Infectivity of a novel type of Cryptosporidium andersoni to laboratory mice

Previously, we reported 'a novel type' of Cryptosporidium andersoni detected from cattle in Japan, and showed that the isolate was infective to mice. In the present study, we examined the patterns of oocyst shedding in both immunocompromised and immunocompetent mice, as well as pathological lesions in the infected mice. After oral inoculation with 1 x 10(6) oocysts, all five severe combined immunodeficiency (SCID) mice began to shed endogenously produced oocysts on day 6 post-inoculation (p.i.). The number of oocysts per day (OPD) reached 1 x 10(6) on day 17 p.i., and an OPD level of 1 x 10(6) to 10(7) was maintained until 91 days p.i. when the mice were sacrificed. In the five immunocompetent mice inoculated with 1 x 10(6) oocysts, the pre-patent and patent periods were 6 and 19 days, respectively, and the maximal OPD level was 1.5 x 10(5) on average. On histological examinations of infected SCID mice, a large number of parasites were present on the surface of the gastric glands of the stomach, but not in other organs examined. In conclusion, the novel type of C. andersoni, which genetically coincides with C. andersoni reported in other countries, is infective to mice, but susceptibility was lower than that of Cryptosporidium muris infecting rodents from the perspective of infectivity to immunocompetent mice.     

Relationship between type 1/type 2 immune responses and occurrence of vertical transmission in BALB/c mice infected with Neospora caninum

To examine the relationship between occurrence of vertical transmission and type 1/type 2 immune responses induced by Neospora caninum infection in BALB/c mice, pregnant (group 1 p) and non-pregnant mice (group 1 np) were inoculated with 2 x 10(6) of the N. caninum parasites and then we examined the vertical transmission rate and production of IFN-gamma and IL-4. We also studied chronically infected mice, which were bred at 4 weeks or more after infection (group 2), and mice inoculated during pregnancy and re-bred at 4 weeks or more after delivery (group 3). In groups 1p, 2 and 3, vertical transmission was observed in 27.4, 41.4, and 50% of the offspring, respectively. The serum IFN-gamma level increased on days 1 and 5 post-inoculation (p.i.) in groups 1 p and 1 np, while no increase level was observed in groups 2 and 3 during pregnancy or after delivery. When the mice in groups 2 and 3 were re-inoculated, all mice showed a transient increase in serum IFN-gamma on day 1 post-re-inoculation. The serum IL-4 level in both of groups 1p and np increased in a similar manner following infection. In group 3, the serum IL-4 level was somewhat higher than that in group 2 after re-inoculation. The anti-N. caninum antibody IgG1 titer in group 3 increased on day 10 post-re-inoculation. These results suggest that the mice infected during pregnancy may acquire a weaker immune response to the parasite than mice infected when they are not pregnant, and that mice infected during pregnancy may show an enhanced type 2 immune response in the recrudescence of the infection.     

Studies on the pathology of dicrocoeliasis and fascioliasis in the goat. II. The histochemistry of bile-duct mucosubstances

Histochemical studies were made on the characteristics of carbohydrate-rich compounds in the bile-duct mucosa of goats infected either with Dicrocoelium dendriticum or with Fasciola hepatica. In a previous study the author showed that a chronic infection with these parasites produces glandular hyperplasia in the walls of bile ducts. This study revealed a definitely increased secretion of qualitatively normal mucosubstances by the hyperplastic epithelial cells.Epithelial mucosubstances in the bile-duct walls were characterized by the presence of periodate-reactive hydroxy groups and both carboxyl and sulphate radicals. The intensity of the PAS reaction was moderate or strong and not definitely inhibited by the acidic or acetyl radicals. Specific enzymatic digestions did not reveal periodate-reactivity produced by glycogen or basophilia due to hyaluronic acid or chondroitin-4 and -6-sulphates in the epithelial cells. On the other hand, sialidase digestion showed that the main part of the carboxymucins was sialidase-labile sialomucins. Their susceptibility towards sialidase was not definitely increased after deacetylation. The single and sequential histochemical methods showed a more intense staining reaction for sulphate radicals in the mucosubstances of the deep glandular and goblet cells than in those of the superficial glands, which were characterized by high concentration of carboxyl groups.Keyword: Dicrocoelium dendriticum, Fasciola hepatica, liver, bile ducts, mucosubstances     

