combined immunodeficient (SCID) mice were inoculated with oocysts of 13 different Caryospora (Protozoa, Apicomplexa) species isolated from the faeces of 10 reptilian and three raptorial bird hosts in attempt to test heteroxenous life cycle pattern. Only three reptilian isolates originated from viperid snakes, namely from Calloselasma rhodostoma, Atheris nitschei and Vipera ursinii induced lethal dermal caryosporosis in SCID mice. Neither clinical signs nor developmental stages were observed in mice infected with further nine caryosporan isolates originated from other reptilian and raptorial bird hosts. Results of this study confirmed that SCID mice represent a useful tool for evaluation of heteroxenous life cycle pattern of caryosporan coccidia and that only the Caryospora species from viperid and crotalid snakes produce dermal caryosporosis in mice

Sexual development of Taenia solium in hamsters from rodent-derived cysticerci.

In order to determine whether Taenia solium can be maintained in the laboratory using rodents as definitive hosts, six nude rats, 20 immunosuppressed Mongolian gerbils and 20 immunosuppressed Syrian hamsters were each inoculated through a stomach tube with three cysticerci recovered from SCID mice. No adult worms of T. solium were found in the intestinal tract of any of these 46 rodents. In addition, five immunosuppressed Syrian hamsters were fed with the same number of cysticerci enclosed in rodent muscles from SCID mice. Two of these hamsters were found to be infected 40 days post-infection, each harbouring a sexually developed worm in the intestinal tract. Although no eggs were produced, prepatent infections may be possible if a longer time was allowed for worm development. Moreover, the maintenance of the life cycle of T. solium in the laboratory using the rodent model can be established.

Susceptibility of severe combined immuno-deficient (SCID) mice to Trypanosoma brucei gambiense and T. b. rhodesiense.

Susceptibility of severe combined immunodeficient (SCID) mice to 7 isolates of Trypanosoma brucei gambiense and 2 isolates of T. b. rhodesiense was examined in terms of their infectivity, course of parasitaemia, packed cell volume (PCV) and survival period in comparison with that of normal immunocompetent (BALB/c) mice. All isolates of T. b. gambiense and T. b. rhodesiense caused high (> 1 x 10(8) parasites/ml) parasitaemia in the SCID mice, the survival periods ranged from 5 to 47 days. On the other hand, 5 of 7 isolates of T. b. gambiense developed chronic infection in the BALB/c mice with sporadic but persistent parasitaemia with less than 5 x 10(6) parasites/ml. All the mice tested in this group survived more than 60 days after infection. In contrast, the 2 remaining isolates of T. b. gambiense and both isolates of T. b. rhodesiense showed high virulence in the BALB/c mice and killed all of them within 30 days after infection. The results demonstrate that the SCID mice, in which functional B- and T-cell-mediated immunities are congenitally lacking, are highly susceptible for 'low-virulence' T. b. gambiense. This makes SCID mice useful tools for the isolation of parasites from T. b. gambiense sleeping sickness patients and the propagation of large amounts of such parasites.

Metastasis of Theileria annulata macroschizont-infected cells in scid mice is mediated by matrix metalloproteinases.

Theileria annulata (Ta)-infected leucocytes are able to disseminate in scid mice. The dose of virulent parasites of the Ta-Ode line required to achieve quantifiable dissemination was found to be 2 x 10(6) cells given i.p. Dissemination was higher on day 11 post-inoculation than on day 18. The attenuated Ta-Ode cells were found to disseminate very poorly compared to their virulent progenitors, which correlates with a marked reduction in matrix metalloproteinase (MMP) expression. A daily i.p. injection of mice with BB94, a synthetic inhibitor of MMPs, almost completely ablated dissemination compared to controls. This provides strong evidence that metastasis of Theileria annulata macroschizont-infected host cells is mediated by host MMPs induced by the parasite. This has important implications for explaining a number of pathological features of tropical theileriosis in cattle.

In vitro hatched oncospheres of Asian Taenia from Korea and Taiwan develop into cysticerci in the peritoneal cavity of female scid (severe combined immunodeficiency) mice.

In vitro hatched (but not activated) oncospheres of Asian Taenia obtained in Korea and Taiwan, prepared by the sodium hypochlorite method, rinsed with sterile PBS several times and adjusted to 5 x 10(4)/0.5 ml PBS, were injected intraperitoneally or subcutaneously into male or female scid mice of 3 different strains. When these scid mice were sacrificed 4 months later, the females harboured fully developed cysticerci either in the peritoneal cavity or under the back skin, whereas males did not. All cysticerci from the peritoneal cavity were easily recovered by rinsing the abdomen with PBS. Although most cysticerci recovered from pig liver usually become calcified within 1-2 months, in female scid mice they all increased in size and were viable. Intraperitoneal (i.p.) injection of in vitro hatched oncospheres is recommended for easier recovery of Asian Taenia metacestodes in laboratory animals.

Human Taenia eggs develop into cysticerci in scid mice.

