Immune responses in pregnant cattle and bovine fetuses following experimental infection with Neospora caninum.

Humoral and cell-mediated immune (CMI) responses [i.e. proliferative responses and gamma interferon (IFN-gamma) production], were elicited in five cows infected between 159 and 169 days of gestation by a combined intravenous-intramuscular inoculation of Neospora caninum tachyzoites. Analysis of antigen-specific immunoglobulin (IgG) subclasses revealed a predominant IgG2 response in two cows, a mixed IgG1-IgG2 response in two other cows and a predominant IgG1 response in one cow. No correlation was found between IgG2 titers and IFN-gamma levels. CD4-T cells were responsible for the CMI responses in peripheral blood mononuclear cells from three infected cows. All five fetuses removed from infected dams at week 9 post-infection (219-231 days of gestation) mounted strong Neospora-specific humoral responses and had a predominant IgG1 response, regardless of their ability to produce IFN-gamma. However, CMI responses were highly variable between fetuses. These data indicate the complexity of the immune mechanisms associated with Neospora infection in both the dams and their fetuses.

A POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation failed to prevent foetal infection in pregnant cattle following i.v./i.m. experimental tachyzoite challenge.

Cattle immunised with a POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation were previously shown to produce interferon (IFN)-gamma at levels similar to those of tachyzoite-infected cattle. In view of the critical role of IFN-gamma in resistance of mice to N. caninum infection, these results prompted us to test the POLYGEN-adjuvanted preparation in pregnant cattle to determine whether it will be able to prevent foetal infection following an experimental tachyzoite challenge. Seven heifers were immunised at 35 and 63 days of gestation with the POLYGEN-adjuvanted preparation, while five heifers were inoculated with POLYGEN alone at the same days of gestation. Four weeks later, all heifers were challenged with a combined i.v./i.m. inoculation of tachyzoites. The same challenge was given to seven unimmunized heifers at the same stage of gestation. An additional unimmunized heifer was inoculated with uninfected monolayer cell culture material. All challenged heifers, immunized and unimmunized, had infected foetuses. Immunized heifers developed both parasite-specific humoral and cellular immune responses, characterised by increased IFAT titres, a predominant IgG1 response, elevated lymphoproliferative response and IFN-gamma production. Following tachyzoite challenge, they developed an anamnestic humoral response and produced similar amounts of IgG1 and IgG2 antibodies, but did not have an anamnestic cellular immune response. The lack of anamnestic cellular immune response and/or the large i.v/i.m tachyzoite inoculum may have contributed to the failure of the preparation.

A modified agglutination test for Neospora caninum: development, optimization, and comparison to the indirect fluorescent-antibody test and enzyme-linked immunosorbent assay.

Current serologic tests used to detect antibodies to Neospora caninum require species-specific secondary antibodies, limiting the number of species that can be tested. In order to examine a wide variety of animal species that may be infected with N. caninum, a modified direct agglutination test (N-MAT) similar to the Toxoplasma gondii modified direct agglutination test (T-MAT) was developed. This test measures the direct agglutination of parasites by N. caninum-specific antibodies in serum, thus eliminating the need for secondary host-specific anti-isotype sera. The N-MAT was compared to the indirect fluorescent-antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA) with a "gold standard" serum panel from species for which secondary antibodies were available (n = 547). All positive samples tested were from animals with histologically confirmed infections. Up to 16 different species were tested. The N-MAT gave a higher sensitivity (100%) and specificity (97%) than the ELISA (74 and 94%, respectively) and had a higher sensitivity but a lower specificity than the IFAT (98 and 99%, respectively). The reduced specificity of the N-MAT was due to false-positive reactions in testing fetal fluids with particulate matter or severely hemolyzed serum. Overall, the N-MAT proved to be highly sensitive and specific for both naturally and experimentally infected animals, highly reproducible between and within readers, easy to use on large sample sizes without requiring special equipment, and useful in testing serum from any species without modification.

