Melissa T. Hines

The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona

Striped skunks, initially negative for antibodies to Sarcocystis neurona, formed sarcocysts in skeletal muscles after inoculation with S. neurona sporocysts collected from a naturally infected Virginia opossum (Didelphis virginiana). Skunks developed antibodies to S. neurona by immunoblot and muscles containing sarcocysts were fed to laboratory-reared opossums which then shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0 x 7.5 microm and each contained four sporozoites and a residuum. Sarcocysts from skunks and sporocysts from opossums fed infected skunk muscle were identified as S. neurona using PCR and DNA sequence analysis. A 2-month-old, S. neurona-naive pony foal was orally inoculated with 5 x 10(5) sporocysts. Commercial immunoblot for antibodies to S. neurona performed using CSF collected from the inoculated pony was low positive at 4 weeks p.i., positive at 6 weeks p.i., and strong positive at 8 weeks p.i. Gamma-interferon gene knockout mice inoculated with skunk/opossum derived sporocysts developed serum antibodies to S. neurona and clinical neurologic disease. Merozoites of S. neurona present in the lung, cerebrum, and cerebellum of mice were detected by immunohistochemistry using polyclonal antibodies to S. neurona. Based on the results of this study, the striped skunk is an intermediate host of S. neurona.

Disseminated hemangiosarcoma in the horse: 35 cases.

Thirty-five cases of disseminated hemangiosarcoma (21 clinical cases and 14 previously reported cases) were reviewed to describe the disease in horses. Hemangiosarcoma occurred in mature, particularly middle-aged horses, with no apparent sex predilection. Thoroughbreds seemed to be overrepresented (13 cases) but a true breed predilection could not be established. The respiratory and musculoskeletal systems were most commonly affected and presenting complaints included dyspnea (26%), subcutaneous or muscular swelling (24%), epistaxis (17%), and lameness (12%). Heart and respiratory rates were usually increased and mucous membrane color was frequently pale or icteric. Capillary refill time and rectal temperature were often normal. Anemia (88%), neutrophilic leukocytosis (62%), and thrombocytopenia (48%) were common. Examination of tissue samples collected by fine-needle aspirate or biopsy established an antemortem diagnosis in 4 horses. The diagnosis was made during postmortem examination in the remaining 31 horses. The lung and pleura (77%), skeletal muscle (46%), and spleen (43%) were most commonly affected. A primary site of tumor involvement could be identified in 22 horses. Hemangiosarcoma should be included as a differential diagnosis for horses with evidence of hemorrhage into body cavities, skeletal muscle, or subcutaneous locations.

Clearance of Virulent but Not Avirulent Rhodococcus equi from the Lungs of Adult Horses Is Associated with Intracytoplasmic Gamma Interferon Production by CD4+ and CD8+ T Lymphocytes

ABSTRACT Rhodococcus equi is a gram-positive bacterium that infects alveolar macrophages and causes rhodococcal pneumonia in horses and humans. The virulence plasmid of R. equi appears to be required for both pathogenicity in the horse and the induction of protective immunity. An understanding of the mechanisms by which virulent R. equi circumvents protective host responses and by which bacteria are ultimately cleared is important for development of an effective vaccine. Six adult horses were challenged with either virulent R. equi or an avirulent, plasmid-cured derivative. By using a flow cytometric method for intracytoplasmic detection of gamma interferon (IFN-γ) in equine bronchoalveolar lavage fluid (BALF) cells, clearance of the virulent strain was shown to be associated with increased numbers of pulmonary CD4+ and CD8+ T lymphocytes producing IFN-γ. There was no change in IFN-γ-positive cells in peripheral blood, suggesting that a type 1 recall response at the site of challenge was protective. The plasmid-cured strain of R. equi was cleared in horses without a significant increase in IFN-γ-producing T lymphocytes in BALF. In contrast to these data, a previous report in foals suggested an immunomodulating role for R. equi virulence plasmid-encoded products in downregulating IFN-γ expression by equine CD4+ T lymphocytes. Intracytoplasmic detection of IFN-γ provides a method to better determine whether modulation of macrophage-activating cytokines by virulent strains occurs uniquely in neonates and contributes to their susceptibility to rhodococcal pneumonia.

