Tick-free Area Research Articles

Comparison of Seroprevalence and Identification of Risk Factors for Theileria equi in Horses From Vector-Free and Infested Areas in Southern Brazil

The apicomplexan hemoprotozoan Theileria equi is a tick-borne pathogen that causes disease in equids, and together with Babesia caballi, causes equine piroplasmosis (EP). Many ticks are associated with EP around the world, and in South America three species may be related: Dermacentor nitens, Amblyomma sculptum, and Rhipicephalus microplus, as they are commonly found in horses. Among the species cited above, only R. microplus is found in Rio Grande do Sul state, Southern Brazil. In addition, this state has the only area legally free of R. microplus in Brazil. This study aimed to compare the seroprevalence for T. equi on farms located in a tick-free area (23 farms, 215 horses) and a tick-infested (25 farms, 141 horses) adjacent areas, as well as to identify potential risk factors for exposure to T. equi. Blood was collected from 356 horses from both areas, and later an indirect enzyme-linked immunosorbent assay was performed to detect anti-T. equi antibodies. Besides the blood collection, questionnaires and interviews were conducted in an attempt to identify potential risk factors. The R. microplus-free zone had 6% seropositive horses against 70% in the tick-infested zone. Previous observation of ticks on horses of the herd increased four times the odds of a horse to be seropositive for T. equi, and by three times if ticks were observed on cattle which share paddocks with horses on the farm. The results showed a large difference in T. equi seroprevalence between tick-infested and free areas, and also emphasized the relevance of R. microplus in the epidemiology of T. equi. The study also reveals the potential of the establishment of a T. equi-free zone for horse breeding in Southern Brazil.

Molecular of Anaplasma marginale Theiler (Rickettsiales: Anaplasmataceae) in horseflies (Diptera: Tabanidae) in Uruguay

Anaplasma marginale is transmitted biologically by infected ticks or mechanically by biting flies and contaminated fomites. In tick-free areas, such as southern Uruguay, horseflies could be the principal vectors of this pathogen for bovines, causing anaplasmosis. The objective of this work was to detect the presence of A. marginale by MSP-5 PCR and Sanger sequencing in the most prevalent species of horseflies obtained using different collection methods in Colonia, Tacuarembó and Paysandú, Uruguay. Eight horsefly species were tested (Dasybasis missionum, Poeciloderas lindneri, Tabanus campestris, T. claripennis, T. fuscofasciatus, T. platensis, T. tacuaremboensis and T. triangulum); four species were found to be positive for A. marginale, with D. missionum and P. lindneri having the most frequent infections, while only one individual each of T. fuscofasciatus and T. tacuaremboensis was positive. Both D. missionum and P. lindneri were positive for A. marginale in tick-free areas, and the implications are discussed in this report.

Bovine immunisation with a recombinant peptide derived from synthetic SBm7462® (Bm86 epitope construct) immunogen for Rhipicephalus microplus control

Rhipicephalus microplusis the most important ectoparasite of livestock in tropical and subtropical areas around the world. Research focused on developing an efficient vaccine for cattle tick control is a high priority. The aim of this study was to evaluate the rSBm7462® peptide (Bm86-B and T cell epitopes) regarding its properties of immunogenicity, protective effect in cattle and efficacy against R. microplus. This peptide was produced by a fermentative process in the yeast culture system of Komagataella (Pichia) pastoris strain Km 71. The vaccination assay was conducted in a tick-free area using non-splenectomised Holstein Friesian calves, separated into immunisation and control groups. These animals individually received the recombinant peptide and the inoculum without peptide using saponin as an adjuvant at three time points. The calves were challenged 21 days after the last immunisation using 4500 larvae per animal. An indirect ELISA was used to identify the IgG kinetics of serum samples from the calves studied. The qPCR was performed to determine the cytokine gene expression from the total RNA of the cultured PBMCs. Histomorphometry of the germinal centres (GCs) was performed measuring slides with haematoxylin-eosin staining of surgically removed lymph nodes from immunised calves. The antibody response showed a significant induction of high-affinity IgGs in calves immunised with the recombinant peptide in comparison to the controls. The kinetics of antibodies in immunised calves showed a significant increase during the experiment. This increase in high-affinity IgGs correlated with a gradual increase of the GC diameter following each peptide vaccination. Cytokine expression profiles demonstrating an adaptive immune response in calves immunised with rSBm7462® confirmed the T-dependent response. Vaccine efficacy was calculated at 72.4 % following the analysis and fecundity of collected adult female ticks, compared between control and vaccinated groups. These findings demonstrate that this new recombinant peptide is an option for control of R. microplus infestations.

