Evaluation of some acute phase proteins in cattle naturally infected with Babesia bigemina

Bovine babesiosis due to Babesia bigemina infection induces systemic inflammation, evidenced by increased sialic acid (SA) levels and declined cholinesterase activity. The current study was undertaken to assess further indicators of the systemic inflammation in the naturally infected cattle. To this end, serum levels of some selected acute phase-proteins (APPs) including serum amyloid A (SAA), haptoglobin (Hp), ceruloplasmin (Cp), and fibrinogen were measured. Additionally, sensitivity and specificity of the APPs were calculated by receiver operating characteristic curve. The correlation among APPs, SA and cholinesterase activity were also assessed. Our previous blood specimens were used to measure APPs. Briefly, the diseased animals were divided into two groups according to the parasitemia: 12 mildly (20 % <) and 8 severely (20 %>) infected animals. Moreover, 10 healthy animals as the control were included. The levels of all APPs were measured to be significantly elevated in a parasitemia burden-dependent fashion as compared to the control. Furthermore, all the APPs showed 100 % specificity, but only SAA and Cp had 100 % sensitivity. A strong and positive correlation was calculated between the APPs and SA; however, cholinesterase activity was inversely correlated with AAPs and SA. In conclusion, inflammatory reactions play a pivotal role in the pathogenesis of bovine babesiosis and APPs can be considered as the potential indicators of inflammation.     

Frozen and fresh Anaplasma centrale vaccines in the protection of cattle against Anaplasma marginale infection

The immunity induced by frozen and fresh Anaplasma centrale vaccines against anaplasmosis caused by A. marginale was tested in 12-month old Friesian steers. A. centrale parasitaemia occurred in all cattle inoculated with both types of vaccine. The average maximal decrease in PCV for the frozen and fresh vaccines was 41.0 and 40.3% respectively. All cattle recovered spontaneously. Vaccinated and control steers of the same age were challenged six months later with doses of 10(6), 10(7) or 10(8) A. marginale organisms. Vaccinated cattle showed average maximal A. marginale parasitemia of 1.2-4.0 versus 10.3-12.0% in control cattle. The average maximal decrease in packed cell volume (PCV) was 33.1 and 30.0% for steers vaccinated with frozen or fresh vaccine, respectively, and 57.4% for the non-vaccinated steers. All vaccinated cattle recovered spontaneously from the A. marginale infection while 7 out of 8 control steers required specific treatment. It thus appears that both frozen and fresh A. centrale vaccines are equally capable of inducing partial protection against infection with A. marginale and of preventing severe red blood cell destruction.     

Vaccination of cattle with Anaplasma marginale derived from tick cell culture and bovine erythrocytes followed by challenge-exposure with infected ticks

Anaplasmosis, a hemolytic disease of cattle caused by the tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been controlled using killed vaccines made with antigen harvested from infected bovine erythrocytes. We recently developed a cell culture system for propagation of A. marginale in a continuous tick cell line. In this study, we performed a cattle trial to compare the bovine response to vaccination with A. marginale harvested from tick cell culture or bovine erythrocytes. All immunized and control cattle were then challenge-exposed by allowing male Dermacentor variabilis infected with A. marginale to feed and transmit the pathogen. Nine yearling cattle (three per group) were used for this study and were immunized with cell culture-derived A. marginale, erythrocyte-derived A. marginale or received adjuvant only to serve as controls. Each vaccine dose contained approximately 2 x 10(10) A. marginale and three immunizations were administered at weeks 1, 4 and 6. At week 8, cattle were challenge-exposed by allowing 60 D. variabilis male that were infected with A. marginale as adults to feed on the cattle. Antibody responses of cattle against major surface proteins (MSP) 1a, 1b and 5, as determined by ELISAs, peaked 2 weeks after the last immunization. Cattle immunized with infected IDE8 cell-derived antigens had a preferential recognition for MSP1b while cattle immunized with erythrocyte-derived antigens had a preferential recognition for MSP1a. Protection efficacy was evaluated using the percent infected erythrocytes (PPE), the packed cell volume (PCV), and the prepatent period. A. marginale-immunized cattle showed lower PPE and higher PCV values when compared to control animals and did not display clinical anaplasmosis. The cell culture-derived A. marginale shows promise for use as antigen in development of a new killed vaccine for anaplasmosis.     

