Future options for brucellosis surveillance in New Zealand beef herds

It is probable that bovine brucellosis will be eradicated from New Zealand by 1990. However, continued surveillance for the disease will have to be maintained for a number of years. This paper examines alternative methods of surveillance in beef herds with a view to ensuring that the most cost effective method is used. The three surveillance methods examined are the present automated complement fixation test, abattoir surveillance, and a system based on a delayed hypersensitivity skin test using a purified Brucella protein antigen. It is concluded that despite the relatively low sensitivity of the skin test the probability of its identifying herds as infected is likely to be greater in practice than abattoir surveil- lance. The skin test is cheaper than the present complement fixation test.     

Monitoring of dairy herds for Brucella abortus infection when prevalence is low

A total of 2,698 dairy herds were surveyed in 1981–1982 in New South Wales and north eastern Victoria in a review of the methods used to monitor them for the presence of Brucella abortus., The methods used to monitor dairy herds were testing of all breeding cows over 1 year of age using the rose bengal test (RBT) and complement fixation test (CFT), the bulk milk ring test (BMRT), and testing of blood samples collected at abattoirs using the RBT and CFT. The surveyed herds had at least one whole herd test, and BMRT was done at regular intervals in the period of the survey. Of the 99 (3.7%) herds that reacted to the BMRT, 91 (3.4%) herds had false positive reactions and 8 (0.3%) herds were declared infected on follow-up herd testing. False-positive reactions were obtained in 22 herds on more than one occasion. Common causes of false positive reactions to the BMRT were thought to be previous vaccination with Strain 19 and sampling in very early or late lactation. Of the 98 (3.63%) herds that reacted to the whole herd serological tests, 80 (2.96%) herds had false-positive reactions and 18 (0.67%) herds were declared infected. Strain 19 vaccination was thought to be an important cause of false-positive reactions. Fifty-three (2.0%) herds showed suspicious reactions on abattoir monitoring but none was declared infected on follow-up testing. Of the 18 herds with infected or equivocal status, the BMRT identified 8. In a further 6 herds, the infected cattle were not in the milking herd. Four other herds had milkers with high CFT titres which could not be confirmed as infected on culture. In no herds were culture positive RBT or CFT reactors from the milking herd detected without the BMRT being positive. The proportion of false-positive reactions to the BMRT was high but the BMRT proved very useful in identifying dairy herds infected with B. abortus, when the prevalence of brucellosis was very low. Aust Vet J, 64 : 97–100     

Haemophilus somnus: a comparison among three serological tests and a serological survey in beef and dairy cattle.

Serological tests for the detection of antibodies against Haemophilus somnus were carried out in herds of beef and dairy cattle using three different techniques: agglutination, complement fixation and counterimmunoelectrophoresis. The agglutination test appeared to detect more seroreactors than the complement fixation and counterimmunoelectrophoresis tests. Results of the three tests indicated that there were more positive reactors in beef cattle and dairy cattle from infected herds than in dairy cattle from clinically normal herds.     

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.     

An intradermal test for the diagnosis of brucellosis in extensively managed cattle herds

The application of a delayed hypersensitivity test for the diagnosis of bovine brucellosis was examined in a series of field experiments. The test is based on the intradermal injection of ‘Brucellin’, a lipopolysaccharide-free protein extract of Brucella abortus strain 45/20.

The Brucellin test was compared with the complement fixation (CF) test in 8656 cows of mixed age and known vaccination and herd status. An intradermal injection of 0.1 ml of the allergen was made in either the cervical region or the caudal fold. The injection site was examined 72 h after administration of Brucellin and any increase in skin thickness of 2 mm was regarded as positive.

When administered into the caudal fold site the Brucellin test had a sensitivity relative to the CF test of between 52 ± 14% and 61 ± 6%. The relative specificity of the test exceeded 99%.

Calfhood vaccination with B. abortus strain 19 did not result in positive Brucellin test results. There was no evidence that the injection of Brucellin induced a serological response.

Despite the low relative sensitivity of the Brucellin test, it is a useful low-cost tool for identifying infected herds. It is a least as effective as slaughterhouse surveillance systems.     

A comparative examination of swine sera for antibody to Aujeszky virus with the conventional and a modified virus-serum neutralization test and a modified direct complement fixation test.

