The intravenous administration of johnin in the diagnosis of paratuberculosis in practice

The diagnostic value of the intravenous injection of 1.6 or 3.2 ml of johnin was evaluated in a total of 116 cattle. The sensitivity was 84% with clinically suspect cattle. A general reaction occurred frequently as well as elevation of body temperature (average 1.47 degrees C). The observance of a general reaction was a good additional source of information when evaluating the test. There was no significant difference in elevation of temperature between the cattle injected with 1.6 ml and those injected with 3.2 ml. A combination of diagnostic techniques such as the complement fixation test, intravenous johnin test, microscopic examination of the faeces and a biopsy examination of the rectal mucosa were necessary, in order to confirm the diagnosis of Johne's disease. In this manner a diagnosis can be confirmed with clinically suspect cattle in 96% of the cases. The sensitivity of the intravenous johnin test was considerably lower in cases of non-clinical Johne's disease.     

Aggregate testing for the evaluation of Johne's disease herd status

This paper examines methods for evaluating herd Johne's disease status that could be used in a survey of the cattle industry. Emphasis is placed on aggregate testing, a process whereby a random sample of cattle from a herd is assessed using an imperfect test, such as an ELISA for detecting antibody in serum. Important aggregate test parameters discussed include: sample size, herd-level sensitivity, herd-level specificity, the number of reactors used for declaring a positive herd result, and the expected within-herd prevalence of disease. Aggregate testing may be useful for several livestock diseases. However, problems arise when it is applied to Johne's disease because of the poor sensitivity of the available diagnostic tests, the low within herd prevalence of infection, and clustering of false positives within a herd.     

Diagnosis of bovine spongiform encephalopathy: A review

Summary

Cows affected with bovine spongiform encephalopathy (BSE) display chronic neurological signs consisting of behavioural changes, abnormalities of posture and movement, and/or hyperaesthesia. At present, there are no laboratory test available to diagnose BSE in the live animal. In this article, we describe the post‐mortem diagnostic examination of brains from BSE‐suspected cattle as currently performed at ID‐Lelystad. The routine laboratory diagnosis of BSE consists of histopathological examination of the brain and detection of the modified prion protein, PrP^BSE, in brain tissue. These tests, however, have the disadvantage of being laborious and time consuming, so that results are available only after several days.

Recently, at ID‐Lelystad a new post‐mortem test has been developed that enables screening of larger volumes of brain samples for PrP^BSE within 1 day. This BSE test is especially suited for slaughterline monitoring. A preliminary validation study has shown that both sensitivity and specificity are 100% compared to the gold diagnostic standard of histopathology.     

Detection of Mycobacterium avium subsp. paratuberculosis in skeletal muscle and blood of ewes from a sheep farm in New Zealand

Abstract

AIM: To determine whether viable Mycobacterium avium subsp. paratuberculosis (Map) is present in skeletal muscle and blood in ewes with and without Johne's disease confirmed histologically.

METHODS: A total of 51 mixed-aged ewes in poor body condition from a farm with a history of clinical Johne's disease were culled and examined at necropsy. BACTEC radiometric culture was performed on samples of skeletal muscle from the biceps femoris, mononuclear cells in peripheral blood (hereafter referred to as blood), and ileum. Histological sections and Ziehl-Neelsen (ZN)-stained impression smears of terminal ileum and mesenteric lymph nodes were examined. Ewes were defined as having confirmed Johne's disease if there was histopathological evidence typical of the disease within the ileum and adjacent lymph nodes.

RESULTS: Eighteen of 21 (86%) ewes with confirmed clinical Johne's disease were culture-positive for Map from sites peripheral to the alimentary tract, comprising 15 from skeletal muscle and 13 from blood. Five of 30 (17%) ewes that did not have Johne's disease were culture-positive, with four from skeletal muscle and one from blood. The likelihood that ewes with confirmed Johne's disease had systemic Map infection compared with ewes without was determined as OR=30 (95% CI=6.3–142.0; p<0.001).