肠艾美耳球虫配子生殖与病理变化

用单个肠艾美耳球虫Eimeria intestimalis Cheissin 1984卵囊感染无球虫兔,获得纯种进行研究。1.肠艾美耳球虫的配子生殖阶段寄生于空肠和回肠,此时宿主组织有较严重病变。12指肠、结肠和盲肠未见虫体,但在感染后264小时见盲肠的个别绒毛内有1～3个配子体,可能属偶然现象。2.感染后180小时发现极少数早期配子体,感染后192小时出现少量配子体寄生在空肠和回肠的绒毛和腺上皮细胞内,感染后216至264小时,绒毛上皮和腺上皮细胞内多为配子体、合子和卵囊所取代。感染后216小时出现极少量卵囊,264小时则见有大量卵囊。3.感染开始时(感染后61～73小时),回肠、空肠绒毛上皮正常;腺上皮细胞出现少量滋养体和裂殖体。96至192小时后,肠绒毛上皮和腺上皮受侵害程度渐趋严重,肠绒毛变矮,绒毛上皮及腺上皮细胞肿大变空,细胞核消失。许多腺泡塌陷。感染后216～264小时,肠绒毛受侵害最为严重,空肠和回肠绒毛萎缩或消失,变为一层矮柱或立方形上皮细胞或全无上皮细胞覆盖的绒毛。固有层均质红染,或颗粒状。肠腺塌陷,数量减少,大小不一。腺腔内见有配子体、合子或卵囊残留,部分腺泡上皮细胞再生,细胞核增生成堆。12指肠、盲肠和结肠正常。     

Some studies on the growth and development of Echinococcus granulosus, camel origin in experimentally infected dogs.

The development of Echinococcus granulosus of camel subspecies in 35 experimentally infected dogs was studied at 3, 7, 10, 13, 18, 23, 35 and 56 days post-infection (d.p.i.). The morphological characteristics of each developmental stage is studied and discussed. Morphological characters included number, total length, shape, arrangement of rosteller hooks and blade length to total length percentage (B1/T1%). In addition, total worm length, scolex, suckers, rostellum, neck and segments were measured and described. Other useful characters are considered to be the position of common genital pore, number and distribution of testes, uterus, shape of ovary and vitelline glands. Generally, the results indicated that the segmentation first appears at 18 d.p.i. Moreover, male and female genitalia could be detected at the same period. However, completely developed genitalia appeared at 56 d.p.i. Histological examination of the small intestine of experimentally infected dogs revealed that the parasites were found in distended and thin wall crypts of Liberkuhn at the periods of 3.7 and 10 d.p.i, while the parasite scoleces were found embeded in the mucosa at the periods of 13, 18 and 23 d.p.i. No significant pathological changes were encountered in both infected and control dogs. The data presented here are the first report of strobilar development of E. granulosus of camel origin in experimentally infected dogs.     

Foot-and-mouth disease virus (FMDV) experimental infection: susceptibility and immune response of adult mice

Adult mice are susceptible to foot-and-mouth disease virus (FMDV) infection only under some experimental conditions. This paper report the results of pathogenesis studies on 4 different strains of mice (CF1, C3H, NIH-nude, BALB-c/J) infected with the cloned and uncloned 0(1)C strain of FMDV. High virus titers were detected in blood and pancreas 12-24 h after infection (p.i.); these persisted for up to 48 h p.i. in CF1 and BALB-c/J mice and 72 h p.i. in the two other mouse strains. Virus titers observed in other organs were lower than those found in blood. In pancreas, and occasionally in salivary glands, oropharynx, heart and testicles, viral antigen was detected by direct immunofluorescent assay. Circulating neutralizing antibodies appeared in CF1 and C3H mice at 72 and 96 h p.i. respectively, and their titers remained unchanged during the 30-day experimental period. Antibodies against viral infection-associated antigen (VIA) were detected for a shorter period. In animals irradiated with 1 LD 50 (total body irradiation), viremia persisted up to 14 days p.i. and a low antibody response was observed which began at the end of viremia. No differences in the response of mice to cloned or uncloned FMDV were observed.     

