Changes in the levels of Cryspovirus during in vitro development of Cryptosporidium parvum.

The purpose of this study was to develop and utilize semi-quantitative RT-PCR and PCR assays for measuring the level of Cryspovirus, the viral symbiont of Cryptosporidium parvum, during in vitro development of the protozoan. Cultures of human carcinoma cells (HCT-8) were inoculated with excysting C. parvum sporozoites, followed by harvest of cells and culture medium at 2-, 24-, 48-, and 72-h post-infection. Changes in viral RNA levels were detected by RT-PCR using primers specific for RNA encoding the 40-kDa capsid protein (CP) or RNA-dependent RNA polymerase (RdRp). Parasite or host DNA was quantified by PCR specific for C. parvum or human glyceraldehyde-3-phosphate dehydrogenase (HuGAPDH). An internal standard (competitor) was incorporated into all assays as a control for PCR inhibition. Intracellular levels of C. parvum DNA increased between 2- and 48-h post-infection, and then decreased at 72 h. Culture medium overlying these C. parvum-infected cells displayed a similar increase in CP and RdRp signal, reaching peak levels at 48 h. However, the CP and RdRp levels in cellular RNA displayed only a modest increase between 2 and 48 h, and exhibited no change (CP) or decreased (RdRp) at 72 h. These data suggest that during the first 48 h of C. parvum in vitro development, Cryspovirus is released into the media overlying cells but remains at fairly constant levels within infected cells.

First report of Enterocytozoon bieneusi in pigs in Brazil.

Although Brazil is the world's fourth largest producer and exporter of pork, there is no information on Enterocytozoon bieneusi in pigs. This study was undertaken to determine the presence of E. bieneusi in pigs in the State of Rio de Janeiro, Brazil. Fecal samples were collected from 91 pigs (1- to 12-month-old) in 10 properties and examined by molecular methods. The presence of E. bieneusi was determined by PCR and all PCR positive specimens were sequenced to determine the genotype by nucleotide sequence analysis of the internal transcribed spacer of the rRNA gene. E. bieneusi was found in pigs in all farms. Fifty four (59.3%) samples were E. bieneusi-positive. A wide genetic diversity was found with 21 genotypes identified, 4 previously reported (O, EbpA, CS-1, and H) and 17 novel genotypes named PigEb1-PigEb17. All 17 novel genotypes identified in this study clustered within the previously designated zoonotic Group 1. The most prevalent genotypes were novel genotypes PigEb2 and PigEb4 (16/91, 17.6%, each). Mixed infections with 2 or 3 genotypes were detected in 13 pigs (24.1%). The high prevalence in pigs observed in this study, the description of two known zoonotic genotypes (EbpA and O), and the report of 17 new genotypes of E. bieneusi, represent an important advancement in the study of the wide genetic diversity of this organism, emphasizing the importance of further research, especially in geographical areas where little or no research has been conducted. The zoonotic risk of these novel genotypes and their importance to other animal species is still unknown, but needs to be further evaluated.

Identity of Sarcocystis species of the water buffalo (Bubalus bubalis) and cattle (Bos taurus) and the suppression of Sarcocystis sinensis as a nomen nudum.

There are uncertainties concerning the identity and host species specificity of Sarcocystis species of the water buffalo (Bubalus bubalis) and cattle (Bos taurus). Currently, in cattle three species are recognized with known endogenous stages, viz.: S. cruzi (with canine definitive host), S. hirsuta (feline definitive host), and S. hominis (primate definitive host). Recently, a fourth Sarcocystis species with an unknown life cycle has been reported from cattle. In the water buffalo, four species of Sarcocystis have been described: S. fusiformis (feline definitive host), S. buffalonis (feline definitive host), S. levinei (canine definitive host), and S. dubeyi (definitive host unknown but not cat or dog). Besides, there are studies of Sarcocystis infections in buffalo and cattle from China with results that are difficult to interpret and validate. For example, some of the studies report transmission of Sarcocystis species between cattle and buffalo, but steps to preclude exogenous exposures were not reported. A species of the water buffalo, 'S. sinensis', was proposed at a Chinese national conference in 1990, and published as an abstract without figures and with no archived type specimens for verification. The International Code of Zoological Nomenclature Articles 9 and 10 state that "abstracts of articles, papers, posters, text of lectures, and similar material when issued primarily to participants at meetings, symposia, colloquia or congress does not constitute published work"; therefore, S. sinensis is a nomen nudum.

