Endogenous transplacental transmission of Neospora hughesi in naturally infected horses

Over a 2-yr study period, we investigated possible endogenous transplacental transmission of Neospora hughesi in 74 mare and foal pairs following the diagnosis of neuronal neosporosis in a weanling foal. Presuckle and postsuckle serum of each foal, serum and colostrum of each periparturient mare, and serum of each mare and foal pair, collected at 3-mo intervals thereafter, were tested for N. hughesi using an indirect fluorescent antibody test (IFAT). Furthermore, whole blood and colostrum samples and placentae were tested for the presence of N. hughesi by real-time PCR. The mares' seroprevalence at foaling based on IFAT (titer ≥ 160) was 52 and 6% in 2006 and 2007, respectively. Colostral antibodies against N. hughesi were detected in 96 and 11% of the mares in the 2-yr study. With the exception of 3 foals, all remaining foals were born seronegative to N. hughesi. Passive transfer of colostral antibodies to N. hughesi was documented in 15 foals. Three foals born from 2 different mares had presuckle antibodies at a titer ranging from 2,560 to 20,480. All 3 foals were born healthy. Two foals were born to the same dam that also gave birth to the weanling diagnosed with neuronal neosporosis in 2005. The third foal was born to a second mare with no previous foaling history at the farm. Seroconversion was documented in 10 foals and 9 mares over the 2-yr study. All blood and colostrum samples tested PCR negative for N. hughesi. Only 1 placenta collected in 2007 from the mare with the 2 congenitally infected foals tested PCR positive for N. hughesi. In conclusion, N. hughesi persisted in this population via endogenous transplacental infection.     

Evaluation and comparison of an indirect fluorescent antibody test for detection of antibodies to Sarcocystis neurona, using serum and cerebrospinal fluid of naturally and experimentally infected, and vaccinated horses

The objectives of this study were to evaluate the accuracy of the indirect fluorescent antibody test (IFAT) using serum and cerebrospinal fluid (CSF) of horses naturally and experimentally infected with Sarcocystis neurona, to assess the correlation between serum and CSF titers, and to determine the effect of S. neurona vaccination on the diagnosis of infection. Using receiver-operating characteristic analysis, the areas under the curve for the IFAT were 0.97 (serum) and 0.99 (CSF). Sensitivity and specificity were 83.3 and 96.9% (serum, cutoff 80) and 100 and 99% (CSF, cutoff 5), respectively. Titer-specific likelihood ratios (LRs) ranged from 0.03 to 187.8 for titers between <10 and 640. Median time to conversion was 22-26 days postinfection (DPI) (serum) and 30 DPI (CSF). The correlation between serum and CSF titers was moderately strong (r = 0.6) at 30 DPI. Percentage of vaccinated antibody-positive horses ranged from 0 to 95% between 0 and 112 days after the second vaccination. Thus, the IFAT was reliable and accurate using serum and CSF. Use of LRs potentially improves clinical decision making. Correlation between serum and CSF titers affects the joint accuracy of the IFAT; therefore, the ratio of serum to CSF titers has potential diagnostic value. The S. neurona vaccine could possibly interfere with equine protozoal myeloencephalitis diagnosis.     

Risk of transplacental transmission of Sarcocystis neurona and Neospora hughesi in California horses

The study objective was to assess the risk of transplacental transmission of Sarcocystis neurona and Neospora hughesi in foals from 4 California farms during 3 foaling seasons. Serum of presuckle foals and serum and colostrum of periparturient mares were tested using indirect fluorescent antibody tests for S. neurona and N. hughesi. Serum antibody titers were < or =10 in 366 presuckle foals tested. There was no serologic or histologic evidence of either parasite in aborted fetuses or placentas examined. Positivity for S. neurona and N. hughesi in mares increased with age. Mares < or =9 yr that originated from Kentucky were 3.8 and 1.4 times more likely to be positive for S. neurona and N. hughesi, respectively, than mares from California. The strength of association between positivity to either parasite and state of birth decreased as age increased. Mares positive for S. neurona and N. hughesi were 2.2 and 1.7 times more likely, respectively, to have a previous abortion than negative mares, adjusted for age and state of birth. The annual mortality rate for mares was 4%. The annual incidence rate of equine protozoal myeloencephalitis was 0.2%. In conclusion, there was no detectable risk of transplacental transmission of S. neurona and N. hughesi. Prevalence of antibodies against both parasites in mares increased with age.     

