Comparison of PCR assay and bacteriological culture method for the detection of Brucella melitensis in stomach content samples of aborted sheep fetuses

The aim of this study was to evaluate the polymerase chain reaction (PCR) assay for detection of Brucella melitensis in stomach content samples of aborted sheep fetuses and to compare its performance with bacteriological culture method. It was also aimed to determine the agreement between PCR and Rose Bengal plate test (RBPT). Materials were collected from aborted sheep from 109 different sheep flocks in the region of Van during the lambing seasons of 2004-2005 and 2005-2006. Stomach contents from 135 aborted sheep fetuses were examined by bacteriological culture and PCR, and 135 sera from these aborting ewes were tested by RBPT. Identification and typing of Brucella strains were performed using standard classification test. B. melitensis biovar 3 was isolated from 26 (19.2%) of foetal stomach contents. B. melitensis was detected by PCR in 29 (21.4%) stomach content samples. Twenty five sera (18.5%) from aborting ewes tested positive by RBPT. The detection limit of B. melitensis 16 M strain by PCR was 1.7 x 10(3) cfu (colony forming units) /ml in spiked stomach contents. Diagnostic sensitivity and specificity of the PCR were detected as 100% and 97.2%, respectively. The agreement between PCR and RBPT was found to be 97%. In conclusion, PCR assay would have an advantage over conventional bacteriological culture method, but in particular for its ability to meet the specificity requirements for the detection of B. melitensis in stomach content samples of aborted sheep fetuses.     

The isolation and serology of Brucella melitensis in a flock of goats in central RSA

Brucella melitensis biotype 1 was isolated in pure culture from the lungs, liver, spleen, kidney, stomach contents, abomasum and brain of an aborted caprine (Boer goat) foetus in the district of Cullinan near Pretoria. The 18 does and 1 ram in the flock of Boer goates were examined serologically by means of the complement fixation (CF) test, using Brucella abortus antigen. Six weeks later they were examined again, using B. abortus as well as B. melitensis biotype 1 antigens. No significant differences were found between the 2 CF tests using B. abortus antigen, or between the results obtained by using the B. abortus and B. melitensis antigens. Twelve goats, showing CF antibody titres, were slaughtered and examined bacteriologically. No relationship was found between the serological and bacteriological results.     

Field evaluation of a PCR for the diagnosis of chlamydial abortion in sheep

A total of 94 vaginal swab samples and 195 serum samples collected from aborted ewes in 15 flocks were examined by pcr and a complement fixation test, respectively. In addition, 172 samples of stomach contents from fetuses from different flocks submitted for the diagnosis of abortion during the four lambing periods between 2000 and 2004 were tested by pcr. Chlamydial dna was detected in seven vaginal swabs obtained from five of the 15 flocks and in six samples of fetal stomach contents. The results of pcr and flock serology for Chlamydia were positive in five of the 15 flocks and negative in eight.     

A live-attenuated and an inactivated chimeric porcine circovirus (PCV)1-2 vaccine are both effective at inducing a humoral immune response and reducing PCV2 viremia and intrauterine infection in female swine of breeding age

The objective of this pilot study was to determine the efficacy of inactivated (1 or 2 dose) and live-attenuated chimeric porcine circovirus (PCV)1-2 vaccines in sows using the PCV2-spiked semen model. Thirty-five sows were randomly divided into 6 groups: negative and positive controls, 1 dose inactivated PCV1-2 vaccine challenged (1-VAC-PCV2), 2 dose inactivated PCV1-2 vaccine challenged (2-VAC-PCV2), 1 dose live-attenuated PCV1-2 vaccine unchallenged (1-LIVE-VAC), and 1 dose live-attenuated PCV1-2 vaccine challenged (1-LIVE-VAC-PCV2). The inactivated PCV1-2 vaccine induced higher levels of PCV2-specific antibodies in dams. All vaccination strategies provided good protection against PCV2 viremia in dams, whereas the majority of the unvaccinated sows were viremic. Four of the 35 dams became pregnant: a negative control, a positive control, a 2-VAC-PCV2 sow, and a 1-LIVE-VAC-PCV2 sow. The PCV2 DNA was detected in 100%, 67%, and 29% of the fetuses obtained from the positive control, inactivated vaccinated, or live-attenuated vaccinated dams, respectively. The PCV2 antigen in hearts was only detectable in the positive control litter (23% of the fetuses). The PCV1-2 DNA was detected in 29% of the fetuses in the litter from the 1-LIVE-VAC-PCV2 dam. Under the conditions of this pilot study, both vaccines protected against PCV2 viremia in breeding age animals; however, vertical transmission was not prevented.     