Detection of Neospora from tissues of experimentally infected rhesus macaques by PCR and specific DNA probe hybridization.

Neospora is a newly recognized Toxoplasma-like cyst-forming coccidian parasite that causes abortion or congenital infections in naturally or experimentally infected animals. In this study, pregnant rhesus macaques were inoculated with culture-derived tachyzoites of a bovine Neospora isolate, and tissue samples from various major organs were collected from dams and fetuses for the detection of parasite DNA by using oligonucleotide primers COC-1 and COC-2 for PCR amplification of a conserved coccidial nuclear small-subunit rRNA gene sequence, and amplification products were confirmed by hybridization with a Neospora-specific DNA probe. PCR products were amplified from DNAs of different fetal monkey tissues, including brain, heart, lung, liver, spleen, skeletal muscle, skin, and placenta. In addition, Neospora DNA was amplified from the brain, heart, and lung tissues of infected rhesus macaque dams. The PCR and probe hybridization system may provide an effective method for the detection of Neospora infection in fetuses and dams from nonhuman primates and may be useful in determining the zoonotic potential of Neospora.

Detection of Neospora sp. from infected bovine tissues by PCR and probe hybridization.

Neospora sp. can cause fetal abortion or neurological disease in congenitally infected calves. Latent tissue stages in infected cows may contribute to vertical transmission of Neospora sp. from dam to offspring in multiple pregnancies. In this investigation, the polymerase chain reaction (PCR) and Neospora-specific assay were employed to detect Neospora sp. by amplification of nuclear small-subunit rRNA gene sequences in infected cattle tissues. Tissues from 11 cattle, including 6 experimentally and 2 naturally infected cows, 1 naturally infected newborn calf, and 2 uninfected control cows, were evaluated in this study. Neospora-specific PCR products were amplified from DNAs of different bovine tissues, including brain, spinal cord, heart, lung, kidney, diaphragm, skeletal muscle, and placenta, as well as amniotic fluid samples of infected cattle. The PCR-based amplification and probe hybridization system proved useful in assessing the location of tissue-stage parasites in naturally and experimentally infected cattle, even when Neospora sp. antibody titers fall below normal cut-off values by an indirect immunofluorescent antibody test.

A novel protein polymorphism differentiates the California serotype of infectious bronchitis from other serotypes common to California.

The California (Cal) serotype of infectious bronchitis virus (IBV) was isolated from layer flocks in southern California in the early 1980s. Since then, it has spread to the broiler-producing regions of central California, where it has been implicated in respiratory disease outbreaks in vaccinated flocks. Lack of a procedure for quickly identifying IBV serotypes in commercial chicken flocks has prevented the causal association of the IBV Cal serotype with respiratory disease outbreaks. A protein polymorphism has been identified in the matrix protein of the Cal serotype; it appears to be unique among other common serotypes of infectious bronchitis virus found in California. This polymorphism can be identified on western blots using raw or concentrated infectious allantoic fluid as the source material. Identification of the Cal serotype and of serotypes in the Mass and Conn groups can be performed rapidly using field samples from suspect flocks. The identification of this polymorphism provides an alternative method for the rapid identification of the Cal serotype of IBV.

Evidence of vertical transmission of Neospora sp infection in dairy cattle.

OBJECTIVE: To determine whether heifers with naturally acquired congenital exposure to Neospora sp would transmit the infection to their offspring during gestation. DESIGN: Prospective cohort study. ANIMALS: Neonatal heifers on a dairy with a history of Neospora sp infections were selected for the study on the basis of their serum titers to Neospora sp, as determined by the use of indirect fluorescent antibody testing. Seropositive heifers (n = 25) had titers > or = 1:5,120 and seronegative heifers (25) had titers < or = 1:80. All heifers were raised and bred on the dairy, and samples were obtained from heifers and their calves at the time of calving. PROCEDURE: Blood samples were tested for Neospora sp antibodies. Histologic evaluations, Neospora sp immunohistochemical examinations, and protozoal culturing were performed on samples obtained from selected offspring (second-generation calves). RESULTS: Seropositive heifers gave birth to calves with titers > or = 1:1,280 to Neospora sp. All offspring from seropositive heifers that were necropsied had evidence of Neospora sp infection. All seronegative heifers and their offspring had titers < 1:80 to Neospora sp. CLINICAL IMPLICATIONS: Congenitally acquired Neospora sp infection can persist in clinically normal heifers and be transmitted transplacentally to their offspring. Vertical transmission can be a way by which neosporosis is maintained in herds.