Effect of Fasciola gigantica infection on adrenal and thyroid glands of riverine buffaloes

Effect of Fasciola gigantica infection on adrenal and thyroid glands was investigated using eight male, yearling Murrah buffaloes. The animals were randomly assigned to two groups of four buffaloes each (Group-A, infected; Group-B, non-infected control). Animals of Group-A were orally infected with 1000 F. gigantica viable metacercariae, keeping other four animals of Group-B as uninfected control. In the infected buffaloes, the clinical signs began appearing from 7th week postinfection (p.i.) and eggs were detected in the faeces between day 93 and 99 (95.5+/-1.25) postinfection (p.i.). The serum cortisol level, revealed a significant (P<0.05) rise during initial stage of the infection, followed by a continuous fall from 12th week onward. Peak cortisol level on 10th week (13.30+/-2.57ngml(-1)) was associated with eosinophilia (11.0+/-0.95%). However, non-infected controls maintained almost uniform cortisol levels (3.97+/-0.15-5.88+/-0.09ngml(-1)) throughout the period of the study. The pathological changes of adrenal glands were correlated with physiological dysfunction of the glands. The levels of T(3) and T(4) were significantly (P<0.05-0.01) low from 14th week onward and were synchronous with in situ migration, growth and development of F. gigantica. Significant reduction in the thyroid hormones was further supported by histopathological evidence of lymphocytic thyroiditis confirming hypothyroidism. A decrease in Hb, PCV, total erythrocyte counts and appearance of reticulocytes in the blood of the infected buffaloes suggested regenerative anemia, which could partly be due to hypothyroidism.     

Diagnosis and manifestation of encephalitozoonosis in mice after experimental infection with different species and application of dexamethasone

Twenty-eight BALB/c mice were infected with different strains of Encephalitozoon species (Encephalitozoon cuniculi II - mouse type, E. cuniculi III - dog type, Encephalitozoon hellem, Encephalitozoon intestinalis). Five of them were infected with E. cuniculi II (mouse type) and simultaneously immunosuppressed with dexamethasone. Clinical signs of encephalitozoonosis were not remarkable. Ascites was found in two mice of dexamethasone-treated group 14 days post-infection (p.i.). The histopathological changes were found mainly in spleen and liver in the form of lymphoepithelioid granuloma. Spores were found in faeces since day 14 p.i. and visualized by Calcoflour White M2R. After cultivation on cellular cultures (VERO E6 - monkey kidney cells, RK-13 - rabbit kidney fibroblasts), the species differentiation was performed by PCR using panmicrosporidial primers (PMP1, PMP2) and specific primers (ECUN-F, ECUN-R, V1, SI-500). The differences were recorded in the immune response of immunocompetent and immunosuppressed mice. At day 60 p.i., the titres of specific antibodies measured by indirect immunofluorescence antibody test were lower (1:4096) in dexamethasone-treated mice when compared with non-immunosuppressed animals (1:8196). The significant increases of antibody titres were recorded in particular infected groups within the experiment (P < 0.01 between day 14 p.i. and day 30 p.i., P < 0.001 between day 14 p.i. and day 60 p.i.). Experimental encephalitozoonosis in non-immunosuppressed and immunosuppressed mice provides a useful model for the study of immune response and lesions associated with these protozoans.     

A pregnant mouse model for bovine Tritrichomonas foetus infection

The economically important effects of Tritrichomonas foetus infection in cattle are abortion and infertility, yet there has not been an animal model to examine the parasite-host interactions during gestation. In this study, 5- and 7- to 8-week-old BALB/cAnNCr, BALB/cJ, and SCID/NCr mice on a BALB/c background were intravaginally infected with T. foetus. All BALB/cAnNCr and BALB/cJ mice, and 89% of SCID/NCr mice sustained infections for 13 weeks, if inoculated before 5 weeks of age. Infection rates were lower in all mouse strains inoculated at 7 weeks of age, although BALB/cAnNCr mice were significantly more susceptible than BALB/cJ or SCID/NCr mice. Vaginal bacterial flora did not account for the variation in mouse-strain susceptibility, although coagulase-negative staphylococci in vaginal flora were associated with failure of T. foetus to infect. As with infected cattle, T. foetus-specific vaginal immunoglobulin (Ig) G and IgA antibodies were elevated after infection. The number and viability of day-10 fetuses were reduced in mice infected at 5 weeks of age and bred 12 weeks after infection. Lesions in pregnant and nonpregnant infected mice, including suppurative and eosinophilic vaginitis; cervicitis; endometritis with distension of the uterine lumen; endometrial ulceration; and glandular ectasia, with neutrophils in the glandular lumen and loss of gland epithelium, were similar to those in cattle. The decidua and placenta were multifocally necrotic. Immunohistochemistry demonstrated trichomonads in vaginal folds and uterine glands, and adjacent to fetal tissues. In summary, experimentally infected BALB/cAnNCr mice showed many pathologic similarities to cattle and may serve as a model to study host-trichomonad interactions.     

