Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs,swine and cattle

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD₅₀) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.     

An evaluation of the anamnestic test for brucellosis in cattle of the northern pastoral area

SUMMARY An investigation of the anamnestic test for brucellosis using Brucella abortus 45/20 vaccine was carried out in 3 groups of weaner cattle on 2 farms in western Queensland. Each group originally consisted of about 500 cattle. They were bled before and at 6 or 10 weeks after vaccination and again in the following year. The serums were tested by the complement fixation (CFT), Rose Bengal (RBT) and indirect haemolysis tests (IHLT). Most of the cattle reacting to one or more of the tests were killed and selected tissues were subjected to bacteriological examination for B. abortus. B. abortus was isolated from 19 of 30 (63%) pre-vaccinal reactors, 23 (24%) of 96 cattle reacting at 6 or 10 weeks after vaccination (the anamnestic test) and 1 (2%) of 50 cattle reacting one year after vaccination. The reactor found to be infected the year after vaccination had high serological titres in each of the 3 serological tests: RBT of 3, CFT of 128 and IHLT of 256. A subsequent test showed the group to be brucellosis-free. The CFT was the most efficient test. In the pre-vaccination tests 17 of 19 infected animals were positive in the CFT compared with 11 positive in the IHLT and 17 in the RBT. In post vaccination tests 22 of 23 infected animals were positive in the CFT compared with 18 in the IHLT and 19 in the RBT. At the pre-vaccinal and anamnestic tests (6 or 10 weeks after vaccination) 19 of 57 (33%) cattle with CF titre of 4 or 8 yielded B. abortus on culture compared with none of 26 cattle with similar titres in the year after vaccination. The interpretation of CF titres in cattle following 45/20 vaccination needs to be re-examined.     

Serological response of cattle after vaccination and challenge with Brucella abortus

New and currently used serological procedures were evaluated using sera from cattle that were challenged with B. abortus S544 (S544) after vaccination with either B. abortus S19 (S19) or B. abortus 45/20 (S45/20) as calves or adults. In animals vaccinated with S19, titres to the indirect haemolysis test (IHLT) rose more slowly, declined more rapidly and involved fewer animals than did titres to the complement fixation test (CFT). In animals vaccinated with S45/20 the rough antigen complement fixation test (RCFT) showed persistent titres. At slaughter the IHLT and CFT were found to be more specific and more sensitive than the Rose Bengal Plate Test (RBPT) and Serum Agglutination Test (SAT) in the detection of cattle infected with B. abortus.     

Estimating the impact on the food chain of changing bovine spongiform encephalopathy (BSE) control measures: The BSE Control Model

The decline in the bovine spongiform encephalopathy (BSE) epidemic in Great Britain (GB) demands a review of control strategies to ensure that they remain proportionate. Amongst controls that are subject to review are those intended to minimise the risk of BSE exposure of consumers through food. Such risk mitigation steps are costly, and the relative impact of each in terms of human exposure to BSE infectivity is not known. This risk assessment, termed the BSE Control Model, aims to estimate by use of stochastic simulation the impact of testing of cattle at slaughter and the removal of Specified Risk Materials (SRM) on potential BSE infectivity consumed. This paper describes the use of the model to investigate the effect of different risk management methods that have been or could be implemented between 2005 and 2010.Our results suggest that the amount of infectivity consumed in 2005 with the Over Thirty Month (OTM) rule in place was a mean of 0.03 bovine oral ID₅₀ (BO ID₅₀). This is an extremely low amount, particularly considering that it would be spread over, on average, 236 infected carcases that would be further sub-divided into portions for human consumption. The highest contributor to the total amount of infectivity consumed per year is spinal contamination at carcase splitting (35%). In 2006 the OTM scheme was discontinued and head meat was again permitted into the food chain. These changes resulted in an increase in the amount of infectivity consumed, rising to an estimated 28 BO ID₅₀ in 2006, and 19 BO ID₅₀ in 2007.In 2008 the age at removal of vertebral column was raised from 24 to 30 months, and an estimated 24 BO ID₅₀ of infectivity was consumed. At the beginning of 2009 the age at testing of cattle was raised to 48 months for healthy slaughter, emergency slaughter and fallen stock. Under these conditions, an estimated mean of 24 BO ID₅₀ will be consumed in 2009, decreasing to 20 BO ID₅₀ in 2010. Even though presented in terms of bovine rather than human oral ID₅₀, such estimates represent an extremely low exposure of the British population. Considerable uncertainty would surround any attempt to try to convert such exposure into estimates of new cases of vCJD, but the most recent estimates of the size of the species barrier between cattle and humans (4000, EFSA, 2006) suggest that there would be few, if any, new cases of vCJD arising from such exposure levels.     

