Borrelia burgdorferi Infection and Lyme Disease in North American Horses: A Consensus Statement.

Borrelia burgdorferi infection is common in horses living in Lyme endemic areas and the geographic range for exposure is increasing. Morbidity after B. burgdorferi infection in horses is unknown. Documented, naturally occurring syndromes attributed to B. burgdorferi infection in horses include neuroborreliosis, uveitis, and cutaneous pseudolymphoma. Although other clinical signs such as lameness and stiffness are reported in horses, these are often not well documented. Diagnosis of Lyme disease is based on exposure to B. burgdorferi, cytology or histopathology of infected fluid or tissue and antigen detection. Treatment of Lyme disease in horses is similar to treatment of humans or small animals but treatment success might not be the same because of species differences in antimicrobial bioavailability and duration of infection before initiation of treatment. There are no approved equine label Lyme vaccines but there is strong evidence that proper vaccination could prevent infection in horses.

Seroprevalence of Borrelia burgdorferi in Horses Presented for Coggins Testing in Southwest Virginia and Change in Positive Test Results Approximately 1 Year Later.

BACKGROUND: Lyme disease can affect people, dogs, and horses, but it remains poorly understood, especially in the horse. Determining the seroprevalence of Borrelia burgdorferi in horses in different geographic areas will enable better understanding of the epidemiology of the disease, thus improving diagnosis and treatment of affected animals. HYPOTHESIS: To determine the seroprevalence of B. burgdorferi in horses in southwest Virginia. ANIMALS: Horses presented for routine Coggins testing from January 2013 to January 2014 had additional blood drawn for Lyme Multiplex Assay testing. METHODS: Of 492 samples collected, 250 samples were analyzed using the Lyme Multiplex Assay. Of the 83 horses that had positive test results to at least 1 outer surface protein (Osp), 63 were available for follow-up testing 5-17 months later (June 2014). RESULTS: Thirty-three percent of horses had positive results for antibodies to at least 1 Osp. Horses with a positive outer surface protein F (OspF) result were older (14.5 ± 0.79) than horses with a negative OspF result (11.6 ± 0.53). Of the horses available for follow-up testing, 63% had the same result as that of the initial test. There was no difference in test result between initial and follow-up testing. CONCLUSIONS: Horses seropositive to B. burgdorferi are common in Virginia, and older horses are more likely to have a positive test result for OspF than younger horses. Follow-up testing indicated that the majority of horses that were positive on initial testing did not have a different test result 5-17 months later.

Equine Protozoal Myeloencephalitis: An Updated Consensus Statement with a Focus on Parasite Biology, Diagnosis, Treatment, and Prevention.

Equine protozoal myeloencephalitis (EPM) remains an important neurologic disease of horses. There are no pathognomonic clinical signs for the disease. Affected horses can have focal or multifocal central nervous system (CNS) disease. EPM can be difficult to diagnose antemortem. It is caused by either of 2 parasites, Sarcocystis neurona and Neospora hughesi, with much less known about N. hughesi. Although risk factors such as transport stress and breed and age correlations have been identified, biologic factors such as genetic predispositions of individual animals, and parasite-specific factors such as strain differences in virulence, remain largely undetermined. This consensus statement update presents current published knowledge of the parasite biology, host immune response, disease pathogenesis, epidemiology, and risk factors. Importantly, the statement provides recommendations for EPM diagnosis, treatment, and prevention.

Immunotherapeutics to prevent the replication of Brucella in a treatment failure mouse model.

Outer membrane vesicles (OMVs) from Brucella melitensis and irradiated Brucella neotomae have been shown to be effective vaccines against a B. melitensis challenge in a mouse model. The present study evaluates the efficacy of these two vaccines as immuno-therapeutics in combination with conventional antibiotics against a B. melitensis infection. BALB/c mice chronically infected with B. melitensis were treated for 4 weeks with doxycycline and gentamicin and vaccinated twice during the course of therapy. Antibiotics in sub-therapeutic concentrations were chosen in such a way that the treatment would result in a therapeutic failure in mice. Although no additive effect of vaccines and antibiotics was seen on the clearance of B. melitensis, mice receiving vaccines along with antibiotics exhibited no Brucella replication post-treatment compared to mice treated only with antibiotics. Administration of irradiated B. neotomae along with antibiotics led to higher production of IFN-γ ex vivo by splenocytes upon stimulation with heat inactivated B. melitensis while no such effect was seen by splenocytes from mice vaccinated with OMVs. OMV vaccinated mice developed significantly higher anti-Brucella IgG antibody titers at the end of the treatment compared to the mice that received only antibiotics. The mice that received only vaccines did not show any significant clearance of Brucella from spleens and livers compared to non-treated control mice. This study suggests that incorporating OMVs or irradiated B. neotomae along with conventional antibiotics might be able to improve therapeutic efficacy and control the progression of disease in treatment failure cases.

