Acute toxicity and tissue distributions of malathion in Ambystoma tigrinum.

The kinetics of the bioaccumulation of malathion (O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate) and the biological impact of exposure for tiger salamanders, Ambystoma tigrinum, were assessed through exposure to soil surface contaminated with 50 microg/cm(2) or 100 microg/cm(2 )malathion and ingestion of an earthworm exposed to soil contaminated with 200 microg/cm(2) malathion. Malathion and malaoxon burdens in salamanders sampled at different times after exposure(s) were measured by gas chromatography in four tissue/organ subgroups: liver, epaxial muscle, pooled viscera (except the liver and brain), and pooled avisceral carcass (muscle, skin, and bone). The total tiger salamander xenobiotic burdens were calculated from these data. The malathion/malaoxon burden 1 day after exposure was greatest in the avisceral carcass and 2 days after exposure was greatest in the viscera. Bioconcentration and bioaccumulation factors remained less than unity throughout the experiment and did not support the hypothesis of bioaccumulation of malathion in the tiger salamander. Biological impact was assessed with a colorimetric brain cholinesterase microassay. Brain cholinesterase activities in salamanders exposed to malathion-contaminated soil (50 microg/cm(2) or 100 microg/cm(2 )malathion) were suppressed approximately 50-65% and 90%, respectively, compared to unexposed controls. The exposed animals did not exhibit overt clinical signs of malathion toxicosis.

The role of transcription in EGF- and FSH-mediated oocyte maturation in vitro.

Understanding mechanisms responsible for meiotic resumption in mammalian oocytes is critical for the identification of strategies to enhance developmental competence of in vitro-matured oocytes. Improvement of in vitro oocyte maturation systems is dependent on a better understanding of mechanisms that regulate oocyte maturation both in vivo and in vitro as well as on the identification of methods to manipulate the meiotic progression of oocytes matured in vitro in a physiological manner. The purpose of this review is two-fold: first, to examine the mechanisms that underlie the acquisition of oocyte developmental competence and regulation of oocyte maturation in vivo and in vitro; second, to present data examining the role of transcription in mediating the ability of EGF and FSH to induce oocyte maturation in vitro. Results presented support the conclusions that (1) EGF-induced oocyte maturation does not require nascent gene transcription in both mice and domestic cats; (2) FSH requires gene transcription to induce oocyte maturation in both species; (3) EGF must be present in the maturation medium to optimize the effectiveness of FSH to promote oocyte maturation; (4) the mechanism used by FSH to induce oocyte maturation in vitro appears to predominate over that used by EGF when both EGF and FSH are present in maturation medium used for either murine or feline cumulus oocyte complexes.

Use of a two-step Percoll gradient for separation of loggerhead sea turtle peripheral blood mononuclear cells.

In order to determine a suitable procedure for isolating peripheral blood mononuclear cells (PBMCs) from loggerhead sea turtles (Caretta caretta), blood was collected using three different anticoagulants (sodium heparin, sodium citrate or potassium EDTA) and separated using a single step commercially-prepared arabinogalactan gradient of 1.077 g/ml density or multiple step Percoll gradients between 1.053 and 1.076 g/ml density (40-60% stock isotonic Percoll suspension). Heparinized blood centrifuged over a two-step 45/55% (1.059/1.070 g/ml) Percoll gradient yielded 99 to 100% mononuclear cells at the 45/55% interface. Mononuclear cell viability ranged from 85 to 97% with cell yields up to 9.2 x 10(6) cells/mL. An unexpected finding was a population of low density granulocytes migrating to 40% (1.053 g/ml) and 45% Percoll layers in the multiple step gradients. These granulocytes could be eliminated from the PBMC preparation by use of the two-step 45/55% Percoll gradient. Isolated PBMCs can be used for cellular immunology and toxicology studies on these threatened marine organisms for which other tissues can usually be obtained only sporadically from post-mortem specimens.

Cloning and sequencing hybrid striped bass (Morone saxatilis x M. chrysops) transforming growth factor-beta (TGF-beta), and development of a reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay to measure TGF-beta mRNA of teleost fish.