Blastocystis tropism in the pig intestine.

Blastocystis has been reported in pig feces but the sites of development in the gastrointestinal tract are unknown. The present study was undertaken to determine predilection sites of Blastocystis in 11 naturally infected pigs examined at 20 weeks of age. At necropsy, feces and contents of the duodenum, jejunum, ileum, and cecum were examined by immunofluorescence (IFA) microscopy and PCR and tissues from these sites as well as the proximal and distal colon were processed for histology from pigs 1 to 5. Feces were examined by IFA microscopy, and segments from the jejunum and ileum were processed for histology from pigs 6 to 11. Multiple sections were cut from each tissue segment, and each was stained with the following: hematoxylin and eosin, polyclonal rabbit antibody to Blastocystis, and ParaFlor B monoclonal antibody to Blastocystis. Blastocystis was detected in feces of all 11 pigs by IFA microscopy and determined by PCR and gene sequencing to be subtype 5 for pigs 1-5. Blastocystis was also detected in the lumen contents removed from the cecum of pigs 1-5 examined by IFA microscopy and in the cecum of pigs 4 and 5 by PCR. Blastocystis was also observed in tissue sections from the jejunum of 7 of the 11 pigs, in the proximal and distal colon of pigs 1-5, and in the cecum of 4 of these 5 pigs but was not detected in the duodenum or ileum of any pigs. In tissue sections, Blastocystis was found primarily in the lumen usually associated with digested food debris, sometimes in close proximity or appearing to adhere to the epithelium, but no stages were found to penetrate the epithelium or the lamina propria.

Glucagon-like peptide 2 therapy reduces negative effects of diarrhea on calf gut.

Damage to the intestinal epithelium reduces nutrient absorption and animal growth, and can have negative long-term health effects on livestock. Because the intestinotropic hormone glucagon-like peptide 2 (GLP-2) has been shown to contribute to gut integrity, reduce inflammation, and improve nutrient absorption, the present study was designed to determine whether administration of GLP-2 to calves with coccidiosis in the first month of life affects intestinal growth and mediates negative effects of the proinflammatory response. Holstein bull calves (n=19) were assigned to 4 treatment groups of 4 to 5 calves each: (1) infected with Eimeria bovis, GLP-2 treated; (2) noninfected, GLP-2 treated; (3) infected with E. bovis, buffer treated; and (4) noninfected, buffer treated. Infected calves received 100,000 to 200,000 sporulated E. bovis oocysts suspended in milk replacer on d 0 of the study. On d 18, calves in the GLP-2 groups received a subcutaneous injection of 50 µg of bovine GLP-2/kg of body weight twice daily for 10 d, and calves in the buffer-treated groups received an equivalent volume of sodium bicarbonate buffer only. On d 28, calves were slaughtered 2h after injection of 5-bromo-2'-deoxyuridine (BrdU). Intestinal tissues were measured and villus height, crypt depth, and BrdU immunostaining were evaluated in segments of the small intestine. Nitrotyrosine immunostaining, a measure of nitro-oxidative damage, was evaluated in the ileum and cecum. No GLP-2 treatment by E. bovis infection interaction was observed for any parameter measured, with the exception of nitrotyrosine immunostaining in the cecum. Large intestinal weight was greater in infected than noninfected calves and with GLP-2 treatment relative to buffer treatment. Calves that received GLP-2 also had greater small intestinal weight but no difference in cell proliferation, as assessed by BrdU labeling, relative to buffer-treated calves. No treatment effects were detected for villus height, crypt depth, or villus height:crypt depth ratio in segments of the small intestine. Protein tyrosine nitration was over 3-fold greater in the ileum and cecum of infected calves relative to noninfected calves, and GLP-2 therapy reduced tyrosine nitration in infected calves by 47% in the ileum and 69% in the cecum relative to buffer-treated calves. Treatment with GLP-2 promotes intestinal growth in neonatal calves and reduces the detrimental effects of nitro-oxidative stress in the ileocecum of calves with coccidiosis.