Serologic and clinical responses of premunized, vaccinated, and previously infected cattle to challenge exposure by two different Anaplasma marginale isolates

Two Anaplasma marginale isolates, one originating in Florida (FAM) and the other from Virginia (VAM), were compared immunologically by cross-challenge exposure of 14 Anaplasma carrier cattle, 8 previously infected cattle, and 6 splenectomized carrier calves. In addition, 28 cattle vaccinated with a commercially available adjuvant killed vaccine and 22 nonvaccinated cattle were challenge exposed with either FAM or VAM. A detectable clinical response was not produced by either FAM or VAM challenge exposure in carrier and previously infected cattle; however, evidence of A marginale growth as characterized by low percentages of parasitemia and increased serum complement-fixation titers was seen in carrier cattle given a heterologous challenge organism and in previously infected cattle inoculated with either homologous or heterologous organisms. Among splenectomized calves, there was virtually no cross protection to the heterologous challenge exposure, whereas a homologous challenge failed to elicit any detectable response. Vaccinated cattle were resistant to VAM exposure, but the clinical response to FAM exposure was severe with a 47% mortality. Most of these cattle displayed typical acute anaplasmosis that was only marginally less severe than that encountered in nonvaccinated cattle.     

Response of cattle upon reexposure to Anaplasma marginale after elimination of chronic carrier infections

Sixteen cattle serotest-negative for anaplasmosis with either no previous exposure (2 animals) or cleared 8 months earlier of their carrier state by chemotherapy (14 animals) were each exposed to Anaplasma marginale. Anaplasma serotest titers were determined by complement-fixation and rapid card agglutination tests conducted during a 63-day trial period. Serologic reactions indicated that all cattle (both groups) were converted to seropositive by the 21st day after exposure. Fluctuations in PCV were seen in the 2 groups between days 21 and 35. However, parasitemia levels were detectable only in the 2 previously unexposed control cattle. Three splenectomized calves, given 10 ml of blood from 3 of the former carrier cattle 14 days after the latter were reexposed, developed severe clinical and hematologic signs of anaplasmosis and seroconverted from negative to positive on both serologic tests. The need to acquire a better understanding of immunity in anaplasmosis is discussed.     

Establishment and characterization of an Oklahoma isolate of Anaplasma marginale in cultured Ixodes scapularis cells

Anaplasma marginale is a tick-borne hemoparasite of cattle worldwide. The Virginia isolate of A. marginale was propagated previously in a cell line derived from embryos of the tick, Ixodes scapularis. The cultured Anaplasma (VA-tc) was passaged continuously for over 4 years and retained its infectivity for cattle and antigenic stability. We report herein the continuous in vitro cultivation of a second isolate of A. marginale derived from a naturally infected cow in Oklahoma (OK-tc). Blood from the infected cow was subinoculated into a splenectomized calf and blood collected at peak parasitemia was frozen, thawed and used as inoculum on confluent tick cell monolayers. Colonies of Anaplasma were apparent in low numbers at 9 days post exposure (PE) and infection in monolayers reached 100% by 4-5 weeks PE. Cultures were passaged by placing supernatant onto fresh tick cell monolayers at a dilution of 1:5 or 1:10. By the third passage development of the OK-tc was similar to that of the VA-tc and a 1:5 dilution resulted in 100% infection in 10-12 days. Inoculation of OK-tc into a splenectomized calf caused clinical anaplasmosis and Dermacentor ticks that fed on this calf transmitted the organism to a second susceptible calf. Major surface proteins (MSPs) 1-5 of the OK-tc were compared with homologous proteins present on VA-tc and the erythrocytic stage of the Oklahoma isolate. The MSPs 1, 2, 4, 5 were conserved on the OK-tc but there was evidence for structural variation in MSP3 between the cultured and erythrocytic stage of Anaplasma. MSP2 and MSP3 were the major proteins recognized by serum from infected cattle. Two-dimensional gels also identified positional differences between VA-tc and OK-tc in MSP2 and MSP3. The OK-tc may have potential to be used as antigen for development of an improved vaccine for anaplasmosis in the South Central United States.     