A transforming growth factor (TGF)-beta was isolated and cloned from hybrid striped bass (Morone saxatilis x M. chrysops) anterior kidney mononuclear cells. This isolate (Genbank accession number AF140363) contains an open reading frame of 1146 bases coding for a 382 amino acid protein most similar to rainbow trout TGF-beta (57.3 and 78.6% identity with precursor and active protein, respectively) and rat TGF-beta 1 (41.1 and 68.8% identity with precursor and active protein, respectively). Consensus primers were demonstrated to amplify specifically by polymerase chain reaction (PCR), a TGF-beta segment from 14 species of teleost fish comprising 10 taxonomic families in 7 orders. A reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay was devised to measure TGF-beta mRNA expression in teleost fish. Higher levels of TGF-beta mRNA expression were detected in mononuclear cells of peripheral blood than from spleen or anterior kidney.

Quantitative polymerase chain reaction for transforming growth factor-beta applied to a field study of fish health in Chesapeake Bay tributaries.

Fish morbidity and mortality events in Chesapeake Bay tributaries have aroused concern over the health of this important aquatic ecosystem. We applied a recently described method for quantifying mRNA of an immunosuppressive cytokine, transforming growth factor-beta (TGF-beta), by reverse transcription quantitative-competitive polymerase chain reaction to a field study of fish health in the Chesapeake Basin, and compared the results to those of a traditional cellular immunoassay macrophage bactericidal activity. We selected the white perch (Morone americana) as the sentinel fish species because of its abundance at all of the collection sites. White perch were sampled from Chesapeake Bay tributaries in June, August, and October 1998. Splenic mononuclear cell TGF-beta mRNA levels increased and anterior kidney macrophage bactericidal activity decreased, particularly in eastern shore tributaries, from June to August and October. The results of the two assays correlated inversely (Kendall's [Tau] b = -0.600; p = 0.0102). The results indicated both temporal and spatial modulation of white perch immune systems in the Chesapeake Basin, and demonstrated the utility of quantitative PCR for TGF-beta as a molecular biomarker for field assessment of teleost fish immune status.

Feline immunodeficiency virus is shed in semen from experimentally and naturally infected cats.

Although a laboratory isolate of feline immunodeficiency virus (FIV), FIV-NCSU1, has been transmitted by artificial insemination in domestic cats, transmission by naturally infected males during mating has not been reported. In order to determine whether virus shedding in semen is unique to the NCSU1 isolate, we analyzed electroejaculates from four specific-pathogen-free males infected with another laboratory strain, FIV-Petaluma, and eight random source males with naturally acquired infections. Seminal cell lysates from the cats infected with the Petaluma isolate were screened by nested polymerase chain reaction amplification for FIV gag DNA. Seminal cells and seminal plasma from these FIV-Petaluma cats were further analyzed for the presence of virus by cocultivation with a feline CD4+ T cell line and Crandell feline kidney cells. Electroejaculates from the naturally infected cats were cocultivated with the T cell line. Our results demonstrated that cell-free FIV was present in seminal plasma from two FIV-Petaluma cats and two naturally infected cats. Cell-associated seminal virus was detected in all of the FIV-Petaluma infected cats and one naturally infected cat. Secretion of viral gag p26 antigen, an indication of active viral replication, was evident in cocultures containing motile sperm purified by a swim-up procedure from a FIV-Petaluma cat. These results confirm that FIV shedding in semen is not restricted to a specific virus isolate. Furthermore, swim-up sperm from FIV-infected cats may be infectious in vitro.

Horizontal transmission of feline immunodeficiency virus with semen from seropositive cats.

