[Show abstract][Hide abstract] ABSTRACT: 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 72kDa protein or a 61kDa 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 48h 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.
Published by Elsevier B.V.
[Show abstract][Hide abstract] ABSTRACT: The epithelial lining of the airway tract and allergen-specific IgE are considered essential controllers of inflammatory responses to allergens. The human low affinity IgE receptor, CD23 (FcɛRII), is capable of transporting IgE or IgE-allergen complexes across the polarized human airway epithelial cell (AEC) monolayer in vitro. However, it remains unknown whether the CD23-dependent IgE transfer pathway in AECs initiates and facilitates allergic inflammation in vivo, and whether inhibition of this pathway attenuates allergic inflammation. To this end, we show that in wild-type (WT) mice, epithelial CD23 transcytosed both IgE and ovalbumin (OVA)-IgE complexes across the airway epithelial barrier, whereas neither type of transcytosis was observed in CD23 knockout (KO) mice. In chimeric mice, OVA sensitization and aerosol challenge of WT/WT (bone-marrow transfer from the WT to WT) or CD23KO/WT (CD23KO to WT) chimeric mice, which express CD23 on radioresistant airway structural cells (mainly epithelial cells) resulted in airway eosinophilia, including collagen deposition and a significant increase in goblet cells, and increased airway hyperreactivity. In contrast, the absence of CD23 expression on airway structural or epithelial cells, but not on hematopoietic cells, in WT/CD23KO (the WT to CD23KO) chimeric mice significantly reduced OVA-driven allergic airway inflammation. In addition, inhalation of the CD23-blocking B3B4 antibody in sensitized WT mice before or during airway challenge suppressed the salient features of asthma, including bronchial hyperreactivity. Taken together, these results identify a previously unproven mechanism in which epithelial CD23 plays a central role in the development of allergic inflammation. Further, our study suggests that functional inhibition of CD23 in the airway is a potential therapeutic approach to inhibit the development of asthma.Mucosal Immunology advance online publication, 18 March 2015; doi:10.1038/mi.2015.16.
[Show abstract][Hide abstract] ABSTRACT: RESUMEN Protección en pollos de engorde alojados con cohortes inmunizados contra Eimeria maxima y E. acervulina. El uso de vacunas con ooquistes vivos es cada vez más importante en el control de la coccidiosis aviar en pollos de engorde. El conocimiento de los mecanismos empleados cuando los pollos ingieren los ooquistes y adquieren inmunidad es importante para optimizar los métodos de aplicación de vacunas vivas. El presente estudio pone a prueba la hipótesis de que pollos no inmunizados previamente pueden ingerir ooquistes mediante el contacto con cama con ooquistes eliminados por pollos cohortes inmunizados. En el experimento 1, pollos de engorde de un día fueron alojados en corrales que contenían cama limpia. En el Ensayo 1, el 100% de los pollos en algunos de los corrales fueron inmunizados con 2.5 × 103 ooquistes de Eimeria acervulina, mientras que en otros corrales se inmunizó sólo al 75% de los pollos y el resto de cohortes dentro de los corrales no fueron inmunizados. Otros corrales contenían pollos que sirvieron como controles no inmunizados y no desafiados, o como controles no inmunizados y desafiados (NIC). En el día 21, las aves se expusieron a un desafío homólogo de 6 × 105 ooquistes. Un segundo ensayo idéntico se llevó a cabo, pero las aves fueron inmunizadas con 500 ooquistes de Eimeria maxima y se desafieron con 3 × 103 ooquistes de E. maxima. En el Experimento 2, el 100% de los pollos en algunos corrales se inmunizaron con 500 oocistos de E. acervulina, mientras que en otros corrales se inmunizaron ya sea el 75% o el 50% de las aves. En el día 14, las aves fueron desafiadas con 1 × 106 ooquistes. El ensayo 2 fue idéntico al ensayo 1, excepto que las aves fueron inmunizadas con 100 ooquistes de E. maxima y desafiados con 1 × 106 ooquistes. En todos los experimentos se midió el aumento de peso, la conversión alimenticia (FCR), carotenoides en plasma y conteos de ooquistes en la cama. En el Experimento 1, el nivel de protección en los grupos que contenían un 25% de los cohortes no inmunizados, de acuerdo a las determinaciones de aumento de peso, nivel de carotenoides, conversión alimenticia y por conteos de ooquistes en la cama, fue idéntico a los grupos que contenían 100% de los pollitos inmunizados. En el Experimento 2, los corrales donde 50% o 75% de las aves fueron inmunizados con E. maxima o con E. acervulina no estuvieron bien protegidos contra disminuciones en la ganancia de peso y de carotenoides plasmáticos, ni contra aumentos en los recuentos de ooquistes en la cama después del desafío en el día 14 en comparación con el grupo control no inmunizado y desafiado. Además, los corrales en donde se inmunizó al 100% de los pollos no estaban bien protegidos en comparación con el grupo control no inmunizado y desafiado y la resistencia a la infección por coccidiosis en pollos inmunizados fue menor que la resistencia observada en los pollos desafiados a los 21 días. En conjunto, estos resultados sugieren que cuando las aves se desafían después de los 21 días, los cohortes están protegidos de los efectos perjudiciales del desafío. Sin embargo, cuando el desafío se realizó a los 14 días, las aves cohortes no estuvieron bien protegidas. Los resultados apoyan la conclusión de que la protección contra la coccidiosis se transmite a las aves cohortes por el contacto con ooquistes en la cama diseminados por los pollos inmunizados, pero esta resistencia puede tomar 14 días para desarrollarse.
