Early IL-4 gene expression in abomasum is associated with resistance to Haemonchus contortus in hair and wool sheep breeds.

Early immune events associated with reduced larval burden remain unclear in parasite-resistant breeds of sheep. Therefore, our objective was to determine breed differences in immune-related gene expression following infection with H. contortus. Gene expression in abomasal tissue and mucosa and in abomasal lymph nodes (ALN) was measured in 24 St. Croix (hair) lambs and 24 Dorset x (Finn-Rambouillet) (wool) lambs at 0 (uninfected), 3, 5 and 7 days after infection with 10 000 L3 H. contortus larvae. Expression of IL-4 in abomasal mucosa was detected on day 3 and increased to day 7 in hair lambs, but was not detectable in wool lambs. Genes that recruit neutrophils (CXCL1) and macrophages (MCP1) were upregulated in abomasal mucosa of hair lambs. Genes associated with alternative macrophage activation (ARG-1) and eosinophil activation (Gal-14) were also upregulated in the abomasal mucosa of hair lambs. Tissue remodeling genes (MMP13, PDGF) and tumour necrosis factor alpha (TNF-α) and MCP1 were upregulated in abomasal tissue of wool lambs; these lambs also had greater expression of forkhead box P3 in ALN. These data indicate a role for early IL-4 expression locally and demonstrate potential downregulation of immunity in wool sheep that could facilitate establishment of H. contortus.

Gene expression profiles of hair and wool sheep reveal importance of Th2 immune mechanisms for increased resistance to.

Management of gastrointestinal parasites is a critical issue for sheep producers worldwide. Increases in the prevalence of drug-resistant worms have complicated parasite control and increased economic losses. Therefore, other methods of parasite control need to be assessed, including the use of genetically resistant animals in breeding programs. Hair sheep breeds such as the St. Croix have greater parasite resistance than conventional wool breeds. However, the immune mechanisms that control parasite resistance in hair or wool breeds have not yet been fully determined, and information on cytokine expression profiles for both wool sheep selected for increased resistance and hair sheep is limited. Our objective was to investigate gene expression differences in 24 parasite-resistant hair and 24 susceptible wool sheep to identify immune effectors associated with resistance to . One-half of the lambs were infected and sacrificed at 3 or 27 d after infection. Remaining lambs were not infected. Breed differences in expression of genes associated with Th1 and Th2 immune responses in lymph nodes and abomasal tissue were determined. Th2-associated genes included IL-4, IL-13, IL-5, IgE, the α chain of the IL-4 receptor, and the α chain of the high-affinity IgE receptor (FcεRI). Th1-associated genes included interferon gamma (IFN-γ), the p35 subunit of IL-12 (IL-12 p35), and the ß1 and ß2 chains of the IL-12 receptor (IL-12 Rß1 and IL-12 Rß2, respectively). In both hair and wool sheep, infection with resulted in greater expression of IgE, IL-13, IL-5, and IL-12 p35 and somewhat reduced expression of IFNγ in lymph nodes. In abomasal tissue, parasite infection resulted in greater IgE, IL-13, FcεRI, and IL-12 p35 expression in infected lambs compared with control lambs. Between breeds, hair sheep had a stronger Th2 response after infection than wool sheep, with increased expression of IgE and IL-13 and decreased expression of IFNγ in lymph nodes and increased expression of IL-13 and decreased expression of IL-12 p35 in abomasal tissue. Expression of IL-4 in lymph nodes did not differ between hair and wool lambs, and IL-4, IL-5, IL-12 Rß1, and IL-12 Rß2 expression was too low to measure at the times sampled in abomasal tissue.

Prevalence of IgG Antibodies to Toxoplasma gondii in Veterinary and Undergraduate Students at Virginia Tech, Blacksburg, Virginia.