Probability of Rhipicephalus microplus introduction into farms by cattle movement using a Bayesian Belief Network

Attempts to eliminate Rhipicephalus microplus from Uruguay have been unsuccessful, and, currently, the country is divided into two areas: a tick-free area and a tick-infested area. In the tick-infested area, different farms face different situations. Some farms are in regions where, due to environmental conditions or a lack of infrastructure, it is difficult to eliminate R. microplus, and the only option is to control it. In contrast, other farms can attempt complete removal. Before deciding whether a farmer should attempt to eliminate R. microplus, the probability of reintroduction must be evaluated. The objective of this study was to develop a probabilistic model based on a Bayesian Belief Network (BBN) to assess the likelihood of a farm becoming infested with R. microplus via the introduction of tick-infested cattle. Only the tick-infested area was considered in the development of this model. Nine variables related to environmental conditions and biosecurity measures, with a focus on cattle movement, were considered. Three different sources of data were used to populate the BBN model: data from the literature; a representative national survey from 2016; and a survey developed to identify biosecurity practices on farms. Model sensitivity and specificity were assessed, and an overall accuracy of 92% was obtained. The model was applied to 33 farms located in the tick-infested area. For one farm, the probability of introduction of R. microplus was 1%; for three farms, the probability was between 21% and 34%; for seven farms, it was between 66% and 76%; and for 22 farms, the probability was greater than 83%. This model was useful for estimating the probability of the introduction of R. microplus into farms, making it possible to assess the impact that the evaluated biosecurity measures have on the probability of introduction and, thus, guiding more objective decision making about the control or elimination of R. microplus from farms.

Implication of Nilgai Antelope (Artiodactyla: Bovidae) in Reinfestations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in South Texas: A Review and Update

The Cattle Fever Tick Eradication Program was the first parasite eradication program of veterinary importance in the United States and is considered to be one of the greatest disease eradication programs of all time. The program's utilization of pasture vacation and dipping of cattle in acaricide has been extremely successful for controlling Rhipicephalus (Boophilus) microplus (Canestrini) and R. (B.) annulatus (Say), collectively known as cattle fever ticks, on cattle along the Texas border with Mexico for decades. However, the increase of white-tailed deer, Odocoileus virginianus (Zimmermann), populations in South Texas over the last 50 yr has compromised the success of the program. R. (B.) microplus and R. (B.) annulatus infestation data have confirmed that O. virginianus can support the maintenance and movement of both species of cattle fever tick within the permanent quarantine or buffer zone in South Texas along the Rio Grande, and also in the cattle fever tick-free area north and east of the buffer zone. Over the last two decades, increasing populations of exotic nilgai antelope, Boselaphus tragocamelus (Pallas), in South Texas have further complicated cattle fever tick eradication efforts. Historical cattle fever tick infestation data, host source data, and geographical data support the continued role of O. virginianus in maintaining reinfestations of R. (B.) microplus and R. (B.) annulatus in South Texas as well as the increasing role of nilgai antelope in cattle fever tick maintenance and dispersal.

Modeling the impact of climate and landscape on the efficacy of white tailed deer vaccination for cattle tick control in northeastern Mexico.

Cattle ticks are distributed worldwide and affect animal health and livestock production. White tailed deer (WTD) sustain and spread cattle tick populations. The aim of this study was to model the efficacy of anti-tick vaccination of WTD to control tick infestations in the absence of cattle vaccination in a territory where both host species coexist and sustain cattle tick populations. Agent-based models that included land cover/landscape properties (patch size, distances to patches) and climatic conditions were built in a GIS environment to simulate WTD vaccine effectiveness under conditions where unvaccinated cattle shared the landscape. Published and validated information on tick life cycle was used to build models describing tick mortality and developmental rates. Data from simulations were applied to a large territory in northeastern Mexico where cattle ticks are endemic and WTD and cattle share substantial portions of the habitat. WTD movements were simulated together with tick population dynamics considering the actual landscape and climatic features. The size of the vegetation patches and the distance between patches were critical for the successful control of tick infestations after WTD vaccination. The presence of well-connected, large vegetation patches proved essential for tick control, since the tick could persist in areas of highly fragmented habitat. The continued application of one yearly vaccination on days 1-70 for three years reduced tick abundance/animal/patch by a factor of 40 and 60 for R. annulatus and R. microplus, respectively when compared to non-vaccinated controls. The study showed that vaccination of WTD alone during three consecutive years could result in the reduction of cattle tick populations in northeastern Mexico. Furthermore, the results of the simulations suggested the possibility of using vaccines to prevent the spread and thus the re-introduction of cattle ticks into tick-free areas.