[Show abstract][Hide abstract] ABSTRACT: 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.
Parasitology Research 02/2015; 114(6). DOI:10.1007/s00436-015-4390-6 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 E. 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 24 hr 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 3 assays. Eimeria 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 24 hr 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.
[Show abstract][Hide abstract] ABSTRACT: The uptake of amino acids is mediated by active transporters located on the basolateral and brush border membranes of intestinal epithelial cells. The current study investigated the expression of amino acid transporters (AAT) and other genes in the intestine of chicks infected with Eimeria maxima. At 7-day postinfection (PI), tissue from each intestinal segment (duodenum, jejunum, and ileum) was taken from birds inoculated with 3 × 10(3) oocysts/bird and processed to recover RNA. Analysis of gene expression was performed using real-time reverse transcription polymerase chain reaction (qRT-PCR). Results were given as relative expression using β2-microglobulin as an endogenous control. All the genes studied were expressed in three segments of the intestines, and expression of the genes was altered by infection with E. maxima. Even though the jejunum is considered the parasite's primary predilection site, there was no segment-related difference in expression of most of the genes studied. The antimicrobial peptide (LEAP2) was downregulated in all three segments of the intestine. The results also demonstrate that transporters associated with brush border membranes were downregulated while transporters associated with the basolateral membranes were upregulated and that E. maxima alters the expression of AAT and LEAP2 throughout the small intestine.
Parasitology Research 09/2014; 113(10). DOI:10.1007/s00436-014-4114-3 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The current study investigates the use of irradiated oocysts to protect broiler chicks, raised on litter, from infection with multiple species of Eimeria. In order to determine the optimum radiation dose for each Eimeria species, 1-day-old chicks were immunized with oocysts of Eimeria maxima, Eimeria acervulina, or Eimeria tenella exposed to gamma radiation ranging from 0-500 Gy. The litter oocyst counts at 7 days postimmunization, and the effect on weight gain following a challenge infection, decreased with an optimum dose between 150-200 Gy. Based on this finding, broiler chicks were immunized with a mixture of E. maxima, E. acervulina, and E tenella that had been exposed to 150 or 200 Gy. This resulted in more than a 100-fold reduction in litter oocyst counts and significant protection from a challenge infection, as measured by improved weight gain and feed conversion ratio (FCR). Immunization of birds with oocysts receiving 200 Gy was less effective in providing protection from a challenge infection. An additional formulation of vaccines containing two different oocyst doses of the three species that had been irradiated with 150 Gy were evaluated in their ability to attenuate oocyst output and convey protection to challenge. Results were similar with both high and low numbers of irradiated oocysts. Immunized chicks shed less oocysts at 7 days postimmunization and were protected from negative effects of challenge infection as measured by FCR, changes in weight gain, lesion scores, and measurement of body composition. However, the level of protection was somewhat less than that achieved by immunization with nonirradiated oocysts. The overall conclusion is that an irradiated oocyst vaccine developed in this study can effectively protect chicks that are raised on litter from challenge infection with multiple species of Eimeria, comparable to vaccines with virulent or precocious strains.
