Development of an auxotrophic, live-attenuated Brucella suis vaccine strain capable of expressing multimeric GnRH.

Feral swine cost around $1.5 billion each year in agricultural, environmental, and personal property damages. They are also the most widespread carriers of the zoonotic disease brucellosis, which threatens both livestock bio-security and public health. Currently, there is no approved vaccine against brucellosis in pigs. This is a preliminary report on the development of a live-attenuated B. suis vaccine that could be employed to deliver heterologous antigens to control swine populations. An attenuated vaccine strain provided significant protection against B. suis challenge in mice. Leucine auxotrophy in the vaccine strain allowed the over-expression of heterologous antigens without the use of antibiotic resistant markers. Vaccinated mice showed the development of antibodies against expressed antigen. Further evaluation is required to assess its ability to cause infertility using the mouse model prior to further testing for use as a tool for feral swine population and disease control.

Effect of copper on the up-regulation/down-regulation of genes, cytotoxicity and ion dissolution for mesoporous bioactive glasses.

In the present study, copper-based (25 - x)CaO - xCuO -10P2O5 - 5B2O3 - 60SiO2 (x = 2.5, 5, 7.5, 10 mol%) mesoporous bioactive glasses (MBGs) were synthesized using the sol-gel technique with cetyltrimethyl ammonium bromide as the structure-directing agent. The live-dead cell count and cytocompatibility of MBGs for J774A.1 murine macrophages have also been investigated for different concentrations of MBGs. The ionic dissolution profile for Ca, P and Si has been evaluated in the simulated body fluid. The effect of copper content as well as the ionic dissolution products on the up-regulation and down-regulation of TGIF-2, HDAC-4, Smurf-1, mir-30c and mir-130a genes for the murine model are investigated using reverse transcription-polymerase chain reaction. Silica and calcium release follows a similar trend as followed by gene up-regulation of TGIF-2, HDAC-4 and mir-30c genes. This indicates that silica and calcium release influence gene expression.

Experimental comparison of hemolytic and nonhemolytic Ornithobacterium rhinotracheale field isolates in vivo.

Ornithobacterium rhinotracheale (ORT) is a nonhemolytic, gram-negative, pleomorphic, rod-shaped bacterium that causes upper and lower respiratory tract disease in poultry. Recently, hemolytic strains of ORT have been isolated with increasing frequency from field outbreaks. A study was conducted to determine whether the hemolytic phenotype is associated with any change in virulence. Briefly, 225 turkey poults, vaccinated against hemorrhagic enteritis at 4 wk of age, were randomly divided into nine replicates housed in separate rooms: three sham treatment controls (25 poults/replicate), three challenged with a nonhemolytic (NH) field isolate (24 poults/replicate), and three challenged with a hemolytic (H) field isolate (24 poults/replicate). Nine days postvaccination, poults were inoculated intratracheally with either 0.2 ml sterile phosphate-buffered saline (PBS), 2 x 10(8) colony-forming units (CFU) of the NH isolate in 0.2 ml PBS, or 2 x 10(8) CFU of the H isolate in 0.2 ml PBS. Serum and body weights were obtained at 0, 7, 14, and 21 days postinoculation (dpi). Tissues were taken for culture and histopathology from five randomly selected poults/replicates at 7, 14, and 21 dpi. When compared with poults inoculated with the H isolate or controls, those inoculated with the NH isolate showed a highly significant depression in weight gain at 7 dpi. NH poults also had significantly higher levels of antibody against ORT at 14 and 21 dpi. Reisolations decreased over time and, by 21 dpi, only the NH phenotype could be found. Based on a Likert-type scale, poults inoculated with the NH isolate had significantly higher histopathologic lesion scores in lung tissue at 7, 14, and 21 dpi. Results suggest that nonhemolytic field isolates are more virulent then hemolytic ones. These findings are unusual because hemolytic phenotypes are often more virulent in other bacterial species.

Synthesis, cytotoxicity, and hydroxyapatite formation in 27-Tris-SBF for sol-gel based CaO-P2O5-SiO2-B2O3-ZnO bioactive glasses.

