Diagnosis of Mycobacterium bovis infection in calves sensitized by mycobacteria of the avium/intracellulare group

Because of the frequent exposure of cattle to mycobacteria of the avium/intracellulare group, an investigation was carried out into the possible repercussions thereof on the diagnosis of bovine tuberculosis. Three calves from a bovine tuberculosis-free herd, scored avian reactors in the gamma-interferon assay for bovine tuberculosis, were sedated and inoculated endotracheally with a virulent Mycobacterium bovis strain. Then, three other avian reactors were housed with the above donor calves. Mycobacterium bovis was isolated from the nasal swabs of the three endotracheally infected, donor calves. On these samples, TB complex-specific polymerase chain reaction (PCR) tests for IS6110 were also positive, albeit with a different time kinetics. The three contact-infected calves showed clear immunological signs of infection; however, their nasal swabs were always PCR-negative and only Mycobacterium avium was isolated. In the endotracheally infected donor calves there was a rise of the gamma-interferon responses to avian and bovine purified protein derivative (PPD) tuberculins, which reached the same stable plateau levels over the whole experiment. The above effect was also observed in the contact-infected calves, even though the response to avian PPD tuberculin always remained at a higher level. By using conventional bovine and avian PPD tuberculins, the comparative intradermal test was generally positive in endotracheally infected, as opposed to contact-infected calves; a positive intradermal test for M. bovis was obtained in two contact-infected calves by different bovine PPD tuberculins based on M. bovis bacillus Calmette-Guerin (BCG) secreted or somatic antigens. It was concluded that M. bovis infection may be concealed for some time in cattle sensitized by mycobacteria of the avium/intracellulare group and that different diagnostic procedures should be adopted for such animals.     

牛分枝杆菌重组蛋白TB27.4的表达、纯化及活性鉴定

为探索TB27.4蛋白在牛结核病鉴别诊断中的作用,本试验以牛分枝杆菌Vallee Ⅲ株基因组DNA为模板,PCR扩增tb27.4全长基因片段,将其定向克隆到原核表达载体pET-32a(+)中,构建重组质粒pET-TB27.4,优化原核表达条件,并用AKTA Purifier对蛋白的纯化条件进行优化。SDS-PAGE结果显示重组蛋白为可溶性表达,且大小与理论值相符,用牛分枝杆菌阳性血清进行Western blotting检测有特异性条带,且可特异性地刺激牛分枝杆菌感染牛外周血淋巴细胞释放大量IFN-γ。结果表明,重组蛋白TB27.4具有良好的B细胞活性和T细胞刺激活性,为进一步研究其在牛结核病诊断中的作用奠定了基础。     

11种牛分枝杆菌抗原在牛结核病诊断中的初步评价

为筛选及评价用于牛结核病诊断的抗原,本试验将CFP-10、ESAT-6、TB10.4、TB27.4、MPT51、MPT63、MPT64、MPB70、MPB83、Rv3872和Ag85B共11种牛分枝杆菌抗原分别作为包被抗原建立间接ELISA方法,比较其对牛结核病的检出率;同时利用豚鼠和牛的皮试试验评价重组蛋白作为皮试试验刺激原的潜力。此外,将重组蛋白分别刺激结核病阳性牛和阴性牛的抗凝血24 h,检测血浆中的IFN-γ水平,评价各重组蛋白作为IFN-γ释放试验刺激原的潜力。结果显示,不同重组蛋白对结核病阳性血清的反应活性不一,MPB70总检出率最高,为59.7%;其次是Ag85B、ESAT-6和MPB83,检出率均在45%以上;MPT51的检出率最低,仅为2.2%。豚鼠和牛皮试试验均显示,单个重组蛋白作为刺激原难以产生令人满意的迟发型过敏反应（delayed type hypersensitivity,DTH）,而TB10.4、TB27.4、MPT64、MPT63或Rv3872作为补充抗原,分别与CFP-10或ESAT-6混合,均可特异性地刺激结核病阳性牛产生较强的DTH反应,且与PPD-B无显著差异（P>0.05）。重组蛋白CFP-10、ESAT-6、TB10.4和MPT51均能刺激结核病牛全血释放一定的IFN-γ,其中CFP-10、CFP-10-ESAT-6串联蛋白和MPT51刺激结核病阳性牛全血释放的IFN-γ显著高于阴性牛（P<0.05）。因此,这11种牛分枝杆菌抗原并不适合单独用于牛结核病的血清学诊断、皮试试验或IFN-γ释放试验,但以CFP-10和ESAT-6为核心,TB10.4、TB27.4、MPT64、MPT63、Rv3872或MPT51作为其补充抗原,均能提高检测敏感性,有作为皮试试验和IFN-γ释放试验特异性刺激原用于牛结核病诊断的潜力。     