Analysis of giardin expression during encystation of Giardia lamblia.

The present study analyzed giardin transcription in trophozoites and cysts during encystation of Giardia lamblia . Encystment was induced using standard methods, and the numbers of trophozoites and cysts were counted at various time points during the process. At all time points, RNA from both stages were assayed for levels of alpha2-, beta-, and delta-giardin mRNA as well as for cyst wall protein 3 (CWP3) mRNA using quantitative RT-PCR. In encystation medium, the number of G. lamblia trophozoites decreased, while the number of cysts increased between 0 and 72 hr. In trophozoites, alpha2- and beta-giardin transcription decreased over time, while delta-giardin transcription remained unchanged during the same time period. CWP3 transcription exhibited a slight increase in trophozoites at 8 hr, followed by a decrease at subsequent time points. Expression of alpha2-giardin increased at 48 hr in cysts followed by decreased expression at 72 hr, while beta- and delta-giardin expression was unchanged during encystation. CWP3 transcription gradually decreased from 24-72 hr in cysts. Consistent with previous studies, giardin proteins appeared to be disassembled into amorphous structures inside cysts during encystation. These findings represent the first analysis of giardin transcription in separate populations of trophozoites and cysts during encystation and indicate differential regulation of giardin mRNA expression by these developmental stages.

Occurrence of Cryptosporidium andersoni in Brazilian cattle.

Feces were collected from 68 dairy cattle, 1 to 12 mo of age, on 12 farms in the municipality of Campos dos Goytacazes, Rio de Janeiro, Brazil, and examined for the presence of Cryptosporidium sp. All samples were subjected to molecular analysis by polymerase chain reaction (nested PCR) of the 18S rRNA. Four positive samples (4.54%) were sequenced and identified as Cryptosporidium andersoni. This species represents a risk for Brazilian cattle because infection can affect cattle productivity. Moreover, C. andersoni is considered a zoonotic species.

Cryptosporidium pig genotype II diagnosed in pigs from the state of Rio De Janeiro, Brazil.

Pigs may represent a source of Cryptosporidium sp. infection to humans. The objective of this study was to identify the Cryptosporidium species present in pigs from the State of Rio de Janeiro, Brazil, and verify what risks pigs represent in the transmission of human cryptosporidiosis, because there is no such information to date in Brazil. Ninety-one samples of pig feces were collected from 10 piggeries in 2 municipalities located in the north and northwest regions of the State of Rio de Janeiro, Brazil. A nested polymerase chain reaction (PCR) protocol to amplify an 830-bp fragment of the small subunit rDNA (SSU rRNA) gene was followed by sequencing of all positive PCR samples. Two samples (2.2%) were Cryptosporidium sp. positive and were identified as pig genotype type II (PGII). This genotype has been observed in an immunocompetent person, in cattle without pigs nearby, and from a potential human source. Its potential for zoonotic transmission is little known and should be rigorously studied.

Detection and comparison of Giardia virus (GLV) from different assemblages of Giardia duodenalis.

Five assemblages of Giardia duodenalis were identified from cysts in cattle, dog, cat, sheep, and reindeer feces using ribosomal DNA (rDNA) sequencing. Assemblage A was present in cattle and reindeer feces, Assemblages C and D were present in dog feces, Assemblage E was present in cattle and sheep feces, and Assemblage F was present in cat feces. Giardia virus, originally referred to as Giardia lamblia virus (GLV), is a double-stranded RNA virus. Primers designed for the GLV capsid protein gene identified GLV sequences in G. lamblia from a reindeer (Assemblage A) and from a dog (Assemblage C). Two distinct GLV sequences were identified in the dog specimen and 1 sequence was identified in the reindeer specimen. None of these GLV sequences was identical with previously published GLV sequences. It appears that GLVs are genetically diverse and that more than 1 virion can be present in a single sample. Because many of the specimens that contained cysts were found to be negative for GLV, it appears that this test for capsid protein is of limited value for the purposes of detecting G. lamblia.

Examination of naturally exposed bottlenose dolphins (Tursiops truncatus) for Microsporidia, Cryptosporidium, and Giardia.