Survival and moulting of Amblyomma variegatum nymphs under cold conditions of the Malagasy highlands

Although Amblyomma variegatum is now regularly recorded up to 1600m in altitude in the Malagasy highlands, where it was previously reported not to persist without a constant supply of ticks introduced from lower infested regions, some parts of the highlands remain tick-free. Studies were carried out to verify whether the cold climate prevailing in these areas in June–September could prevent the survival and moulting of nymphs, the tick life stage present in the environment at this period. Cohorts of engorged A. variegatum nymphs were released from June to August in six different sites (three in 2010, altitudes 1200–1415m; three in 2011, altitudes 1585–1960m) which were reported to be either tick-infested (two in 2010, one in 2011) or tick-free. The ticks were placed in cages driven into the soil and open at the bottom so that they could hide in the soil or root network. Of the 1975 nymphs released in 2010 and the 1494 released in 2011, 86% and 85% were recovered, respectively. Twenty to 23% of the recovered ticks were dead, and some of them were obviously predated; predation also likely contributed to the disappearance of the non-recovered ticks. When the rainy season started in October, 59% of the newly moulted adults were still alive in the cages. The moulting period lasted up to 20weeks, depending on the site and release period. As verified in 2011, unfed nymphs could also survive the cold season. Various A. variegatum life stages are thus able to survive the adverse cold and/or dry seasons: unfed nymphs, engorged nymphs in developmental diapause, moulted adults in behavioural diapause as observed previously. Strong variation in mortality and recovery rates was observed between cages, highlighting the importance of the micro-environment and micro-climate for tick survival. The minimum temperature recorded in the field sites varied from 1.1°C to 6.8°C, but the tick-free sites were not the coldest ones; they were, however, those for which the temperature remained below 10°C for the longest time over the study period. Recovery and mortality rates in the tick-free sites were similar to those of the tick-infested sites: the temperatures recorded during the study periods did not prevent ticks from surviving and moulting although it did delay the metamorphosis. Low temperature alone can therefore not explain the persistence of tick-free areas in the highlands. To further monitor survival, cohorts of engorged nymphs were also maintained in an incubator at 3.6°C, 6.2°C or 12.8°C. More than 50% mortality was observed after 6days at 3.6°C, and after 15days at 6.2°C, whereas 18days at 12.8°C only delayed moulting. The collected survival, moulting and climatic data presented in this study should help to develop a predictive model to assess the distribution of A. variegatum according to climate characteristics.

Genetic diversity of Anaplasma marginale in Argentina

Bovine anaplasmosis caused by Anaplasma marginale is a worldwide major constraint to cattle production. The A. marginale major surface protein 1 alpha ( msp1α) gene contains a variable number of tandem repeats in the amino terminal region and has been used for the characterization of pathogen genetic diversity. This study reports the first characterization of A. marginale genetic diversity in Argentina based on msp1α genotypes and its putative relationship with Rhipicephalus ( Boophilus) microplus infestations. Herein, we analyzed whole blood bovine samples from anaplasmosis outbreaks in R. microplus infested (9 samples) and eradicated/free (14 samples) regions. Sequence analysis revealed the existence of 15 different msp1α genotypes with 31 different repeat units. Six new repeat sequences were discovered in this study and 13/31 (42%) repeats were unique to Argentinean strains. The analysis of msp1α repeat sequences according to R. microplus infestations resulted in three repeat groups: (i) found in tick-infested regions (20 repeats), (ii) found in tick free regions (6 repeats) and (iii) randomly distributed (5 repeats). Moreover, A. marginale msp1α genetic diversity was higher in tick-infested regions than in tick free areas. These results, together with previous evidence suggesting that A. marginale msp1α repeat units co-evolved with the tick vector, might represent an evidence of the role of tick-mediated transmission for the generation of pathogen genetic diversity.

