Experimental infection of pregnant ewes with enteric and abortion-source Chlamydophila abortus

Two groups of pregnant ewes were experimentally infected oronasally in midpregnancy. A faecal and an abortion-source isolate of Chlamydophila abortus were used. They were derived from a healthy ewe from a flock with no history of abortion, and from an aborted foetus in a farm with enzootic abortion. As assessed by modified Ziehl-Neelsen (MZN) staining, egg culture, antigen ELISA, the Clearview test and immunohistochemistry, inoculation resulted in placental and/or foetal infection in all ewes. Histopathology revealed placentitis in two and four ewes inoculated with the enteric or abortion-source isolate, respectively, in addition, these samples were immunohistochemically positive for chlamydial antigen. All six ewes infected with the enteric isolate and five of seven ewes infected with the abortion-source isolate showed evidence for a serological response by an indirect ELISA or CFT. Neither chlamydiae nor lesions were detected in the placentae and lambs of the uninfected control ewes, which remained seronegative. Our results suggest that enteric C. abortus can be associated with placental and foetal lesions in sheep.     

Rechallenge of previously-infected pregnant ewes with Chlamydophila abortus

In an attempt to ascertain the means whereby previous exposure to Chlamydophila (C.) abortus can protect against the re-occurrence of enzootic abortion of ewes (EAE), ten previously-exposed ewes were intravenously rechallenged with a large infective dose of C. abortus during pregnancy. The patterns of development of chlamydial placentitis and its sequelae closely resembled that observed following first-time challenge of previously-na?ve ewes, although placentitis appeared to develop more slowly following rechallenge infection and none of the rechallenged ewes aborted. Chorioallantoic and foetal pathology and foetal immune responses were qualitatively similar whilst the local maternal response to C. abortus infection of the endometrium did not appear to differ in rechallenged and first-time challenged sheep. This demonstrates that if C. abortus reaches the foetal side of the placenta, a stereotypical response is elicited, regardless of the status of maternal immunity. Therefore it appears that in natural circumstances, acquired immunity of the dam protects against the re-occurrence of EAE by preventing the causative agent from reaching the susceptible foetal trophoblast.     

Rechallenge of previously-infected pregnant ewes with Chlamydophila abortus

We studied some behavioural, clinical, biochemical and haematological variables in Desert (Najdi) sheep and goats subjected to the acute stressful stimulus of isolation from the flock, and the influence of pretreatment with xylazine (n = 6) or sodium betaine (n = 6). The isolation stress resulted in increased vocalization and in variable and statistically nonsignificant increases in heart, pulse and respiratory rates. Isolation caused significant increases in the plasma concentrations of cortisol (from about 35.2 to about 83.8 mmol/L) and glucose (from 3.1 to 4.2 mmol/L), and a decrease in that of magnesium (from 0.82 to 0.65 mmol/L). The endogenous thiocyanate concentration was unaffected. The isolation stress also significantly decreased the haematocrit (PCV), and the number of lymphocytes, and increased the concentration of haemoglobin. Pretreatment of sheep and goats with xylazine at a dose of 0.01 mg/kg by the intravenous route significantly ameliorated the effects induced by the stressful stimulus. The effects of pretreatment of the two species with sodium betaine (10 mg/kg) produced variable and nonsignificant effects. There were no significant differences between sheep and goats in the responses to the isolation stress, except in vocalization, which was greater in sheep than in goats.     

