Multilocus Sequence Typing of Eimeria maxima in Commercial Broiler Flocks.

About 35% of all broiler flocks in the United States receive an anticoccidial vaccine, but it is not possible to easily differentiate Eimeria vaccine strains from Eimeria field isolates. Being able to do that would allow using vaccines in a more targeted way. The objective of this study was to collect Eimeria maxima isolates from broiler flocks that received anticoccidial feed additives and flocks that had been vaccinated against coccidia and then test them with a multilocus sequencing typing (MLST) scheme developed for this study. Fecal samples were obtained from commercial broiler flocks in Alabama and Tennessee. Oocyst counts in samples tended to be lower in flocks receiving anticoccidial feed additives and higher in vaccinated flocks. Selected samples were screened for presence of E. maxima by quantitative PCR, and Eimeria spp. composition was investigated by next-generation amplicon sequencing (NGAS) in 37 E. maxima positive samples. Other detected Eimeria spp. besides E. maxima were Eimeria acervulina in 35 samples, Eimeria praecox in 23 samples, Eimeria mitis or Eimeria mivati in 17 samples, and Eimeria necatrix or Eimeria tenella in 10 samples. Six partial E. maxima genes (dnaJ domain containing protein, 70-kDa heat shock protein, prolyl endopeptidase, regulator of chromosome condensation domain containing protein, serine carboxypeptidase, and vacuolar proton-translocating ATPase subunit) of 46 samples were sequenced. The MLST scheme was able to differentiate two vaccines from each other. Three of 17 samples from vaccinated flocks differed from the vaccine used in the flock, while 16 of 29 samples from unvaccinated flocks differed from the vaccine. However, there was also a large number of low-quality, ambiguous chromatograms and negative PCRs for the selected genes. If and when more advanced, possibly next-generation sequencing-based methods will be developed, the genes should be considered as targets.

Tipificación por secuenciación multilocus de Eimeria maxima en parvadas comerciales de pollos de engorde. Alrededor del 35% de todas las parvadas de pollos de engorde en los Estados Unidos recibe una vacuna anticoccidial, pero no es posible diferenciar fácilmente las cepas vacunales de Eimeria de los aislados de campo de Eimeria. La posibilidad de diferenciar entre cepas vacunales y de campo permitiría usar vacunas de una manera más específica. El objetivo de este estudio fue recolectar aislamientos de Eimeria maxima de parvadas de pollos de engorde que recibieron aditivos alimenticios anticoccidiales y parvadas que habían sido vacunadas contra coccidia y luego analizarlos con un esquema de tipificación por secuenciación multilocus (MLST) desarrollado para este estudio. Las muestras fecales se obtuvieron de parvadas comerciales de pollos de engorde en Alabama y Tennessee. Los conteos de ooquistes en las muestras tendieron a ser más bajos en las parvadas que recibieron aditivos alimenticios anticoccidiales y más altos en las parvadas vacunadas. Las muestras seleccionadas se examinaron para determinar la presencia de E. maxima mediante PCR cualitativa, y Eimeria spp. la composición se investigó mediante secuenciación de amplicones de próxima generación (NGAS) en 37 muestras positivas de E. maxima. Además de E. máxima, otras Eimeria spp detectadas, fueron Eimeria acervulina en 35 muestras, Eimeria praecox en 23 muestras, Eimeria mitis o Eimeria mivati en 17 muestras, y Eimeria necatrix o Eimeria tenella en 10 muestras. Se secuenciaron seis genes parciales de E. maxima (proteína que contiene al dominio dnaJ, proteína de choque térmico de 70 kDa, prolil endopeptidasa, proteína que contiene al regulador del dominio de condensación cromosómica, serina carboxipeptidasa y la subunidad de ATPasa vacuolar translocadora de protones) de 46 muestras. El esquema MLST pudo diferenciar dos vacunas entre sí. Tres de 17 muestras de parvadas vacunadas diferían de la vacuna utilizada en la parvada, mientras que 16 de 29 muestras de parvadas no vacunadas diferían de la vacuna. Sin embargo, también hubo una gran cantidad de cromatogramas ambiguos y de baja calidad y PCR negativos para los genes seleccionados. En Cuando se desarrollen métodos más avanzados, posiblemente de próxima generación, basados en la secuenciación, estos genes deben considerarse como objetivos.

