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.     

Immunoglobulin concentrations and bovine enterovirus inhibitors in fetal bovine fluids.

Fluids from 53 bovine fetuses ranging in age from 90 to 240 days were examined for immunoglobulin G (IgG) and immunoglobulin M (IgM) and neutralizing activity to ten bovine viruses. Non-specific inhibitors to bovine enteroviruses were found in serum, allantoic, and amniotic fluids of most samples tested. In most cases, serum IgG were within normal values. Neither IgG nor IgM was detected in amniotic fluids, whereas 2 samples of allantoic fluid contained traces of IgG.     

Abnormalities of foetal fluids in sheep: two case reports

SUMMARY Two cases of abnormal accumulation of foetal fluids in sheep are described. In the first case, a ewe, at 64 days of pregnancy, had 2 sacs of fluid, one at each of the tips of the pregnant and non-pregnant horns. Neither sac contained a foetus. The composition of the fluids differed from those of the amniotic and allantoic fluids associated with a single foetus. The composition resembled that of foetal plasma. We conclude that the 2 sacs were amniotic sacs from conceptuses that had been resorbed, and that at this early (0.4) stage of pregnancy, amniotic fluid can accumulate in the absence of a viable foetus.
The second case was a rare case of hydrallantois in a ewe at 100 days of pregnancy. The volume of allantoic fluid (1800 mL) was more than 10 times normal. The composition was normal, except for high chloride and calcium concentrations and a very low creatinine concentration. The foetus and the volume and composition of the amniotic fluid were normal. The hydrallantois might have occurred as a result of some alteration in function of the allantoic membrane, perhaps as a result of altered hormonal status of the ewe.     

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.     

TRANSMISSION OF A MUCOSAL DISEASE VIRUS INFECTION BETWEEN SHEEP

The transmission of mucosal disease virus (MDV) from infected ewes and their lambs to susceptible sheep was investigated. MDV was recovered from the amniotic fluid of an infected pregnant ewe and from the blood, nasal swabs and urine of hairy lambs. MDV infection was transmitted either at lambing, from infective foetal fluids or lambs, or later as a result of contact with a surviving with a hairy lamb and either aborted or gave birth to an infected hairy lamb. Adult sheep and 12-months-old sheep were less readily infected than were newborn lambs. Pregnant ewes were infected by contact with a hairy lamb and either aborted or gave birth to an infected hairy lamb. A method to minimise spread of the infection in the field is suggested.     

Experimental Neospora Caninum Infection in Pregnant Dairy Heifers Raises Concentrations of Pregnancy‐Associated Glycoproteins 1 and 2 in Foetal Fluids

Plasma concentrations of PAG‐1 are used for pregnancy diagnosis and as a marker of placental/foetal well‐being, while those of PAG‐2 may be an indicator of abortion risk in Neospora caninum‐infected cows. Studies have shown that N. caninum infection modifies PAG‐1 and PAG‐2 patterns in maternal blood plasma. However, no prior work has examined the effects of N. caninum infection on concentrations of PAGs in foetal fluids. In this study, PAG‐1, PAG‐2 and pH levels were determined in the amniotic and allantoic fluids of foetuses collected at 152 days of gestation from control uninfected dams and from dams experimentally infected with N. caninum on Day 110 of gestation. Foetal fluids from infected foetuses had significantly higher PAG‐2 concentrations (p = 0.026) and pH values (p = 0.02) than fluids from non‐infected foetuses. In infected foetuses, significantly higher concentrations of PAG‐1 (p < 0.001) and PAG‐2 (p < 0.001) were detected in fluid samples showing antibodies against N. caninum than those without antibodies. Moreover, pH values were significantly higher (p = 0.011) in foetal fluid samples with antibodies than in samples from non‐infected foetuses. In conclusion, this is the first report on the effect of N. caninum infection on PAG levels in foetal fluids. Our results indicate that following the experimental infection of dams with N. caninum on Day 110 of gestation, foetal fluids collected from the infected foetuses of these dams featured higher PAG‐1 and PAG‐2 levels and pH values than fluids from non‐infected controls, provided that the samples tested showed the presence of antibodies. The clinical implications of these findings are that following infection with N. caninum, most cows will experience some level of placental damage and that this injury correlates with foetal fluid PAG levels and pH.     

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.     

Cytology of bovine foetal fluids.

Cell contents of foetal fluids obtained separately from amniotic and allantoic cavities of live cows by transvaginal amniocentesis and by aspiration from pregnant uteri immediately after slaughter were studied using Papanicolaou's staining procedure. The samples obtained belonged to cows which were between ten to 24 weeks of gestation. Cell scrapings from foetal and membranous surfaces normally in contact with the foetal fluids were also stained simultaneously with foetal fluid smears by Papanicolaou's method to determine the probable origin of the cells detected in the amniotic and allantoic cavities. The cell types were classified according to morphology and tinctorial patterns and their origin was deduced on the basis of similarity to cell scrapings from the surface layers of foetus and foetal cavities. The evaluation of foetal fluid cells revealed that a majority of cells are of foetal origin and that the cytological features of these cells may be useful in assessing the condition of the foetus.     

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.     