Canine and bovine Neospora caninum control sera examined for cross-reactivity using Neospora caninum and Neospora hughesi indirect fluorescent antibody tests

Neospora caninum is a well known protozoan parasite of domestic and wild animals. Neospora hughesi is a closely related protozoan with an unknown life cycle, host range, and infection prevalence. Many serologic surveys of N. caninum have been performed without consideration of potential cross-reactions with N. hughesi, which could confound results. The aim of this study was to investigate whether postexposure sera from animals experimentally infected with N. caninum exhibit significant reactivity differences when tested using N. caninum and N. hughesi Immunofluorescent Antibody Tests (IFAT). Pre- and postinfection serum samples from 10 dogs, 20 calves, and 17 cows were tested by dual IFATs. All pre-exposure samples for N. caninum tested seronegative for both organisms. All postexposure samples that were seropositive for N. caninum were also positive for N. hughesi, although N. hughesi antibody titers were usually 1 dilution lower (P < 0.02). Serologic surveys for N. caninum may be confounded by cross-reacting titers with N. hughesi, but true positive N. caninum antibody titers are greater than, or equal to, cross-reacting N. hughesi antibody titers.     

Recombinant NhSAG1 ELISA: a sensitive and specific assay for detecting antibodies against Neospora hughesi in equine serum

Neospora hughesi is a recently identified cause of equine protozoal myeloencephalitis. However, the significance of this parasite is poorly understood. An enzyme-linked immunosorbent assay (ELISA) with a recombinant form of the N. hughesi 29-kDa surface antigen (rNhSAG1) was developed for serodiagnosis of equine N. hughesi infections. Parallel ELISA analysis showed that animals immunized or infected with N. hughesi exhibited greater antibody reactivity with rNhSAG1 than with the Neospora caninum homolog, rNcSAG1. The rNhSAG1 ELISA showed 94.4% sensitivity and 95.0% specificity when compared with N. hughesi western blot results for 1,006 samples. The N. hughesi seroprevalence was 3.4% for the 1,917 samples tested by ELISA, which is less than earlier reports. Importantly, western blot analysis of ELISA-positive sera revealed only 18 true seropositive samples for an even lower seroprevalence of 0.9%. These results imply that Neospora spp. infections are uncommon in horses. The sensitivity and specificity exhibited by the rNhSAG1 ELISA suggest that it has a potential use for serodiagnosis of N. hughesi infection in equids. Furthermore, the high-throughput capability of the ELISA will allow for screening large sample sets, which should provide a better understanding of N. hughesi epidemiology.     

Avidity of anti-neurocytoskeletal antibodies in cerebrospinal fluid and serum

Antibodies have different avidities that can be evaluated using modified enzyme-linked immunosorbent assay (ELISA) techniques. We determined levels and avidities of antibodies to light (NFL) and medium (NFM) subunits of neurofilaments and tau protein in serum and cerebrospinal fluid (CSF) from 26 patients and anti-tau antibody levels and their avidities in 20 multiple sclerosis (MS) patients and 20 age- and sex-matched controls. Each sample was analyzed using both standard ELISA and also using a similar ELISA protocol with the addition of urea. The avidities of anti-neurocytoskeletal antibodies were higher in the CSF than those in serum (anti-NFL, p < 0.0001; anti-tau, p < 0.01; anti-NFM, n.s.). There was no relationship between avidities in serum and CSF for individual anti-neurocytoskeletal antibodies. We did not observe the relationship among the avidities of various anti-neurocytoskeletal antibodies. The avidities of anti-tau antibodies in the CSF were significantly higher in the MS patients than those in the controls (p < 0.0001). The study demonstrates the differences in avidities of CSF or serum neurocytoskeletal antibodies measured as the urea resistance by ELISA method. Avidity determination of anti-neurocytoskeletal antibodies could contribute to the evaluation of the immunological status of patients.     

Antigen recognition by serum antibodies in non-human primates experimentally infected with Mycobacterium tuberculosis

Tuberculosis is a significant threat to non-human primates and their caretakers. The diagnosis of tuberculosis in living non-human primates is currently based on the tuberculin skin test, which is cumbersome and sometimes inaccurate. Development of an accurate serodiagnostic test requires identification of the key antigens of Mycobacterium tuberculosis involved in antibody production. When sequential serum samples obtained from 17 cynomolgus, rhesus, and African green monkeys up to seven months since experimental infection with M. tuberculosis Erdman were screened for antibody against purified proteins of M. tuberculosis, three highly seroreactive antigens were identified. One protein, ESAT-6, reacted with sera from all infected animals. Two additional proteins, alpha-crystallin and MTSA-10, were recognized by sera from approximately 90% of infected animals. Time course analysis of antibody production indicated that the earliest response was usually to ESAT-6 alone or to ESAT-6 and other antigen(s). These results provide experimental evidence of the potential value of ESAT-6 as an antigen for use in serodiagnosis of tuberculosis in non-human primates.     