A single mid-gestation exposure to TCDD yields a postnatal autoimmune signature, differing by sex, in early geriatric C57BL/6 mice.

We recently observed an autoimmune profile in 24-week-old C57BL/6 mice that received a 2.5 or 5.0µg/kg mid-gestation dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Mustafa et al., 2008). The clinical signs were consistent with a lupus-like syndrome and included: increased autoantibody levels, renal IgG and C3 immune complex deposition with associated inflammation, and increased peripheral Vß(+) T cells. No studies currently exist following the progression of such disease into middle or advanced ages, when human autoimmune diseases may manifest. Therefore in the present study, littermates of mice from the previous 24 week prenatal TCDD study were allowed to age to 48 weeks, considered early geriatric in mice. Similarities and differences in the disease profile based on age and sex were observed. Peripheral autoreactive Vß(+) T cells were increased in both sexes at 48 weeks, in contrast to males only at 24 weeks. Activated T cells from 48-week-old prenatal TCDD females over-produced the pro-inflammatory cytokine IFN-γ while males over-produced IL-10, effects again not seen at 24 weeks. Splenic transitional-2 B cells (CD21(int)CD24(hi)) were increased in males while transitional-1 B cells (CD23(neg) CD1(neg)) were increased in females at 48 weeks. Autoantibodies to cardiolipin and CD138(+) spleen plasma cells were significantly increased in the aged males but not females. Anti-IgG and anti-C3 immune complex renal deposition were also significantly increased in the prenatal TCDD males but not females. These selective changes in the aged male mice may be noteworthy, in that the prevalence of SLE in humans shifts dramatically toward males with aging. The collective findings in aged mice suggest that prenatal TCDD permanently biases the postnatal immune response in C57BL/6 mice toward autoimmunity, and support a significant B cell component to the induced renal autoimmune disease.

Influence of age on surfactant isolated from healthy horses maintained on pasture.

BACKGROUND: Surfactant alterations are described in horses after exercise, anesthesia, and prolonged transport, in horses with recurrent airway obstruction, and in neonatal foals. The effect of horse age or bronchoalveolar lavage fluid (BALF) sample characteristics on surfactant is unknown. OBJECTIVES: To evaluate surfactant phospholipid composition and function in healthy horses, and to investigate the influence of age and BALF sample characteristics on surfactant. ANIMALS: Seventeen healthy horses 6-25 years of age maintained on pasture year-round. METHODS: BALF was collected by standard procedures and was assessed for recovery volume, nucleated cell count (NCC), and cytology. Cell-free BALF was separated into crude surfactant pellet (CSP) and surfactant supernatant (Supe) by ultracentrifugation. Phospholipid and protein content were determined from both fractions. CSP phospholipid composition was analyzed by high-performance liquid chromatography with an evaporative light scatter detector. Surface tension of CSP was evaluated with a pulsating bubble surfactometer. Regression analysis was used to evaluate associations between age, BALF sample characteristics, and surfactant variables. RESULTS: Results and conclusions were derived from 15 horses. Increasing age was associated with decreased phospholipid content in CSP but not Supe. Age did not affect protein content of CSP or Supe, or surfactant phospholipid composition or function. Age-related surfactant changes were unaffected by BALF recovery percentage, NCC, and cytological profile. CONCLUSIONS AND CLINICAL IMPORTANCE: Older horses have decreased surfactant phospholipid content, which might be because of age-related pulmonary changes. Surfactant composition is unaffected by BALF sample characteristics at a BALF recovery percentage of at least 50%.

Role of lung surfactant in respiratory disease: current knowledge in large animal medicine.

