Publications by authors named "Mark C Jenkins"

45 Publications

The effects of tributyrin supplementation on weight gain and intestinal gene expression in broiler chickens during Eimeria maxima-induced coccidiosis.

Poult Sci 2021 Apr 18;100(4):100984. Epub 2021 Jan 18.

Animal Biosciences and Biotechnology Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Beltsville, MD 20705, USA. Electronic address:

Butyrate is a feed additive that has been shown to have antibacterial properties and improve gut health in broilers. Here, we examined the performance and gene expression changes in the ileum of tributyrin-supplemented broilers infected with coccidia. Ninety-six, Ross 708 broilers were fed either a control corn-soybean-based diet (-BE) or a diet supplemented with 0.25% (w/w) tributyrin (+BE). Birds were further divided into groups that were inoculated with Eimeria maxima oocysts (EM) or sham-inoculated (C) on day 21 posthatch. At 7 d postinfection (7 d PI), the peak of pathology in E. maxima infection, tributyrin-supplemented birds had significantly improved feed conversion ratios (FCR, P < 0.05) and body weight gain (BWG, P < 0.05) compared with -BE-infected birds, despite both groups having similar feed intake (FI, P > 0.05). However, at 10 d post-infection (10 d PI) no significant effects of feed type or infection were observed. Gene expression in the ileum was examined for insights into possible effects of infection and tributyrin supplementation on genes encoding proteins related to immunity, digestion, and gut barrier integrity. Among immune-related genes examined, IL-1B and LEAP2 were only significantly affected at 7 d PI. Transcription of genes related to digestion (APN, MCT1, FABP2, and MUC2) were primarily influenced by infection at 7 d PI and tributyrin supplementation (FABP2 and MUC2) at 10 d PI. With exception of ZO1, tight junction genes were affected by either infection or feed type at 7 d PI. At 10 d PI, only CLDN1 was not affected by either infection or feed type. Overall tributyrin shows promise as a supplement to improve performance during coccidiosis in broiler chickens; however, its effect on gene expression and mode of action requires further research.
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http://dx.doi.org/10.1016/j.psj.2021.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921011PMC
April 2021

Correlation Between Clostridium perfringens Alpha- and NetB-Toxin and Chick Mortality in Commercial Broiler Farms During Different Anticoccidial Control Programs.

Avian Dis 2020 09;64(3):401-406

Mountaire Farms Inc., Millsboro, DE 19966.

The purpose of the present study was to determine whether a correlation existed between chick mortality and the presence of Clostridium perfringens alpha-toxin and NetB-toxin genes (cpa and netB) in C. perfringens recovered from litter in commercial broiler houses. Because coccidiosis predisposes chickens to necrotic enteritis, the concentration of Eimeria oocysts in these samples was measured, and the numbers were used in similar correlation analyses. Litter samples were collected at 0, 2, and 4 wk growout from six broiler farms (18 houses total) during an anticoccidial drug (ACD) control program and from nine broiler farms (23 houses total) during an Eimeria vaccine (VAC) control program. Of these, litter samples were collected from five farms during both ACD and VAC programs. The litter samples were processed for Eimeria oocyst and C. perfringens spore enumerations by standard parasitologic and microbiologic techniques. DNA was also extracted for C. perfringens DNA for PCR detection of genes coding for alpha- and NetB-toxin. A general trend during the ACD programs was a transient decrease in both Eimeria maxima and non-E. maxima (Eamipt) numbers at 2 wk growout. The pattern was slightly different during VAC with E. maxima and Eamipt levels increasing over time. Average concentrations of C. perfringens in litter were highest at 2 wk (∼105-106 spores/g) during ACD and at placement during VAC (∼105-106 spores/g). During the ACD program, a strong correlation was observed between 0 and 3-wk chick mortality and the presence at placement (0 wk) of netB (r = 0.42-0.48) or cpa (r = 0.55-0.67). A very strong correlation was observed in 0-5-wk chick mortality and the presence of netB at 4 wk growout (0.73-0.95). During a VAC program, a strong correlation was only observed between the presence of netB at placement and 0-1-wk chick mortality (r = 0.67).
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http://dx.doi.org/10.1637/aviandiseases-D-19-00118DOI Listing
September 2020

Mucosal Delivery of a Self-destructing Salmonella-Based Vaccine Inducing Immunity Against Eimeria.

Avian Dis 2020 09;64(3):254-268

Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University, Tempe, AZ 85287-5401.

A programmed self-destructive Salmonella vaccine delivery system was developed to facilitate efficient colonization in host tissues that allows release of the bacterial cell contents after lysis to stimulate mucosal, systemic, and cellular immunities against a diversity of pathogens. Adoption and modification of these technological improvements could form part of an integrated strategy for cost-effective control and prevention of infectious diseases, including those caused by parasitic pathogens. Avian coccidiosis is a common poultry disease caused by Eimeria. Coccidiosis has been controlled by medicating feed with anticoccidial drugs or administering vaccines containing low doses of virulent or attenuated Eimeria oocysts. Problems of drug resistance and nonuniform administration of these Eimeria resulting in variable immunity are prompting efforts to develop recombinant Eimeria vaccines. In this study, we designed, constructed, and evaluated a self-destructing recombinant attenuated Salmonella vaccine (RASV) lysis strain synthesizing the Eimeria tenella SO7 antigen. We showed that the RASV lysis strain χ11791(pYA5293) with a ΔsifA mutation enabling escape from the Salmonella-containing vesicle (or endosome) successfully colonized chicken lymphoid tissues and induced strong mucosal and cell-mediated immunities, which are critically important for protection against Eimeria challenge. The results from animal clinical trials show that this vaccine strain significantly increased food conversion efficiency and protection against weight gain depression after challenge with 105E. tenella oocysts with concomitant decreased oocyst output. More importantly, the programmed regulated lysis feature designed into this RASV strain promotes bacterial self-clearance from the host, lessening persistence of vaccine strains in vivo and survival if excreted, which is a critically important advantage in a vaccine for livestock animals. Our approach should provide a safe, cost-effective, and efficacious vaccine to control coccidiosis upon addition of additional protective Eimeria antigens. These improved RASVs can also be modified for use to control other parasitic diseases infecting other animal species.
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http://dx.doi.org/10.1637/aviandiseases-D-19-00159DOI Listing
September 2020

