An Intradermal Test to Detect Latent Warble (Hypoderma spp.) Infection in Cattle

An intradermal test was developed to screen cattle for infection with the first-instar larvae of the warble flies Hypoderma lineatum and H. bovis. The diagnostic antigen, prepared from the first-instar larvae of H. lineatum, produced a distinct dermal reaction in cattle infected with the first-instar larvae of either species, but not in cattle in which the infection could not be confirmed later either on necropsy or by the appearance of warbles in the back. The reaction to the antigen was unpredictable in cattle with warbles in their backs. The diagnostic property of the antigen was also demonstrated in rabbits and guinea pigs artificially sensitized to the antigen.     

Immunity,safety and protection of an Adenovirus 5 prime - Modified Vaccinia virus Ankara boost subunit vaccine against Mycobacterium avium subspecies paratuberculosis infection in calves

Vaccination is the most cost effective control measure for Johne’s disease caused by Mycobacterium avium subspecies paratuberculosis (MAP) but currently available whole cell killed formulations have limited efficacy and are incompatible with the diagnosis of bovine tuberculosis by tuberculin skin test. We have evaluated the utility of a viral delivery regimen of non-replicative human Adenovirus 5 and Modified Vaccinia virus Ankara recombinant for early entry MAP specific antigens (HAV) to show protection against challenge in a calf model and extensively screened for differential immunological markers associated with protection. We have shown that HAV vaccination was well tolerated, could be detected using a differentiation of infected and vaccinated animals (DIVA) test, showed no cross-reactivity with tuberculin and provided a degree of protection against challenge evidenced by a lack of faecal shedding in vaccinated animals that persisted throughout the 7 month infection period. Calves given HAV vaccination had significant priming and boosting of MAP derived antigen (PPD-J) specific CD4⁺, CD8⁺ IFN-γ producing T-cell populations and, upon challenge, developed early specific Th17 related immune responses, enhanced IFN-γ responses and retained a high MAP killing capacity in blood. During later phases post MAP challenge, PPD-J antigen specific IFN-γ and Th17 responses in HAV vaccinated animals corresponded with improvements in peripheral bacteraemia. By contrast a lack of IFN-γ, induction of FoxP3+ T cells and increased IL-1β and IL-10 secretion were indicative of progressive infection in Sham vaccinated animals. We conclude that HAV vaccination shows excellent promise as a new tool for improving control of MAP infection in cattle.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0112-9) contains supplementary material, which is available to authorized users.     

Repeat tuberculin skin testing leads to desensitisation in naturally infected tuberculous cattle which is associated with elevated interleukin-10 and decreased interleukin-1 beta responses

The principal surveillance tool used to control bovine tuberculosis in cattle is the removal of animals that provide a positive response to the tuberculin skin-test. In this study we performed a longitudinal investigation of the immunological and diagnostic consequences of repeated short-interval skin-tests in cattle naturally infected with Mycobacterium bovis. Tuberculin skin-test positive cattle were subjected to up to four further intradermal comparative cervical skin-tests at approximately 60-day intervals. A significant progressive reduction in the strength of the skin-test was observed after successive tests. In contrast, the magnitude of interferon-γ (IFN-γ) responses was not influenced by repeat skin-testing either transiently around the time of each skin-test or longitudinally following repeated tests. A significant boost in blood interleukin-10 (IL-10) production was observed within 3 days following each skin-test although the magnitude of this boosted response returned to lower levels by day 10 post-test. The application of a novel multiplex assay to simultaneously measure seven cytokines and chemokines also identified that skin-testing resulted in a significant and progressive reduction in antigen specific interleukin-1β (IL-1β) whilst confirming stable IFN-γ and elevated IL-10 responses in the blood. Therefore, we have demonstrated that in cattle naturally infected with M. bovis, repeat short-interval skin-testing can lead to a progressive reduction in skin-test responsiveness which has potential negative consequences for the detection of infected animals with marginal or inconclusive skin-test responses. The desensitising effect is associated with decreased IL-1β and elevated IL-10 responses, but importantly, does not influence antigen specific IFN-γ responses.     

Comparison of fluorescent antibody tests for tuberculosis and paratuberculosis with antigens coupled to insoluble spheres or taken up by macrophages

Sera of 106 cattle from farms with histories of Mycobacterium johnei infection and sera from 15 human tuberculous patients as well as a number of control sera were examined by means of two different fluorescent antibody tests (FAT) for the occurrence of antibodies against M. johnei and M. tuberculosis respectively. The antigens used were PPD johnin and PPD tuberculin. In the macrophage uptake FAT (MU/FAT) mouse macrophages after phagocytosis of the tuberculins served as the matrix; in the tests performed using the defined antigen substrate spheres (DASS) system, Sepharose beads activated by CNBr were used for the matrix. A good correlation was found between the results of the DASS/FAT and those of the MU/FAT, which is known to be a sensitive and specific test in the diagnosis of Johne's disease in cattle. It is suggested that the FAT, with utilization of the DASS system, might have good prospects for routine examination for antibodies against species of Mycobacterium.     