CaO-P2O5-SiO2-B2O3-ZnO bioactive glasses were prepared via an optimized sol-gel method. The current investigation was focused on producing novel zinc based calcium phosphoborosilicate glasses and to evaluate their mechanical, rheological, and biocompatible properties. The morphology and composition of these glasses were studied using X-ray diffraction (XRD) and scanning electron microscopy (SEM). The particle size, mechanical and flexural strength was also determined. Furthermore, the zeta potential of all the glasses were determined to estimate their flocculation tendency. The thermal analysis and weight loss measurements were carried out using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) respectively. For assessing the in-vitro bioactive character of synthesized glasses, the ability for apatite formation on their surface upon their immersion in simulated body fluid (SBF) was checked using SEM and pH measurements. MTS assay cytotoxicity assay and live-dead cell viability test were conducted on J774A.1 cells murine macrophage cells for different glass concentrations.

Immunotherapeutics to prevent the replication of Brucella in a treatment failure mouse model.

Outer membrane vesicles (OMVs) from Brucella melitensis and irradiated Brucella neotomae have been shown to be effective vaccines against a B. melitensis challenge in a mouse model. The present study evaluates the efficacy of these two vaccines as immuno-therapeutics in combination with conventional antibiotics against a B. melitensis infection. BALB/c mice chronically infected with B. melitensis were treated for 4 weeks with doxycycline and gentamicin and vaccinated twice during the course of therapy. Antibiotics in sub-therapeutic concentrations were chosen in such a way that the treatment would result in a therapeutic failure in mice. Although no additive effect of vaccines and antibiotics was seen on the clearance of B. melitensis, mice receiving vaccines along with antibiotics exhibited no Brucella replication post-treatment compared to mice treated only with antibiotics. Administration of irradiated B. neotomae along with antibiotics led to higher production of IFN-γ ex vivo by splenocytes upon stimulation with heat inactivated B. melitensis while no such effect was seen by splenocytes from mice vaccinated with OMVs. OMV vaccinated mice developed significantly higher anti-Brucella IgG antibody titers at the end of the treatment compared to the mice that received only antibiotics. The mice that received only vaccines did not show any significant clearance of Brucella from spleens and livers compared to non-treated control mice. This study suggests that incorporating OMVs or irradiated B. neotomae along with conventional antibiotics might be able to improve therapeutic efficacy and control the progression of disease in treatment failure cases.

Detection of Salmonella Enteritidis in asymptomatic carrier animals: comparison of quantitative real-time PCR and bacteriological culture methods.

Quantification of Salmonella in asymptomatic carrier animals can be used to assess microbial risk and monitor the level of contamination in domestic animals used for food production. We examined the sensitivity, specificity and accuracy of real-time qPCR, without pre-enrichment or selective enrichment stages, for the quantification of S. enterica serovar Enteritidis in resistant mice, as a model of asymptomatic carrier animal. The results were compared with those obtained by traditional bacteriological culture methods, the gold standard test. Two hundred and forty-three samples, including spleen, liver, mesenteric lymph nodes, portions of intestine, intestinal content of the ileocecal portion, and feces, were collected from a group of 27 C57BL/6 mice, that had been intragastrically inoculated with high doses of S. enterica serovar Enteritidis. The real-time qPCR assay presented a consistent linearity of the standard curve (r(2) = 0.999), with very low differences between melting temperatures, and low coefficients of variation in intra- (< 1%) and interassay (< 2%) comparisons. The primers were highly specific; there was no amplification with other Salmonella serovars or with DNA from uninfected tissues and feces from mice. The detection limit of the technique was defined as 32 copies of S. enterica serovar Enteritidis. A sensitivity of 90%, a specificity of 77% and an accuracy of 79% were obtained. The higher sensitivity of PCR was reflected in a kappa coefficient of 0.41, showing moderate agreement between tests. We conclude that real-time qPCR is a good alternative for diagnostic scanning in asymptomatic carrier animals, due to its high sensitivity and rapidity.

Efficacy of amphiphilic core-shell nanostructures encapsulating gentamicin in an in vitro salmonella and listeria intracellular infection model.

Core-shell nanostructures with nonionic amphiphilic shells and ionic cores encapsulating gentamicin were designed for therapy against intracellular pathogens, including Salmonella and Listeria. Flow cytometry and confocal microscopy showed that their uptake into J774A.1 macrophages proceeded mainly by fluid-phase endocytosis and clathrin-mediated pathways. The nanostructures were nontoxic in vitro at doses of 50 to 250 microg/ml, and they significantly reduced the amounts of intracellular Salmonella (0.53 log) and Listeria (3.16 log), thereby suggesting effective transport into the cells.