Comparison of the sensitivity of in vitro and in vivo tests for detection of the presence of a bovine viral diarrhoea virus type 1 strain

Veterinary vaccines are usually tested for the absence of contaminants. However, the quality control does not always imply that vaccines are not contaminated as, for example, illustrated by the bovine herpes virus 1 (BHV1) vaccine used in The Netherlands in 1999 that contained a small amount of bovine viral diarrhoea virus (BVDV1). Thousands of cows were vaccinated with BHV1 vaccine batches, and the question arose as to whether these small amounts of BVDV1, most likely not detected with in vitro tests, could have infected cattle. More in general, the question was whether the outcome of the in vitro tests, i.e. the in vitro infectivity, was indicative for the infectivity for cattle, i.e. the in vivo infectivity. We therefore carried out in vitro experiments to determine the sensitivity of a BVDV1 isolation assay. In addition, we performed two animal experiments, in which we estimated the lowest dose needed to infect calves with BVDV1. We extrapolated the experimental in vitro and in vivo results from a tissue culture infectious dose (TCID50) to a cattle infectious dose (CID50). We observed a partial response in the calves inoculated with this dose: four out of six calves turned out to be infected. In the tissue culture test, all 20 samples tested negative. The response in vivo, however, was not significantly higher than the in vitro response, which implies that no difference in susceptibility was observed between the animal test and the tissue culture test. Based on the results in our experiments, some cattle may have been infected with BVDV1 after the application of the contaminated BHV1 vaccine during the vaccination campaign. The question remains that how many cattle received contaminated vaccine, and became infected with BVDV1.     

Comparison of the Q-fever complement fixation test and two commercial enzyme-linked immunosorbent assays for the detection of serum antibodies against Coxiella burnetii (Q-fever) in ruminants: Recommendations for use of serological tests on imported animals in New Zealand

Abstract

AIM: To make valid recommendations on the use of serological test methods for the detection of serum antibodies in ruminants against Coxiella burnetii (Q-fever), by comparing the performance of the complement fixation test (CFT) and two ELISA, and by identifying reasons for discrepancies between the test methods.

METHODS: A total of 73 serum samples from infected cattle, 69 from infected goats, and 100 samples from non-infected cattle and 57 samples from non-infected sheep, as well as 95 samples from infected cattle herds (mix of seropositive and seronegative samples), were tested using the CFT, the IDEXX ELISA (I-ELISA) and the Pourquier ELISA (P-ELISA). A mixed panel of 12 serum samples from sheep from inter-laboratory proficiency testing (proficiency panel) was also tested using the CFT and both ELISA, and further investigated using IgG- and IgM-specific ELISA.

RESULTS: Generally, the two commercial ELISA were more sensitive than the CFT for the detection of infected ruminants. Good agreement between ELISA for positive and negative results was found for samples from the infected herd, while results for the positive panels varied between the two ELISA. For the total of the positive serum panels, the I-ELISA detected 95% of samples as positive or suspicious, while the P-ELISA detected only 81%. In the P-ELISA, more samples were considered suspicious (18%) than in the I-ELISA (14%). All sera from noninfected sheep and cattle tested negative in the serological test methods employed, except for one positive sample from a sheep in the P-ELISA. Further investigation revealed that a CFT-positive but ELISA-negative result was due to high IgM and low IgG reactivity.

CONCLUSIONS: The two commercial ELISA were more sensitive than the CFT in all panels from infected ruminants. However, they could only detect IgG. The I-ELISA should be the serological test method of choice for cattle, sheep and goats for import testing of animals into New Zealand because it was more sensitive than the P-ELISA and was equally specific to the PELISA and the CFT. For other animal species, such as deer and camelids, the CFT should still be used since none of the ELISA has been evaluated for these species. This study has shown that the two commercial ELISA will detect the majority of infected ruminants but may miss animals that have not developed an IgG response.     