Research Note: Effect of butyric acid glycerol esters on ileal and cecal mucosal and luminal microbiota in chickens challenged with Eimeria maxima.

Poult Sci 2020 Oct 3;99(10):5143-5148. Epub 2020 Jul 3.

United States Department of Agriculture, Agricultural Research Service, Animal Biosciences and Biotechnology Laboratory, Beltsville, MD 20705, U.S.A.

Coccidiosis is one of the most prevalent diseases seen in the poultry industry leading to excessive economic losses. The aim of this study was to investigate the effect of butyric acid glycerol esters (BE) on the ileal and cecal microbiota in birds challenged with Eimeria maxima (EM). Ross 708 male broilers were fed a diet supplemented with 0 (control) or 0.25% BE from day 1. On day 21, half of the birds were infected with 10 EM oocysts. For determing microbiota, ileal and cecal contents and epithelial scrapings were collected at 7 and 10 D postinfection (PI). Alpha diversity of bacterial communities was mostly affected (P < 0.05) by time PI and EM infection. The richness of luminal bacterial populations in the ileum and ceca was affected (P < 0.05) by addition of BE and by time PI × EM × BE interaction, respectively. In the ileal and cecal luminal and mucosal bacterial communities, permutational multivariate analysis of variance (PERMANOVA, unweighted UniFrac) showed significant (P < 0.05) differences because of time PI and interaction between time PI, EM, and BE. Significant (P < 0.05) differences in taxonomic composition at the family level were observed in microbiota of luminal and mucosal populations of the ileum and ceca owing to time PI, EM, BE, and their interactions. The bacterial community present in the cecal lumen was characterized by the lowest number of differential bacteria, whereas the cecal mucosal community was characterized by the highest number of differentially abundant bacteria. In conclusion, our results show that EM infection and time PI has the biggest impact on microbial diversity in the chicken gut. The presence of BE in the diet had a limited effect on gut microbiota.
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http://dx.doi.org/10.1016/j.psj.2020.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598111PMC
October 2020

Exploring Genomes to Understand Population Biology: Recent Progress and Future Opportunities.

Genes (Basel) 2020 09 21;11(9). Epub 2020 Sep 21.

Animal Parasitic Diseases Laboratory, Building 1040, Agricultural Research Service, USDA, Beltsville, MD 20705, USA.

, protozoan parasites from the phylum Apicomplexa, can cause the enteric disease coccidiosis in all farmed animals. Coccidiosis is commonly considered to be most significant in poultry; due in part to the vast number of chickens produced in the World each year, their short generation time, and the narrow profit margins associated with their production. Control of has long been dominated by routine chemoprophylaxis, but has been supplemented or replaced by live parasite vaccination in a minority of production sectors. However, public and legislative demands for reduced drug use in food production is now driving dramatic change, replacing reliance on relatively indiscriminate anticoccidial drugs with vaccines that are species-, and in some examples, strain-specific. Unfortunately, the consequences of deleterious selection on population structure and genome evolution incurred by exposure to anticoccidial drugs or vaccines are unclear. Genome sequence assemblies were published in 2014 for all seven species that infect chickens, stimulating the first population genetics studies for these economically important parasites. Here, we review current knowledge of eimerian genomes and highlight challenges posed by the discovery of new, genetically cryptic operational taxonomic units (OTUs) circulating in chicken populations. As sequencing technologies evolve understanding of eimerian genomes will improve, with notable utility for studies of biology, diversity and opportunities for control.
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http://dx.doi.org/10.3390/genes11091103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564333PMC
September 2020

Excystation of Eimeria acervulina, E. maxima, and E. tenella differs in response to trypsin and chymotrypsin and the presence of reducing agents DTT and TCEP.

Mol Biochem Parasitol 2019 10 10;233:111219. Epub 2019 Sep 10.

Animal Parasitic Diseases Laboratory, NEA, Agricultural Research Service, USDA, Beltsville, MD, 20705, United States.