Persistent infection of turkeys with an avirulent strain of turkey hemorrhagic enteritis virus.

The Virginia avirulent strain (VAS) of turkey hemorrhagic enteritis virus (THEV), which is commonly used in live vaccines for commercial turkeys, was studied to determine characteristics of infection. It has been observed that turkeys infected with the VAS maintain protective antibody levels in excess of 20 wk postvaccination. It is theorized that this immune response is modulated by either a persistent or latent infection. A series of studies have been undertaken to determine changes in virus location and serology over time. A trial was also conducted to evaluate the effect of corticosteroid administration on viral recrudescence, and an attempt was made to isolate live virus from tissues of birds 10 wk postinfection (pi). Antibody titers were determined by enzyme-linked immunosorbent assay, and PCR was used to detect viral DNA. Histopathology was performed on formalin-fixed paraffinized tissues. Viral DNA was detected in various tissues through 15 wk pi in the presence of high antibody titers. Viral DNA was detected at 3-5 days pi in the spleens of susceptible turkeys inoculated with tissues collected from infected birds at 10 wk pi. It is unknown whether the viral DNA is associated with live virus or rather is the result of persistent maintenance of the viral genome within lymphoid/macrophage target cells. Future studies will test for viral RNA in order to confirm the presence of replicating THEV. Regardless of the actual status of the THEV DNA detected at 10-15 wk pi, it is clear that THEV does not cause a simple acute infection. The characteristics of THEV infection are identical to the nonlytic persistent infections seen in human adenoviruses, and therefore THEV may serve as a model for the study of virus-cell interactions mediating persistence.

Wild boars as sources for infectious diseases in livestock and humans.

Wild boars (Sus scrofa) are indigenous in many countries in the world. These free-living swine are known reservoirs for a number of viruses, bacteria and parasites that are transmissible to domestic animals and humans. Changes of human habitation to suburban areas, increased use of lands for agricultural purposes, increased hunting activities and consumption of wild boar meat have increased the chances of exposure of wild boars to domestic animals and humans. Wild boars can act as reservoirs for many important infectious diseases in domestic animals, such as classical swine fever, brucellosis and trichinellosis, and in humans, diseases such as hepatitis E, tuberculosis, leptospirosis and trichinellosis. For examples, wild boars are reservoirs for hepatitis E virus, and cluster cases of hepatitis E have been reported in Japan of humans who consumed wild boar meat. In Canada, an outbreak of trichinellosis was linked to the consumption of wild boar meat. The incidence of tuberculosis owing to Mycobacterium bovis has increased in wild boars, thus posing a potential concern for infections in livestock and humans. It has also been documented that six hunters contracted Brucella suis infections from wild swine in Florida. This article discusses the prevalence and risk of infectious agents in wild boars and their potential transmission to livestock and humans.

In vitro trafficking and efficacy of core-shell nanostructures for treating intracellular Salmonella infections.

Nanostructures encapsulating gentamicin and having either amphiphilic (N1) or hydrophilic (N2) surfaces were designed. Flow cytometry and confocal microscopy studies demonstrated a higher rate of uptake for amphiphilic surfaces. A majority of N1 were localized in the cytoplasm, whereas N2 colocalized with the endosomes/lysosomes. Colocalization was not observed between nanostructures and intracellular Salmonella bacteria. However, significant in vitro reductions in bacterial counts (0.44 log10) were observed after incubation with N1, suggesting that the surface property of the nanostructure influences intracellular bacterial clearance.

Immune responses and protection against experimental challenge after vaccination of bison with Brucella abortus strain RB51 or RB51 overexpressing superoxide dismutase and glycosyltransferase genes.

