Gastric amebiasis due to Entamoeba histolytica in a Dama wallaby (Macropus eugenii)

A 1.5-year-old captive female Dama wallaby (Macropus eugenii) died after a 3-month period of progressive weight loss, anorexia, bloat, and diarrhea. Histopathologic examination revealed numerous Entamoeba histolytica trophozoites within the gastric mucosa and, less frequently, gastric submucosa and submucosal vessels. Immunofluorescent antibody testing confirmed the identity of the trophozoites as E. histolytica. The trophozoites were associated with mild glandular epithelial necrosis, mucosal erosions, and lymphoplasmacytic inflammation. E. histolytica most commonly causes necrotizing and ulcerative colitis in humans and captive nonhuman primates, and it causes necrotizing and ulcerative gastritis in nonhuman primates with sacculated stomachs adapted for leaf fermentation. Rare cases of gastric amebiasis also have been been reported in captive macropods, which also have complex sacculated stomachs. To our knowledge, this is the first report confirming E. histolytica as the cause of gastric amebiasis in a wallaby. The zoonotic potential of this infection in macropods is uncertain.     

Porcine colon explants in the study of innate immune response to Entamoeba histolytica

Human amebiasis is caused by the protozoan Entamoeba histolytica. This protozoan is responsible for muco-hemorrhagic diarrhoea and liver abscess in affected populations. E. histolytica can be asymptomatic commensally confined to the intestinal lumen or can result in invasion of the colonic mucosa leading to ulceration and/or liver abscesses. Recently, human colonic explants have been identified as valuable in the study of host-parasite interactions. Here we investigated the potential of porcine colonic explants as an alternative to human tissues which are far less available. Porcine colonic explants were cultured with two strains of E. histolytica, one virulent (HM1:IMSS) and one avirulent (Rahman). Results from histopathological and real-time PCR analysis showed that porcine explants cultured with virulent ameba trophozoites react similarly to their human counterparts with an invasion of the tissue by the trophozoites and the triggering of typical innate immune response against the parasite. On the contrary, explants cultured with avirulent ameba trophozoites were preserved. The study open the way to the use of porcine colonic explants in the study of the complex interactions between the parasite and the host.     

凹甲陆龟溶组织内阿米巴原虫病的病理组织学观察

对患溶组织内阿米巴原虫病而死亡的凹甲陆龟进行了系统的病理组织学观察，该病的病变主要发生在消化道、肝、心脏、肾、脾等部位，尤以胃肠道和肝病变最为明显，表现为卡他性一坏死性胃肠炎和坏死性一肉芽肿性肝炎，胃肠黏膜出血、水肿、上皮细胞变性、坏死脱落，形成溃疡灶；肝细胞水泡变性，细胞核裂解，肝小叶内可见大小不等的结节性坏死灶，并伴有明显的炎症反应。     

Survival and morphologic changes of entodiniomorphid ciliate Troglodytella abrassarti in chimpanzee feces

Entodiniomorphid ciliates occur in the hindgut of both captive and wild African great apes. These ciliates do not form cysts, and therefore they are more susceptible for degradation. This present study focused on the survival, quantification, and decomposition processes of Troglodytella abrassarti trophozoites in the feces of captive chimpanzees. Fecal samples were examined using wet mounts and the merthiolate-iodine-formaldehyde concentration method, and the number of ciliates was expressed as ciliates per gram, which did not differ when examined from three different samples of the same feces. Trophozoites of T. abrassarti survived 5-15 hr after defecation at 25 degrees C under aerobic conditions. Decomposition of trophozoites began immediately after defecation; however, most of the trophozoites had a compact shape and visible cilia. Trophozoites, although without cilia, can be detected in the feces 55-65 hr after defecation, although most of the trophozoites were fragmented. The total number of ciliates in the sample started to decrease 35-55 hr after defecation. The absence of entodiniomorphid ciliates in fecal samples could not be caused by delayed feces fixation; instead, the absence was due to low sensitivity of coproscopic techniques. However, because of quick morphologic changes of trophozoites, accurate identification of ciliates in older samples may be difficult or even impossible.     