Phenotypic characterisation of cell populations in the brains of horses experimentally infected with West Nile virus.

BACKGROUND: West Nile virus (WNV), a mosquito borne member of the Flaviviridae, is one of the most commonly diagnosed agents of viral encephalitis in horses and people worldwide. OBJECTIVES: A cassette of markers for formalin-fixed paraffin-embedded tissue and an archive of tissues from experimental infections in the horse were used to investigate the equine neuroimmune response to WNV meningoencephalomyelitis to phenotype the early response to WNV infection in the horse. STUDY DESIGN: Quantitative analysis using archived tissue from experimentally infected horses. METHODS: The thalamus and hindbrain from 2 groups of 6 horses were compared and consisted of a culture positive tissues from WNV experimentally horses, in the other, normal horses. Formalin-fixed paraffin-embedded tissue from the thalamus and hindbrain were immunolabeled for microglia, astrocytes, B cells, macrophages/neutrophils, CD3+ T cells. Fresh frozen tissues were immunolabeled for CD4+ and CD8+ T lymphocyte cell markers. Cell counts were obtained using a computer software program. Differences, after meeting assumptions of abnormality, were computed using a general linear model with a Tukey test (P<0.05) for pairwise comparisons. RESULTS: In WNV-challenged horses, Iba-1+ microglia, CD3+ T lymphocyte and MAC387+ macrophage staining were significantly increased. The T cell response for the WNV-challenged horses was mixed, composed of CD4+ and CD8+ T lymphocytes. A limited astrocyte response was also observed in WNV-challenged horses, and MAC387+ and B cells were the least abundant cell populations. MAIN LIMITATIONS: The results of this study were limited by a single collection time post-infection. Furthermore, a comprehensive analysis of cellular phenotypes is needed for naturally infected horses. Unfortunately, in clinical horses, there is high variability of sampling in terms of days post-infection and tissue handling. CONCLUSIONS: The data show that WNV-challenged horses recruit a mixed T cell population at the onset of neurologic disease.

Genomewide association study reveals a risk locus for equine metabolic syndrome in the Arabian horse.

Equine obesity can cause life-threatening secondary chronic conditions, similar to those in humans and other animal species. Equine metabolic syndrome (EMS), primarily characterized by hyperinsulinemia, is often present in obese horses and ponies. Due to clinical similarities to conditions such as pituitary pars intermedia dysfunction (formerly equine Cushing's disease), conclusive diagnosis of EMS often proves challenging. Aside from changes in diet and exercise, few targeted treatments are available for EMS, emphasizing the need for early identification of at-risk individuals to enable implementation of preventative measures. A genomewide association study (GWAS) using Arabian horses with a history of severe laminitis secondary to EMS revealed significant genetic markers near a single candidate gene () that may play a role in cholesterol homeostasis. The best marker, BIEC2-263524 (chr14:69276814 T > C), was correlated with elevated insulin values and increased frequency of laminitis ( = 0.0024 and = 9.663 × 10, respectively). In a second population of Arabian horses, the BIEC2-263524 marker maintained its associations with higher modified insulin-to-glucose ratio (MIRG) values ( = 0.0056) and BCS ( = 0.0063). Screening of the predicted coding regions by sequencing identified a polymorphic guanine homopolymer and 5 haplotypes in the 3' untranslated region (UTR). An 11 guanine (11-G) allele at was correlated with elevated insulin values in the GWAS population ( = 0.0008) and, in the second population, elevated MIRG and increased BCS > 6.5 ( = 0.0055 and = 0.0162, respectively). The BIEC2-263524-C and the 3' UTR -11(G) polymorphisms were correlated at a 98% frequency, indicating strong linkage disequilibrium across this 150-kb haplotype. Assays for these markers could diagnose horses with a genetic predisposition to develop obesity. Additionally, discovery of FAM174A function may improve our understanding of the etiology of this troubling illness in the horse and warrants investigation of this locus for a role in metabolic- and obesity-related disorders of other species.