Infection and Seroconversion of Susceptible Animals Introduced into a Babesiosis Endemic Area

The study aimed to determine the incubation period of Babesia sp. infection in naive cattle and to monitor the serological response once exposed to natural Boophilus microplus (Rhipicephalus microplus)-infested paddocks. The study was carried out on a farm located in Veracruz, Mexico. Five groups of five steers were relocated every 3 months from a tick-free area to a tick-infested paddock. Animals were introduced in October, January, April, July, and October. Blood samples were taken daily until day 21 to determine packed cell volume (PCV), percentage of parasitized erythrocytes (PPE), and antibody titers to Babesia bigemina and B. bovis by the indirect fluorescent antibody procedure. Detection of Babesia in blood was also performed by species-specific PCR. The estimated incubation period was 6-14 days post introduction to paddocks (PIP), with fever (41 degrees C) for at least 3 days. PCV decreased by >25% and Babesia parasites were observed during the clinical phase of the disease. The highest individual PPEs (0.44% and 0.22% for B. bovis and B. bigemina, respectively) were observed from animals introduced in October. The four other groups showed a mean PPE ranging from 0.002-0.146% at day 14 PIP. All animals were detected as PCR positive between 8-14 days PIP. The highest antibody titers were 1:3328. The environmental conditions were favorable throughout the year for tick reproduction as the farm showed enzootic stability and hyperendemic conditions for bovine babesiosis. In this type of farm, strategic tick control could be accompanied by babesiosis vaccination, particularly for cattle relocated from tick-free areas.

Differential Bos taurus cattle response to Babesia bovis infection

Bovine babesiosis is a tick-borne disease caused by Babesia spp. haemoprotozoans. The disease is of great importance at tick enzootic unstable areas and hampers cattle production in several developing countries. The available immunisation alternatives are pre-immunition and attenuated vaccines. Despite being efficient and protective, they are unsafe as they use cattle blood cells as inoculum and may potentially spread other diseases. Another alternative to help in babesiosis control would be the identification of genetically resistant cattle to Babesia bovis infection. The objective of this work was to phenotype cattle based on primary response against B. bovis infection. Two-hundred and forty half-sib Hereford and Aberdeen Angus heifers (120 animals from each breed), 12–18-month-old naïve cattle, originated from a tick-free area in Southern Brazil, were used in the experiment. Animals were monitored following an inoculation with 1 × 10 7 B. bovis parasitised erythrocytes. Results showed three different phenotypes: 1—‘susceptible’, animals with babesiosis clinical signs that received treatment to avoid death; 2—‘intermediate’, animals with clinical signs: parasitaemia, ≥21.5% reduction in packed cell volume (PCV) and increase in body temperature when compared to their pre-challenge physiological parameters, no specific treatment was needed as animals self recovered from the disease, and 3—‘resistant’, animals without clinical signs that showed B. bovis presence in blood smears, <21.5% PCV reductions, with little or no increase in body temperature and no need for babesiosis treatment. The frequencies of each phenotype were: 45.4, 26.7, and 27.9%, respectively, demonstrating the existence of phenotypic variation for B. bovis in Bos taurus cattle.

Efficacy of a Single Doramectin Injection Against Adult Female &lt;I&gt;Boophilus microplus&lt;/I&gt; (Acari: Ixodidae) in the Final Stages of Engorgement Before Detachment

The efficacy of injectable doramectin applied at 200 microg/kg was evaluated against adult female Boophilus microplus (Canestrini) (Acari: Ixodidae) in the later stages of engorgement before detachment. Lethal levels of doramectin in the serum of treated cattle (9.7-36.6 ppb) were reached and sustained within 24 h after treatment. However, treatment at 19 or 20 d postinfestation allowed greater tick survival and reproductive capability (IF) than at 18 d postinfestation, indicating that a significant portion of the ticks were able to engorge and detach before obtaining a lethal dose of doramectin. Thus, treatment at 18 d after infestation provided significantly higher overall control (99.5%) than treatment at 19 or 20 d postinfestation (95.8 and 89.1%, respectively). Analysis of control on a daily basis demonstrated that treatment at 18 d postinfestation provided > 99% on each day of the evaluation. Conversely, treatment at 19 or 20 d postinfestation produced levels of control ranging from 22.6 to 85.6% during the first 2 d of female detachment, and > or = 99% control was not achieved until after the fourth day of female detachment, where it remained throughout the study. Therefore, application of injectable doramectin at < or = 18 d after tick infestation was the only treatment regime considered acceptable for use in the U.S. Boophilus Eradication Program. Treatment intervals > 18 d postinfestation could pose a substantial risk of dispersing viable ticks to tick-free areas outside the permanent quarantine zone that has been established along the Texas-Mexico border.