IFN-gamma diagnostic tests in the context of bovine mycobacterial infections in Belgium

In countries where cattle tuberculosis caused by Mycobacterium bovis (Mbov) and paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (Mptb) are present, testing strategies for the Mbov eradication have to discriminate between these two infections. Present indirect tests are based on the analysis of the specific cellular immune response (DTH, IFN-gamma) against crude mycobacterial antigens (avian and bovine PPD). In this study, we compared the evolution of the IFN-gamma responses of animals experimentally infected with Mbov, Mptb, or inoculated with Mycobacterium phlei. Mbov inoculation induced a strong IFN-gamma response that allows rapid classification of the status of the animals following interpretation criteria set up by us. Experimental inoculation with M. phlei induced sensitisation to mycobacterial antigens as detected by the IFN-gamma test but these reactions were of short duration, therefore, repeated testing allows us to define these animals as aspecific reactors. IFN-gamma response induced after oral inoculation of calves with Mptb was of low intensity and ratio of responses measured against avian versus bovine PPD did not allow a clear diagnostic at least for the six first month of infection.     

Comparison of purified protein derivatives and effect of skin testing on results of a commercial gamma interferon assay for diagnosis of tuberculosis in cattle.

Purified protein derivatives (PPD) prepared in the USA were compared with those prepared in Australia by a private company (CSL Veterinary) for use with a commercial gamma interferon (gamma-IFN) assay for diagnosis of bovine tuberculosis. The effect of skin testing on results of the gamma-IFN assay was determined, and results were compared when blood samples were stimulated with PPD within 2 hours and after 24 hours of sample collection. Twenty cattle that were sensitized by subcutaneous injection of heat-killed Mycobacterium bovis were randomly divided into 3 groups. Cattle in group A were tested with the caudal fold skin test (CFT) on day 0 and the comparative cervical skin test (CCT) on day 7. Cattle in group B were tested with the CFT on day 0 and the CCT on day 63, and group C cattle were not skin tested. Blood samples for the gamma-IFN assay were collected at various times throughout the study period. Optical density (OD) values for the gamma-IFN assay were not significantly different when blood samples were stimulated with US avian PPD and CSL avian PPD. However, OD values were significantly higher for US bovine PPD than for CSL bovine PPD. However, the final interpretation of the gamma-IFN assay was usually the same when using either US or CSL PPD. In addition, OD values for the gamma-IFN assay were significantly higher for blood samples collected after sensitized cattle were skin tested than for samples collected from the same cattle before skin testing or from cattle not skin tested. The OD values for blood samples stimulated within 2 hours of sample collection were significantly higher than for samples stimulated 24 hours after sample collection. However, OD values for all PPD-stimulated samples from sensitized cattle were significantly higher in samples collected 3 days after skin testing and stimulated 24 hours after collection than for samples from the same animals collected before skin testing and stimulated within 2 hours of sample collection. Results of this study indicate that PPD prepared in the USA or Australia can be used to stimulate blood samples for the gamma-IFN assay. Skin testing cattle prior to collection of blood for the gamma-IFN assay boosts production of gamma-IFN by lymphocytes from cattle that have had prior exposure to M. bovis antigens. Use of the gamma-IFN assay in conjunction with skin testing may improve detection of cattle infected with M. bovis. In addition, the increase in production of gamma-IFN after skin testing will permit greater flexibility in conducting the assay because samples can be stimulated after they have been shipped overnight rather than only on the day of sample collection.     

Assessment of defined antigens for the diagnosis of bovine tuberculosis in skin test-reactor cattle

The continued use of purified protein derivative (PPD) tuberculin is considered to be the main factor which limits the specificity of diagnostic tests for bovine tuberculosis (TB). This study evaluated a whole blood interferon-gamma (IFN-gamma) assay and compared the diagnostic potential of PPD with two tuberculosis-specific antigens, ESAT-6 and MPB70. To provide estimates of sensitivity and specificity, responses were measured in 180 skin test-reacting cattle, of which 131 were confirmed as tuberculous, and in 128 cattle from TB-free herds. For the skin test reactors, there was a positive correlation between the IFN-gamma responses to PPD from Mycobacterium bovis (PPDB) and PPD from Mycobacterium avium (PPDA), indicating cross-reactivity between these complex antigens which are the basis of the skin test. In comparisons of the ESAT-6 IFN-gamma test with a PPD IFN-gamma test (using PPDB compared with PPDA), there was a decrease in sensitivity (76.3 per cent vs 89.3 per cent), but a clear increase in specificity (99.2 per cent vs 92.2 per cent). The provision of high specificity, even with lower sensitivity, offers major benefits for testing in areas with a low incidence of TB.     

