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Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314. A pair of mAb (CC313 and CC314) was identified that together could be used to detect both recombinant and native bovine IL-4 by ELISA and a luminometric detection method was applied to the ELISA. Using this method native bovine IL-4 was detected in supernatants of PBMC stimulated with mitogens. In addition, high level secretion of IL-4 by Fasciola hepatica specific Th2 clones, but not by a Babesia bovis specific Th1 clone, was confirmed. The ELISA was also able to detect recombinant ovine IL-4. The pair of mAb used for ELISA could also be used for the detection of IL-4 spot forming cells by ELISPOT. In addition intracytoplasmic expression of IL-4 could be detected. The ability to detect ruminant IL-4 by three methods: ELISA, ELISPOT and by flow cytometric analysis of intracytoplasmic expression will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

For many vector-transmitted protozoal parasites, immunological control of acute infection leads to a state of persistent infection during which parasitemias may cycle unnoticed in infected but otherwise clinically healthy animals. Achieving persistent infection is a strategy that favors parasitism, since both host and, therefore, parasite survive, and endemically infected animal populations provide a reservoir of parasites continually available for subsequent transmission. Examples of the major economically important protozoan pathogens that cause persistent infection in mammals include the related Theileria and Babesia parasites as well as Trypanosoma species. Control of acute infection and maintenance of clinical immunity against subsequent infection are determined by the interplay of innate and acquired immune responses. This review will focus on approaches taken to gain an understanding of the molecular basis for innate and acquired immunity against the hemoprotozoan parasite of cattle, Babesia bovis. Knowledge of mechanisms used by the parasite to survive within infected cattle from acute to persistent infection combined with definition of the correlates of protective immunity in cattle should be applicable to designing effective vaccines.

Tick-borne transmission of ehrlichial pathogens requires rickettsemic reservoir hosts to maintain a population of infected vectors. Persistence in their respective mammalian hosts appears to be a common feature of the tick-transmitted ehrlichiae. How infection persists in immunocompetent hosts is unknown. In this review, we describe studies on Anaplasma marginale, an ehrlichial pathogen of cattle, that support antigenic variation as a primary mechanism of persistence.

Fasciolosis is an economically significant disease of ruminants, caused by infection with the digenetic trematodes, Fasciola hepatica and F. gigantica. Some vaccination trials using irradiated metacercariae or isolated proteins have been shown to afford significant protection. However, the mechanisms of specific immunity against this pathogen have not been elucidated. We have identified thioredoxin, a tegument antigen of F. hepatica, among several proteins that are common to both the juvenile and adult fluke within the mammalian host and have undertaken studies to characterize bovine T cell responses to recombinant thioredoxin protein (FH 2020). Peripheral blood mononuclear cells from immune cattle proliferated specifically to crude F. hepatica antigenic extract but not to FH 2020. However, after repeated stimulation of lymphocytes by alternating crude extract and FH 2020, FH 2020-specific proliferation by T cell lines was observed. T cell clones were subsequently generated and found to respond specifically but weakly to both crude antigen and FH 2020. Thioredoxin appears to be only weakly antigenic for bovine T cells and is, therefore, an unpromising candidate for inducing resistance to F. hepatica.

Interleukin 4 (IL-4) is a pleotropic cytokine affecting a wide range of cell types in both the mouse and the human. These activities include regulation of the growth and differentiation of both T and B lymphocytes. The activities of IL-4 in nonprimate, nonmurine systems are not well established. Herein, we demonstrate in the bovine system that IL-4 upregulates production of IgM, IgG1, and IgE in the presence of a variety of costimulators including anti-IgM, Staphylococcus aureus cowan strain I, and pokeweed mitogen. IgE responses are potentiated by the addition of IL-2 to IL-4. Culture of bovine B lymphocytes with IL-4 in the absence of additional costimulators resulted in the increased surface expression of CD23 (low-affinity Fc epsilon RII), IgM, IL-2R, and MHC class II in a dose-dependent manner. IL-4 alone increased basal levels of proliferation of bulk peripheral blood mononuclear cells but in the presence of Con A inhibited proliferation. In contrast to the activities of IL-4 in the murine system, proliferation of TH1- and TH2-like clones was inhibited in a dose-dependent manner as assessed by antigen-or IL-2-driven in vitro proliferative responses. These observations are consistent with the role of IL-4 as a key player in regulation of both T and B cell responses.

Ulnar artery aneurysms and pseudoaneurysms are rare lesions that usually occur distal to the wrist and cause symptoms as a result of embolization and not rupture. An elderly woman presented with acute rupture of an ulnar artery pseudoaneurysm proximal to the wrist, which caused severe neurologic compromise as a result of bleeding into Guyon's canal and the carpal tunnel. The patient had a remote wrist fracture resulting in a deformity that allowed the ulnar head to be in proximity to the ulnar artery. Rupture of the volar capsule with chronic abrasion of the artery eventually led to pseudoaneurysm formation and subsequent rupture. Emergency operative treatment with excision of the pseudoaneurysm, ulnar artery ligation, and repair of the volar capsule resulted in complete neurologic recovery without vascular compromise. This case is unique because of the proximal location of the pseudoaneurysm and the presentation with rupture. The anatomy, pathogenesis, and treatment options are discussed. Excision of the pseudoaneurysm with repair of the torn volar capsule is recommended to avoid recurrent problems. Vascular reconstruction is needed in cases where radial arterial flow is inadequate.