Corticosteroid immunosuppression and monoclonal antibody-mediated CD5 ÷ T lymphocyte depletion in normal and equine infectious anaemia virus-carrier horses

The immune control of chronic equine infectious anaemia (EIA) lentiviral infection was investigated by specifically depleting CD5+ T lymphocytes in vivo with monoclonal antibody (MAb) or by immunosuppression with corticosteroids. MAb was given at 25 to 50 mg/day intravenously for 11 days. Murine IgG1 anti-equine CD2 MAb (n = 2 horses) or IgG1 (n = 2) and IgG2a control MAb (n = 2 normal; 2 EIA-infected) did not deplete CD2+ T lymphocytes in horses. Horses given murine IgG2a anti-CD5 MAb HB19A (n = 4 normal; 5 EIA-infected) had depletion of peripheral blood CD5+ T lymphocytes during treatment. These horses, however, maintained a residual population of CD2+ T lymphocytes [15 (+/- 3)% of pretreatment numbers] that did not express CD5 but expressed either CD4 or CD8. These antigenically modulated CD5- T lymphocytes responded normally in vivo to intradermal inoculation with phytohaemagglutinin and in vitro to allogeneic leukocyte stimulation in one-way mixed lymphocyte reactions. EIA virus-infected horses (n = 5) did not develop recrudescent viraemia or disease following in vivo CD5+ T lymphocyte depletion. Immunosuppression of EIA virus-infected horses with corticosteroids (1 mg/kg body weight/day, intravenously for 9 days) resulted in detectable recrudescent EIA viraemia in 6/11 horses (55%) and recrudescent disease in 9/11 horses (82%). Normal horses (n = 3) treated with corticosteroids developed no clinical disease. These results demonstrate that the use of murine IgG2a MAbs to appropriate equine lymphocyte antigens will facilitate in vivo investigation of the role of T lymphocyte subpopulations in the control of EIA or other important equine diseases.

Analysis of anamnestic immune responses in adult horses and priming in neonates induced by a DNA vaccine expressing the vapA gene of Rhodococcus equi

Rhodococcus equi remains one of the most important pathogens of early life in horses, yet conventional vaccines to prevent rhodococcal pneumonia have not been successful. DNA vaccination offers an alternative to conventional vaccines with specific advantages for immunization of neonates. We developed a DNA vaccine expressing the vapA gene (pVR1055vapA) that induced an anamnestic response characterized by virulence associated protein A (VapA)-specific IgG antibodies in sera and bronchoalveolar lavage fluid (BALF) as well as VapA-specific proliferation of pulmonary lymphocytes when tested in adult ponies. In contrast, none of the adults receiving the control plasmid responded. To determine if pVR1055vapA induced VapA-specific responses in the foal, the targeted age group for vaccination against R. equi, 10 naïve foals were randomly assigned at birth to two groups of five. At 8-15 days of age (day 1), foals were vaccinated by intranasal and intradermal (i.d.) routes with either pVR1055vapA or the negative control pVR1055vapA_rev. All foals were DNA boosted at day 14 and protein boosted at day 30 with either recombinant VapA or recombinant CAT (control group). Prior to the protein boost, neither group developed VapA-specific immune responses. However, at day 45, two of the VR1055vapA-vaccinated foals had increased titers of VapA-specific IgGb, IgM and IgGa in the sera, and IgG in the BALF. The induction of the opsonizing isotypes IgGa and IgGb has been previously shown to be associated with protection against R. equi. No VapA-specific immune responses were detected in the control group. This study indicates that the DNA vaccine effectively stimulates anamnestic systemic and pulmonary immune responses in adult horses. The results in foals suggest that the DNA vaccine also primed a subset of immunized neonates. These data support further development and modification to produce a DNA vaccine to more effectively prime neonatal foals.

Immunity to Rhodococcus equi: antigen-specific recall responses in the lungs of adult horses.