Release of sporozoites from Eimeria oocysts/sporocysts is an essential step in the intracellular development of the parasite in its host. Little is known about this process except that elevated temperature (∼ 40 °C) plus trypsin and bile salts are required for sporozoite to escape from sporocysts. In this study, it was found that adding a reducing agent, either dithiothreitol (DTT) or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), increased the lifespan of sporozoites released from Eimeria maxima. While the addition of DTT or TCEP affected the apparent molecular weight of trypsin, it did not interfere with excystation of E. maxima, but rather had a positive effect on the number of viable sporozoites present after release. This effect was time-dependent in that the number of intact sporozoites at 15 and 30 min after excystation was similar between untreated and DTT- or TCEP-treated sporocysts. However, by 45-60 min, virtually no sporozoites were observed in excystation fluid not containing DTT or TCEP. Of interest is that this effect appeared to be Eimeria species-dependent. Eimeria acervulina and E. tenella sporozoites remained viable for at least 60 min after excystation in the absence of DTT or TCEP. The effect of DTT and TCEP on chymotrypsin was also studied with all 3 Eimeria species because there is some evidence that chymotrypsin is an effective excystation enzyme. Indeed, E. maxima sporozoites excysting from sporocysts with chymotrypsin in the presence of DTT or TCEP remained viable for at least 60 min after release, unlike excystation done in the absence of these reducing agents. Chymotrypsin was capable of excysting E. acervulina in the presence or absence of DTT or TCEP. Of interest, is that chymotrypsin was ineffective in the excystation of E. tenella. These findings suggest that trypsin and chymotrypsin have differential effects on sporozoite excystation and that reducing agents may alter sites on the enzyme that affect sporozoite viability, but not release from sporocysts.
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http://dx.doi.org/10.1016/j.molbiopara.2019.111219DOI Listing
October 2019

Effect of Urbanization on Neospora caninum Seroprevalence in White-Tailed Deer (Odocoileus virginianus).

Ecohealth 2019 03 9;16(1):109-115. Epub 2019 Jan 9.

Department of Veterinary Preventive Medicine, Ohio State University, 1920 Coffey Rd., Columbus, OH, 43210, USA.

The protozoan Neospora caninum is transmitted between domestic and wildlife species. Urbanized environments and deer density may facilitate this transmission and play a critical role in the spillover of N. caninum from domestic animals to wildlife. White-tailed deer (Odocoileus virginianus; WTD) are an important intermediate host for maintaining the sylvatic cycle of N. caninum in the USA. Here, we assayed serum samples from 444 WTD from a nature reservation across a suburban to urban gradient in Ohio, USA. Antibodies to N. caninum were found by using a recombinant NcGRA6 ELISA in 23.6% (105/444). Significant risk factors for seropositivity were age class and urbanization. Deer from urbanized environments were at greater odds of being seropositive (89/323, 27.6%) than those from suburban habitats (16/121, 13.2%), and this difference persisted when adjusting for age and sex. Age was also a significant risk factor with adults at greater odds to be seropositive than fawns and yearlings. We speculate the main route of exposure in WTD is ingestion of N. caninum oocysts from contaminated environments and urbanized habitats facilitate this exposure.
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http://dx.doi.org/10.1007/s10393-018-1390-xDOI Listing
March 2019

Incorporation of a recombinant Eimeria maxima IMP1 antigen into nanoparticles confers protective immunity against E. Maxima challenge infection.

Vaccine 2018 02;36(8):1126-1131

Department of Microbiology, Southern Illinois University, United States.

The purpose of this study was to determine if conjugating a recombinant Eimeria maxima protein, namely EmaxIMP1, into 20 nm polystyrene nanoparticles (NP) could improve the level of protective immunity against E. maxima challenge infection. Recombinant EmaxIMP1 was expressed in Escherichia coli as a poly-His fusion protein, purified by NiNTA chromatography, and conjugated to 20 nm polystyrene NP (NP-EmaxIMP1). NP-EMaxIMP1 or control non-recombinant (NP-NR) protein were delivered per os to newly-hatched broiler chicks with subsequent booster immunizations at 3 and 21 days of age. In battery cage studies (n = 4), chickens immunized with NP-EMaxIMP1 displayed complete protection as measured by weight gain (WG) against E. maxima challenge compared to chickens immunized with NP-NR. WG in the NP-EMaxIMP1-immunized groups was identical to WG in chickens that were not infected with E. maxima infected chickens. In floor pen studies (n = 2), chickens immunized with NP-EMaxIMP1 displayed partial protection as measured by WG against E. maxima challenge compared to chickens immunized with NP-NR. In order to understand the basis for immune stimulation, newly-hatched chicks were inoculated per os with NP-EMaxIMP1 or NP-NR protein, and the small intestine, bursa, and spleen, were examined for NP localization at 1 h and 6 h post-inoculation. Within 1 h, both NP-EMaxIMP1 and NP-NR were observed in all 3 tissues. An increase was observed in the level of NP-EmaxIMP1 and NP-NR in all tissues at 6 h post-inoculation. These data indicate that 20 nm NP-EmaxIMP1 or NP-NR reached deeper tissues within hours of oral inoculation and elicited complete to partial immunity against E. maxima challenge infection.
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http://dx.doi.org/10.1016/j.vaccine.2017.11.014DOI Listing
February 2018

Experimental Neospora caninum infection in chickens (Gallus gallus domesticus) with oocysts and tachyzoites of two recent isolates reveals resistance to infection.

Int J Parasitol 2018 02 10;48(2):117-123. Epub 2017 Sep 10.