[Show abstract][Hide abstract] ABSTRACT: Macrophage migration inhibitory factor (MIF) is recognized as a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. Our group has identified both chicken and Eimeria MIFs, and characterized their function in enhancing innate immune responses during inflammation. In this study, we report that chicken CD74 (ChCD74), a type II transmembrane protein, functions as a macrophage surface receptor that binds to MIF molecules. First, to examine the binding of MIF to chicken monocytes/macrophages, fresh isolated chicken peripheral blood mononuclear cells (PBMCs) were stimulated with rChIFN-γ and then incubated with recombinant chicken MIF (rChMIF). Immunofluorescence staining with anti-ChMIF followed by flow cytometry revealed the binding of MIF to stimulated PBMCs. To verify that ChCD74 acts as a surface receptor for MIF molecules, full-length ChCD74p41 was cloned, expressed and its recombinant protein (rChCD74p41) transiently over-expressed with green fluorescent protein in chicken fibroblast DF-1 cells. Fluorescence analysis revealed a higher population of cells double positive for CD74p41 and rChMIF, indicating the binding of rChMIF to DF-1 cells via rChCD74p41. Using a similar approach, it was found that Eimeria MIF (EMIF), which is secreted by Eimeria sp. during infection, bound to chicken macrophages via ChCD74p41 as a surface receptor. Together, this study provides conclusive evidence that both host and parasite MIF molecules bind to chicken macrophages via the surface receptor ChCD74.
Developmental and comparative immunology 07/2014; 47(2):319-326. · 2.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Abstract Previous studies comparing the genome sequences of Cryptosporidium parvum with C. hominis identified a number of highly divergent genes that might reflect positive selection for host specificity. In the present study, the C. parvum DNA sequence cgd8-5370, that encodes a protein whose amino acid sequence differs appreciably from its homologue in C. hominis, was cloned by PCR and expressed as a recombinant protein in Escherichia coli. Antisera raised against the recombinant cgd8-5370 antigen strongly recognized a unique 33 kDa protein in immunoblots from reducing and non-reducing SDS-PAGE of native C. parvum protein. However, anti-Cp33 sera did not recognize the native 33 kDa homologue in C. hominis. In an immunofluorescence assay (IFA), anti-Cp33 serum recognized an antigen in the anterior end of air-dried C. parvum sporozoites, but failed to bind at any sites in C. hominis sporozoites, indicating its specificity for C. parvum. IFA staining of live C. parvum sporozoites with anti-Cp33 serum failed to bind to the parasite, indicating that the CP33 antigen is not on the sporozoite surface, which is consistent with topology predictions based on the encoded amino acid sequence. RT-PCR analysis of cgd8-5370 mRNA before or during C. parvum oocyst excystation revealed transcripts only in excysting sporozoites. Thus, Cp33 represents one of a small number of proteins shown to differentiate C. parvum from C. hominis sporozoites and oocysts.
Journal of Parasitology 03/2014; 100(4). DOI:10.1645/13-433.1 · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 PCR and confirmed diagnosis. This is the first report of isolation of N. caninum from tissues of any wild canid host.
[Show abstract][Hide abstract] ABSTRACT: Control of avian coccidiosis is increasingly being achieved by the administration of low doses of Eimeria oocysts to newly hatched chicks. The purpose of this study was to test the efficacy of gel beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine to protect broilers raised in contact with litter. Newly hatched chicks were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel beads. Control, 1-day-old chicks were given an equivalent number of Eimeria oocysts (10(3) total) by oral gavage or received no vaccine (nonimmunized controls). All chicks were raised in floor-pen cages in direct contact with litter. At 4 wk of age, all chickens and a control nonimmunized group received a high-dose E. acervulina, E. maxima, and E. tenella challenge infection. Chickens immunized with Eimeria oocysts in gel beads or by spray vaccination displayed significantly (P < 0.05) greater weight gain (WG) compared to nonimmunized controls. Feed conversion ratio (FCR) also showed a significant (P < 0.05) improvement in both groups relative to nonimmunized controls. Moreover, WG and FCR in both groups was not significantly different (P > 0.05) from chickens immunized by oral gavage or from nonimmunized, noninfected controls. Oocyst excretion after Eimeria challenge by all immunized groups was about 10-fold less than in nonimmunized controls. These findings indicate that immunization efficacy of gel beads and spray vaccination is improved by raising immunized chicks in contact with litter.
