Controlling bovine leukemia virus in dairy herds by identifying and removing cows with the highest proviral load and lymphocyte counts.

The objective of this field trial was to reduce bovine leukemia virus (BLV) transmission and prevalence in commercial dairy herds using proviral load (PVL) and lymphocyte count (LC) measurements as indicators of the most infectious animals for culling or segregation. Bovine leukemia virus causes lymphoma in <5% of infected cattle, and increased lymphocyte counts (lymphocytosis) in about one-third. Recent research has shown that dairy cows infected with BLV have altered immune function associated with decreases in milk production and lifespan. Recent findings show that a minority of infected cattle have PVL concentrations in blood and other body fluids of over 1,000 times that of other infected cattle. In combination with a high LC, these animals are thought to be responsible for most transmission of BLV in a herd. Milk or blood samples from adult cows in our 3 Midwestern dairy farm field trials were tested semiannually with ELISA for BLV antibodies, and ELISA-positive cattle were then retested using a blood LC and a quantitative PCR test for PVL to identify the animals presumed to be most infectious. Herd managers were encouraged to consider PVL and LC status when making cull decisions, and to segregate cows with the highest PVL and LC from their BLV ELISA-negative herd mates where possible. After 2 to 2.5 yr of this intervention, the incidence risk of new infections decreased in all 3 herds combined, from 13.8 to 2.2, and the overall herd prevalence decreased in all 3 herds combined from 62.0 to 20.7%, suggesting that this approach can efficiently reduce BLV transmission as well as prevalence. This is encouraging, because a very low prevalence of BLV infection would make it economically feasible to cull the remaining ELISA-positive cattle, as was achieved in national eradication programs in other countries decades ago.

Infectious feline herpesvirus detected in distant bone and tendon following mucosal inoculation of specific pathogen-free cats.

Emerging evidence suggests that cats infected with feline herpesvirus-1 (FHV-1) may experience a brief viremic phase. The objective of this study was to determine whether natural routes of FHV-1 inoculation could result in viremic transmission of infectious virus to connective tissues (cortical bone, tendon). Three specific pathogen-free cats were experimentally inoculated with FHV-1 via a combined mucosal (oronasal, ocular) route. Cats were euthanized at the peak of clinical signs to aseptically harvest tissues (cortical bone, tendon, trachea/tongue) for co-culture with a susceptible cell line to promote spread of infectious virus. Viral infection of Crandall-Rees feline kidney cells was microscopically visualized by cytopathic effect (CPE). Additionally, co-culture DNA was extracted either at the point of CPE or 16 days of culture without evidence of CPE, to amplify FHV-1 glycoprotein B gene using real-time PCR. Infectious virus was detected in distant cortical bone (two cats, moderate to severe clinical signs) and tendon (one cat, severe clinical signs). Direct infection of mucosal (trachea, tongue) tissues also was confirmed in these two cats. In contrast, all co-cultured tissues from the third cat (mild clinical signs) were negative for FHV-1 by CPE and PCR. Results of this study demonstrated that early primary FHV-1 viremia may be distributed to distant connective tissues.

Prevalence of bovine herpesvirus-4 infection in cats in Central Michigan.

The gammaherpesvirus bovine herpesvirus-4 (BHV-4) has been isolated from a wide variety of animals, including lions and domestic cats. Although BHV-4 antibodies have been detected in normal cats and cats with urinary disorders, the epidemiology and pathogenic role of BHV-4 in cats is unknown. The purpose of this study was to determine the prevalence of BHV-4 antibodies and viral nucleic acid in a population of free-roaming cats. Plasma and peripheral blood leukocyte samples were collected from 52 male and 52 female free-roaming cats impounded at a regional animal control facility in Central Michigan. Plasma concentrations of BHV-4 antibodies were measured with an indirect fluorescent antibody test. Peripheral blood leukocyte DNA was isolated, and a 2-stage polymerase chain reaction with heminested primers delineating a conserved portion of the BHV-4 glycoprotein B gene homologue was used to amplify BHV-4-specific DNA sequences. BHV-4 antibodies were detected in 38 (73%) male and 23 (44%) female cats. Seropositive cats were significantly more likely to be male than female (odds ratio = 3.22; P = .007). Cell-associated viremia was detected in 17 (33%) male and 11 (21%) female cats. Of the 61 seropositive cats, 23 (38%) had a detectable viremia; only 5 (12%) seronegative cats had detectable viremia. Seropositive cats were significantly more likely to be viremic than seronegative cats (OR = 4.30: P = .009). Our results suggest that BHV-4 infection may be more widespread in certain cat populations than previously reported. Furthermore, many cats seropositive for BHV-4 antibodies have a concurrent cell-associated viremia.