Epizootic of fatal amebiasis among exhibited snakes: epidemiologic, pathologic, and chemotherapeutic considerations.

An epizootic of reptilian amebiasis seems to have caused the death of 15 to 16 large and valuable captive snakes (boas, pythons, and anacondas) occupying one of 5 large display dioramas in the Steinhart Aquarium of the California Academy of Science, Golden Gate Park, San Francisco. Subsequent review of previous snake deaths in the colony indicated that of 464 snakes that had died since early 1969, 89 snakes had intestinal or hepatic lesions, and 80 of these snakes had pathologic features which involved severe intestinal ulceration, hemorrhage, and massive enteritis, with or without hepatic necrosis and destruction, condition compatible with Entamoeba invadens infection. The present epizootic began in November, 1972, with the death by acute enteritis of a red-tailed boa constrictor (Boa constrictor amarali) and was followed by the loss of 15 other large boids and pythonids. The affected snakes became immobile, refused to feed, and began to die 10 weeks after the death of the red-tailed boa. Seven boa constrictors, 4 pythons, and 4 anacondas from the same diorama died during the ensuing 10 weeks. Entamoeba invadens trophozoites were identified in the stool of the remaining living snake, a 3-m boa constrictor, and in the liver and the intestinal tissue of 1 of the dead boas examined microscopically. The parasite was also found in the stool of a giant Burmese python (Python molurus bivittatus) that died in the adjacent diorama and in the tissues of a blue-tongued skink (Tiliqua scincoides), separately housed, that died of enteritis during this period. Amebic cysts were recovered from turtle and alligator fecal samples taken from a central "swamp," or reservoir, draining the dioramas, water that is returned to the snake display areas after passage through a biological sand-gravel filter and ultraviolet radiation exposure. Cultures from these stools were positive and proved lethal to an experimentally infected boa constrictor. Treatment of the surviving snake in the affected diorama with metronidazole at the dose rate of 275 mg/kg proved rapidly effective; toxicosis was not observed. Other snakes and lizards suspected of having the infection were similarly treated and returned to normal behavior and feeding patterns. Epidemiologic considerations review the probable mode of introduction and spread of this highly lethal snake pathogen and recommendations are made for avoiding infection, prophylactic treatment, and handling of similar epizootics when they do occur among captive reptiles in aquariums, zoos, and research laboratories.     

Cryptosporidium muris-like infection in stomach of cynomolgus monkeys (Macaca fascicularis)

Abstract. Protozoa were present in routine sections of the gastric fundus of 15 cynomolgus monkeys (Macaca fascicularis) that were being studied in three toxicity studies with novel immunosuppressive agents. Upon detailed light microscopic and ultrastructural evaluation, all stages of parasite development (trophozoites, schizonts, gamonts, and oocysts) were seen and they structurally resembled Cryptosporidium muris, which normally is found in stomachs of rodents. Cryptosporidia were primarily present in the upper one third of fundic glands that were often concurrently colonized by a Helicobacter heilmannii-like organism; however, no clear correlation was found between bacterial burden and the number of protozoa. The primarily mononuclear cellular infiltrate appeared to coincide with the presence of protozoa only in a few animals. Changes in mucous epithelial cells mainly occurred in animals that were part of a 39-week study. Mucous epithelial cells in affected glands contained an increased amount of mucus composed of predominantly acid mucosubstances compared to the normally present neutral mucosubstances. C. muris-like protozoa are newly recognized etiologies for opportunistic infections in the stomach of immunocompromized nonhuman primates. This is the first report of C. muris-like parasite in stomachs of monkeys.     

Pentatrichomonas hominis infection in four kittens

Four purebred domestic cats examined because of diarrhea were found to have Pentatrichomonas hominis, a rarely reported trichomonad parasite, in their feces. Treatment with a combination of metronidazole and enrofloxacin tended to improve consistency of the feces, whereas treatment with metronidazole alone reduced the number of P hominis trophozoites in fecal smears but did not necessarily result in an improvement in clinical signs. Two cats were euthanatized. Necropsy revealed lymphoplasmacytic enterocolitis with eosinophils and eosinophilic globular leukocytes, neutrophils in the mucosa of the colon and within intraluminal contents of the cecum, and P hominis trophozoites in intraluminal contents of the colon and cecum.     