Identification of Chlamydophila abortus and the development of lesions in placental tissues of experimentally infected sheep

Chlamydophila (C.) abortus is a major cause of infectious abortion in sheep in many countries. Twenty-one pregnant sheep were experimentally infected intranasally with C. abortus at 70 days of gestation (dg). Thereafter, a number of animals were killed at weekly intervals and a post-mortem examination was carried out. Evidence of chlamydial infection in the placenta was determined by isolation of the bacterium by tissue culture and detection of C. abortus DNA by real-time polymerase chain reaction (real-time PCR). In addition, histopathological changes in the placenta were assessed, as was the detection of chlamydial antigen by immunohistochemistry (IHC). Evidence of placental infection was observed as early as 2 weeks after inoculation, and while only relatively low numbers of bacteria were isolated by culture and/or detected by real-time PCR prior to 113-114dg, at 119-121dg, it was more numerous. This study, using the four criteria for assessment of infection, showed that while C. abortus gained access to the placenta as early as 85dg, characteristic histopathological changes were not apparent until 119/121dg. While the chronology of when the bacterium arrived in the placenta and subsequent lesion development is remarkable for its consistency this paper provides more reliable data on the former which in turn now allows study of the factors that permit its access to this tissue and govern its multiplication and the ensuing triggering of damage.     

Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus

Chlamydophila abortus, the aetiological agent of enzootic abortion of ewes (EAE), replicates in trophoblast cells leading to their destruction and dissemination of the bacterium to foetal organs. To further understand the pathogenesis of EAE, amniotic and allantoic fluids were collected from experimentally infected pregnant ewes at 30 (7 samples from each fluid), 35 (8 samples from each fluid), 40 (10 samples from each fluid) and 43 (6 amniotic fluids and 7 allantoic fluids) days post-infection to determine pathogen numbers and other markers of infection. Whilst experimentally infected ewes had characteristic placental lesions, only two amniotic and seven allantoic fluid samples were positive for C. abortus by real-time PCR. In contrast, all amniotic and allantoic fluids were positive for immunoglobulin. Immunoglobulins were generally detected earlier in allantoic fluid than in amniotic fluid and the numbers of samples containing immunoglobulins increased as infection progressed. IgG in amniotic and allantoic fluids was shown to be specific for C. abortus, and reacted with the major outer membrane proteins, polymorphic outer membrane protein and macrophage infectivity potentiator protein. A comparison of two-dimensional immunoblots using purified IgG from the allantoic fluid, amniotic fluid, ewe serum and foetal serum of a C. abortus infected animal at 40 days post infection indicated a pattern of reactivity intermediate between that of the ewe serum and the foetal serum. Results suggest that a maternal source of immunoglobulin is predominant at 30 days post-infection but that foetal derived antibodies may be contributed at a later stage.     

Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period, respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 10⁵ tripomastigotes. Clinical examination, determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid, blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented severe infection and died in the 34^th and 35^th days post-infection (dpi), respectively; but both fetuses were recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130^th day (30 dpi) of pregnancy; and Ewe 6 aborted one fetus in the 140^th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV, body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes.     

Immunopathology of Chlamydophila abortus infection in sheep and mice

Chlamydophila abortus targets the placenta, causing tissue damage, inflammation and abortion (enzootic abortion of ewes). It is one of the main infectious causes of abortion in ewes, resulting in major economic losses to agricultural industries worldwide. Although ruminants and pigs are the principal hosts, humans are also susceptible to infection. Control of disease requires a host inflammatory response, which is likely to contribute to pathology and abortion. Mouse models have been widely used to provide insight into the role of specific immune cells in controlling infection and disease. The use of such model systems for investigating the mechanisms of abortion, latency, persistence, and immunity to reinfection will result in the identification of novel vaccine control strategies for sheep.     

Effect of feeding lasalocid to pregnant ewes experimentally infected with Toxoplasma gondii.

Sixteen 50 day gestational ewes were fed lasalocid at the rate of 30 g t-1 and were orally inoculated with 100 infective Toxoplasma gondii oocysts 5 days after beginning feeding of lasalocid. Seventeen control ewes were similarly inoculated with T. gondii and were not fed lasalocid. The rate of abortion and neonatal mortality in both treated and untreated ewes was similar, indicating that feeding lasalocid was not effective in preventing T. gondii abortion in sheep.     

Prevalence of Chlamydophila abortus infection in domesticated ruminants in Taiwan.