Diagnostic performance and application of a real-time PCR assay for the detection of Salmonella in fecal samples collected from hospitalized horses with or without signs of gastrointestinal tract disease.

The main objective of this study was to assess the diagnostic performance of a real-time polymerase chain reaction (PCR) assay for the detection of Salmonella in fecal samples collected from hospitalized horses with or without signs of gastrointestinal (GI) tract disease. The PCR assay used primers and a probe that targeted the invA gene of Salmonella. Assuming a sensitivity of 100% and a specificity of 96.6%, and a disease prevalence of 2%, 5%, and 10-15% in study horses, the PCR assay had a high (100%) negative predictive value, and a positive predictive value that ranged from 37% in horses without signs of GI disease that tested Salmonella culture-negative, to 60% in horses with signs of GI disease that tested Salmonella culture-negative, to 76-83% in horses with signs of GI disease that tested Salmonella culture-positive. This study provides evidence that the real-time PCR that targets the Salmonella invA gene can be used as a screening test for the detection of Salmonella in feces of hospitalized horses with signs of GI disease. Horses that test PCR-positive can be tested in series using bacteriologic culture to reduce false positive results or to provide additional data (e.g., antibiogram and serotyping data) that can be used to identify potential nosocomial Salmonella infections.

Safety and efficacy of recombinant activated factor VII for refractory hemorrhage in pediatric patients on extracorporeal membrane oxygenation: a single center review.

BACKGROUND: Medically refractory hemorrhage in patients on ECMO (extracorporeal membrane oxygenation) support can have catastrophic complications. Recombinant-Activated Factor VII (rFVIIa; NovoSeven®) may provide lifesaving hemostasis; however, there are reports of catastrophic thrombosis related to its administration. OBJECTIVE: This review attempts to add safety and efficacy data to existing literature regarding the use of rFVIIa for refractory hemorrhage in pediatric patients on ECMO support. Design/ METHODS: A retrospective chart review was performed for all pediatric patients on ECMO who received rFVIIa for refractory hemorrhage from 2004 to 2009. Data was extracted for each refractory bleeding event, including patient blood loss and transfused blood products in the 6 hours before the first dose, between rFVIIa doses and in the 6 hours after the final dose. For purposes of data collection, a hemorrhagic event was defined as new onset hemorrhage or a hemorrhage occurring at least 12 hours after the most recent dose of rFVIIa. RESULTS: In total, seven patients aged 1 month to 15 years received rFVIIa for 14 different hemorrhagic events. There was no significant difference in blood loss or blood product transfusion associated with rFVIIa administration. There was one patient-related and one ECMO-related complication temporally associated with rFVIIa administration: decreased ECMO circuit oxygenator efficiency and the development of an intra-gastric clot requiring surgical evacuation. CONCLUSION: These data suggest limited efficacy for rFVIIa use for refractory hemorrhage in pediatric patients on ECMO support. There were two non-catastrophic complications temporally associated with its administration.

Environmental risk factors associated with West Nile virus clinical disease in Florida horses.

The objective of this study was to examine the extrinsic risk factors of West Nile virus (WNV) clinical disease in Florida horses as established from confirmed and negative horses tested within the state from 2001 to 2003. An Arboviral Case Information Form (ACF) was submitted by a referring veterinarian at the time of testing to the Florida Department of Agriculture and Consumer Services on every horse suspected of a viral encephalitis in Florida. A follow-up survey that focused on arbovirus prevention and farm ecology was created and mailed to the owner of each tested horse. Data from the follow-up survey indicated peak WNV prevalence in the late summer months in Florida. Quarter horses were the most commonly affected breed. The WNV vaccine was highly protective and natural water on the property also had a protective association. Factors that increased the risk of WNV to horses were the use of fans and a stable construction of solid wood or cement. Some risk indicators were dead birds on the property and other ill animals on the property. Data from this retrospective study have helped identify factors associated with WNV transmission in equines in Florida. Horses that have not been vaccinated and show clinical signs of arboviral infection from June to November should be tested for WNV. Horses that have been vaccinated and show clinical signs should be tested when the vaccination was administered within 1 month or greater than 6 months prior to the onset of clinical symptoms associated with WN infection.