Evidence of hepatitis A infection in immature rhesus monkeys

Forty immature (less than 2 years old) rhesus monkeys (Macaca mulatta) with marked increases in aspartate and alanine aminotransferase activities were examined. Serological and histopathological evaluations were done to determine if affected animals were infected with hepatitis A virus. Although no clinical signs of illness were noted in any of the monkeys, an excellent correlation was found between the increased serum aminotransferase values and seropositivity with the acute phase (IgM) HAVAB-M antibody. Histopathological evaluations of livers of selected animals revealed hepatic lesions consistent with those in chimpanzees and marmosets infected with hepatitis A virus: generalized activation of sinusoidal lining cells, focal hepatocellular necrosis with occasional acidophilic bodies, and cuffs of mononuclear cells in the portal areas. Although no animals were seropositive for HAVAB upon receipt from the breeding colony, a total of ten of 18 animals for which serological data were available had seroconverted to HAVAB positivity during the 4-month observation period. These results are consistent with hepatitis A infection in immature rhesus monkeys and indicate the potential significance of serological testing in animals in which hepatic function is being evaluated.     

Effect of a Large Dose of Di (2-ethylhexyl) phthalate (DEHP) on Hepatic Peroxisome in Cynomolgus Monkeys (Macaca Fascicularis)

To elucidate the effect of a large dose of di (2-ethylhexyl) phthalate (DEHP), a plasticizer and peroxisome proliferator-activated receptor-α (PPARα) agonist, on hepatic peroxisomes, we orally administered 1,000 mg/kg/day, once daily, to 3 male and 4 female cynomolgus monkeys for 28 days consecutively. Light-microscopic and electron microscopic examinations of the liver were carried out in conjunction with measurement of the hepatic fatty acid β-oxidation system (FAOS), carnitine acetyltransferase (CAT) and carnitine palmitoyltransferase (CPT) activities, which are peroxisomal and/or mitochondrial enzyme activities. Electron microscopically, enlargement of the mitochondria was observed with lamellar orientation of the cristae along the major axis. Although the number of peroxisomes showed a tendency to increase when compared with those in a biopsied specimen before treatment, no abnormality in morphology was observed. A slight increase in CPT activity was noted at termination. No changes were noted in hepatic FAOS or CAT activity. In conclusion, although repeated oral treatment of cynomolgus monkeys with a large dose of DEHP induced a subtle increase in the numbers of peroxisomes with slight enlargements of the mitochondria, this low-sensitivity response to peroxisome proliferators in cynomolgus monkeys was considered to be closer to the response in humans than that in rodents.     

An outbreak of melioidosis in imported primates in Britain.

An outbreak of melioidosis, a bacterial infection caused by Pseudomonas pseudomallei, was identified in a batch of feral cynomolgus monkeys (Macaca fascicularis) imported to Britain from the Philippines. Thirteen confirmed or possible cases occurred among a batch of 50 animals. Subsequent investigations revealed that the infection was uncommon among imported primates from a variety of sources, although three other cases were identified in monkeys imported from Indonesia. The majority of the affected monkeys had splenic abscesses, and hepatic abscesses and infections of the soft tissues and skin were also frequently observed. Most of the infected animals had no clinical signs despite extensive abscesses, and the presence of infection was only suspected when they were shown to have serum antibodies to P pseudomallei by an enzyme-linked immunosorbent assay. Although there was no evidence of cross infection of other animals or human handlers, this outbreak is a reminder of the dangers of working with wild-caught primates and the potential for the establishment of environmental foci of melioidosis.     