Department of Preventive Veterinary Medicine and Animal Health, School of Veterinary Medicine, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, CEP 05508-000 São Paulo, SP, Brazil. Electronic address:

The importance of birds in the biological cycle of Neospora caninum is not clear. We report unsuccessful Neospora infection in chickens (Gallus gallus domesticus) using two isolates of N. caninum. In experiment #1, 30 White Leghorn chickens were orally inoculated with viable N. caninum oocysts (NC-SP1 isolate, 200 oocysts per bird) via the crop at 21days of age. Groups of three birds were euthanised at intervals of 7days (a total of 9weeks) and one group was challenged with the same oocyst dose at 37daysp.i. and observed for 11weeks. Blood samples were collected weekly, and sera were tested using IFAT. Chicken tissues were collected for PCR, quantitative PCR and immunohistochemistry. Two dogs approximately 45days of age were fed with tissues from chickens euthanised at 138 and 159daysp.i. The results indicated that the chickens were resistant to neosporosis as revealed by failure to seroconvert, to detect parasite DNA or N. caninum antigen by immunohistochemistry in inoculated bird tissues, and by no oocyst excretion by the dogs fed avian tissues. Similar results were obtained in experiment #2, in which 34 1-week-old chickens were each s.c. inoculated with 100,000 tachyzoites of the NcWTDMn1 isolate of N. caninum. The chickens were euthanised on days 7, 15, 22, 28, 36 and 60p.i. At necropsy, all tissues and serum from each bird were collected. All chickens remained asymptomatic, and N. caninum antigen was not detected by immunohistochemistry. Seven chickens euthanised at day 60p.i. demonstrated low (1:25 dilution) levels of antibodies by using the Neospora agglutination test. Two 12-week-old dogs fed tissues pooled from 10 inoculated chickens euthanised at day 60p.i. did not excrete N. caninum oocysts. This investigation indicates that chickens are resistant to experimental infection by N. caninum.
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http://dx.doi.org/10.1016/j.ijpara.2017.07.004DOI Listing
February 2018

Eimeria Oocyst Concentrations and Species Composition in Litter from Commercial Broiler Farms During Anticoccidial Drug or Live Eimeria Oocyst Vaccine Control Programs.

Avian Dis 2017 Jun;61(2):214-220

B Mountaire Farms, Inc., Millsboro, DE 19966.

The purpose of this study was to determine if Eimeria oocyst concentrations and species composition in commercial broiler house litter changed during different cycles of anticoccidial drug (ACD) or live Eimeria oocyst vaccine (VAC) control programs and if there was a correlation between Eimeria oocyst levels and broiler performance. Litter samples were collected from a total of 15 different broiler farms encompassing a total of 45 individual houses during at least one complete grow-out cycle over a 21-mo period. Of these 15 broiler farms, three were followed for the entire 21-mo period spanning three ACD and four VAC cycles. Samples were collected at 2, 4, and 7-8 wk of grow-out corresponding to starter, grower, and withdraw periods of the ACD cycle. On a number of occasions, litter samples were obtained just prior to chick placement. Eimeria oocysts were isolated from all samples, counted by microscopy, and extracted for DNA to identify Eimeria species by ITS1 PCR. In general, Eimeria oocyst concentration in litter reached peak levels at 2-4 wk of grow-out regardless of coccidiosis control measure being used. However, peak oocyst numbers were sometimes delayed until 7-8 wk, indicating some level of Eimeria spp. drug resistance or incomplete vaccine coverage. Eimeria maxima , Eimeria acervulina , Eimeria praecox, and Eimeria tenella were generally present in all samples, and no difference in the species composition was noted between houses on a particular farm. While Eimeria species composition was similar among houses, Eimeria spp. oocyst levels exhibited sporadic peaks in one house of a given location's houses. Of particular interest was the observed correlation between E. maxima oocyst abundance and chick mortality. However, no correlation was observed in E. maxima oocyst levels, and the performance parameters adjusted feed conversion ratio and average daily weight gain. This study showed that understanding the dynamics of Eimeria spp. oocyst levels and species composition in litter during ACD or VAC programs may provide insight into the effectiveness of coccidiosis control measures in commercial broiler production.
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http://dx.doi.org/10.1637/11578-010317-Reg.1DOI Listing
June 2017

Differences in fecundity of Eimeria maxima strains exhibiting different levels of pathogenicity in its avian host.

Vet Parasitol 2017 Mar 17;236:1-6. Epub 2017 Jan 17.

Animal Parasitic Diseases Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA.

Eimeria maxima is one of the most pathogenic species of avian coccidia, yet it is unknown why different E. maxima strains differ in the pathogenic effects they cause in chickens. The purpose of this study was to determine if a more pathogenic E. maxima strain (APU1) was also more fecund than a less pathogenic E. maxima strain (APU2). At identical doses, E. maxima APU1 always produces greater intestinal lesions and lower weight gain compared to E. maxima APU2. Using a dose response study, median and mean intestinal lesion scores in E. maxima APU1-infected chickens were greater by a score of 1-1.5 compared to chickens infected with E. maxima APU2. Likewise, weight gain depression in E. maxima APU1-infected chickens was 20-25% greater (equivalent to 110-130g body weight) than in E. maxima APU2-infected chickens. In order to understand the underlying cause of these observed clinical effects, 120 broiler chicks (5 oocyst levels, 6 replicates/level) were inoculated with various doses of E. maxima APU1 or APU2 oocysts. The dynamics of oocyst shedding was investigated by collecting fecal material every 12h from 114 to 210h post-inoculation (p.i.) and every 24h thereafter from 210 to 306h, and then processed for measuring E. maxima oocyst output. Oocysts were first observed at 138h p.i., and time of peak oocyst production was nearly identical for both E. maxima APU1 and APU2 around 150-162h. Total oocyst production was 1.1-2.6 fold higher at all dose levels for E. maxima APU1 compared to E. maxima APU2, being significantly higher (P<0.05) at the log 1.5 dose level. Other groups of chickens were infected with higher doses of E. maxima APU1 or APU2 oocysts, and intestinal lesions were assessed by histology at 72, 96, 120, and 144h p.i. Although schizonts, gamonts, and oocysts were observed at expected time-points, no obvious differences were noted in lesions induced by the two E. maxima strains. This study showed that the greater fecundity of E. maxima APU1 compared to E. maxima APU2 explains in part the observed differences in pathogenicity of the two E. maxima strains, but that other factors may contribute to differences in observed clinical effects.
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http://dx.doi.org/10.1016/j.vetpar.2017.01.009DOI Listing
March 2017

Evaluation of ionophore sensitivity of Eimeria acervulina and Eimeria maxima isolated from the Algerian to Jijel province poultry farms.