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.     

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.     

Optimization of in-house ELISA based on recombinant major sperm protein (rMSP) of Dictyocaulus viviparus for the detection of lungworm infection in cattle

The aim of this study was to optimize an in-house ELISA based on a recombinant version of the major sperm protein (MSP) of Dictyocaulus viviparus for routine diagnosis of lungworm infection in cattle. A recombinant MSP (rMSP) was cloned into pGEX-6P-1 vector and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (DE3) chemically competent cells. The product was then employed as capture antigen in an ELISA, and validated against 304 samples of known status (216 negative and 88 positive) in which the antibody levels in sera had also been measured earlier with a commercial ELISA kit (Ceditest® lungworm ELISA). The receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity as 97.7% (95% confidence interval [CI]: 91.9–99.7%) and 98.1% (CI: 95.3–99.5%), respectively. The results from the in-house rMSP-based ELISA were compared with results obtained on both fecal examination and the Ceditest® lungworm ELISA. Rising antibody levels in sera of experimentally infected calves were observed between 21 and 28 days post infection, when patency was also confirmed by the presence of larvae in feces. Notably, using the in-house rMSP-based ELISA infection was confirmed in calves shedding larvae approximately 3–4 weeks post inoculation, while using the Ceditest® lungworm ELISA those animals remained negative. Additionally, 251 sera samples from calves naturally exposed to the parasites on pasture were used to evaluate the test. In in-house rMSP-based ELISA no cross-reactions were observed with sera from calves infected with the gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora), even though the presence of eggs in the feces was confirmed. Overall, the in-house rMSP-based ELISA optimized in this study showed excellent diagnostic performance for detection of lungworm infection in cattle.     

Evaluation of the enzyme-linked immunosorbent assay in the detection of cattle infected with Brucella abortus

One group of 51 cattle was vaccinated with B. abortus S19 (S19) and a further 51 cattle were vaccinated with B. abortus S45/20 (S45/20). Forty-eight cattle (24 from each group) and a control group of 12 cattle were subsequently challenged with B. abortus S544. The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgM antibodies in these groups. All cattle vaccinated with S19 had high levels of IgG and IgM, but the S45/20 vaccine produced detectable antibody in only a few cattle. In those cattle where the challenge induced infection, the mean levels of IgG and IgM were much higher than those of the uninfected cattle in the same groups. When the isolation of B. abortus was compared at slaughter with the serological results, the ELISA, when used to detect specific IgG, was more sensitive but less specific than the serum agglutination test, complement fixation test and indirect haemolysis test, and more sensitive and more specific than the Rose Bengal test.     

Isolation of Theileria mutans from Kenyan buffalo,and transmission by Amblyomma gemma

Blood from 2 buffalo harbouring Theileria organisms was inoculated into a splenectomized Ayrshire calf. The calf developed an infection which extended over a long period. The infection was transmitted to two cattle with Amblyomma gemma by transstadial transmission between the larvae and nymphs. Severe anaemia developed in these cattle and correlated with the parasitaemia. Schizonts morphologically characteristic of T. mutans were detected for short periods in the lymphoid cells of cattle infected by the ticks. The antigens and sera prepared from the cattle reacted with T. mutans sera and antigens in the indirect fluorescent antibody test. After recovery from the primary parasitaemia, the cattle had detectable organisms and antibodies to T. mutans for more than two years.     

Trypanosomes

Most immunological studies on trypanosomes have been made on species in the subgenus Trypanozoon which includes those causing sleeping sickness in man, but members of the subgenera Duttonella and Nannomonas are more important as the cause of disease in cattle. Wherever possible, studies on these subgenera in cattle are discussed in this paper. The antigenic structure of trypanosomes is complex and common antigens between species, strains and populations occur in homogenates. Antigens variation is responsible for trypanosome surface alter during infection and this antigenic variation is responsible for many of the immunological problems of trypanosmiasis. The characteristics humoral response to infection is a rapid and sustained rise in IgM followed by a later increase in IgG but little is known about cell-mediated immunity in trypanosomiasis. The altered immunoligical state of the infected animal may be of significance in the pathogenesis of the disease in which anemia is marked and kinin production occurs. Possible methods of stimulating protective immunity are discussed and the suppression of immune response to other antigens is analysed.     