Progress in the development of vaccines and diagnostic reagents to control tuberculosis in cattle

The sharp rise of bovine tuberculosis (TB) in Great Britain and the continuing problem of wild life reservoirs in countries such as New Zealand and Great Britain have resulted in increased research efforts into the disease. Two of the goals of this research are to develop (1) cattle vaccines against TB and (2) associated diagnostic reagents that can differentiate between vaccinated and infected animals (differential diagnosis). This review summarises recent progress and describes efforts to increase the protective efficacy of the only potential TB vaccine currently available, Mycobacterium bovis BCG, and to develop specific reagents for differential diagnosis. Vaccination strategies based on DNA or protein subunit vaccination, vaccination with live viral vectors as well as heterologous prime-boost scenarios are discussed. In addition, we outline results from studies aimed at developing diagnostic reagents to allow the distinction of vaccinated from infected animals, for example antigens that are not expressed by vaccines like Mycobacterium bovis Bacille-Calmette-Guérin, but recognised strongly in Mycobacterium bovis infected cattle.     

Antibodies to mycobacteria in cattle not infected with Mycobacterium bovis

An indirect anti-IgG enzyme-linked immunosorbent assay (ELISA) using a whole cell sonicate of Mycobacterium bovis as the coating antigen, was used to detect anti-mycobacterial antibodies in cattle not infected with M. bovis. False positive M. bovis ELISA scores were produced in 6 cattle experimentally inoculated with Mycobacterium avium-intracellulare-scrofulaceum (MAIS) serovars 2, 8, 9, 14 and 18 and Mycobacterium flavescens, respectively. False positive ELISA results were also found in 39.5% of cattle from which other mycobacteria were cultured and in 56.4% of necropsied cattle with other pathological conditions. No M. bovis was cultured from these animals. Other groups of animals, with no pathological conditions, which had been tuberculin-tested negative, tuberculin-tested positive and never tuberculin tested showed positive ELISA results in 15.4%, 73.6% and 42.4% of the respective groups. The variation of these non-specific responses in uninfected cattle highlights the need for careful selection of negative controls in evaluating ELISAs for the diagnosis of bovine tuberculosis.     

Unexpected high responses to tuberculin skin-test in farmed red deer: implications for tuberculosis control

Tuberculosis (TB) in deer is a serious zoonotic disease of worldwide distribution. Detection of infected animals is usually performed using single or comparative skin-testing (SST/CST), although false responses due to sensitization to other mycobacteria may occur, hampering diagnostic specificity. We describe the evolution of the responses to the SST, CST and to an in-house serological assay in a red deer farm subjected to regular TB testing in southern Spain in an attempt to understand the dynamics of possible non-specific reactions occurring under field conditions. We performed 2288 skin-tests and ELISAs in nine sampling periods between May 2009 and January 2011. In May 2010, a strong increase in skin fold thickness in response to avian purified protein derivative (PPD) (mean=4.0mm, 95% CI=3.5-4.5) and bovine PPD (mean=1.8mm, 95% CI=1.6-2.0) was observed in yearling deer hinds (n=150), compared to values recorded for the same individuals in November 2009 (avian PPD: mean=0.7 mm, 95% CI=0.6-0.8 and bovine PPD: mean=0.7 mm, 95% CI=0.6-0.7) and in January 2011 (avian PPD: mean=2.2mm, 95% CI=1.9-2.4 and bovine PPD: mean=1.1mm, 95% CI=1.0-1.2). Using SST, 54 animals (36%) of the yearlings tested in May 2010 would have been classified as positive reactors, while none of them was positive in the CST. The five animals with highest skin fold increases to mycobacterial antigens were culled and subjected to post-mortem analysis, which confirmed the absence of Mycobacterium tuberculosis complex (MTBC) infection but demonstrated the presence of environmental mycobacteria and closely related bacteria in four out of the five analyzed animals. Our results demonstrated how non-specific responses to mycobacterial antigens can adversely affect the specificity of TB diagnosis based on the SST. Thus, once TB infection has been ruled out using confirmatory techniques, application of comparative diagnostic tests is highly advisable to maximize test specificity and avoid the slaughter of false positive reactors.     