Effects of a 98% solution of glycerol or sterilization with ethylene oxide on FeLV in bone allografts and effects on bone incorporation of allografts in cats.

OBJECTIVES: To compare virucidal effects and bone incorporation properties of cortical bone allografts transplanted into specific-pathogen-free (SPF) cats. Allografts consisted of untreated bone from a SPF cat (negative-control group) and bone from 5 FeLV-infected cats that was subjected to sterilization with ethylene oxide (ETO), preservation with glycerol, or no treatment (positive-control group). SAMPLE POPULATION: Bones from the aforementioned groups and twenty 8-week-old SPF cats (5 cats/group) implanted with an allograft from 1 of the aforementioned groups. PROCEDURE: After implantation, blood samples were collected weekly to monitor FeLV p27 antigen and antibody titers. Quantification of FeLV provirus was performed on blood samples at weeks 0, 4, and 8 and donor bone samples at time of implantation. Cats were euthanatized 8 weeks after transplantation, and graft sites were evaluated. RESULTS: All results for negative-control cats were negative. All ETO group cats had negative results for antigen and provirus in blood, whereas 1 cat had a low antibody titer. Although 3 ETO-treated allografts were positive for provirus, the DNA appeared denatured. One cat in the glycerol group had positive results for all tests in blood samples. All glycerol-preserved allografts were positive when tested for provirus. All results for positive-control group cats were positive. Differences in incorporation of bone grafts were not observed. CONCLUSIONS AND CLINICAL RELEVANCE: Glycerol preservation of FeLV-infected bone allografts did not eliminate transmission of retrovirus to recipients. In contrast, ETO sterilization appeared to denature DNA and prevent infection. Treatments did not affect incorporation of bone grafts in young cats.

Calcium oxalate crystalluria in a goat.

As part of a routine health evaluation of an 8-month-old female Nubian goat, serum biochemical analyses and urinalysis were performed. Most serum biochemical values including concentrations of blood calcium and indicators of urinary system dysfunction, such as blood urea nitrogen, creatinine, and phosphorous concentrations, were within reference ranges. An aliquot of voided urine was hypersthenuric and acidic and contained numerous typical cuboidal-bipyramidal calcium oxalate dihydrate crystals and unique rectangular parallelepiped crystals that were confirmed by energy dispersive X-ray microanalysis as being of calcium oxalate dihydrate composition. We hypothesize that the calcium oxalate crystals resulted from a diet containing calcium and oxalic acid. Treatment was not administered, and the goat remained healthy during the ensuing year.

A potentially misleading presentation and course of acute myelomonocytic leukemia in a dog.

A nine-year-old, castrated male golden retriever had lethargy, fever, massive peripheral lymphadenomegaly, hepatosplenomegaly, and pale mucous membranes. There was a marked leukocytosis (456.3 x 10(3) cells/microliter) with 99% blasts; a moderate, nonregenerative anemia; and marked thrombocytopenia. A tentative diagnosis of acute lymphocytic leukemia was made pending results of cytochemical staining. Despite the severity of the laboratory and clinical findings, the dog exhibited a partial response to an induction chemotherapy protocol commonly used for lymphoma. Subsequent cytochemical staining of the original blood and bone-marrow samples resulted in a revised diagnosis of acute myelomonocytic leukemia (AML-M4). Clinicopathological findings, response to treatment, and clinical outcome in this case of canine AML-M4 are discussed.

Retroviral transmission in bone allotransplantation. The effects of tissue processing.

