HISTOMONAS MELEAGRIDIS INFECTIONS IN TURKEYS IN THE USA: A CENTURY OF PROGRESS, RESURGENCE, AND TRIBUTE TO ITS EARLY INVESTIGATORS, THEOBALD SMITH, ERNST TYZZER, AND EVERETT LUND.

Histomoniasis, caused by the protozoan, Histomonas meleagridis, is an economically important disease of turkeys, and it also affects several other species of domesticated and wild Galliformes, including chickens. Under natural conditions, the parasite is transmitted through eggs of a nematode, Heterakis gallinarum, that shares its hosts with Hi. meleagridis. The protozoan infects tissues of both male and female He. gallinarum and eventually is carried within the worm egg. Histomonas meleagridis more readily infects and develops in chickens, and the proximity of chicken farms is a major risk factor for outbreaks in turkeys. Chemoprophylaxis had controlled Hi. meleagridis in turkeys very successfully, but histomoniasis has recently reemerged in turkeys because anti-histomonal drugs are no longer permitted by the United States Food and Drug Administration because of the concerns for residual toxins in poultry meat. Horizontal transmission of the protozoan in the absence of worm eggs remains a mystery because the flagellate trophozoite excreted in the feces of turkeys is not viable for any length of time. A proposed resistant stage of the protozoan has not yet been conclusively demonstrated. Here we review the discovery of the protozoan and the current status of the disease and its control.

An optimized assay for detecting Encephalitozoon intestinalis and Enterocytozoon bieneusi in dairy calf feces using polymerase chain reaction technology.

The purpose of this study was to optimize primary and nested polymerase chain reaction (PCR) assays for detecting the microsporidia Encephalitozoon intestinalis and Enterocytozoon bieneusi in fecal samples from dairy calves. PCR for these microsporidia were compared to immunofluorescence assays (IFA) based on commercially available monoclonal antibodies specific for outer wall proteins of Enc. intestinalis or Ent. bieneusi. Fecal samples were collected from 15 dairy calves and processed by molecular sieving followed by salt floatation to recover Enc. intestinalis and Ent. bieneusi spores. An aliquot of the final supernatant was applied to glass slides for IFA testing; another aliquot was extracted for total DNA using a QIAamp Stool Mini-Kit for primary and nested Enc. intestinalis- and Ent. bieneusi-specific PCR analysis. Internal standards were generated for both Enc. intestinalis and Ent. bieneusi PCR assays to control for false negative reactions due to the presence of inhibitors commonly found in fecal samples. Using the commercial MicrosporIFA (Waterborne, Inc.) as the gold standard, the optimized Enc. intestinalis PCR method provided 85.7% sensitivity and 100% specificity with a kappa value = 0.865. Likewise, using the commercial BienusiGlo IFA (Waterborne, Inc.) as the gold standard, the optimized Ent. bieneusi PCR method provided 83.3% sensitivity and 100% specificity with a kappa value = 0.857. Sequencing of amplicons from both PCR assays confirmed the presence of Enc. intestinalis or Ent. bieneusi. In conclusion, our optimized assays for recovering and detecting Enc. intestinalis or Ent. bieneusi in feces from dairy calves provides a valuable alternative to traditional IFA methods that require expertise to identify extremely small microsporidia spores (~ 2.0 µm). Our assays also improve upon existing molecular detection techniques for these microsporidia by incorporating an internal standard to control for false negative reactions.

Viable Eimeria oocysts in poultry house litter at the time of chick placement.