Detection of occurrence of a recent genetic bottleneck event in Indian hill cattle breed Bargur using microsatellite markers

The effective number of breedable individuals is a crucial determinant for maintaining genetic variability within a population. The population of Bargur, the hill cattle of South India, has gone down drastically by more than 93 % in the past three decades, and only a few thousand animals are available at present. The present study was undertaken to evaluate Bargur cattle for mutation drift equilibrium and to detect the occurrence of recent genetic bottleneck event, if any, in this population. About 50 unrelated animals, true to the type, were sampled and genotyped at 25 microsatellite loci. The mean observed heterozygosity (0.808?±?0.023) was higher than the mean expected heterozygosity (0.762?±?0.008) with 15 out of 25 microsatellite loci exhibiting heterozygosity excess when assumed under Hardy–Weinberg equilibrium. To evaluate Bargur cattle for mutation drift equilibrium, three tests were performed under three different mutation models, viz., infinite allele model (IAM), stepwise mutation model (SMM) and two-phase model (TPM). The observed gene diversity (H _e) and expected equilibrium gene diversity (H _eq) were estimated under different models of microsatellite evolution. All the 25 loci were found to exhibit gene diversity excess under IAM and TPM, while 22 loci were having gene diversity excess under SMM. All the three statistical tests, viz., sign test, standardized differences test, and Wilcoxon sign rank test, revealed significant (P?<?0.01) deviation of Bargur cattle population from mutation-drift equilibrium under all the three models of mutation. Furthermore, the qualitative test of allele frequency distribution in Bargur cattle population revealed a strong mode shift from the normal L-shaped form suggesting that the population had experienced genetic bottleneck in the recent past. The occurrence of genetic bottleneck might have led to the loss of several rare alleles in the population, which point towards the need for efforts to conserve this important cattle germplasm. The present study is the first report in demonstrating the genetic basis of demographic bottleneck in an Indian cattle population.     

Multiple diagnostic tests to identify cattle with Bovine viral diarrhea virus and duration of positive test results in persistently infected cattle

Several tests for Bovine viral diarrhea virus (BVDV) were applied to samples collected monthly from December 20, 2005, through November 27, 2006 (day 0 to day 342) from 12 persistently infected (PI) cattle with BVDV subtypes found in US cattle: BVDV-1a, BVDV-1b, and BVDV-2a. The samples included clotted blood for serum, nasal swabs, and fresh and formalin-fixed ear notches. The tests were as follows: titration of infectious virus in serum and nasal swabs; antigen-capture (AC) enzyme-linked immunosorbent assay (ELISA), or ACE, on serum, nasal swabs, and fresh ear notches; gel-based polymerase chain reaction (PCR) testing of serum, nasal swabs, and fresh ear notches; immunohistochemical (IHC) testing of formalin-fixed ear notches; and serologic testing for BVDV antibodies in serum. Of the 12 animals starting the study, 3 died with mucosal disease. The ACE and IHC tests on ear notches had positive results throughout the study, as did the ACE and PCR tests on serum. There was detectable virus in nasal swabs from all the cattle throughout the study except for a few samples that were toxic to cell cultures. The serum had a virus titer ≥ log₁₀ 1.60 in all samples from all the cattle except for 3 collections from 1 animal. Although there were several equivocal results, the PCR test most often had positive results. The BVDV antibodies were due to vaccination or exposure to heterologous strains and did not appear to interfere with any BVDV test. These findings illustrate that PI cattle may be identified by several tests, but differentiation of PI cattle from cattle with acute BVDV infection requires additional testing, especially of blood samples and nasal swabs positive on initial testing. Also, calves PI with BVDV are continual shedders of infectious virus, as shown by the infectivity of nasal swabs over the 11-mo study.     

