Amelioration of virulent Babesia bovis infection in calves by administration of the nitric oxide synthase inhibitor aminoguanidine.

Calves undergoing initial infection with a virulent strain of the haemoprotozoan parasite Babesia bovis were treated with aminoguanidine (AG), an inhibitor of the inducible form of nitric oxide synthase (iNOS). The mean maximum parasitaemia of the AG treated calves was significantly lower than that of the control cattle. In addition, the febrile response and decrease in packed cell volume (PCV) observed during acute infection were significantly ameliorated in the AG treated cattle relative to the controls. However, AG had no effect on the multiplication of B. bovis in the microaerophilous stationary-phase (MASP) in-vitro culture system. These results provide evidence of a role for nitric oxide (NO) produced in response to acute infection in the pathology of bovine babesiosis.

Anaplasma marginale: effect of the treatment of cattle with an interferon gamma-neutralizing monoclonal antibody or the nitric oxide synthetase inhibitor aminoguanidine on the course of infection.

Cattle undergoing initial infection with the rickettsia Anaplasma marginale were treated with either a monoclonal antibody (MoAb) with neutralizing activity for bovine interferon gamma (IFN-gamma) or aminoguanidine (AG), a specific inhibitor of the inducible form of nitric oxide synthetase (iNOS). Plasma levels of MoAb and AG were measured over the time of administration. The course of A. marginale infection was not altered in the MoAb-treated cattle relative to untreated controls. In cattle treated with AG however, A. marginale infection was significantly ameliorated, as judged by lower parasite levels and decreased anaemia in these cattle relative to the controls. The implications of these findings in relation to the basis for immunity against this economically important haemoparasite are discussed.

Increased resistance to Anaplasma marginale infection in cattle chronically infected with Theileria buffeli (syn. T. orientalis).

Calves chronically infected with the benign haemoprotozoan parasite Theileria buffeli (syn. T. orientalis) and T. buffeli-free calves were experimentally infected with virulent Anaplasma marginale. The daily mean maximum parasitaemia in the T. buffeli-carrier calves was lower and delayed relative to that of the Theileria-free calves. Anaemia was less marked in the Theileria infected calves, although this difference was not statistically significant. The susceptibility of Theileria-carrier and Theileria-free older cattle to virulent A. marginale infection was also investigated. The mean maximum parasitaemia observed in the Theileria-infected cattle was significantly lower than that of the Theileria-free cattle and the time to maximum parasitaemia was increased significantly in the Theileria-infected relative to the Theileria-free cattle. Of the Theileria-carrier cattle, 33% exhibited maximum parasitaemias of less than 0.1% infected erythrocytes and no clinical anaemia as a result of A. marginale infection. In contrast, the lowest maximum parasitaemia observed in the Theileria-free cattle was 7%. The percentage of cattle requiring treatment to prevent mortality due to anaemia was 50% and 91% in the Theileria-infected and Theileria-free cattle respectively. For the duration of increasing A. marginale parasitaemia, the level of Theileria in carrier cattle was significantly depressed or undetectable. Following the resolution of peak A. marginale parasitaemia, the level of Theileria parasites increased rapidly to become significantly higher than that prior to infection and then decreased gradually to a level similar to that prior to infection. The mechanism of the increased resistance to A. marginale infection conferred by T. buffeli-carrier state is unknown, but is likely to involve non-specific cell-mediated immunity, as no serological cross-reactivity exists between these two highly divergent parasite species. The susceptibility of relatively mature cattle to clinical anaplasmosis under field conditions is likely to be significantly affected by the widespread distribution and common occurrence of T. buffeli throughout the range of A. marginale in Australia, Africa and southeast Asia.

Vaccination against Babesia bovis: T cells from protected and unprotected animals show different cytokine profiles.