Toxoplasma gondii is a globally distributed parasitic protozoan that infects humans and other warm-blooded vertebrates. Felids are the only definitive host for T. gondii, and they excrete oocysts in their faeces. The national prevalence in humans is declining in the United States. This zoonotic organism is of particular interest due to its importance in pregnant women, in individuals with altered immune systems, and in reactivated ocular infections. Exposure to the parasite in humans is usually associated with consumption of raw or undercooked meat or by accidental ingestion of oocysts. It was hypothesized that veterinary students would have a greater chance at exposure to the parasite than an average population of undergraduate students due to increased contact with cats who are infected. A commercially available ELISA was used to examine serum samples from 336 students (252 veterinary students and 84 undergraduate students) at Virginia Polytechnic Institute and State University and the Virginia-Maryland Regional College of Veterinary Medicine for serum IgG antibodies to T. gondii antigen. The prevalence of T. gondii in these subjects was 5.6% in veterinary school students (n = 252) and 2.4% in undergraduates (n = 84). There was no significant difference (P > 0.05) in the prevalence of T. gondii antibodies in veterinary versus undergraduate students. The overall prevalence of 4.8% in all students in this study reflects the continuing decline of antibodies to T. gondii in humans in the United States.

The effect of vitamin E supplementation on an experimental Haemonchus contortus infection in lambs.

The objective of this study was to investigate the effect of vitamin E supplementation on an experimental Haemonchus contortus infection in lambs. Twenty lambs were stratified into two treatment groups based on fecal egg count. Worm-free lambs, 28-32 weeks of age, were supplemented with vitamin E (d-α-tocopherol) for 12 weeks following the recommendations of the National Research Council for the minimum daily requirement (control; 5.3 IU/kg body weight (BW)/day (d), n=10) or the requirement for optimal immune function (VE10; 10 IU/kg BW/d, n=10). Five weeks following initiation of vitamin E supplementation, lambs were infected with 10,000 H. contortus third stage larvae. Samples were taken weekly to quantify serum α-tocopherol, serum total non-specific immunoglobulin (Ig)G, whole worm antigen specific IgG, packed cell volume (PCV), and fecal egg count (FEC). Expression of cytokine genes IFN-λ and IL-4 were measured in peripheral blood collected prior to slaughter. Lambs were necropsied six weeks after infection and the α-tocopherol concentration of liver, muscle and lymph node were measured as well as abomasal worm burden and histologic evaluation of the abomasum for inflammation and enumeration of eosinophils and globule leukocytes. The livers of VE10 lambs contained slightly more α-tocopherol than control lambs. No differences were observed in serum, muscle or lymph node α-tocopherol concentration, serum IgG or peripheral mRNA expression of IL-4 or IFN-λ between control and VE10 lambs. However, lambs supplemented at 10IU/kg BW/d had a lower PCV reduction, FEC and worm burden 49% less than control lambs. Worm burden was negatively correlated with eosinophil (-0.720, P<0.05) and globule leukocyte count (-0.867, P<0.05). Strong positive correlations were observed within the inflammatory cell response in VE10 lambs that was absent in control lambs. These data indicate that additional vitamin E supplementation resulted in lower worm burden and greater recruitment of innate effector cells to the site of infection. Further studies are necessary to elucidate the mechanism by which vitamin E affects greater recruitment of innate effector cells to the abomasum during gastrointestinal nematode infection of lambs.

The use of redberry juniper (Juniperus pinchotii) to reduce Haemonchus contortus fecal egg counts and increase ivermectin efficacy.

Objectives of this study were to determine if a redberry juniper-based diet can reduce fecal egg counts (FEC) and increase ivermectin (IVM) efficacy in IVM-resistant Haemonchus contortus. Predominant genera present were Haemonchus (range 45-100%) and Trichostrongylus (range 0-47%). The FEC reduction for IVM in the ewe flock was 25% (95% confidence intervals 79% to -162%) and confirmed IVM resistance. After natural infection was established, Barbados Blackbelly and St. Croix lambs (n=64, 6 months old) were randomly assigned to pens and fed a pelleted treatment diet (4 pens/treatment and 8 lambs/pen) consisting of traditional feed ingredients mixed with either 30% hay (CNTL) or 30% ground juniper leaves and stems (JUN). Lambs were fed during two periods: Period 1 (days 0-28) and Period 2 (days 28-42). On day 28, half of the lambs from each treatment and pen were treated with IVM orally (0.2 mg/kg), creating four treatment groups: lambs fed CNTL or JUN and either not treated (CNTLn, JUNn) or treated (CNTLi, JUNi) with IVM. During Period 1, lambs fed CNTL had greater (P<0.001) average daily gain than lambs fed JUN, which was probably caused by the CNTL diet having greater protein and less acid detergent fiber, lignin, and condensed tannins than the JUN diet. Lambs had similar (P>0.46) FEC on days 0 and 28, but lambs fed JUN had 69.1% lower (P<0.001) FEC on day 15 as compared to lambs fed CNTL. During Period 2, CNTLi lambs had greater (P<0.05) average daily gain than JUNn and JUNi lambs. Lambs fed JUN and treated with IVM (JUNi) had 66%, 65%, and 61% lower (P<0.05) FEC as compared to CNTLn, CNTLi, and JUNn lambs, respectively. Results suggest that feeding lambs a diet containing 30% redberry juniper reduced FEC and increased IVM efficacy by 65% (JUNi vs. CNTLi). Specific mechanisms involved in increasing IVM efficacy by feeding diets containing bioactive compounds warrants further investigation.