Analysis by ELISA and Western blotting of antibody reactivities in cattle infected with Mycobacterium paratuberculosis after absorption of serum with M phlei

Preabsorption of cattle serum with Mycobacterium phlei was of value in eliminating falsely positive reactions in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against M paratuberculosis. Specific antibody titres from 16 animals naturally infected with M paratuberculosis were unaffected by absorption. Analysis by Western blotting indicated that a different set of antigens of M paratuberculosis were recognised by serum from falsely positive reactors compared with that from animals with established infection. After experimental infection the time required for seroconversion in the ELISA in nine calves lay between 10 and 28 months, although one animal had not seroconverted after 30 months when the experiment ended. All animals shed M paratuberculosis in their faeces before seroconversion.     

Comparison between a gamma-IFN assay and intradermal tuberculin test for the diagnosis of bovine tuberculosis in field trials in Brazil.

Bovine tuberculosis is a major problem in Brazil. The intradermal tuberculin test is the standard test for detection of bovine tuberculosis in Brazil but can lack both sensitivity and specificity. The purpose of this study was to compare a bovine gamma-IFN assay with the tuberculin test under field conditions in Brazil. A total of 1632 animals from 13 dairy farms were tested using the single cervical tuberculin test (SCTT). Among those animals, about 15% of each herd, 220 in total, represented a high-risk group and were selected to be tested using the gamma-IFN test. Of the 1632 animals tested, 207 presented significant reactions representing 12.7% of the cattle studied. In the selected group the number of animals positive by the gamma-IFN assay was 126/220 (57.3%) and the total number of reactive cows on SCTT was 106/220 (48.2%). The real number of infected cattle, following standards, was 120/220 (54.5%). From these results the relative sensitivity rate of gamma-IFN test was 100% including the false-positive results and 88.3% for the SCTT--a significant (P < 0.01) difference in favour of the gamma-IFN test of 11.7%. The gamma-IFN assay also identified some positive animals 60-120 days earlier than the SCTT. In conclusion, we believe that the gamma-IFN assay can be used alone or in combination with the SCTT, as a valuable tool for the control of bovine tuberculosis in the Brazilian national herd.     

Development and evaluation of an enzyme-linked immunosorbent assay for detection of bovine antibodies to epizootic hemorrhagic disease of deer viruses.

An indirect enzyme-linked immunosorbent assay (I.ELISA) is described for detection of bovine serum antibody to epizootic hemorrhagic diseases of deer virus (EHDV). Serum samples, at a dilution of 1:200, were incubated with group-specific EHDV antigens, pre-adsorbed to microtiter plates. Bound antibodies were detected by a murine monoclonal antibody to bovine immunoglobulin (Ig)G1 (heavy-chain specific) conjugated with horseradish peroxidase. The performance of the I.ELISA in detecting antibodies to EHDV in sequential serum samples from calves experimentally infected with serotypes 1,2,3 and 4 was evaluated. The I.ELISA detected EHDV antibodies from 14 days postinfection when seroconversion by the standard agar gel immunodiffusion (AGID) test was also evident. The group-specific antibodies to EHDV increased exponentially during the first two to four weeks postinfection and remained relatively stable for about 12 months in some calves. Unlike observations with the AGID test, no reaction was seen in the I.ELISA between blue-tongue virus (BTV) antigen and sera from calves given a single dose of EHDV. The performance of the I.ELISA and AGID were compared using 3,135 AGID negative bovine field sera from herds in Ontario, Alberta and British Columbia and 130 AGID positive samples collected from cattle in 1987 and 1988 during and after outbreaks of EHD in the Okanagan Valley, British Columbia. The specificity and sensitivity of the assay relative to the AGID test were 99.3% and 91.5% respectively, with an overall agreement of 99.0% between the tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and partial purification of serologically defined Boophilus microplus larval antigens by natural ectoparasite exposure

In an effort to identify life-stage specific Boophilus microplus proteins that elicit a humoral response in cattle, soluble proteins were extracted from 10- to 14-day-old larvae and subsequently fractionated by size-exclusion chromatography and reverse-phase high pressure liquid chromatography. Several antigens were identified by Western blotting as potentially shared with other ixodid tick species since antibodies to these proteins were present in sera of calves not previously exposed to B. microplus. Six putative B. microplus-specific antigens were identified by antibodies in the sera of calves repeatedly exposed to B. microplus larvae. One of the antigens, a 19.1 kDa protein, was used in the development of a diagnostic kELISA for previous exposure to B. microplus. The 19.1 kDa protein did not have tryptic protease activity or inhibit bovine trypsin activity, but appeared to be allergenic in that a partially pure fraction elicited immediate-type hypersensitivity responses in calves previously exposed to B. microplus.     