Rhodococcal pneumonia is an important disease of young horses that is not seen in immunocompetent adults. Since all foals are normally exposed to Rhodococcus equi in their environment, we hypothesized that most develop protective immune responses. Furthermore, these antigen-specific responses were hypothesized to operate throughout adult life to prevent rhodococcal pneumonia. A better understanding of the mechanisms of immune clearance in adult horses would help define the requirements for an effective vaccine in foals. Adult horses were challenged with virulent R. equi by intrabronchial inoculation into the right lung, and pulmonary immune responses were followed for 2 weeks by bronchoalveolar lavage. Local responses in the inoculated right lung were compared to the uninfected left lung and peripheral blood. Challenged horses rapidly cleared R. equi infection without significant clinical signs. Clearance of bacteria was associated with increased mononuclear cells in bronchoalveolar lavage fluid (primarily lymphocytes) and inversion of the normal macrophage:lymphocyte ratio. There was no significant increase in neutrophils at 7 days post-challenge. Flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that clearance correlated with significant increases in pulmonary T-lymphocytes, both CD4+ and CD8+. Prior to challenge, most adult horses demonstrated low proliferative responses when pulmonary lymphocytes were stimulated with soluble R. equi ex vivo. However, clearance was associated with marked increases in lymphoproliferative responses to soluble R. equi antigen and recombinant VapA, a virulence associated protein of R. equi and candidate immunogen. These results are compatible with previous work in mice which showed that both CD4+ and CD8+ T-cells play a role in immune clearance of R. equi. Recognition of VapA in association with clearance lends further support to its testing as an immunogen. Importantly, the cellular responses to R. equi challenge were relatively compartmentalized. Responses were more marked and the sensitivity to antigen dose was increased at the site of challenge. The blood, including peripheral blood mononuclear cells, was an insensitive indicator of local pulmonary responses.

Rhodococcus equi-Specific Cytotoxic T Lymphocytes in Immune Horses and Development in Asymptomatic Foals

ABSTRACT Rhodococcus equi is an important cause of pneumonia in young horses; however, adult horses are immune due to their ability to mount protective recall responses. In this study, the hypothesis that R. equi-specific cytotoxic T lymphocytes (CTL) are present in the lung of immune horses was tested. Bronchoalveolar lavage (BAL)-derived pulmonary T lymphocytes stimulated with R. equi lysed infected alveolar macrophages and peripheral blood adherent cells (PBAC). As with CTL obtained from the blood, killing of R. equi-infected targets by pulmonary effectors was not restricted by equine lymphocyte alloantigen-A (ELA-A; classical major histocompatibility complex class I), suggesting a novel or nonclassical method of antigen presentation. To determine whether or not CTL activity coincided with the age-associated susceptibility to rhodococcal pneumonia, CTL were evaluated in foals. R. equi-stimulated peripheral blood mononuclear cells (PBMC) from 3-week-old foals were unable to lyse either autologous perinatal or mismatched adult PBAC targets. The defect was not with the perinatal targets, as adult CTL effectors efficiently killed infected targets from 3-week-old foals. In contrast, significant CTL activity was present in three of five foals at 6 weeks of age, and significant specific lysis was induced by PBMC from all foals at 8 weeks of age. As with adults, lysis was ELA-A unrestricted. Two previously described monoclonal antibodies, BCD1b3 and CD1F2/1B12.1, were used to examine the expression of CD1, a nonclassical antigen-presenting molecule, on CTL targets. These antibodies cross-reacted with both foal and adult PBAC. However, neither antibody bound alveolar macrophages, suggesting that the R. equi-specific, major histocompatibility complex-unrestricted lysis is not restricted by a surface molecule identified by these antibodies.

Monoclonal Antibodies to the Equine CD2 T Lymphocyte Marker, to a Pan-Granulocyte/Monocyte Marker and to a Unique Pan-B Lymphocyte Marker