Vaccination of cattle against the haemoprotozoan parasite, Babesia bovis, with the recombinant antigen 11C5 resulted in 9 of 15 cattle being protected against challenge infection. The cellular immune responses of protected and unprotected cattle were compared in order to identify differences in response. No differences were observed in the pattern of change in various blood leukocyte populations throughout challenge infection. FACScan analysis revealed an increase in the proportion of cells bearing the CD2 marker in both protected and unprotected cattle over the course of infection. There were no observable differences in the frequency of various cell-surface markers between the unprotected and protected cattle. During the period of patent parasitaemia, in vitro cultures of peripheral blood mononuclear cells (PBMC) from protected cattle produced significantly more TNF-alpha (P < 0.05) than cultures from unprotected cattle. TNF-alpha concentrations remained at pre-challenge levels until day 10, when levels in the unvaccinated control and vaccinated/unprotected animals dropped. By peak parasitaemia, TNF-alpha production in vitro was significantly greater (P < 0.05) in cultures of PBMCs from protected cattle. Interferon production showed an initial peak at day 5 in all cattle, followed by a decrease and a second peak at days 10-13 in protected cattle only, which coincided with resolution of the infection.

Anaplasma marginale: effect of challenge of cattle with varying doses of infected erythrocytes.

Three groups, each of 6 Hereford cattle, were infected by the i.v. inoculation of 10(10), 10(8) or 10(6) Anaplasma marginale-infected erythrocytes. The mean time taken to reach a 1% parasitaemia was 7.3, 13.9 and 19.9 days in the 10(10), 10(8)and 10(6) infection dose groups, respectively. The rates of increase in parasitaemias during the exponential phase of parasite multiplication were similar for the 3 groups (doubling time 0.9 days). The exponential increase of the parasitaemia in the 10(10) dose group extended to a higher level or 10(6) dose groups (to approximately 10% compared with 3%). The mean maximum parasitaemia attained in the 10(10), 10(8), and 10(6) infection dose groups was 23.7, 14.7 and 8.7%, respectively> The time taken to reach the treatment criterion (packed cell volume decrease to 15% or lower) from a 1% parasitaemia was similar for the 3 groups. These results showed that the pathological outcome (anaemia) of anaplasmosis were similar over the 10,000-fold infective dose range tested.

Anaplasma marginale: detection of carrier cattle by PCR-ELISA.

A highly sensitive and specific polymerase chain reaction (PCR) based assay for the detection of the minute levels of Anaplasma marginale present in the blood of long-term carrier cattle was developed. A simple lysis method was used to remove most of the haemoglobin from the blood to facilitate direct input of samples into the PCR reactions without prior purification of the DNA. PCR product was detected by enzyme-linked immunosorbent assay (ELISA) to simplify the processing of large numbers of samples. The sensitivity limit of the PCR-ELISA was 0.00015% parasitaemia (24 infected erythrocytes per microlitre of blood). No cross-reactivity of the assay was observed when A. marginale-negative blood infected with Babesia bovis or Theileria orientalis was tested. The PCR-ELISA was shown to be 92% efficient in the detection of long-term A. marginale carrier cattle. No false-positive results were obtained. These results compared favourably with 2 serological assays for detection of A. marginale carrier cattle (card agglutination test and ELISA) which were applied to the same experimental animals.

Peripheral blood lymphocyte proliferative responses in cattle infected with or vaccinated against Anaplasma marginale.

An assay was developed for measurement of the peripheral blood lymphocyte proliferative response (PBLPR) in cattle infected with or immunised against Anaplasma marginale. PBLPR was not evident in all cattle that had recovered from A. marginale infection. However, A. marginale-sensitised lymphocytes were detected in the spleens of all immune cattle tested in the absence of detectable PBLPR. During the course of initial infection, cattle exhibited detectable PBLPR for a period corresponding with and up to 2 weeks after patent parasitaemia, followed by a second, usually larger peak in PBLPR corresponding to the time of sub-clinical relapse of cattle. Analysis of the PBLPR of A. marginale chronically infected cattle demonstrated highly variable PBLPR between individuals and over time. A positive PBLPR was induced in cattle by vaccination using a crude A. marginale antigen preparation. The PBLPR of vaccinated cattle subsequently infected with A. marginale was markedly different from that of naive cattle, with reduced PBLPR being associated with the onset of parasitaemia. The antigen used in the PBLPR assay was inactivated by proteolysis. Proteolysis also abolished immunity that had been induced in cattle vaccinated using the antigen preparation. A marginale-sensitised PBL did not proliferate in response to antigen from the heterologous species A. centrale. A. centrale-sensitised PBL, however, responded to A. marginale antigen. Interferon-gamma (IFN-gamma) was detected in PBLPR-assay supernatants and was associated with a strong PBLPR.