Protección contra babesiosis con una vacuna mixta de B.bovis y B.bigemina derivada de cultivo in vitro en una confrontación de campo. II Inmunización en una área endémica

The protection conferred by a combined in vitro culture-derived Babesia bovis and B. bigemina vaccine to susceptible Bos taurus bulls in a babesiosis endemic area was evaluated. Participating animals were over 18 months of age coming from a tick free area. Twenty animals were allocated at random into two groups of 10 animals each and taken to a tick endemic area. One week after arrival, 10 of them were vaccinated i.m. with an experimental Babesia spp. immunogen at a dose of 1 x 107 infected erythrocytes of each species. The 10 control animals were inoculated with uninfected red blood cells. All the animals were kept from the day of arrival until d 21 post vaccination in a pen free of Boophilus ticks and then released to a tick infested paddock where they remained until study',s completion. Twelve days after the start of the tick challenge, nine of the control animals presented poor physical condition, and also severe signs of babesiosis i.e. rectal temperature (RT) of more than 41 oC, a packed cell volume (PCV) drop of 46 % and presence of both species of Babesia in blood stained smears and had to be treated in order to avert their death. On the other hand, although the vaccinated animals presented a RT of 40.4 oC and a PCV drop of 40.7 %, only three animals showed poor physical condition and had to be treated on d 22 post introduction to the infected paddock. It can be concluded that a combined B. bovis and B. bigemina vaccine is able to confer a 70 % protection to bovines introduced to Babesia infected areas

Immunization of cattle with synthetic peptides derived from the Boophilus microplus gut protein (Bm86)

Three synthetic peptides (SBm4912, SBm7462 and SBm19733), derived from the Bm86 glycoprotein from Boophilus microplus gut, were constructed and used to immunize cattle from a tick-free area. The immunized animals received three subcutaneous doses of the peptides, with saponin as adjuvant, at 30-day intervals. The immune response was evaluated by IgG elicited against the peptides by the detection of anti-Bm86 specific antibodies in situ and by Western blotting analysis. After tick challenge, reduction in the number, weight and oviposition capacity of engorged females was observed in the tick population that had fed on immunized animals. The results pointed a high efficacy (81.05%) for the SBm7462 synthetic peptide in relation to the others ( p<0.01), demonstrating the efficiency of the immune response elicited by synthetic peptides to control the cattle tick B. microplus.

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle.

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.

Detection of IgM antibodies against Babesia bovis in cattle

The diagnosis of acute babesiosis by direct examination of blood smears has some limitations and the indirect serological methods currently in use are designed for detection of IgG, which may not be detectable at an early stage of infection. There is a need, therefore, for rapid and reliable procedures to diagnose acute infections. An ELISA system using a crude antigenic preparation of Babesia bovis was standardized for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Serum samples of cattle imported from tick-free areas collected before and during an immunization process were used to validate the tests. The specificity was 94% and sensitivity 100%. Specific IgM antibodies against B. bovis first appeared on the 11th day post-inoculation (p.i.) in animals infested with Boophilus microplus ticks and on the 19th day p.i. in animals which had been inoculated with infected blood. Antibody titers decreased after Day 33; however, all animals remained positive until the end of the experiment (124 days).

Correlation between antibodies to Cowdria ruminantium (rickettsiales) in cattle and the distribution of Amblyomma vector ticks in Zimbabwe.

Cowdriosis, caused by Cowdria ruminantium, is transmitted by Amblyomma ticks, which are widely distributed in Zimbabwe. To assess the distribution of this disease in Zimbabwe, cattle either exposed to Amblyomma ticks or maintained in areas free from these ticks were tested for antibodies to Cowdria. A total of 324 sera were tested using competitive ELISA and the indirect fluorescent antibody test (IFAT). At diptanks in Amblyomma-infested areas 52% (n = 95) and 26% (n = 47) of sera were positive by cELISA and IFAT, respectively. At diptanks in Amblyomma-free areas 11% (n = 125) and 10% (n = 134) of sera were positive by cELISA and IFAT, respectively. The results were significantly different between Amblyomma-infested and tick-free areas (chi 2 = 24.73, P < or = 0.005 for IFAT and chi 2 = 57.53, P < or = 0.005 for cELISA). High background readings in field sera, possibly due to cross-reactive antibodies to Ehrlichia spp., complicated the determination of a realistic cut-off point, especially in cELISA. On the basis of the distribution of Amblyomma ticks, currently a large part of Zimbabwe can be considered endemic for the disease.

