Barbara M. Daft

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.

A retrospective study of circovirus infection in pigeons: nine cases (1986-1993)

Circovirus infections were diagnosed in 12 pigeons from the United States, 4 pigeons from Australia, and 1 pigeon from Canada (1986-1993). Circovirus was identified by electron microscopic examination of basophilic botryoid cytoplasmic inclusions that had a histologic appearance similar to that of psittacine beak and feather disease virus inclusions. Inclusions were seen in splenic, bursal, gut-associated, and bronchus-associated lymphoid tissue macrophages and in bursal epithelial cells. Inclusions were composed of paracrystalline arrays of tightly packed, nonenveloped icosahedral virions 14-17 nm in diameter. Histologic changes in the spleens ranged from lymphofollicular hyperplasia with mild discrete lymphocellular necrosis to lymphoid depletion and diffuse histiocytosis. Lesions in the bursa of Fabricius ranged from mild lymphocellular necrosis to severe cystic bursal atrophy. Remaining histologic findings coincided with concurrent bacterial, viral, fungal, and parasitic infections. Immunoperoxidase staining and DNA in situ hybridization demonstrated that pigeon circovirus is distinct from psittacine beak and feather disease virus; however, both viruses apparently share some homologous DNA sequences. Clinical and diagnostic findings indicate that pigeon circovirus may be similar to psittacine beak and feather disease virus with respect to acquired immunodeficiency and subsequent multiple secondary infections.

Comparison of a Serum Indirect Fluorescent Antibody Test with Two Western Blot Tests for the Diagnosis of Equine Protozoal Myeloencephalitis

A serum indirect fluorescent antibody test (IFAT) was compared with a Western blot (WB) and a modified Western blot (mWB) for diagnosis of equine protozoal myeloencephalitis (EPM). Using receiver-operating characteristic (ROC) analysis, the area under the curve of the IFAT was greater than the area under the curves of the WB and the mWB (P = 0.025 and P = 0.044, respectively). There was no statistically significant difference between the areas under the curves of the WBs (P > 0.05). On the basis of an arbitrarily chosen cut-off titer for a positive test result of 1:80 for the IFAT and interpreting weak positive WB results as positive test results, the sensitivities and 95% confidence intervals (CI) of all 3 tests were identical and equal to 88.9% (51.8–99.7%). The specificities and 95% CIs of the IFAT, WB, and mWB test were 100% (91–100%), 87.2% (72.6–95.7%), and 69.2% (52.4–83%), respectively. The overall accuracy of the IFAT was shown to be better than that of the WBs and, therefore, the test has potential for use in the diagnosis of EPM caused by Sarcocystis neurona.

Pathology and diagnosis of Coxiella burnetii infection in a goat herd.

Albert OM, Gonder JR, Papale J, Craft JL, Dohlman HG, Reid MC, Sunderman FW Jr: Induction of ocular neoplasms in Fischer rats by intraocular injection of nickel subsulfide. Invest Ophthalmol Vis Sci 22:768-782, 1982 2 Apple OJ, Rabb MF: Ocular Pathology, pp. 406-443. CV Mosby Co., St. Louis, MO, 1985 3 Jacobiec FA, Font RL: Orbit. In: Ophthalmic Pathology, ed. Spencer WH, 3rd ed., vol. 3., pp. 2459-2860. WB Saunders Co., Philadelphia, PA, 1986 4 Jitawi SA, Cochran AJ, Cancilla PA, Wen D-R: The expression of S-I 00 protein and neuron-specific enolase in meningiomas. Dis Markers 6: 109-117, 1988 5 Moore BW: A soluble protein characteristic of the nervous system. Biochem Biophys Res Commun 19:739-744, 1965

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.

Lyme Neuroborreliosis in 2 Horses

Lyme neuroborreliosis—characterized as chronic, necrosuppurative to nonsuppurative, perivascular to diffuse meningoradiculoneuritis—was diagnosed in 2 horses with progressive neurologic disease. In 1 horse, Borrelia burgdorferi sensu stricto was identified by polymerase chain reaction amplification of B burgdorferi sensu stricto–specific gene targets (ospA, ospC, flaB, dbpA, arp). Highest spirochetal burdens were in tissues with inflammation, including spinal cord, muscle, and joint capsule. Sequence analysis of ospA, ospC, and flaB revealed 99.9% sequence identity to the respective genes in B burgdorferi strain 297, an isolate from a human case of neuroborreliosis. In both horses, spirochetes were visualized in affected tissues with Steiner silver impregnation and by immunohistochemistry, predominantly within the dense collagenous tissue of the dura mater and leptomeninges.