Molecular and serological detection of A. centrale- and A. marginale-infected cattle grazing within an endemic area

A reverse line blot hybridization (RLB) one-stage nested PCR (nPCR) for Anaplasma centrale and a nested PCR for Anaplasma marginale were used to detect infected cattle grazing within an endemic region in Israel. A novel set of PCR primers and oligonucleotide probes based on a 16S ribosomal RNA gene was designed for RLB detection of both Anaplasma species, and the performance of the molecular assays compared. The immunofluorescent antibody test (IFA) was used to detect antibodies to both Anaplasma species, whereas, a highly sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) was used to detect antibodies in A. centrale-vaccinated cattle. The RLB and the nested PCR procedures showed bacteremia with sensitivity of 50 infected erythrocytes per milliliter. Up to 93% of the A. centrale vaccinates carried specific antibodies that were detected by cELISA, and up to 71% of the vaccinated cattle were found to be naturally infected with A. marginale according to the PCR and the RLB assays. Nevertheless, no severe outbreaks of A. marginale infection occurred among vaccinated herds in this endemic region. It appears that both, molecular tools and serology are useful for evaluation of the vaccine efficacy. In the light of wide natural field infection with A. marginale, strong recommendations to continue the A. centrale vaccination program regime will continue until a new generation of non-blood-based vaccine will be developed.     

The correlations among serum tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and sialic acids with peripheral lymphocytes in bovine tropical theileriosis

The infection with protozoan parasite Theileria annulata induces changes triggering the activation and/or proliferation of the host lymphocytes. In order to find out the possible correlations among peripheral circulatory lymphocytes, cytokine activities and the level of sialic acids, 50 dairy Holstein cattle, naturally infected with T. annulata, were divided into 4 subgroups according to their parasitemia rates (<1%, 1–3%, 3–5% and >5%). Also, ten non-infected cattle were sampled as control group. Blood samples were taken from jugular vein into acid citrate dextrose-containing tubes for measuring hematological parameters and B and T (CD₄ and CD₈) cell populations and without anticoagulant for TNF-α, IFN-γ and sialic acid concentrations. Remarkable decreases observed in red blood cells (RBCs), white blood cells (WBCs) and packed cell volume (PCV) in infected cattle compared to healthy ones (P < 0.05). Also, with increase in parasitemia rate, total lymphocytes and monocytes alleviated in the diseased groups. By contrast, total neutrohpils and the concentrations of TNF-α, IFN-γ and total sialic acids were significantly elevated (P < 0.05) in infected animals. Accordingly, the circulatory populations of CD₄ and CD₈ T cells and B cells showed a substantial decrease, while a significant increase was observed in T (CD₄ and CD₈) cells in cattle infected with <1% parasitemia rates. Decreased circulatory T cell population shows the ineffective responses of T cells to the stimulatory cytokines such as IFN-γ or TNF-α. On the other hand, the elevation of cytokines (particularly IFN-γ) and sialic acids have presumably an inhibitory role on circulatory B cell population in infected cattle. In addition, a high level of sialic acid concentration indicates the probable role of sialic acid to regulate the parasite-host cell adhesion during sporozoites invasion.     

Bovine anaplasmosis: susceptibility of seronegative cows from an infected herd to experimental infection with Anaplasma marginale

Adult cows from an Anaplasma marginale-infected herd that were negative to the A marginale rapid card agglutination (RCA) and complement fixation (CF) tests for 1 to 4 years developed acute anaplasmosis after inoculation with 0.5 ml of blood from an A marginale carrier cow. The test cattle were as susceptible as the control cattle of similar ages. Also, 2 cows that had seroconverted from RCA/CF-positive to RCA/CF-negative status naturally were fully susceptible to anaplasmosis when they were experimentally infected. Results of the study indicated that indigenous seronegative cattle in anaplasmosis-enzootic regions probably do not have acquired or natural immunity to A marginale infection.     