Estimation of the potential demand for a bovine babesiosis and anaplasmosis vaccine in argentina

A survey to estimate the potential demand for a bovine babesiosis and anaplasmosis vaccine was carried out by a postal questionnaire sent to 4407 veterinarians in the north and centre of Argentina. Twenty-three percent of the questionnaires (1047) were answered; 317 (7.2% of the total, 30% of those answered) pointed out the occurrence of the diseases and/or probable demand for the vaccine. The minimum and maximum demands were 407 892 and 982 300 doses for anaplasmosis, and 272 496 and 631 400 doses for babesiosis, respectively. The results presented from here onwards are based on the minimum demand. Sixty-eight and 48% of the anaplasmosis and babesiosis vaccine demand, respectively, came from veterinarians living in the Boophilus microplus-free area, while the remainder came from the tick-infested area, reflecting the fact that many farm owners from the tick-infested area seek professional assistance from veterinarians living in the tick-free area. Most of the demand for both vaccines was aimed at breeding cattle (77.2%), 14.5% at dairy cattle and the rest at studs (8.3%). Veterinarians living in the tick-infested area demanded vaccine for breeding cattle in a higher proportion than veterinarians from the tick-free area, whose demand was greater for dairy cattle. In the tick-free area, the demand for anaplasmosis vaccine was twice that for babesiosis vaccine, whereas in the tick-infested area the demand was equal. This agrees with the fact that the anaplasmosis-stricken area extends beyond the tick-infested region. Sixty percent of the respondents preferred a vaccine frozen in liquid nitrogen, whilst the rest preferred a refrigerated one, the first group accounted for 73% of the total demand. Fifty-six percent of the demand occurred between March and June with the peak in April, which reflects the main weaning date. The elasticity of the demand is discussed in relation to the probable benefits to be accrued by the farmers who make use of it. The annual mortality rates due to both babesiosis and anaplasmosis were estimated at 1.5 and 3.5% in the milk herds of Salta and Tucumán, respectively. In the beef herds of these two provinces, the mortality rate due to both diseases has been estimated at 3.5%. Using these figures and current prices, the systematic application of the vaccine in calves would render a benefit-cost ratio between 4.6 and 9.0, which would warrant its use in the herds of the areas at risk, i.e. in enzootic instability.

A microplate enzyme‐linked immunorsorbent assay for measuring antibody to Anaplasma marginale in cattle serum

An enzyme-linked immunosorbent assay (ELISA) method is described for measuring antibody against Anaplasma marginale in cattle serum. This method was more sensitive and objective than a previously described ELISA method for A. marginale and possible reasons for this are discussed. All 83 cattle experimentally infected with A. marginale (81) or A. centrale (2) developed demonstrable specific antibody but the serums of 98.8% of 839 cattle from cattle tick-free areas did not react by ELISA; 378 serums containing antibody to Babesia bovis were tested for cross reactions in the A. marginale ELISA. There were no significant cross-reactions except when cattle had been inoculated at least twice with B. bovis-infected erythrocytes, presumably due to antibodies reacting with erythrocyte material in the ELISA antigen. The ELISA detected antibodies for more than 3 years after infection, at least 2 years longer than did a complement fixation test. When A. marginale infections in cattle were eliminated by long acting oxytetracycline, their serums ceased to react by ELISA. An ELISA score for serum antibody level was shown to have a statistically significant correlation with ELISA titre.

The irradiation of [formula omitted]. II. The immunogenicity of irradiated blood parasites for intact cattle and splenectomised calves

Intraerythrocytic forms of B. bovis were exposed to 350 Grays (Gy) γ irradiation and were then injected intravenously into intact two and three year old Hereford steers. One of 15 steers died on initial infection and subsequently six steers were given a virulent heterologous challenge three weeks after recovery; all six animals were highly immune. The remaining eight animals were kept under quarantine conditions for 10 months and were then challenged with a different virulent heterologous strain of B. bovis . Seven of eight were highly immune, but one animal died. Subsequently a further 12 steers were injected intravenously with 1 × 10 8 irradiated organisms. All showed only mild transient clinical signs. After 12 months quarantine in a tick-free area these animals were then challenged with a virulent heterologous strain and all 12 were shown to be highly immune. Irradiation reduced the infective dose from 1 × 10 8 to 2.5 × 10 3 parasites. These parasites multiplied at the same rate, and achieved the same maximum parasitaemia as the parent non-irradiated strain, but the disease produced by them was not severe. A dose of 2.5 × 10 3 non-irradiated paasites was lethal to all of the four animals which received it. It was concluded that irradiation had produced a predominantly avirulent parasite population.

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