Safety of an attenuated West Nile virus vaccine, live Flavivirus chimera in horses.

REASON FOR PERFORMING STUDY: West Nile virus (WNV) infection is endemic and able to cause disease in naive hosts. It is necessary therefore to evaluate the safety of new vaccines. OBJECTIVES: To establish: 1) the safety of a modified live Flavivirus/West Nile virus (WN-FV) chimera by administration of an overdose and testing for shed of vaccine virus and spread to uninoculated sentinel horses; 2) that this vaccine did not become pathogenic once passaged in horses; and 3) vaccine safety under field conditions. METHODS: There were 3 protocols: 1) In the overdose/shed and spread study, horses were vaccinated with a 100x immunogenicity overdose of WN-FV chimera vaccine and housed with sentinel horses. 2) A reversion to virulence study, where horses were vaccinated with a 20x immunogenicity overdose of WN-FV chimera vaccine. Horses in both studies were evaluated for abnormal health conditions and samples obtained to detect virus, seroconversion and dissemination into tissues. 3) In a field safety test 919 healthy horses of various ages, breeds and sex were used. RESULTS: Vaccination did not result in site or systemic reactions in either experimental or field-injected horses. There was no shed of vaccine virus, no detection of vaccine virus into tissue and no reversion to virulence with passage. CONCLUSIONS: WN-FV chimera vaccine is safe to use in horses with no evidence of ill effects from very high doses of vaccine. There was no evidence of reversion to virulence. In addition, administration of this vaccine to several hundred horses that may have been previously exposed to WNV or WNV vaccine resulted in no untoward reactions. POTENTIAL RELEVANCE: These studies establish that this live attenuated Flavivirus chimera is safe to use for immunoprophylaxis against WNV disease in horses.

Efficacy, duration, and onset of immunogenicity of a West Nile virus vaccine, live Flavivirus chimera, in horses with a clinical disease challenge model.

REASON FOR PERFORMING STUDY: West Nile virus (WNF) is a Flavivirus responsible for a life-threatening neurological disease in man and horses. Development of improved vaccines against Flavivirus infections is therefore important. OBJECTIVES: To establish that a single immunogenicity dose of live Flavivirus chimera (WN-FV) vaccine protects horses from the disease and it induces a protective immune response, and to determine the duration of the protective immunity. METHODS: Clinical signs were compared between vaccinated (VACC) and control (CTRL) horses after an intrathecal WNV challenge given at 10 or 28 days, or 12 months post vaccination. RESULTS: Challenge of horses in the immunogenicity study at Day 28 post vaccination resulted in severe clinical signs of WNV infection in 10/10 control (CTRL) compared to 1/20 vaccinated (VACC) horses (P<0.01). None of the VACC horses developed viraemia and minimal histopathology was noted. Duration of immunity (DPI) was established at 12 months post vaccination. Eight of 10 CTRL exhibited severe clinical signs of infection compared to 1 of 9 VACC horses (P<0.05). There was a significant reduction in the occurrence of viraemia and histopathology lesion in VACC horses relative to CTRL horses. Horses challenged at Day 10 post vaccination experienced moderate or severe clinical signs of WNV infection in 3/3 CTRL compared to 5/6 VACC horses (P<0.05). CONCLUSIONS: This novel WN-FV chimera vaccine generates a protective immune response to WNV infection in horses that is demonstrated 10 days after a single vaccination and lasts for up to one year. POTENTIAL RELEVANCE: This is the first USDA licensed equine WNV vaccine to utilise a severe challenge model that produces the same WNV disease observed under field conditions to obtain a label claim for prevention of viraemia and aid in the prevention of WNV disease and encephalitis with a duration of immunity of 12 months.