The AIDS virus of cat species, feline immunodeficiency virus (FIV), has been used extensively as an animal model of HIV-1 infection. This felid lentivirus shares many molecular and biochemical traits with HIV-1 and causes similar immunologic and clinical perturbations, most notably CD4+ cell loss, impaired cell-mediated immunity and increased susceptibility to opportunistic pathogens. Previous reports have shown that FIV is transmitted horizontally by biting and vertically in utero and through nursing. Our objective was to determine whether FIV could be venereally transmitted in domestic cats. In the first experiment, susceptibility of the female reproductive tract to mucosal transmission of the FIV isolate, NCSU1, was demonstrated via intravaginal inoculation with infected cultured cells. We next identified virus in electroejaculates from asymptomatic, chronically FIV-NCSU1-infected, adult males. A fragment of FIV gag provirus DNA was detected by nested polymerase chain reaction (PCR) in nonfractionated seminal cells and in swim-up sperm preparations. Additionally, replication-competent virus was isolated from cell-free seminal plasma and seminal cells by co-cultivation with a feline CD4+ T-cell line. In the third study, queens were artificially inseminated via an intrauterine laparoscopic technique with electroejaculates from FIV-NCSU1-infected males. Of six inseminations carried out with fresh semen, three resulted in infection of queens. Lastly, immunohistochemical studies identified potential virus target cell populations in normal female reproductive tissues. In conclusion, these experiments indicate that FIV infection in domestic cats may provide a unique small animal model of sexual transmission of HIV-1.

Cytologic identification of Toxoplasma gondii in bronchoalveolar lavage fluid of experimentally infected cats.

OBJECTIVE: To determine whether it was possible to retrieve organisms, by means of bronchoalveolar lavage (BAL), from cats inoculated with Toxoplasma gondii. DESIGN: Experimental study. ANIMALS: 27 cats. Sixteen of the 27 were experimentally infected with feline immunodeficiency virus. PROCEDURE: All cats were inoculated with T gondii tachyzoites. Cats were grouped on the basis of feline immunodeficiency virus status and route (IV or intra-arterial) and number of tachyzoites administered. Bronchoalveolar lavage was performed by means of a standard technique. Lavage fluid was evaluated cytologically for tachyzoites. RESULTS: Clinical signs of toxoplasmosis varied widely among individual cats, but were generally most pronounced in group-1 and -2 cats (n = 5 each) and less pronounced in group-3 (n = 5) cats. Group-4 and -5 cats (n = 6 each) did not have clinical signs of toxoplasmosis. In 14 of the 15 cats in groups 1, 2, and 3, tachyzoites were detected in BAL fluid collected 7 days after inoculation. Tachyzoites were detected 14 days after inoculation in the single cat without tachyzoites 7 days after inoculation. A necropsy was performed on 9 of these cats, and tachyzoites were identified histologically in 4 of the 9. Tachyzoites were not detected in BAL fluid collected 3 days (n = 6) or 7 days (n = 6) after inoculation from the 12 cats in groups 4 and 5. Tachyzoites were not identified histologically in any of these 12 cats. CLINICAL IMPLICATIONS: BAL may be useful in the diagnosis of toxoplasmosis, particularly in cats with signs of pulmonary involvement.

Transmission of feline immunodeficiency virus in domestic cats via artificial insemination.

The objective of this study was to determine whether semen from male domestic cats infected with feline immunodeficiency virus (FIV) can transmit virus to females. Twelve inseminations were performed by an intrauterine laparoscopic technique with fresh or cryopreserved electroejaculates from asymptomatic males chronically infected with the NCSU1 strain of FIV. Of six inseminations performed with fresh semen, three resulted in infection of queens, as indicated by seroconversion, expression of FIV gag provirus in peripheral blood leukocytes, and reduced peripheral CD4+/CD8+ T-lymphocyte ratios. None of the six inseminates with thawed cryopreserved semen resulted in infection. Two infected queens and one uninfected queen became pregnant. Virus was not evident in the seven offspring. We conclude that FIV can be transmitted horizontally by artificial insemination with fresh semen.

Effect of FIV infection on lung inflammatory cell populations recovered by bronchoalveolar lavage.