The purpose of this study was to determine if Eimeria oocysts recovered from litter at the time of chick placement in commercial broiler houses contained oocysts that were infectious for chickens. Over 100 litter samples were collected from 30 poultry farms representing a total of 60 different broiler houses with 9 houses sampled more than once over 1.5 yr. The samples were collected just before the placement of newly hatched chicks and after an anticoccidial drug (ACD) or Eimeria vaccine (VAC) program, and processed for counting oocysts followed by Eimeria species determination using ITS1 PCR. Broiler chicks were inoculated with recovered Eimeria oocysts to determine if the litter oocysts were viable and capable of causing patent infection. At placement, E. maxima (Emax) oocysts were detected in 70 of 75 houses after ACD program and 46 of 47 houses after VAC program. Eimeria acervulina, E. praecox, and/or E. tenella (Eapt) were detected in 75 of 75 houses after ACD program and 47 of 47 houses after VAC program. Viability testing revealed that 33.0% of broiler houses contained viable Emax oocysts, while 46.9% contained viable Eapt oocysts. During VAC programs, the concentration of Emax oocysts at placement and the total number of Emax oocysts shed by chickens in viability studies showed a very strong correlation (r = 0.83). Likewise, during ACD programs, the concentration of Eapt oocysts at placement and the total number of Eapt oocysts shed by chickens in the viability study showed a strong correlation (r = 0.62). In general, Eimeria oocyst levels at placement and number of viable oocysts shed by chickens in the viability study were similar among houses on the same farm. However, the number of Eimeria oocysts shed in the viability studies was considerably less than expected based on the number of oocysts given. These data suggest that nearly 100% of all poultry houses contain Emax and Eapt oocysts at placement with 30 to 50% of the houses containing viable Eimeria oocysts, thus possibly representing a source of the protozoa to newly hatched chicks.

Differential expression of intestinal nutrient transporters and host defense peptides in Eimeria maxima-infected Fayoumi and Ross chickens.

Fayoumi chickens are believed to be more disease resistant compared to commercial broiler chickens. The objective of this study was to compare mRNA expression of intestinal nutrient transporters, digestive enzymes, and host defense peptides (HDP) between Eimeria maxima-challenged Fayoumi and Ross broiler chickens. At 21 d of age, Ross broilers and Fayoumi lines M5.1 and M15.2 were challenged with 1,000 E. maxima oocysts. Control birds were not challenged. Duodenum, jejunum, and ileum were sampled (n = 6) at 7 d post challenge. Gene expression was analyzed using relative quantification PCR. Data were analyzed by ANOVA and significance level was set at P < 0.05. There was numerical, but not statistically significant, differential weight gain depression for Ross (15%) and Fayoumi lines M5.1 (21%) and M15.2 (22%) and significant line-specific changes in gene expression. For nutrient transporters, there was downregulation of mRNA for the brush border membrane, amino acid transporters b0,+AT/rBAT, BoAT, and EAAT3 in different segments of the small intestine of Ross and both lines of Fayoumi chickens, indicating that E. maxima challenge likely caused a decrease in nutrient uptake. For HDP, there was downregulation of avian beta defensin (AvBD) 1, 6, 10, 12, and 13 mRNA in the jejunum of the 2 Fayoumi lines, but no change in the Ross broilers. In the duodenum, there was upregulation of AvBD10 mRNA in the Ross and both Fayoumi lines and additionally upregulation of AvBD11, 12, and 13 mRNA in only Fayoumi line M15.2. Liver expressed antimicrobial peptide 2 (LEAP2) mRNA was downregulated in the duodenum and jejunum of Ross and Fayoumi line M5.1 but not in Fayoumi line M15.2. The homeostatic, non-challenged levels of AvBD mRNA were greater in Fayoumi line M15.2 than Ross and Fayoumi line M5.1 in the duodenum and ileum. This study demonstrates tissue- and genetic line-specific transcriptional responses to E. maxima, highlights novel potential candidate genes for response to coccidiosis, and confirms a role for several previously reported genes in response to coccidiosis.

Re-evaluation of the life cycle of Eimeria maxima Tyzzer, 1929 in chickens (Gallus domesticus).

A time-course study was conducted to resolve discrepancies in the literature and better define aspects of the Eimeria maxima life cycle such, as sites of development and both morphology and number of asexual stages. Broiler chickens were inoculated orally with five million E. maxima oocysts (APU1), and were necropsied at regular intervals from 12 to 120 h p.i. Small intestine tissue sections and smears were examined for developmental stages. The jejunum contained the highest numbers of developmental stages. At 12 h p.i., sporozoites were observed inside a parasitophorous vacuole (PV) in the epithelial villi and the lamina propria. By 24 h, sporozoites enclosed by a PV were observed in enterocytes of the glands of Lieberkühn. At 48 h p.i., sporozoites, elongated immature and mature schizonts, were all seen in the glands with merozoites budding off from a residual body. By 60 h, second-generation, sausage-shaped schizonts containing up to 12 merozoites were observed around a residual body in the villar tip of invaded enterocytes. At 72 and 96 h, profuse schizogony associated with third- and fourth-generation schizonts was observed throughout the villus. At 120 h, another generation (fifth) of schizonts were seen in villar tips as well as in subepithelium where gamonts and oocysts were also present; a few gamonts were in epithelium. Our finding of maximum parasitization of E. maxima in jejunum is important because this region is critical for nutrient absorption and weight gain.