The Occurrence of Avian Influenza A Subtype H6N2 in Commercial Layer Flocks in Southern California (2000–02): Clinicopathologic Findings

Abstract Between February 2000 and February 2002, the California Animal Health and Food Safety Laboratory System diagnosed 26 cases of low-pathogenic H6N2 avian influenza from 12 commercial egg-laying farms. The most common gross and histologic lesions observed in infected chickens were fibrinous yolk peritonitis, salpingitis, oophoritis, and nephritis. Edema of the mesentery of the oviduct and pale, swollen kidneys were also observed. Mortality in infected flocks ranged from 0.25% to 3%, and egg production dropped 7% to 40%.

Cytokine gene signatures in neural tissue of horses with equine protozoal myeloencephalitis or equine herpes type 1 myeloencephalopathy

This study was designed to determine the relative levels of gene transcription of selected pathogens and cytokines in the brain and spinal cord of 12 horses with equine protozoal myeloencephalitis (epm), 11 with equine herpesvirus type 1 (ehv-1) myeloencephalopathy, and 12 healthy control horses by applying a real time pcr to the formalin-fixed and paraffin-embedded tissues. Total rna was extracted from each tissue, transcribed to complementary dna (cdna) and assayed for Sarcocystis neurona, Neospora hughesi, ehv-1, equine gapdh (housekeeping gene), tumour necrosis factor (tnf)-α, interferon (ifn)-γ, interleukin (il)-1β, il-2, il-4, il-6, il-8, il-10 and il-12 p40. S neurona cdna was detected in the neural tissue from all 12 horses with epm, and two of them also had amplifiable cdna of N hughesi. The relative levels of transcription of protozoal cdna ranged from 1 to 461 times baseline (mean 123). All the horses with ehv-1 myeloencephalopathy had positive viral signals by pcr with relative levels of transcription ranging from 1 to 1618 times baseline (mean 275). All the control horses tested negative for S neurona, N hughesi and ehv-1 cdna. The cytokine profiles of each disease indicated a balance between pro- and anti-inflammatory markers. In the horses with epm the pro-inflammatory Th1 cytokines (il-8, tnf-α and ifn-γ) were commonly expressed but the anti-inflammatory Th2 cytokines (il-4, il-6 and il-10) were absent or rare. In the horses with ehv-1 the proinflammatory cytokine il-8 was commonly expressed, but il-10 and ifn-γ were not, and tnf-α was rare. Tissue from the control horses expressed only the gene gapdh.

QUALITATIVE EVALUATION OF SELECTIVE TESTS FOR DETECTION OF NEOSPORA HUGHESI ANTIBODIES IN SERUM AND CEREBROSPINAL FLUID OF EXPERIMENTALLY INFECTED HORSES

Neospora hughesi is a newly recognized protozoan pathogen in horses that causes a myeloencephalitis similar to Sarcocystis neurona. There are no validated serologic tests using the gold standard sera that are currently available to detect specific N. hughesi antibodies and, thus, no tests available to detect antemortem exposure or estimate seroprevalence in the horse. The objectives of the present study were to establish a bank of gold standard equine sera through experimental infections with N. hughesi and to assess several serologic tests for the detection of related protozoan antibodies. Seven horses were inoculated with N. hughesi tachyzoites, and 7 horses received uninfected cell culture material. The horses were monitored, and blood and cerebrospinal fluid were collected repeatedly over a 4-mo period. With the sera, 4 different serologic techniques were evaluated, including a whole-parasite lysate enzyme-linked immunosorbent assay (ELISA), a recombinant protein ELISA, a modified direct agglutination test, and an indirect fluorescent antibody test. Qualitative and quantitative evaluation of the results showed that the N. hughesi indirect fluorescent antibody test (IFAT) consistently discriminated between experimentally infected and noninfected horses, using a cutoff of 1:640. Sera from 3 naturally infected horses had titers >1:640. Cerebrospinal fluid in all but 1 infected horse had very low N. hughesi IFAT titers (<1:160), starting at postinoculation day 30.

Tracheal aspergillosis in 6 1/2-week-old chickens caused by Aspergillus flavus.

A case of localized tracheal aspergillosis in 6 1/2-week-old single-comb white leghorn pullets caused by Aspergillus flavus is documented. Yellow caseous plaques adherent to the mucosal surface of the tracheas were observed grossly. In several tracheas, the plaques occluded the lumina, and the surrounding tracheal walls were reddened. Histologically, the mucosa was necrotic and infiltrated with macrophages, and fibroplasia was evident in the subadjacent tracheal wall. The lumen of the trachea was almost completely occluded by a combination of fungal mycelia and pyogranulomatous exudate. Portions of tracheal cartilage were elevated into the lumen of the trachea. Other than a sudden increase in mortality to 0.5% per day, there was no evidence of disease in the flock. Depletion of bursal lymphocytes, with concomitant cryptosporidiosis, was evident on histological examination. Acute infectious bursal disease was diagnosed in the succeeding flock at this ranch based upon serology and typical histology.