Sera of pigs from élite breeding herds, of boars and sows collected at slaughter-houses, and of pigs from herds known to be infected, were examined for antibody to Aujeszky virus. The conventional and a modified virus-neutralizing antibody (VNA) test and a modified direct complement fixation (CF) test were employed. In simultaneous titrations of positive sera the modified VNA test gave titers approx. 4 log2 units above the titers obtained by the conventional test. The conventional VNA test was found insufficiently sensitive. Unspecific neutralization in the modified VNA test was infrequent in serum dilution 1/2 and rare in dilution 1/4. The GF tests on sera of slaughter sows and animals from known infected herds showed a remarkable consistency with the VNA tests. Inconsistent results were obtained with but few sera. Abt. 5 % of the sera could not be examined because of complement fixation with control antigen.     

Application of an enzyme-linked immunosorbent assay in the final stages of a bovine brucellosis eradication program

Bovine field serums from the Australian brucellosis eradication program were used to compare 2 enzyme linked immunosorbent assays (ELISA) with the complement fixation test (CFT) and Rose Bengal test (RBT). One ELISA used an anti-bovine IgG horseradish peroxidase conjugate (ELISA 1) and the other a monoclonal anti-bovine Ig alkaline phosphatase conjugate (ELISA 2). When compared with the CFT, the ELISA 2 like the ELISA 1 lacked specificity in B. abortus vaccinated herds but the ELISA 2 was more specific than the ELISA 1 in previously infected herds and equally as specific as the ELISA 1 in nonvaccinated Brucella free herds. In this study the ELISA 2 proved more sensitive than the CFT, RBT and ELISA 1 particularly in herds where B. abortus biotype 2 was present. The value of using the ELISA 2 in conjunction with the CFT in an eradication program is discussed.     

Demonstrating freedom from disease using multiple complex data sources 2: case study--classical swine fever in Denmark

A method for quantitative evaluation of surveillance for disease freedom has been presented in the accompanying paper (Martin et al., 2007). This paper presents an application of the methods, using as an example surveillance for classical swine fever (CSF) in Denmark in 2005. A scenario tree model is presented for the abattoir-based serology component of the Danish CSF surveillance system, in which blood samples are collected in an ad hoc abattoir sampling process, from adult pigs originating in breeding herds in Denmark. The model incorporates effects of targeting (differential risk of seropositivity) associated with age and location (county), and disease clustering within herds. A surveillance time period of one month was used in the analysis. Records for the year 2005 were analysed, representing 25,332 samples from 3528 herds; all were negative for CSF-specific antibodies. Design prevalences of 0.1-1% of herds and 5% of animals within an infected herd were used. The estimated mean surveillance system component (SSC) sensitivities (probability that the SSC would give a positive outcome given the animals processed and that the country is infected at the design prevalences) per month were 0.18, 0.63 and 0.86, for among-herd design prevalences of 0.001, 0.005 and 0.01. The probabilities that the population was free from CSF at each of these design prevalences, after a year of accumulated negative surveillance data, were 0.91, 1.00 and 1.00. Targeting adults and herds from South Jutland was estimated to give approximately 1.9, 1.6 and 1.4 times the surveillance sensitivity of a proportionally representative sampling program for these three among-herd design prevalences.     

Rotavirus infections associated with diarrhoea in calves in Egypt

The successful isolation and identification of rotavirus from newborn calves with diarrhoea is reported for the first time in Egypt. From 25 faecal samples taken from diarrhoeic calves, ten virus isolates were found to give cytopathogenic effects on bovine embryonic kidney cells. Three of the isolates were identified as rotavirus using fluorescent antibody staining, serum-neutralization, complement fixation and agar gel precipitation. The complement fixation test revealed the presence of rotavirus antibodies in 18 of 105 serum samples obtained from other calves slaughtered at Cairo abattoir.     

Potential infection-control benefit for Ireland from pre-movement testing of cattle for tuberculosis