Development of an ELISA system for detection of anti-Anaplasma marginale antibodies in cattle in Brazil

An ELISA test was developed for detecting antibodies against Anaplasma marginale in bovine sera. Four antigenic preparations were produced from infected red blood cells. Some aliquots of this preparation were stored at -70 degrees C with 30% DMSO in phosphate-buffered saline (PBS) and others were lysed with 0.9% NH4Cl and stored at -20 degrees C. Typical anaplasmal structures were seen by electron microscopy in the antigenic preparations containing the erythrocytes that had been stored with DMSO. The performance of the ELISA test was evaluated by testing 298 positive serum samples collected from immunized cattle, 39 negative serum samples collected from cattle imported from areas free of A. marginale and 50 samples collected from cattle naturally infected in the field. The test gave a specificity of 94.87% and a sensitivity of 100%.     

Evaluation of cytokines and sialic acids contents in horses naturally infected with Theileria equi

This study was undertaken to assess the effects of T. equi infection on serum concentrations of some important cytokines including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-1 beta (IL-1β), IL-1α, IL-4, IL-6, IL-8, IL-10, IL-12α, IL-12β, IL-18, as well as total, protein and lipid binding sialic acids (TSA, PBSA and LBSA). Furthermore, any probable relation among the parasitemia, cytokines and sialic acids (SAs) were calculated using Pearson correlation and simple linear regression. Almost 300 draft horses (Kurdish-breed) with age of 3–4 years old from north-west of Iran were examined and an infected group comprised of 28 mares, naturally infected with T. equi, was identified and divided into 3 subgroups according to their parasitemia rates (low <1 %, moderate 1–3 % and high 3–5 %). Twenty healthy horses were considered as a control. Characterization and differentiation of piroplasmosis were conducted using routine hematological procedures and specific PCR assay. The results revealed a significant increase (P < 0.05) in all of the cytokines and SAs in a parasitic burden-dependent fashion. Additionally, a strong and positive relation was detected among the parasitemia, cytokines and SAs. Conclusively, T. equi infection is associated with induction of severe inflammatory processes in horses and SA plays a pivotal role in pathophysiology of the disease as it is tightly correlated with the parasitemia rate.     

Radioimmunoassay for Anaplasma marginale antibodies in cattle

A radioimmunoassay is described for use in the detection of Anaplasma marginale antibodies in cattle sera. Optimal sensitivity and specificity were obtained by using 2 antigens, an A marginale antigen and a RBC antigen (obtained before infection was established) from the same calf. In addition, sera were preabsorbed with RBC from healthy cattle and with sonicated Babesia bovis. Of 86 sera obtained from cattle with A marginale infection (as determined by blood smear examination or by results of subinoculation of blood from such infected cattle into splenectomized calves), 85 had positive results by use of this test. Of 100 sera obtained from cattle raised in an anaplasmosis-free area, 98 yielded negative results, and sera obtained from 35 cattle (97 sera) infected with B bigemina and from 18 cattle infected with Theileria orientalis yielded negative results. By use of this test, 99 of 100 sera obtained from cattle with B bovis infection were negative for A marginale. Anaplasma marginale antibodies were detected in 18 cattle that had been pastured in a Boophilus microplus-free area for 2 years after natural infection. After 3 years, 16 of these cattle were still seropositive for A marginale. Sixteen cattle pastured in a Bo microplus-infested area had detectable antibody against A marginale 27 months after initial infection with A marginale. Sensitivity and specificity of the test were assessed as 98.8% for each.     

