Biology of Isospora spp. from Humans, Nonhuman Primates, and ...

Biology of Isospora spp. from humans,nonhuman primates, and domestic animals.D S Lindsay, J P Dubey and B L BlagburnClin. Microbiol. Rev. 1997, 10(1):19.CONTENT ALERTSUpdated information and services can be found at:http://cmr.asm.org/content/10/1/19These include:Receive: RSS Feeds, eTOCs, free email alerts (when new articlescite this article), more»Downloaded from http://cmr.asm.org/on February 5, 2014 by guestInformation about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtmlTo subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/

20 LINDSAY ET AL. CLIN. MICROBIOL. REV.Isospora species can cause serious disease in humans andnursing pigs. Clinical disease is seldom seen in nonhumanprimates, dogs, or cats. Isospora species do not produce diseasein horses, domestic ruminants, rabbits, or domestic poultry,and reports of isosporan oocysts in the feces of these hostsprobably represent pseudoparasites that originated in feedcontaminated with wild-bird feces.TAXONOMIC PROBLEMSFIG. 1. Sporulated oocysts of I. belli. (a) Oocyst containing two sporocysts(arrows). Note the oocyst wall (open arrow), the sporozoites (S) in the sporocysts,and the nucleus of a sporozoite (arrowhead). (b) A Caryospora-like oocystof I. belli containing one sporocyst (arrow). Note the oocyst wall (open arrow), asporozoite (S), and sporocyst residuum (R). The oocysts are unstained. Magnification,1,900. Courtesy of Donald Duszynski, University of New Mexico.host feces. Most oocysts are excreted unsporulated and mustundergo a developmental period (sporulation) outside the hostbefore they are sporulated and become infectious. Sporulatedoocysts of Isospora species are characterized by having twosporocysts. Each sporocyst contains four sporozoites (Fig. 1a).The sporocyst may or may not have a Stieda body. A Stiedabody is a proteinaceous plug found at one end of the sporocyst.A sub-Stieda body may be present directly beneath the Stiedabody. Life cycle studies indicate that species of Isospora with aStieda body are generally monoxenous and confined to theintestines whereas species that lack a Stieda body often can useparatenic hosts, may have latent stages in the host, and may befacultatively heteroxenous. All important and valid species ofIsospora that infect humans, nonhuman primates, dogs, cats,and domesticated mammals lack a Stieda body in their sporocysts.Generic names of Levinia (34) and Cystoisospora (64)have been proposed for the Isospora species that utilizeparatenic hosts, but these generic names have not gained widespreadacceptance.About 248 species of Isospora had been described prior to1986 (95). Most of these species are known only from oocystsfound in the feces of the host animal. Until life cycle andcross-transmission studies are conducted to determine moreabout the biology of these species, the species validity of manyof these coccidians is questionable.The sporulated oocysts of Isospora species resemble thesporulated oocysts of the related genera, Toxoplasma, Hammondia,Besnoitia, Frenkelia, and Sarcocystis. This resemblanceled to much confusion during the period from the late 1800s tothe mid-1970s, when the life cycle of these parasites was notknown. We will consider the two most notable examples inwhich these problems cause confusion.Isospora hominisHuman isosporiasis is caused by Isospora belli, which is a truemember of the genus Isospora (187). Many early reports ofhuman coccidiosis refer to infection with a parasite describedas Isospora hominis. I. hominis is actually a species of Sarcocystis,and the name is a synonym for Sarcocystis hominis or S.suihominis, species acquired by ingesting rare or raw infectedbeef or pork, respectively. There is no structural means ofdifferentiating these two species of Sarcocystis in human fecalsamples or in intestinal biopsy specimens. Intestinal sarcocystosisin humans can be a serious disease (18), unlike in otheranimals, which normally show no clinical signs. In many earlyreports, it is impossible to determine whether the authors aredescribing I. belli or a Sarcocystis species. An example of thisconfusion can be found in the pioneering work on coccidiosis,Coccidia and Coccidiosis of Domesticated, Game and LaboratoryAnimals and of Man, by E. R. Becker, published in 1934(7). Becker includes line drawings that demonstrate sporulationof I. belli but refers to the parasite as I. hominis. The onlyway one can be certain if early authors are describing I. belli orI. hominis (Sarcocystis) is to examine the drawings or photomicrographsif present. If none are included, a definitive identificationmay not be possible.Isospora bigeminaIn the early and mid-1900s, it was thought that dogs and catsshared the same species of coccidia (7, 8). The name Coccidiumbigemina had been given by Stiles in 1891 to a parasitedeveloping in the lamina propria of a dog (39). The organismwas placed in the genus Isospora in 1906. Based on the locationof the sporocysts, the parasite observed by Stiles was obviouslya species of Sarcocystis. Wenyon believed that there were two“races” of I. bigemina that could be differentiated based on sizeand called them the large and small races of I. bigemina (188).The large race developed in the lamina propria and was excretedas sporulated oocysts or sporocysts (i.e., a Sarcocystisspecies), whereas the small race developed in the epithelialcells of the small intestine and was excreted as unsporulatedoocysts. It is clear now that the small race of I. bigemina in dogsis actually Hammondia heydorni, an obligatorily heteroxenousparasite (81). It is impossible to determine what the small raceof I. bigemina in cats actually was because its oocysts are structurallyindistinguishable from those of Toxoplasma gondii,Hammondia hammondi, and Besnoitia species. Reports of thelarge race of I. bigemina in cats, other animals, and humansalso actually refer to Sarcocystis species.Downloaded from http://cmr.asm.org/ on February 5, 2014 by guest

Downloaded from http://cmr.asm.org/ on February 5, 2014 by guestFIG. 2. Developmental stages of I. rivolta in cats and mice. (A, B, G to J, M, and N) Smears fixed in methanol and stained with Giemsa. (C to F) Sections stainedwith iron hematoxylin (C, D, F) and by the PAS reaction (E). (K and L) Smears not fixed or stained. (A and C) Division of meronts by endodyogeny (arrow). (B) Animmature meront with four nuclei. (D) Two multinucleated meronts (arrows) in the same parasitophorous vacuole. (E) PAS-positive granules (arrow) in merozoites.(F) Meronts with different-sized merozoites (arrows). (G) An immature microgamont with many nuclei (arrow). (H) Several mature microgametes (arrow). (I)Macrogamont with a large nucleus (arrow) and prominent nucleolus. (J) An unsporulated oocyst. (K) Unsporulated oocyst containing a contracted sporont. (L)Sporulated oocyst containing two sporocysts with sporozoites (arrows). (M) Extraintestinal zoites in the mesenteric lymph node of a cat. One zoite is in a host cell(arrow), and one has ruptured out of its host cell (arrowhead). (N) Extraintestinal tissue cyst containing a single zoite (arrow) in the mesenteric lymph node of a mouse.Magnifications, 2,300. Reprinted with permission of the publisher from reference 38.22