Human interferon alpha fails to inhibit the development of Babesia bigemina and Anaplasma marginale infections in cattle.

Studies were undertaken to determine whether human interferon alpha (HuIFN-alpha) administered orally could inhibit the development of clinical disease caused by the intraerythrocytic protozoan Babesia bigemina and the intraerythrocytic rickettsia Anaplasma marginale in cattle. HuIFN-alpha did not inhibit intraerythrocytic multiplication of either of the two parasites, suggesting that there is no role for HuIFN-alpha administered orally in the control of these pathogens.

Anaplasma marginale: failure of sera from immune cattle to confer protection in passive-transfer experiments.

High levels of immunity to Anaplasma marginale were induced in cattle either by vaccination using sonically disrupted A. marginale-infected erythrocytes or by repeated infection with different strains of the rickettsia. In both instances, high levels of anti-A. marginale antibody were detected in the sera of the immune cattle by immunoblotting. Serum from one animal that had been made immune by repeated infection was transferred intravenously to A. marginale-susceptible calves (three non-splenectomised and two splenectomised) undergoing initial A. marginale infection at serum doses of 2-10 ml/kg. Neither the course nor the outcome of infection as indicated by the parasite levels attained or the level of anaemia induced was altered in the calves that received the immune serum relative to the course or outcome of infection in control calves (two non-splenectomised and two splenectomised) that received serum from an two splenectomised) that received serum from an A. marginale-naive donor animal. In a similar experiment, a pool of sera from four steers that had been vaccinated with sonically disrupted A. marginale initial bodies was transfused into two intact A. marginale-susceptible calves during the early stage of A. marginale infection at a dose of 10 ml/kg. No difference was observed in the course or outcome of infection in these calves relative to the course or outcome of infection in the two non-splenectomised calves that were transfused with non-immune serum.(ABSTRACT TRUNCATED AT 250 WORDS)

A sensitive ELISA technique for the diagnosis of Anaplasma marginale infections.

A sensitive enzyme-linked immunosorbent assay (ELISA) technique using a horse radish peroxidase conjugate is described for measuring Anaplasma marginale antibodies in bovine serum. This technique utilizes two antigen preparations, a 'negative' antigen derived from an animal prior to infection and a 'positive' antigen derived from A. marginale-infected red cells from the same animal following infection. This markedly reduces cross-reactions which are a result of isoantigens. Absorbance values obtained using the 'negative' antigen are subtracted from those obtained using the 'positive' antigen to give a net figure. Of 100 A. marginale-positive sera tested no false negative results were obtained. All 11 animals maintained tick-free after initial diagnosis of naturally transmitted anaplasmosis were positive 3 years later, 15 A. marginale-infected animals maintained with ticks were positive 27 months after initial infection and a further 26 animals infected with A. marginale by blood inoculation were positive 3 months later. Three per cent of negative sera, 2% of B. bovis and 4% of B. bigemina-positive sera gave positive reactions.

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.

Protection of Babesia bigemina-immune animals against subsequent challenge with virulent Babesia bovis.