Acute phase proteins response in cats naturally infected by hemotropic mycoplasmas

Information about the acute phase proteins (APP) response in cats naturally infected with hemoplasmas and in cats co-infected with different species of hemoplasmas is lacking.This study evaluated serum amyloid A (SAA), haptoglobin (Hp) and albumin in 48 cats naturally infected with hemoplasmas, including 25 with Candidatus Mycoplasma haemominutum and 23 co-infected with different hemoplasmas agents; and in 10 healthy control cats.Infected cats had significantly higher Hp and lower albumin than controls. Symptomatic cats had significantly higher SAA and Hp, and lower albumin than asymptomatic animals, and also than controls. Asymptomatic cats had significantly higher Hp than controls. Concentrations of APP were not significantly different between single infected and co-infected cats.According with these results, hemoplasmosis should be considered when alterations in APP are detected in diseased cats with compatible clinical signs. Furthermore, a subclinical infection should be considered in apparently healthy cats from endemic areas with increased Hp.     

Transmission of Anaplasma marginale Theiler by males of Dermacentor andersoni Stiles fed on an Idaho field-infected, chronic carrier cow

The role of ticks and carrier cattle in epizootics of bovine anaplasmosis was further clarified by demonstrating unequivocally, for the first time, that male ticks fed on a chronic carrier cow naturally infected with Anaplasma marginale can transmit this parasite intrastadially and biologically when subsequently fed on susceptible cattle. These data indicate that field epizootics of acute anaplasmosis may be initiated by males of tick vector species that feed on carrier cattle and subsequently transfer to susceptible cattle.     

Evaluation of antioxidant status and oxidative stress in sheep naturally infected with Babesia ovis

The present study aimed to assess antioxidant status and oxidative stress in sheep naturally infected with Babesia ovis. Red blood cell (RBC) count, hemoglobin (Hb) concentration, packed cell volume (PCV), piroplasm parasitemia percentage, malondialdehyde (MDA) concentration, erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activities and total antioxidant capacity (TAC) were determined in 52 sheep naturally infected with B. ovis as well as same number of healthy sheep in West-Azerbaijan province, Iran. Microscopic examination of Giemsa-stained peripheral blood smears revealed B. ovis infection. The parasitological diagnosis was confirmed using polymerase chain reaction (PCR) analysis by amplifying a partial small subunit ribosomal RNA (ssu rRNA) gene sequence of B. ovis of 52 diseased sheep, 18 (34.61%), 11 (21.15%), 16 (30.76%) and 7 (13.48%) had <1%, 1-2%, 2-3% and >3% parasitemia, respectively. Compared to controls, the activities of erythrocyte GSH-Px, SOD, TAC and CAT showed a significant decrease, whereas the concentration of MDA in erythrocytes of infected sheep increased significantly. Parasitemia rate was positively correlated with MDA and negatively correlated with PCV, SOD, CAT, GSH-Px and TAC. Also, MDA was negatively correlated with PCV, SOD, catalase, GSH-Px and TAC. The study demonstrated that B. ovis plays an important role in the occurrence of oxidative damage to RBCs and anemia in ovine babesiosis.     

Antibodies to Anaplasma marginale major surface proteins 1a and 1b inhibit infectivity for cultured tick cells

Major surface protein 1 (MSP1) of the cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) is a complex of two proteins, MSP1a and MSP1b. Previous studies demonstrated that MSP1a and MSP1b are adhesins for bovine erythrocytes, while only MSP1a proved to be an adhesin for tick cells. In this study, a tick cell culture system for propagation of A. marginale was used to develop an infection inhibition assay for testing the ability of antisera to block infection of A. marginale for cultured tick cells. A. marginale derived from cell culture was incubated with various antisera prior to inoculation onto cell monolayers. The monolayers were harvested 7 days post-inoculation and A. marginale in the cultures was quantified using an antigen detection ELISA. Antisera tested in the infection inhibition assay were derived from persistently infected cattle, from cattle immunized with A. marginale purified from bovine erythrocytes, and from rabbits and cattle that were immunized with the recombinant MSP1a, MSP1b and MSP1 complex. Antibodies from cattle persistently infected with A. marginale, cattle immunized with A. marginale from bovine erythrocytes or cattle immunized with the recombinant MSP1 complex did not inhibit the infectivity of A. marginale for tick cells. Antiserum from rabbits immunized with MSP1a and MSP1b (individually or combined) reduced infection of both the Virginia and Oklahoma isolates of A. marginale for tick cells by 25-70%. Likewise, antisera from cattle immunized with recombinant MSP1a or MSP1b inhibited infection of tick cells by 26-37%. These results further confirm the role of MSP1 complex proteins in infection of tick cells. Lack of inhibition of infection by antisera from naturally infected cattle or cattle immunized with whole organisms suggests that the bovine immune response is not directed toward blocking infection of A. marginale for tick cells and may contribute to the continued infectivity of the pathogen for ticks.     