VOL. 10, 1997 BIOLOGY OF ISOSPORA SPP. 23reason, it is more accurate to refer to the host as a paratenicrather than an intermediate host.Transmission electron microscopy reveals that the sporozoitesare inside a parasitophorous vacuole (PV) (14, 42, 129)(Fig. 3). The appearance of the contents of the PV changesduring the course of infection. At 1 day postinoculation (p.i.)sporozoites are surrounded by a PV membrane that has a wavyappearance, and the PV contains numerous vesicles. By 7 daysp.i., there is an electron-dense granular layer immediately beneaththe PV membrane. Filaments or tubules may also bepresent in this layer. It is this granular layer that appears as athick wall by light microscopy. Membrane-bound, electrondensegranules, apparently of host cell origin, are present atthe margins of the PV membrane. The sporozoite lies in thecenter of the cyst. Sporozoites increase in size during thecourse of infection and accumulate polysaccharide granules intheir cytoplasm. It is because of the presence of these polysaccharide(amylopectin?) granules that the sporozoites stain positivelyin the periodic acid-Schiff (PAS) reaction. The crystalloidbodies of sporozoites remain intact during the course ofthe infection.Disease does not occur in paratenic hosts (38). Parasitesremain viable for at least 23 months in extraintestinal tissues ofmice (38). When the definitive host ingests a paratenic host,the subsequent prepatent period may be shorter than wheninfections are initiated by oocysts. The number of oocysts producedby the definitive host and the patent period are similarto those in oocyst-induced infections (43). The tissues of paratenichosts are not infectious for other paratenic hosts (38).An interesting interaction occurs in mice experimentally infectedwith I. felis and then challenged with Babesia microti. Miceinfected with I. felis 28 days before infection with B. microti do notdevelop B. microti antibodies but are completely resistant to infectionwith B. microti (176). Partial resistance to B. microti can beachieved by transfer of spleen cells from mice infected with I. felis.Treatment of I. felis-infected mice with a monoclonal antibody toL3T4 cells increases their susceptibility to B. microti infection(176). These results suggest that cell-mediated immunity is involvedin the observed nonspecific resistance.DEVELOPMENT IN VITROSeveral mammalian Isospora species have been grown in cellcultures (54, 56, 57, 58, 102, 107). Primary cell cultures fromthe host animal generally support the most numerous and mostchronologically advanced parasite stages. Sporozoites are obtainedfrom excysted oocysts and used as an inoculum. Sporozoitespenetrate host cells and undergo several divisions byendodyogeny. In primary porcine and bovine cell cultures,binucleate meronts and merozoites of I. suis were motile andwere observed to exit and enter host cells (102). No noticeableill effects were observed in the host cells. Only I. rivolta and I.suis have produced multinucleate meronts (with more than twonuclei) in cell cultures, and these meronts did not reach maturity(54, 102). Sexual stages and oocysts do not develop in cellcultures. Continuous cultivation of an Isospora species has notbeen achieved in cell culture.I. felis, I. rivolta, and I. suis will develop from sporozoites tounsporulated oocysts in the chorioallantoic membrane of developingchicken embryos (3, 71, 105). Development is usuallylimited to the tissues of the chorioallantoic membrane, butmeronts of I. felis have been seen in the livers and intestines ofchicken embryos that have been chemically immunosuppressed(71). Although complete development has been obtained,the in ovo system has not gained widespread use becausefew oocysts are obtained and they do not sporulate.DIAGNOSIS OF COCCIDIAL INFECTIONSCoccidia are often members of the normal fauna of animalhosts, and the mere presence of oocysts in the feces is notalways indicative of clinical infection (103). Demonstration ofoocysts in fecal samples is the method of choice for identifyingcoccidian infections in animals. Fecal flotation in Sheather’ssugar solution (500 g of sugar, 320 ml of water, 6.5 g of phenol)is most often used, but other flotation solutions such as zincsulfate or saturated sodium chloride can be used. If largeamounts of fecal fat are present, other concentration techniquessuch as formalin-ether or ethyl acetate sedimentationmay be more applicable because of the removal of fecal fat bythe solvents. No special stains are needed to observe the oocysts.However, special stains are often used to identify humaninfections with I. belli.The diagnosis of coccidiosis in animals is based on clinicalsigns (diarrhea), history, evaluation for potential copathogens,and demonstration of coccidial oocysts of a pathogenic speciesin the animals’ feces. Knowing the actual numbers of oocystspresent in the feces is of little help in determining if clinicaldisease is present.Demonstration of parasite stages in tissue samples collectedat necropsy in animal infections or in intestinal biopsy specimensor samples collected at autopsy in human infections isalso suitable for obtaining a diagnosis. Special stains are oflittle value in identifying coccidial stages. Familiarity with theappearance of the stages is far more useful in locating them inhistological samples (Fig. 2).ISOSPORA INFECTIONS OF HUMANSI. natalensis has been reported in humans (48), but little isknown about this parasite. It was found in the feces of a21-year-old patient suffering from amebic dysentery and otherprotozoal and helminth infections. Oocysts of I. natalensis wereobserved on four consecutive days (after the patient had beentreated for amebic dysentery), and the I. natalensis infectionwas self-limiting. Infection with this parasite has apparently notbeen observed since 1953, when it was described. Its oocystsresemble those of the I. ohioensis complex seen in dogs, I.rivolta of cats, and I. suis of pigs, but they are slightly larger(Table 1).I. chilensis described from humans in South America is nota valid name; it is a species of Sarcocystis. As mentioned above,I. hominis is also no longer considered a valid name because ittoo is a species of Sarcocystis.Three cases of infection with a coccidian species believed tobe an isosporan were reported from humans in Papua NewGuinea (4). The oocysts were excreted unsporulated, werespherical, and measured 8.5 m in diameter. Sporulation wasslow, taking about 2 weeks, and the final proportion of oocyststhat sporulated was only about 10%. The sporocysts of thiscoccidium were illustrated in drawings with no Stieda body, butthere appears to be a Stieda body in the photomicrographs thataccompany the description. The parasite is probably a speciesof Cyclospora, a recently recognized coccidial pathogen of humansthat has two sporocysts with Stieda and sub-Stieda bodiesthat enclose two sporozoites (144).ISOSPORA BELLI INFECTIONSI. belli infections are essentially cosmopolitan in distributionbut are more common in tropical and subtropical regions,especially Haiti, Mexico, Brazil, El Salvador, tropical Africa,the Middle East, and Southeast Asia (53, 88, 164). Pigs, dogs,Downloaded from http://cmr.asm.org/ on February 5, 2014 by guest

24 LINDSAY ET AL. CLIN. MICROBIOL. REV.Downloaded from http://cmr.asm.org/ on February 5, 2014 by guest

VOL. 10, 1997 BIOLOGY OF ISOSPORA SPP. 25FIG. 3. Stages of I. ohioensis in lymphoid cells of the mesenteric lymph nodes of mice. (A) Zoite in a smear, 4 days after infection. Magnification, 1,650. (B)Electron micrograph of the crystalloid body 5 days after infection. Note the regular arrangement of units. Magnification, 71,300. (C) Electron micrograph of a zoitein the region of the nucleus 7 days after infection. The parasitophorous vacuole (PV) is filled nearly completely by granular material (GM). Magnification, 23,800.