Vaccination is a tool that could be beneficial in managing the high prevalence of brucellosis in free-ranging bison in Yellowstone National Park. In this study, we characterized immunologic responses and protection against experimental challenge after vaccination of bison with Brucella abortus strain RB51 (RB51) or a recombinant RB51 strain overexpressing superoxide dismutase (sodC) and glycosyltransferase (wboA) genes (RB51+sodC,wboA). Bison were vaccinated with saline only or with 4.6 x 10(10) CFU of RB51 or 7.4 x 10(10) CFU of RB51+sodC,wboA (n = eight animals/treatment). Bison vaccinated with RB51 or RB51+sodC,wboA had greater (P < 0.05) antibody responses, proliferative responses, and production of gamma interferon to RB51 after vaccination than did nonvaccinates. However, bison vaccinated with RB51+sodC,wboA cleared the vaccine strain from draining lymph nodes faster than bison vaccinated with the parental RB51 strain. Immunologic responses of bison vaccinated with RB51+sodC,wboA were similar to responses of bison vaccinated with RB51. Pregnant bison were intraconjunctivally challenged in midgestation with 10(7) CFU of B. abortus strain 2308. Bison vaccinated with RB51, but not RB51+sodC,wboA vaccinates, had greater protection from abortion, fetal/uterine, mammary, or maternal infection than nonvaccinates. Our data suggest that the RB51+sodC,wboA strain is less efficacious as a calfhood vaccine for bison than the parental RB51 strain. Our data also suggest that the RB51 vaccine is a currently available management tool that could be utilized to help reduce brucellosis in free-ranging bison.

Optimization of the use of C57BL/6 mice as a laboratory animal model for Neospora caninum vaccine studies.

The development and testing of vaccines for Neospora caninum in mice require challenge studies to demonstrate a reduction in clinical signs or prevention of vertical transmission of the parasite after vaccination. Genetic susceptibility to N. caninum varies with the strain of mice. In this study, C57BL/6 mice were evaluated as a model for Neospora vaccine studies. A lethal challenge model was developed and the LD(50) was determined to be 1.5 x 10(7)N. caninum tachyzoites/mouse, delivered intraperitoneally. Brain lesions encountered in sections from sub-lethally challenged mice were scored on the basis of severity and total number of lesions to develop a histopathological scoring system for vaccine efficacy. A vertical transmission model for N. caninum vaccine studies was developed by studying mice that were infected either 2 weeks prior to mating or between days 12 and 14 of pregnancy. It was found that infection prior to mating reduced the average number of pups per litter. DNA extracted from fetal tissue was examined by a N. caninum specific polymerase chain reaction (PCR). The rate of vertical transmission was 0, 100 and 90.5% for the uninfected controls, mice infected during pregnancy and mice infected before mating, respectively. This study demonstrates that the C57BL/6 strain of mice is a good model for N. caninum vaccine studies because it is possible to establish a clear-cut lethal challenge model in C57BL/6 mice and they transmit the disease to their offspring efficiently.

Curli production and genetic relationships among Escherichia coli from cases of bovine mastitis.

Curli are adhesive surface structures produced by some Escherichia coli and Salmonella strains that bind host proteins and activate inflammatory mediators. In this study, 61 E. coli isolates from 36 clinical cases of bovine mastitis were characterized using enterobacterial repetitive intergenic consensus-PCR and screened for their ability to produce curli. Effect of curli production on case recovery, based on a return to precase milk yield, was investigated for a subset of 43 isolates from 20 quarters of 19 cows. Thirty-five (57%) of 61 isolates were curli positive. Fifty-eight of the 61 isolates clustered into 2 clonal groups at 52% genetic similarity. Genetically diverse E. coli isolates were simultaneously cultured from individual cases. Twenty-three isolates from 13 cows were clustered in clonal group I, of which 5 cases (38%) were curli positive; 35 isolates from 22 cows were clustered in clonal group II, of which 15 cases (68%) were curli positive. No association was found between genetic similarity and phenotypic curli expression of isolates from cows with clinical E. coli mastitis cases. Phenotypic curli expression in isolates did not affect recovery of cows' milk yield to premastitis production levels.

Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum.

Neospora caninum, an apicomplexan parasite, is a leading cause of bovine abortions worldwide. The efficacy of gamma-irradiated N. caninum strain NC-1 tachyzoites as a vaccine for neosporosis was assessed in C57BL6 mice. A dose of 528 Gy of gamma irradiation was sufficient to arrest replication but not host cell penetration by tachyzoites. Female C57BL6 mice were vaccinated with two intraperitoneal inoculations of 1 x 10(6) irradiated tachyzoites at 4-wk intervals. When stimulated with N. caninum tachyzoite lysates, splenocytes of vaccinated mice, cultured 5 and 10 wk after vaccination, secreted significant (P<0.05) levels of interferon gamma, interleukin (IL)-10, and small amounts of IL-4. Antibody isotype-specific ELISA of sera from vaccinated mice exhibited both IgG1 and IgG2a isotypes of antibodies. Vaccinated mice were challenged intraperitoneally with 2 x 10(7)N. caninum tachyzoites. All vaccinated mice remained healthy and showed no obvious signs of neosporosis up to the 25th day post-challenge when the study was terminated. All unvaccinated control mice died within 1 wk of infection. Gamma-irradiated N. caninum tachyzoites can serve as an effective, attenuated vaccine for N. caninum.