Comparative efficacies of three commercially available vaccines against West Nile Virus (WNV) in a short-duration challenge trial involving an equine WNV encephalitis model.

We used a severe challenge model that produces clinical West Nile virus (WNV) disease to test the efficacy of three commercially available equine WNV vaccines in horses. Twenty-four healthy, WNV-seronegative horses of varying ages and genders were placed, in random and blind manner, into three trial groups consisting of eight horses each; two horses in each group received (i) an inactivated WNV vaccine (K-WN), (ii) a modified-live vaccine (CP-WN) containing the WNV prM and E proteins expressed by a canarypox vector, (iii) a live-chimera vaccine (WN-FV) containing WNV prM and E proteins expressed in a YF17D vector, or (iv) a diluent control. Challenge by this model caused grave neurological signs, viremia, moderate to severe histopathologic lesions in the brain and spinal cord, and an outcome of 0% survivorship in all six control horses. In contrast, challenge in horses at between 28 days postvaccination with the chimera vaccine and 56 days postvaccination with the commercial inactivated or modified-live vaccine resulted in 100% survivorship (protection from the onset of WNV encephalitis and viremia). Horses vaccinated with the live-chimera vaccine showed significantly fewer clinical signs than did the control horses (P

[An efficient mutational method for photosynthetic bacteria].

The pigment and auxotrophic mutants of Rhodobacter sphaeroides Y6 were obtained by treatment with ethyl methanesulfonate (EMS) followed by lithium chloride (LiCI). Treatment with 0.081 M EPS and subsequent treatment with 0.071 M LiCI resulted in 12% higher frequency of pigment mutations than application of 0.081 M EMS alone; the frequency of auxotrophic mutations increased 2.5-fold when treatment with lithium chloride was applied. A blue shift 10 nm was recorded in the absorption spectrum of carotenoids form YM5-3 green mutant; considerable accumulation of neurosporine was revealed by HPLC and mass spectrometry. The method is efficient for isolating mutants of photosynthetic bacteria.

Leukoencephalitis associated with selective viral replication in the brain of a pony with experimental chronic equine infectious anemia virus infection.

Neurologic disease occurs sporadically in horses infected with the equine infectious anemia virus (EIAV). This report describes a case of clinically severe neurologic disease in a pony experimentally infected with EIAV. This pony did not have fever or anemia, which are the characteristic clinical signs of disease. The histopathologic changes were characterized as lymphohistiocytic periventricular leukoencephalitis. Polymerase chain reaction and in situ hybridization data showed that the brain lesions were directly associated with viral replication and that high-level viral replication occurred selectively within the lesion and not in other tissues. These findings suggest that EIAV-associated neurologic disease is the direct result of viral replication.

Fate of patulin in the presence of the yeast Saccharomyces cerevisiae.

Patulin is known to become analytically non-detectable during the production of cider from contaminated apple juice. The fate of [14C]-labelled patulin during the alcoholic fermentation of apple juice was studied. Three commercial cider strains of Saccharomyces cerevisiae degraded patulin during active fermentative growth, but not when growing aerobically. The products of patulin degradation were more polar than patulin itself and remained in the clarified fermented cider. Patulin did not appear to bind to yeast cells or apple juice sediment in these model experiments. HPLC analysis of patulin-spiked fermentations showed the appearance of two major metabolites, one of which corresponded by both TLC and HPLC to E-ascladiol prepared by the chemical reduction of patulin using sodium borohydride. Using a diode array detector, both metabolites had a lambda(max) = 271 nm, identical to that of ascladiol. The nmr spectrum of a crude preparation of these metabolites showed signals corresponding to those of the E-ascladiol prepared chemically and a weaker set of signals corresponding to those reported in the literature for Z-ascladiol.

Neutralization of maternal IL-4 modulates congenital protozoal transmission: comparison of innate versus acquired immune responses.