(D) Zoite in a smear of mesenteric lymph node, 4 days after infection. Giemsa stain was used. Magnification, 1,650. (E) Electron micrograph of a zoite 14 days afterinfection. The PV has an electron-lucent space (ES) and granular material (GM). Magnification, 23,800. Other abbreviations: A, amylopectin; CH, chromatin; CR,crystalloid body; DK, dark granules; HC, host cell; IT, intravacuolar tubules; LP, limiting membrane of the PV; NH, host cell nucleus; MN, micronemes; PA, zoite;PE, three-layered pellicle of zoite; R, rhoptries; SL, tissue cyst wall. Reprinted with permission of the publisher from reference 42.mice, rats, rabbits, guinea pigs, and rhesus monkeys are notsuitable definitive hosts (61, 87); however, in one study, patentinfections were reported in two of three gibbons (193). Thislack of susceptibility has led some researchers to discountanimals as reservoirs (90). However, it is not known if these orother animals may serve as paratenic hosts for I. belli. The roleof paratenic hosts in the transmission of I. belli needs to beinvestigated to establish whether modes of transmission otherthan by contaminated food or water exist. The existence ofparatenic hosts may help explain infections occurring in areaswhere sanitation is adequate.Life Cycle of I. belliOocysts are passed in feces unsporulated or partially sporulated(sporoblast stage). They can sporulate in less than 24 h(133). Oocysts are elongate and ellipsoidal with slightly taperedends, or one end may be tapered and the other end blunt(Fig. 1; Table 1). The patent period is not known. It may be aslittle as 15 days in some patients (127). Chronic infectionsdevelop in some patients, and oocysts are excreted for severalmonths to years. In one case, an apparently immunocompetentindividual had symptoms that were present for 26 years andhad I. belli infection documented on several occasions over a10-year period.All life cycle stages typical of Isospora species have beenobserved by light and transmission electron microscopy (16,149, 179). The number of asexual types present has not beendetermined. If the life cycle is similar to that of other carnivore/omnivoreIsospora species, the first asexual division wouldbe by endodyogeny. Division by endodyogeny probably occursrepeatedly. Endogenous stages are located in enterocytes liningthe villi of the small intestine and rarely in those in thelarge intestine (16, 149, 179). Endogenous stages are seldomfound in other locations such as enterocytes lining the crypts orin cells in the lamina propria. Extraintestinal infections havebeen observed in AIDS patients (see below) and probably alsooccur in immunocompetent patients.Intestinal Infections in AIDS PatientsDiarrhea produced by I. belli in AIDS patients is often veryfluid and secretory-like and leads to dehydration requiringhospitalization. Fever and weight loss are also common findings.Other opportunistic pathogens are also common in thesepatients. Intestinal lesions induced by I. belli and the responsesto chemotherapy are usually similar to those in immunocompetentpatients.In an extensive 8-year surveillance program of AIDS patientsin Los Angeles County (164), I. belli was found in 127(1%) of 16,351 patients. The prevalence of infection was highestamong foreign-born patients, especially patients from ElSalvador (7.4%) or Mexico (5.4%) or of other Hispanic ethnicity(2.9%). Patients between the ages of 14 and 24 weremore likely to have I. belli infection than were older patients.Patients with a history of Pneumocystis carinii pneumonia wereless likely to have I. belli infection. The authors concluded thatisosporiasis among AIDS patients in Los Angeles may be relatedto travel exposure and/or recent immigration from LatinAmerican countries. Additionally, the use of trimethoprim(TMP)-sulfamethoxazole (SMX) for the treatment or preventionof P. carinii pneumonia may effectively prevent the acquisitionof primary I. belli infection or the recrudescence ofexisting I. belli infection. It was recommended that physicianshave an increased index of suspicion for I. belli in AIDS patientswith diarrhea who have immigrated from or traveled toLatin America, are Hispanics born in the United States, areyoung adults, or have not received prophylaxis with TMP-SMXfor P. carinii. Additionally, it was suggested that AIDS patientstraveling to Latin America and other developing countries beadvised of the potential for food-borne and waterborne acquisitionof I. belli infection and consider taking TMP-SMX chemoprophylaxis.I. belli infection was observed in 20 (15%) of 131 AIDSpatients with opportunistic infections at Port-au-Prince, Haiti(28). Stool samples collected from 170 siblings, friends, andsexual partners were negative. No demographic or laboratorycharacteristics distinguished patients with AIDS and I. bellifrom patients with AIDS and other opportunistic infections. Inanother study, three of three patients with I. belli infectionwere from Haiti and lived in the United States at the time ofthe study (190).Nine (19%) of 46 patients from Zaire with chronic diarrheaand suspected of having AIDS had I. belli (80). Eight of thenine I. belli-positive patients were later confirmed to haveAIDS. I. belli was found in 13 (9.9%) of 81 AIDS patientsexamined at a reference center in Sao Paulo, Brazil (158).Stool samples from 81 immunocompetent individuals werenegative for I. belli. Three (5%) of 60 AIDS patients examinedin Catalinya, Spain, were positive for I. belli oocysts (155).A pregnant AIDS patient with I. belli diarrhea diagnosed at5.5 months of pregnancy delivered a live human immunodeficiencyvirus-positive infant (147). Her sexual partner was alsoTABLE 1. Measurements of oocysts of Isospora speciesfrom mammalsSpeciesHostDimensions (m) of:Oocysts aSporocysts aI. belli Humans 23–36 by 12–17 12–14 by 7–9I. natalensis Humans 24–30 by 21–25 17 by 12I. arctopitheci NH primates b 21–30 by 21–25 13–21 by 10–16I. callimico NH primates 13–21 by 12–17 10–13 by 7–9I. endocallimici NH primates 25–31 by 21–27 15–20 by 10–15I. scorzai NH primates 23 by 20 14 by 9I. canis Dogs 34–40 by 28–32 18–21 by 15–18I. ohioensis Dogs 19–27 by 18–23 15–19 by 10–13I. burrowsi Dogs 17–22 by 16–19 12–16 by 8–11I. rivolta Cats 18–28 by 16–23 14–16 by 10–13I. felis Cats 38–51 by 27–39 20–26 by 17–22I. suis Pigs 17–25 by 16–21 11–14 by 8–11a Measurements represent the range unless none was reported.b NH primates, nonhuman primates.Downloaded from http://cmr.asm.org/ on February 5, 2014 by guest

26 LINDSAY ET AL. CLIN. MICROBIOL. REV.positive for I. belli. Treatment with TMP-SMX never eliminatedthe I. belli infection.Extraintestinal Infections in AIDS PatientsTwo reports of disseminated extraintestinal isosporiasis inpatients with AIDS have been published (130, 149). The firstpatient was a 38-year-old white male homosexual who wasexamined at the National Institutes of Health, Bethesda, Md.(149). He initially presented to a local hospital with a history ofprogressive dyspnea and fever; he also complained of dysphagia,nausea, vomiting, and brown watery diarrhea (eight ornine episodes daily). He had lost 20 lb (9.17 kg) in 2 months(15% of his body weight). P. carinii pneumonia and oropharyngealcandidiasis were noted, and he was treated with TMP-SMX and pentamidine. His condition improved, and he wasdischarged 24 days after admission. He subsequently was readmittedcomplaining of nausea, vomiting, and diarrhea. Hewas diagnosed as having Giardia lamblia infection and wastreated with metronidazole. Five months after his initial hospitalization,he was diagnosed as having I. belli and Entamoebahistolytica infection. He was treated with TMP-SMX, metronidazole,and diodoquin. Three months later he presented withdyspnea, fever, diarrhea, and generalized wasting. Cytomegaloviruspneumonia was demonstrated at this time. Repeatedstool examinations were negative. He died 2 weeks later. Atautopsy, the body demonstrated severe cachexia, focally consolidatedlungs, multiple small intestinal foci of multifocal erythemaand hemorrhage, ulcerated cecal lesions up to 5 mmacross, and enlarged mesenteric, periaortic, and mediastinallymph nodes. Microscopically, disseminated cytomegalovirusinfection involving the lungs, intestines, adrenal glands, mesentericlymph nodes, and, to a lesser extent, liver and pancreaswas observed. Mycobacterium kansasii was cultured from theliver and spleen, although no granulomas were observed intissue sections.Microscopic findings associated with I. belli infection wereobserved in the lymph nodes and walls of both the small andlarge intestines. Marked lymphocytic depletion was observedin the lymph nodes, and foci of granuloma-like histiocytic proliferationwere seen in the mesenteric, periaortic, and mediastinallymph nodes. Intracellular zoites were observed in thecytoplasm of histiocytes. The parasites were surrounded by athick eosinophilic