Expression of host defense peptides in the intestine of Eimeria-challenged chickens.

Avian coccidiosis is caused by the intracellular protozoan Eimeria, which produces intestinal lesions leading to weight gain depression. Current control methods include vaccination and anticoccidial drugs. An alternative approach involves modulating the immune system. The objective of this study was to profile the expression of host defense peptides such as avian beta-defensins (AvBDs) and liver expressed antimicrobial peptide 2 (LEAP2), which are part of the innate immune system. The mRNA expression of AvBD family members 1, 6, 8, 10, 11, 12, and 13 and LEAP2 was examined in chickens challenged with either E. acervulina, E. maxima, or E. tenella. The duodenum, jejunum, ileum, and ceca were collected 7 d post challenge. In study 1, E. acervulina challenge resulted in down-regulation of AvBD1, AvBD6, AvBD10, AvBD11, AvBD12, and AvBD13 in the duodenum. E. maxima challenge caused down-regulation of AvBD6, AvBD10, and AvBD11 in the duodenum, down-regulation of AvBD10 in the jejunum, but up-regulation of AvBD8 and AvBD13 in the ceca. E. tenella challenge showed no change in AvBD expression in any tissue. In study 2, which involved challenge with only E. maxima, there was down-regulation of AvBD1 in the ileum, AvBD11 in the jejunum and ileum, and LEAP2 in all 3 segments of the small intestine. The expression of LEAP2 was further examined by in situ hybridization in the jejunum of chickens from study 2. LEAP2 mRNA was expressed similarly in the enterocytes lining the villi, but not in the crypts of control and Eimeria challenged chickens. The lengths of the villi in the Eimeria challenged chickens were less than those in the control chickens, which may in part account for the observed down-regulation of LEAP2 mRNA quantified by PCR. Overall, the AvBD response to Eimeria challenge was not consistent; whereas LEAP2 was consistently down-regulated, which suggests that LEAP2 plays an important role in modulating an Eimeria infection.

Changes in expression of an antimicrobial peptide, digestive enzymes, and nutrient transporters in the intestine of E. praecox-infected chickens.

Coccidiosis is a major intestinal disease of poultry, caused by several species of the protozoan Eimeria. The objective of this study was to examine changes in expression of digestive enzymes, nutrient transporters, and an antimicrobial peptide following an Eimeria praecox challenge of chickens at days 3 and 6 post-infection. Gene expression was determined by real-time PCR and analyzed by one-way ANOVA. In the duodenum, the primary site of E. praecox infection, a number of genes were downregulated at both d3 and d6 post-infection. These genes included liver expressed antimicrobial peptide 2 (LEAP2), the cationic (CAT1), anionic (EAAT3), and L-type (LAT1) amino acid transporters, the peptide transporter PepT1 and the zinc transporter ZnT1. Other transporters were downregulated either at d3 or d6. At both d3 and d6, there was downregulation of B(o)AT and CAT1 in the jejunum and downregulation of LEAP2 and LAT1 in the ileum. LEAP2, EAAT3, and ZnT1 have been found to be downregulated following challenge with other Eimeria species, suggesting a common cellular response to Eimeria.

Characterization of the Eimeria maxima sporozoite surface protein IMP1.

The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection.

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.

Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers.

Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. The site of invasion and lesions in the intestine is species-specific, for example E. acervulina affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain. The growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to compare the expression of digestive enzymes, nutrient transporters and an antimicrobial peptide in broilers challenged with either E. acervulina, E. maxima or E. tenella. The genes examined included digestive enzymes (APN and SI), peptide and amino acid transporters (PepT1, ASCT1, b(0,+)AT/rBAT, B(0)AT, CAT1, CAT2, EAAT3, LAT1, y(+)LAT1 and y(+)LAT2), sugar transporters (GLUT1, GLUT2, GLUT5 and SGLT1), zinc transporter (ZnT1) and an antimicrobial peptide (LEAP2). Duodenum, jejunum, ileum and ceca were collected 7 days post challenge. E. acervulina challenge resulted in downregulation of various nutrient transporters or LEAP2 in the duodenum and ceca, but not the jejunum or ileum. E. maxima challenge produced both downregulation and upregulation of nutrient transporters and LEAP2 in all three segments of the small intestine and ceca. E. tenella challenge resulted in the downregulation and upregulation of nutrient transporters and LEAP2 in the jejunum, ileum and ceca, but not the duodenum. At the respective target tissue, E. acervulina, E. maxima and E. tenella infection caused common downregulation of APN, b(0,+)AT, rBAT, EAAT3, SI, GLUT2, GLUT5, ZnT1 and LEAP2. The downregulation of nutrient transporters would result in a decrease in the efficiency of protein and polysaccharide digestion and uptake, which may partially explain the weight loss. The downregulation of nutrient transporters may also be a cellular response to reduced expression of the host defense protein LEAP2, which would diminish intracellular pools of nutrients and inhibit pathogen replication.

A rapid method for determining salinomycin and monensin sensitivity in Eimeria tenella.

Standard methods of determining the ionophore sensitivity of Eimeria rely on infecting chickens with an isolate or a mixture of Eimeria spp. oocysts in the presence of different anti-coccidial drugs. The purpose of this study was to develop a rapid in vitro method for assessing salinomycin and monensin sensitivity in Eimeria tenella. Cultures of MDBK cells were grown to 85% confluency, and then inoculated with excysted E. tenella laboratory strain (APU-1) sporozoites in the presence of different concentrations of salinomycin or monensin. At various timepoints, the monolayers were fixed for counting intraceullar sporozoites, or were subjected to DNA extraction, followed by molecular analysis using quantitative (qPCR) or semi-quantitative PCR (sqPCR). Preliminary experiments showed that 24h was the optimum time for harvesting the E. tenella-infected cell cultures. The average number of E. tenella sporozoites relative to untreated controls displayed a linear decrease between 0.3 and 33.0 µg/ml salinomycin and between 0.3 and 3.3 µg/ml monensin. A similar pattern was observed in the relative amount of E. tenella DNA as measured by sqPCR. A linear decrease in the relative amount of E. tenella DNA was observed over the entire range of salinomycin and monensin concentrations as measured by qPCR possibly reflecting the greater sensitivity of this assay. Comparison of sporozoite counting, sqPCR, and qPCR signals using a criterion of 50% inhibition in sporozoite numbers or level of PCR amplification product showed good agreement between the three assays. E. tenella field isolates (FS-1 and FS-2) displaying resistance to salinomycin and monensin were evaluated in the in vitro assay using qPCR and sqPCR. Compared to E. tenella APU-1, the E. tenella FS-1 and FS-2 isolates showed higher levels of E. tenella DNA at 24h by both qPCR and sqPCR. This in vitro assay represents a significant advance in developing rapid, cost-effective methods for assessing ionophore sensitivity in E. tenella.

Expression of digestive enzymes and nutrient transporters in Eimeria acervulina-challenged layers and broilers.