The transmission of a retrovirus through transplantation of processed bone allografts was studied using the feline leukemia virus. The long bones of 4 previously infected donor cats were harvested and assigned to 1 of 3 treatment groups: single freeze/thaw cycle, double freeze/thaw cycle, or double freeze/thaw cycle with water flush to remove bone marrow. Cortical bone grafts and corticocancellous bone grafts from each treatment group were transplanted into individual specific-pathogen-free recipients. Samples of plasma were obtained weekly from all recipients and were tested with an enzyme-linked immunosorbent assay to detect viral antigen. For animals that tested consistently negative for viral antigen, plasma samples also were tested for antiviral antibody to feline leukemia virus measured by live cell immunofluorescence. The results of the antigen and antibody testing revealed that all of the cortical and corticocancellous bone allografts in each of the 3 treatment groups transmitted virus. The ability of the treated bone allografts to transmit a feline retrovirus suggests that routine processing and removal of bone marrow may not inhibit their ability to transmit other retroviruses, such as the human immunodeficiency virus.

Retroviral transmission by the transplantation of connective-tissue allografts. An experimental study.

The transmission of a retrovirus by the transplantation of allografts of connective tissues was studied in a feline model with use of the feline leukemia virus, a retrovirus with a replication cycle and pathological characteristics similar to those of the human immunodeficiency virus. The retrovirus was used to infect four specific-pathogen-free cats that were subsequently used as tissue donors. Fresh allografts of menisci, patellar ligaments, and patellar ligament and bone composites were harvested from infected donors and were transplanted into the knee joints of twelve specific-pathogen-free cats. A fresh cancellous-bone allograft was transplanted into the proximal part of the tibia of four additional specific-pathogen-free cats, which served as positive control animals. Additional grafts from infected donors were harvested and were stored at -80 degrees Celsius for ten weeks. A fresh-frozen graft was then transplanted into the knee of twelve other specific-pathogen-free cats. Samples of plasma were obtained weekly from all twenty-eight cats and were tested with both an enzyme-linked immunosorbent assay to detect the presence of viral antigen and an immunofluorescent antibody assay to determine exposure to the virus. All types of fresh and fresh-frozen connective-tissue allografts from the infected donors resulted in transmission of the retrovirus to the recipient cats. The recipients had evidence of viral antigen or rising antibody titers as early as two weeks after the transplantation. Histological examination of specimens of the allografts revealed normal incorporation of the transplanted tissues, with no sign of rejection of the graft.

Lipopolysaccharide induces monocyte chemoattractant protein production by rat mesangial cells.

Lipopolysaccharide, a potent pro-inflammatory constituent of bacterial cell walls, is capable of promoting glomerular inflammation, by both activating circulating inflammatory cells and local interactions with renal parenchymal cells. We sought to determine whether lipopolysaccharide was capable of promoting glomerular inflammation by directly stimulating mesangial cell production of monocyte chemoattractant protein 1, a recently described cytokine capable of eliciting recruitment of mononuclear phagocytes into inflammatory foci. Northern hybridization analysis revealed dose and time-dependent induction of mRNA coding for monocyte chemoattractant protein 1 in quiescent rat mesangial cells treated with lipopolysaccharide. Lipopolysaccharide-elicited induction of monocyte chemoattractant protein mRNA was detectable after 1 hour and persisted for at least 30 hours. Media isolated from rat mesangial cell cultures stimulated by lipopolysaccharide possessed monocyte chemotactic activity that was detectable at 8 hours and peaked at 24 hours; an antimonocyte chemoattractant protein antibody blocked 87% of this chemotactic activity. We suggest that lipopolysaccharide, released from bacterial cell walls, promotes glomerular inflammation by stimulating mesangial cell production of monocyte chemoattractant protein 1.

Pre- and postexposure chemoprophylaxis: evidence that 3'-azido-3'-dideoxythymidine inhibits feline leukemia virus disease by a drug-induced vaccine response.