Serological relationship of a Japanese Babesia species and Babesia bigemina by the complement fixation and capillary-tube agglutination tests

The complement fixation (CF) test and the capillary-tube agglutination (CA) test were used to study the antigenic relationship between Babesia bigemina and the large Babesia species frequently infecting cattle in Japan. The CF antigen was prepared from parasitized erythrocytes by extraction with distilled water. The CA antigen was prepared from parasitized erythrocytes by mild sonification of mixtures of Babesia and erythrocyte stroma, following lysis of the erythrocytes with hypotonic saline solution. All the sera used were collected from experimentally-infected cattle. Cross reaction was demonstrated between the Japanese Babesia species and B. bigemina. There was, however, a difference of two dilutions in titer between homologous and heterologous antibody in the CF test, and a difference of more than three tubes in titer between both antibodies in the CA test. It was possible, therefore, to distinguish the Japanese Babesia species from B. bigemina by the CF and CA tests.     

Serum antibodies to the catalase antigen of Aspergillus fumigatus in cattle

Aspergillus fumigatus is an important agent of mycotic infection in cattle and a potent source of antigens. However, the efficacy of serological diagnosis of aspergillosis in cattle remains controversial. Corbel (1972) and Knudtson et al. (1974) considered a precipitin assay useful as a supplementary test in the diagnosis of mycotic abortion, whereas Wiseman et al. (1984) found the specificity too low to justify its routine use. We have studied 1) the antibody response to the catalase antigen of A. fumigatus in experimentally infected cattle and 2) the prevalence of catalase antibodies and A. fumigatus precipitins in healthy and diseased cattle. The aim was to ascertain how far detection of antibodies to a defined fungal antigen can contribute to the often difficult diagnosis of mycosis.     

Use of peripheral blood leukocytes in the study of cell-mediated immunity in bovine anaplasmosis—A review

Peripheral blood leukocytes separated by erythrocyte aggregation and density-gradient centrifugation were used in tests to measure the cell-mediated response in bovine anaplasmosis. The leukocyte migration inhibition test was apparently related to the host protective immune response. Leukocyte stimulation by a specific anaplasma antigen elicits positive but low level stimulation indices in cultures of cells collected from cattle exposed to live anaplasma. There appears to be a nonspecific enhancement of blastogenesis in cultures of leukocytes from cattle injected with inactivated-oil fortified Anaplasma marginale vaccine. Cytotoxicity tests indicated that both leukocytes and antibodies are active in clearance of anaplasma infected erythrocytes from the circulatory system.     

Circulating antibodies and blood histamine in cattle after treatment against hypodermosis

Studies have been made of the effect of treatment with an organophosphorus insecticide on calves with or without Hypoderma infestation. A close relationship has been demonstrated between variations of circulating antibodies and blood histamine levels. It is suggested that histamine is released following an immunological reaction due to the formation of complexes between antigenic proteins of the dying larvae and circulating antibodies. Other etiologic agents such as intrinsic larval toxicity were also examined.The small variations of histamine levels suggest that histamine plays only a minor part in the mediation of symptoms of anaphylaxis often observed in parasitised cattle after preventive treatment against hypodermosis.     

Genetic differentiation of Mycobacterium bovis and Mycobacterium tuberculosis isolated from cattle and human sources in,Egypt (Suez Canal area)

Bovine tuberculosis is a devastating illness in cattle and it has the ability to transmit causing severe troubles in human. Mycobacterium bovis (M. bovis) infection in human indeed becomes increasingly critical especially in developing countries. Early diagnosis is very important to control and limit its spreading. The aim of this study is to examine the genetic differentiation and possibilities of transmission between cattle and human. Lymph node and sputum samples were collected from cattle and patients showing tuberculin test positive; respectively for phenotypic identification and for molecular examination by detection of IS6110 and oxyR genes which are specific for MTC and M. bovis; respectively. The phenotypic identification of sputum samples showed 80 % positive by both stain and culture, while, lymph nodes revealed 66 % and 84 % positive by stain and culture method; respectively. Alignment of oxyR gene sequences of M. tuberculosis and M. bovis was used as a feature for differentiation between the 2 genes in these two genetically closely similar microorganisms showed 99 % identities between the 2 genes. Alignment and phylogenetic analysis of Mpb70 gene sequences from animal and human origin showed very high relatedness (99.32 %) to each other confirming that the zoonotic transmission is most probably occurred.     

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsa-stained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a co-agglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.     

Serologic and virologic investigations into pestivirus infection in wild and domestic ruminants in the Pyrenees (NE Spain)

An outbreak of disease associated to a border disease virus was described in the Southern chamois (Rupicapra pyrenaica) in Spain in 2002. Sera and/or spleen samples from 57 mouflon, 15 red deer, 21 roe deer, 3 fallow deer, 55 sheep, 32 cattle, and 68 goats sharing the chamois habitat were studied. An antibody ELISA test yielded an inconclusive result in 2 mouflon and positive results in 5 goat sera. Comparative virus neutralization tests were performed on the 2 inconclusive mouflons, 3 of the 5 seropositive goats, 55 sheep and 32 cattle, using 6 pestivirus strains. Positive results were obtained in 1 mouflon, 2 goats, 69% of sheep and 78% of cattle. Virological investigations performed with an antigen ELISA test yielded negative results in 21 goats and 39 mouflons, the result in 1 mouflon being inconclusive. PCR performed on 12 goats and the inconclusive mouflon gave negative results. These results suggested that it is unlikely that chamois BDV is infecting wild and domestic ruminants.     