Effects of high pressure processing on hatching of eggs of the zoonotic rat tapeworm Hymenolepis diminuta.

High hydrostatic pressure processing (HPP) is an effective non-thermal treatment to remove pathogens from a variety of food and food products. It has been extensively examined using prokaryotic organisms but has had limited study on eukaryotic organisms. Treatment using HPP has been shown to be effective in inactivating nematode larvae in food and Ascaris suum eggs. Nothing is known on the efficacy of HPP on tapeworm cysts or eggs. Eggs of important zoonotic tapeworms including Echinococcus and Taenia spp. can potentially contaminate water and food intended for human consumption. The present study examined the efficacy of HPP on the viability of Hymenolepis diminuta eggs. Efficacy of HPP treatment was measured using an egg hatch assay in two experiments. One thousand unhatched H. diminuta eggs in Hanks balanced salt solution were packaged in sealable bags and exposed to 100-600megapascals (MPa; 1MPa=10atm=147psi) for 60s in a commercial HPP unit. Positive (no HPP) and negative (No HPP but frozen/thawed) controls were examined in each experiment. None of the HPP untreated and frozen eggs (negative controls) were able to hatch or exclude trypan blue when placed in the hatching solution in experiment 1 or 2. HPP untreated and nonfrozen eggs (positive controls) hatched and excluded trypan blue; 75% were positive in experiment 1 and 80% were positive in experiment 2. No hatched eggs were observed when they were exposed to 300-600MPa for 60s. Treatment at 400MPa and above caused rupturing of the oncosphere. Results from this study indicate that HPP is a possible method to inactivate tapeworm eggs and that the susceptibility of tapeworm eggs to HPP is similar to or greater than that of nematode eggs or tissue larvae.

Effect of vitamin E supplementation on naturally acquired parasitic infection in lambs.

Gastrointestinal nematode infections cause substantial economic losses in pasture-based sheep farming worldwide. Host nutritional status has been identified as a key component of immune function. While vitamin E supplementation is known to have broad-spectrum effects on immune function in livestock, to our knowledge, there are no reports on the effect of vitamin E supplementation on trichostrongylid parasite infections in lambs. This study evaluated the effect of parenteral vitamin E supplementation on naturally acquired parasite infection in lambs. Twenty-seven spring lambs were sequentially assigned to receive injections of vitamin E (15 or 30 IU D-α-tocopherol/kg body weight (BW) or placebo, every two weeks, from birth to 28 weeks of age. Blood was collected at weeks 0, 2, 4, 8, 12, 16, 20, 24, and 28 to determine serum α-tocopherol concentration. Once the youngest animal reached 15 weeks of age all lambs were dewormed and grazed together on a pasture known to be contaminated with trichostrongylid larvae. Fecal egg count and blood packed cell volume (%) were determined on each lamb immediately prior to deworming and for the first seven weeks of pasture infection. Lambs were euthanized when they reached 28 weeks of age for determination of parasite worm burdens. Vitamin E supplementation at 30 IU/kg BW increased serum α-tocopherol over that of placebo (P<0.001) however, there was no effect of vitamin E supplementation on liver (P=0.804) or muscle (P=0.16) α-tocopherol content. There was no effect of vitamin E supplementation on fecal egg counts, packed cell volume, worm burden or nematode species distribution. Nematode genera identified were Haemonchus (30%), Trichostrongylus (42%), Nematodirus (27%), Strongyloides sp. (<1%), and Aonchotheca sp. (<1%). These results indicate that biweekly injections of vitamin E at 15 and 30 IU d-α-tocopherol/kg BW, had no effect on parasitological parameters used in the study to assess gastrointestinal nematode infection.