cyst wall in hematoxylin-and-eosin-stainedsections. The cyst wall was PAS positive. Most of the infectedcells contained only one zoite; however, some contained two orthree. Examination of the intestinal tissues demonstrated intraepithelialasexual and sexual stages of I. belli and occasionallymerozoites that appeared to be in cells in the laminapropria. Numerous I. belli oocysts were observed in scrapingsobtained from the intestine.The second case was observed in a 30-year-old black womanwho was a native of Burkina Faso but had lived in France for2 years (130). She initially presented with fever, diarrhea, andweight loss. She was found to have esophageal candidiasis andI. belli infection. The I. belli infection was treated with TMP-SMX (200 mg/day), and the diarrhea resolved within a week.She was placed on maintenance therapy of 100 mg of TMP-SMX daily but suffered eight episodes of recurrent infectiondiagnosed by stool examination or duodenal biopsy over thenext 3 years. Examination of the biopsy specimens demonstratedsevere villous atrophy and meronts, gamonts, and oocystsof I. belli within enterocytes. Gamonts and merozoite-likestages were observed in the lamina propria. No other pathogenswere observed in the biopsy specimens. An autopsy conductedafter her death revealed cachexia. The abdominal cavityFIG. 4. Tissue cysts of I. belli in the spleen of an AIDS patient. A longitudinalview and a cross-section of tissue cysts are present. Note the tissue cyst wall(arrows) and the nucleus (open arrow) of one zoite. Magnification, 1,000.contained 0.5 liter of serous ascitic fluid. The liver, spleen, andmesenteric lymph nodes were enlarged. The small intestineand colonic mucosa were pale and atrophic, but no ulcerationsor perforations were present. No gross lesions were observedin the omentum or other tissues. Examination of samples collectedat autopsy revealed stages of I. belli in the intestine,mesenteric and mediastinal lymph nodes, liver, and spleen(Fig. 4). The extraintestinal stages were always observed assingle organisms that did not stain with acid-fast stains. Thetissue cyst wall was PAS negative, but the enclosed zoite containedPAS-positive granules. The tissue cyst wall did not stainby the Gomori-Grocott method. Massive infection was observedin the lymph nodes in association with plasmacytosisand some eosinophils but no granulomatous reaction. Parasiteswere usually grouped in clusters in the paracortical areas or thelumen of the sinus. Few parasites were observed in Kupffercells or within macrophages located in portal areas. No involvementof the biliary system was noted. A moderate steatosisand cholestasis was also observed. The spleen had I. bellitissue cysts in the red and white pulp; the cysts were associatedwith congestion and atrophy of the white pulp.Notable differences in the light microscopic findings in thesetwo patients are the presence of more than one zoite within atissue cyst observed in the first patient and the lack of PASreactivity of the tissue cyst wall in the second. Additionally, nogranulomatous reaction was observed in the lymph nodes inthe second patient. It was believed that the concurrent cytomegalovirusinfection helped lead to dissemination of the parasitein the first patient. However, cytomegalovirus or other intestinalpathogens were not documented in the second patient.Transmission electron microscopy was used to examine portionsof lymph nodes in both patients, and the findings weresimilar. The zoites were in a PV within the cytoplasm of histiocytes.Organelles typical of coccidial sporozoites/merozoiteswith a crystalloid body and polysaccharide granules werepresent. An electron-dense granular layer was seen immediatelybeneath the PV membrane. This layer probably composedthe tissue cyst wall observed by light microscopy. Theultrastructural features of these tissue cysts observed in thelymph nodes of humans are similar to the tissue cysts observedin mice inoculated with I. felis and I. ohioensis.A recent study (23) examined the submicroscopic appearanceof I. belli infection in a 30-year-old white female intravenousdrug user from Italy who had AIDS. Her symptoms wereDownloaded from http://cmr.asm.org/ on February 5, 2014 by guest

VOL. 10, 1997 BIOLOGY OF ISOSPORA SPP. 27watery, nonbloody diarrhea and fever. She was treated withTMP-SMX, and her diarrhea stopped in 2 days. No otherclinical data were presented. Ultrastructural examination ofsmall intestinal biopsy specimens taken at the duodenojejunaljunction demonstrated trophozoites, merozoites, meronts, andmacrogamonts in epithelial cells. Occasionally, merozoiteswere observed in the intestinal lumen, in the lamina propria,and within lymphatic channels. The demonstration of merozoitesin lymphatic channels documents a means of their disseminationto lymph nodes and to other tissues. The authors consideredthat their findings of extracellular merozoites mightindicate that I. belli is not strictly an intracellular parasite. Thisconsideration is erroneous, because it is well documented thatmotile stages of Isospora can leave host cells and invade newhost cells (110). This movement is a normal part of the lifecycle, and these fortuitous observations of extracellular stagesare not indicative of extended extracellular survival by theseforms of the parasite. It is interesting that the photomicrographof a merozoite in a lymphatic channel (Fig. 6 in reference23) appears to be a tissue cyst. The merozoite is surroundedby electron-dense material identical to that seen intissue cysts in lymph nodes.Asexual and sexual stages and oocysts of I. belli have beenobserved in the bile duct epithelium of an AIDS patient withacalculous cholecystitis (8a). No lymph nodes were examinedin this patient, and the relationship between bile duct infectionsand disseminated infections with tissue cysts is presentlynot known.Infections in Other Immunocompromised HostsClinical disease in I. belli infections is usually more severe inimmunocompromised patients than in immunocompetent patients.I. belli has been observed in patients with concurrentHodgkin’s disease (16), non-Hodgkin’s lymphoproliferativedisease (72), human T-cell leukemia virus type 1-associatedadult T-cell leukemia (68), and acute lymphoblastic leukemia(189). These patients respond to specific anti-I. belli treatment(see below).It was suggested in one report that treatment with prednisolone(60 mg/day for 13 days) led to transient immunosuppressionand severe I. belli infection in one patient (134). Thepatient recovered without specific treatment.Infections in Immunocompetent HostsI. belli causes serious and sometimes fatal disease in immunocompetenthumans. Symptoms of I. belli infection includediarrhea, steatorrhea, headache, fever, malaise, abdominalpain, vomiting, dehydration, and weight loss (16, 75, 85, 98,179). Blood is not usually present in the feces. Eosinophilia isobserved in some patients. The disease is often chronic, withparasites present in the feces or biopsy specimens for severalmonths to years. Recurrences are common.Experimental infections demonstrate that fever begins 8days after ingestion of oocysts and lasts for about 8 days (120).Nonbloody diarrhea begins 7 to 9 days after ingestion of oocysts.The prepatent period is 10 to 11 days, and oocysts areexcreted for 32 to 38 days. No oocysts were excreted when onevolunteer attempted to reinfect himself 33 days after ingestionof oocysts, indicating that immunity had developed.Disease is more severe in infants (98) and young children(178) than in adults. A 6-month-old male infant in Californiahad I. belli infection that terminated fatally after 30 weeks ofcontinuous total parental nutrition (98). The disease was characterizedby severe diarrhea (1 to 3 liters daily) due to choleralikehypersecretion of intraluminal fluid. Little clinical responseto surgical, dietary, or antibiotic treatments wasobserved. An 18-month-old female in Thailand was admittedto hospital with severe dehydration, inappetence, and weakness(178). She