The use of a brucella protein antigen in dermal hypersensitivity as an adjunct method to diagnose bovine brucellosis

A total of 191 cows in 3 herds were injected with a brucella protein antigen in an effort to compare serologic and bacteriologic findings with delayed hypersensitivity and to possibly detect incubative brucellosis. Using cultural and serologic results, the responses to the BPA antigen were inferior to serologic tests in the diagnosis of infection. None of the 23 skin test positive/seronegative cows were positive on subsequent herd blood tests.     

Use of delayed-type hypersensitivity test to diagnose brucellosis in calves born to infected dams

Calves (n = 2) born to dams with experimentally induced brucellosis, and calves (n = 4) born to dams with naturally occurring infection were examined by the delayed-type hypersensitivity (DTH) test for possible B. abortus infection. The results were compared with the serum agglutination test, complement fixation test, and Coombs test. Five calves were nursed by their dams for 8-10 weeks after birth. One calf was separated from its dam and fed artificial milk. Three to five months after birth, four calves tested seropositive in the serologic tests. Antibodies were detected in one calf as early as 1 week after birth. The calf fed on artificial milk was seronegative 4-5 weeks after birth. All calves reacted to the DTH test antigen from week 12 until the end of the experiment, even though serologic tests were negative. We conclude that the DTH test is a valuable technique for diagnosing Brucella in calves born to infected dams.     

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.     

Detection of brucellosis in diary herds after an outbreak of the disease using a delayed-type hypersensitivity test

A study of 14 herds was conducted to assess the skin delayed-type hypersensitivity (SDTH) test used in combination with the serum agglutination test and complement fixation test to curtail the spread of brucellosis. The tests were used to screen dairy herds suspected of brucellosis, and herds located near infected herds. The herds were tested every 5–6 weeks for 6 months. Cattle with a palpable skin swelling at the injection site of the allergen were considered as brucellosis suspects and were slaughtered. Supramammary lymph nodes of these cattle were collected and examined bacteriologically to determine their infection status.

Brucellosis was detected and confirmed bacteriologically in three seronegative herds in which cattle tested positive with the SDTH test. Brucellosis was also confirmed bacteriologically in five seropositive herds in which cattle tested positive with the SDTH test. Brucellosis was not confirmed bacteriologically in one seronegative and four seropositive herds in which cattle reacted positively with the SDTH test. One herd reacted negatively in all tests. Because the SDTH test is able to detect latent carriers of Brucella organisms in seronegative herds, we recommend that the SDTH test be used in combination with serologic tests to curtail the spread of brucellosis.     

The efficacy of the skin delayed-type hypersensitivity using a Brucellin prepared from a mucoid strain of Brucella abortus to detect brucellosis

Eight-hundred-and-ninety-six cattle belonging to herds officially designated Brucella-free, and 190 cattle belonging to infected herds were tested with the skin delayed-type hypersensitivity (SDTH) test, using brucellin (273) prepared from a mucoid strain of Brucella abortus. An increase in skinfold thickness > or = 2 mm was considered a positive SDTH test. The serum agglutination test, complement fixation test and bacteriological examination were used to confirm SDTH test results. Results show that 6 of the 896 uninfected cattle tested positive in the SDTH test, indicating a 99.3% specificity. Of the 44 cattle that tested serologically or bacteriologically positive, 33 tested positive in the SDTH test, indicating a 75% sensitivity. The value of the SDTH test was demonstrated by its ability to detect infection earlier than serological tests, and by confirming infection in cattle with ambiguous serological test results. An increase in skinfold thickness > or = 1 mm in cattle in suspected herds should not be ignored, as it may indicate specific sensitization. We recommend the use of the SDTH test in combination with serological tests for early diagnosis of brucellosis in cattle.     

Evaluation of a delayed-type hypersensitivity test for the diagnosis of Brucella abortus infection in cattle

The delayed-type hypersensitivity (DTH) test was used to diagnose brucellosis in two cows experimentally induced with brucellosis, and 176 dairy cows from a farm suspected of brucellosis. DTH test results were compared with results of the milk ring test, the serum agglutination test, the complement fixation test and the Coombs test. Cows positive in the DTH test and in one of the other tests were examined bacteriologically. In experimentally infected animals the DTH test was positive 10 days after infection, 1-4 weeks before serologic tests indicated brucellosis. Although the DTH test was positive during the whole experiment, on the one occasion when serologic titres were high, it was negative. Of the 176 dairy cows, 45 were positive in one or more serologic tests. In twelve cows (29%) the diagnosis was inconclusive because they were positive in only one of the serologic tests. In these cases the DTH test confirmed the infection. Three cows with high serologic response tested negative in the DTH test. B. abortus was isolated from 13 of 15 cows examined. We conclude that when serologic results are ambiguous, the DTH test is a useful additional technique for diagnosing brucellosis.     