Differences in immune parameters are associated with resistance to Haemonchus contortus in Caribbean hair sheep.

Caribbean hair sheep are more resistant to gastrointestinal nematodes than conventional wool breeds, but mechanisms that confer resistance are not fully understood. This study compared immune effector cell populations and antibody concentrations in 12 hair and 12 wool lambs infected with the abomasal parasite Haemonchus contortus and sacrificed at 3 or 27 days post-infection (p.i.) and 14 uninfected animals of each breed. Faecal egg counts were over 2.5-fold higher (P = 0.12) and packed cell volumes approximately 8% lower (P < 0.10) in infected wool lambs. Abomasal lymph nodes were heavier in infected animals (P < 0.05) and infected hair sheep had larger lymph nodes than infected wool sheep (P < 0.05). Tissue eosinophil concentrations were likewise larger (P = 0.07) in hair compared with wool sheep at 3 days p.i. Circulating levels of IgE and IgA in uninfected lambs were higher in hair sheep (P < 0.05) and during infection, hair sheep had higher serum IgA than wool sheep at 3, 5, and 21 days p.i. (P < 0.05). Serum IgE in infected lambs did not differ between breeds, but concentrations of IgE in lymph nodes were higher (P < 0.01) at 27 days p.i. in infected hair sheep.

Toxoplasma gondii and Trypanosoma cruzi antibodies in dogs from Virginia.

Toxoplasma gondii and Trypanosoma cruzi are zoonotic protozoan parasites that cause disseminated infections in many vertebrate species. The present study determined the seroprevalence of T. gondii and Tr. cruzi in a population of dogs from Virginia. Serum samples were tested from 90 domestic dogs collected from animal shelters in Virginia. Using an indirect immunofluorescent antibody test, sera were examined at a 1 : 50 dilution and antibodies to T. gondii were found in 19 dogs (21%). Antibodies to Tr. cruzi were determined by qualitative immunochromatographic dipstick assay. One (1%) of the 90 dogs had Tr. cruzi antibodies and it was also seropositive for T. gondii. Our findings indicate that dogs are frequently exposed to T. gondii in Virginia, but that antibodies to Tr. cruzi are rare in the same geographical region.

Microarray analysis reveals difference in gene expression profiles of hair and wool sheep infected with Haemonchus contortus.

Sheep infected with the abomasal parasite, Haemonchus contortus, have reduced growth rates, decreased wool production, and anemia, and heavy infections may result in death. Anthelmintic treatment can remove worms, but the cost of treatment and prevalence of drug-resistant worms has led to greater focus on genetic resistance of the host to parasitism. Variation in parasite resistance exists within and among sheep breeds, and Caribbean hair sheep have greater resistance than most conventional wool breeds. Our objective was to investigate differences in gene expression between 24 parasite-resistant hair and 24 susceptible wool lambs to determine genetic mechanisms involved in resistance to H. contortus. Half of the animals of each breed were infected and sacrificed at 3 or 27 days post-infection; the remaining animals were uninfected controls. Breed differences in abomasum and abomasal lymph node tissue gene expression were assessed using bovine cDNA microarrays. Over 60 transcripts differed between breeds for each tissue and infection status. Genes differentially expressed between hair and wool sheep 3 days PI were assessed for gene function and mechanisms for greater immune cell infiltration, abomasal tissue repair, Th17 response, and anticoagulation were present in parasite-resistant hair sheep. By 27 days PI, hair sheep had greater expression of genes involved in gut motility, inflammatory cytokines, and cell proliferation and differentiation compared to wool sheep. Changes in these processes indicate Caribbean hair sheep have a stronger inflammatory response when infected with H. contortus which may facilitate the increased parasite resistance observed in these sheep.

Variation in Eimeria oocyst count and species composition in weanling beef heifers.