Epidemiology of hepatitis E virus in indoor-captive cynomolgus monkey colony

A serological survey of hepatitis E virus (HEV) antibody was conducted using 202 adult captive cynomolgus monkeys, who did not show any clinical signs of acute hepatitis. Out of these, 44 monkeys were sero-positive for anti-HEV IgG and all monkeys were negative for anti-HEV IgM. All positive monkeys came from either Vietnam or China, but none from the Philippines, Indonesia, or our facility. Selected 12 monkeys out of positive monkeys from Vietnam, including 9 positive and 3 negative, revealed mostly within the reference ranges for alanine aminotransferase (ALT) and asparatate aminotransferase (AST) by serum biochemistries. Their titers of anti-HEV IgG did not correlate with the concentrations of ALT and AST. Moreover, HEV-RNA could not be detected from any fecal specimens of the 12 monkeys. Thus, monkeys with anti-HEV IgG sero-positive did not seem to be source of the HEV-pollution, because 1) sero-positive monkeys did not excrete HEV-RNA from their feces, and 2) monkeys from the Philippines and Indonesia have remained to be sero-negative for anti-HEV IgG, even if the monkeys were kept in same animal room of our facility. From these results, it could be inferred that primary infection of HEV occurred in the exported countries, but not in our colony. The contamination of HEV in indoor-captive monkeys could be prevented by precise quarantine tests, including ELISA for detecting anti-HEV and RT-PCR for HEV RNA.     

地鼠肾细胞弓形虫培养的实验研究

本文报道用正常地鼠肾细胞进行弓形虫培养的实验研究。接种虫体２×１０￣６个时，第２天细胞出现病变，第４天及第６天虫体繁殖逐渐增多，第７天虫体比原增殖达２６倍，到第９天以后虫体增殖逐渐减少。我们还对不同稀释度（１：２０，１：４０，１：８０，１：１６０）的弓形虫抗原量接种细胞进行观察，结果显示接种１：２０抗原液的一组，第５天观察到细胞严重受损，而接种１：１６０抗原液的一组则细胞变化不明显，展示抗原的接种量与虫体增殖有关。本研粉虽然比小白鼠传代腹水培养工作量大，但可获得足够量的抗原，而且地鼠肾细胞培养，取材方便。此法既可作制备抗原和分离用，又可传代、保种，是一种较好的方法。     

Detection of Echinococcus multilocularis infection in a colony of cynomolgus monkeys (Macaca fascicularis) using serology and ultrasonography.

Five animals in a colony of cynomolgus monkeys (Macaca fascicularis) died or were euthanatized because of alveolar echinococcosis, during a period of 5 years. The remainder of the colony was screened for possible infection with Echinococcus multilocularis, using serology and ultrasonography. A total of 46 animals out of a group of 55 were examined. The presence of anti-Em2 antibodies analyzed with enzyme-linked immunosorbent assay was demonstrated in 3 monkeys. In 2 of these 3 monkeys, multilocular structures compatible with metacestodal cysts in the liver were identified, using ultrasonography. The presence of alveolar echinococcosis was subsequently confirmed at postmortem examination in 1 animal. The other animals are still alive. Two other monkeys were negative in the serological examination but had cystic structures in the liver, which were identified as bile duct cysts at postmortem examination in 1 animal. The other monkey is still alive. These findings suggest that serology for antibodies against the Em2 antigen may represent a useful method in identifying animals that might be infected with E. multilocularis and are therefore at risk of developing fatal alveolar echinococcosis.     

The binding and distribution of albendazole and its principal metabolites in Giardia duodenalis

Trophozoites of the protozoan parasite Giardia duodenalis were exposed to various albendazole concentrations for 4 h, washed, fixed and incubated with antibodies raised against albendazole and its two major metabolites albendazole sulphoxide and albendazole sulphone. Tubulin antibodies were also used. A peroxidase- or FITC-conjugated secondary antibody was used to detect the primary antibody with transmission electron microscopy or confocal laser scanning microscopy, respectively. Albendazole, a benzimidazole compound, was detected in the mid-dorsal region of trophozoites, albendazole sulphoxide in the posterior-dorsal region and albendazole sulphone in clusters above the median bodies. Tubulin was recognised in the ventral disk. This is the first indication that G. duodenalis may be capable of metabolising albendazole and the potential path of the metabolised drug traced within the trophozoite. Fluorescence measurements revealed that albendazole sulphoxide binding decreased and albendazole sulphone binding increased with exposure of the trophozoites to increasing albendazole concentration. This indicates that if albendazole was being metabolised by trophozoites, it occurred to a greater extent following exposure to higher albendazole concentrations.     

