[show abstract][hide abstract] ABSTRACT: 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.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 07/2012; 24(5):846-54. · 1.18 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Six ovine fetal brains were harvested 33 to 35 days postchallenge from 5 ewes, each of which was given 3000 Toxoplasma gondii oocysts on day 90 of pregnancy. Histopathologic examination of transverse sections taken at 13 levels in the fetal brains revealed the presence of toxoplasmosis-related lesions in all 6 brains. However, lesions were not randomly distributed (P = .007); they were most numerous at the level of the optic tract, the rostral margin of the pons, and 4 mm caudal to the ansate sulcus and were absent in all sections at the level of the caudal cerebellum. Lesion distribution may be due to hemodynamic factors, differences in the expression of endothelial surface receptor molecules at the level of the blood-brain barrier, or the presence of localized permissive/inhibitory factors within the brain. The results have implications for the selection of areas of brain from aborted ovine fetuses to be examined histopathologically for laboratory diagnosis.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Reproductive Immunology 06/2011; 90(2):214-9. · 2.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Veterinary Immunology and Immunopathology 03/2011; 140(1-2):1-9. · 1.88 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Little is known of the common diseases of hunting dogs or of the reasons why they are culled. To address these questions, necropsy examinations were conducted on 52 hounds aged 1.5-12 years (mean 6.5 ± 2.5 years) and culled from 10 Irish hunting kennels over a 3-year period. Progressive systemic disease was seen in six dogs only and encompassed individual cases of tuberculosis caused by Mycobacterium bovis, bronchioalveolar carcinoma with metastasis to regional lymph nodes, renal amyloidosis, suppurative pneumonia, extramedullary plasmacytoma in the atrial wall of the heart and foreign body-induced hepatitis with focal peritonitis. Single or multiple localized tumours were identified in five dogs and, apart from the aforementioned, included two cutaneous haemangiomas, a trichoepithelioma, a lipoma and a mammary ductal adenoma. Three dogs were culled for lameness; one of these dogs had torn musculature, another had cellulitis and the third had a healed fracture of the tibia and fibula. Chronic renal changes were present in 48% of the dogs and included focal proliferative, exudative or crescentic glomerulonephritis (33%) or low-grade interstitial inflammatory changes (50%). The most frequently diagnosed skin lesions reported in this study were mild healed decubitus ulcers (33%), scars (33%) and stereotypic dermatitis (13%). These findings indicate that hounds are likely to be culled for reasons other than the presence of disease in most cases. In addition, this survey highlights different disease patterns in hounds than are typically observed in pet dogs.
Journal of comparative pathology 01/2011; 145(1):59-67. · 1.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Phylogenetic analysis was performed on the haemagglutinin and neuraminidase subtype N2 genes of low-pathogenic avian influenza viruses (LPAIVs) detected in Ireland between 2003 and 2007. Nucleotide sequences were compared to previously published sequences from the National Centre for Biotechnology Information. Sequences from viruses of the same subtype isolated in different years were compared to examine the possibility that LPAIVs may have been maintained in Ireland from year to year. All viruses had closest identity with published sequences of European lineage, supporting the conclusion that LPAIVs had been introduced to Ireland by dabbling ducks that had migrated from Europe. The data suggested that different subtypes of virus had been introduced each year. However, there was evidence that some LPAIVs may have been maintained in the sedentary waterfowl population for consecutive seasons. Furthermore, almost identical H6 and H10 sequences with different N types were found in isolates from the same season, suggesting that reassortment had occurred.
Epidemiology and Infection 10/2010; 139(8):1191-201. · 2.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] 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 x 10(6) 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.
[show abstract][hide abstract] ABSTRACT: A neuropathologic survey was conducted on mink brains from the 5 licensed mink farms in Ireland. The survey was part of a transmissible spongiform encephalopathy surveillance study. Aleutian disease (AD) was present on 4 of the 5 farms (80%). Neuropathologic features of nonsuppurative meningoencephalitis were common in mink from the 4 affected farms but were absent in the mink from the fifth farm, which was free of AD. The meningoencephalitis was characterized by infiltrates of lymphocytes and plasma cells, which were present in meninges, perivascular spaces, and the brain parenchyma. Fibrinoid necrotizing arteritis was seen in 11 mink brains, all of which were obtained from a single farm. Aleutian mink disease virus (AMDV) sequences for the capsid protein VP2 were obtained from brain samples from all affected farms. Although containing previously unreported amino acid residues, similarities with European and North American isolates were observed in the hypervariable regions within VP2, suggesting Irish AMDV is related to those isolates. The predicted amino acid residues, suspected of conferring pathogenicity at certain positions of the VP2 sequence, were present in the viral nucleic acid sequences.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 01/2010; 22(1):101-5. · 1.18 Impact Factor
[show abstract][hide abstract] ABSTRACT: Specimens for the detection of avian influenza virus (AIV) were collected from 1937 waterfowl on the Wexford Sloblands, a major wetland reserve in southeast Ireland, between January 2003 and September 2007. During the same period, 1404 waterfowl were sampled at other locations in Ireland. Specimens were tested either by virus isolation or real-time reverse transcriptase polymerase chain reaction (rtRT-PCR). A total of 32 isolates of AIV, comprising nine subtypes, was obtained from specimens from the Sloblands compared with just one isolate from elsewhere in Ireland. Samples from nine other waterfowl, five of which were from the Sloblands, tested positive for AIV by rtRT-PCR. Ecological factors are likely to have contributed to the higher detection rate of AIV at the Sloblands compared with the rest of Ireland. It was concluded that targeted surveillance at such sites is a cost-effective means of monitoring the circulation of new AIVs in waterfowl, whereas widespread opportunistic sampling is unproductive and wasteful of resources.