Vet Parasitol 2016 Jul 14;224:77-81. Epub 2016 May 14.

Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, ARS, USDA, Beltsville, MD 20705, USA. Electronic address:

The present study represents the first description of ionophore resistance in recovered from commercial Algerian (Jijel-Algeria) broiler farms. Microscopy and intervening transcribed sequence 1 PCR (ITS1 PCR) revealed only 2 Eimeria species present in litter from these farms- namely Eimeria acervulina and Eimeria maxima. A pool of these isolates were evaluated in broiler chickens (Cobb 500) for sensitivity to 5 anticoccidial compounds-diclazuril (1ppm), lasalocid (125ppm), monensin (125ppm), narasin (70ppm) and salinomycin (60ppm). As indicated by anticoccidial sensitivity profiles based on lesion scores and anticoccidial index (ACI), complete resistance to monensin and narasin, partial resistance to salinomycin and lasalocid, and complete sensitivity to diclazuril was observed. While lack of sensitivity to monensin is not surprising given its use for years as the sole anticoccidial compound, the resistance to monoether (narasin) and polyether (lasalocid) ionophores suggests that cross-resistance has developed in a segment of the Eimeria population. The fairly uniform Eimeria species composition among all poultry farms suggests that E. acervulina and E. maxima more rapidly develop resistance to ionophore drugs.
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http://dx.doi.org/10.1016/j.vetpar.2016.04.040DOI Listing
July 2016

Protection of Broiler Chicks Housed with Immunized Cohorts Against Infection with Eimeria maxima and E. acervulina.

Avian Dis 2015 Mar;59(1):98-105

The use of live oocyst vaccines is becoming increasingly important in the control of avian coccidiosis in broilers. Knowledge of the mechanisms employed when chicks uptake oocysts and become immune is important for optimizing delivery of live vaccines. The current study tests the hypothesis that chicks not initially immunized may ingest oocysts by contact with litter containing oocysts shed by immunized cohorts. In Experiment 1, day-old broiler chicks were housed in pens containing clean litter. In Trial 1, 100% of chicks in some pens were immunized with 2.5 X 10(3) Eimeria acervulina oocysts while in other pens only 75% of chicks were immunized and remaining cohorts within the pens were not immunized. Other pens contained chicks that served as nonimmunized nonchallenged controls or nonimmunized challenged controls (NIC). On day 21, birds were given a homologous challenge of 6 X 10(5) oocysts. A second identical trial was conducted, except birds were immunized with 500 Eimeria maxima oocysts and were challenged with 3 X 10(3) E. maxima oocysts. In Experiment 2, 100% of chicks in some pens were immunized with 500 E. acervulina oocysts while in other pens either 75% or 50% of the birds were immunized. On day 14, birds were challenged with 1 X 10(6) oocysts. Trial 2 was identical to Trial 1 except that birds were immunized with 100 E. maxima oocysts and challenged with 1 X 10(6) oocysts. For all experiments weight gain, feed conversion ratio (FCR), plasma carotenoids, and litter oocyst counts were measured. In Experiment 1, the level of protection in groups containing 25% nonimmunized cohorts, as measured by weight gain, carotenoid level, FCR, and oocyst litter counts, was identical to groups containing 100% immunized chicks. In Experiment 2, pens where 50% or 75% of birds were immunized with either E. maxima or E. acervulina were not well protected from decreases in weight gain and plasma carotenoids nor from increases in litter oocyst counts following a challenge infection administered on day 14 relative to NIC. In addition, pens of birds where 100% of chicks were immunized were not well protected compared to NIC, and resistance to coccidiosis infection in immunized chicks was less than resistance in chicks challenged at 21 days. These results in total suggest that, when birds are challenged after 21 days, cohorts are protected from detrimental effects of challenge infection. However, when challenge infection is given at 14 days, cohorts are not well protected. The results support a conclusion that protection to coccidiosis is conveyed to cohorts by contact with oocysts shed into the litter by immunized chicks, but this resistance may take 14 days to develop.
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http://dx.doi.org/10.1637/10958-101014-regDOI Listing
March 2015

Evaluation of an experimental irradiated oocyst vaccine to protect broiler chicks against avian coccidiosis.

Avian Dis 2014 Sep;58(3):391-7

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.
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http://dx.doi.org/10.1637/10679-092613-Reg.1DOI Listing
September 2014

Expression of nutrient transporters in duodenum, jejunum, and ileum of Eimeria maxima-infected broiler chickens.

Parasitol Res 2014 Oct 6;113(10):3891-4. Epub 2014 Sep 6.

Animal Parasitic Diseases Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Beltsville, MD, 20705, USA,

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.
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http://dx.doi.org/10.1007/s00436-014-4114-3DOI Listing
October 2014

Both host and parasite MIF molecules bind to chicken macrophages via CD74 surface receptor.

Dev Comp Immunol 2014 Dec 30;47(2):319-26. Epub 2014 Jul 30.