The effect of the host lymphocyte lysosomal system on the developing intracellular Theileria annulata sporozoites

The fate of developing intracellular Theileria annulata sporozoites within bovine peripheral blood lymphocytes (PBL) was investigated in vitro using acid phosphatase ultracytochemistry. The intracellular sporozoites, while they developed into trophozoites, fed, and transformed into schizonts, provoked intense host lysosomal activity as early as 30 min after they were mixed with lymphocytes. Although viable developing trophozoites failed to fuse with lysosomal vesicles, thereby avoiding enzymatic digestion, the dead parasites readily formed 'phagolysosomes', resulting in lysosomal degranulation and trophozoite digestion. Possible factors responsible for the observed lack of lysosomal fusion with viable trophozoites are discussed.     

Spontaneously occurring hepatocellular neoplasia in adolescent cynomolgus monkeys (Macaca fascicularis)

Spontaneous hepatic neoplasms were identified in two adolescent (<5 years of age) male cynomolgus monkeys (Macaca fascicularis). Monkey No. 1 had a solitary hepatocellular carcinoma (HCC). Monkey No. 2 had multiple discrete tumors consisting of several poorly circumscribed HCCs and a mixed hepatocholangiocellular carcinoma (MHC). Metastases were not evident in either monkey. Histochemical and immunohistochemical stains were used to assess phenotypic alterations in the tumors. Many or most neoplastic hepatocytes (NHs) of both monkeys stained positive for low-molecular-weight cytokeratin (LMWCK), cytokeratin (CK) 8, and CK 18. In monkey No. 1, small aggregates of NHs were positive for carcinoembryonic antigen (CEA), glutathione S-transferase-pi (GST), and alpha-fetoprotein (AFP), but NHs were uniformly negative for CK 7. NHs in monkey No. 2 were negative for CEA and AFP but were multifocally positive for GST and CK 7. Broad-spectrum cytokeratin (BSCK), high-molecular-weight cytokeratin (HMWCK), and CK 19 did not react with NHs of either animal. Neoplastic cells forming ductlike structures in the MHC of monkey No. 2 stained with LMWCK, CK 7, CK8, CK 18, BSCK, and GST but not with HMWCK or CK 19. Tumors in both monkeys had enhanced pericellular fibronectin staining. Nonneoplastic parenchyma of both monkeys contained multiple discrete foci of cellular alteration and scattered aggregates of hepatocytes with strong cytoplasmic staining for fibronectin. Staining patterns of these tumors demonstrate immunophenotypic heterogeneity of the neoplastic cells within individual tumors and variability among tumors. This information may serve as a useful reference for others encountering similar lesions in primates.     

Trichomonad gastritis in rhesus macaques (Macaca mulatta) infected with simian immunodeficiency virus

In a retrospective study, 51 cases of gastritis (14%) were identified from among 341 necropsies performed on simian immunodeficiency virus (SIV)-infected rhesus macaques (Macaca mulatta) at the New England Primate Research Center from 1993 to 2001. Protozoa were seen in the stomach of 13 monkeys (25%) with gastritis. Two histopathologic manifestations of gastritis were observed: seven cases of lymphoplasmacytic gastritis with trichomonad trophozoites within lumens of gastric glands and four cases of necrosuppurative gastritis containing intralesional periodic acid-Schiff-positive protozoa; two cases of gastritis had morphologic features of both types of gastritis. In instances of necrosuppurative and combined lymphoplasmacytic and necrosuppurative gastritis, protozoa were 4-35 microm in diameter and round to tear-shaped. Because of the unusual morphology of the protozoa in these latter cases, transmission electron microscopy and polymerase chain reaction (PCR) were used to further identify these organisms. The protozoa were definitively identified as Tritrichomonas in all cases on the basis of ultrastructural characteristics (flagella and undulating membranes) and amplification of a 347-bp product of the 5.8S ribosomal RNA gene of Tritrichomonas foetus, Tritrichomonas suis and Tritrichomonas mobilensis by PCR using DNA extracted from stomach tissue. On the basis of these observations, we conclude that Tritrichomonas can be a significant cofactor in the development of necrosuppurative gastritis in SIV-infected rhesus macaques.     