Human immunodeficiency virus (HIV) and ovine progressive pneumonia virus have been associated with lymphocytic pneumonitis. Pulmonary cell populations in cats infected with feline immunodeficiency virus (FIV) were evaluated by bronchoalveolar lavage (BAL) to identify changes associated with lentivirus infection in this species. Bronchoalveolar lavage was performed through an endotracheal tube using 15 ml kg-1 body weight of sterile 0.9% sodium chloride solution. Results of BAL fluid cytologic analysis from 19 cats experimentally infected with FIV for at least 8 months were compared with results from 34 uninfected cats. Infected cats had significantly higher total cell counts and relative neutrophil counts (P < 0.01). Lymphocytosis did not occur. Bronchoalveolar lavage fluid was collected from nine additional cats prior to, and 2, 6, and 17-18 weeks following infection with FIV. Neither neutrophilia nor lymphocytosis was associated with FIV infection in these cats.

Characterization of phocid herpesvirus-1 and -2 as putative alpha- and gammaherpesviruses of North American and European pinnipeds.

To study the relationships between herpesvirus recently isolated from different pinniped species, antigenic and genetic analyses were performed. First, herpesviruses isolated from North American harbour seals (Phoca vitulina), a Californian sea lion (Zalophus californianus) and a European grey seal (Halichoerus grypus) were examined in an enzyme immunoassay (EIA) with a panel of monoclonal antibodies which had previously been shown to allow typing of herpesviruses from European harbour seals into two distinct virus types: phocid herpesvirus type-1 and type-2 (PhHV-1 and PhHV-2). The EIA data showed that all but one of the isolates from seals ranging in United States coastal waters were PhHV-2-like while the European grey seal herpesvirus was PhHV-1-like. Genetic characterization was facilitated by PCR analysis using primers based on conserved regions of the glycoprotein B and D (gB and gD) genes of the antigenically closely related canid (CHV) and felid (FHV) herpesvirus. Specific amplified products were obtained with five isolates antigenically characterized as PhHV-1-like but not with five PhHV-2-like isolates. Sequence analysis of the PCR products confirmed greatest similarity to members of the genus Varicellovirus of the Alphaherpesvirinae and in particular to CHV. Sequence analysis of two EcoRI fragments of the PhHV-2 genome (European isolate 7848) revealed greatest similarity to gammaherpesviruses and in particular equine herpesvirus-2. Although an unambiguous subgrouping was not feasible, this is the first evidence that PhHV-2 may be a putative gammaherpesvirus of pinnipeds.

Detection of feline immunodeficiency virus in semen from seropositive domestic cats (Felis catus).

Electroejaculates from experimentally infected domestic cats were evaluated for the presence of feline immunodeficiency virus (FIV). Virus was isolated from cell-free seminal plasma and seminal cells by cocultivation with a feline interleukin-2-dependent CD4+ T-cell line, in which productive infection was demonstrated by syncytium formation and FIV gag p26 antigen secretion. In addition, an 868-bp segment of the FIV gag provirus gene was identified in cocultured cells by PCR and Southern analysis. A 582-bp fragment of the FIV gag provirus genome was detected by nested PCR and Southern analysis in nonfractionated seminal cells and in sperm purified by a swim-up procedure. This is the first report describing the detection of replication-competent FIV in cell-free and cell-associated forms in domestic cat semen.

Tumor necrosis factor-alpha responses are depressed and interleukin-6 responses unaltered in feline immunodeficiency virus infected cats.

Feline immunodeficiency virus (FIV), a lentivirus similar to HIV, causes an acquired immunodeficiency syndrome in cats. Similar to human immunodeficiency virus (HIV), the pathogenesis of FIV is associated with dysregulation of the cytokine network. While alterations in tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) expression have been reported in HIV-infected patients, changes attributable to HIV and those caused by cofactors such as secondary infections cannot always be readily distinguished. This study evaluated the effect of FIV infection on TNF-alpha and IL-6 production in cats not exposed to other potential cofactors such as secondary infections. TNF-alpha and IL-6 activities were evaluated in bronchoalveolar lavage (BAL) cells from FIV-infected and uninfected specific pathogen free (SPF) cats. Supernatants from lipopolysaccharide (LPS)-stimulated BAL cells from uninfected SPF cats had high levels of TNF-alpha and IL-6 activity, while stimulated BAL cell supernatants from FIV-infected SPF cats had significantly lower levels of TNF-alpha but unaltered IL-6 activity. Similarly, Con A/phorbol myristate acetate (PMA) stimulated non-adherent (NA-) peripheral blood mononuclear cells (PBMC) from FIV infected cats synthesized less TNF-alpha than similarly treated NA-PBMC from uninfected cats. Feline immunodeficiency virus could be recovered from the culture supernatants of BAL cells from infected cats by co-cultivation with susceptible lymphocytes. In situ hybridization identified FIV mRNA in a small fraction of alveolar macrophages in the BAL cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytologic characterization of bronchoalveolar lavage fluid collected through an endotracheal tube in cats.