Serological crossreactivity between Brucella abortus and Yersinia enterocolitica 0:9 III. Specificity of the in vitro antigen-specific gamma interferon test for bovine brucellosis diagnosis in experimentally Yersinia enterocolitica 0:9-infected cattle

The course of immunological reaction in 10 Yersinia enterocolitica 0:9 experimentally-infected heifers was followed using the conventional brucellosis tests complement fixation test (CFT), serum agglutination test (SAT) and brucella card test (BCT), and a recently developed Brucella antigen-specific gamma interferon (IFN-γ) test. Initially, the animals were exposed orally to 10¹⁰ colony-forming units (CFU) of Y. enterocolitica 0:9. Four weeks later, they were inoculated intravenously with 10⁸ CFU of Y. enterocolitica 0:9 cells. After oral inoculation, the response in the conventional brucellosis tests was minimal. Only after intravenous inoculation were CFT and SAT titres and BCT reactions comparable to natural, false positive brucellosis reactors. After oral exposure the Brucellergen-stimulated release of IFN-γ peaked at values above the cut-off stimulation index of 2.5 in 80% of the heifers. After intravenous inoculation, stimulation indices above 2.5 were present in only 10% of the animals. Two B. abortus infected control cattle showed stimulation indices of 3.1 and 3.4, and a negative control animal exhibited a stimulation index of 1.0. These findings show, in contrast to a previous study, that the Brucellergen-specific IFN-γ assay cannot be used as a specific and discriminatory test for B. abortus infections.     

Applied serology in the latter stages of the eradication of bovine brucellosis

Late in the program to eradicate bovine brucellosis from Western Australia, Rose Bengal test (RBT) and complement fixation test (CFT) results on the serums from 2,307 cattle (from herds where infection was still present after a minimum of 3 complete herd tests) showed that 327 were positive in the CFT and 246 were positive in the RBT (p less than 0.001). Subsequent testing by the RBT, CFT and the indirect haemolysis test (IHLT) of 722 serums from cattle slaughtered as part of infected herds showed that of 177 cattle positive on culture, 138 were positive in all 3 tests, 9 were negative in all 3 tests and no animal positive on culture had a reaction only in the RBT. In the 177 cattle from which B. abortus was isolated, positive reactions in the CFT occurred in the serums of 159 of them. Application of the RBT as a screening test followed by a confirmatory CFT would have resulted in 149 of the 177 cattle being positive and application of the CFT/IHLT (double test) on the serums of all cattle in the herds would have resulted in 168 or the 177 being regarded as positive.     

A comparison of standard serological tests for the diagnosis of bovine brucellosis in Canada.

Six agglutination and two complement fixation tests were compared with respect to specificity, sensitivity and relative sensitivity for the serodiagnosis of bovine brucellosis. Based on 1051 sera from brucellosis free herds, the specificity of the tests was 98.9% for the buffered plate antigen test (BPAT), 99.2% and 99.3% for the standard tube and plate agglutination tests (STAT and SPAT), respectively, and 99.8% for the 2-mercaptoethanol test (2MET). On this small sample, the rose bengal plate test (RBPT), card test (CARD) and the complement fixation test (CFT) correctly classed all sera as negative. On a sample of 167 culture positive cattle, the sensitivities of the tests were CFT: 79.0%, BPAT: 75.4, RBPT: 74.9%, CARD: 74.3%, SPAT: 73.1%, STAT: 68.9%, and 2MET: 59.9%. All tests combined detected only 82% of these infected cattle. Analysis of the relative sensitivity of the six agglutination tests gave the following ranking: BPAT greater than RBPT greater than CARD greater than SPAT greater than STAT. The 2MET ranked between the BPAT and RBPT or between the RBPT and CARD depending on the analysis used. The use of the BPAT as a screening test is recommended provided that a test of high specificity and sensitivity such as the CFT is used to confirm screening test reactions.     

Recovery threshold of Toxocara canis eggs from soil

The purpose of this study was to evaluate the threshold of Toxocara canis eggs form soil samples through utilisation of a centrifuge-flotation technique (CFT). Aliquots of soil (1 g each) were artificially contaminated with known numbers of T. canis eggs (1, 10, 25, 50, 100, and 200 eggs). The threshold was evaluated based on a CFT using zinc sulphate (Zn₂SO₄) and sodium nitrate (Na₂NO₃) solutions at a specific gravity of 1.20. The number of eggs recovered was directly proportional to the number of eggs employed to seed the soil. Both solutions enabled full recovery of samples containing merely three eggs; only Zn₂SO₄ demonstrated efficiency in soil contaminated with a single egg. A recovery rate of 100% was obtained for all tests with samples containing 10 and 25 eggs for Zn₂SO₄ and Na₂NO₃, respectively_. There was no difference in the mean number of recovered eggs regarding either the efficacy of the solutions or the repetition of evaluations in the same trial (p > 0.05). Therefore, the CFT is efficient for the detection of Toxocara eggs, even in samples containing low egg numbers.     