had four or five diarrhetic bowel movementsdaily. She responded to treatment with electrolytes and TMP-SMX, and her diarrhea ceased within 5 days.Microscopic Lesions Due to I. belliThe main microscopic changes are villous atrophy and crypthyperplasia (16, 149, 179). Eosinophils may be present in thelamina propria in large numbers approaching those seen ineosinophilic enteritis. Plasma cells, lymphocytes, and polymorphonuclearleukocytes (PMNs) are present in increased numbers.The lymphatics may be dilated.DiagnosisThe Sheather sugar flotation method is an excellent methodfor detecting oocysts of I. belli (26, 115). The unsporulatedoocysts of I. belli are readily visible unstained by light microscopy.Oocysts are in a slightly higher plane of focus than otherparasite cysts or ova (49). Flotation methods are superior todirect fecal smears for detecting oocysts (53). Sedimentationconcentration methods are also more sensitive than directsmears. Charcot-Leyden crystals may (70, 88, 131, 162) or maynot (163) be present in stool samples that contain I. bellioocysts.Stained fecal smears made from concentrated samples mayaid in the detection of I. belli oocysts (17, 92, 115, 137, 145).The modified acid-fast stain produces pink-staining oocyststhat contain bright red sporonts or sporoblasts (137). Oocystsstained by the auramine-rhodamine procedure fluoresce brightyellow (115). When the Giemsa stain is used, both the oocystsand sporoblasts stain pale blue. The heated safranin-methyleneblue technique produces oocysts that are orange-red (17). Thetrichrome stain is of little use (92).Duodenal aspirates (100, 179), the duodenal string test(190), and small intestinal biopsies (179) are also useful insuspected cases in which oocysts are not found in stool samples.I. belli oocysts are observed in duodenal aspirates and inmucus collected in the string test. Developmental stages of I.belli can be identified in enterocytes in small intestinal biopsyspecimens. Some biopsy samples may be negative for developmentalstages but contain characteristic lesions. Likewise, oocystsmay be present in stool samples from some biopsy-negativepatients (63). Routine histological staining methods aresatisfactory for demonstrating parasite stages. Many of theparasites will be in vacuoles, making them readily identifiable.I. belli can cause disease with relatively few stages of the parasitepresent and can be missed on small intestinal biopsy.TreatmentMany agents have been used to treat I. belli infections. Combinationsof protozoal dihydrofolate reductase/thymidylatesynthase inhibitors (TMP or pyrimethamine) with sulfonamides(SMX, sulfadiazine, or sulfadioxine) have generallyproven effective. Treatment with TMP-SMX has been usedmost often (23, 28, 62, 88, 92, 147, 178, 189). One study examinedthe TMP-SMX treatment of a group of 32 Haitian AIDSpatients. The patients ranged in age from 24 to 55 years old.They had a history of chronic intermittent diarrhea with amean duration of 7.9 months (range, 2 to 26 months). Thediarrhea was liquid, and 2 to 10 stools were excreted a day. Thepatients also had a history of diffuse, crampy abdominal pain,nausea, and intermittent fever. Of the 32 patients, 28 requiredDownloaded from http://cmr.asm.org/ on February 5, 2014 by guest

28 LINDSAY ET AL. CLIN. MICROBIOL. REV.oral or intravenous rehydration before or during the first 3days of the study. The patients were treated with oral TMP(160 mg)-SMX (800 mg) four times a day for 10 days. Diarrheaand abdominal pain stopped 1 to 6 days (mean, 2.5 days) aftertreatment. All stool samples examined after the end of treatmentwere negative. At the end of the study, the prophylaxis ofI. belli infection was examined in these patients. Ten patientsreceived placebo orally three times a week, 10 received TMP(160 mg)-SMX (800 mg) orally three times a week, and 12received pyrimethamine (25 mg)-sulfadioxine (500 mg) orallyonce a week. Of the 10 patients given placebo, 5 developedrecurrent I. belli infection in 1 to 3.5 months and were retreatedwith TMP-SMX for 10 days with favorable outcomes.None of the patients given pyrimethamine-sulfadioxine hadrelapses, and 1 of the patients given TMP-SMX developed anasymptomatic I. belli infection. Severe pruritus developed in 1patient in each drug treatment group, resulting in the terminationof treatment.Pyrimethamine-sulfadoxine has been used less frequentlythan TMP-SMX but also gives prompt clinical response andeliminates the parasite when used (70, 133). Pyrimethaminesulfadiazineis also effective in treating I. belli infection (132,179). Pyrimethamine used alone is also effective in patientswith sulfonamide allergies (186).Macrolide antibiotics have marginal efficacy in treating I.belli enteritis. Sirimamycin given at 1.5 g twice daily initiallyprovided clinical improvement in a Haitian AIDS patient whodid not respond to TMP-SMX, furazolidone, or tetracyclinetreatments for I. belli enteritis (66). The response to treatmentlasted about a month, and then the patient relapsed. A treatmentcourse with pyrimethamine-sulfadiazine was initiated afterthe relapse, but little improvement was observed. Roxithromycin(2.5 mg/kg every 12 h) was used successfully to treat anAfrican AIDS patient who was suffering from chronic I. belliinduceddiarrhea that did not respond to TMP-SMX or pyrimethaminetreatments (136). Roxithromycin was given orallyfor 15 days, and the diarrhea became intermittent and lesssevere. Although diarrhea requiring hospitalization occurredtwice during the 2 months after treatment, no I. belli oocystswere observed in stool samples.Treatment with anti-giardial agents such as metronidazole,tinidazole, quinacrine, and furazolidone is probably of littlevalue (19, 175, 179, 186). However, some cases of apparentlysuccessful treatment with metronidazole have been reported(62, 72).Administration of the antimalarial compounds primaquinephosphate and chloroquine phosphate gave temporary relief ofchronic I. belli infection in an immunocompetent patient aftera 2-week treatment course (179). Intestinal biopsy specimensand duodenal aspirates were negative. The patient relapsed in1 month, and biopsy and aspirate specimens were positive forI. belli.Veterinary anticoccidial drugs have been used with somesuccess in treating I. belli infections in humans. Amprolium wasused in an AIDS patient in the Netherlands who was sufferingfrom severe diarrhea (181) and for whom treatment with pyrimethamine-sulfadiazinewas stopped because of pancytopenia.Spiramycin had been only partially effective. Amproliumwas given orally beginning at 10 mg/kg and increasing to 90mg/kg. The frequency of diarrhea lessened after 6 days oftreatment. Amprolium treatment was stopped on day 7 becauseof polyneuropathy but was reinitiated on day 20 at areduced dose of 30 mg/kg. The stool became normal by day 28of treatment, and no oocysts were present after day 35. Diclazurilwas used in a trial to treat eight AIDS patients with I. bellidiarrhea in Kinshasa, Zaire (89). Each patient received 200 mgof diclazuril orally for 7 days. Oocysts were eliminated from thestools by 2 to 3 days. Diarrhea completely stopped in four ofthe eight patients, but severe diarrhea persisted in one patient.Oocysts were present in the stools of one of three patientsexamined more than 1 month later. Diarrhea and oocyst excretionrecurred at 47 days after treatment.ISOSPORA INFECTIONS OF NONHUMAN PRIMATESLittle is known about the coccidial infections of nonhumanprimates. Most of the Isospora species recorded are knownonly by their oocyst structure (Table 1).I. callimico was isolated from the feces of a Goeldi’s marmoset(Callimico goeldi) at a laboratory animal facility in Baltimore,Md. (Table 1) (84). The oocysts were excreted for 7days and sporulated in 2 days.I. endocallimici was isolated from the feces of five Goeldi’smarmosets from the Tulane University Delta Regional PrimateResearch Center in Louisiana (Table 1) (46). Two of theanimals were born at the center, and three were exported