[Show abstract][Hide abstract] ABSTRACT: The present study is the first characterization of Neospora caninum macrophage migration inhibitory factor (NcMIF). BLAST-N analysis of NcMIF revealed high similarity (87%) to the Toxoplasma gondii MIF. NcMIF was cloned and expressed in Escherichia coli in 3 forms, NcMIF (mature protein), NcMIFm (mutation of proline-2 to glycine), and NcMIFhis (addition of a polyhistidine tag at the N-terminus). None of these recombinant NcMIFs (rNcMIF) had tautomerase, oxidoreductase, or immunologic regulatory activities. rNcMIF was unable to compete with recombinant human MIF for a MIF receptor (CD74), suggesting that NcMIF does not bind to this MIF receptor. The glycine substitution for proline-2 of NcMIF resulted in increased retention time on SEC-HPLC and decreased formation of dimers and trimers. The addition of N-terminal HIS-tag led to increased formation of trimers. Immunofluorescence staining demonstrated that NcMIF was localized to the apical end of N. caninum tachyzoites. Immunoelectron microscopy further revealed that NcMIF was present in the micronemes, rhoptries, dense granules, and nuclei. NcMIF was abundant in the tachyzoite lysate and present in excretory and secretory antigen (ESAg) preparations. Total and secretory NcMIF was more abundant in a non-pathologic clone, Ncts-8, than in the wild type isolate (NC1). Furthermore, NcMIF release by the both isolates was increased in the presence of calcium ionophore. This differential production of NcMIF by the pathologic and non-pathologic isolates of N. caninum may suggest a critical role of this molecule in the infectious pathogenesis of this parasite.
[Show abstract][Hide abstract] ABSTRACT: A recently completed analysis of Eimeria maxima transcriptome identified a gene with homology to sequences expressed by E. tenella and E. acervulina but lacking homology with other organisms including other apicomplexans. This gene, designated Eimeria-specific protein (ESP), codes for a protein with a predicted molecular weight of 19 kDa. The ESP gene was cloned and the recombinant protein expressed in bacteria and purified for preparation of specific antisera. Quantitative RT-PCR showed transcription of ESP was low in unsporulated oocysts and after 24 h of sporulation. However, transcription nearly doubled after 48 h of sporulation and reached its highest levels in sporozoites (SZ) and merozoites (MZ). The protein was detectable by Western blot in both sporulated oocysts and in SZ and MZ. Immuno-localization by light microscopy identified ESP in paired structures in the anterior of SZ and MZ. Immuno-localization by electron microscopy identified ESP in MZ rhoptries but no specific staining of any SZ structures was detected. In addition, localization studies on intestinal sections recovered from birds 120-h post-infection indicates that oocysts do not stain with anti-ESP but staining of microgametocytes and developing oocysts was observed. The results indicate that ESP is associated with the rhoptry of E. maxima and that the protein may have functions in other developmental stages.
Parasitology Research 07/2013; 112(10). DOI:10.1007/s00436-013-3518-9 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Macrophage migration inhibitory factor (MIF) is recognized as a soluble factor produced by sensitized T lymphocytes and inhibits the random migration of macrophages. Recent research shows a more prominent role of MIF as a multi-functional cytokine mediating both innate and adaptive immune responses. Our group has identified both chicken and Eimeria MIF, and characterized its molecular function in enhancing innate immune responses during inflammation. In this study, we report that chicken CD74 (ChCD74), a type II transmembrane protein, functions as a macrophage surface receptor that binds to MIF molecules. The results of flow cytometry and fluorescent microscopy showed that incubation of macrophages with recombinant chicken MIF (rChMIF) led to binding of rChMIF on the surface of HTC cells. To verify that ChCD74 acts as a surface receptor for MIF molecules, the recombinant form of ChCD74 (rChCD74) was transiently over-expressed with green fluorescent protein at its N-terminus in HEC cells. Fluorescence analysis showed that incubation of HTC cells transiently over-expressing rChCD74 with rChMIF showed co-localization of the two molecules. These results were confirmed by immunoprecipitation assay, indicating their close interaction. Since Eimeria sp. also produce and secrete Eimeria MIF (EMIF) molecules during infection, we examined the binding of rEMIF to chicken macrophages via ChCD74. Our analysis showed binding of rEMIF to chicken macrophages via rChCD74 as a surface receptor. Together, this study concludes that both host and parasite MIF molecules bind to chicken macrophages via the surface receptor ChCD74.