CONCLUSION: The prevalence of Map infection of skeletal muscle and blood in ewes with confirmed Johne's disease was 71% and 62% respectively, and in unaffected ewes was 13% for muscle and 3% for blood.

CLINICAL RELEVANCE: Skeletal muscle and blood are potential sources of exposure of humans to Map, and the risk appears higher from sheep with Johne's disease.     

Application of different methods for the diagnosis of experimental paratuberculosis in goats

The diagnosis of subclinical paratuberculosis is still considered a major problem worldwide. As part of investigating diagnostic strategies for the paratuberculosis infection, sequential results of various diagnostic methods in a progressive experimental infection in goats were evaluated. Twenty-three goat kids were divided into three groups: the infected, contact and control, comprising 10, five and eight goats respectively. Animals of the infected group were orally inoculated on seven occasions with 5 ml of inoculum containing 2 x 10(9)Mycobacterium avium ssp. paratuberculosis per ml. Lymphoycte proliferation test using johnin PPD detected paratuberculosis infection from 60 days post-infection (DPI) onwards. The johnin PPD was found to be a better antigen for the proliferative assays as compared with the sonicated antigen. The faecal smear examination with acid-fast staining detected more goats as positive than bacterial culture and polymerase chain reaction (PCR). Lipoarabinomannan enzyme-linked immunosorbent assay (ELISA) started detecting infected goats from 150 DPI onwards followed by indirect ELISA and agar gel immunodiffusion from 180 DPI onwards. Histological examination was confirmatory and detected five infected goats as positive.     

Loop-mediated isothermal amplification (LAMP) test for specific and rapid detection of Brucella abortus in cattle

Background: Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level.

Objective: To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle.

Methods: LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR).

Results: The developed LAMP assay works at 61 °C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB® green dye I was used to visualize the result with naked eye.

Conclusion: The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.     

Diagnostic value of intravenously administered johnin purified protein derivative (PPD) in bovine paratuberculosis

The diagnostic value of intravenously administered johnin purified protein derivative (PPD) was studied in 45 cattle of different age, coming from herds infected by, or free from, Mycobacterium paratuberculosis. In addition to observing the clinical symptoms, the animals' sera were assayed for specific antibodies by the complement fixation (CFT) and immunodiffusion (AGID) tests. The blastogenic transformation of peripheral lymphocytes was determined on the basis of 3HTdR incorporation. Changes in the neutrophilic leucocyte/lymphocyte ratio of the blood were also monitored. Detection of the pathogen in the faeces was attempted by microscopic examination and by culturing. Combined evaluation of responses elicited by intravenously administered johnin PPD can be a valuable aid in recognizing infected animals, particularly those among the heifer progeny of infected cows.     

Intravenous johnin and tuberculin tests in cattle vaccinated with Mycobacterium paratuberculosis cells and subsequently inoculated with Mycobacterium bovis.

Calves at 30 days of age were vaccinated with a killed whole-cell Mycobacterium paratuberculosis vaccine. Four months later, these calves were inoculated with Mycobacterium bovis. The intravenous tuberculin and johnin tests were applied both before and after inoculation. The results of the hematologic investigation had extremes at both high and low values and were too unsuitable for statistical analysis. The intravenous tuberculin test is considered unsuitable for diagnosis of bovine tuberculosis in cattle vaccinated against paratuberculosis.     

Comparison of subjective and objective test evaluations for use of Mycobacterium phlei-adsorbed serum in a dot-enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in cattle.