Influence of maternal obesity on fetal development in pigs

Fetuses from linebred lean (L) and linebred obese (O) and reciprocal crossmatings were examined at 110 d of gestation for line, maternal and heterotic effects. There was no significant heterotic effect for any trait measured. A significant maternal effect was observed for adipose tissue lipoprotein lipase (LPL) activity and for serum triglycerides. The enzyme activity and triglycerides concentration were higher in fetuses from O dams than in fetuses from L dams. In a lipid clearance test, no maternal effect was observed for changes in serum concentrations of triglycerides and free fatty acids or in optical density (associated with the disappearance of injected Liposyn). Linebred O fetuses exhibited higher LPL activity in both the biceps femoris muscle and sc adipose tissue compared with linebred L fetuses. The LPL activity of the adipose tissue was higher than that of the skeletal muscle. The percentage of dry matter, percentage of triglycerides and protein/DNA were higher in the muscle of linebred O fetuses than in that of linebred L fetuses. Based on tissue LPL activity and on muscle compositional traits, linebred O fetuses were more mature at 110 d of gestation than were the linebred L fetuses. Maternal obesity had little detectable influence on fetal development of the pig when measured at 110 d of gestation.     

布鲁氏菌omp28基因的克隆、表达及反应原性分析

本研究克隆了羊种布鲁氏菌16M株、羊种布鲁氏菌M28株、犬种布鲁氏菌、绵羊附睾种布鲁氏菌、牛种布鲁氏菌A19株、猪种布鲁氏菌S2株的omp28基因并对以上不同种菌株的omp28基因序列及编码的氨基酸序列进行了比对,结果显示不同种布鲁氏菌omp28基因之间仅6个碱基不同,而且只有2个氨基酸不同,亲水性分析结果显示两处氨基酸的差异对蛋白亲水性不造成影响.将羊种布鲁氏菌16M的omp28基因亚克隆到pET32a中表达,OMP28在低温下诱导以可溶性形式高效表达.Westem-blot结果显示OMP28反应原性良好,是布鲁氏菌病诊断抗原的可能选择.     

Fetal and maternal immunologic manifestations of intrauterine adenovirus SV-20 infection

Rhesus monkey fetuses of either immune or nonimmune dams were inoculated in utero with Adenovirus SV-20 (AdSV-20), a virus capable of inducing fetal pneumonia, and studied immunologically at various intervals. AdSV-20 infection at 90–100 days gestational age resulted in absolute lymphopenia in a few fetuses, reduced numbers of peripheral blood lymphocytes (PBL) which formed rosettes with sheep erythrocytes (ERL) and reduced complement-receptor lymphocytes (CRL) in a majority, while Fc fragment-receptor lymphocytes (FcRL) were occasionally increased. There was a tendency for depression of ERL and CRL early in infection of 120–130-day fetuses, followed by stimulation of these populations and FcRL in later phases. Maternal immunity did not protect against these effects of AdSV-20 infection in fetuses. Immune and nonimmune dams were spared adverse clinical effects and had no changes in lymphoid cell populations following inoculation of their fetuses.Despite precocious production of circulating IgM, fetuses of nonimmune dams had little or no demonstrable anti-AdSV-20 serum neutralizing (SN) antibody, indicating that the ability to develop an effective immune response was suppressed or had not been acquired at the gestational ages studied. Nonimmune dams displayed little evidence of seroconversion following inoculation of their fetuses with AdSV-20, except in those dams whose fetuses died in utero, whereby there was a significant antibody response. SN antibody titers of immune dams were not boostered substantially subsequent to inoculation of their fetuses, and fetal SN titers were lower than maternal titers, suggesting absence of an active fetal antibody response in this group also. Direct inoculation of AdSV-20 into 90–130-day rhesus monkey fetuses provided a model system for immunologic study of fetal infection, probably involving complex fetal-maternal interactions, in a situation where the infected, viable fetus and its dam appeared to be microbiologically isolated from one another.     

Preliminary evidence for a diagnostic immunoglobulin G1 antibody response among culture-positive cows vaccinated with Brucella abortus strain 19 and challenge exposed with strain 2308

The sera of cows inoculated with Brucella abortus have a characteristically high titer of immunoglobulin (Ig) G1 antibodies to a soluble brucella antigen compared with sera of noninoculated vaccinated cattle. Concentrations of antigen-specific IgG1 were greater than 10-fold higher than those for IgG2, even though total IgG2 concentrations were higher than total IgG1 concentrations. Increases in IgG1 antibodies to Brucella abortus soluble antigen were detected shortly after vaccination in those cows from which strain 19 was isolated and by 28 weeks in cows from which strain 2308 was isolated. Increases in specific antibodies were not paralleled by increases in either total IgG1 or total IgG2 concentrations. Rather, there was a 15-fold to greater than 200-fold increase in specific activity, with up to 16% of the IgG1 specific for the brucella antigen used in the assay. Thus, measurement of changes in total IgG1 concentrations is not a reliable method to identify brucellosis-associated anti-Brucella abortus soluble antigen activity. Only one cow in a panel of 10 selected for detailed study showed a false-positive IgG1 titer, whereas some serologic assays showed as many as 4 or 5 false-positives. Results of the complement-fixation test, among the battery of serologic tests used for detection of brucellosis, best agreed with the occurrence of increased IgG1 antibody levels.     