The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated haemolytic anaemia

Background

Diagnosing canine immune-mediated haemolytic anaemia (IMHA) is often challenging because all currently available tests have their limitations. Dogs with IMHA often have an increased erythrocyte osmotic fragility (OF), a characteristic that is sometimes used in the diagnosis of IMHA. Since the classic osmotic fragility test (COFT) is time-consuming and requires specialized equipment, an easy and less labour-intensive rapid osmotic fragility test (ROFT) has been used in some countries, but its diagnostic value has not yet been investigated.This study aimed to evaluate erythrocyte osmotic fragility in dogs with and without IMHA, to compare results of the classic (COFT) and rapid (ROFT) test and to assess the value of the ROFT as diagnostic test for canine IMHA.Nineteen dogs with IMHA (group 1a), 21 anaemic dogs without IMHA (group 1b), 8 dogs with microcytosis (group 2), 13 hyperlipemic dogs (group 3), 10 dogs with lymphoma (group 4), 8 dogs with an infection (group 5) and 13 healthy dogs (group 6) were included.In all dogs, blood smear examination, in-saline auto-agglutination test, Coombs’ test, COFT and ROFT were performed. In the COFT, OF5, OF50 and OF90 were defined as the NaCl concentrations at which respectively 5, 50 and 90% of erythrocytes were haemolysed.

Results

Compared with healthy dogs, OF5 and OF50 were significantly higher in group 1a (P < 0.001) and OF5 was significantly higher in group 3 (P = 0.0266). The ROFT was positive in 17 dogs with IMHA, 10 hyperlipemic dogs, one anaemic dog without IMHA and one healthy dog.

Conclusions

Osmotic fragility was increased in the majority of dogs with IMHA and in dogs with hyperlipidemia, but not in dogs with microcytosis, lymphoma or an infection. Although more detailed information was obtained about the osmotic fragility by using the COFT, the COFT and ROFT gave similar results. The ROFT does not require specialized equipment, is rapid and easy to perform and can be used easily in daily practice. Although, the ROFT cannot replace other diagnostic tests, it may be a valuable additional tool to diagnose canine IMHA.     

Evaluation of bovine cutaneous delayed-type hypersensitivity (DTH) to various test antigens and a mitogen using several adjuvants

The Bacillus Calmette Guerin (BCG)-induced/purified protein derivative (PPD)-elicited tuberculin skin test is a reliable measure of cell-mediated immune response (CMIR), specifically delayed-type hypersensitivity (DTH); however, its use in livestock may confound diagnosis of Mycobacterium tuberculosis. Therefore, various alternative antigen/adjuvant combinations were evaluated as inducers of DTH that were compared to the BCG/PPD test system with the purpose of finding a skin DTH protocol that does not cross-react with the tuberculin test and allows identification of high and low CMIR responder phenotypes. Specifically, 30 non-lactating cows (five/treatment) were sensitized on day 0 with mycobacteria [BCG, M. tuberculosis or Mycobacterium phlei cell wall extract (MCWE)], and ovalbumin (OVA) emulsified in Freund's complete adjuvant (FCA), non-ulcerative Freund's adjuvant (NUFA), complete NUFA or MCWE. On day 21, cows were injected intradermally with various test antigens including PPD tuberculin, phlein, and OVA. Phosphate buffered saline was included as the negative control and the T-cell mitogen phytohemagglutinin (PHA) was also administered. Double skin-fold thickness was evaluated before and at 6, 24, and 48 h post-injection. Skin biopsies were taken at 24 and 48 h to assess oedema, necrosis, and inflammatory cell infiltration. BCG/PPD and M. phlei/phlein treatments when given with a Freund's adjuvant induced equivalent DTH with peak reactions at 24-48 h after antigen injection. Cows receiving NUFA had fewer injection site granulomas than FCA or CNUFA treatments. The change in skin thickness response to PHA peaked at 6 h. Only cows receiving mycobacteria in NUFA had skin response to OVA, which peaked 6-24 h post-injection. Only sites tested with PPD or phlein had significantly higher lymphocyte infiltration than control, whereas neutrophils were significantly higher at PHA test sites and eosinophils predominated at the PHA test sites. Macrophages were significantly more numerous at the PPD and/or phlein test sites in treatment groups that received killed mycobacteria in a Freund's adjuvant and/or with BCG, and at the PHA test sites in all treatment groups. It was concluded that the M. phlei/phlein system did induce DTH and was similar to the DTH induced by the BCG/PPD system when MCWE was administered with a Freund's adjuvant. Therefore, this protocol is suitable for detecting high/low CMIR responders in research herds. However, cross-reaction to PPD was evident following induction of DTH using M. phlei. Hence, this protocol does not alleviate the problem of artificial induction of DTH cross-reactivity and would not be suitable for commercial herds where tuberculin testing is required.     