Bronchoalveolar lavage was performed through an endotracheal tube in 34 specific-pathogen-free cats to determine expected values for bronchoalveolar lavage fluid cytologic analysis, using this method of collection. Saline solution for lavage was instilled in 3 separate aliquots at a volume of 5 ml/kg of body weight each. Analysis of sequential aliquots was performed to investigate the differences in cell counts among the 3 fractions. The effect of combining aliquots, including or omitting the first fraction, was evaluated to determine whether all aliquots could be combined for analysis without substantially affecting results. The total number of nucleated cells retrieved from each cat ranged from 0.9 to 31.1 x 10(6). Most of these cells were macrophages (78 +/- 15%, mean +/- SD) and eosinophils (16 +/- 14%). The first aliquot had the greatest number of epithelial cells, and the lowest total nucleated cell count and relative and absolute eosinophil counts. Differences among aliquots also were identified for relative and absolute macrophage counts, relative and absolute neutrophil counts, and absolute lymphocyte count. Statistically significant differences were found for many of the cell counts when values from the combination of the second and third aliquots were compared with values from the combination of all 3 aliquots. Magnitude of the differences was small, and these differences were not believed to be of practical consequence.

Feline immunodeficiency virus can be experimentally transmitted via milk during acute maternal infection.

Postnatal transmission of feline immunodeficiency virus (FIV) in neonates nursed by acutely infected mothers and infection resulting from oral inoculation of kittens with FIV were evaluated. Ten of 16 kittens nursed by four queens with FIV infection established immediately postpartum developed FIV infection. Five of 11 neonates orally administered cell-free FIV culture supernatant developed FIV infection. Kittens that developed FIV infection had greater proportions of CD4+ and Pan-T+ lymphocytes at birth than negative kittens. Infectious virus was recovered from the milk of acutely infected mothers. We conclude that FIV may be experimentally transmitted via milk from queens with acute infections and that oral administration of FIV to neonatal kittens results in infection.

Pathogenesis of ovine lentivirus-induced arthritis: phenotypic evaluation of T lymphocytes in synovial fluid, synovium, and peripheral circulation.

Sheep and goats develop a chronic, progressive arthritis reminiscent of rheumatoid arthritis, but caused by lentiviruses related to human immunodeficiency virus. The distribution of T lymphocytes in peripheral circulation of two infected sheep with arthritis, one infected sheep with interstitial pneumonia, three asymptomatic sheep, and three uninfected sheep was evaluated. Sheep with clinical disease have depressed ratios of CD4/CD8 lymphocytes in peripheral circulation compared to asymptomatic and uninfected animals. In one sheep, the depressed ratio was due to an absolute increase in CD8-positive lymphocytes. The predominant lymphocyte populations in both synovial fluid and synovium from this animal were also CD8 positive. Macrophages were the other predominant cell population in synovial fluid and were infected with lentivirus. Little cell-free virus was detected in the synovial fluid, although 1 in 400 cells was infected as determined by infectious center assays. Infected cells in the synovial fluid had a reduction in virus gene expression compared to infected cells in peripheral circulation. This reduction in virus gene expression may be due to the presence of interferon-like activity in the synovial fluid.

Pathogenesis of lentivirus-induced arthritis. A review.

While it has been known for some years that there is an association between lentiviruses and slowly progressive joint diseases in ruminants, the realization that the human immunodeficiency virus, the cause of AIDS, is a lentivirus has made this group of virus the focus of a considerable research effort. The manifestations of lentivirus infection in animals are discussed and reference is made to the possibility of using them as models for human rheumatoid arthritis and for AIDS.