Avian coccidiosis is a disease caused by intestinal protozoa in the genus Eimeria. Clinical signs of coccidiosis include intestinal lesions and reduced feed efficiency and BW gain. This growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to examine the differential expression of digestive enzymes, transporters of amino acids, peptides, sugars, and minerals, and an antimicrobial peptide in the small intestine of Eimeria acervulina-infected broilers and layers. Uninfected broilers and layers, in general, expressed these genes at comparable levels. Some differences included 3-fold and 2-fold greater expression of the peptide transporter PepT1 and the antimicrobial peptide LEAP2 (liver expressed antimicrobial peptide 2), respectively, in the jejunum of layers compared with broilers and 17-fold greater expression of LEAP2 in the duodenum of broilers compared with layers. In the duodenum of Eimeria-infected broilers and layers, there was downregulation of aminopeptidase N; sucrase-isomaltase; the neutral, cationic, and anionic amino acid transporters b(o,+)AT/rBAT, B(o)AT, CAT2, and EAAT3; the sugar transporter GLUT2; the zinc transporter ZnT1; and LEAP2. In the jejunum of infected layers there was downregulation of many of the same genes as in the duodenum plus downregulation of PepT1, b(o,+)AT/rBAT, and the y(+) L system amino acid transporters y(+) LAT1 and y(+) LAT2. In the ileum of infected layers there was downregulation of CAT2, y(+)LAT1, the L type amino acid transporter LAT1, and the sugar transporter GLUT1, and upregulation of APN, PepT1, the sodium glucose transporter SGLT4, and LEAP2. In E. acervulina-infected broilers, there were no gene expression changes in the jejunum and ileum. These changes in intestinal digestive enzyme and nutrient transporter gene expression may result in a decrease in the efficiency of protein digestion, uptake of important amino acids and sugars, and disruption of mineral balance that may affect intestinal cell metabolism and Eimeria replication.

Isolation of viable Neospora caninum from brains of wild gray wolves (Canis lupus).

Neospora caninum is a common cause of abortion in cattle worldwide. Canids, including the dog and the dingo (Canis familiaris), the coyote (Canis latrans), and the gray wolf (Canis lupus) are its definitive hosts that can excrete environmentally resistant oocysts in the environment, but also can act as intermediate hosts, harboring tissue stages of the parasite. In an attempt to isolate viable N. caninum from tissues of naturally infected wolves, brain and heart tissue from 109 wolves from Minnesota were bioassayed in mice. Viable N. caninum (NcWolfMn1, NcWolfMn2) was isolated from the brains of two wolves by bioassays in interferon gamma gene knockout mice. DNA obtained from culture-derived N. caninum tachyzoites of the two isolates were analyzed by N. caninum-specific Nc5 polymerase chain reaction and confirmed diagnosis. This is the first report of isolation of N. caninum from tissues of any wild canid host.

Congenital transmission of Neospora caninum in white-tailed deer (Odocoileus virginianus).

Neosporosis is an important cause of bovine abortion worldwide. Many aspects of transmission of Neospora caninum in nature are unknown. The white-tailed deer (Odocoileus virginianus) is considered one of the most important wildlife reservoirs of N. caninum in the USA. During the hunting seasons of 2008, 2009, and 2010, brains of 155 white-tailed deer fetuses were bioassayed in mice for protozoal isolation. Viable N. caninum (NcWTDMn1, NcWTDMn2) was isolated from the brains of two fetuses by bioassays in mice, and subsequent propagation in cell culture. Dams of these two infected fetuses had antibodies to N. caninum by Neospora agglutination test at 1:100 serum dilution. DNA obtained from culture-derived N. caninum tachyzoites of the two isolates with Nc5 PCR confirmed diagnosis. Results prove congenital transmission of N. caninum in the white tailed deer for the first time.

Modulation of the intestinal environment, innate immune response, and barrier function by dietary threonine and purified fiber during a coccidiosis challenge in broiler chicks.