Gerbil model of acute neosporosis.

Experimental infections with the NC-1 strain of Neospora caninum were conducted in gerbils (Meriones unguiculatus) to determine their acute responses to experimental intraperitoneal infection. Five groups of five female gerbils were used and they were intraperitoneally infected with 1x10(6), 2x10(6), 3x10(6), 4x10(6) or 5x10(6) tachyzoites. Gerbils in all groups developed clinical signs of neosporosis which consisted of inactivity 4-5 days post-inoculation. Morbidity and mortality were observed in all groups. Grossly there was a clear fibrinous exudate in the abdominal cavity and adhesions of the spleen and pancreas to the stomach in gerbils suffering from acute neosporosis. The LD50 was calculated as 9.3x10(5) tachyzoites per gerbil. The results indicate that gerbils can be used as a suitable model of acute neosporosis. This model can be used to screen candidate treatments and to test the efficacy of vaccines for neosporosis without the need to use histology or PCR to demonstrate treatment efficacy.

Pathogenic and fecal Escherichia coil strains from turkeys in a commercial operation.

The biochemical phenotypes and antimicrobial susceptibility patterns of 105 clinical Escherichia coli isolates from flocks with colibacillosis in a turkey operation were compared with 1104 fecal E. coli isolates from 20 flocks in that operation. Clinical isolates and 194 fecal isolates with biochemical phenotypes or minimum inhibitory concentrations for gentamicin and sulfamethoxazole similar to clinical isolates were tested for somatic antigens and the potential virulence genes hylE, iss, tsh, and K1. The predominant biochemical phenotype of clinical isolates contained 21 isolates including 14 isolates belonging to serogroup 078 with barely detectable beta-D-glucuronidase activity. Thirty-five fecal isolates had biochemical phenotypes matching common phenotypes of clinical isolates. Sixty-six (63%) clinical isolates exhibited intermediate susceptibility or resistance to gentamicin and sulfamethoxazole compared with 265 (24%) fecal isolates (P < 0.001). Seventy-seven clinical isolates reacted with O-antisera, of which 51 (66%) belonged to the following serogroups: O1, O2, O8, O25, O78, O114, and O119. In comparison, 8 of 35 (23%) fecal isolates subtyped on the basis of biochemical phenotype belonged to these serogroups and four of 167 (2%) fecal isolates subtyped on the basis of their antimicrobial resistance patterns belonged to these serogroups. Iss, K1, and tsh genes were detected more often among clinical isolates than these fecal isolates (P < 0.05). In summary, a small subgroup of E. coli strains caused most colibacillosis infections in this operation. These strains existed at low concentration in normal fecal flora of healthy turkeys in intensively raised flocks. The data suggest that colibacillosis in turkey operations may be due to endogenous infections caused by specialized pathogens.

Effects of long-term estrogen treatment on IFN-gamma, IL-2 and IL-4 gene expression and protein synthesis in spleen and thymus of normal C57BL/6 mice.

Estrogens have been shown to markedly modulate the immune system. One mechanism by which estrogens could modulate the immune system is by regulating cytokines, an aspect not well-studied thus far. To address this issue, normal C57BL/6 orchiectomized mice were given estrogen and its effects on selected cytokines, interferon-gamma (IFN-gamma), interleukin 2 (IL-2) and IL-4 in lymphocytes from a developmental organ (thymus) and a mature lymphoid organ (spleen) examined. Estrogen significantly increased IFN-gamma and IL-2 mRNA in concanavalin-A (Con-A) activated thymocytes, splenic lymphocytes, and in enriched splenic T cells. Estrogen had no marked effect on IL-4 mRNA. While estrogen increased IFN-gamma mRNA in Con-A activated unseparated splenic lymphocytes and enriched splenic T cells, a numerical increase in IFN-gamma was noticed only in the supernatants of Con-A activated unseparated splenic lymphocytes, but not in enriched splenic T cells. This suggests that for optimal secretion of IFN-gamma in estrogen-treated mice, co-stimulatory signals from antigen presenting cells are needed. Gender differences in IFN-gamma and IL-2 mRNA were also evident. Con-A activated splenic lymphocytes from gonadal-intact, untreated female had a pattern of numerical increase in IFN-gamma mRNA, and IFN-gamma and IL-2 protein levels compared to their male counterparts. Taken together, our data suggests that estrogens regulate the expression of cytokines, which could account in part, for the gender differences in immune capabilities.