The benefits of postexposure 3'-azido-3'-dideoxythymidine (AZT) prophylaxis following human immunodeficiency virus exposure are unknown. We describe a comprehensive assessment of pre- and postexposure AZT therapy in the feline leukemia virus (FeLV)-cat model for AIDS which included in vitro testing, an in vivo dose-response titration, a postexposure treatment study, plasma drug concentration determinations, and evaluation of the immune response to FeLV. In in vitro studies, AZT prevented FeLV infection of a feline T-lymphoid cell line, giving 50 and 90% inhibition concentrations of 4.6 and 11.1 mM, respectively. In all of the in vivo efficacy studies, AZT was administered by continuous subcutaneous infusion for 28 days. AZT toxicity was excessive at a dosage of 120 mg/kg of body weight per day, causing acute anemia, but AZT was tolerable at 60 mg/kg/day. In preexposure studies, AZT was efficacious in preventing chronic antigenemia at a dosage of > or = 15 mg/kg/day, at which plasma AZT concentrations averaged between 0.51 and 0.81 micrograms/ml (2.13 and 3.03 microM). As a postexposure treatment, at 60 mg/kg/day, AZT prevented chronic FeLV antigenemia when treatment was started up to 96 h post-virus inoculation (p.i.), but not when treatment was started at 192 h p.i. The 4-day period between 96 and 192 h p.i. appears to be critical for establishing chronic viremia. It is presumed that the increase in virus load between 4 and 8 days p.i. was able to overwhelm the immunologic functions responsible for containment of FeLV infection, even though AZT therapy effectively controlled viremia during the treatment period. The antibody response to FeLV varied depending on the time of AZT treatment initiation relative to virus challenge. When AZT treatment was started 48 h before or 8 h after FeLV challenge, antibodies to FeLV were not detected until after AZT treatment was discontinued at 28 days p.i. Following AZT treatment, however, antibody titers rapidly increased at a rate suggestive of a secondary immune response. When AZT treatment was initiate at later time points relative to virus challenge (24, 48, and 96 h p.i.), antibodies to FeLV became detectable during the treatment period. These results indicate that AZT treatment does not completely prevent FeLV infection, even when treatment begins before virus challenge, and that immune sensitization to FeLV proceeds during the prophylactic drug treatment period.

Prophylactic and therapeutic effects of phosphonoformate against feline leukemia virus in vitro.

Phosphonoformate (PFA), a noncompetitive inhibitor of reverse transcriptase (RT), inhibited feline leukemia virus (FeLV) infection of 2 feline cell lines and inhibited progeny virus RT activity in a chronically FeLV-infected cell line. Feline leukemia virus infection of 3201 cells, an FeLV-negative lymphoma cell line, was inhibited by greater than 70% at a concentration of only 1 microM PFA and by greater than 90% at concentrations of 64 to 256 microM PFA, as evidenced by RT activity. However, FeLV antigen expression by 3201 cells remained relatively constant over noncytotoxic concentrations of PFA. Because the persistence of viral antigen expression with concomitant suppression of RT activity appears to be unique and because 3201 cells express small amounts of an endogenous retrovirus (RD-114) and contain endogenous FeLV proviral sequences, a possible role of endogenous retroviruses acting as helper viruses was suggested. Feline leukemia virus infection of 81C cells, a sarcoma-positive, leukemia-negative fibroblast cell line, was inhibited by greater than 50% at a concentration of 64 microM PFA and by greater than 98% at concentrations of 256 to 512 microM PFA, as indicated by suppression of focus formation. The feline lymphoid cell line FL-74 is a large producer of FeLV. When FL-74 cells were cultured in the presence of 256 microM PFA, virus production (virus budding and viral antigen) was not affected, but progeny virus lost RT activity and infectivity. Direct addition of PFA (256 microM) to FeLV also reduced RT activity and infectivity. These data indicate that PFA can directly and rapidly inactivate retrovirus independent of cellular processing, presumably by inhibiting RT.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of antiviral activity and toxicity of dextran sulfate in feline leukemia virus-infected cats.

The feline leukemia virus (FeLV) disease model was used to conduct a toxicity and antiretrovirus efficacy trial of dextran sulfate (DS; molecular mass, 7,000 to 8,000 Da). In vitro, FeLV infection of feline lymphoid cells was inhibited by 10 micrograms of DS per ml. DS was administered to cats by continuous intravenous infusion at doses of 600, 120, 24, or 4.8 mg/kg of body weight per day, beginning 24 h before FeLV challenge. Doses of 24 mg/kg/day and more were excessively toxic, causing intestinal lesions and death. Similar changes were observed in unchallenged animals receiving 24 mg/kg/day, indicating that toxicity was DS mediated. The dosage of 4.8 mg/kg/day was subtoxic but did not prevent the induction and persistence of FeLV viremia. The results demonstrate that DS by continuous intravenous infusion is excessively toxic at high doses and ineffective at preventing FeLV infection at a subtoxic dose in the FeLV cat model.