The intermediate hosts for Taenia saginata and T. solium are cattle and pigs (and humans for the latter), respectively. In vitro-hatched (but not activated) oncospheres of both Asian Taenia (T. saginata asiatica, a new subspecies of T. saginata or T. asiatica, a new species) and T. solium injected subcutaneously into the backs of mice with severe combined immunodeficiency (scid) developed into fully matured cysticerci. Five-month-old metacestodes of Asian Taenia had no hooklets and were bigger in size than those previously reported and similar to those of T. saginata. Their morphology suggested that the cysticerci were more advanced than those in the intermediate host animals. It is suggested that scid mice are valuable experimental animal models for studying human taeniid cestode infections.

Features of Schistosoma mansoni infection in SCID mice.

Features of Schistosoma mansoni infection in SCID mice, which lack functional T- and B-lymphocytes, were investigated. The retarded development of parasites as well as reduction of liver egg recovery in SCID mice was significantly lower than those in congenic counterpart C.B-17 mice. Furthermore, the rate of parasite recovery from SCID mice with primary infection was always lower than that from C.B-17 mice by 20%, showing the innate resistance to S. mansoni infection. SCID mice vaccinated with UV-attenuated S. mansoni cercariae did not show protective immunity against a homologous challenge infection. The present innate resistance exhibited in SCID mice is discussed in relation to cell mediated immunity of macrophage activation by IFN-gamma which would not involve T-lymphocytes but is initiated by IL-12 and TNF-alpha cytokines. SCID mice may provide novel information on the host-parasite relationship in schistosome infections.

Parenteral strobilar development of Echinococcus multilocularis in scid mice.

Parenteral strobilation of Echinococcus multilocularis was observed in acid (severe combined immuno-deficient) mice after intracerebral, subcutaneous and intraperitoneal inoculation with protoscoleces. Evaginated protoscoleces and segmented worms were obtained at the inoculated sites. Most worms recovered from peritoneal cavity of scid mice were encapsulated by connective tissue and granulocytes but showed a maximum of 3 proglottids, elongation of genital primordia and vesiculation. Viability of worms recovered from the subcutaneous tissue and peritoneal cavity of scid mice were higher (69.1-91.4%) than those from the immunologically normal C.B-17 (4.0-48.0%) control mice. However, viabilities of worms from the cerebrum of both scid and C.B-17 were almost the same (87.7-94.4%). Worms recovered from scid mice showed further development of reproductive organs when transplanted into the small intestine of prednisolone treated golden hamster. These findings suggest that the parenteral milieu of scid mice allows adult development of E. multilocularis protoscoleces.

In vitro excystation of Spironucleus muris.

In vitro excystation of Spironucleus muris cysts, purified by sequential sucrose and Percoll gradients from mouse feces, was studied. Three in vitro excystation procedures, used for Giardia, were assessed to determine the most useful method. Excystation was monitored by light microscopy and subsequently characterized by transmission and scanning electron microscopy. Spironucleus muris excysted routinely at a level greater than 90% when induced in Hanks' balanced salt solution containing sodium bicarbonate at pH 2.0 and transferred to Tyrodes' salt solution as an excystation medium. Similarly, high rates of excystation were recorded after induction of S. muris cysts in 0.1 M potassium phosphate buffer (pH 7.0) with sodium bicarbonate and excystation in trypticase-yeast extract-iron medium (TYI medium) or phosphate-buffered saline. A lower rate and percentage of excystation were observed after induction of S. muris cysts in an aqueous hydrochloric acid solution (pH 2.0) followed by excystation in TYI medium. All excystation methods produced extremely active S. muris trophozoites with normal morphology. Nonexcysting S. muris cysts have a wall composed of an outer fibrous and an inner membranous portion. Following induction, numerous vesicles appeared in the peritrophic space. Excystation began by the cyst wall opening at one pole, and the anterior part of the trophozoite protruding from the cyst wall. The trophozoite emerged progressively from the cyst wall and the empty cyst wall appeared to collapse. Excysted trophozoites exhibited normal morphological features of S. muris trophozoites isolated from the mouse intestine.

Use of the Bo-RBC-SCID mouse model for isolation of a Babesia parasite from grazing calves in Japan.

SCID mice with circulating bovine red blood cells (Bo-RBC-SCID mice) were used to isolate Babesia parasites from grazing calves in Japan. Although the initial blood samples obtained from the calves contained both Babesia and Theileria parasites, we were able to isolate only the Babesia parasites by repeated blood passages in Bo-RBC-SCID mice, taking advantage of the much more rapid growth of Babesia than of Theileria. The parasites isolated had characteristics of large-type Babesia, showing typical paired pyriform morphology. The Bo-RBC-SCID mice infected with the Babesia parasites developed a high level of parasitemia, showing severe clinical symptoms characterized by hemoglobinuria, jaundice, and hemolytic anemia. In addition, some mice exhibited nervous symptoms, particularly paralysis of both posterior limbs. The results demonstrated that the Bo-RBC-SCID mouse model was useful not only for isolating Babesia from cattle but also for studying the disease caused by the Babesia infection.

In vivo sequestration of Plasmodium falciparum-infected human erythrocytes: a severe combined immunodeficiency mouse model for cerebral malaria.