fromPeru. No transmission or life cycle studies have been conductedwith these species.I. scorzai was isolated from the feces of a Uakari monkey(Cacajao rubicundus) that was housed in the London Zoo, andthe parasite was transmitted to another monkey, Cebus nigrivittatus(2). The life cycle of I. scorzai is not known. Experimentallyinoculated kittens did not excrete oocysts.I. cebi was isolated from the feces of a Cebus albifrons fromthe Alto Magdalena region of Colombia (119). The sporocystsof this species have Stieda bodies, indicating that it is a pseudoparasiteof avian origin. A similar Isospora species was isolatedfrom the feces of a Bonnet monkey (Macaca radiata) atthe Delhi Zoo in India but was not named (9).Isospora paponis was isolated from Chacma baboons (Papioursinus) (125). Oocysts sporulated endogenously in the smallintestines, indicating that this is a Sarcocystis species. Additionally,sporulated oocysts of this species have been seen in theskeletal muscles of Chacma baboons (126). Chimpanzees (Pantroglodytes) can also serve as definitive hosts for Sarcocystisspecies, and reports of Isospora sporocysts in their feces actuallydescribe Sarcocystis sporocysts.I. arctopitheci InfectionsI. arctopitheci has been studied more than the other coccidiaof nonhuman primates (76–78, 140). Hendricks conductedcross-transmission studies with this parasite and claimed tohave successfully transmitted it to members of six genera ofNew World nonhuman primates, four families of carnivores,and one marsupial species (77). This is an unusually large anddiverse definitive host range, and further experimental studiesare needed to confirm or deny these initial findings.The endogenous life cycle of I. arctopitheci occurs in thesmall intestine (140). Developmental stages are located in enterocyteson the distal two-thirds of the villi, and parasitedensities are greatest in the jejunum. Asexual multiplicationwas found to be exclusively by endodyogeny, and eosinophilicbodies were present in gamonts (140). The prepatent periodwas about 7 days, but the patent period was not reported.Extraintestinal stages were not seen in the definitive host.Experimental studies indicate that I. arctopitheci can bepathogenic (140). Of 13 titi marmosets (Saguinus geoffroyi), 4died after being inoculated with 1 10 5 to 2 10 5 oocysts. Noclinical signs were seen in marmosets that died 3 and 5 days p.i.Bloody diarrhea was seen in two marmosets that died 7 daysp.i. All nine other marmosets remained normal. The micro-Downloaded from http://cmr.asm.org/ on February 5, 2014 by guest

VOL. 10, 1997 BIOLOGY OF ISOSPORA SPP. 29scopic lesions observed were necrosis of apical enterocyteswith exposure of the lamina propria.Diagnosis and TreatmentDiagnosis is made by finding the characteristic oocysts (Table1) in fecal samples. Fecal flotation with Sheather’s sugarsolution is recommended as a reliable and sensitive technique.Sedimentation or other concentration techniques are also adequate.Most Isospora infections in nonhuman primates are subclinical.We are unaware of any reports on the treatment of Isosporainfections in nonhuman primates. Agents used in humansor veterinary products may be of some value.ISOSPORA INFECTIONS OF DOGS AND CATSInfections of DogsSeveral species of Isospora infect dogs (Table 1). Cats arenot the definitive hosts for Isospora species found in dogs (32).Young dogs are more likely to be infected, and surveys indicatethat 3 to 38% of dogs are positive for coccidial oocysts (91).Stray dogs are more likely to be infected than are dogs withowners because stray dogs must hunt for food and thereforehave more exposure to infected paratenic hosts.It is unclear if coccidiosis is a serious problem in dogs (103,146). Diarrhea associated with the presence of coccidial oocystsin young dogs occurs, but the clinical significance is notestablished because of the possibility of concurrent bacterial orviral infections. Published reports of naturally occurring caninecoccidiosis are few (24, 44, 141), and further studies on naturalcases are needed before firm conclusions can be made. Experimentalinfections have not usually been associated with disease.I. canis infections. I. canis has the largest oocysts of thecanine Isospora species and is the only species that can bediagnosed by microscopical examination of oocysts (Table 1).I. canis develops in cells in the lamina propria of the posteriorsmall intestine (93). Three asexual types are present, and thefirst asexual division is probably by endodyogeny. Theprepatent period is 9 days. The length of the patent period hasnot been determined.Disease was not produced in 25 6-week-old or 6 8-week-oldpups inoculated with 1 10 5 to 1.5 10 5 I. canis oocysts (93).Solid immunity follows primary infection, and no oocysts aredischarged after challenge (6). It has been suggested that thestress of weaning and shipping may enhance I. canis infections(93). This suggestion needs further investigation because theseoutbreaks of coccidially associated diarrhea may be related toa decrease in immunity and reactivation of latent extraintestinalstages with subsequent intestinal infection and clinical signsof disease.The I. ohioensis complex. Three Isospora species havingsmaller oocysts than I. canis can be found in dogs: I. ohioensis,I. burrowsi (Table 1), and I. neorivolta. Because they cannot beseparated based on oocyst structure and because I. ohioensiswas the first named, these oocysts are often referred to as I.ohioensis-like (44) or members of the I. ohioensis complex.I. ohioensis develops in enterocytes in the small intestine,cecum, and colon of dogs (35). Two asexual types are recognized,and division by endodyogeny is observed. The prepatentperiod is 5 days, and the length of the patent period is notknown. The parasite can cause disease in experimentally infected7-day-old pups but not weaned pups or young dogs (36).Diarrhea was the major clinical sign seen in the 7-day-old pups.Microscopic changes were villous atrophy, necrosis of apicalenterocytes, and cryptitis. Dogs developed an immunity thatlasted for about 2 months.I. burrowsi develops in enterocytes and cells in the laminapropria in the posterior small intestine (180). Two asexualtypes are present. Division by endodyogeny has not been recordedbut probably occurs. The prepatent period is 6 days,and oocysts are excreted for 9 to 15 days.I. neorivolta develops in cells in the lamina propria in theposterior small intestine (41). Four asexual types are recognized,and division by endodyogeny is observed. The prepatentperiod is 6 days, and oocysts are excreted for 13 to 23 days.Little is known about the pathogenesis of I. burrowsi or I.neorivolta infection in dogs. Neither caused disease in experimentalinfections of dogs (41, 116, 180).Because the significance of diarrhea caused by coccidia indogs is unclear, the treatment of the condition is also unclear.Suspected clinical cases can be treated with a variety of drugsused alone or in combination (see below).Infections of CatsI. rivolta and I. felis infect cats. Dogs do not serve as definitivehosts for these species (159). Both feline Isospora specieshave extraintestinal stages in the feline definitive host and in avariety of paratenic hosts. From 3 to 36% of cats examinedexcrete oocysts (91). Stray cats are more likely to excrete oocysts.Coccidiosis in cats is not thought to be a common problem(191) and is usually seen only in naturally infected kittensin which other disease-causing agents may be present. Thedrugs used to treat dogs are used to treat kittens.I. rivolta infections. I. rivolta develops in enterocytes in thesmall intestine (38). Three structural types of asexual stagesare present. The first asexual division is by endodyogeny. Theprepatent period is 4 to 7 days, and oocysts are excreted formore than 14 days.Experimentally, I. rivolta can cause disease in newborn kittens(38). Diarrhea occurs 3 to 4 days after administration of1 10 5 or 1 10 6 sporocysts. Microscopic changes consist