Prevalence of antibodies to Neospora caninum in horses in North America

Serum samples from 296 horses slaughtered for food in the United States were tested for antibodies to Neospora caninum by the Neospora-agglutination test (NAT). Antibodies were found in 69 (23.3%) horses with titers of 1:40 (19 horses), 1:80 (19 horses), 1:100 (3 horses), 1:200 (7 horses), 1:400 (4 horses), and 1:800 (17 horses). This is the first serologic survey for N. caninum antibodies in horses.     

Detection of antibodies to Angiostrongylus cantonensis in serum and cerebrospinal fluid of patients with eosinophilic meningitis

Adult and young adult antigens of Angiostrongylus cantonensis were purified by immuno-affinity chromatography and used to detect antibody in serum and cerebrospinal fluid (CSF), by enzyme-linked immunosorbent assay (ELISA), in cases of human eosinophilic meningitis or meningoencephalitis. The levels of IgG, IgA, IgM and IgE antibodies to A. cantonensis in these patients were higher than levels in control subjects. Antibodies in patients detected against adult and young adult worm antigens of A. cantonensis did not differ significantly. Significantly higher IgM and IgE antibody levels were observed in serum compared with CSF from infected patients (Student's t-test, P less than 0.05). Both adult and young adult A. cantonensis antigens proved to be highly sensitive in ELISA for serum antibodies; however, the sensitivity was significantly lower in tests on CSF.     

Direct PCR detection of phytoplasmas in experimentally infected insects

Phytoplasmas in leafhoppers have been detected by PCR using chrysanthemum yellows (CY)-infected chrysanthemum as source plants, and two cicadellid Deltocephalinae species, Macrosteles quadripunctulatus and Euscelis incisus, as vectors. Three different primer pairs were used; two of these are universal and have been designed on conserved sequences of the 16S rRNA gene of phytoplasmas, and one was designed on extrachromosomal DNA of a severe strain of western aster yellows phytoplasma. They were used to amplify CY DNA obtained by two different extraction procedures; one was extraction with cetyl-trimethyl-ammonium-bromide (CTAB), and the other was boiling in Tris-EDTA buffer. The chromosomal primers amplified phytoplasma-specific bands only from “CTAB” samples, while the plasmid primers were successful with both procedures. Amplification of phytoplasma DNA was possible from as little as 1/10000 of total DNA extracted from a single hopper. Failure to amplify phytoplasma DNA from insects stored at –20^oC for 2 yr suggested that some kind of inhibition develops during long term tissue storage. Direct PCR appeared a very specific, sensitive and rapid method to detect phytoplasmas in fresh leafhoppers, provided that a proper combination of extraction and amplification procedures was used.     

Prevalence of antibodies to Neospora sp. in horses from Alabama and characterisation of an isolate recovered from a naturally infected horse [corrected

An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of the 536 serum samples. Endpoint titres for the positive samples were 1:50 (35/6.5%), 1:100 (19/3.5%), 1:200 (7/1.3%) and 1:1600 (1/0.2%). Tachyzoites were first seen in cultured bovine turbinate cells 32 days after inoculation with spinal cord homogenates from the horse with CNS disease. Tachyzoites reacted with known N. caninum-positive serum from horses, cows, dogs and mice, but did not react with murine anti-Toxoplasma gondii or equine anti-Sarcocystis neurona serum. Ultrastructural features of tachyzoites and results of comparison of tachyzoite immunodominant proteins revealed that they were identical to those of N. hughesi, a species described recently from a naturally infected horse. The isolate recovered from the naturally infected horse in the present study (designated NA1) is thought to be an isolate of N. hughesi, although confirmation of this awaits additional molecular characterisation. These results provide some additional evidence that N. hughesi is a valid species and that Neospora infections in horses may occur in widely separated geographic regions of the United States.     

Effects of miltefosine treatment in fibroblast cell cultures and in mice experimentally infected with Neospora caninum tachyzoites

Miltefosine was investigated for its activity against Neospora caninum tachyzoites in vitro, and was shown to inhibit the proliferation of N. caninum tachyzoites cultured in human foreskin fibroblasts (HFF) with an IC50 of 5·2 μM. Treatment of infected cells with 25 μM miltefosine for a period of 10 h had only a parasitostatic effect, while after 20 h of treatment parasiticidal effects were observed. This was confirmed by transmission electron microscopy of N. caninum-infected and miltefosine-treated HFF. Administration of miltefosine to N. caninum-infected Balb/c female mice at 40 mg/kg/day for 14 days resulted in 6 out of 10 mice exhibiting weight loss, ruffled coat and apathy between days 7 and 13 post-infection. In the group that received placebo, only 2 out of 8 mice succumbed to infection, but the cerebral burden was significantly higher compared to the miltefosine treatment group. In a second experiment, the time-span of treatment was reduced to 5 days, and mice were maintained without further treatment for 4 weeks. Only 2 out of 9 mice in the miltefosine treatment group exhibited signs of disease, while 8 out of 10 mice succumbed to infection in the placebo group. These results showed that miltefosine hampered the dissemination of parasites into the CNS during experimental N. caninum infection in mice.     