The natural history of Anaplasma marginale

The intracellular pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), described by Sir Arnold Theiler in 1910, is endemic worldwide in tropical and subtropical areas. Infection of cattle with A. marginale causes bovine anaplasmosis, a mild to severe hemolytic disease that results in considerable economic loss to both dairy and beef industries. Transmission of A. marginale to cattle occurs biologically by ticks and mechanically by biting flies and by blood-contaminated fomites. Both male ticks and cattle hosts become persistently infected with A. marginale and serve as reservoirs of infection. While erythrocytes are the major site of infection in cattle, A. marginale undergoes a complex developmental cycle in ticks that begins by infection of gut cells, and transmission to susceptible hosts occurs from salivary glands during feeding. Major surface proteins (MSPs) play a crucial role in the interaction of A. marginale with host cells, and include adhesion proteins and MSPs from multigene families that undergo antigenic change and selection in cattle, thus contributing to maintenance of persistent infections. Many geographic strains of A. marginale have been identified worldwide, which vary in genotype, antigenic composition, morphology and infectivity for ticks. Isolates of A. marginale may be maintained by independent transmission events and a mechanism of infection/exclusion in cattle and ticks. The increasing numbers of A. marginale genotypes identified in some geographic regions most likely resulted from intensive cattle movement. However, concurrent A. marginale strain infections in cattle was reported, but these strains were more distantly related. Phylogenetic studies of selected geographic isolates of A. marginale, using msp4 and msp1α, provided information about the biogeography and evolution of A. marginale, and msp1α genotypes appear to have evolved under positive selection pressure. Live and killed vaccines have been used for control of anaplasmosis and both types of vaccines have advantages and disadvantages. Vaccines have effectively prevented clinical anaplasmosis in cattle but have failed to block A. marginale infection. Vaccines are needed that can prevent clinical disease and, simultaneously, prevent infection in cattle and ticks, thus eliminating these hosts as reservoirs of infection. Advances in genomics, proteomics, immunology and biochemical and molecular technologies during the last decade have been applied to research on A. marginale and related organisms, and the recent development of a cell culture system for A. marginale has provided a format for studying the pathogen/tick interface. Recent advancements and new research methodologies should provide additional opportunities for development of new strategies for control and prevention of bovine anaplasmosis.     

A comparative study of ELISA and other methods for the detection of Brucella antibodies in bovine sera

Enzyme-linked immunosorbent assay (ELISA), using β-galactosidase and a fluorigenic substrate, was used for the detection of antibodies to Brucella abortus in bovine sera.Among 677 animals from 9 brucellosis-free herds, none reacted in the ELISA. Among 785 animals from 23 brucellosis-infected herds, 336 were positive in ELISA, 229 in the slow agglutination test (SAT), 185 in the complement fixation test (CFT), and 165 in the Rose-Bengal test (RBT).Experimental infections were conducted with two B. abortus strains. At slaughter on day 101, after intraconjunctival infection of heifers with B. abortus strain 19 organisms, 3 animals were positive in the SAT, 3 in the CFT, 4 in the RBT and 11 in the ELISA, and Brucella organisms could be cultivated from 10 animals; among these, 2 scored positive in the SAT, 3 in the CFT, 3 in the RBT and 8 in the ELISA test. Seventeen heifers were infected with organisms of B. abortus strain 2308. On day 101, 11 heifers were found to be carriers, all of which yielded positive results in the CFT, RBT and ELISA tests, but not in the SAT.     

Biological properties of viral haemorrhagic septicaemia (VHS) virus: Adsorption and replication

Adsorption and growth of VHS virus of trout were studied in cultures of RTG-2 and FHM cells.Within 1 h virus infectivity in the culture fluid decreased by 90%. No measurable adsorption took place after this time. A higher adsorption rate was achieved by pretreatment of cell cultures with 10 μg zinc sulphate and 10 or 50 μg diethyl-aminoethyl (DEAE) dextran.In both cell lines small amounts of viral antigen could be detected in the perinuclear region by the immunofluorescence technique as early as 3 h after inoculation. Infectivity titres of the culture supernatants started to rise 8–10 h post-infection and reached their maximum after 18 h. In both cell lines, 50% or more of the total infectivity remained cell associated, the yields of free virus obtained from FHM cells being 0.5 log₁₀ TCID₅₀ lower.     