This study is to (1) investigate the prevalence of Chlamydophila abortus infection in cows and goats in Taiwan, and (2) compare the genetic properties of Taiwanese isolates with abortion strains from other sources. Approximately 71% of aborted cows and 58% of aborted does had IgG against C. abortus in their sera. The seroprevalence rate in cows may be overestimated, because a certain degree of cross-reactivity with C. pecorum cannot be ruled out. Only 22.7% (from aborted cows) and 33.3% (from aborted dogs) of vaginal swabs that tested positive by polymerase chain reaction led to successful isolation of C. abortus by inoculation into chicken embryos, equivalent to 7.1% and 7.9% of isolation rates, respectively. The major outer membrane protein gene of 15 Taiwanese abortion isolates was compared with that of various strains by restriction fragment length polymorphism (RFLP) and nucleotide sequencing. Restriction enzyme CfoI was able to distinguish Taiwanese ruminant isolates, which have identical RFLP patterns, from C. felis (feline) and C. psittaci (avian) strains. Taiwanese isolates had 98.8-100% homology with known ruminant abortion strains and were phylogenetically closest to bovine LW508 strain.     

Monitoring clinical outcomes,pathological changes and shedding of Chlamydophila abortus following experimental challenge of periparturient ewes utilizing the natural route of infection

Enzootic abortion of ewes (EAE) caused by Chlamydophila abortus is an important disease resulting in significant lamb loss in most sheep producing countries. Ewes are considered to be naturally infected with C. abortus via the oral–nasal route and may become persistent carriers, shedding during subsequent oestrous cycles and at lambing. The aim of this study was to monitor the clinical outcomes, pathological changes and shedding of C. abortus in 18 periparturient orally infected sheep for two breeding seasons. In the first season, C. abortus was detected by real-time PCR (rt-PCR) in 13/18 conjunctival swabs at oestrus. Three out of the 15 pregnant ewes gave birth to 1 live and 1 dead lamb, and 2 of them aborted. Following parturition/abortion, C. abortus was detected in 12/15 vaginal swabs and in all the collected foetal membranes. However, only those membranes containing high copy numbers of the bacterium displayed the EAE typical lesions. In the second season, none of the 13 pregnant ewes aborted, and 5 of them gave birth to dead or weak lambs. C. abortus was not detected in conjunctival or vaginal swabs at oestrus or parturition. The bacterium was detected at low levels in 36% of the foetal membranes, but with no evidence of histopathological lesions. These results indicate that C. abortus can be detected in a large proportion of animals during the first pregnancy after oral infection. However, this proportion is reduced at the subsequent breeding season, confirming the occurrence of a chronic low level persistent infection in post-abortion/lambing ewes.     

Chlamydophila abortus infection in the mouse: a useful model of the ovine disease

Chlamydophila (C.) abortus is an obligate intracellular bacterium able to colonize the placenta of several species of mammals, which may induce abortion in the last third of pregnancy. The infection affects mainly small ruminants resulting in major economic losses in farming industries worldwide. Furthermore, its zoonotic risk has been reported in pregnant farmers or abattoir workers. Mouse models have been widely used to study both the pathology of the disease and the role of immune cells in controlling infection. Moreover, this animal experimental model has been considered a useful tool to evaluate new vaccine candidates and adjuvants that could prevent abortion and reduce fetal death. Future studies using these models will provide and reveal information about the precise mechanisms in the immune response against C. abortus and will increase the knowledge about poorly understood issues such as chlamydial persistence.     