Kinetics of antibodies and antigens in serum of mice experimentally infected with Echinostoma caproni (Trematoda: Echinostomatidae)

The present study reports on the kinetics of antibodies and antigens in serum of mice experimentally infected with 75 metacercariae of Echinostoma caproni during the first 12 wk postinfection (wpi). Antibody titers in the serum of mice were determined by an indirect enzyme-linked immunosorbent assay (ELISA) using excretory/secretory (ES) antigens of E. caproni. The early detection of antibodies against ES antigens of E. caproni is feasible using indirect ELISA. Mice developed significant antibody responses at 2 wpi, and the values progressively increased until the end of the experiment. This may be related to the intestinal absorption of adult worm antigens that induces humoral responses. The presence of E. caproni circulating antigens was determined by a capture ELISA based on polyclonal rabbit antibodies against ES antigens of E. caproni. High levels of seroantigens in mice were detected by 1-2 wpi, probably because of the local inflammatory responses in mice induced by the adult worms. A drop in circulating antigen levels was observed at 9 wpi, which could reflect changes in the intestinal tissues over the course of the infection.     

Antigen, antibody and immune complex detection in serum samples from rats experimentally infected with Strongyloides venezuelensis

In order to establish an antigen, antibody and immune complex detection by enzyme-linked immunosorbent assay (ELISA) in serum samples, normal or immunocompromised Wistar rats experimentally infected with Strongyloides venezuelensis were used. The microtitre plates were coated with IgG anti-S. venezuelensis for antigen and immune complex detection and with alkaline parasite extract for antibody detection. Analysis revealed at least 12.5 μg/mL of S. venezuelensis specific antigens in serum samples. Assay for antigen detection was not a good approach for evaluating infection in normal or immunocompromised rats. In normal rats IgG specific for S. venezuelensis was preferentially detected during the first 13 days post-infection (p.i.) and immune complex detection was significantly reduced in 21 p.i. day. On the other hand, in immunocompromised rats, IgG and immune complex were detected during the entire kinetic (5, 8, 13 and 21 p.i). These results suggest that immune complex screening seems to be an alternative for early strongyloidiasis diagnosis in immunocompromised individuals.     

Evaluation of the usefulness of the ELISA method for determining the level of enterovirus antibodies in serum and cerebrospinal fluid

The usefulness of the methods was compared: complement fixation test (CFT), neutralization test (NT) and ELISA IgG and IgM against enteroviruses for the evaluation of specific immune reaction in sera and cerebrospinal fluid (CSF) samples of patients with confirmed enterovirus infections. The criteria were established for the assessment of ELISA results in rapid diagnosis of enterovirus neuroinfections. The criteria accepted by the producer lowered the sensitivity of the method and the possibility of recognition of local synthesis of antibodies in the CNS. The use of serum negative in CFT and negative CSF as reference for the determination made possible using of that kit for rapid diagnosis of neuroinfections. The modified ELISA IgG test makes possible determination of antibodies in CSF and serum, and accepting the generally recognized criteria for local production of antibodies in the CNS the ELISA test makes possible rapid diagnosis of neuroinfections which is not possible by other methods.     

Diagnosis of encephalitozoonosis in experimentally infected rabbits by intradermal and immunofluorescence tests.

The indirect immunofluorescence antibody test was performed on serial blood samples from eight young New Zealand White rabbits with experimental encephalitozoonosis. The test showed seroconversion in six of the eight infected rabbits by the 8th day after inoculation and in all rabbits by the 15th day. Antibody titers reached a peak by about the 36th day after inoculation and remained significantly elevated until the termination of the experiment at 84 days after inoculation. None of four sham-inoculated rabbits showed an immunofluorescence response by the 60th day after inoculation. Immunofluorescence and intradermal test responses were compared before infection and at the 60th day after inoculation in a total of 32 experimentally infected rabbits. Both tests were equally effective (100%) in detecting infected animals. Six of eight (first group) and 22 of 24 (second group) experimentally infected rabbits were confirmed histologically to have lesions compatible with encephalitozoonosis. No cross reactions were observed between Encephalitozoon cuniculi and Toxoplasma gondii, Eimeria perforans, or Eimeria stiedai by intradermal test or immunofluorescence test.