IL-4 levels were modulated in mice to test the hypothesis that induction of a maternal type 1 response would decrease the frequency of congenital Neospora caninum transmission. This hypothesis tested the relationship between IL-4 and both innate and adaptive immunity utilizing two basic experimental designs. In the first, maternal IL-4 was neutralized with mAb during pregnancy in naive mice concomitant with initial, virulent infection. In the second, maternal IL-4 was neutralized before pregnancy concomitant with a priming inoculation consisting of live, avirulent N. caninum tachyzoites followed by virulent challenge during subsequent gestation. In mice that were naive before pregnancy, neutralization of IL-4 during gestational challenge did not result in decreased congenital transmission as measured by PCR performed on 1-day-old neonatal mice. In mice that were primed and modulated before pregnancy, congenital transmission from gestational challenge was significantly decreased compared with control mice. Reduction in transmission constituted a decrease in the numbers of mice transmitting N. caninum and a lower frequency of transmission by individual dams (p < 0.05). Decreased congenital transmission was associated with significantly lower levels of maternal splenocyte IL-4 secretion, lower IL-4 mRNA levels, and higher levels of IFN-gamma secretion. Protected mice had significantly decreased Neospora-specific IgG1 compared with nonmodulated mice. These studies define a relationship between maternal Ag-specific immunity and the frequency of congenital transmission and demonstrate that modulation of type 2 cytokine responses can change the frequency of congenital protozoal transmission.

Interferon-gamma and interleukin-12 mediate protection to acute Neospora caninum infection in BALB/c mice.

The type of immune response required to protect mice against clinical disease during acute Neospora caninum challenge was investigated in BALB/c mice. Groups of female BALB/c mice were infected i.p. with N. caninum tachyzoites concomitant with either: (1) antibody to interferon-gamma; (2) recombinant murine interleukin-12; or (3) recombinant murine interleukin-12 plus antibody to interferon-gamma. Mice treated with anti-interferon-gamma alone had increased morbidity/mortality, decreased body weight, increased foci of liver necrosis and increased numbers of N. caninum tachyzoites in the lung by 7 days p.i. compared with controls. Increased disease and parasite load in the anti-interferon-gamma-treated mice was associated with antigen-specific antibody IgG1 > IgG2a and a three-fold decreased ratio of antigen-specific interferon-gamma:interleukin-4. Mice treated with recombinant murine interleukin-12 had decreased encephalitis and brain parasite load at 3 weeks p.i. compared with control mice treated with PBS. In recombinant murine interleukin-12-treated mice, decreased brain lesions and parasite load were associated with antigen-specific antibody IgG2a > IgG1 and a three-fold increased ratio of antigen-specific interferon-gamma:interleukin-4 from splenocytes; the interleukin-12 effect was dependent upon interferon-gamma, as indicated by concomitant in vivo interferon-gamma neutralisation. By 6 weeks p.i. with N. caninum, there were no differences in brain lesions and parasite load between interleukin-12- and PBS-treated groups, indicating that the effects of interleukin-12 on driving a protective type 1 response were transient. These data indicate a role for interferon-gamma, interleukin-12 and type 1 immune responses in control of acute neosporosis in mice.

Detection by PCR of Neospora caninum in fetal tissues from spontaneous bovine abortions.

The routine diagnosis of Neospora caninum abortion is based upon histopathologic changes in fetal tissues and identification of tissue parasites by immunohistochemistry. Confirmation of N. caninum infection by immunohistochemistry has low sensitivity. In the present study, we examined the utility of PCR in detecting N. caninum infection in fetal tissues from spontaneous bovine abortion. DNA was obtained from fresh and formalin-fixed tissues from 61 bovine fetuses submitted for abortion diagnosis. Histopathology and immunohistochemistry determined the true status of N. caninum infection in each fetus. In formalin-fixed paraffin-embedded tissues, PCR detected N. caninum DNA in 13 of 13 true-positive fetuses (100%) and in 1 of 16 true-negative fetuses (6%). In fresh or frozen tissues, PCR detected N. caninum DNA in 10 of 13 true-positive fetuses (77%) and 0 of 11 true-negative fetuses (0%). PCR also detected N. caninum DNA in 6 of 8 fetuses that had typical lesions of N. caninum but were immunohistochemistry negative, indicating a higher sensitivity of PCR in comparison to that of immunohistochemistry. N. caninum DNA was amplified most consistently from brain tissue. PCR detection of N. caninum DNA in formalin-fixed, paraffin-embedded tissues was superior to that in fresh tissues, presumably because of the increased accuracy of sample selection inherent in histologic specimens.