Age-related differences in phosphonoformate-induced bone toxicity in cats.

Phosphonoformate (PFA), a monophosphonate pyrophosphate analog, caused plasma biochemical and bone histomorphologic abnormalities in cats given 1,000 mg/kg/day as a continuous intravenous infusion for 14 days. Plasma biochemical alterations observed in young cats (10 weeks old) treated with PFA included increased calcium and decreased phosphorus, alkaline phosphatase, and calcitriol. Young cats treated with PFA developed rickets-like lesions characterized by widened growth plates, increased osteoid, and failure of mineralization. In addition, area of mineralized trabecular bone was decreased. Osteoclast size was increased whereas osteoclast perimeter and number were unaffected in young PFA-treated cats. Plasma alkaline phosphatase was decreased in adult cats (greater than or equal to 1 year old) treated with PFA but changes in calcium, calcitriol, and immunoreactive parathyroid hormone were highly variable and not significantly different. Adult cats treated with PFA exhibited osteomalacia characterized by increased osteoid area, perimeter, and width with failure of mineralization. In addition, static resorption indices were increased in PFA-treated adult cats but area of mineralized trabecular bone was not decreased. The monophosphonate PFA inhibited bone mineralization in young and adult cats similar to bisphosphonate treatment in other species. Because PFA is currently in phase I trials for use in AIDS, results of this study suggest a need to evaluate patients treated with PFA for metabolic bone disease.

Monocyte chemotaxis and activating factor production by keratinocytes in response to IFN-gamma.

Monocytes accumulate in the epidermis and along the dermo-epidermal junction in several different inflammatory skin diseases. To determine whether human epidermal keratinocytes elaborate a specific chemotaxin responsible for the accumulation of monocytes at these anatomic sites, monocyte chemotactic activity in conditioned 16-h cultured keratinocyte supernatants were assayed using human peripheral blood monocytes as the target cell. Dilutional analysis revealed directed monocyte migration in IFN-gamma-treated (100 U/ml) keratinocyte supernatants (80% maximal FMLP response) which was 10-fold more than IFN-gamma itself or untreated keratinocyte activity alone. Gel filtration chromatography revealed that this activity eluted just ahead of a 12.5-kDa molecular mass marker. Blocking studies demonstrated that a rabbit polyclonal antibody to monocyte chemotaxis and activating factor (MCAF) inhibited all monocyte chemotaxis by greater than 80%. Keratinocytes were metabolically labeled with 35S-cysteine/methionine, and after 16 h incubation the supernatants immunoprecipitated with the same anti-MCAF antibody. MCAF was detected as a protein doublet of 12 and 9 kDa only in IFN-gamma-treated (100 U/ml) keratinocyte supernatants. Incubation with IFN-gamma and TNF-alpha (250 U/ml) in combination resulted in increased production of MCAF protein. By Northern blot analysis, MCAF mRNA was constitutively expressed in keratinocytes and upregulated only in the presence of IFN-gamma. TNF-alpha, IL-1 beta, transforming growth factor-beta and phorbol esters had no positive or negative influence on MCAF mRNA. These studies demonstrate that biologically active MCAF is elaborated by human epidermal keratinocytes upon activation by IFN-gamma, a cytokine also required for the induction of adherence between monocytes and keratinocytes. Keratinocyte-derived MCAF is likely to be important in the regulation of cutaneous monocyte trafficking and may also be responsible for the recruitment of Langerhans cells and dermal dendrocytes, which share many phenotypic features with monocytes/macrophages, to their anatomic locations in skin.

Chylothorax in cats: 37 cases (1969-1989).