Deletion of wboA enhances activation of the lectin pathway of complement in Brucella abortus and Brucella melitensis.

Brucella spp. are gram-negative intracellular pathogens that survive and multiply within phagocytic cells of their hosts. Smooth organisms present O polysaccharides (OPS) on their surface. These OPS help the bacteria avoid the bactericidal action of serum. The wboA gene, coding for the enzyme glycosyltransferase, is essential for the synthesis of O chain in Brucella. In this study, the sensitivity to serum of smooth, virulent Brucella melitensis 16M and B. abortus 2308, rough wboA mutants VTRM1, RA1, and WRR51 derived from these two Brucella species, and the B. abortus vaccine strain RB51 was assayed using normal nonimmune human serum (NHS). The deposition of complement components and mannose-binding lectin (MBL) on the bacterial surface was detected by flow cytometry. Rough B. abortus mutants were more sensitive to the bactericidal action of NHS than were rough B. melitensis mutants. Complement components were deposited on smooth strains at a slower rate compared to rough strains. Deposition of iC3b and C5b-9 and bacterial killing occurred when bacteria were treated with C1q-depleted, but not with C2-depleted serum or NHS in the presence of Mg-EGTA. These results indicate that (i) OPS-deficient strains derived from B. melitensis 16M are more resistant to the bactericidal action of NHS than OPS-deficient strains derived from B. abortus 2308, (ii) both the classical and the MBL-mediated pathways are involved in complement deposition and complement-mediated killing of Brucella, and (iii) the alternative pathway is not activated by smooth or rough brucellae.

Molecular comparison of the dense granule proteins GRA6 and GRA7 of Neospora hughesi and Neospora caninum.

Neospora hughesi is a recently described apicomplexan parasite that has been associated with several cases of equine protozoal myeloencephalitis. The biology of this new parasite is just beginning to be defined. Towards this understanding, we report important differences between the nucleotide and deduced amino acid sequences of the dense granule proteins GRA6 and GRA7 of N. hughesi and Neospora caninum. This information can be used to differentiate the two species and contribute to further understanding of the prevalence and biology of N. hughesi. The newly defined proteins of N. hughesi are referred to as NhGRA6 and NhGRA7 in keeping with the protocol for naming homologous proteins of the Apicomplexa. Genes of the two dense granule proteins of N. hughesi (isolate Nh-A1) and four different isolates of N. caninum were isolated via PCR and their DNA sequences were determined. Computer analysis indicated that the two gene sequences were identical among all four N. caninum isolates. However, the gene for NhGRA6 was found to be 96 nucleotides longer at the 3' end than that of NcGRA6, resulting in a protein product that is 32 amino acids larger than NcGRA6. Two tandem repeat sequences were identified at the 3' end of the NhGRA6 gene. These repeat sequences contributed to the lengthening of the carboxy terminus of NhGRA6 in comparison with that of NcGRA6. The larger size of NhGRA6 was further confirmed by Western blot analysis in which NcGRA6 monospecific antibodies recognised a protein of approximately 42 kDa in N. hughesi whole tachyzoite preparation but a protein of 37 kDa in N. caninum whole tachyzoite preparation. Analysis of GRA7 gene sequences indicated a 6 and 14.8% difference at nucleotide and amino acid sequence level, respectively, between NcGRA7 and NhGRA7. Despite the same number of residues in the deduced amino acid sequences of all the GRA7 proteins, Western blot analysis indicated a difference in the migration pattern of NhGRA7 in comparison with NcGRA7. Results of our study indicate that diagnostic tests based on differences in dense granule sequences and antigenicity may have potential to differentiate between N. hughesi and N. caninum. Such diagnostic tests would be valuable tools to aid in our understanding of the epidemiology of these parasites. Additionally, dense granule proteins are immunogenic and they may have potential as use in recombinant vaccines against neosporosis.