Comparison of intracerebral parasite load, lesion development, and systemic cytokines in mouse strains infected with Neospora caninum.

Neospora caninum, an apicomplexan parasite closely related to Toxoplasma gondii, causes abortion, stillbirths, and congenital neurologic disease in multiple animal species. The present study focuses on the development of encephalitis and intracerebral parasite load that occurs 6 wk postinfection (PI). Utilizing BALB/c, C57BL/6, and B10.D2 mice, an initial investigation was undertaken to determine the relative resistance of inbred strains to N. caninum-induced encephalitis. Relative resistance was defined in terms of central nervous system lesion development and parasite load. Based on other protozoal infections in mice, it was hypothesized that BALB/c and C57BL/6 should be contrasting in their relative resistance to N. caninum, with BALB/c and congenic B10.D2 mice less susceptible than C57BL/6 mice. Contrary to expectation, BALB/c and C57BL/6 were both highly susceptible to the development of N. caninum-induced encephalitis, whereas B10.D2 mice were resistant. Both BALB/c mice and C57BL/6 mice had significantly higher numbers of brain lesions and intracerebral tachyzoites than B10.D2 mice. Resistance in B10.D2 was associated with a high interferon (IFN)-gamma: interleukin (IL)-4 ratio from antigen-stimulated splenocytes, whereas susceptibility in C57BL/6 and BALB/c mice corresponded with a low splenocyte IFN-gamma: IL-4 ratio. In vivo measurement of Neospora-specific isotype antibodies demonstrated predominately IgG2a in serum from B10.D2 mice and IgG1 in serum from BALB/c and C57BL/6 mice. In conclusion, susceptibility of mice to N. caninum is unique compared to other protozoal diseases. The present study also demonstrates that parasite load is a fundamental measurement for evaluating disease induced by N. caninum and that a type 1 cytokine response may be necessary for regulation of this parameter.

Enzymology of oxidation of tropic acid to phenylacetic acid in metabolism of atropine by Pseudomonas sp. strain AT3.

Pseudomonas sp. strain AT3 grew with dl-tropic acid, the aromatic component of the alkaloid atropine, as the sole source of carbon and energy. Tropic acid-grown cells rapidly oxidized the growth substrate, phenylacetaldehyde, and phenylacetic acid. Crude cell extracts, prepared from dl-tropic acid-grown cells, contained two NAD+-linked dehydrogenases which were separated by ion-exchange chromatography and shown to be specific for their respective substrates, dl-tropic acid and phenylacetaldehyde. Phenylacetaldehyde dehydrogenase was relatively unstable. The stable tropic acid dehydrogenase was purified to homogeneity by a combination of ion-exchange, molecular-sieve, and affinity chromatography. It had a pH optimum of 9.5 and was equally active with both enantiomers of tropic acid, and at this pH, phenylacetaldehyde was the only detectable product of tropic acid oxidation. The formation of phenylacetaldehyde from tropic acid requires, in addition to dehydrogenation, a decarboxylation step. By analogy with NAD+-specific isocitrate and malate dehydrogenases, phenylmalonic semialdehyde, a 3-oxoacid, would be expected to be the precursor of phenylacetaldehyde. Other workers have established that isocitrate and malate dehydrogenases catalyze the decarboxylation of enzyme-bound or added 3-oxoacid intermediates, a reaction that requires Mn2+ or Mg2+ ions. Studies with tropic acid dehydrogenase were hampered by lack of availability of phenylmalonic semialdehyde, but in the absence of added divalent metal ions, both enantiomers of tropic acid were completely oxidized and we have not, by a number of approaches, found any evidence for the transient accumulation of phenylmalonic semialdehyde.

Fetal loss in BALB/C mice infected with Neospora caninum.