Rectal fecal samples were collected daily on 10 consecutive days in November 2004 from 11 weaned beef heifers to assess daily variation in fecal oocyst count and species composition. Subsequent samples were collected from the same animals on 15 April 2005 and 9 June 2005. Oocyst numbers were determined by the modified McMaster's test, and species were identified by examination of oocysts recovered with the Wisconsin sugar flotation technique. Soil samples were collected from the heifer pasture on 8 June 2005, and oocysts were quantified and identified to species. Mean fecal oocyst counts varied little at all sampling dates ranging from 134-377 oocysts/g. Ten Eimeria spp. were identified in fecal samples collected in November and April and 11 in June. Eimeria bovis was the most common species identified at all samplings. Mean species composition showed little variation during the 10-day sampling period in November, remained similar in April, and varied slightly in June. Twelve Eimeria spp. were identified in soil samples in proportions similar to those seen in fecal samples. The results indicate that clinically normal weanling beef heifers are likely to be infected with a diverse, but relatively stable, community of Eimeria spp.

Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum.

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.

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

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

Inheritance of fecal egg count and packed cell volume and their relationship with production traits in sheep infected with Haemonchus contortus.

This study describes responses to artificial infection with Haemonchus contortus in ewes and lambs of 50% Dorset, 25% Rambouillet, and 25% Finn-sheep ancestry and provides estimates of genetic parameters for measures of parasite resistance. One hundred ninety-eight ewes out of 64 sires, and 386 lambs out of 25 sires were evaluated in autumn and spring of 2 yr. Ewes were dewormed shortly after weaning their lambs and lambs were dewormed at about 120 d of age. One week after deworming, ewes and lambs were dosed with approximately 10,000 infective larvae of H. contortus. After infection, BW, fecal egg counts (FEC), and packed cell volume (PCV) were measured weekly for 7 wk in lambs kept in drylot and fortnightly for 11 wk in ewes on pasture. Summary traits were defined as initial PCV, mean BW across all times, and means for PCV (MPCV) and log-transformed FEC (MLFEC) at wk 3 to 7 after infection for lambs and wk 3 to 11 after infection for ewes. Ewes and lambs did not lose weight overall in any year or season, but there was no consistent effect of year or lambing season on mean LFEC or mean PCV during infection in either ewes or lambs. Yearling ewes were less resistant to infection than older ewes, with lower PCV (P < 0.05) and higher LFEC (P < 0.05). During infection, PCV was positively correlated with BW and negatively correlated with LFEC in both ewes and lambs. In lambs, heritabilities were 0.39 (P < 0.01) for PCV, 0.10 (P < 0.05) for LFEC across all measurement times, and 0.19 (P < 0.01) for three measures of LFEC taken at the peak of infection. Heritability estimates for ewes were 0.15 (P < 0.05) for PCV and 0.31 (P < 0.01) for LFEC. Repeatabilities for LFEC and PCV across measurement times were moderate in ewes and lambs. Correlations between dam and lamb records for MLFEC were generally low, suggesting different mechanisms of resistance in lambs and nonlactating ewes. Ewes with higher genetic merit for growth as lambs were less resistant to infection as adults, but genetic merit for fertility and prolificacy were not related to parasite resistance. Lambs with higher genetic merit for body weight were more resistant to infection. Selection for resistance to H. contortus is therefore possible and should not adversely affect growth of lambs and fertility of ewes in this production environment.

Performance of hair sheep composite breeds: resistance of lambs to Haemonchus contortus.