Experimental infection of pregnant goats with swine fever virus

Thirteen pregnant goats were inoculated intravenously with the ALD strain of virulent swine fever (SF) virus on Days 64-84 of gestation. Dams showed transient and mild viremia, and produced high serum neutralizing (SN) antibody after inoculation. Six inoculated dams were reared until parturition occurred and bore six apparently normal, one apparently normal but dead, one mummified and three edematous kids. Neutralizing antibody was demonstrated in the pre-colostral sera obtained from all normal kids, but no SF virus was isolated from any of them. The other seven dams were killed on post-inoculation days (PID) 5-61, and fetuses, placenta and amnion were tested for the virus and SN antibody. All fetuses of five dams examined within PID 40 were positive for SF virus, but negative for SN antibody. SF virus was also isolated from one of three fetuses examined on PID 61. Conversely, the other two fetuses examined on PID 61 were negative for SF virus, but positive for SN antibody. Furthermore, SF virus was isolated from the placenta and amnion of all the dams.     

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 indirect hemagglutination test for the diagnosis of Eperythrozoon suis infection in swine.

An indirect hemagglutination test was developed to detect naturally occurring eperythrozoonosis in swine and to follow experimentally induced infections serologically. The antigen used was prepared from the plasma of acutely infected, splenectomized pigs. The test was specific and reliable in detecting swine latently infected with the disease.     

Serogroups of Campylobacter fetus and Campylobacter jejuni isolated in cases of ovine abortion

Of 38 aborted ovine fetuses from 23 sheep flocks 29 C. fetus subsp. fetus and 22 C. jejuni were isolated and examined biochemically and serologically for heat-stable antigens. Serologic examinations were carried out by passive haemagglutination test. In case of C. fetus subsp. fetus strains alkaline antigen extraction was used. Antisera to two serogroups of C. fetus and to Penner serotype reference strains 1 to 60 were produced in rabbits. Abortion was caused in 18 (78.3%) flocks by C. fetus subsp. fetus and in 5 (21.7%) flocks by C. jejuni. Six C. fetus subsp. fetus strains grew well at both 43 and 25 degrees C. With one exception all C. fetus subsp. fetus were resistant, whereas all 29 C. fetus subsp. venerealis strains were sensitive to 30 micrograms/ml cefoxitin and cefamandole. These two cephalosporins can be used to differentiate the two subspecies of C. fetus. Passive haemagglutination test using alkaline antigen extraction is a proper method for the examination of heat-stable antigens of both C. fetus subspecies. Out of 24 C. fetus subsp. fetus strains 13 belonged to serogroup A(01), and 11 to serogroup B(02). C. jejuni strains examined belonged to Penner serogroup 1 (6 strains), to serogroup 5 (4 strains) and to serogroup 8 (4 strains).     

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.     

Serologic examination of aborted ovine and bovine fetal fluids for the diagnosis of border disease, bluetongue, bovine viral diarrhea, and leptospiral infections

Fetal serum from most of 994 bovine and 553 ovine aborted fetuses was tested serologically for antibodies to border disease (BD), bovine viral diarrhea (BVD), and bluetongue (BT) viruses, and to Leptospira sp., and the results were compared with the results of isolation procedures, fluorescent antibody tests (FAT), and histologic examinations of the same fetuses. Antibodies to BT virus were not found in any of the 994 bovine and 553 ovine fetuses. Antibody titers to BVD virus were present in 39 of 966 bovine fetuses tested, and BVD virus was detected in 4 of the 39. Four of 74 fetuses in which the BVD virus was detected by FAT or isolation had titers to BVD virus. Microagglutination (MAT) titers to 1 or more of 5 serovars of leptospires were present in 52 of 773 bovine fetal sera tested. Leptospires were not detected by FAT in any bovine fetuses that had leptospiral antibody titers. Leptospires were detected by FAT in 15 aborted calves, and none of these had MAT titers. Antibody titers to BD virus were present in 80 of 486 fetal lamb sera tested, and the virus was detected by FAT or isolation in 3 of the 80 fetuses. Border disease virus was detected in 14 of 486 fetal lambs tested. Twelve of the 14 were tested serologically and 3 had titers to BD virus. Leptospiral antibody titers were present in 27 of 326 ovine fetal sera tested. Leptospires were not detected in any of the 326 ovine fetuses tested by FAT.     