A complementary diagnosis of naturally occurring tuberculosis in water buffaloes (Bubalus bubalis) in Rio de Janeiro using a MPB70-ELISA, Brazil

As tuberculosis is still a worldwide infection and buffalo breeding represents an important economic activity in various countries, the purpose of this study was to employ an enzyme-linked immunosorbent assay (ELISA) using MPB70 as a capture antigen for the diagnosis of naturally occurring tuberculosis in water buffaloes in Brazil. After the introduction of newly acquired cattle onto a tuberculosis (TB) free farm, an outbreak of TB was recorded in a mixed herd comprising water buffaloes (21) and cattle (46). The entire herd was tested by intradermal tuberculin injection (ITT) and positive animals were slaughtered and tested by culture, polymerase chain reaction (PCR), and ELISA. From the 21 buffaloes sampled, three were reactive by ITT. All the three had positive culture and ELISA, while PCR was positive in two of them. Besides that, one ITT-negative buffalo was slaughtered and presented positive results by both culture and ELISA, and was considered as anergic. Although there were only few animals, those findings demonstrate the diagnostic usefulness of an MPB70-ELISA to correctly detect Mycobacterium bovis tuberculosis in water buffaloes.     

Ante mortem diagnosis of tuberculosis in cattle: a review of the tuberculin tests, gamma-interferon assay and other ancillary diagnostic techniques

The early, preclinical stages of bovine TB can be detected in live animals by the use of tests of cellular immunity (the skin, gamma-interferon and lymphocyte transformation tests). Tests of humoral (antibody) immunity, Mycobacterium bovis PCR probes on early tissue cultures or live cattle specimens, and tests based on "electronic nose" technology have been developed more recently. The key measure of diagnostic test accuracy is the relationship between sensitivity and specificity, which determines the false-positive and false-negative proportions. None of the tests currently available for the diagnosis of bovine TB allow a perfectly accurate determination of the M. bovis infection status of cattle. Although various factors can reduce the sensitivity and specificity of the skin tests, these remain the primary ante mortem diagnostic tools for TB in cattle, providing a cost-effective and reliable means of screening entire cattle populations. Despite the inescapable limitations of existing diagnostic tests, bovine TB has been effectively eradicated from many developed countries and regions with the implementation of sound programmes of regular tuberculin skin testing and removal of reactors, coupled with slaughterhouse surveillance for undetected infections, repeat testing and culling of infected herds, cattle movement restrictions to prevent introduction of infected animals and occasional slaughter of entire herds with intractable breakdowns. This is likely to remain the mainstay of bovine TB control programmes for the foreseeable future. Additionally, newer ancillary in vitro diagnostic assays are now available to TB control programme managers to supplement the skin tests in defined circumstances according to the specific disease situation in each country or region. The strategic deployment of ancillary in vitro tests alongside the primary skin tests has enhanced the detection of M. bovis-infected cattle and reduced the number of animals slaughtered as false positives.     

Serologic cross-reactivity of antibodies against Borrelia theileri, Borrelia burgdorferi, and Borrelia coriaceae in cattle.

OBJECTIVE: To evaluate the immune response induced by Borrelia theileri infection and to determine whether B theileri induces cross-reacting antibodies to other bovine borreliae. ANIMALS: Two 3-month-old calves, 1 of which was splenectomized. PROCEDURE: Calves were exposed to Boophilus microplus infected with B theileri. Rectal temperature, PCV, bacteremia, and clinical signs of infection were monitored. Serum was obtained weekly and used to evaluate the humoral response to homologous antigen and B burgdorferi and B coriaceae, using an indirect fluorescent antibody (IFA) test, and to B burgdorferi, using a commercially available ELISA. The identity of cross-reacting antigens was explored, using monoclonal antibodies to genus- and species-specific antigens in an IFA test. RESULTS: B theileri-infected calves produced antibodies that cross-reacted with B burgdorferi and B coriaceae whole-cell antigens. Borrelia theileri whole-cell antigen was recognized by genus-specific monoclonal antibody H9724 but not by species-specific antibody H5332. False-positive reactions were not observed when serum from B theileri-infected calves was tested by use of the ELISA for B burgdorferi. CONCLUSIONS: B theileri induces humoral responses in infected cattle that can be confused with those of other borrelial infections. Care must be taken to definitively distinguish between the various borreliae that may cause disease in cattle. CLINICAL RELEVANCE: Serologic cross-reactivity must be taken into account when making a serodiagnosis of Lyme borreliosis or epizootic abortion in epidemiologic studies involving cattle.     