Indirect fluorescent antibody test for the detection of antibodies to Echinococcus granulosus in experimentally infected pups

Adult parasites of Echinococcus granulosus of buffalo origin were used as antigen in the indirect fluorescent antibody test (IFA) for detection of antibodies to E. granulosus in experimentally infected pups. The technique permitted detection of antibodies on Day 5 post-infection (p.i.) and up to Day 80 p.i. in infected animals. The antigen-antibody reaction was characterized by the appearance of a specific brilliant greenish yellow fluorescence on the embryophore of mature eggs present within and outside the gravid segment of the cestode. A maximum antibody titre of 1:320 at Day 50 p.i. was observed in the infected pups, coinciding with maturation of adult worms in the intestine of the host.     

Relationship between egg output and parasitic burden in lambs experimentally infected with different doses of Dicrocoelium dendriticum (Digenea)

The relationship between egg elimination and parasitic burden was studied in two groups of 12 lambs experimentally infected with 1000 and 3000 Dicrocoelium dendriticum metacercariae, respectively. Half the animals in each group were slaughtered 2 months post-infection (p.i.) and the other half 6 months p.i. In order to detect and follow elimination of D. dendriticum eggs by the lambs, faeces samples collection started one and a half months p.i. and continued fortnightly until the end of the experiment. Egg elimination was first detected between days 49 and 79 p.i. (mean = 59 +/- 1.6 SE). Mean eggs per gram (epg) was higher in the lambs infected with 3000 metacercariae (347.2 +/- 42.4 epg) than in those infected with 1000 (194.8 +/- 14.4), although no significant differences were detected between both groups using the Student 't' test. Egg elimination was higher in the faeces samples taken in the afternoon (mean = 357.8 +/- 47.6 epg) than in those from the morning (mean = 215.7 +/- 21.3). The percentage of metacercariae which became established as worms was higher in the animals dosed with 1000 metacercariae (21.6%) than in those infected with 3000 (16.3%). The number of worms recovered on necropsy of each animal varied between 30 and 2063 (mean = 346.6 +/- 80.5) and their length between 2.6 and 7.1 mm (mean = 5.2 +/- 0.1). The mean number of parasites for lambs infected with 3000 metacercariae (489.3 +/- 163.1) was higher than that obtained from those dosed with 1000 (215.7 +/- 41.4), although more worms were collected in some cases from the lambs infected with the latter dose than the former. In general there was an increase in the number of epg eliminated as days p.i. and parasitic burden increased. A positive relationship was observed via the correlation coefficient between the number of epg eliminated by each of the lambs throughout the experiment and that of worms recovered. This relationship was more intense on considering only the number of epg eliminated between days 120 and 180 p.i.     

Infectivity and pathogenicity of Cryptosporidium andersoni to a novel host, southern multimammate mouse (Mastomys coucha)

The infectivity and pathogenicity of Cryptosporidium andersoni (bovine isolate) for neonatal and adult southern multimammate mice (Mastomys coucha) was studied using transmission experiments. C. andersoni isolate used in this study was not infective for BALB/c mice, but experimental infection proved susceptibility of neonatal and adult M. coucha to the infection. The prepatent period was 20-24 days, the patent period varied between 46 and 59 days. No signs of clinical illness or macroscopic findings were detected in infected animals. Cryptosporidium developmental stages were detected only in the glandular part of the stomach of M. coucha in histological sections stained with Wolbach's modification of Giemsa and using immunofluorecence. Histopathological changes were characterized by dilatation and epithelial metaplasia of infected gastric glands without inflammatory response in the lamina propria. Neonatal M. coucha were more susceptible to C. andersoni infection than adults. M. coucha seems to be a useful laboratory model for study of C. andersoni infection.     

