Direct isolation of the agent of enzootic abortion of ewes (Chlamydia psittaci) in cell cultures

Isolation of Chlamydia psittaci in McCoy cell coverslip cultures from clinical material from field cases of enzootic abortion of ewes proved more sensitive than diagnosis by examination of smears stained by Ziehl-Neelsen. Enzootic abortion could be diagnosed in the absence of fetal membranes by culture of fetal lung or liver tissue.     

Chlamydia psittaci: is tonsillar tissue the portal of entry in ovine enzootic abortion?

Ewes at 70 days of gestation were exposed by various routes to a culture of Chlamydia psittaci. Chlamydial infection of fetal tissues, generally accompanied by abortion, was observed only in four of six ewes inoculated via the tonsillar crypts and in five of seven ewes injected subcutaneously; most of these also developed antibodies to C psittaci. Seroconversion after normal lambing was also observed in most ewes inoculated orally or by stomach tube, despite failure to detect chlamydia in them.     

Rapid detection of Chlamydia psittaci in vaginal swabs of aborted ewes and goats by enzyme linked immunosorbent assay (ELISA)

An enzyme linked immunosorbent assay (ELISA) for the detection of Chlamydia psittaci in vaginal swabs of aborted ewes and goats has been developed using microtiter plates coated with sheep anti-Chlamydia immunoglobulin G. This technique was compared to the direct isolation of the agent by plaque assay on McCoy cells. Among 89 specimens from animals in infected flocks, 58 were positive by both methods, seven were only positive by ELISA, and nine others were only positive by direct isolation (plaque assay). None of the 75 specimens from animals in healthy flocks gave a positive response in ELISA or the plaque assay. Unlike direct isolation in cell culture, the ELISA technique permitted the detection of Chlamydia even in the absence of special care in sampling and conservation of specimens.     

A simple staining method for the identification of chlamydial elementary bodies in the fetal membranes of sheep affected by ovine enzootic abortion.

A dark-ground methylene blue (DGMB) staining method was used to demonstrate chlamydial elementary bodies in fetal membranes of sheep affected by Chlamydia psittaci. Before evaluation on material from clinically affected animals, the DGMB method was compared with modified Ziehl-Neelsen (MZN) and dark-ground Giemsa (DGG) staining methods for its ability to demonstrate chlamydial elementary bodies in hens' eggs which had been experimentally infected with C. psittaci. DGMB was more specific in its staining of chlamydial elementary bodies than DGG or MZN. The DGMB method was found to be a more reliable technique for the examination of fetal membranes from sheep affected with C. psittaci than DGG or MZN. Those samples diagnosed as positive using the DGMB showed a good correlation with those diagnosed as positive on macroscopic examination.     

High prevalence of chlamydial (Chlamydophila psittaci) infection in fetal membranes of aborted equine fetuses

Seventy-seven cases of equine abortion from 49 Hungarian farms that occurred between 1998 and 2000 were investigated for the presence of chlamydiae by immunohistochemistry, PCR and/or MZN staining. Evidence of the presence of these bacteria was obtained in 64 cases (83.1%) from 41 (83.7%) different farms. Partial ompA gene sequencing of PCR products revealed that the agent was Chlamydophila psittaci. Based on the findings of microbial diagnosis, pathology and case history, chlamydial infection was considered to be the most likely cause of abortion in at least 11 (14.3%) cases. In the remaining 53 Chlamydophila-positive cases, either other bacterial and viral agents (n = 22 or 28.6%) as well as non-infectious factors (n = 14 or 18.2%) were identified as more probable primary causes of disease, or the role of chlamydiae remained unclear because lesions in fetuses and fetal membranes were absent (n = 17 or 22.1%). When chlamydial antigen was detected in aborted equine placental tissue using immunohistochemistry it was seen only in the chorionic epithelial cells, but not in other parts of the fetal membranes nor in any of the fetal tissues. In conclusion, chlamydial infection of the genital tract should be considered a possible factor in equine reproductive disorders.     

Selenium and vitamin E effect on antibody production of sheep vaccinated against enzootic abortion (Chlamydia psittaci)

The effect of selenium (Se) and vitamin E (vit E) on antibody production of sheep vaccinated against Chlamydia psittaci (ovis) was investigated. Thirty-two sheep, one year old, seronegative to Chlamydia infection, vaccinated against enterotoxemia and dewormed were used. Injectable sodium selenite (0.1 mg/kg b.w.) was given twice to animals of the first group (gSe), with a three week interval. The sheep of the second group (gE) received 1 g vit E each orally, six times at weekly intervals. The animals of the third group (gSeE) were given Se and vit E in doses and routes of administration as in gSe and gE. The animals of the fourth group served as controls (gC) and injected normal saline. The first vaccination was made at the time that the second Se injection was given. Revaccination was made two weeks later. The experiment lasted 29 weeks. The results indicated that Se alone led to a significant increase of Chlamydia antibody response (P < 0.05), but not when it was given in combination with vit E. Animals that received vit E (gE) had much lower titres, just above of those of the controls.     