Coccidiosis is a major contributor to economic losses in the poultry industry due to its detrimental effects on growth performance and nutrient utilization. We hypothesized that the combined effects of supplemental dietary Thr and purified fiber may modulate the intestinal environment and positively affect intestinal immune responses and barrier function in broiler chicks infected with Eimeria maxima. A Thr-deficient basal diet (3.1 g of Thr/kg of diet) was supplemented with 70 g/kg of silica sand (control) or high-methoxy pectin and 1 of 2 concentrations of Thr (1.8 or 5.3 g/kg of diet; 4 diets total), and fed to chicks from hatch to d 16 posthatch. On d 10 posthatch, chicks received 0.5 mL of distilled water or an acute dose of Eimeria maxima (1.5 × 10(3) sporulated oocytes) with 6 replicate pens of 6 chicks per each of 8 treatment combinations (4 diets and 2 inoculation states). Body weight gain, feed intake, and G:F increased (P < 0.01) with addition of 5.3 g of Thr/kg of diet. Eimeria maxima schizonts were present only in intestinal tissue sampled from infected birds (P < 0.01). Weights of cecal digesta were highest (P < 0.01) in pectin-fed birds, and ceca with the heaviest weights also had the highest concentrations of total short-chain fatty acids. Expression of interleukin-12 in ileal mucosa was highest (P < 0.01) in infected birds receiving the control diet with 5.3 g of supplemental Thr/kg. In cecal tonsils, interferon-γ expression was highest in infected birds receiving the control diet (fiber × infection, P < 0.05); interferon-γ expression was lowest in infected birds fed the high Thr diet (Thr × infection, P < 0.05). There were no differences due to infection or Thr supplementation for cytokine expression in birds fed pectin-containing treatments. Overall, we conclude that although pectin has some protective function against coccidiosis, Thr supplementation had the greatest effect on intestinal immune response and maintenance of near normal growth in young broiler chicks infected with E. maxima.

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.

Does gravity cause load-bearing bridges in colloidal and granular systems?

We study structures which can bear loads, "bridges", in particulate packings. To investigate the relationship between bridges and gravity, we experimentally determine bridge statistics in colloidal packings. We vary the effective magnitude and direction of gravity, volume fraction, and interactions, and find that the bridge size distributions depend only on the mean number of neighbors. We identify a universal distribution, in agreement with simulation results for granulars, suggesting that applied loads merely exploit preexisting bridges, which are inherent in dense packings.

Gray wolf (Canis lupus) is a natural definitive host for Neospora caninum.

The gray wolf (Canis lupus) was found to be a new natural definitive host for Neospora caninum. Neospora-like oocysts were found microscopically in the feces of three of 73 wolves from Minnesota examined at necropsy. N. caninum-specific DNA was amplified from the oocysts of all three wolves. Oocysts from one wolf were infective for the gamma interferon gene knock out (KO) mice. Viable N. caninum (designated NcWolfUS1) was isolated in cell cultures seeded with tissue homogenate from the infected mouse. Typical thick walled tissue cysts were found in outbred mice inoculated with the parasite from the KO mouse. Tissue stages in mice stained positively with N. caninum-specific polyclonal antibodies. Our observation suggests that wolves may be an important link in the sylvatic cycle of N. caninum.

Effect of corn distillers dried grains with solubles and Eimeria acervulina infection on growth performance and the intestinal microbiota of young chicks.

Chicks were used to determine whether dietary corn distillers dried grains with solubles (DDGS) may prevent or ameliorate Eimeria acervulina (EA) infection. The experiment had a completely randomized design with a factorial arrangement of 3 diets (inclusion of 0, 10, or 20% DDGS) × 2 challenge treatments: inoculation with distilled water or with 10(6) sporulated EA oocysts. Each treatment was replicated with 8 pens of 5 chicks each. Experimental diets were fed from 7 to 21 d of age. Inoculation occurred on d 10 of age, considered postinoculation (PI) d 0. Feed intake and BW were measured on PI d 0, 7, and 14. Excreta samples were collected on PI d 0, 5 to 10, 12, and 14 to detect oocysts. On PI d 14, mucosal samples were collected for the analysis of bacterial populations by denaturing gradient gel electrophoresis, using the V3 region of bacterial 16S ribosome. The EA challenge reduced (P < 0.001) ADG by 17%, ADFI by 12%, and G:F by 6% from PI d 0 to 7, and by smaller percentages from PI d 7 to 14. Diet and challenge treatments did not interact in the chick performance, so dietary DDGS did not alleviate EA infection. Oocysts in excreta were detected PI only in EA chicks and no dietary effects were found. Cecal bacterial population was changed (P < 0.05) by effect of dietary DDGS and EA infection. The cecal bacterial diversity among chicks within treatments and homogeneity among chicks within treatments were reduced by EA infection (P = 0.02 to 0.001) and increased by feeding 10% DDGS (diet quadratic, P < 0.001). In summary, feeding up to 20% DDGS to young chicks did not prevent or ameliorate EA infection. Changes in cecal microbiota of chicks fed 10% DDGS can be interpreted as beneficial for intestinal health.