Sheila Worrall

Detection and quantification of Toxoplasma gondii in ovine maternal and foetal tissues from experimentally infected pregnant ewes using real-time PCR.

A real-time PCR (rt-PCR) targeting the 529-bp repeat element (RE) of Toxoplasma gondii was used to detect and quantify the parasite burden in maternal and foetal tissues in 18 seronegative ewes infected with 3000 toxoplasma oocysts on day 90 of pregnancy. The infected ewes were sacrificed in groups of 4-6 at 21, 25, 33 and 35 days post-challenge. Ten sham inoculated pregnant ewes were used as controls. T. gondii was not detected in the control ewes or their foeti. The parasite was only detected in the maternal tissues in a few of the challenged ewes on a small number of occasions where it was identified in spleen and uterine lymph nodes. T. gondii was detected in the foetal spleen and liver at the early sacrifice times but only sporadically thereafter. In the case of amniotic, allantoic and foetal aqueous humor samples T. gondii was only detected on a small number of occasions. However, it was found in the majority of the foetal lung and placentome samples throughout the study period, while placentomes and foetal brains contained high levels of the parasite during the later stages. Histopathological examination of placentome and brain tissue from the foeti in the present study revealed a strong correlation between histopathological lesions and quantities of the parasite DNA detected. These results indicate that the cotyledonary component of the foetal membranes is the sample of choice for the diagnosis of T. gondii by rt-PCR, followed by foetal lung and brain.

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.

Application of quantitative real-time polymerase chain reaction for the diagnosis of toxoplasmosis and enzootic abortion of ewes

Toxoplasma gondii and Chlamydophila abortus are the 2 most common infectious causes of ovine abortion worldwide. These obligate intracellular pathogens are associated with severe placentitis leading to abortion or stillbirth in pregnant ewes, and resulting in significant economic losses. The objectives of the current study were the development, validation, and application of a duplex real-time polymerase chain reaction (PCR) assay capable of quantifying the burden of infection by T. gondii and C. abortus in material submitted for diagnostic purposes. The validation was carried out using samples from ewes experimentally infected with these organisms. Based on the numbers of genome copies detected, an arbitrary cutoff level was established to correlate with significant pathological changes sufficient to give rise to abortion. When the PCR assay was applied to samples from 66 Irish farms with naturally occurring outbreaks of ovine abortion, toxoplasmosis and enzootic abortion of ewes (EAE) accounted for 14% and 20% of the farms, respectively, while on 6% of the farms, there was evidence of dual infection. When standard diagnostic techniques including histopathological examination, serological analysis, chlamydial antigen detection, and bacteriological culture, were used on samples from the same farms, toxoplasmosis was diagnosed in 17% of farms, and EAE in 12%; dual infection was diagnosed on 3% of the farms. In general, good agreement was found between the PCR and the standard methods. The duplex real-time PCR assay developed in this study has proved to be a very sensitive and rapid tool that might provide a valuable addition to the methods currently available for routine diagnosis of ovine abortions.

Identification of immunologically relevant proteins of Chlamydophila abortus using sera from experimentally infected pregnant ewes.

ABSTRACT Chlamydophila abortus is an intracellular pathogen and the etiological agent of enzootic abortion of ewes (EAE). C. abortus has a biphasic development cycle; extracellular infectious elementary bodies (EB) attach and penetrate host cells, where they give rise to intracellular, metabolically active reticulate bodies (RB). RB divide by binary fission and subsequently mature to EB, which, on rupture of infected cells, are released to infect new host cells. Pregnant ewes were challenged with 2 × 106 inclusion forming units (IFU) of C. abortus cultured in yolk sac (comprising both EB and RB). Serum samples were collected at 0, 7, 14, 21, 27, 30, 35, 40, and 43 days postinfection (dpi) and used to identify antigens of C. abortus expressed during disease. Additionally, sera from fetal lambs were collected at 30, 35, 40, and 43 dpi. All serum samples collected from experimentally infected pregnant ewes reacted specifically with several antigens of EB as determined by one-dimensional (1-D) and 2-D gel electrophoresis; reactive antigens identified by mass spectrometry included the major outer membrane protein (MOMP), polymorphic outer membrane protein (POMP), and macrophage infectivity potentiator (MIP) lipoprotein.

Immunohistochemical detection of IgM and IgG in lung tissue of dogs with leptospiral pulmonary haemorrhage syndrome (LPHS).