Possibility of Neospora caninum infection by venereal transmission in CB-17 scid mice

CB-17 scid and BALB/c male mice were inoculated intraperitoneally with Neospora caninum to examine the possibility of its venereal transmission. Some of these mice were killed on days 7 and 20 post-inoculation to examine the genital organs for presence of the parasite. The remaining scid male mice were housed with non-infected female mice from day 7 p.i. and kept with them for 14 days. These scid mice died between days 28 and 35 p.i. N. caninum DNA was detected in the testis of mice on days 7 and 20 p.i. by PCR and tachyzoite viability was determined by bioassay conducted by means of mouse inoculation. Microscopically, fewer tachyzoites were detected in the testis obtained on day 20 p.i., than in other organs. The inoculated BALB/c male mice survived until the end of the experiment with no clinical signs and N. caninum DNA was detected in the testis on day 7 p.i. but not on day 14 p.i. Five of eight female scid mice housed with infected males became pregnant. Tachyzoites were detected in three of these mice and their neonates (n=3, 5 and 13, respectively). In three non-pregnant mice, no parasite was detected. Two of the four female BALB/c mice housed with infected male scid mice became pregnant but the parasite was not detected in them or in the neonates (n=3 and 13, respectively). These results indicate that the tachyzoites were present in the genital organs of the immunodeficient mice from day 7 p.i. and suggest that transmission may occur through mating with male mice.     

Prevalence and pathogenicity of Cryptosporidium andersoni in one herd of beef cattle

Over a 35-week period from January to July 2002, a breed of Hereford beef cattle (H) and their hybrids were monitored. Five hundred and ninety-nine individual fecal samples from calves and 96 samples from their mothers were examined. First excretion of Cryptosporidium andersoni oocysts in calves was found in the 9th week after the start of calving (a calf 63-day old). The prevalence of C. andersoni in calves ranged from 11.1 to 92.9% depending on age. The mean prevalence in their mothers was found to be 43.8%. The size of oocysts was 8.48 +/- 0.78 x 6.41 +/- 0.59 microm. Infection intensity in calves ranged from 32 000 to 4 375 000 oocysts per gram (OPG) and in mothers from 78 000 to 2 552 000 OPG. Three cases of abomasal cryptosporidiosis slaughtered at the age of 81, 157 and 236 days were examined histologically and ultrastructurally [transmission electron microscopy (TEM) and scanning electron microscopy (SEM)]. Cryptosporidium infection of the abomasum was located in the upper half of the mucosal glands in the plicae spirales of the fundus, corpus and near the ostium omasoabomasicum. Cryptosporidia were not located in the glandular epithelium of the pars pylorica in the abomasum minimally 10 cm from pylorus. Histopathological changes in the site of cryptosporidial infection in the abomasum had a non-inflammatory character and included distinctive dilatation of infected parts of the glands with atrophy and metaplasia of the glandular epithelial cells, goblet cell activation and mucus hyperproduction. The TEM revealed a relatively small number of Cryptosporidium life cycle stages attached to glandular epithelial cells. In SEM the inner mucosal abomasal surface appeared swollen but was never infected by cryptosporidia.     

Adrenal and thyroid dysfunctions in experimental Trypanosoma congolense infection in cattle.

Severe pathological changes were observed in the adrenal and thyroid glands of Zebu (Bos indicus) heifers infected with Trypanosoma congolense. In the adrenal glands, severe inflammatory changes characterised by mononuclear cellular infiltration in the subcapsular areas, zona glomerulosa, zona fasciculata and zona reticularis were observed. In addition, there were hyperaemia, haemorrhage and hyperplasia resulting in increased adrenal weight in the two heifers slaughtered on Days 50 and 70 post-infection (p.i.). In the medullary areas of these two heifers, there was only mononuclear cellular infiltration. In the two heifers slaughtered on Day 113, localised mononuclear cellular infiltration was observed in addition to cortical cell depletion and fibrosis, resulting in reduced adrenal weight when compared with the controls. In the thyroid glands of the infected heifers, gross enlargement of the follicles filled with pale staining colloids were observed in association with squamous metaplasia of the follicular epithelium and fibroplasia. Serum cortisol concentrations showed an appreciable but non-significant increase (P less than 0.05) between Weeks 2 and 6 p.i. in the infected heifers (5.6 +/- 0.86 ng ml-1 vs. 4.4 +/- 0.34 ng ml-1) when compared with the uninfected controls or the pre-infection level. Thereafter, cortisol levels declined though non-significantly throughout the rest of the study. However, there was a steady increase towards normal levels from Week 12 p.i. until the end of the experiment. Changes in the basal serum concentrations of thyroxine (T4) were also measured weekly. There was a progressive decrease in the levels of T4 from the third until the 11th week p.i., when the decrease became significant (P less than 0.05) and remained so until the 16th week when the experiment ended.     