ofcongestion, erosion of enterocytes, villous atrophy, and cryptitis.No disease was seen in 10- to 13-week-old kittens inoculatedwith up to 10 5 oocysts. Cats develop immunity to infection,but it is not complete because some oocysts are shed afterchallenge (38).I. felis infections. I. felis develops in enterocytes in the smallintestine and occasionally the cecum (161). Three structuraltypes of asexual stages are recognized. The first asexual divisionis by endodyogeny. The prepatent period is 7 to 11 days,and oocysts are excreted for about 11 days.Experimental studies indicate that I. felis is not pathogenicfor cats over 1 month of age (83, 161). Few signs of disease areseen in 6- to 13-week-old cats given 1 10 5 to 1.5 10 5oocysts. Mild microscopic changes consisting of congestion,erosion of superficial enterocytes, and neutrophil infiltrationmay be seen. Four-week-old kittens are the most susceptible,and enteritis, emaciation, and death can occur after inoculationof 10 5 oocysts (1).Cats develop immunity to I. felis, because after infection,they have no or decreased oocyst production when challengedwith I. felis oocysts. Studies indicate that cats infected naturallywith I. felis develop lower antibody titers than do those experimentallyinoculated with I. felis (142). If these cats are challengedwith Toxoplasma gondii, they will develop an antibodytiter to T. gondii and demonstrate an anamnestic response to I.felis antigen. A 22-kDa peptide on sporozoites is the major I.felis protein antigen recognized by immune feline serum (143).Peptides of 22, 45, 58, and 62 kDa on T. gondii tachyzoites orDownloaded from http://cmr.asm.org/ on February 5, 2014 by guest

30 LINDSAY ET AL. CLIN. MICROBIOL. REV.sporozoites are recognized by I. felis immune feline serum.Absorption of I. felis immune serum with these T. gondii stagesremoves reactivity of the 45-, 58-, and 62-kDa peptides, implyingthat the 22-kDa peptide is specific to I. felis.I. felis and T. gondii have evolved an unusual relationship inthe feline definitive host (20, 33, 37). Cats that have previouslyrecovered from a T. gondii infection will reexcrete T. gondiioocysts if they receive a primary challenge with I. felis oocysts.Cats that have a primary I. felis infection followed by a primaryT. gondii infection develop strong immunity to T. gondii andwill not reexcrete T. gondii oocysts if challenged with I. felisoocysts. The biological significance or mechanism of this relationshipis unknown.DiagnosisFecal flotation with Sheather’s sugar solution is the recommendedmethod. It is important to examine stools for bacterialand viral agents that cause disease in these animals becausecoccidiosis is usually asymptomatic. Dogs are coprophagic andoften will have oocysts from other animal feces in their samples.It is important to recognize these pseudoparasites. Themost common of these are Eimeria species from ruminants,rabbits, or rodents. These oocysts will not be in the two-celledstage as is common for Isospora species. They often will haveornamentations, such as micropyle caps or dark thick walls,that are not found on Isospora oocysts. Isospora oocysts thatcontain sporocysts with Stieda bodies are also pseudoparasites.Cats may also have coccidial pseudoparasites in their fecesfrom the ingestion of prey.TreatmentSulfadimethoxine given at 50 mg/kg orally once a day for 10to 14 days will eliminate oocyst excretion in most dogs and cats(104, 191). The combination of ormetoprim (11 mg/kg) andsulfadimethoxine (55 mg/kg) given orally for up to 23 days hasbeen used effectively in dogs (45). Amprolium given orallyonce a day at 300 to 400 mg/kg for 5 days or 110 to 220 mg/kgfor 7 to 12 days is effective in treating coccidiosis in dogs. Otheragents such as furazolidone, quinacrine, and metronidazoleprobably are of little clinical value.ISOSPORA SUIS INFECTIONS OF PIGSThe actual number of valid species of coccidia that infectswine is unknown because most are known only from thesporulated-oocyst stage. Levine and Ivens list 13 named speciesof Eimeria and 3 species of Isospora isolated from swine (97).I. suis, I. almataensis, and I. neyrai are the species of Isosporaisolated from swine. I. almataensis and I. neyrai are known onlyfrom oocysts in the feces. I. almataensis is most probably acombination of a bird Isospora sp. and I. suis.Biester and Murray described I. suis from pigs in 1934 (10–12). However, it was not recognized as a major cause of diseasein nursing piglets until the early 1970s (167, 171). This probablyreflects the modernization of the swine production industryand the use of confinement facilities for the farrowing (birthing)of piglets.Clinical Signs and PathogenicityCoccidiosis in pigs is a severe disease of nursing piglets (52,168). I. suis is the cause of neonatal porcine coccidiosis (167).There are no reports of coccidiosis caused by I. almataensis orI. neyrai. Eimeria species do not cause clinical coccidiosis innursing pigs (106). Neonatal porcine coccidiosis caused by I.suis is ubiquitous where pigs are farrowed in confinement (21,25, 31, 51, 99, 139, 152, 156, 157) and is responsible for 15 to20% of the cases of piglet diarrhea seen at diagnostic laboratoriesin the United States, Canada, and other countries. Outbreaksof coccidiosis occur year-round. I. suis can be seen innursing piglets suffering from other neonatal diarrheal diseases,and it increases the severity of disease caused by theseagents (135, 150, 151, 171).Infected piglets develop diarrhea at 7 to 14 days of age. Thediarrhea is yellowish to gray and initially pasty but becomesfluid after 2 to 3 days; blood is never present if I. suis is the onlyinfectious agent. If blood is present, other agents are involvedas primary or copathogens. Piglets become covered with diarrheticfeces, causing them to stay damp and smell like souredmilk. They become lethargic but continue to nurse. Infectionsfail to respond to commonly used antibiotics. Piglets within alitter and all litters in the farrowing house are not equallyaffected by coccidiosis. Morbidity is high, and mortality is moderate.Microscopic changes consist of villous atrophy, villousfusion, necrotic enteritis, and crypt hyperplasia (52, 74, 173,183). Experimental studies indicate that the development ofclinical disease and microscopic lesions are dependent uponthe number of oocysts inoculated and the age at which pigletsare inoculated (13, 86, 112, 153, 172, 173). Doses of 5 10 4oocysts or less generally produce diarrhea but no mortality inyoung (1- to 3-day-old) piglets, doses of 7 10 4 to 3 10 5oocysts cause low to moderate mortality, and doses of 4 10 5or greater cause high mortality in young piglets. Weight gainsof infected piglets are depressed (111).There is some evidence that I. suis may cause postweaningdiarrhea in 5- to 6-week-old piglets (138), with diarrhea beginning4 to 7 days after the piglets are weaned. Morbidity is 80 to90%, but mortality is very low.Endogenous stages are found throughout the small intestineand occasionally in the cecum and colon (73, 113, 123, 173).Parasite densities are highest in the jejunum and ileum. Developmentalstages are found in enterocytes. Two types ofmeronts are produced (113). Type 1 meronts are binucleateand divide by endodyogeny (122, 123), whereas type 2 merontsare multinucleate. Both types of meronts are motile and canactively exit and penetrate host cells (110). The prepatentperiod is 4 to 5 days, and the patent period is 2 weeks or longer.Several peaks in oocyst numbers can occur during the patentperiod (21, 73, 184). Extraintestinal stages of I. suis have notbeen found by microscopic examination of tissues from infectedpiglets or in experimentally inoculated mice (73, 123,148, 170). Oral feeding of mouse or pig tissues was inconclusivein one study (170). Transmission occurred following intraperitonealinoculation of intestinal lymph node homogenates orhomogenates of pooled spleen and liver from pigs inoculated 1or 2 days previously with large numbers (5 10 7 and 1 10 7oocysts, respectively) of I. suis oocysts (73). The prepatentperiod was 10 to 12 days in the recipient pigs.The role of viral and bacterial copathogens with I. suis hasbeen examined experimentally (5, 185). The responses of gnotobioticand conventional pigs to I. suis and rotavirus coinfectionare similar (185). The degree of observed clinical diseaseis more severe when the pathogens are administered concurrentlythan when either is given singly. Both the virus and theparasite develop preferentially in the enterocytes of the centraland distal portion of the villi in the small intestine, and competitionfor a suitable host cell is believed to be the cause of theobserved increase in clinical disease and microscopic lesions.An established I. suis infection will interfere with the establishmentof a Salmonella typhimurium infection (5). The increasedgut motility and destruction of host cells probablyDownloaded from http://cmr.asm.org/ on February 5, 2014 by guest