Avian Immunobiology Laboratory, Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.dci.2014.07.021DOI Listing
December 2014

The use of dual-energy X-ray absorptiometry to assess the impact of Eimeria infections in broiler chicks.

Avian Dis 2013 Jun;57(2):199-204

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.
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http://dx.doi.org/10.1637/10392-092812-Reg.1DOI Listing
June 2013

A highly divergent 33 kDa Cryptosporidium parvum antigen.

J Parasitol 2014 Aug 6;100(4):527-31. Epub 2014 Mar 6.

Environmental, Microbial, and Food Safety Laboratory, ARS, USDA, Beltsville, Maryland 20705;

Previous studies comparing the genome sequences of Cryptosporidium parvum with Cryptosporidium 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, which 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.
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http://dx.doi.org/10.1645/13-433.1DOI Listing
August 2014

Protecting chickens against coccidiosis in floor pens by administering Eimeria oocysts using gel beads or spray vaccination.

Avian Dis 2013 Sep;57(3):622-6

Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, ARS, USDA, Beltsville, MD 20705, USA.

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.
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http://dx.doi.org/10.1637/10516-022213-Reg.1DOI Listing
September 2013

Characterization and localization of an Eimeria-specific protein in Eimeria maxima.

Parasitol Res 2013 Oct 3;112(10):3401-8. Epub 2013 Jul 3.

Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, USDA/ARS, Beltsville, MD, 20705, USA,

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.
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http://dx.doi.org/10.1007/s00436-013-3518-9DOI Listing
October 2013

Differing susceptibilities of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts to desiccation.

J Parasitol 2013 Oct 25;99(5):899-902. Epub 2013 Apr 25.

Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, ARS, USDA, Beltsville, Maryland 20705; * Animal Biosciences & Biotechnology Laboratory, Beltsville Agricultural Research Center, ARS, USDA Beltsville, Maryland 20705.

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 Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts to dessication. Sporulated E. acervulina, E. maxima, or E. tenella oocysts were incorporated into gelatin beads and incubated at 32 C 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. Eimeria 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 time points. 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.
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http://dx.doi.org/10.1645/13-192.1DOI Listing
October 2013

Macrophage migration inhibitory factor (MIF) of the protozoan parasite Eimeria influences the components of the immune system of its host, the chicken.

Parasitol Res 2013 May 23;112(5):1935-44. Epub 2013 Feb 23.

Beltsville Agricultural Research Center, USDA/ARS, 10300 Baltimore Ave., Beltsville, MD 20705, USA.

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.
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http://dx.doi.org/10.1007/s00436-013-3345-zDOI Listing
May 2013

Molecular characterization and immunological roles of avian IL-22 and its soluble receptor IL-22 binding protein.

Cytokine 2012 Dec 11;60(3):815-27. Epub 2012 Sep 11.

Avian Immunobiology Laboratory, Department of Animal and Poultry Sciences, Virginia Tech, Blacksburg, VA 24061, USA.

As a member of the interleukin (IL)-10 family, IL-22 is an important mediator in modulating tissue responses during inflammation. Through activation of STAT3-signaling cascades, IL-22 induces proliferative and anti-apoptotic pathways, as well as antimicrobial peptides (AMPs), that help prevent tissue damage and aid in its repair. This study reports the cloning and expression of recombinant chicken IL-22 (rChIL-22) and its soluble receptor, rChIL22BP, and characterization of biological effects of rChIL-22 during inflammatory responses. Similar to observations with mammalian IL-22, purified rChIL-22 had no effect on either peripheral blood mononuclear cells (PBMCs) or lymphocytes. This was due to the low expression of the receptor ChIL22RA1 chain compared to ChIL10RB chain. rChIL-22 alone did not affect chicken embryo kidney cells (CEKCs); however, co-stimulation of CEKCs with LPS and rChIL-22 enhanced the production of pro-inflammatory cytokines, chemokines and AMPs. Furthermore, rChIL-22 alone stimulated and induced acute phase reactants in chicken embryo liver cells (CELCs). These effects of rChIL-22 were abolished by pre-incubation of rChIL-22 with rChIL22BP. Together, this study indicates an important role of ChIL-22 on epithelial cells and hepatocytes during inflammation.
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http://dx.doi.org/10.1016/j.cyto.2012.08.005DOI Listing
December 2012

Gel-Bead Delivery of Eimeria oocysts protects chickens against coccidiosis.

Avian Dis 2012 Jun;56(2):306-9

Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, Unites States Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA.

Vaccines composed of either virulent or attenuated Eimeria spp. oocysts have been developed as an alternative to medication of feed with ionophore drugs or synthetic chemicals. The purpose of this study was to evaluate the use of gel-beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine against coccidiosis. Newly hatched chicks (Gallus gallus domesticus) were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel-beads. Control day-old chicks were given an equivalent number of Eimeria oocysts (10(4) total) by oral gavage. After 3 days, chicks were randomly assigned to individual cages, and feces were collected between days 5 and 8 postinfection. All samples were processed for total Eimeria oocysts. 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. Oocyst excretion by chicks fed gel-beads or inoculated by oral gavage was 10- to 100-fold greater than that of chicks spray-vaccinated with the Eimeria oocysts mixture (log 6.3-6.6 vs. log 4.8). Subsequent protection against challenge as measured by weight gain and feed conversion efficiency was significantly greater (P < 0.05) in gel-bead and oral gavage groups compared with spray-vaccinated or nonimmunized groups. Also, gel-bead and oral gavage groups showed no significant difference (P > 0.05) in weight gain and feed conversion efficiency compared with nonchallenged controls. These findings indicate that incorporation of Eimeria spp. oocysts in gel-beads may represent an effective way to deliver live oocyst vaccines to day-old chicks for preventing subsequent outbreaks of coccidiosis in the field.
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http://dx.doi.org/10.1637/9940-092111-Reg.1DOI Listing
June 2012

Gene expression during excystation of Cryptosporidium parvum oocysts.