Serologic diagnosis of toxoplasmosis in experimentally infected pregnant goats and transplacentally infected kids

Eight pregnant goats were inoculated orally with 10 to 1,000 oocysts of Toxoplasma gondii at 83 to 102 days of gestation. Serum samples from the goats and from the kids born to them were analyzed, using the Sabin-Feldman dye test (DT), a commercially available modified agglutination test (MAT), and a latex agglutination test. Six of the does were observed for greater than 1 year; during this time, they delivered twice. All does developed DT and MAT antibody titers of greater than or equal to 1:2,048 within 29 days after inoculation, and the high titers persisted through the 2nd pregnancy; therefore, serologic results alone should not be relied on for the diagnosis of T gondii-induced abortion in does. On the other hand, all transplacentally infected kids had DT or MAT antibody titers of 1:2,048 before ingesting colostrum, indicating the usefulness of serologic evaluation of the fetus or stillborn kid in the diagnosis of abortion. Antibody was not found in the sera of noninfected kids born to Toxoplasma-infected does. The passively acquired colostral antibody declined by 5 months. Therefore, specific antibody found in adult goats is probably actively acquired. The commercially available MAT was simple, sensitive, and reliable for the diagnosis of caprine toxoplasmosis. The latex agglutination test needs further improvement, as titers rarely exceeded 1:256.     

Protection of pregnant mice against placental and splenic infection by three strains of Chlamydophila abortus with a live 1B vaccine

The efficacy of a live 1B vaccine against three strains of Chlamydophila abortus, AB16, LLG and POS, was assessed in pregnant mice in terms of the reduction in the levels of infection recorded in their placentas, fetuses and spleens. The vaccine was more effective against the AB16 strain than against the LLG and POS strains, suggesting that there are antigenic differences between the three strains.     

Prevalence and risk factors associated with Chlamydophila abortus infection in dairy herds in Jordan

A cross-sectional study was carried out to determine seroprevalence and to identify risk factors associated with Chlamydophila abortus infection in 62 nonvaccinated dairy herds (671 cows) in Jordan between January and June 2007. Information regarding herd management was recorded through a personal interview with farmers. Antibodies against C. abortus were detected using an ELISA test kit. Chi-square analysis and multivariable logistic regression model were used to identify risk factors associated with C. abortus seropositivity. The true prevalence of antibodies against C. abortus in individual cows and cattle herds were 19.9?% and 66.3?%, respectively. Univariable Chi-square analysis revealed three variables with P????0.25 that were further offered to multivariable logistic regression analysis. Small-sized herds were identified as a risk factor for seropositivity to C. abortus, while sweeping followed by water hosing and using disinfectants were identified as protective factors. Cows in the age groups of >8 and ??10?years old and >2 and ??6?years old had the highest and lowest significant seroprevalence to C. abortus, respectively. Results of this study indicated that C. abortus is highly prevalent in Jordan's dairy herds and Chlamydophila infection could be controlled by applying strict biosecurity measures in the dairy farms.     

Experimental Brucella ovis infection in pregnant ewes.

Forty yearling Brucella-free ewes were inoculated with Brucella ovis by the conjunctival route in mid or late first pregnancy. Only a few ewes excreted B ovis during pregnancy and gave birth to stillborn lambs, but most of them excreted the organism at lambing or during lactation. One of the 11 lambs which were born alive but died before they were weaned was found to be infected postmortem. In contrast, none of the 46 surviving lambs which were reared in isolation until adulthood, was found to be infected. At weaning, the 40 ewes were mated again with five Brucella-free rams. Although many of the ewes excreted B ovis, none of the rams was found to be infected when necropsied after mating. Most of the ewes that became pregnant, all having excreted B ovis during their first pregnancy, cleared the infection during the second pregnancy. However, three remained persistently infected and excreted B ovis in their milk throughout the second lactation. None of the lambs born to these three ewes was found to be infected when necropsied at weaning.     