Use of a dot-ELISA with serum adsorbed with Mycobacterium phlei or with nonadsorbed serum was compared. In addition, results attained using visual observation were compared with those obtained using a densitometer. Infection status of cattle was determined by results of culture of feces from a number of cattle with various degrees of exposure (low prevalence and test-negative) and disease manifestation (clinical suspect vs subclinical infection). Two paratuberculosis-negative herds, fecal culture-confirmed clinically suspect cases of paratuberculosis, and cows from 2 paratuberculosis-infected herds with diagnosis confirmed on the farm (low infection rate) were tested. Significant (P less than 0.05) increase in the dot-ELISA response was found in cattle with heavy M paratuberculosis shedding when nonadsorbed and adsorbed sera were used, compared with the response in cattle that were fecal culture-negative or were shedding M paratuberculosis at lower amounts. Paratuberculosis was diagnosed by visual determination in 29 of 44 (65.9%) of fecal culture-positive, clinically suspect cattle when nonadsorbed serum was used. Results of the visual test were negative in 85 of 93 (91.4%) of the fecal culture-negative cattle when nonadsorbed serum was used. However, when using M phlei-adsorbed serum, the sensitivity of the visual determination decreased to 34.1% (15/44), and the specificity increased to 97.8% (91/93).(ABSTRACT TRUNCATED AT 250 WORDS)     

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 evidence for exposure to bovine viral diarrhoea virus in sheep co-grazed with beef cattle in New Zealand

ABSTRACT

Aims: To determine whether sheep that co-grazed with cattle that were suspected to be positive for bovine viral diarrhoea (BVD) virus had serological evidence of exposure to the virus.

Methods: Eighteen commercial farms that routinely co-grazed cattle and sheep in the same paddocks were recruited through purposive sampling. The recruiting veterinarians identified nine farms with cattle herds that were known or highly suspected to be positive for BVD and nine farms that were considered to be free of BVD. Blood samples were taken from 15 ewes aged 1 year on each farm and samples were submitted to a commercial diagnostic laboratory to test for antibodies against pestiviruses using an ELISA. All samples that were positive were then tested using a virus neutralisation test (VNT)for antibodies against BVD virus.

Results: Of the 270 blood samples, 17 were positive for pestivirus antibodies by ELISA and these originated from two farms that were known or suspected to have BVD virus-positive cattle. None of the samples from the nine flocks co-grazed with cattle herds that were known or suspected to be BVD virus-negative were positive for pestivirus antibodies. Within the two positive farms, 2/15 samples from the first farm and 15/15 samples from the second farm were antibody-positive. When the 17 positive blood samples were submitted for VNT, all 15 samples from the second farm tested positive for BVD virus antibodies with the highest titre being 1:512.

Conclusions and clinical relevance: In this small sample of New Zealand sheep and beef farms with suspected BVD infection in cattle, there was evidence of pestivirus exposure in co-grazed sheep. Although we were unable to confirm the origin of the exposure in these sheep, these findings highlight that farmers who are trying to eradicate BVD from their cattle should be mindful that the infection may also be circulating in sheep, and both populations should be considered a possible risk to each other for generating transient and persistent infections. Further work is needed to estimate the true prevalence of New Zealand sheep flocks that are affected by BVD and the associated economic impacts.     

The detection of Mycobacterium paratuberculosis in bovine faeces by isolation and the comparison of isolation with the examination of stained smears by light microscopy

The detection of Mycobacterium paratuberculosis organisms in bovine faeces by isolation was compared with that by the microscopical examination of Ziehl-Neelsen stained faecal smears for the presence of clumps of acid-fast M. paratuberculosis organisms. Faeces were obtained from cattle naturally or experimentally infected with M. paratuberculosis as well as from uninfected cattle. Microscopical examination was an unreliable method for the detection of M. paratuberculosis organisms since the organisms were only detected in 99 (=55.9%) of 177 culturally positive faecal samples. In addition, clumps of acid-fast organisms indistinguishable from M. paratuberculosis were also observed in three of 18 samples from cattle free from Johne's disease and in 18 of 37 culturally negative samples from paratuberculous cattle. When M. paratuberculosis organisms were added to faeces from an uninfected cow, results showed that isolation attempts should be positive when 15 or more M. paratuberculosis organisms per gram of faeces are present.     