Lung surfactant is produced by type II alveolar cells as a mixture of phospholipids, surfactant proteins, and neutral lipids. Surfactant lowers alveolar surface tension and is crucial for the prevention of alveolar collapse. In addition, surfactant contributes to smaller airway patency and improves mucociliary clearance. Surfactant-specific proteins are part of the innate immune defense mechanisms of the lung. Lung surfactant alterations have been described in a number of respiratory diseases. Surfactant deficiency (quantitative deficit of surfactant) in premature animals causes neonatal respiratory distress syndrome. Surfactant dysfunction (qualitative changes in surfactant) has been implicated in the pathophysiology of acute respiratory distress syndrome and asthma. Analysis of surfactant from amniotic fluid allows assessment of fetal lung maturity (FLM) in the human fetus and exogenous surfactant replacement therapy is part of the standard care in premature human infants. In contrast to human medicine, use and success of FLM testing or surfactant replacement therapy remain limited in veterinary medicine. Lung surfactant has been studied in large animal models of human disease. However, only a few reports exist on lung surfactant alterations in naturally occurring respiratory disease in large animals. This article gives a general review on the role of lung surfactant in respiratory disease followed by an overview of our current knowledge on surfactant in large animal veterinary medicine.

Viability and function in lymphocytes cultured from the horse, chicken, and mouse: effects of different leukocyte enrichment techniques.

Methods of lymphocyte enrichment tend to vary across species, with the most common techniques employed being density-gradient separation and erythrocyte lysis buffer enrichment. In this study, we assessed lymphocyte viability and proliferation of avian, equine, and murine lymphocytes enriched by a commercial density-gradient technique and under identical, standardized culture conditions. The results of this study clearly show that, under identical enrichment and culture conditions, lymphocyte viability and function can be quite different among the equine, bird, and mouse species. Secondly, the type of enrichment technique employed in the mouse can impact the quality of the immune data generated.

An enhanced postnatal autoimmune profile in 24 week-old C57BL/6 mice developmentally exposed to TCDD.

Developmental exposure of mice to the environmental contaminant and AhR agonist, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), causes persistent postnatal suppression of T cell-mediated immune responses. The extent to which prenatal TCDD may induce or exacerbate postnatal autoimmune disease remains unknown. In the present study, time-pregnant high affinity AhR C57BL/6 mice received a single oral administration of 0, 2.5, or 5 microg/kg TCDD on gestation day (gd) 12. Offspring of these mice (n=5/gender/treatment) were evaluated at 24 weeks-of-age and showed considerable immune dysregulation that was often gender-specific. Decreased thymic weight and percentages of CD4(+)CD8(+) thymocytes, and increased CD4(+)CD8(-) thymocytes, were present in the female but not male offspring. Males but not females showed decreased CD4(-)CD8(+) T cells, and increased Vbeta3(+) and Vbeta17a(+) T cells, in the spleen. Males but not females also showed increased percentages of bone marrow CD24(-)B220(+) B cell progenitors. Antibody titers to dsDNA, ssDNA and cardiolipin displayed increasing trends in both male and female mice, reaching significance for anti-dsDNA in both genders and for ssDNA in males at 5 microg/kg TCDD. Immunofluorescent staining of IgG and C3 deposition in kidney glomeruli increased in both genders of prenatal TCDD-exposed mice, suggestive of early stages of autoimmune glomerulonephritis. Collectively, these results show that exposure to TCDD during immune system development causes persistent humoral immune dysregulation as well as altered cell-mediated responses, and induces an adult profile of changes suggestive of increased risk for autoimmune disease.

Impact of different cell isolation techniques on lymphocyte viability and function.

The outcome of immunological assays is markedly influenced by the method of isolation of lymphocytes. It is, therefore, important to comparatively assess various techniques of isolation of lymphocytes, an aspect thus far not thoroughly addressed. In particular, the potential of isolation techniques to influence cell recovery, viability, and function has not yet been evaluated. These studies were designed to determine the effect of different mechanical tissue dissociation methods on the viability and function of lymphocytes. Following spleen and thymus removal, the lymphoid organs were dissociated by one of four different tissue dissociation techniques: metallic screen, sheer force slide, commercial stomacher, or plunger-screen. Cells were then enumerated and a trypan blue exclusion technique and 7-amino-actinomycin D (7-AAD) were both employed to assess viability. Mitogen-induced lymphocyte proliferation was measured using the Alamar Blue assay. Cell viability and lymphocyte surface antigen expression were assessed using flow cytometry. No significant differences in lymphocyte viability, morphology, or surface antigen expression were observed among the different techniques. Likewise, cellular apoptosis and necrosis were comparable across all the techniques. However, mitogen induced splenic T-cell proliferation was higher in cells collected using the metallic screen and plunger-screen isolation methods as compared to the sheer force slide or commercial stomacher procedures. These data suggest that cell recovery, morphology, and viability are not affected by isolation techniques. However, lymphocyte function, as assessed by mitogen induced proliferation, was negatively affected by the sheer force slide or commercial stomacher isolation techniques.