Relationship of hepatic lipidosis to health and performance in dairy cattle

In a field study of 80 cows in 9 dairy herds, serial liver biopsies were performed over the peripartum period to determine degree of hepatic lipidosis. Cattle were separated into categories of mild, moderate, and severe hepatic lipidosis on the basis of maximal amounts of hepatic triglyceride that accumulated during this period. Number of cattle with mild, moderate, and severe hepatic lipidosis were 52, 16, and 12, respectively. Cattle with severe hepatic lipidosis had greater concentrations of hepatic triglyceride before calving and after parturition, and greater serum nonesterified fatty acid concentrations and body condition loss after parturition than cattle with mild hepatic lipidosis. Rate of disease and culling and death rate because of disease were greater in cattle with severe hepatic lipidosis. Cattle with severe hepatic lipidosis had reproductive performance equal to clinically normal cattle; however, cattle with moderate hepatic lipidosis had increased days to conception, possibly related to greater milk production.     

An electron microscopic study of intra-erythrocytic stages of Babesia bovis in the brain capillaries of infected splenectomized calves.

Splenectomized vaccine donor calves undergoing primary reactions to Babesia bovis infections may develop cerebral babesiosis which leads to death if not treated in time. A brain biopsy was performed on an artificially-infected animal showing nervous symptoms and the tissue was immediately processed for electron microscopic examination. Virtually every erythrocyte in the brain capillaries sectioned was infected with B. bovis. Intra-erythrocytic merozoites, trophozoites and dividing trophozoites were indentified. Important features of the piriform merozoites included a reduced apical complex consisting of the anterior polar ring, microtubules, rhoptries and micronemes. Unidentified membrane-bound bodies, mostly spherical in shape, were observed anterior to the nucleus. The trophozoites showed very little structural differentiation and no food vacuoles or micropores could be detected. Each trophozoite produced 2 identical merozoites and the parent cell became totally incorporated in the daughter merozoites in the multiplication process. Projections were seen radiating from the surface of infected erythrocytes which appeared to adhere to other surfaces on contact. This probably resulted in the sludging of infected erythrocytes in the capillaries. The latter observations coincide with those described for Babesia argentina.     

神经退行性疾病转基因猴模型的构建及相关技术体系建立

非人灵长类动物因与人有最近亲缘关系而成为研究人类生命领域最高级模式动物。本研究采用慢病毒载体技术结合胚胎工程技术,拟建立一套高效简便转基因猴制作技术体系,建立小脑共济失调转基因猴模型,为该病研究提供最为理想动物模型,同时为建立其他疾病转基因猴模型提供技术路线。利用卵泡浆内单精子显微注射技术(Introeytoplasmiesperm injection,ICSI)技术总共构建88个体外受精胚胎,经过体外培养共计32枚发育到原核期并进行病毒注射后移入7只受体猴输卵管内。B超妊娠检测,怀孕3只,其中:1只未能发育到期中间流产,1只发育到期生1死胎,1只顺产生1只活试管猴,经过人工喂养奶粉后健康存活,但经检测转基因为阴性。本研究虽然未能通过病毒注射获得阳性转基因猴,但对食蟹猴(Macaca fascicularis)超数排卵及B超监测卵泡发育技术、卵母细胞回收及体外成熟培养技术、电刺激采精及精子获能技术、食蟹猴体外受精技术和胚胎体外培养技术以及胚胎移植等技术体系进行多次探索和优化,并成功建立相关技术平台,将为后续利用试管猴技术结合高效的TALEN和CRISPR/Cas9等基因编辑工具获得各种疾病模型奠定基础。