The suitability of mice as a model for reproductive loss due to Neospora caninum infection was investigated. Groups of mice were infected with 2 x 10(6) N. caninum before pregnancy (10 days) and during pregnancy (days 5 and 10 of gestation). In mice infected before and during early pregnancy, fetal loss was evaluated throughout gestation, and pregnancy loss was evaluated by enumeration of fetal resorptions and total fetuses. In mice infected before pregnancy, no difference was present in resorptions between infected and control mice, although litter size was decreased in the infected mice (P < 0.05). In mice infected during early pregnancy (day 5 gestation) and examined temporally throughout gestation, resorptions were increased in the infected mice compared to the control mice (P < 0.05). In mice infected at 5 days gestation and examined at one time point (day 14 of gestation), the resorption rate for infected mice was 33% and 12% for controls (P < 0.05). Routine histopathologic examination and immunohistochemistry using N. caninum-specific antisera did not identify tachyzoites in placental and fetal tissues during the pre- and early pregnancy infections. In mice infected late midgestation (day 10), N. caninum tachyzoites were identified in placenta and fetal muscle and neural tissue. In the placenta, there was multifocal necrosis and hemorrhage with intralesional tachyzoites. Tachyzoites in fetal tissues were not associated with pathologic changes. No reproductive loss was associated with mice infected late in gestation. These data demonstrate that mice can be used as a model for the study of fetal resorption and congenital infection associated with N. caninum.

Evaluation of fetal infection and abortion in pregnant ponies experimentally infected with Ehrlichia risticii.

Fetal infectivity of Ehrlichia risticii was investigated in 19 ponies that were E risticii negative on the basis of results of an indirect fluorescent antibody (IFA) test. Thirteen pregnant ponies were infected by IV administration of E risticii between 90 and 180 days of gestation. Six pregnant ponies served as noninfected controls. Each infected pony had clinical signs of equine monocytic ehrlichiosis, was confirmed to be ehrlichemic, and developed an IFA titer to E risticii. Two infected ponies became recumbent, were unresponsive to supportive care, and were euthanatized. After recovery from clinical illness, the remaining ponies were observed throughout gestation for reproductive abnormalities. On abortion, each fetus was necropsied and tissue specimens from the liver, bone marrow, spleen, colon, and mesenteric lymph nodes were inoculated into canine monocyte cell cultures. Six infected ponies aborted at a mean 217 days of gestation, which was between postinoculation days 65 and 111. Five fetuses were recovered for evaluation, and E risticii was isolated from 4 of them. All 5 fetuses recovered had similar histologic finding, including enterocolitis, periportal hepatitis, and lymphoid hyperplasia with necrosis of the mesenteric lymph nodes and spleen. All 5 fetuses tested negative for IgG to E risticii, although 3 had low IgM titer to E risticii. The remaining 5 infected ponies had normal parturition. Presuckle IFA titer to E risticii was measured in 4 of the term foals, and results for 3 were positive. Two foals from infected ponies were monitored for 6 months and daily gain in body weight was comparable to that of a control foal. None of the control ponies became ill or seroconverted during the clinical illness phase, and none aborted throughout gestation Two control ponies seroconverted to E risticii 6 weeks before parturition. Results of this study indicate that E ristcii is a primary abortifacient under experimental conditions.

Transformations of morphine alkaloids by Pseudomonas putida M10.

The oxidation of morphine by washed-cell incubations of Pseudomonas putida M10 gave rise to a large number of transformation products including hydromorphone (dihydromorphinone), 14 beta-hydroxymorphine, 14 beta-hydroxymorphinone, and dihydromorphine. Similarly, in incubations with oxymorphone (14 beta-hydroxydihydromorphinone) as substrate, the major transformation product was identified as oxymorphol (14 beta-hydroxydihydromorphine). The identities of all these biological products were confirmed by mass spectrometry and 1H nuclear magnetic resonance spectroscopy. This is the first report describing structural evidence for the biological synthesis of 14 beta-hydroxymorphine and 14 beta-hydroxymorphinone. These products have applications as intermediates in the synthesis of semisynthetic opiate drugs.