The negative effects of the residues of ivermectin in cattle dung using a sustained-release bolus on Aphodius constans (Duft.) (Coleoptera: Aphodiidae)

This paper reports the findings of two trials into the effects of the treatment of cattle with ivermectin slow-release (SR) bolus on the larval development of the dung beetle Aphodius constans Duft. Rectal faecal samples were collected prior to treatment and every 3 and 2 weeks in a first and second trial, respectively, and up to 156 days post-administration of the SR bolus. Faecal ivermectin concentration reached a peak at 63 days post-treatment (1427 ng g(-1)) and ivermectin was detected up to 147 days post-treatment in the first trial (7.2 ng g(-1)). First stage larvae of A. constans were reared with control or contaminated dung and adult beetles were counted after emergence. In the first trial, the comparison of pairwise samples showed that ivermectin prevented the development of larval A. constans until day 105, while at day 135 the rate of emergence was still significantly lower than the corresponding series of control (p < 0.05). In the second trial, the difference between control and treated series remained significant until 143 days post-treatment, with no emergence until 128 days post-administration of SR bolus to cattle. These results show the negative effect of ivermectin on the development of larval A. constans, even at a low concentration (38.4 ng g(-1)). The administration of ivermectin sustained-release bolus to cattle was highly effective in killing dung beetle larvae for approximately 143 days after treatment. The results were similar when dung was obtained from a single animal kept alone, or from a blending of faecal pats obtained from a group of animals kept in field conditions during the whole trial period.     

Serodiagnosis of Metastrongylus apri infection of pigs by immunofluorescence

The lyophilised first stage larvae of Metastrongylus apri were used as antigen in the indirect fluorescent antibody test for the diagnosis of the infection of pigs. The larvae were recovered from the embryonated eggs discharged by the gravid females of the nematode in vitro. Ten pigs were used in the test. Serial serum samples were taken of seven pigs, experimentally infected with varying doses of M. apri larvae, all of which were found to discharge eggs of the nematode in their faeces after infection was established. The other three pigs were uninfected and used as controls.Positive cuticular fluorescence of the larvae was first detected with the serum samples obtained between 14 and 33 days post-infection. This fluorescence persisted with subsequent serum samples up to 85 days post-infection. Pronounced and uniform cuticular fluorescence was generally observed with the serum dilutions of up to $\text{1}\text{10}$. Earlier post-infection serum samples either exhibited no cuticular fluorescence or gave non-brilliant fluorescence. The pre-infection serum of these pigs and also serum samples obtained from the three uninfected control pigs did not give cuticular fluorescence even at the initial serum dilution of $\text{1}\text{5}$.     

Efficacy of Strychnos spinosa (Lam.) and Solanum incanum L. aqueous fruit extracts against cattle ticks

The efficacy of Solanum incanum and Strychnos spinosa aqueous fruit extracts was evaluated against cattle ticks in on-station experiments and laboratory tick bioassays. In the on-station experiment using cattle, fruit extracts were applied at three concentrations 5, 10, and 20 % (w/v) and compared with a commercial acaricide, Tickbuster® (amitraz) spray (positive control) and no treatment (negative control). The treatments were applied at weekly intervals for 6 weeks as surface sprays on 32 Mashona cattle in a completely randomized design experiment. Ticks on individual cattle were identified, counted, and recorded daily. Peripheral blood samples were collected for parasite screening. In the laboratory, tick bioassays were conducted at four concentrations, 5, 10, 20, and 40 % (w/v) fruit extracts compared to Tickbuster® (amitraz) spray (positive control) and distilled water (negative control). The extracts were incubated with Rhipicephalus (Boophilus) decoloratus tick larvae and mortalities for each treatment level recorded after 24 and 48 h. The 5 % Solanum incanum treatment had higher efficacy ratio (P?<?0.05) than the other fruit extract concentrations of the same plant species. Efficacy ratio was higher (P?<?0.05) in the 5 % S. spinosa-treated cattle than in the untreated control but lower (P?<?0.05) than that for the amitraz treatment. The bioassays indicated that there was a high efficacy ratio for the lowest fruit extract concentrations when ticks were exposed to acaricidal treatments for 48 h compared to 24 h. Overall, the results indicate that Solanum incanum and Strychnos spinosa individually have some acaricidal effect.