Leptospiral pulmonary haemorrhage syndrome (LPHS) is a severe form of leptospirosis. Pathogenic mechanisms are poorly understood. Lung tissues from 26 dogs with LPHS, 5 dogs with pulmonary haemorrhage due to other causes and 6 healthy lungs were labelled for IgG (n=26), IgM (n=25) and leptospiral antigens (n=26). Three general staining patterns for IgG/IgM were observed in lungs of dogs with LPHS with most tissues showing more than one staining pattern: (1) alveolar septal wall staining, (2) staining favouring alveolar surfaces and (3) staining of intra-alveolar fluid. Healthy control lung showed no staining, whereas haemorrhagic lung from dogs not infected with Leptospira showed staining of intra-alveolar fluid and occasionally alveolar septa. Leptospiral antigens were not detected. We conclude that deposition of IgG/IgM is demonstrable in the majority of canine lungs with naturally occurring LPHS, similar to what has been described in other species. Our findings suggest involvement of the host humoral immunity in the pathogenesis of LPHS and provide further evidence to support the dog as a natural disease model for human LPHS.

Detection of Toxoplasma gondii antigens reactive with antibodies from serum, amniotic, and allantoic fluids from experimentally infected pregnant ewes

Toxoplasma gondii, an intracellular protozoan parasite, is one of the major causes of infectious abortion in sheep. To further understand the pathogenesis of toxoplasmosis, serum, amniotic and allantoic fluids and foetal stomach contents were collected from experimentally infected pregnant ewes to determine pathogen numbers and other markers of infection. Fifteen pregnant ewes (90 days of gestation) were each orally inoculated with 3000 sporulated oocysts of T. gondii. Serum samples were collected weekly following challenge. Amniotic and allantoic fluids and foetal stomach contents were collected at 21, 25, 28, 33 and 35 days post-infection. Characteristic placental lesions were detected in 1 of 4 challenged ewes at day 25, 3 of 4 challenged ewes at day 28 and in all challenged ewes at days 33 and 35 post-infection. T. gondii was detected only sporadically in amniotic and allantoic fluids before 35 days of infection, by real-time PCR, and only in ewes with placental lesions. At 35 days post-infection, high numbers of parasite were detected in both amniotic and allantoic fluids. An increase in the number of fluids from challenged animals with IgM and IgG was detected over time, except for IgG in allantoic fluid, which was detected in all samples from day 21 post-infection. IgG in amniotic and allantoic fluids was shown to be specific for T. gondii, and reacted with antigens with an apparent molecular mass of approximately 22 kDa and 30 kDa. Results suggest a maternal source of immunoglobulin in the allantoic fluid and a foetal source of immunoglobulin in the amniotic fluid early in infection but that both sources may contribute immunoglobulin to both fluids at a later stage.

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.

Interferon-γ expression in trophoblast cells in pregnant ewes challenged with Chlamydophila abortus

Pregnant ewes were challenged with Chlamydia abortus at 91-98 days of gestation and euthanised at 14, 21 and 28 days post-challenge. IFNγ mRNA labelling appeared to be co-localised with Chlamydial lipopolysaccharide within trophoblast cells in discrete areas lining the primary villi in the limbus and hilar zone of the placentomes from challenged sheep on days 21 and 28 post-infection. The presence of IFNγ was also demonstrated by immunohistochemistry. No labelling was seen in tissues from the non-infected ewes. The presence of IFNγ in trophoblast cells from infected ewes may indicate an attempt to restrict the replication of the organism and be an important trigger for the inflammatory responses that develop on the fetal side of the placenta in enzootic abortion.

The expression of mucin genes and the presence of mucin gene products in the equine endometrium

In the equine reproductive tract, little is known about mucin gene expression and the role of mucins in barrier function and host-cell interaction. The aims of the study were to identify equine orthologs of mammalian mucin genes using available equine sequence data, to profile expression of equine orthologous mucin genes in the endometrium using reverse transcriptase polymerase chain reaction (RT-PCR), to determine spatial expression patterns of mucin genes using in situ hybridisation, and to confirm the presence of mucin gene products using Western blotting and equine-specific mucin antibodies during oestrus and dioestrus. While the mucin gene expression pattern in equine endometrium is similar to that of other mammals, several mucins appear to be uniquely expressed in this tissue (eqMUC3B, 7, 18, and 20) and one is hormonally regulated (eqMUC3B).