Bottlenose dolphins (Tursiops truncatus) captured in the estuarine waters off the coasts of South Carolina and Florida were examined for the presence of Microsporidia, Cryptosporidium sp., and Giardia sp. DNA extracted from feces or rectal swabs was amplified by polymerase chain reaction using parasite-specific small subunit ribosomal RNA gene primers. All positive specimens were subjected to gene sequence analysis. Of 83 dolphins, 17 were positive for Microsporidia. None was positive for Cryptosporidium or Giardia. Gene sequence data for each of the positive specimens were compared with data in GenBank. Fourteen specimens were found similar to, but not identical to, the microsporidian species Kabatana takedai, Tetramicra brevifilum, and Microgemma tinca, reported from fish, and possibly represent parasites of fish eaten by dolphins. Gene sequence data from 3 other specimens had approximately 87% similarity to Enterocytozoon bieneusi, a species known primarily to infect humans and a variety of terrestrial mammals, including livestock, companion animals, and wildlife. It is not clear if these specimens represent a species from a terrestrial source or a closely related species unique to dolphins. There were neither clinical signs nor age- or gender-related patterns apparent with the presence of these organisms.

Fecundity of Cryptosporidium parvum is correlated with intracellular levels of the viral symbiont CPV.

Differences in the virulence and fecundity of Cryptosporidium parvum isolates have been observed by several researchers studying cryptosporidiosis. The purpose of the present study was to determine if there was a correlation between intracellular levels of the viral symbiont CPV in C. parvum and fecundity of two isolates of the parasite, namely C. parvum Beltsville (B) and C. parvum Iowa (I). Dairy calves infected with 10(6)C. parvum-B excreted 5-fold more oocysts compared with calves infected with the same number of C. parvum-I oocysts. The increased fecundity of the former strain was corroborated by semi-quantitative PCR assay of DNA isolated from cell cultures infected with either C. parvum-B or C. parvum-I. Quantitative reverse transcriptase-PCR analysis of viral RNA revealed a 3-fold greater number of CPV in C. parvum-B compared with C. parvum-I oocysts. These findings may indicate a role for CPV in fecundity and possibly virulence of C. parvum.

Enterocytozoon bieneusi in mature dairy cattle on farms in the eastern United States.

Fecal specimens were obtained from mature milking cows on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Polymerase chain reaction (PCR)-positive specimens for Enterocytozoon bieneusi were found in 24 of 541 cows examined (4.4%) and on 12 of 14 farms. The prevalence of E. bieneusi varied considerably from farm to farm, with the lowest prevalence (2.3%) on FL-2 and the highest prevalence (12.5%) on VT-2. None of the cows exhibited signs of diarrhea. All PCR-positive specimens were sequenced to determine the genotype of E. bieneusi. Five genotypes were identified. Three were identified as cattle-specific genotypes previously reported as BEB1, BEB2, and BEB4, and two new genotypes, BEB 6 and BEB7, were found. None have been reported to infect humans.

Enterocytozoon bieneusi genotypes in dairy cattle in the eastern United States.

Fecal specimens were obtained from 12-24-month-old dairy heifers on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. PCR positive specimens for Enterocytozoon bieneusi were found in 131 of 571 heifers examined (23%) and on all the farms visited. The prevalence of E. bieneusi varied considerably across farms, with the lowest prevalence (4.7%) on MD-2 and the highest prevalence (37.8%) on NY-2. All PCR positive specimens that amplified the ITS region as well as a portion of the flanking large and small subunit ribosomal RNA genes were sequenced to determine the genotype(s) of the E. bieneusi present and six genotypes were identified. Most were identified as cattle-specific genotypes, previously reported from cattle as BEB1, BEB2, BEB3, and BEB4. Two isolates were genetically identical or similar to E. bienesusi reported as the human pathogens Peru 6 and Peru 9 (or D) genotypes. Although our data demonstrate the presence of zoonotic genotypes in cattle, most genotypes found in cattle were host specific.

A comparison of several serologic tests to detect antibodies to Toxoplasma gondii in naturally exposed bottlenose dolphins (Tursiops truncatus).