Pre-movement testing for bovine tuberculosis (BTB) was compulsory in Ireland until 1996. We determined the proportion of herd restrictions (losing BTB-free status) attributable to the recent introduction of an infected bovid; described events between restoration of BTB-free status (de-restriction) and the next herd-level test for BTB; estimated the proportion of undetected infected cattle present at de-restriction; identified high-risk movements between herds (movements most likely to involve infected cattle); and determined the potential yield of infected cattle discovered (or herds that would not lose their BTB-free status) by pre-movement testing, relative to the numbers of cattle and herds tested. We used national data for all 6252 herds with a new BTB restriction in the 12 months from 1 April 2003 and 3947 herds declared BTB-free in the 12 months from 1 October 2001. We identified higher-risk animals from our logistic generalized estimating-equation models. We attributed 6-7% of current herd restrictions to the recent introduction of an infected animal. There were considerable changes to herd structure between de-restriction and the next full-herd test, and infection was detected in 10% of herds at the first assessment (full-herd test or abattoir surveillance) following de-restriction. Following movement from a de-restricted herd, the odds of an animal being positive at the next test increased with increasing time in the source herd prior to movement, increasing time between de-restriction and the next full-herd test and increasing severity of the source herd restriction. The odds decreased with increasing size of the source herd. We estimated that 15.9 destination-herd restrictions per year could be prevented for every 10,000 cattle tested pre-movement and that 3.3 destination-herd restrictions per year could be prevented for every 100 source herds tested pre-movement. The yield per pre-movement test can be increased by focusing on high-risk movements; however, this would result in a substantial decrease in the total number of potential restrictions identified.     

A comparative study of the complement fixation test for mycoplasma hyopneumoniae in SPF and non-SPF herds of swine in Illinois

Serum samples from 458 swine from eight specific pathogen-free (SPF) herds swine were compared to samples from 178 swine from eight non-SPF herds in Illinois using the complement fixation (CF) test for Mycoplasma hyopneumoniae antibodies. In SPF herds, 9.4% of the samples gave positive test results, compared to 51.7% of the samples from non-SPF herds. The range of reactor rates in SPF herds was 0–26.9% and in non-SPF herds 3.4–100%. Used on herd basis with some modification, the CF test should be useful in monitoring state and nationally accredited SPF swine herds.     

Risk-based surveillance of antimicrobial residues in pigs – Identification of potential risk indicators

Around 20,000 samples are analysed each year for the presence of antibacterial residues in Danish finisher pigs, and between zero and five samples are detected positive above the maximum residue level (MRL). The intention was to develop a risk-based surveillance programme involving fewer samples while ensuring equal sensitivity. Therefore, risk indicators were searched for. Data were obtained from the Danish slaughterhouse database covering the period from July 2010 to December 2012. Residues were found or suspected in 17 incidents. In nine of these, the farmer had called in to prevent the pigs from being slaughtered. Hence, eight suspect cases were found through the surveillance programme, and two of these were above MRL. For these eight case herds, the number of pigs slaughtered and the number in which each of the following lesions were found were included in a statistical analysis: chronic pleuritis, tail bite, chronic pericarditis, chronic pneumonia, chronic peritonitis, osteomyelitis, abscess in hindquarters, abscess in leg/toe and abscess in forequarters. Only chronic pleuritis was associated with the presence of residues. Next, data from all herds delivering pigs for slaughter to the same abattoir were included covering a 3-month period prior to the residue finding. The prevalence of chronic pleuritis was on average 1.7 times higher in the eight case herds compared to all other herds. In two herds, the prevalence was significantly higher (P ≤ 0.05), and in one herd substantially higher, but only borderline significant (P = 0.1). In the remaining herds, the prevalence did not differ from the other herds delivering pigs to the abattoir. This indicates that chronic pleuritis might be considered as a risk indicator for use in surveillance. Other risk indicators/factors – reported in the cases where the farmers called in – were inadequate marking of treated animals and incorrect use of medication dispensers. These factors are not suited for use in surveillance and should be dealt with otherwise.     

A comparison of five serological tests for bovine brucellosis.

Five serological assays: the buffered plate antigen test, the standard tube agglutination test, the complement fixation test, the hemolysis-in-gel test and the indirect enzyme immunoassay were diagnostically evaluated. Test data consisted of results from 1208 cattle in brucellosis-free herds, 1578 cattle in reactor herds of unknown infection status and 174 cattle from which Brucella abortus had been cultured. The complement fixation test had the highest specificity in both nonvaccinated and vaccinated cattle. The indirect enzyme immunoassay, if interpreted at a high threshold, also exhibited a high specificity in both groups of cattle. The hemolysis-in-gel test had a very high specificity when used in nonvaccinated cattle but quite a low specificity among vaccinates. With the exception of the complement fixation test, all tests had high sensitivities if interpreted at the minimum threshold. However, the sensitivities of the standard tube agglutination test and indirect enzyme immunoassay, when interpreted at high thresholds were comparable to that of the complement fixation test. A kappa statistic was used to measure the agreement between the various tests. In general the kappa statistics were quite low, suggesting that the various tests may detect different antibody isotypes. There was however, good agreement between the buffered plate antigen test and standard tube agglutination test (the two agglutination tests evaluated) and between the complement fixation test and the indirect enzyme immunoassay when interpreted at a high threshold. With the exception of the buffered plate antigen test, all tests were evaluated as confirmatory tests by estimating their specificity and sensitivity on screening-test positive samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