Sensitivity and specificity of the complement fixation test for detection of cattle persistently infected with Anaplasma marginale.

The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs.     

Cytokine gene expression profiles in peripheral blood mononuclear cells from Neospora caninum naturally infected dams throughout gestation

Neospora caninum is a major cause of abortion in cattle but it is not known why some infected animals suffer abortion while others do not. An essential role in protective immunity against N. caninum has been proposed for Th1 cytokines such as IFN-γ and IL-12 although cytokine patterns in N. caninum infected pregnant cattle have been scarcely addressed. In this study, gene expression of the cytokines IFN-γ, IL-12, IL-10, IL-4 and TNF-α was analyzed by real time RT-PCR in peripheral blood mononuclear cells in N. caninum naturally infected dams throughout pregnancy. Blood samples were drawn from 18 cows (13 N. caninum seropositive and 5 N. caninum seronegative) on Days 45, 90, 120, 150, 180 and 210 of pregnancy or until abortion. Four seropositive animals aborted. Compared to the seronegative animals, N. caninum infected dams showed up-regulated mRNA levels of the Th1 cytokines, IFN-γ, TNF-α and IL-12p40, along with up-regulation of the T regulatory (Treg) cytokine IL-10. In contrast, expression levels of IL-4 (Th2 cytokine) did not differ significantly among the different groups throughout the study period. Our findings indicate clear differences in peripheral blood cytokine gene expression levels during pregnancy between animals naturally infected with N. caninum and seronegative control animals. To the best of our knowledge, this is the first study to examine the gene expression of Th1, Th2 and regulatory cytokines in the peripheral blood of pregnant cows naturally infected with N. caninum.     

Characteristics of an attenuatedAnaplasma marginale of deer origin as an anaplasmosis vaccine

A fully virulent field Anaplasma marginale isolate, passaged to a splenectomised white tailed deer, lost virulence for cattle after 18 months in the non-bovine host. This attenuated A. marginale produced a mild response in four 18-month-old Holstein steers characterised by an average 2.4% parasitaemia, a 13% drop in PCV and a positive serological response. All vaccinated steers were immune to challenge with a fully virulent, heterologous A. marginale when compared with non-vaccinated controls.     

Concomitant infection of cattle with the vaccine strain Anaplasma marginale ss centrale and field strains of A. marginale

Bovine anaplasmosis, caused by Anaplasma marginale, the intraerythrocytic rickettsia, is controlled by vaccination with live Anaplasma marginale ss centrale (A. centrale), a subspecies of relatively low pathogenicity. We have experimentally demonstrated that an animal primarily infected with A. marginale, or with the related vaccine subspecies A. centrale can be infected with the heterologous subspecies, and carries both bacteria. The co-infection was detected in experimentally cross-infected calves for up to 3 months after the last inoculation with the heterologous subspecies. The occurrence of characteristic cyclic rickettsemia of A. centrale and A. marginale was observed by examination of Giemsa-stained blood smears, or by the presence of specific rickettsial DNA confirmed in PCR assays based on specific msp1a and msp4 for A. marginale, and on specifically designed msp3 and msp4 primers for A. centrale. Sequence analysis of msp4-specific fragments for each subspecies revealed the presence of dual infection in both calves on days 30 and 60 after cross-inoculation with the heterologous Anaplasma subspecies. The experimental cross-infection of calves clearly demonstrated that the concept of "infection exclusion" does not apply to Anaplasma infection in cattle; as there was no infection exclusion of A. marginale in A. centrale-infected cattle, and vice versa. The present results confirmed our previous findings that cattle grazing in an anaplasmosis-endemic field were subject to concomitant infection with both the vaccine A. centrale and the field A. marginale strains.