Comparative evaluation of eight serological assays for diagnosing Chlamydophila abortus infection in sheep

Chlamydophila abortus is one of the principal causes of late-term abortion (enzootic abortion of ewes or EAE) in sheep across Europe. Serological diagnosis of EAE is routinely carried out by the complement fixation test, although the interpretation of results can often be difficult because of cross reaction with Chlamydophila pecorum, which also commonly infects sheep. The purpose of this study was to evaluate and compare four ELISAs developed at Moredun Research Institute and based on whole C. abortus elementary bodies (EBs), an outer membrane preparation of the whole organism (SolPr) and two recombinant polymorphic outer membrane protein fragments (rOMP90-3 and rOMP90-4), with 3 commercial tests, the CHEKIT Chlamydophila Abortus, Pourquier ELISA Chlamydophila abortus and ImmunoComb Ovine Chlamydophila Antibody tests. The tests were evaluated using a panel of 202 sera from experimentally and naturally infected animals, as well as from EAE-free flocks. The EB, SolPr and CHEKIT ELISAs performed similarly to the CFT, all lacking in specificity by cross reacting with sera from C. pecorum infected animals. The ImmunoComb also lacked specificity with C. pecorum sera, but also badly cross reacted with sera from EAE-free flocks. The rOMP90-3, rOMP90-4 and Pourquier ELISAs were the most specific, although the Pourquier test appeared less sensitive with sera from naturally infected animals. Overall, the rOMP90-3 ELISA performed the best, with high sensitivity (96.8%) and no cross reaction with sera from C. pecorum infected animals or from EAE-free flocks (100% specificity) and so would be a suitable alternative to the CFT for the serological diagnosis of EAE.     

一起山羊流产衣原体感染的诊断

陕西省某山羊养殖场发生了一起山羊流产病,经流行病学调查,临床症状和病理剖检观察进行初步诊断;利用布鲁菌虎红平板凝集试验、衣原体间接血凝试剂盒对10份流产母羊血清检测;对病料进行细菌学检测;依据GenBank收录的山羊流产性衣原体基因组设计1对特异性引物,通过PCR方法从病料中扩增衣原体特异性片段。结果表明,根据临床症状和病理变化初步怀疑为布鲁菌和鹦鹉热亲衣原体感染;10份血清检测结果为布鲁菌病血清全部阴性,衣原体感染血清全部为阳性;细菌学检测结果为阴性;PCR结果获得523bp基因片段,测序结果与鹦鹉热亲衣原体100%相似。依据流行病学调查,临床症状和病理剖检观察,病原学和血清学诊断,最终确诊该病为鹦鹉热亲衣原体引起的山羊地方流行性流产。     

Seroprevalence and associated risk factors of Chlamydia abortus infection in ewes in Tunisia

Enzootic abortion of ewes (EAE) caused by Chlamydia abortus is a disease of ruminants that results in serious economic losses in livestock industry. The zoonotic potential of the pathogen adds a public health concern on the efforts to control the disease. We report herein a cross-sectional study that was conducted during the lambing season (June and July) in Tunisia to estimate the seroprevalence of C. abortus infection in large sheep herds with abortion history. A total of 803 ewes were sampled and tested using indirect enzyme linked immunosorbent assay (iELISA). The overall apparent seroprevalence at herd and individual levels were 58 % (95 %CI = 39–74.5 %) and 6.6 % (95 %CI = 4.9–8.3 %), respectively. Significant risk factors investigated using univariate and multivariate analyses were history of infertility (OR = 5.7; 95 %CI = 3.05–10.66), the number of reproductive ewes (OR = 2.1; 95 %CI = 1.12–3.94), the control of new animals at introduction (OR = 4.35; 95 %CI = 2.46–7.68), the sharing of drinking water (OR = 2.18; 95 %CI = 1.22–3.9), the exchange of breeding males (OR = 2.56; 95 %CI = 1.003–6.54), the disposal of abortion materials without precaution (OR = 4.36; 95 %CI = 2.42–7.87), the lack of lambing barn (OR = 2.39; 95 %CI = 1.13–5.04), the non-application of hygienic post-abortion measures (OR = 10.35; 95 %CI = 5.28–20.26) and the manure management (OR = 11.35; 95 %CI = 3.26–39.48). To the best of our knowledge, this is the first sero-epidemiological survey conducted on an abortive disease in Tunisian ewes that investigated the risk factors of C. abortus infection.