In vitro and in vivo evaluation of lysozyme extracts of virulent Mycobacterium bovis in guinea pigs and calves

Extracts of Mycobacterium bovis ATCC 19210 were prepared from cells following treatment with acetone 3 times, ethyl alcohol-ether 3 times (1:1, v/v), and chloroform 3 times. Cells were dried and suspended in 0.05M Tris-HCl (pH 7.5) containing lysozyme (1 mg/50 mg of dried cells). One aliquot of the lysozyme extract was filter-sterilized and 1 aliquot of the lysozyme extract was autoclaved. Delayed-type hypersensitivity responses elicited in sensitized guinea pigs, using the filter-sterilized lysozyme extract, were significantly greater than responses elicited using the autoclaved lysozyme extract (P less than 0.01). The filter-sterilized lysozyme extract and a purified protein derivative (PPD) of M bovis, at equal protein concentrations, elicited comparable delayed-type hypersensitivity responses in sensitized guinea pigs. Significant differences were not detected between the mean enzyme-linked immunosorbent assay (ELISA) values on sera collected from calves before exposure to M bovis, using each of the lysozyme extracts or the M bovis PPD (P greater than 0.05). Significant differences were detected when ELISA values obtained using each of the antigens on post-exposure serum were compared with ELISA values on serum collected from calves before exposure to M bovis (P less than 0.01). Differences were not detected in mean ELISA values on sera from cattle collected 12 months after exposure to M bovis, using each of the lysozyme extracts or M bovis PPD (P greater than 0.05); however, 8 of 8 calves were identified as positive on ELISA, using the filter-sterilized lysozyme extract, 7 of 8 calves were positive, using M bovis PPD, and 7 of 8 calves were positive, using the autoclaved lysozyme extract.     

Experimental reproduction of winter dysentery in lactating cows using BCV -- comparison with BCV infection in milk-fed calves

Infection models were developed for adult cows and for young calves using the same strain of bovine coronavirus (BCV), which for the first time allows experimental reproduction of winter dysentery (WD) in seronegative lactating cows. The cattle were infected through direct contact with an experimentally inoculated calf. All experimental cattle shed faecal BCV with development of diarrhoea, being profusely watery with small amounts of blood in the most severely affected animals, including both cows and calves. The cows, in contrast to the calves, showed depressed general condition and appetite leading to a marked decrease in milk yield. Further age-associated differences were a shorter incubation period in the two youngest calves, but with milder fever and milder decrease in white blood cell counts. These findings shed light on the apparent epidemiological differences between WD and calf BCV diarrhoea suggesting that, (1) the same strains of BCV cause natural outbreaks of calf diarrhoea and WD, (2) seronegative cows are more severely affected by the infection than seronegative conventionally reared calves, and (3) unaffected general condition in diarrhoeic calves may lead to underestimation of the occurrence of calf diarrhoea in WD outbreaks.In response to infection, all cattle produced early interferon type 1 in serum and, except for one calf, in nasal secretions. A finding not previously reported is the detection of interferon type 1 responses in bovine milk. All cattle developed high IgM antibody responses and long-lasting IgA antibody responses both systemically and locally. The serum IgM antibody responses came earlier in most of the calves than in the cows. Prolonged IgM antibody responses were detected in serum and milk, while those in nasal secretions were much shorter. BCV-specific IgA was present in nasal secretions from all cattle throughout the 6 months follow-up. The IgA antibody response in serum was detected up to 17 months post-infection and the duration showed an age-related variation indicating a more prominent IgA memory in the adult cattle and in the older calves than in the younger ones. BCV-specific IgG was detected in all cattle during the experimental period of up to 22 months. In conclusion, WD was reproduced in seronegative lactating cows. The cows showed a more severe general diseases than seronegative calves infected concurrently. Very long-lasting IgA antibody responses were detected both systemically and locally.     

Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.