VOL. 10, 1997 BIOLOGY OF ISOSPORA SPP. 31interfere with the ability of the bacterium to colonize the intestinalmucosa.ImmunityPiglets that recover from I. suis infection exhibit a strongdegree of resistance to reinfection. No clinical signs developafter challenge, and few or no oocysts are excreted in the feces(174). Colostral antibodies against I. suis do not protect pigletsfrom developing clinical coccidiosis (177). Antibody levels inserum peak about 1 week after primary infection, and a secondaryantibody response occurs following challenge infection.Serum antibodies against I. suis do not recognize sporozoitesof Eimeria debliecki, E. neodebliecki, E. scabra,orE. porci in anindirect fluorescent-antibody test. Lymphocyte migration inhibitionresponses of pigs that are immune to I. suis are significantlylower than those of controls when soluble or particulateI. suis sporozoite antigens are used. Polymorphonuclear leukocyte(PMN) chemotactic factors were generated by lymphocytesfrom piglets inoculated with I. suis and incubated withsoluble or particulate sporozoite antigens. Lymphocytes fromcontrol pigs did not produce chemotactic factors for PMNsafter incubation with I. suis sporozoite antigens, and the antigensalone were not chemotactic for PMNs.EpidemiologyThe epidemiology of neonatal porcine isosporiasis is puzzling.Sows are often infected with Eimeria species, but theprevalence of I. suis is usually less than 5% (50, 69, 114, 182).The sow is a logical source of infection for newborn piglets, butstudies conducted in the United States have failed to demonstrateI. suis oocysts in a significant number of sows (50, 114,168). I. suis oocysts were not found in the feces of 77 sowsexamined from 7 farms with a problem of neonatal coccidiosiscaused by I. suis, and only 1 of 172 sows examined from 27farms without a history of neonatal coccidiosis was positive(114). Eimeria oocysts were found in 91% of these sows. Inanother study, sows from two farms with neonatal coccidiosisin piglets were examined on a daily basis for about 1 week priorto farrowing, at farrowing, and for about 2 weeks postfarrowing(168). I. suis oocysts were not found in these sows; however,piglets nursing from these sows developed coccidiosis and excretedI. suis oocysts at 4 to 8 days of age. Microscopic examinationof milk samples and placentas from these sows werenegative for parasites. Once I. suis is established on a farm, itis probably maintained by infection of piglets from the contaminatedfarrowing crate.DiagnosisDiagnosis is based on a clinical history suggestive of coccidiosisand the demonstration of I. suis oocysts in fecal samplesor the demonstration of developmental stages in mucosalsmears or histological sections obtained from necropsy specimens(101). Samples for oocyst identification should be takenfrom pigs that have had diarrhea for 2 days or more becauseclinical signs often appear before oocysts are excreted in thefeces (173). Pasty fecal samples are likely to contain moreoocysts than are liquid samples. Fecal fat makes identificationof oocysts in Sheather’s sugar flotation preparations difficult. Asolution of saturated sodium chloride and glucose has beenrecommended as an alternative flotation medium (79).The use of mucosal imprints stained with any Giemsa-typestain is a reliable method for diagnosing porcine coccidiosis(101). Imprints should be taken from the jejunum or ileumbecause these are the sites where parasite densities are highest.The presence of paired merozoites indicative of division byendodyogeny is characteristic for I. suis in pigs (101). Histologicalsections taken from the jejunum or ileum also containdevelopmental stages in the enterocytes.Treatment and ControlAnticoccidial treatment of piglets has generally proven unrewarding.Nursing piglets do not eat or drink enough to makeantibiotics added to feed or water useful. Catching each pigletfor dosing is time-consuming and labor-intensive and probablypractical only on small farms. Controlled studies indicate thatamprolium, monensin, and furazolidone are not effective inpreventing coccidiosis in nursing piglets (29, 67). Toltrazurildoes show promise as an effective means of preventing coccidiosisin nursing piglets (30). When 20 to 30 mg of toltrazuril/kg wasgiven orally as a single dose to 3- to 6-day-old piglets, coccidiosiswas reduced from 71 to 22%. The severity of diarrhea and oocystexcretion was reduced in toltrazuril-treated piglets.Lasalocid and halofuginone have been evaluated in earlyweanedpigs experimentally infected with I. suis (118, 124).Lasalocid given at 150 mg/kg of feed prevented weight loss inpigs but did not prevent oocyst excretion. These pigs developedstrong immunity to reinfection. Halofuginone given at 6 mg/kgof feed inhibited oocyst production but caused reduced weightgains due to poor feed intake. The halofuginone-treated pigsdid not develop strong immunity to challenge infection.Improved sanitation is the best means of controlling neonatalcoccidiosis (50). Feeding anticoccidial agents to sows is notrecommended because they are not the source of fecal oocystsfor their nursing piglets. Commercially available disinfectantsdo not inhibit the development of I. suis oocysts when used atthe concentrations recommended by the manufacturers (169).Once the oocysts sporulate, they are even more resistant todisinfectants. Steam cleaning is effective in killing sporulatedand unsporulated oocysts and is an effective means of decreasingpiglet exposure to infective I. suis oocysts. Additional preventivemeasures are for farm workers to limit their access toinfected piglets to prevent crate-to-crate spread of infection viatheir boots. Also, flies and other insects should be controlled toprevent them from mechanically carrying oocysts from crate tocrate. Supportive measures such as providing water or electrolytesolutions in piglet waters may be of value in preventingdehydration in clinically infected piglets.FUTURE DIRECTIONSFuture studies on human I. belli infections need to examinethe latent tissue cyst stage of the parasite. It is most probablyresponsible for the recrudescence of clinical disease which isobserved in AIDS and other patients. It is obvious that thetissue cyst stage is not killed by treatment with TMP-SMX. Anin vitro culture system needs to be developed to examine potentialanticoccidial agents against I. belli and to study tissuecyst biology. Experimental inoculation of rodents with I. bellioocysts needs to be examined to determine whether latentinfections develop in these animals. If rodents are found to beparatenic hosts, potentially other food animals may also becapable of transmitting the infection.Additional studies on dog and cat Isospora spp. should beconducted to determine if these coccidia are truly pathogenicin them. Studies with neonatal porcine coccidiosis should focuson identifying effective treatments and vaccines.REFERENCES1. Andrews, J. M. 1926. Coccidiosis in mammals. Am. J. Hyg. 6:784–794.2. Arcay, L. 1967. 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