Equine Culicoides hypersensitivity: evaluation of a skin test and of humoral response

Intradermal tests were carried out on 18 horses with clinical signs of Culicoides hypersensitivity (CHS) and 23 horses without clinical signs of CHS, and sera from these horses were analysed by SDS-PAGE and Western blotting (W-B). Intradermal injections of 0.1 ml of 25 microg/microl sterile Culicoides extract, 0.1 ml of 1:10,000 histamine (positive control) and 0.1 ml of physiological saline (negative control) were made in the dermis of the middle region of the neck. Analysis of reactions indicated that a 1 cm wheal and a skinfold thickness >10% at 24 h represented a valid cut-off between horses with and without CHS. In these conditions the test, even in winter when clinical signs were absent, had 100% sensitivity and specificity. The W-B was performed after running Culicoides extract on a 12% polyacrylamide gel. The test revealed the presence of several bands with molecular weight ranging from 6 to 200 kDa. In particular, a band of 65 kDa was predominantly found in hypersensitive horses by using an anti-IgE antibody while in normal horses the same band was mainly detected by using an anti-IgG antibody. Our results demonstrated that the skin test is a valid diagnostic test, with high sensitivity and specificity and that the band of about 65 kDa probably corresponds to the allergen involved in the pathogenesis of CHS.     

Observations on the use of glyceryl guaiacolate as an adjunct to general anaesthesia in horses

Twenty-one horses undergoing clinical surgery and diagnostic procedures received 15% glyceryl guaiacolate followed by a rapid intravenous injection of a thiobarbiturate for induction of anaesthesia. Premedication was with atropine and acepromazine. Induction was smooth and free from problems apart from transient apnoea in some horses. Maintenance of anaesthesia was with oxygen and halothane administered by means of a closed circle system with soda-lime absorber and with the vaporiser out of circuit. During the period immediately following induction, the heart rate increased and the respiratory rate fell. Blood gas estimations were carried out on 6 horses during anaesthesia. These horses showed respiratory acidosis. Arterial blood oxygen tension values were above those reported in conscious horses. Use of glyceryl guaiacolate in this way provides a safe induction and enables transition to a stable maintenance period which is followed by a quiet and uneventful recovery.     

Use of the brucellosis card test for screening cattle in Saskatchewan.

One group of 28,714 bovine sera were tested by both the brucellosis tube serum agglutination test and the brucellosis card test. The tube serum agglutination test confirmed 99.8% of the negative brucellosis card test results. The brucellosis card test identified 63% of the tube serum agglutination test reactors. In a second group of 496 sera reacting to either the tube serum agglutination test, complement fixation test, plate serum agglutination test or acid antigen serum agglutination test the brucellosis card test identified 99.1% of the complement fixation test positive sera and 91.3% of the sera reacting to any of the other serological tests. The brucellosis card test showed satisfactory agreement with both the complement fixation test and tube serum agglutination test. It appears to be a useful screening test in operations involving large numbers of animals since under these conditions the reactors can be quickly identified and isolated.     

Delayed-type hypersensitivity test for assessing tick-immune status of cattle in Zambia

Delayed-type hypersensitivity skin reactions were used to assess the tick resistance status of Tonga calves in Zambia. The antigen used in the tests was a homogenate of unfed nymphal Rhipicephalus appendiculatus which had been shown to give protective immunity in guinea pigs to adult female R appendiculatus. There was a significant negative correlation between the intensity of the reactions and the total number of ticks (Amblyomma variegatum, R appendiculatus, Hyalomma truncatum, Boophilus decoloratus and Rhipicephalus species) on the animals.