High prevalence of chlamydial (Chlamydophila psittaci) infection in fetal membranes of aborted equine fetuses

Seventy-seven cases of equine abortion from 49 Hungarian farms that occurred between 1998 and 2000 were investigated for the presence of chlamydiae by immunohistochemistry, PCR and/or MZN staining. Evidence of the presence of these bacteria was obtained in 64 cases (83.1%) from 41 (83.7%) different farms. Partial ompA gene sequencing of PCR products revealed that the agent was Chlamydophila psittaci. Based on the findings of microbial diagnosis, pathology and case history, chlamydial infection was considered to be the most likely cause of abortion in at least 11 (14.3%) cases. In the remaining 53 Chlamydophila-positive cases, either other bacterial and viral agents (n = 22 or 28.6%) as well as non-infectious factors (n = 14 or 18.2%) were identified as more probable primary causes of disease, or the role of chlamydiae remained unclear because lesions in fetuses and fetal membranes were absent (n = 17 or 22.1%). When chlamydial antigen was detected in aborted equine placental tissue using immunohistochemistry it was seen only in the chorionic epithelial cells, but not in other parts of the fetal membranes nor in any of the fetal tissues. In conclusion, chlamydial infection of the genital tract should be considered a possible factor in equine reproductive disorders.     

Protection of ewes vaccinated with A22 strain Chlamydia psittaci (ovis) against challenge in pregnancy with homologous and heterologous strains of the organism

Fifty ewes were randomly divided into four groups. Groups A and B were vaccinated with an experimental vaccine derived from the A22 isolate of Chlamydia psittaci (ovis), an isolate known to cause ovine enzootic abortion (OEA). Groups C and D were unvaccinated controls. In mid-pregnancy, animals in group A and C were challenged with live A22 C. psittaci (ovis) and those in B and D were challenged with a field isolate of the organism (BS) against which the commercially available A22 vaccine appeared to offer poor protection. In group A, three animals showed clinical signs of OEA and six excreted chlamydiae. In group B, five ewes showed clinical signs of OEA and excreted chlamydiae. In group C, three ewes had clinical signs of OEA but seven excreted chlamydiae. In group D, all 11 ewes showed clinical signs of OEA and excreted chlamydiae in the products of parturition. This group produced only four live lambs with an average weight of viable lamb per ewe of 1.4 kg, whereas the other groups each produced 12 or 13 lambs with an average weight of viable lamb per ewe of more than 4 kg. The BS isolate was much more virulent than the A22 isolate for unvaccinated, pregnant ewes. However, the A22 vaccine offered significant protection against the heterologous BS isolate although on this occasion it did not appear to alter the course of disease produced by the less pathogenic A22 isolate.     

Protective adaptive immunity to Chlamydophila abortus infection and control of ovine enzootic abortion (OEA)

Ovine enzootic abortion (OEA) remains a major problem in sheep-rearing countries despite the availability of protective vaccines. The causative agent, Chlamydophila abortus, is a Gram-negative bacterium that can induce a persistent, subclinical infection in non-pregnant sheep. The development of a new safe, effective and practical vaccine requires a detailed understanding of host-pathogen interactions and the identification of clear correlates of protection. Since disease (abortion) is only observed during pregnancy, the nature of host immunity to C. abortus and the specialised immunological features that permit maternal acceptance of the semi-allogeneic fetus are central to the pathogenesis of OEA. We review the current literature on persistence of C. abortus, host immunity to infection and mechanisms of abortion. We identify the key outstanding questions surrounding OEA and discuss the current knowledge gaps with a view to developing improved control strategies.     