Cloning and characterization of two recombinant Neospora protein fragments and their use in serodiagnosis of bovine neosporosis.

Bovine neosporosis causes fetal abortion and/or congenital neurologic disease in cattle. For the serodiagnosis of this parasitic disease, two immunodominant clones from a bovine Neospora lambda gt11 library were identified, characterized, and expressed as recombinant proteins for the development of an enzyme-linked immunosorbent assay (ELISA). These two clones, designated N54 and N57, were 29 and 20 kDa, respectively, when expressed as histidine fusion proteins from the pRSET expression vector. Antibodies to recombinant protein N54 recognized five major bands from a Neospora tachyzoite lysate with molecular masses of 97, 87, 77, 67, and 64 kDa. Antibodies to recombinant protein N57 recognized four primary bands with molecular masses of 34, 31, 30, and 28 kDa. When a defined "gold standard" panel of bovine sera from confirmed Neospora-positive and Neospora-negative cattle were characterized by immunoblotting, 57 of the 60 Neospora-positive serum samples recognized proteins with the molecular masses of the N54 heptuplet. Binding to the N57 quadruplet was more variable. The same gold standard panel was used to evaluate and compare an N54-based ELISA, an N57-based ELISA, and a whole-tachyzoite lysate-based ELISA. The sensitivities and specificities were 95 and 96% (N54 ELISA), 82 and 93% (N57 ELISA), and 74 and 93% (lysate ELISA). Thus, compared to the whole-tachyzoite lysate-based ELISA, both recombinant-protein-based ELISAs had higher sensitivities and higher or the same specificities and can be used to replace the whole-tachyzoite lysate ELISA for the serodiagnosis of bovine neosporosis.

Diagnosis of bovine fetal Neospora infection with an indirect fluorescent antibody test.

Fetal fluids from 138 spontaneously aborted bovine fetuses were examined for the presence of antibodies against Neospora antigens by means of an indirect fluorescent antibody test (IFAT). The fetuses were divided into group 1, consisting of 74 fetuses with confirmed or presumptive fetal neosporosis, and group 2, consisting of 64 fetuses with either no aetiological diagnosis, presumptive diagnoses of non-Neospora infections or non-infectious diseases, or fetuses with confirmed diagnoses of other fetal diseases. Thirty-seven of the 74 fetuses in group 1 had detectable titres of antibody to Neospora; approximately 21 per cent of the fetuses between three and five months gestation, 56 per cent of these between six and seven months gestation, and 93 per cent of these between eight and nine months gestation, had detectable titres of antibody. Only one of the 64 fetuses in group 2 had a detectable titre of antibody to Neospora.

Experimental fetal and transplacental Neospora infection in the nonhuman primate.