Use of BACTEC radiometric culture method and polymerase chain reaction for the rapid screening of faeces and tissues for Mycobacterium paratuberculosis

SUMMARY The BACTEC radiometric culture method for detection of Mycobacterium paratuberculosis was evaluated on faeces from cattle on a farm in quarantine for Johne's disease. A multiplex polymerase chain reaction (PCR) based on the IS900 sequence specific for M paratuberculosis and a genus specific 16S rRNA region was developed and used to test cultures showing evidence of mycobacterial growth in the BACTEC liquid radiometric culture medium. Using the BACTEC - PCR combination, confirmation of M paratuberculosis from faeces and tissue of clinically affected animals was achieved within 2 to 4 weeks and 1 week, respectively, a substantial improvement on traditional culture and identification methods. The PCR provided rapid exclusion of M paratuberculosis when other Mycobacterium spp were grown. The radiometric culture medium proved to be very sensitive for culturing Mycobacterium spp.     

Using indirect ELISA to assess different antigens for the serodiagnosis of Fasciola gigantica infection in cattle, sheep and donkeys

An indirect enzyme-linked immunosorbent assay (iELISA) was evaluated for its diagnostic capability in detecting antibodies against Fasciola gigantica infection in cattle, sheep and donkeys sera using crude worm, excretory–secretory and glutathione S-transferase antigens prepared from adult liver fluke. Presence of F. gigantica worms at post-mortem examination of cattle, sheep and donkey’s livers was taken as a gold standard for the evaluation of the assay. The diagnostic sensitivity, specificity and accuracy percentages of iELISA were determined for each antigen. Excretory–secretory antigen gave the best results for the serodiagnosis of F. gigantica infection in cattle, sheep and donkeys using iELISA with diagnostic sensitivity percentages of 93.3%, 94.9% and 93.3%, respectively, while the specificity percentages were 96.7%, 97.2% and 96.3%, respectively, whereas the accuracy percentages were 95%, 96% and 95.7%, respectively. The diagnostic sensitivity percentages of iELISA using crude worm antigen were 96.7%, 100% and 93.3%, respectively, while the specificity percentages were 80%, 83.3% and 85.2%, respectively, whereas the accuracy percentages were 88.3%, 86.7% and 87%, respectively. The diagnostic sensitivity percentages of iELISA using glutathione S-transferase antigen were 66.7%, 71.8% and 60%, respectively, while the specificity percentages were 70%, 77.8% and 77.8%, respectively, whereas the accuracy percentages were 68.3%, 74.7% and 73.9%, respectively. Conclusively, excretory–secretory antigen dependent iELISA can be used as a reliable serodiagnostic test for F. gigantica infection in cattle, sheep and donkeys.     

Comparison of IS900 tissue PCR, bacterial culture, johnin and serological tests for diagnosis of naturally occurring paratuberculosis in goats

Comparative efficacy of an IS900 tissue PCR, bacterial culture, johnin, agar-gel immunodiffusion (AGID) and absorbed-ELISA tests was investigated in 43 goats naturally infected with paratuberculosis. On histological examination, tissue sections from all animals showed typical granulomatous inflammatory changes. The lesions were classified as multibacillary (MB) (n=30), which had diffuse granulomatous lesions with abundant acid-fast bacilli (AFB), and paucibacillary (PB) (n=13), which had focal or multifocal granulomatous lesions with few AFB. The sensitivities of johnin test, tissue culture, faecal culture, tissue PCR, AGID and ELISA were 68% (17/25), 100% (30/30), 84.6% (22/26), 100% (30/30), 96.2% (25/26) and 100% (26/26) in MB goats, and 88.8 (8/9), 46.1% (8/13), 40% (4/10), 61.5% (8/13), 50% (5/10), and 70% (7/10) in PB goats, respectively. Except for the johnin test, which showed higher sensitivity in PB goats, all other tests displayed significantly higher sensitivities in MB goats. The results indicate the usefulness of tissue PCR, culture and serological tests in the diagnosis of clinically affected paratuberculous goats, especially with multibacillary pathology.     