Case records of 37 cats with chylothorax examined at 2 institutions were retrospectively evaluated. Dyspnea and coughing were the most common abnormalities noticed by the owners, and most cats were dyspneic on initial examination. There was no statistically significant difference in the gender distribution of cats studied when compared with reference populations; however, purebred cats appeared to be overrepresented in the study population. Four of the cats had unilateral pleural effusion (2 left side, 2 right side) and 9 cats had effusions that were primarily, but not exclusively, on the right side. Surgery was performed on 20 cats. Fifteen cats underwent thoracic duct or cisterna chyli ligation; 20% had complete resolution of pleural fluid. There was no significant difference in the survival rate of cats that underwent thoracic duct ligation and those that were treated by other means. Six cats had mesenteric lymphangiography performed; 2 cats had normal results, and the remainder had various degrees of thoracic lymphangiectasia. Two cats in which pleuroperitoneal shunts were placed and 2 of 3 cats that underwent pleurodesis were euthanatized or died after surgery.

In vitro and in vivo evidence that the antiviral activity of 2',3'-dideoxycytidine is target cell dependent in a feline retrovirus animal model.

2',3'-Dideoxycytidine (DDC) was evaluated for prophylactic antiviral activity in vitro and in vivo, using the feline leukemia virus (FeLV)-cat animal model. In vitro antiviral activity of DDC against FeLV was dependent upon the target cell used for infection. DDC (5 to 10 microM) inhibited FeLV infection of feline lymphoid cells by greater than 80%, while 6.07 to 12.13 microM DDC was required to similarly inhibit infection of feline fibroblasts. However, 43 to 384 microM DDC was needed to inhibit FeLV infection of primary bone marrow cells by greater than 80%. These in vitro results suggest that, although relatively low doses of DDC may be adequate to prevent infection of feline lymphoid cells, 8- to 80-times-higher doses may be necessary to block infection of bone marrow cells, a primary target cell type for FeLV infection. In vivo studies with DDC consisted of pharmacokinetic and toxicity determinations and evaluation of the prophylactic antiviral activity against FeLV in cats. Clearance and half-life values for DDC in cats were 6.5 ml/min per kg and 54.7 min, respectively. In the prophylactic studies, DDC was administered by continuous intravenous infusion at doses of 22, 15, 10, and 5 mg/kg per h for 28 days in most animals. Cats were challenged intravenously with FeLV 1 to 3 days after drug treatment began. Doses of 22 and 15 mg/kg per h were extremely toxic, causing death in 8 of 10 cats. The mg/kg per h dose was slightly toxic, causing chronic progressive thrombocytopenia over the 28-day treatment period. Of 10 cats given 10 to 5 mg of DDC per kg per h, only one was completely protected from FeLV antigenemia. However, conversion to positive FeLV antigenemia status was delayed by 2 to 7 weeks in seven of nine remaining animals. Interestingly, FeLV infection of bone marrow cells, as indicated by FELV antigen in peripheral blood neutrophils, was only slightly delayed by 0 to 2 weeks, except in the case of the one protected cat, and usually preceded conversion to antigenemia. This pattern of neutrophils becoming antigen positive before detection of antigenemia was not seen in FeLV challenge control animals and indicates that the antiviral activity of DDC may be incomplete during DDC treatment. Results of our in vitro and in vivo studies suggest that feline bone marrow cells may remain partially susceptible to FeLV infection at tolerated doses, while other somatic target tissues (i.e., lymphoid or epithelial tissues) may be protected from infection. Incomplete inhibition of FeLV infection permitted focal bone marrow infection to develop in cats given DDC. These loci of infection served as virus reservoirs which, subsequent to discontinuation of DDC treatment, permitted spread of infection to tissues previously protected during treatment.

Age-related differences in pharmacokinetics of phosphonoformate in cats.

Phosphonoformate (PFA) is a simple PPi analog which inhibits the activities of a variety of viral DNA polymerase, RNA polymerase, and reverse transcriptase enzymes. PFA is a topical and parenteral treatment for human herpesvirus infections and is currently in phase I trials for treatment of acquired immunodeficiency syndrome. Pharmacokinetic properties of PFA in young (growing) and adult specific-pathogen-free cats were compared. Mean PFA clearance from plasma was twofold higher in young cats (7.52 ml/min per kg of body weight) than in adult cats (3.70 ml/min per kg). Higher PFA clearance from plasma observed in young cats may result from higher renal clearance or enhanced accumulation of PFA in bone tissue of young versus adult cats. No plasma protein binding of PFA was observed. Mean oral bioavailability was 35% in young cats. These data indicate that age-related differences in PFA clearance from plasma occur in cats.