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. In a previous study, 138 of 141 (97.8%) bottlenose dolphins (Tursiops truncatus) from the coasts of Florida and California had antibodies to T. gondii by the modified agglutination test (MAT). Although the MAT has been found to be highly sensitive and specific for T. gondii antibodies from several species of terrestrial animals, it has not yet been validated for T. gondii infections in marine mammals. Furthermore, T. gondii has yet not been isolated from dolphins. In the present study, sera from 146 (60 from the 2004 samples and 86 from the 2003 samples) T. truncatus from the coastal areas of South Carolina and Florida were tested for antibodies to T. gondii. Sera from 2004 were tested by the MAT, the indirect fluorescent antibody test (IFAT), the Sabin-Feldman dye test (DT), an indirect hemagglutination test (IHAT), an enzyme-linked immunosorbent assay (ELISA), and Western blot. All 60 dolphins were seropositive, with MAT titers of 1:20 in 3, 1:40 in 19, 1:80 in 29, 1:160 in 2, 1:1,280 in 3, 1:2,560 in 2, and 1:5,120 or higher in 2, and these results were confirmed in another laboratory. The DT titers of these dolphins were <1:10 in 53, 1:800 in 3, 1:1,600 in 2, and 1:3,200 in 2. The IHAT titers were <1:64 in 52, 1:128 in 1, 1:512 in 2, and 1:2,048 in 5. The IFAT titers were <1:20 in 3, 1:20 in 11, 1:40 in 36, 1:80 in 2, 1:160 in 1, and 1:320 or higher in 7. All 7 DT-positive dolphins had high MAT titers, but 2 were negative by the IHAT. Western blot results closely followed MAT results; ELISA results matched MAT results, which were 1:40 or higher. In sera from the 2003 samples, MAT antibodies were found in 86 of 86 dolphins with titers of 1:25 in 29, 1:50 in 23, 1:100 in 27, 1:200 in 3, 1:1,600 in 1, and 1:3,200 in 3; these sera were not tested by other means. Overall, MAT antibodies were found in all 146 dolphin sera tested. Because marine mammals are considered sentinel animals indicative of contamination of the coastal and marine waters by T. gondii oocysts, serologically positive infections need to be validated by the detection of T. gondii organisms in the tissues of seropositive animals.

Effect of hydrogen peroxide and other protease inhibitors on Cryptosporidium parvum excystation and in vitro development.

This study was undertaken to observe the effects of hydrogen peroxide on Cryptosporidium parvum oocysts with respect to protease activity in comparison to known protease inhibitors. In assessing the possible mechanisms of action of hydrogen peroxide, treatment effectiveness was analyzed using 3 assays and the potential roles of proteases and cations were considered. Treatment of C. parvum oocysts with hydrogen peroxide inhibited protease activity up to 50% compared with untreated controls. Treatment of oocysts with chemicals that affect sulfhydryls, including N-ethylmaleimide and dithiolthreitol, inhibited protease activity by >90%. Treatment of oocysts with these chemicals, along with the protease inhibitors, phenylmethylsulfonyl fluoride (PMSF), ethylenediamine-tetraacetic acid, and cystatin, inhibited protease activity as well as in vitro excystation and infection in a cell culture assay. Several mechanisms may result in the successful inhibition of infection and excystation by hydrogen peroxide treatment, including: oxidation of oocyst wall proteins or lipids, chelating of cations necessary for infection, or hydroxyl radical-induced DNA damage to sporozoites, or both.

Characterization and potential use of a Cryptosporidium parvum virus (CPV) antigen for detecting C. parvum oocysts.

The purpose of this study was to characterize the viral symbiont (CPV) of Cryptosporidium parvum sporozoites and evaluate the CPV capsid protein (CPV40) as a target for sensitive detection of the parasite. Recombinant CPV40 was produced in Escherichia coli, purified by affinity chromatography, and used to prepare polyclonal rabbit sera specific for the viral capsid protein. Anti-rCPV40 recognized a 40 kDa and a 30 kDa protein in C. parvum oocysts and appeared to localize to the apical end of the parasite. Anti-rCPV40 serum was capable of detecting as few as 1 C. parvum oocyst in a dot blot assay, the sensitivity being at least 1000-fold greater than sera reactive with total native C. parvum oocyst protein or specific for the 41 kDa oocyst surface antigen. Water samples were seeded with C. parvum oocysts and incubated at 4, 20, or 25 degrees C for greater than 3 months to determine if CPV levels were correlated with oocyst infectivity. Samples were removed monthly and subjected to mouse and cell culture infectivity, as well as PCR analysis for infectivity and viral particle presence. While sporozoite infectivity declined by more than 75% after 1 month at 25 degrees C, the CPV signal was similar to that of control samples at 4 degrees C. By 3 months at 20 degrees C, the C. parvum oocysts were found to be non-infectious, but retained a high CPV signal. This study indicates that CPV is an excellent target for sensitive detection of C. parvum oocysts in water, but may persist for an indefinite time after oocysts become non-infectious.