Parasitol Res 2011 Aug 5;109(2):509-13. Epub 2011 Mar 5.

Environmental Microbiology and Food Safety Laboratory, Agricultural Research Service, USDA, Beltsville, MD 20705, USA.

The present study describes transcription of mRNA from genes encoding metabolic or structural proteins during excystation of Cryptosporidium parvum oocysts. RNA was harvested from C. parvum oocysts before excystation, and at 5, 10, and 15 min during excystation. Subtractive cDNA libraries were prepared by using mRNA from non-excysted C. parvum oocysts to "subtract out" mRNA from excysting oocysts. The "subtracted" cDNA was used to prepare libraries enriched for transcripts possibly involved in excystation. From these libraries, over 1,000 expressed sequence tags (ESTs) were analyzed by DNA sequencing followed by BLAST-N and BLAST-X analysis. While several gene products involved in cell metabolism and cell signaling were consistently recovered, transcription levels, as reflected by the relative number of cDNA sequences (19.2% total), were highly up-regulated in genes coding for structural proteins such as Cp2, CpTSP, CpHC10, and CpSAg. Moreover, of the greater than 1,000 clones analyzed, a high percentage (12.3%) of ESTs detected in excysting oocysts were for hypothetical C. parvum proteins (CpHyP), whose functions are presently unknown.
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http://dx.doi.org/10.1007/s00436-011-2308-5DOI Listing
August 2011

Immunization of female BALB/c mice with Neospora cyclophilin and/or NcSRS2 elicits specific antibody response and prevents against challenge infection by Neospora caninum.

Vaccine 2011 Mar 31;29(13):2392-9. Epub 2011 Jan 31.

Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, USDA/ARS, Beltsville, MD 20705, USA.

Neospora caninum is the causal agent of bovine neosporosis which results in high levels of abortion. The present study determined the protective efficacy of two Neospora antigens--Neospora cyclophilin (NcCyP) and NcSRS2. The ability of native NcCyP to upregulate mouse IFN-γ was also confirmed in this study. Recombinant NcCyP or NcSRS2 were tested either alone or in combination and formulated with adjuvant ImmuMax-SR and CpG. Female BALB/c mice (n=15) of 10-12 weeks of age were immunized s.c. twice over a 2-week interval with vaccines containing either NcCyP (20 μg/dose) alone, NcSRS2 (20 μg/dose) alone, NcCyP plus NcSRS2, or non-recombinant bacterial antigen (NR) in 2 separate trials. All mice were challenge-infected 3 weeks following the booster immunization and necropsied 3 weeks after the challenge infection. Brain and serum were collected and Nc-specific DNA sequence in brain tissue and antibodies in serum were analyzed by PCR or ELISA/Western blotting. Results showed that mice vaccinated with rNcCyP, rNcSRS2, or both rNcCyP and rNcSRS2 responded with high levels of NcCyP or NcSRS2 specific antibodies. Overall, mice received vaccines formulated with either rNcCyP or rNcCyP and rNcSRS2 had a higher (p<0.01) percent protection when compared to the mock- or non-vaccinated mice. The group immunized with rNcSRS2 alone exhibited slightly lower levels of protection, which was higher (p<0.05) than that of the non-vaccinated group but did not differ (p=0.06) from that of the mock-vaccinated group. The results of the present study indicate that NcCyP is a highly efficacious vaccine candidate which may be useful in protection against Neospora infection.
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http://dx.doi.org/10.1016/j.vaccine.2011.01.041DOI Listing
March 2011

Expression of Toll-like receptors and antimicrobial peptides during Eimeria praecox infection in chickens.

Exp Parasitol 2011 Mar 19;127(3):714-8. Epub 2010 Dec 19.

Avian Immunobiology Laboratory, Department of Animal & Poultry Sciences, Virginia Tech, 3170 Litton-Reaves Hall, Blacksburg, VA 24061, USA.

Intestinal colonization of avian species by Eimeria parasites results in the enteric disease, coccidiosis. A study was carried out to assess the immunologic effects of Eimeria praecox infection on the gut of infected chickens. In Experiment 1, birds were orally gavaged with 50,000 E. praecox oocysts; in Experiment 2, an infection dosage of 500,000 E. praecox oocysts was used. Duodenal and jejunal intestinal sections were sampled consecutively on days 1-7 post-infection. Intestinal expression of innate immune gene transcripts was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Analysis of relative gene expression in Experiment 1 revealed an increase (P<0.05) in duodenal Toll-like receptor (TLR)3 expression on days 4 and 6 post-infection. TLR15 expression was significantly decreased in the duodenum of infected birds on day 2, and significantly increased on day 6 post-infection. In Experiment 2, TLR3 was significantly downregulated in the duodenum on day 7 post-infection; however, no significant results were observed in terms of TLR15 expression. TLR4 also exhibited decreased expression (P<0.05) on day 7 post-infection in both intestinal sections. Regarding antimicrobial peptide expression; in the first experiment, expression of liver-expressed antimicrobial peptide-2 (LEAP-2) in infected birds was significantly decreased in the duodenum on days 3 and 4, and in the jejunum on day 4. Similarly, Experiment 2 resulted in depression of LEAP-2 (P<0.05) on days 3-5 in the duodenum. In Experiment 1, cathelicidin antimicrobial peptide (CATHL3) was downregulated (P<0.05) in the jejunum of infected chickens on day 3 post-infection; however, CATHL3 results were non-significant in Experiment 2. Based on the differing results observed in each experiment, it was concluded that both TLR and antimicrobial peptide expression, and thus immunity may be dependent on infection load.
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http://dx.doi.org/10.1016/j.exppara.2010.12.002DOI Listing
March 2011

Molecular cloning and functional characterization of the avian macrophage migration inhibitory factor (MIF).