BACKGROUND: Neospora is a newly recognized Toxoplasma-like protozoan that causes spontaneous abortion and/or neonatal disease in a wide range of animals. The purpose of this study was to determine the susceptibility of primates to Neospora infection. EXPERIMENTAL DESIGN: In experiment 1, two rhesus macaque fetuses were inoculated in utero at gestational day 65 with 1 x 10(6) culture-derived Neospora tachyzoites. A control fetus was given uninfected vehicle. The fetuses were removed by hysterotomy between 13 and 22 days postinoculation. In experiment 2, two pregnant macaques were inoculated intramuscularly and intravenously on gestational day 43 with a total of 1.6 x 10(7) culture-derived tachyzoites. A pregnant control macaque was given uninfected vehicle. The fetuses were removed by hysterotomy between 67 to 70 days postinoculation. Fetal tissues were collected for in vitro parasite isolation, histopathology, and Neospora immunohistochemistry. Fetal blood was examined for Neospora-specific antibody titers using an indirect fluorescent antibody test. RESULTS: Neospora infections were confirmed in all fetuses that received tachyzoites either directly or via transplacental infection. In experiment 1, infected fetuses had reduced amniotic fluid volumes, marked protozoal amnionitis and dermatitis, and a mild multifocal encephalitis. Infected fetuses from experiment 2 had a chronic multifocal necrotizing nonsuppurative meningoencephalitis with microcavitation, that was confined to the cerebrum, and a mild multifocal necrotizing amnionitis. In both experiments, Neospora tachyzoites were detected in association with lesions in fetal tissues by immunohistochemistry, and the parasites were reisolated in vitro. IgG Neospora antibody titers were detected in blood from all infected fetuses, whereas Neospora-specific IgM and IgA titers were found in one and three fetuses, respectively. CONCLUSIONS: Results indicate that nonhuman primates are susceptible to transplacental Neospora infection. The fetal lesions after transplacental infection are similar to those induced by transplacental Toxoplasma infections in primates.

Experimental reproduction of bovine fetal Neospora infection and death with a bovine Neospora isolate.

Studies were conducted to determine the pathogenic potential of the recently isolated bovine Neospora protozoa (BPA-1) for the bovine fetus. Cows chosen for study had Neospora titers < 160 using an indirect immunofluorescent antibody (IFA) test. Four experimental groups were studied. In group 1, 2 fetuses were inoculated in utero at 118 days gestation with culture-derived Neospora tachyzoites. A pregnant control cow was housed in the same pen, observed daily and screened serologically for evidence of exposure to Neospora. In group 2, 2 cows were infected with Neospora tachyzoites at 138 or 161 days gestation, and 1 control cow was given uninfected cell culture suspension simultaneously at 154 days gestation. Groups 3 (85 days gestation) and 4 (120 days gestation) each consisted of 2 cows infected with Neospora tachyzoites and 1 control cow given uninfected material at the same stage of gestation. Dead fetuses were surgically removed from the infected cows in group 1 on postinfection day (PID) 17. The histopathology was compatible with protozoal fetal infection, and protozoa were identified by immunohistochemistry. Viable fetuses were removed surgically from cows in group 2 on PID 28-30. The histopathology was compatible with protozoal fetal infection, protozoa were identified by immunoperoxidase techniques, and Neospora tachyzoites were reisolated in vitro from tissues of the 2 infected fetuses. In groups 3 and 4, the control fetus and 1 infected fetus were removed surgically between PID 26 and PID 33. The remaining infected cows were observed until fetal death or abortion occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro isolation and characterization of a Neospora sp. from aborted bovine foetuses.

A Neospora sp. was isolated from the brains of two aborted bovine foetuses and grown continuously in vitro in bovine cell cultures. A comparison of the antigenic reactivity of in vitro cultivated tachyzoites with polyclonal antisera to Neospora caninum, Hammondia hammondi or Toxoplasma gondii revealed that the bovine protozoal isolates were similar to N. caninum and antigenically distinct from T. gondii. Tachyzoites of both bovine isolates had similar ultrastructural features, including an apical polar ring, conoid, electron-dense rhoptries and micronemes. The orientation of the micronemes, presence of micropores and a large number of electron-dense granules in the posterior portion of the bovine isolate tachyzoites differed from previous descriptions of N. caninum in vivo. Tachyzoites of the bovine isolates were ultrastructurally more similar to in vitro cultivated N. caninum tachyzoites than to tachyzoites of T. gondii or H. hammondi. The antigenic and ultrastructural similarities between N. caninum and the protozoal parasites isolated from aborted bovine foetuses in this study support the proposition that these parasites belong to the genus Neospora.