Use of enoyl coenzyme A hydratase of Mycobacterium avium subsp. paratuberculosis for the serological diagnosis of Johne's disease

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), remains difficult to control because of the lack of specific and sensitive diagnostic tests. In order to improve the specificity of sero-diagnosis for JD, the phage display library derived from genomic DNA of MAP was immunoscreened to identify novel antigenic targets. We selected a clone using antibodies from MAP experimentally infected cattle, and annotated its coding sequence as MAP1197 in the MAP genome, which encoded “echA12_2” in the MAP protein (Map-echA) belonging to Enoyl-CoA hydratase, known as a crotonase enzyme. The Map-echA was expressed in Esherichia coli and purified as a histidine-tag recombinant protein (rMap-echA), and the diagnostic potential of the protein was further evaluated by enzyme-linked immunosorbent assays (ELISA). Antibody responses to rMap-echA were higher in MAP-infected cattle than in uninfected cattle. The specificity of the Map-echA ELISA was also confirmed by evaluation with hyper-immune sera against various kinds of Mycobacterium species. Furthermore, in all experimentally infected cattle the antibody against rMap-echA was detected 2–7 months earlier than by a commercially available ELISA kit. These results suggested that Map-echA can be used as a specific and sensitive serological diagnostic antigen for the detection of MAP infection.     

The use of skin delayed‐type hypersensitivity as an adjunct test to diagnose brucellosis in cattle: A review

Summary

Brucellosis, caused by bacteria of the genus Brucella, is a contagious disease that causes economic loss to owners of domestic animals due to loss of progeny and milk yield. Because cattle, sheep, goats, and to a lesser extent pigs are considered to be the source of human brucellosis, serological tests have been used to screen domestic animals for antibodies against Brucella. Although the serological tests helped to eradicate brucellosis in many countries, serological tests are not always adequate to detect latent carriers of Brucella. Therefore, the use of the skin delayed‐type hypersensitivity (SDTH) test, which is independent of circulating antibodies, might improve the diagnosis of brucellosis. In the literature, however, there are conflicting reports as to the value of the SDTH test for the diagnosis of brucellosis. Some studies consider the test unreliable, whereas others advocate its use because it detects brucellosis earlier than serological tests. The objectives of this study were therefore to assess the characteristics of the SDTH test, to select a Brucella strain that will yield a suitable brucellin for use in the field, and to determine whether the use of serological tests in combination with the SDTH test improves the detection of brucellosis. The results of this study clearly show that the SDTH test detects latent carriers of Brucella and confirms brucellosis in cattle with ambiguous serological test results. Brucellins prepared from smooth or mucoid strains of Brucella are better suited for use in the field than brucellins prepared from rough strains because they detect brucellosis in cattle with acute as well as chronic infection. The SDTH test is highly specific (99.3% specificity), and repeated testing of naive cattle or cattle infected with microorganisms that serologically cross‐react with Brucella does not sensitize cattle to subsequent SDTH tests. However, it is possible that some naive cattle may serologically react to the injection of brucellin. The effect of these serological reactions on the sero‐diagnosis of brucellosis is limited, because cattle may only now and then react serologically either with the serum agglutination test (SAT) or the complement fixation test (CFT). Nevertheless, cattle infected with microorganisms that serologically cross‐react with Brucella may test seropositive for brucellosis 4 to 7 weeks after injection of brucellin, depending on the cross‐reacting microorganism. The value of the SDTH test for the diagnosis of brucellosis was demonstrated after an outbreak of brucellosis. When the SDTH test was used in combination with SAT and CFT at diagnostic threshold ≥2 mm or ≥1 mm (increase in skinfold thickness), respectively, 39/44 (88%) or 42/44 (95%) of the infected cattle were detected compared with only 27/44 (61%) when SAT and CFT were used. When cattle in areas of low prevalence or in areas free from brucellosis are tested with the SDTH test an increase ≥2 mm in skinfold thickness should be considered indicative of infection. When the control and eradication of brucellosis is based on test‐and‐slaughter, an increase of ≥1 mm in skinfold thickness should be considered indicative of infection. Repeated serological testing complemented with the SDTH test in this programme will shorten the quarantine (movement control) period of a suspect herd, limiting the financial loss incurred during outbreaks of the disease. Consequently, since the SDTH test usually does not interfere with the serological diagnosis and can safely be used to establish the infection status of cattle in a suspect herd, it is opportune to consider adding the SDTH test to the procedure currently used to diagnose brucellosis in individual animals.     