This study was designed to evaluate breed differences in resistance to Haemonchus contortus in lambs. A total of 181 ewe lambs representing crossbred Dorsets (DO) and Dorpers (DP; out of 1/2-Dorset, 1/4-Rambouillet, 1/4-Finnsheep ewes) and straight-bred Katahdins (KT) were evaluated over 3 yr. An additional 144 DO, DP, KT, and Barbados Blackbelly x St. Croix (HH) wethers were evaluated over 2 yr. Lambs were weaned at 60 to 90 d of age. After deworming at about 4 mo of age, ewe lambs received approximately 10,000 infective larvae and were evaluated for parasite resistance in drylot, whereas wethers were evaluated on pasture under conditions of natural infection. Each sex was analyzed separately. Egg counts per gram of feces (FEC), log-transformed FEC (LFEC), packed cell volumes (%), and body weights (kg) measured at 3, 4, 5, and 6 wk after deworming and reinfection were analyzed. Breed influenced all traits (P < 0.05) except BW in ewe lambs and PCV in wethers. Year and week influenced (P < 0.05) all traits. At most times, DP had the highest FEC, DO had the lowest PCV, and KT and HH had lower FEC and higher PCV than either DO or DP. Clearly, Dorper sheep were not more resistant to parasites than DO, but they were able to cope with infection better by maintaining similar or higher (P < 0.05) PCV and similar BW. Katahdin and HH were more resistant, with lower FEC (P < 0.05) than DO or DP. Breed differences were more apparent when infection levels were higher; DO and DP were less affected when infection levels were low and when animals were on a better plane of nutrition. Caribbean hair sheep originated in hot, humid regions of West Africa. They and the derivative Katahdin breed seem relatively resistant to parasitism. The Dorper, in contrast, was developed in more arid regions and exhibited little parasite resistance.

Prevalence of agglutinating antibodies to Sarcocystis neurona in raccoons, Procyon lotor, from the United States.

Equine protozoal myeloencephalitis (EPM) is the most important protozoal disease of horses in North America and it is caused by Sarcocystis neurona. Natural cases of encephalitis due to S. neurona have been reported in raccoons, Procyon lotor. We examined 99 raccoons for agglutinating antibodies to S. neurona using the S. neurona agglutination test (SAT) employing formalin-fixed merozoites as antigen. Raccoons originated in Florida (N=24, collected in 1996), New Jersey (N=25, collected in 1993), Pennsylvania (N=25, collected in 1999), and Massachusetts (N=25, collected in 1993 and 1994). We found that 58 (58.6%) of the 99 raccoons were positive for antibodies to S. neurona using the SAT; 44 of 99 raccoons (44%) had titers of > or =1:500. This prevalence is similar to the reported seroprevalence of 33-60% for S. neurona antibodies in horses from the United States using the Western blot test.

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.

Neospora hughesi: experimental infections in mice, gerbils, and dogs.

Neospora hughesi is a recently described cause of equine protozoal myeloencephalitis (EPM). A rodent model for pathogenicity would facilitate development of therapies to be used in horses. In the present study, we examined the susceptibility of BALB/c gamma-interferon gene knockout (gamma-INFKO), BALB/c, CD-1, and C57BL/6 strains of mice and gerbils to infection with tachyzoites of the Nh-A1 strain of N. hughesi isolated from a horse from AL, USA. Only the gamma-IFNKO mice developed severe clinical disease following infection with N. hughesi and died 19-25 days after infection and exhibited severe cardiac lesions. In contrast, experimental infection of gamma-INFKO mice with tachyzoites of the NC-1 or NC-Liverpool strains of Neospora caninum resulted in deaths 8-10 days after infection. The most severe lesions were in the livers, spleens, and lungs of these mice. Gerbils inoculated with N. hughesi did not develop clinical disease, had few microscopic lesions, but did seroconvert. Two dogs fed the brains of mice, shown to contain N. hughesi tissue stages by cell culture and gamma-IFNKO mouse bioassay, did not shed N. caninum-like oocysts over a 23 days observation period. The marked difference in pathogenicity between the two species of Neospora in gamma-IFNKO mice, and lack of oocyst excretion by dogs fed N. hughesi infected mice provide additional evidence that the species distinction between N. caninum and N. hughesi is valid.

Multiple anthelmintic resistance in a goat herd.

Anthelmintic resistance was monitored over a 30 month period within a goat herd in eastern Virginia, USA. Resistance to ivermectin, levamisole and benzimidazole drugs was detected in Haemonchus contortus using the fecal egg count reduction test (FECRT). When levamisole use was discontinued for 1 year, susceptibility to levamisole appeared to return. Although a single treatment with fenbendazole was able to reduce fecal egg counts by only 50%, two doses administered in a 12 h interval increased efficacy to 92%, however, confidence intervals indicated that resistance was still present. When fecal egg counts were determined the following year after several treatment using this protocol, the efficacy of fenbendazole had fallen again to 57% reduction in fecal egg counts. The predominant genus present in cultured composite fecal samples was Haemonchus. Trichostrongylus, Cooperia and Teladorsagia were also present in smaller numbers.