An evaluation of Irish cattle herds with inconclusive serological evidence of bovine brucellosis

Since 1998, there has been a steady decline in herd restrictions and de-populations in Ireland due to bovine brucellosis. There is concern that the interpretation of laboratory results may become increasingly problematic, as brucellosis prevalence falls in Ireland. Therefore, the purpose of the current study was to evaluate the infection status of Irish herds and animals with inconclusive serological evidence of bovine brucellosis. During 12 months from September 1, 2004, laboratory and observational epidemiological data were collected from all Irish herds where animal testing identified at least one animal with a complement fixation test (CFT) reading greater than zero and/or a positive result to the indirect enzyme-linked immunosorbent assay (iELISA). Due to the observational nature of the study, we have robust estimates of the relative, but not the absolute, performance of the CFT, iELISA and brucellin skin test (BST). Herds were divided into three categories (Group A, B or C) on the basis of test results at initial assessment. A total of 639 herds were enrolled into the study, and observed for at least two years following enrolment. A rising CFT titre, with a CFT reading of 111 International CFT Units (IU) or greater at the subsequent blood test, was generally associated with herds where other evidence of infection was also available. Knowledge of the CFT reading at the initial and a subsequent blood test proved useful in distinguishing false-positive and true-positive brucellosis results. There was poor correlation between the CFT and iELISA results, and between the CFT and BST results. As a result of this study, national policy has been modified to include re-sampling of all animals with CFT readings of 20 IU or greater. This project has also led to a reduction in the number of herds restricted, as well as restriction duration. It has also contributed to a reduction in the number of herds listed for contiguous tests, and therefore the potential for contiguity testing of false positive results.     

The Dutch Brucella abortus monitoring programme for cattle: the impact of false-positive serological reactions and comparison of serological tests

The Dutch national Brucella abortus eradication programme for cattle started in 1959. Sporadic cases occurred yearly until 1995; the last infected herd was culled in 1996. In August 1999 the Netherlands was declared officially free of bovine brucellosis by the European Union. Before 1999, the programme to monitor the official Brucella-free status of bovine herds was primarily based on periodical testing of dairy herds with the milk ring test (MRT) and serological testing of all animals older than 1 year of age from non-dairy herds, using the micro-agglutination test (MAT) as screening test. In addition, serum samples of cattle that aborted were tested with the MAT. The high number of false positive reactions in both tests and the serum agglutination test (SAT) and complement fixation test (CFT) used for confirmation seemed to result in unnecessary blockade of herds, subsequent testing and slaughter of animals. For this reason, a validation study was performed in which three indirect enzyme-linked immunosorbent assays (ELISAs), the CFT and the SAT were compared using a panel of sera from brucellosis-free cattle, sera from experimentally infected cattle, and sera from cattle experimentally infected with bacteria which are known to induce cross-reactive antibodies (Pasteurella, Salmonella, Yersinia, and Escherichia). Moreover, four ELISAs and the MRT were compared using a panel of 1000 bulk milk samples from Brucella-free herds and 12 milk samples from Brucella abortus- infected cattle. It is concluded that the ELISA obtained from ID-Lelystad is the most suitable test to monitor the brucelosis free status of herds because it gives rise to fewer false-positive reactions than the SAT.     

Screening of Hungarian cattle herds for Mycoplasma mycoides subspecies mycoides small colony infection with negative results

At abattoirs and farms, 1248 sera were collected from animals representing 121 farms, and examined by complement fixation test using Mycoplasma mycoides subspecies mycoides small colony type (MmmSC) antigen. All sera were negative except seven from four farms, giving ++ reactions in the serum dilution of 1:10. On retesting, these sera and additional 30 sera collected repeatedly in both farms gave negative results. In isolation attempts, 953 lung samples collected from slaughtered cattle at the same abattoirs, and 326 nasal swabs collected from 11 herds proved to be negative for the presence of MmmSC, but M. bovis was isolated frequently. In the small farms 23.95% of the animals had pleurisy and/or pneumonia while in the large herds 34.69% had lesions. DNA extracted from 50 nasal swabs and 430 lung samples was examined by polymerase chain reaction (PCR) using M. mycoides cluster-specific primers. DNA from further 325 lung samples was tested by the more specific M. mycoides subspecies mycoides small colony/large colony/capri specific primers and 196 samples by nested PCR specific for MmmSC. All gave negative results. The detection level of cluster-specific primers and the more specific primers was 33.4 pg of DNA, whereas that of nested PCR was 0.33 pg.     