Epidemiology and Infection 10/2008; 137(4):464-72. · 2.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: An appreciation of the complexities of placental structure and function is essential to understanding the pathogenesis of infectious placentitis and abortion. This review aims to illustrate aspects of ovine pregnancy and placentation that will assist both the research worker and the diagnostic pathologist. Morphologically, the ovine placenta is classified as being chorioallantoic, villous, cotyledonary and synepitheliochorial. Apposition of foetal and maternal tissues in early pregnancy eventually leads to the formation of the definitive placenta. Physiological features of placentation that are essential to normal pregnancy and foetal development include modulation of immune responses at the placental interface, increasing placental bloodflow to allow for increasing foetal demand and the secretion of hormones for the recognition and maintenance of pregnancy. Descriptions of the morphology of the near-term placenta in a normal pregnancy and of the foetal membranes that are voided during normal parturition provide the proper context for understanding the morphological changes associated with placentitis and how these changes are likely to affect placental function.
[show abstract][hide abstract] ABSTRACT: Brains from 100 horses, aged 2-25 years, were systematically examined by histopathology at 46 different neuroanatomical sites. The horses were sourced from a slaughterhouse (group A, n = 57), from a kennel that collected dead animals, and from 2 diagnostic laboratories (group B, n = 43). All horses from group A and 26 horses from group B were examined by a veterinarian in the period before death. None of the horses were known to exhibit clinical signs suggestive of neurologic disease. Among the main changes identified were vacuolation in the neuropil (n = 73), neurons (n = 32), white matter (n = 31), and focal perivascular lymphoid cell infiltrates (n = 35). Spheroids were frequently seen (n = 91), and 10 horses each had more than 10 spheroids in the cuneate or gracile nucleus. Statistically significant age-related changes noted included intraneuronal (n = 97) and glial or extracellular lipofuscin deposition (n = 41), hemosiderin deposition around blood vessels (n = 60), and calcium depositions (n = 24). One horse had low-grade nonsuppurative meningoencephalitis; Alzheimer type II cells were detected in the brains of 2 horses. Hyalinized vessel walls in the cerebellum were observed in 1 horse. It was concluded that some histopathologic changes are a frequent feature in equine brains, which has implications for the pathologists involved in equine neurology and disease surveillance.
[show abstract][hide abstract] ABSTRACT: Samples of brain and lymphoid tissues from 1107 meat and bone meal-fed, culled adult pigs from 24 Irish farms were examined for evidence of transmissible spongiform encephalopathy (TSE) by histopathological, immunohistochemical and Western blotting techniques. No evidence of deposits of abnormal prion protein suggesting the presence of TSE was found. Neuropil vacuolation was apparent in the rostral colliculus in 64 per cent of the brains examined and neuronal vacuolation was present in the dorsal vagal nucleus in 15.4 per cent of the brains. However, similar lesions have been described in pigs used as controls in a bovine spongiform encephalopathy challenge experiment. Age-related changes were also observed, including spheroids in the funicular nucleus of 24.5 per cent of the pigs, deposits of lipofuscin in the trigeminal neurons of 13.75 per cent, and mineral deposits in the walls of vessels in the dorsal vagal nucleus of 0.6 per cent. Low-grade non-suppurative inflammatory changes of uncertain origin were observed in 4 per cent of the animals.
The Veterinary record 08/2006; 159(5):137-42. · 1.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: Placentae from 13 pregnant ewes infected intravenously with Chlamydophila abortus, together with placentae from nine uninfected control ewes, were examined at 14, 21 or 28 days post-inoculation (p.i.). Chlamydial inclusions were present in the trophoblast at 14 days p.i. and were widespread by 21 days p.i. Chorioallantoic lesions (oedema, arteritis and thrombosis) were severe at 28 days p.i., the changes being particularly marked in the membrane surrounding placentomes. Lymphocytes constituted only a small proportion of the cellular infiltrate in the chorioallantois; neutrophil infiltration of the chorionic surface was evident where the trophoblast layer had sloughed, whereas macrophages represented the predominant cell type in the deeper stroma. In contrast, on the maternal side of the placenta, chlamydial inclusions were sparse at all timepoints, and even at 28 days p.i., lesions were restricted to focal endometritis at the placentomal limbus and occasional foci of septal necrosis. T lymphocytes were numerous within endometrial and septal lesions, the infiltrate consistently containing more CD8(+) than CD4(+) cells. The fetal response to chlamydial invasion of the placenta was innate in character, whereas the maternal response appeared to represent an acquired, chlamydia-specific immune response.
Journal of Comparative Pathology 01/2006; 135(2-3):83-92. · 1.38 Impact Factor