Current vaccines for bovine hemoparasites utilize live attenuated organisms or virulent organisms administered concurrently with antiparasitic drugs. Although such vaccines can be effective, for most hemoparasites the mechanisms of acquired resistance to challenge infection with heterologous parasite isolates have not been clearly defined. Selection of potentially protective antigens has traditionally made use of antibodies to identify immunodominant proteins. However, numerous studies have indicated that induction of high antibody titers neither predicts the ability of an antigen to confer protective immunity nor correlates with protection. Because successful parasites have evolved antibody evasion tactics, alternative strategies to identify protective immunogens should be used. Through the elaboration of cytokines, T helper 1-(Th1)-like T cells and macrophages mediate protective immunity against many intracellular parasites, and therefore most likely play an important role in protective immunity against bovine hemoparasites. CD4+ T cell clones specific for soluble or membrane antigens of either Theileria parva schizonts or Babesia bovis merozoites were therefore employed to identify parasite antigens that elicit strong Th cell responses in vitro. Soluble cytosolic parasite antigen was fractionated by gel filtration, anion exchange chromatography or hydroxylapatite chromatography, or a combination thereof, and fractions were tested for the ability to induce proliferation of Th cell clones. This procedure enabled the identification of stimulatory fractions containing T. parva proteins of approximately 10 and 24 kDa. Antisera raised against the purified 24 kDa band reacted with a native schizont protein of approximately 30 kDa. Babesia bovis-specific Th cell clones tested against fractionated soluble Babesia bovis merozoite antigen revealed the presence of at least five distinct antigenic epitopes. Proteins separated by gel filtration revealed four patterns of reactivity, and proteins separated by anion exchange revealed two patterns of reactivity when selected T cell clones were assayed for stimulation by antigenic fractions. Studies using a continuous-flow electrophoresis apparatus have indicated the feasibility of identifying T cell-stimulatory proteins from parasite membranes as well as from the cytosolic fraction of B. bovis merozoites. The Th cell clones reactive with these different hemoparasites expressed either unrestricted or Th1 cytokine profiles, and were generally characterized by the production of high levels of IFN-gamma. A comprehensive study of T cell and macrophage responses to defined parasite antigens will help elucidate the reasons for vaccine failure or success, and provide clues to the mechanisms of acquired immunity that are needed for vaccine development.

Functional studies with WC1+ gamma/delta T cell lines were performed to clarify the role of this subpopulation of gamma/delta T cells in the in vitro immune response to Babesia bovis. As CD4+ T cells decreased and gamma/delta T cells increased in B. bovis-stimulated T cell lines, antigen-specific proliferation declined to background levels. One irradiated gamma/delta T cell line inhibited proliferation of autologous Th1 cells, although unirradiated gamma/delta T cells either synergized with or had no effect on Th cell proliferation. gamma/delta T cells were not cytolytic for bovine alpha/beta T cells, but expressed natural killer (NK)-like cytotoxicity when assayed on xenogeneic NK-sensitive target cells. The gamma/delta T cells were IL-2 dependent and expressed IFN-gamma and TNF-alpha, but not TNF-beta, IL-2, or IL-4 mRNA. Together, these results raise the possibility that WC1+ gamma/delta T cells respond in vitro to autoantigens present on CD4+ T cells or to cytokines secreted by activated CD4+ T cells, resulting in modulation of the CD4+ T cell response and outgrowth of the gamma/delta T cells in parasite-stimulated lines.

This study set out to develop a rapid method for the isolation of schizonts from Theileria parva-infected bovine and buffalo lymphoblastoid cells. Parasitized lymphoblastoid cells were lysed by treatment with the cytolytic toxins, aerolysin and Ah-1 hemolysin, produced by Aeromonas hydrophila, and the schizonts were separated by Percoll density-gradient centrifugation. Light and electron microscopic examination showed that the isolated schizonts from lymphoblastoid cells infected with T. parva (Muguga) retained their normal morphology and were essentially free from host cell components. The schizonts also retained antigens as recognized by a series of anti-schizont monoclonal antibodies. The concentrations of toxin and Ficoll 400 in the lysis buffer which gave optimal cell lysis varied for 10 different infected cell lines tested.

The ankle is the most frequently injured major joint, and ankle sprain is the most frequent sports injury. If the injured ankle is managed correctly from the beginning, pain and swelling and the resultant disability will be kept to a minimum. Early return to participation predisposes the athlete to reinjury. Not only must strength and flexibility be restored, but the athlete must also be pain free and have regained his or her proprioceptive abilities.