Dev Comp Immunol 2010 Sep 20;34(9):1021-32. Epub 2010 May 20.

Avian Immunobiology Laboratory, Department of Animal and Poultry Sciences, Virginia Tech, 3170 Litton-Reaves, Blacksburg, VA 24061, United States.

Macrophage migration inhibitory factor (MIF) is recognized as a soluble factor produced by sensitized T lymphocytes and inhibits the random migration of macrophages. Recent studies have revealed a more prominent role for MIF as a multi-functional cytokine mediating both innate and adaptive immune responses. This study describes the cloning and functional characterization of avian MIF in an effort to better understand its role in innate and adaptive immunity, and potential use in poultry health applications. The full-length avian MIF gene was amplified from stimulated chicken lymphocytes and cloned into a prokaryotic expression vector. The confirmed 115 amino acid sequence of avian MIF has 71% identity with human and murine MIF. The bacterially expressed avian recombinant MIF (rChMIF) was purified, followed by endotoxin removal, and then tested by chemotactic assay and quantitative real-time PCR (qRT-PCR). Diff-Quick staining revealed a substantial decrease in migration of macrophages in the presence of 0.01microg/ml rChMIF. qRT-PCR analysis revealed that the presence of rChMIF enhanced levels of IL-1beta and iNOS during PBMCs stimulation with LPS. Additionally, the Con A-stimulated lymphocytes showed enhanced interferon (IFN)-gamma and IL-2 transcripts in the presence of rChMIF. Interestingly, addition of rChMIF to the stimulated PBMCs, in the presence of lymphocytes, showed anti-inflammatory function of rChMIF. To our knowledge, this study represents the first report for the functional characterization of avian MIF, demonstrating the inhibition of macrophage migration, similar to mammalian MIF, and the mediation of inflammatory responses during antigenic stimulation.
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http://dx.doi.org/10.1016/j.dci.2010.05.005DOI Listing
September 2010

Neospora caninum: cloning and expression of a gene coding for cytokine-inducing profilin.

Exp Parasitol 2010 Aug 6;125(4):357-62. Epub 2010 Mar 6.

Animal Parasitic Diseases Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705, USA.

Profilins are actin-binding proteins that in Toxoplasma gondii stimulate innate immunity in mice by binding Toll-like receptors (TLR) on dendritic cells (DC) leading to release of inflammatory cytokines, primarily IL-12 and IFN-gamma. The purpose of the present study was to characterize Neospora caninum profilin, termed NcProfilin. Recombinant NcProfilin was purified by affinity chromatography, and used to prepare specific antisera to allow characterization of native NcProfilin antigen in N. caninum tachyzoites. By immunoblotting, recombinant NcProfilin is 22kDa, and is similar in size to the respective 22kDa native protein. Immunofluorescence and immunoelectron microscopy localized native NcProfilin to the apical end of N. caninum tachyzoites. Incubation of recombinant NcProfilin with spleen cells from BALB/c mice induced release of IFN-gamma. Also, injection of BALB/c mice with purified rNcProfilin elicited a strong IFN-gamma and IL-12 responses at 6 and 24h after injection indicating that NcProfilin may be an important protein in regulation of cytokine responses to N. caninum.
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http://dx.doi.org/10.1016/j.exppara.2010.03.001DOI Listing
August 2010

Genomic analysis of Eimeria spp. populations in relation to performance levels of broiler chicken farms in Arkansas and North Carolina.

J Parasitol 2009 Aug;95(4):871-80

Animal Parasitic Diseases Lab, United States Department of Agriculture, 10300 Baltimore Ave., Beltsville, Maryland 20705, USA.

The impact of coccidiosis outbreaks on the productivity of broiler chicken farms can be substantial, depending on the severity of disease caused by particular species and strains of Eimeria. We examined the genetic diversity of Eimeria species present in commercial broiler farms in relation to their performance level. Four groups of broiler chicken farms in Arkansas (AR) and North Carolina (NC), having either high or low performance levels, were sampled for Eimeria spp. oocysts. We amplified gDNA from oocysts by using genus-specific primers targeting 18S ribosomal RNA, the first and second internal transcribed spacer regions, and cytochrome c oxidase subunit I as the established species-specific primers. Eimeria spp. diversity was not homogenous among the 4 farm groups, with less-pathogenic species (E. mitis and E. mivati-like) associated with AR and NC high-performance farms, respectively, and a pathogenic species (E. brunetti) associated with AR low-performance farms. Sequence analyses identified multiple E. maxima and E. mitis genetic variants, from which 2 E. maxima variants were unique to low-performance farms. Distinct populations of sequences at the NC high-performance farms were identified as E. mivati-like, based on homology searches. Our study demonstrated the utility of analyzing multiple genomic loci to assess composition and polymorphisms of Eimeria spp. populations.
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http://dx.doi.org/10.1645/GE-1898.1DOI Listing
August 2009