Heartwater (Cowdria ruminantium infection) on São Tomé

Summary

The supernatant obtained after centrifugation of an emulsion of engorged female Amblyomma astrion ticks, collected from cattle on the West African island of São Tomé, was injected intravenously into a goat, which contracted heartwater. This confirmation of the existence of the disease on the island makes strict control measures necessary if present efforts at improving livestock production by imported exotic cattle are to be successful.     

Some observations on reproductive performance in selected commercial beef herds

Abstract

Extract

Although the opinion has been expressed (Andrews, 1971 Andrews, E. D. 1971. Cobalt deficiency in sheep and cattle. N.Z. Dept. Agric. Bull., : 180–180.  [Google Scholar]) that cobalt deficiency among cattle has virtually disappeared in New Zealand and that specimens for laboratory examination are rarely required, 15 to 20% of the liver samples submitted by veterinarians to the Animal Health Reference Laboratory for vitamin B₁₂ assay are from cattle. Diagnostic criteria for cobalt (vitamin B₁₂) deficiency cy have long been established for sheep but not for cattle In view of the continuing requests for vitamin B₁₂ analyses on bovine livers, it was desirable to establish normal values for clinically normal cattle so that results of routine diagnostic analyses could be more readily interpreted.     

First report of the use of mucosal swabs of the palatine tonsillar crypt for detection of Mycoplasma bovis in naturally infected calves

ABSTRACT

Aims: To compare detection by real-time PCR of DNA from Mycoplasma bovis on mucosal swabs taken from the palatine tonsillar crypt and the mainstem bronchi of clinically asymptomatic calves after slaughter.

Methods: We compared the sensitivity of mucosal swabs taken from two sites: the palatine tonsillar crypt and the mainstem bronchi. Paired samples were taken post-mortem at slaughter from 55 clinically well calves from an infected herd and were tested by real-time PCR for the presence of M. bovis-specific DNA.

Results: Mycoplasma bovis DNA was detected in 51 palatine tonsillar crypt swabs (92.7 (95% CI?=?82.4–98.0)%) and seven mainstem bronchial swabs (12.7 (95% CI?=?5.3–24.5)%). All seven calves with positive mainstem bronchial swabs also had positive palatine tonsillar crypt swabs.

Conclusions: When compared to mucosal swabs of the mainstem bronchi, mucosal swabs of the palatine tonsillar crypt were seven times more sensitive for the post-mortem detection of M. bovis DNA. The viability of detected M. bovis was not assessed, because any cattle carrying viable or non-viable M. bovis DNA were determined to be a potential risk to eradication. Palatine tonsillar crypt mucosa may be a useful anatomical site for real-time PCR detection of M. bovis DNA in naturally infected calves. More work is needed to define the persistence and viability of M. bovis at this anatomical site.

Clinical relevance: The results of this study helped form the basis of surveillance tools used in M. bovis control and eradication efforts. Familiarity with these results may help veterinarians better communicate with their clients about the science behind the eradication efforts.