Clinical and immunological responses of ewes following vaccination with an experimental formalin-inactivated Chlamydia psittaci (ovis) vaccine and subsequent challenge with the live organism during pregnancy

The protection afforded by an experimental, killed, adjuvanted vaccine derived from the A22 strain of Chlamydia psittaci (ovis) against ovine enzootic abortion was studied. The vaccine was used undiluted (group A), at a dilution of 10(-3) (group B) and at a dilution of 10(-6) (group C). A fourth control group (group D) was inoculated with all components of the vaccine except the chlamydial antigen. A group of rams (group R) was also vaccinated with the chlamydial antigen diluted to 10(-3). Animals were challenged 70 days after mating with the A22 strain of C. psittaci (ovis) and were studied throughout pregnancy and the subsequent lambing period. Their cell-mediated immune responses were examined using a skin test and their humoral immune responses were studied using an ELISA. Tests for excretion of chlamydiae in their faeces and genital tract during pregnancy and after parturition and in the faeces of their lambs were made. The reproductive performance of the ewes was assessed by calculating the average weight of lambs produced per ewe in each group. The experimental vaccine protected the ewes in groups A and B against challenge with C. psittaci (ovis) as none showed clinical signs of OEA or excreted chlamydiae. The average weight of lambs produced per ewe in both groups was greater than 4 kg. Both groups seroconverted after vaccination but not all of them were positive to the skin test. The experimental vaccine at 10(-6) dilution of antigen did not protect the ewes as three of 10 ewes displayed clinical OEA and excreted chlamydiae in the products of parturition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of an enzyme immunoassay for detection of Chlamydia psittaci in vaginal secretions, placentas, and fetal tissues from aborting ewes

A commercially available enzyme immunoassay (EIA) for the detection of Chlamydia trachomatis in human urogenital and conjunctival specimens was compared with isolation in cell culture for the detection of Chlamydia psittaci in vaginal and placental swabs from aborting ewes and swabs of aborted fetal tissues. The EIA on vaginal swabs collected from 10 ewes experimentally infected with C. psittaci had a sensitivity of 85.7% and a specificity of 85.7%. Vaginal swabs collected at the time of abortion or within 3 days were the best samples for detection of chlamydial infection. The 29 vaginal swabs collected during this period from experimentally infected ewes were all strongly EIA-positive, and chlamydia were isolated from 28. The EIA on vaginal swabs from 78 field cases of abortion had a sensitivity of 78.0% and a specificity of 76.8%. The EIA on swabs of cotyledons from 65 placentas had a sensitivity of 100% and a specificity of 75.0% compared with isolation in cell culture. The EIA on 57 swabs of fetal tissues or body fluids from 10 aborted fetuses or weak lambs from experimentally infected ewes had a sensitivity of 26.6% and a specificity of 88.1% compared with isolation in cell culture. Limitations of the EIA are discussed.     

The relationship between nasal myiasis and the prevalence of enzootic nasal tumours and the effects of treatment of Oestrus ovis and milk production in dairy ewes of Roquefort cheese area

Infection by Oestrus ovis is common in Lacaune dairy ewes of Roquefort cheese area (Aveyron, France). It is believed by local breeders that there is a close relationship between nasal myiasis and the incidence of enzootic nasal tumour. In order to check these anecdotal reports, a serological survey was done on 658 breeding ewes before turn-out and 897 breeding and primiparous (hoggets) ewes at the end of the grazing season. By the time of sampling, it was clear whether the sheep were infected at the end of the winter or had been re-infected over summer.In April and September, 40.7 and 26.3%, respectively, were free of O. ovis infection, indicating that the autumn treatment was not completely effective and that O. ovis adult flies were circulating during the summer in many flocks. There were no differences in the incidence of adenocarcinoma between the groups indicating that there is no relationship between O. ovis infection and the presence of the cancer.Differences in milk production between the three groups were not statistically significant (Anova test P>0.05). In flocks where 1-5% of the ewes were infected or in non-infected flocks, ewes produced 3.6 and 8.56%, respectively, more milk than ewes from flocks where more than 5% of animals were infected. For primiparous ewes, the differences were of 8.5 and 12.24%.     

Evaluation of an ELISA to detect antibodies to maedi-visna virus in individual and pooled samples of milk from sheep

An elisa was used to detect antibodies to maedi-visna virus in samples of serum and milk from individual sheep; the results obtained indicated that the elisa can be used to detect antibodies in milk. The assay was also applied to samples of bulk-tank milk; a standard curve was created and used to calculate the seroprevalence of maedi-visna in 11 flocks of sheep and the results were compared with the results obtained by applying the elisa to individual serum samples. There was good agreement between the seroprevalences calculated from the standard curve for bulk-tank milk and from the individual serum samples.     

Use of egg yolk in serological tests (ELISA and HI) to detect antibody to Newcastle disease, infectious bronchitis, and Mycoplasma gallisepticum

Serum and yolks from commercial flocks and from hens exposed to Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and Mycoplasma gallisepticum (MG) were tested for immunoglobulin G antibody by the enzyme-linked immunosorbent assay (ELISA) and the hemagglutination-inhibition (HI) test. Yolks prepared by chloroform extraction and low-speed centrifugation performed well in the serological tests used and were a suitable alternative to serum for antibody determination by the ELISA for NDV, IBV, and MG and by HI test for NDV.