Cerebral malaria is a fatal complication of infection by Plasmodium falciparum in man. The neurological symptoms that characterize this form of malarial disease are accompanied by the adhesion of infected erythrocytes to the vasculature of the brain. To study this phenomenon in vivo, an acute phase severe combined immunodeficiency (SCID) mouse model was developed in which sequestration of P. falciparum-infected human erythrocytes took place. During acute cerebral malaria in humans, the expression of intercellular adhesion molecule-1 (ICAM-1) is induced in vascular endothelium by inflammatory reactions. Acute phase ICAM-1 expression can also be obtained in SCID mice. The endothelium of the midbrain region was the most responsive to such inflammatory stimulus. It is noteworthy that the reticular formation in the midbrain controls the level of consciousness, and loss of consciousness is a symptom of cerebral malaria. We found that infected human erythrocytes were retained 24 times more than normal erythrocytes in ICAM-1-positive mouse brain. Sequestration to the brain was reduced by anti-ICAM-1 antibodies. These in vivo results were confirmed by the binding of P. falciparum-infected erythrocytes to the ICAM-1-positive endothelium in tissue sections of mouse brain. We conclude that the SCID mouse serves as a versatile in vivo model that allows the study of P. falciparum-infected erythrocyte adhesion as it occurs in human cerebral malaria. Upregulation of ICAM-1 expression in the region of the midbrain correlates with increased retention of malaria-infected erythrocytes and with the symptoms of cerebral malaria.

Factors regulating natural transmission of Plasmodium berghei to the mosquito vector, and the cloning of a transmission-blocking immunogen.

Naturally occurring factors that regulate the infectivity of P. berghei infected rodent hosts to the mosquito vector in vivo have been compared in T.O., Balb/C and immunodeficient SCID mice. No detectable differences in infectivity were observed suggesting B and T cell mediated factors are not involved. Further studies investigated roles for macrophage colony stimulating factors, the cytokines IFN gamma and TNF alpha, of neutrophils, and of nitric oxide in the SCID mouse, but have failed to demonstrate an important role in vivo for any factor examined. Differences between these results and those obtained in vitro on the human and primate parasites must therefore be explained by biological differences between the parasite/host combinations, or by technical differences in experimental designs. Induced immunity to the ookinete surface antigen Pbs 21 of P. berghei can totally block the transmission of the parasite from the gametocyte infected host to the vector. We have cloned the gene encoding Pbs 21 and shown it bears striking structural similarities to Pfs 25, Pgs 25 and more particularly Pgs 28 in that it has a high cysteine content (9.5%), 4 EGF-like domains and hydrophobic amino-'signal'--and carboxyl-'anchor' sequences. The encoding gene is on chromosome 5 and is found also in P. chabaudi, P. yoelii and P. vinckei.

Spontaneous and experimental infections in scid and scid/beige mice.

Severe combined immunodeficient (scid) mice are valuable animals to study a variety of physiologic and disease processes. Their capacity to support multiple tissue xenografts permits these mice to be used as intermediate models for host-specific, fastidious organisms for which a small animal model has not been available previously. However, because they are unable to mount a normal immune response, they are very susceptible to a variety of primary and opportunistic microbial pathogens. Fatal, naturally occurring infections with bacteria such as Proteus mirabilis, Streptococcus viridans, and Escherichia coli have been observed. In addition, based on observations after experimental or naturally occurring viral infections, scid and scid/beige mice have been shown to be very susceptible to infections with viruses such as mouse hepatitis virus, Sendai virus, and murine respiratory virus, with resulting mortality. Of the parasitic infections, Pneumocystis carinii is a relatively common contaminant of the respiratory tracts of scid mice and may complicate research projects, particularly experimental respiratory tract infections. In view of the enhanced susceptibility of these mice to infections of this type, it is essential that they be housed under optimal conditions, which include implementing stringent management practices and a functional barrier system.

The Bo-RBC-SCID mouse model for evaluating the efficacy of anti-theilerial drugs.

We have previously developed a mouse model which allowed the proliferation of Theileria sergenti in severe combined immunodeficiency (SCID) mice with circulating bovine erythrocytes (Bo-RBC). In the present study, this model was utilized to test the efficacy of anti-theilerial drugs. Bo-RBC-SCID mice were created by giving periodic transfusions of T. sergenti-free Bo-RBC, and subsequently infecting with T. sergenti. Three anti-protozoal compounds, Pamaquine (Yamanouchi Pharmaceutical Co. Ltd), Ganaseg (Japan CIBA-GEIGY Ltd) and Buparvaquone (Coopers Animal Health Ltd), were subcutaneously administered into the mice at doses recommended for cattle therapy. Blood examinations demonstrated that all three drugs significantly reduced the level of parasitemia although Ganaseg was effective only at a dose five times higher than that recommended for cattle therapy. Administration of the drugs neither caused any sign of acute toxicity nor changed the rate of Bo-RBC in the SCID mice's circulating blood cells. The results indicate that the Bo-RBC-SCID mouse model may offer a useful in vivo system for evaluating the efficacy of anti-protozoal drugs against T. sergenti.