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

Summary

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.     

In vitro isolation and characterization of bovine Neospora caninum in Korea

The brains of nine aborted bovine fetuses and two newborn calves born from dams suspected to be infected with Neospora caninum were homogenized and inoculated into Vero cells. All fetuses and calves were from cows determined as seropositive to N. caninum by an IFA test. Sera and thoracic fluids of all fetuses and calves also revealed high antibody titer to N. caninum by IFAT ranging from 1:800 to 1:3200. N. caninum was isolated from the brains of one aborted fetus and one newborn calf when the brain homogenates were grown continuously in Vero cell culture. N. caninum tachyzoites, giemsa-positive, were first observed on Days 45 and 56 postinoculation in the newborn calf and the aborted fetus, respectively. The isolates (KBA-1 and KBA-2) were morphologically and ultrastructurally similar to previously published Neospora isolates. The isolated parasites were confirmed as N. caninum by means of the antigenic reactivities, immunostaining, PCR and southern blotting, and electron microscopy.     

Bovine leukemia virus: Duration of BLV colostral antibodies in calves from commercial dairy herds

A study was conducted to determine the duration of colostral antibodies to bovine leukemia virus (BLV) in diary calves from commercial dairy farms. Sera of pregnant dams from four different dairy farms and sera of the calves of these dams were analyzed for BLV antibodies using the agar gel immunodiffusion (AGID) test. Precolostral serum samples were collected from the female calves of known BLV serologically positive dams. Postcolostral serum samples of the same calves were collected on Day 2 and biweekly until 2 consecutively negative AGID tests performed 4 weeks apart were obtained. Subsequently, the biweekly serum samples from each calf were analyzed quantitatively for BLV antibodies with the AGID test. End-point titers were determined using phosphate buffered saline to make two-fold dilutions. A logarithmic transformation of the inverse of the end-point titer was used to determine the regression line of antibody decay for each calf. Estimated weighted regression analysis was used to determine the least-squares regression line for 27 of the 38 calves sampled. The duration of colostral antibodies was calculated as 71 days using the prediction equation. The minium and maximum durations of colostral antibodies were 14 days and 147 days, respectively. The half-life of the antibodies was 36.05 days. Factors affecting the duration of BLV colostral antibodies and the practical applicability of this study were discussed.     

Isolation of Getah virus from dead fetuses extracted from a naturally infected sow in Japan.

Three viruses producing a cytopathic effect in cell culture were isolated from dead fetuses extracted from a naturally infected sow, and were found to be serologically identical by neutralization tests. One of the viruses was cloned and named the Sakura strain. The Sakura strain was identified as Getah virus by cross-neutralization tests.     

Studies on the pathogenesis of epizootic bovine abortion

Ornithodoros coriaceus Koch ticks were fed on 37 pregnant cows. The fetuses were obtained from the cows at 23 to 126 days after maternal tick exposure. Characteristic lesions of epizootic bovine abortion were observed only in those fetuses recovered 100 days more or after maternal tick exposure. Fetuses collected between 50 and 100 days showed mild-to-moderate lymphoid and mononuclear cell hyperplasia. Reaction in fetuses studied less than 50 days after maternal tick exposure were mild. Lesions could not be seen in 2 of the youngest fetuses. Increases in serum immunoglobulin concentrations were present only in those fetuses examined 80 days or more after their dams had been exposed to ticks. The specificity of the immunoglobulins could not be determined. Sera from 12 fetuses tested failed to fix complement in tests for group-specific chlamydial antibodies. A wide variety of microbiological cultivation attempts were made to recover the causative agent of epizootic bovine abortion from these fetuses; however, no agent was recovered regularly, and chlamydial organisms were not recovered from any. The significance of 2 recovered agents, apparently viral, is still to be determined. Fetal tissues, both frozen and fresh, collected from fetuses of dams exposed to a feeding of ticks were capable of reproducing the disease after inoculation into pregnant cows or directly into fetuses.     

Enzyme-linked immunosorbent assay for detecting antibodies to Aujeszky's disease virus in pigs

An indirect micro enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Aujeszky's disease virus in pigs is described. A control antigen prepared from infected cells was included for each serum tested. Of 243 sera from serologically positive farms, 175 (72 per cent) and 147 (60 per cent) were positive by the ELISA test and microtitre serum neutralisation test, respectively. Failure to include a control antigen for each serum would have resulted in 14 sera (6 per cent) being differently recorded. Results for sera from experimental and field infections indicated that seroconversion was more quickly detected by the ELISA test than the microtitre serum neutralisation test. In addition to greater sensitivity the ELISA test has other advantages over the serum neutralisation test. ELISA is a rapid, cheap test which is not dependent on a continuous supply of cell cultures and which can be readily automated.