OBSERVATIONS ON SINGLE BRUCELLOSIS REACTORS IN LARGE HERDS

Over a 3 year period specimens were collected from single reacting cattle to the complement fixation test for brucellosis from large herds in north Queensland. Twenty-four such reactors were destroyed for culture, and Brucella abortus was isolated on 3 occasions. The existence of such low levels of Br. abortus in large herds should not be overlooked in the eradication campaign. Explanations were found for 17 of the single reactors, and included introduction of cattle to the herd, exposure to adjoining infected herds or vaccination with Strain 19.     

Use of an enzyme-linked immunosorbent assay in a bovine brucellosis eradication program

An enzyme-linked immunosorbent assay (ELISA) was developed and was compared with the complement fixation test (CFT) in a bovine brucellosis eradication program. The ELISA detected significantly more reactors than the CFT in both strain 19 vaccinated infected herds (1.79% versus 1.14%) and non-vaccinated infected herds (4.2% versus 3.59%) but not in either vaccinated or non-vaccinated brucella-free herds. The specificity for both tests in brucella-free herds was greater than 0.998. The specificity and sensitivity of the ELISA were compared with those of 3 other tests (the Rose Bengal test; the indirect haemolysis test [IHLT] and the CFT) on serum from 151 animals cultured at slaughter. The calculated specificity of the ELISA in this infected group was lower than for both the CFT and the IHLT (0.58 versus 0.67 versus 0.75). The sensitivity however was much greater (1.0 versus 0.73 versus 0.71). The value of the ELISA when used in an eradication program is discussed.     

Bovine tuberculosis: prevalence and diagnostic efficacy of routine meat inspection procedure in Woldiya municipality abattoir north Wollo zone, Ethiopia

Bovine tuberculosis (BTB) is a widespread and endemic disease of cattle in Ethiopia posing a significant threat to public health. Regular surveillance by skin test, bacteriology, and molecular methods is not feasible due to lack of resources. Thus, routine abattoir (RA) inspection will continue to play a key role for national surveillance. A cross-sectional study was conducted at Woldiya municipal abattoir from April 1, 2009 to April 5, 2010 to estimate the prevalence of BTB in slaughtered cattle on the basis of detailed abattoir inspection and to compare efficacy of RA inspection with respect to detailed abattoir inspection and isolation and identification of Mycobacterium. Diagnostic accuracies (with corresponding measures of statistical uncertainty) were determined by computing test property statistics (sensitivity and specificity). Agreement between RA and detailed abattoir inspections was measured using kappa statistics. Out of 1,029 slaughtered heads of cattle examined during the study period, 63 (6.12 %) and 15 (1.45 %) were diagnosed with gross tuberculous lesions by detailed abattoir meat inspections and RA meat inspections, respectively, making a prevalence of 6.12 % (95 % CI: 5.2–7.1) on the basis of detailed abattoir inspection. About 59.45 % of tuberculous lesions were observed in the lungs and associated lymph nodes, whereas 35.13 % lesions were from the lymph nodes of the head. From 63 cattle suspected with tuberculosis (TB) based on detailed abattoir meat inspection, nine (19.05 %) were identified as Mycobacterium bovis, while three (4.8 %) as Mycobacterium tuberculosis. The sensitivity of RA meat inspection was 23.8 % in comparison to the detailed abattoir meat inspection and 25 % in comparison to culture, respectively. Poor agreement (k?=?0.37) was seen between RA meat examination and detailed abattoir meat examination methods. Similarly, poor agreement (k?=?0.013) was seen between RA meat examination and culture results. In conclusion, relatively higher prevalence (6.12 %) was recorded in Woldiya municipal abattoir on the basis of detailed Abattoir inspection and RA meat inspection protocols currently utilized in Ethiopia which are insufficient to detect the majority (76.19 %) of TB lesions at the gross level, which indicates the magnitude of meat borne zoonotic TB as an ongoing risk to public health. Detailed abattoir inspection protocols were demonstrated to improve the detection level by approximately fourfold. In conclusion, routine meat inspections have limitations in detecting BTB-suggestive lesions which indicate the magnitude of meat-borne zoonotic TB as an ongoing risk to public health.