Two groups of cattle, one previously exposed to Babesia bigemina and one not, were challenged with Babesia bovis. The group previously infected with Babesia bigemina was only mildly affected upon challenge with B. bovis, whereas four of five of the other group were severely affected. Immunoblotting studies performed in both homologous and heterologous systems showed that there were polypeptides of similar molecular weight in both species, but species-specific polypeptides were demonstrated only in B. bovis by the homologous B. bovis reaction. B. bovis antisera reacted avidly with B. bigemina-infected erythrocytes in fluorescent-antibody assays. In contrast, B. bigemina antisera did not cross-react with B. bovis-infected erythrocytes. Two groups of splenectomized calves were immunized with an enriched antigen fraction of B. bigemina. A third group was immunized by infection with B. bigemina and treatment with a drug. One of the groups of calves immunized with the antigenic fraction of B. bigemina, the group immunized by infection with B. bigemina, and a control group were challenged with B. bovis. All control calves died, whereas 50% of the calves immunized by infection with B. bigemina and 75% of the animals immunized with the B. bigemina antigen survived. The second group immunized with the B. bigemina antigen and a control group were challenged with B. bigemina. All control animals died by day 6, whereas 50% of the vaccinates survived, the deaths occurring on days 8 and 11. The nature of the probable protective mechanism is discussed.

The economics of cattle tick control in dry tropical Australia.

The economics of strategic dipping compared to nil treatment of cattle ticks (Boophilus microplus) on Droughtmaster cattle was assessed using a partial budget analysis. The analysis was based on reported experimental data which showed a bodyweight gain advantage from strategic dipping of 45 kg/head for growing cattle and 35 kg/head for breeding cows. Costs of dipping were calculated using 3 acaricide costs, that is 5.9, 20.9 and 62.7 per head and allowances were made for mustering, maintenance of facilities and annual cost of asset purchase under an intensive farm management system similar to the reported experimental conditions. The net gain of benefits over costs per annum for each acaricide cost was $927, $810 and $483 per 100 breeders and their progeny. Breakeven beef prices at which it was worth dipping were found to be 61, 69 and 94 per kg dressed weight depending on the cost of acaricide used for dipping. All prices and costs are expressed in 1981 dollars of purchasing power.

A comparison of direct fluorescent antibody and Giemsa staining for the post-mortem diagnosis of anaplasmosis.

Direct fluorescent antibody (DFA) and Giemsa staining of Anaplasma marginale were compared in smears collected serially at post-mortem (PM) from 11 experimentally infected calves. Once smears had been prepared and air-dried they could be held for at least 5 days before staining with either technique with no noticeable change in staining quality. DFA staining was more sensitive in detecting anaplasms in smears than Giemsa staining. Anaplasma spp could be differentiated from Babesia bovis and B. bigemina by DFA staining but there were cross reactions between A. marginale and A. centrale. Blood smears prepared from subcutaneous vessels in the legs provided better diagnostic material than kidney, heart and lung smears. Brain smears were not suitable for PM diagnosis using either staining technique.

The duration of latent infection and functional immunity in droughtmaster and hereford cattle following natural infection with Babesia argentina and Babesia bigemina.

Tne Droughtmaster and 9 Hereford cattle were born in an enzootic babesiasis area and became naturally infected with Babesia argentina and B.bigemina during a 3 year period. They were then kept free of cattle ticks (Boophilus microplus) for the remainder of the experiment. Annually for the next 3 years their individual infection status with Babesia was determined by sub-inoculation of blood into splenectomised calves. At the end of this period the functional immunity of all cattle was challenged by blood inoculation of heterologous strains of B. argentina and B. bigemina. Infection with B. argentina persisted in all Herefords for 2 years and in 7 for 3 years after they had been freed of B. microplus. The number of Droughtmasters with detectable B. argentina infection progressively declined, and at the end of 3 years only 2 of 10 were still infected. No Herefords were shown to be infected with B. bigemina following 1 year's freedom from B. microplus but latent B. bigemina infection of at least 2 year's duration was demonstrated in one of the Droughtmasters. A marked degree of resistance was apparent in all cattle when they were challenged with an heterologous strain of B. argentina. There were no differences between the response to challenge of the Herefords and Droughtmasters nor between the reactions of cattle which had apparently naturally sterilised B. argentina infection and those which were still infected. The heterologous strain of B. bigemina produced parasitaemia in the majority of animals but only minimal fever and anaemia resulted with no significant differences between the breeds.