Infectivity of microsporidia spores stored in seawater at environmental temperatures.

To determine how long spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis remain viable in seawater at environmental temperatures, culture-derived spores were stored in 10, 20, and 30 ppt artificial seawater at 10 and 20 C. At intervals of 1, 2, 4, 8, and 12 wk, spores were tested for infectivity in monolayer cultures of Madin Darby bovine kidney cells. Spores of E. hellem appeared the most robust, some remaining infectious in 30 ppt seawater at 10 C for 12 wk and in 30 ppt seawater at 20 C for 2 wk. Those of E. intestinalis were slightly less robust, remaining infectious in 30 ppt seawater at 10 and 20 C for 1 and 2 wk, respectively. Spores of E. cuniculi remained infectious in 10 ppt seawater at 10 and 20 C for 2 wk but not at higher salinities. These findings indicate that the spores of the 3 species of Encephalitozoon vary in their ability to remain viable when exposed to a conservative range of salinities and temperatures found in nature but, based strictly on salinity and temperature, can potentially remain infectious long enough to become widely dispersed in estuarine and coastal waters.

Zoonotic protozoa in the marine environment: a threat to aquatic mammals and public health.

This collection of abstracts provides an account of four presentations at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP)(held in New Orleans, LA, USA from 10­14 August 2003) in a symposium session on zoonotic protozoan parasites found in the marine environment and chaired by Ronald Fayer and David Lindsay.The focus was on three genera of parasites of veterinary and public health concern­Toxoplasma,Giardia, and Cryptosporidium with emphasis on their epidemiology in the marine environment.

A redescription of Cryptosporidium galli Pavlasek, 1999 (Apicomplexa: Cryptosporidiidae) from birds.

Cryptosporidium galli Pavlasek, 1999, described from the feces of birds, is redescribed with additional molecular and biological data. Oocysts are ellipsoidal, are passed fully sporulated, lack sporocysts, and measure 8.25 x 6.3 microm (range 8.0-8.5 x 6.2-6.4 microm) with a length-width ratio of 1.30 (n = 50). Oocysts are structurally similar to those of Cryptosporidium baileyi described from chickens, but in addition to being considerably larger than oocysts of C. baileyi, these oocysts infect the proventriculus in a variety of birds and not the respiratory tract. Oocysts were successfully transmitted from chickens to chickens, and morphologically similar oocysts also were observed in a variety of exotic and wild birds (Order Passeriformes, Phasianidae, Fringillidae, and Icteridae). Molecular and phylogenetic analyses at the 18S rRNA, HSP70, and actin gene loci demonstrate that this species is genetically distinct from all known species and genotypes of Cryptosporidium and, thus, was named C. galli.

First detection of microsporidia in dairy calves in North America.

Fecal specimens were obtained from a total of 413 dairy calves from farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. After removal of fecal debris by sieving and density gradient centrifugation, specimens were examined by fluorescence microscopy, polymerase chain reaction (PCR), and DNA sequencing analysis for the presence of microsporidia. Microscopic examination revealed no spores. PCR using generic primers for microsporidia revealed 70 positive calves. PCR was then conducted using specific primers for Enterocytozoon bieneusi, the most frequently found microsporidian in human infections. These primers revealed 13 positive calves from six farms in five states. DNA sequencing analysis of the 13 E. bieneusi-positive specimens confirmed the PCR results and indicated 96.8-99.8% similarity with E. bieneusi sequences in GenBank. This is the first report of E. bieneusi in cattle in North America.