Effects of positive results for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA on results of caudal fold tuberculin test and interferon-gamma assay for tuberculosis in cattle

OBJECTIVE: To determine whether cattle testing positive for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA were more likely to have false-positive responses on the caudal fold tuberculin (CFT) test or interferon-gamma (IFN-gamma) assay for Mycobacterium bovis than cattle testing negative for M paratuberculosis. ANIMALS: 1043 cattle from 10 herds in Michigan. PROCEDURE: Feces and blood samples for plasma were collected from cattle > or =24 months old on the day the CFT test was read. Fecal samples were submitted for microbial culture for M paratuberculosis. Plasma samples were tested for antibody against M paratuberculosis, and IFN-gamma after stimulation with purified protein derivative tuberculin from M bovis or M avium. RESULTS: Of 1043 cattle, 180 (17.3%) had positive CFT test results (suspects) and 8 (0.8%) had positive IFN-gamma assay results after stimulation with purified protein derivative tuberculin from M bovis. Forty-five (4.3%) and 115 (11.0%) cattle tested positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA, respectively. Cattle with positive responses for M paratuberculosis appeared to have an increased likelihood of false-positive results on the CFT test, although this association was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: No significant association was detected among cattle testing positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA and positive CFT test and IFN-gamma assay results for M bovis.     

Saponin Adjuvants. IV. Evaluation of The Adjuvant Quil a in the Vaccination of Cattle Against Foot-And-Mouth Disease

The saponin adjuvant Quil A has been investigated in the vaccination of cattle against foot-and-mouth disease. Using a Frenkel type vaccine a dose-response relationship has been established between Quil A and neutralizing antibody titres. Ten ml of vaccine was combined with 0, 50, 200, 800, and 3200 µg of Quil A. The combinations were each injected into 4 animals. The local reaction on the site of injection produced by injection of the vaccine alone and in combination with different doses of Quil A has been estimated. On this basis a therapeutical dose at 1 mg of Quil A has been estimated to combine maximum adjuvant effect with a minimum of adverse reactions. This dose has been tested in the vaccination of cattle with FMD vaccines derived from BHK suspension cell virus of type O and A respectively. The vaccines were tested in 10 ml and 5 ml doses with or without Quil A, and each in 4 animals. It is concluded that Quil A is a valuable adjuvant for use in the induction of neutralizing antibodies against foot-and-mouth disease in cattle.     

An evaluation of the delayed-type hypersensitivity test for diagnosing brucellosis in individual cattle: a field study

A field study was conducted to evaluate the delayed-type hypersensitivity (DTH) test in diagnosing brucellosis in cattle, in particular the diagnosis of infection in individual cows. A total of 93 cows that were negative, suspect, or positive to the serum agglutination test (SAT), complement fixation test (CFT), or the milk ring test (MRT) were subjected to the DTH test. The cows were then slaughtered and the supramammary lymph nodes were collected for bacteriologic examination. In 989 cows the DTH test, MRT and serologic tests were negative. When the DTH test results were compared with bacteriologic results, 12 of the 93 cows with CFT titres greater than 1:200 tested negative in the DTH test while bacteriologic results were positive. The sensitivity of the DTH test (calculated on the remaining 81 cows) was 100%; the specificity was 83%. The sensitivity of the DTH test (calculated on 93 cows) was 81%; the specificity was 83%. The sensitivity and specificity of the DTH test correlated well with those of the CFT (86-83%). We conclude that the DTH test is very sensitive, and specific enough to diagnose brucellosis in individual cows. The DTH test should be used in combination with serologic tests in the diagnosis of brucellosis.     

Studies on the infectivity of Theileria annulata infected nymphs,adults and ground tissues of the tick Hyalomma anatolicum

The infectivity of unfed nymphs, adults and ground tissue suspensions of the cattle tick, Hyalomma anatolicum, infected with Theileria annulata was investigated using susceptible calves. Sporozoites of T. annulata introduced into the host by the above mentioned tick stages were able to induced clinical theileriasis in calves. The nature of the disease was the same, whether it was induced by sporozoites injected via 200 infected nymphs or 20 adults (both sexes in equal numbers). This suggests a direct correlation between the infectivity of the ticks and the volume of T. annulata-infected blood sucked by them during their preceding instars.