Murine monoclonal antibodies, HB88A, B29A and DH59B separately identify the CD2 T lymphocyte molecule, a unique pan-B lymphocyte surface marker and a pan-granulocyte/monocyte surface molecule, respectively, in the horse. Specificity was shown by two-color immunofluorescent flow cytometry and immunofluorescent microscopy. MAb HB88A reacted with a 52 kDa pan-T lymphocyte molecule present on 75% +/- 7 of peripheral blood lymphocytes (PBL) (n = 15 horses). It also reacted with lymphocytes restricted to T lymphocyte dependent areas of lymph node and spleen. Specificity of mAb HB88A to CD2 was demonstrated by its reactivity to COS7 cells which expressed a transfected 1.5 kb equine lymphocyte c-DNA clone having 77.5% overall sequence homology with human CD2 c-DNA. MAb B29A reacted with a pan-B lymphocyte specific cell surface complex, 143, 72, 50, 40, 27 and 14.5 kDa, present on 19% +/- 7 of PBL (n = 15 horses). This complex has not been described in the horse or other species. MAb DH59B reacted with a 96 kDa pan-granulocyte/monocyte specific surface protein and identified macrophages and Kupffer cells in equine tissue sections. Together these mAbs can be used to identify and quantitate the major constituents of equine leukocytes.

Effects of a polymerized ultrapurified bovine hemoglobin blood substitute administered to ponies with normovolemic anemia.

The development of ultrapurified hemoglobin-based oxygen carriers has eliminated many problems associated with whole-blood transfusions in other species. We hypothesized that the administration of polymerized ultrapurified bovine hemoglobin (PUBH) would result in improved hemodynamic parameters in ponies with normovolemic anemia without adverse effects on renal function or coagulation times. Normovolemic anemia was induced in 6 healthy adult ponies. Over a 3-day period, at least 45 mL/kg of whole blood was withdrawn from each pony until a target PCV of <12% was attained. Plasma was separated from the red blood cells via centrifugation and readministered to the ponies on each day. After the final plasma transfusion, 15 mL/kg of hetastarch (control, n = 6) or 15 mL/kg of PUBH (treatment, n = 6) was administered at 10 mL/kg/h IV. Administration of PUBH at a rate of 10 mL/kg/h was not associated with any adverse effects in 5 of the 6 ponies. One pony experienced an anaphylactoid reaction during infusion of PUBH. The reaction, characterized by intense pruritus, tachycardia, and tachypnea resolved shortly after stopping the infusion. Ponies receiving PUBH had significantly lower cardiac indices (P = .03) and heart rates (P = .002) than control animals. A significantly greater increase in central venous pressure was observed in the PUBH group compared to the hetastarch group (P = .02). No adverse renal or coagulation effects were observed with PUBH infusion. These results suggest that PUBH improves hemodynamics and oxygen transport parameters in horses experiencing normovolemic anemia. Patients should be monitored closely during infusion for any adverse reactions.

Pharmacokinetics of Butorphanol and Evaluation of Physiologic and Behavioral Effects after Intravenous and Intramuscular Administration to Neonatal Foals

BACKGROUND Despite frequent clinical use, information about the pharmacokinetics (PK), clinical effects, and safety of butorphanol in foals is not available. OBJECTIVES The purpose of this study was to determine the PK of butorphanol in neonatal foals after IV and IM administration; to determine whether administration of butorphanol results in physiologic or behavioral changes in neonatal foals; and to describe adverse effects associated with its use in neonatal foals. ANIMALS Six healthy mixed breed pony foals between 3 and 12 days of age were used. METHODS In a 3-way crossover design, foals received butorphanol (IV and IM, at 0.05 mg/kg) and IV saline (control group). Butorphanol concentrations were determined by high-performance liquid chromatography and analyzed using a noncompartmental PK model. Physiologic data were obtained at specified intervals after drug administration. Pedometers were used to evaluate locomotor activity. Behavioral data were obtained using a 2-hour real-time video recording. RESULTS The terminal half-life of butorphanol was 2.1 hours and C0 was 33.2 +/- 12.1 ng/mL after IV injection. For IM injection, Cmax and Tmax were 20.1 +/- 3.5 ng/mL and 5.9 +/- 2.1 minutes, respectively. Bioavailability was 66.1 +/- 11.9%. There were minimal effects on vital signs. Foals that received butorphanol spent significantly more time nursing than control foals and appeared sedated. CONCLUSIONS AND CLINICAL IMPORTANCE The disposition of butorphanol in neonatal foals differs from that in adult horses. The main behavioral effects after butorphanol administration to neonatal foals were sedation and increased feeding behavior.