Poultry Science Association Annual Meeting, San Diego, CA, US; 07/2013
[Show abstract][Hide abstract] ABSTRACT: A number of parameters have been used to assess the impact ofcoccidiosis on chickens in clinical settings as well as in experimental studies. However, a rapid way to determine body composition would be useful to evaluate or compare responses to coccidia and could give further insight into the metabolic impact of infection. The current study evaluates the use of dual X-ray absorptiometry (DEXA) to determine the impact of coccidiosis on body composition in chicks receiving inoculations with single or mixed species of Eimeria. Chicks infected with Eimeria maxima, Eimeria acervulina, or Eimeria tenella had altered parameters of body composition as measured by DEXA at 6 days postinfection (PI). The greatest effects were noted in birds infected with E. acervulina or E. maxima, where lean mass and fat were reduced from control values about 75% and 85%, respectively. In chicks infected with E. tenella, tissue and fat were reduced about 10%. Bone mineral content (BMC) was about 75% of control values in birds infected with E. acervulina or E. maxima, but only E. acervulina altered bone mineral density (BMD). The decreases in BMC and BMD are likely due to malabsorption. In chicks receiving a mixed coccidian infection, all DEXA parameters were significantly decreased at 8 days PI compared with age-matched controls. As with single infections, BMD and BMC were significantly depressed (P < 0.05). Values of all DEXA parameters were near 92% of control values by day 16 PI. Analysis of all birds in the current study indicates DEXA tissue weight slightly underestimated the gravimetrically measured weight by about 3%. The current results demonstrate that DEXA is a potentially important tool for the rapid evaluation of the effect of coccidiosis on broiler chicks and suggest it can be useful for evaluation of vaccines and other disease controls.
[Show abstract][Hide abstract] ABSTRACT: Abstract Outbreaks of avian coccidiosis may occur when susceptible chickens are raised on litter containing viable Eimeria oocysts. The purpose of this study was to compare the relative sensitivities of E. acervulina, E. maxima, and E. tenella oocysts to dessication. Sporulated E. acervulina, E. maxima, or E. tenella oocysts were incorporated into gelatin beads, and incubated at 32oC for 0, 1, 2, or 3 days. In vitro oocyst excystation rates were measured for each combination of Eimeria species and incubation time. Day-old broiler chicks were allowed to ingest the oocysts-containing beads, and total oocyst production was measured from days 5-8 post-inoculation. Although no effect on excystation was observed, E. maxima oocysts displayed greater resistance to drying compared to E. acervulina and E. tenella oocysts. Eimeria acervulina oocyst production decreased 100-fold after 1-2 days incubation. E. tenella oocysts were slightly more resistant to drying in that a 100-fold decrease in oocyst production was delayed until 2 days. For both E. acervulina and E. tenella, very few oocysts were observed after 3 days incubation. Eimeria maxima oocyst production remained high at all timepoints. Subsequent studies revealed E. maxima oocyst production was ablated only after 5 days incubation. These findings may explain in part the observed prevalence of E. maxima in litter from commercial poultry operations.
Journal of Parasitology 04/2013; 99(5). DOI:10.1645/13-192.1 · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Macrophage migration inhibitory factor (MIF) is a soluble factor produced by sensitized T lymphocytes that inhibits the random migration of macrophages. Homologues of MIF from invertebrates have been identified, making it an interesting molecule from a functional perspective. In the present study, the localization of a parasite MIF protein as well as its effect on the host was characterized. Western blot analysis shows that Eimeria MIF (EMIF) is found during all parasite developmental stages tested. Transmission electron microscopy shows that MIF is distributed throughout cytosol and nucleus of Eimeria acervulina merozoites. Immunohistochemical analysis suggests that EMIF may be released into the surrounding tissues as early as 24 h after infection, while later during oocyst formation, MIF expression is localized to areas immediately surrounding the oocysts, as well as in wall-forming bodies. The chemotaxis assay revealed an inhibitory function of EMIF on chicken monocyte migration. Quantitative real-time PCR was performed to examine the effect of EMIF on host immune system by measuring the transcripts of inflammatory mediators. An ex vivo stimulation study showed that E. acervulina MIF (EaMIF) enhanced expression of pro-inflammatory cytokines and chemokines in the presence of lipopolysaccharide (LPS). Furthermore, sequential treatment of adherent peripheral blood mononuclear cells with EaMIF, chicken MIF, and LPS in 2-h intervals led to the highest levels of interleukin (IL)-1B, chemokine CCLi3, IL-18, and interferon-gamma mRNA expression. This study shows that parasite MIF is widely expressed and may have potential effects on the immune system of the host.
Parasitology Research 02/2013; 112(5). DOI:10.1007/s00436-013-3345-z · 2.10 Impact Factor