鼠类机会性寄生虫感染的实验研究

将住家附近诱捕到的74只黄胸鼠、43只褐家鼠和56只小家鼠随机分为实验Ⅱ组和对照组。实验Ⅰ组90只野鼠饮用1.0/L醋酸地塞米松水溶液；实验Ⅱ组66只野鼠给予皮下注射醋酸可的松；对照组不作任何处理。结果从实验Ⅰ组和实验II组的野鼠粪便和各种组织内共分离出5种机会性寄生虫，即微小隐孢子虫，结肠小袋纤毛虫，溶组织内阿米巴，弓形虫和卡氏肺孢子虫。然后将分离的虫体分别制成接种物，接种各种实验动物。接种后.经免疫处理的实验动物均出现典型的临床症状或死亡；未经免疫处理的实验动物则呈隐性感染。结果证明在机体免疫功能低下时.实验动物接种机会性病体后均能获得感染。提示我们，鼠类机会性寄生虫可能成为人体和其他动物感染的传染源，在公共卫生学上有重要意义。     

日本血吸虫童虫冷冻保藏复苏后生活史循环的研究

本文报告了日本血吸虫童虫经液氮冻藏复苏后的生活史循环的情况。童虫冻藏３６天和５０天后腹腔注射感染小鼠，亲代成虫回收率为９．０和８．９％。感染鼠肝内的子代虫卵孵出的子１代毛蚴感染钉螺，感染率为５９和６７％；平均每个钉螺第１次逸出的子１代尾蚴数为８００～９００条。子１代尾蚴感染小鼠后，其子１代成虫回收率为５５和５６％。合抱雌、雄成虫的体长、性腺的发育和虫体对宿主的致病力与正常途径感染者无明显差异。     

Ultrastructure and frequency of mastitis caused by ovine progressive pneumonia virus infection in sheep

Twenty-five sheep, experimentally (n = 15) or naturally (n = 6) infected with ovine progressive pneumonia virus and noninfected controls (n = 4), were evaluated for histological and ultrastructural lesions of mastitis. Histologically, nine of 15 experimentally infected sheep and all six naturally infected sheep had lympho-plasmacytic mastitis. Severity of the lesion increased with length of time after infection. Periductal lymphatic nodules were seen in five sheep experimentally infected for 2.8 years or longer and in five naturally infected sheep that were 3.7 years old or older. Ultrastructurally, responses to ovine progressive pneumonia virus were diffuse lympho-plasmacytic infiltrates in glandular interstitium, lymphocytic and occasional plasmacytic infiltrates in ductal walls and lumens, lymphoblasts surrounded by small lymphocytes in glandular interstitium, and degeneration of epithelium releasing cells and cellular debris into the lumen. Based on the prevalence of lesions, the mammary tissue was more susceptible to ovine progressive pneumonia virus than other target organs: lung, brain, and synovium. Lesions did not differ between breeds of sheep. Ovine progressive pneumonia virus was not seen in the mammary tissue but was isolated from 15 of 17 mammary glands.     

Global metabolomic analysis of blood from mice infected with Brucella abortus

To better understanding Brucella abortus infection, serum metabolites of B. abortus-infected and -uninfected mice were analyzed and twenty-one metabolites were tentatively identified at 3 and 14 days post-infection (d.p.i.). Level of most lysophosphatidylcholines (LPCs) was found to increase in infected mice at 3 d.p.i., while it was decreased at 14 d.p.i. as compared to uninfected mice. In contrast, acylcarnitines were initially reduced at 3 d.p.i then elevated after two-weeks of infection, while hydroxysanthine was increased at 14 d.p.i. in infected mice. Our findings suggest that the significant changes in LPCs and other identified metabolites may serve as potential biomarkers in acute phase of B. abortus infection.