Anatomy, physiology and pathology of the amniotic and allantoic compartments in the sheep and cow

In both cows and sheep the allantoic fluid is confined to 2 sacs connected by a flattened isthmus, one in the non-pregnant horn and one in the upper part of the pregnant horn. The chorion encloses the amniotic and allantoic compartments, forming the amniochorion and chorioallantois, respectively. In the last third of gestation the compositions of both amniotic and allantoic fluids differ substantially from each other and from those of foetal urine, and maternal and foetal plasma. There is less variation in composition than in volume for a given gestational age. Abnormalities of volume are more common in cows than sheep, and hydrallantois is more common than hydramnois. Data obtained from both physiological experiments and pathological cases suggest that the foetal membranes play an important role in the regulation of composition and volume of foetal fluids. Evidence is presented that the permeability of the membranes to various solutes, as well as their capacity to produce and respond to a number of hormones, can affect the foetal fluid composition and/or volume. Progesterone, oestrogens and prolactin are some of the hormones known to affect foetal fluids. Foetal adrenal insufficiency has been associated with hydramnios implying a lack of hormones from this gland in this disease. The changes in allantoic fluid composition from normal to that closely resembling maternal/foetal extracellular fluid, in hydrallantois, suggests an alteration of membrane function as an aetiology and the continued production of fluid, after removal of the foetus in some cases, favours this hypothesis.     

EQUINE HERPESVIRUSES: Experimental Infecton of a Foetus with Type 2

Intrauterine infection of pregnant mare with equine herpesvirus type 2 (EHV 2) did not result in foetal abortion, stillbirth or recognisable disease. Collection of uterine fluid by allantocentesis or amniocentesis 107 days after inoculation confirmed that intrauterine infection was established. EHV 2 was isolated from both allantoic and amniotic fluid separately collected at the time of elective Caesarean section 156 days after inoculation and virus neutralising antibody to EHV 2 was present in the foal's presuckle serum at birth. A very mild clinical disease, characterised by a scant, mucous oculo-nasal discharge was observed between 4 and 11 days after birth. EHV 2 was isolated from 22 nasal swabs taken between birth and 65 days of age, on which day the foal was infected with equine adenovirus. EHV 2 was not isolated from 6 nasal swabs collected from 66 to 71 days of age.     

The protein composition of sheep foetal fluids

Amniotic fluid contains several proteins including alpha-foetoprotein derived from the foetal plasma, but lacks the higher molecular weight alpha2- and gamma-globulins. The protein composition of the fluid, like that of the serum, remains unchanged after the first month of gestation, and is buffered from the influence of the allantoic fluid by the amnion. Allantoic fluid contains a significantly higher concentration and a greater variety of proteins; it is not a simple transudate of either foetal or maternal plasma, and though containing proteins derived from both sources, it is deficient in albumin but relatively abundant in gamma-globulins. The latter comprises gamma-foetoprotein and mature type IgG. It is believed that maternal IgG may reach the allantoic sac in appreciable amount, but its transmission to the amniotic sac and thence to the foetus may be prevented by the impermeability of the amnion.     

Molecular detection of Chlamydophila abortus in post-abortion sheep at oestrus and subsequent lambing

Enzootic abortion of ewes (EAE), resulting from infection with the bacterium Chlamydophila abortus (C. abortus), is a major cause of lamb loss in Europe. The purpose of this study was to assess the potential impact of the shedding of organisms in post-abortion ewes at oestrus and subsequent lambing on the epidemiology of EAE. Using a newly developed C. abortus specific real-time PCR assay, few chlamydial genomes could be detected in vaginal swabs taken from post-abortion ewes at oestrus. At subsequent parturition, all ewes lambed normally with no macroscopic or microbiological evidence of infection. Real-time PCR analysis of placental samples identified very few or no chlamydial genomes, which contrasted significantly with samples taken at the time of abortion, where an average of 2.7x10(7) chlamydial genomes per microgram of total tissue DNA was detected. Few genomes could also be detected from vaginal and cervical tissue samples and lymph nodes taken post-mortem. The results, although not discounting the possibility of a chronic low level persistent infection in post-abortion ewes, suggest that the low levels of chlamydial DNA detected during the periovulation period and at lambing do not significantly impact on the epidemiology of EAE. In terms of flock management, the products of abortion should be considered the major and principal source of infection for transmission to na?ve ewes.     

Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera

Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.     

Variations in the concentrations of the immunoglobulins IgG1, IgG2, IgM and IgA in sheep. 1. Changes in the blood serum and in the milk of sheep of different breeds and cross-breeds during lactation

A total of 103 ewes from the breeds Black mutt., Texel, Finn. L., Heidschnucke and the crossbreeds Texel x Finn. L. and Black. mutt. x Finn. L. was studied. Blood samples were drawn at days 1, 7, 21 and 42 and milk samples at the 4th, 12th, 24th and 72nd hour after the onset of lactation and subsequently on days 7, 21 and 42. The concentrations of the immunoglobulins IgG1, IgG2, IgM and IgA were assayed in serum and milk. The following results were obtained: 1. The total immunoglobulin contents in the serum was not significantly different between breeds. 2. All ewes showed a rise in serum immunoglobulin concentrations by about one third over the first six weeks of lactation. Between 50-60% of this increase were on the account of IgG1. 3. The serum concentration of IgG1 and IgG1 rose as of the third day, those of IgM as of day 21 after lambing. 4. The rise in serum immunoglobulin concentration continued after the weaning of the lambs. 5. The ratio of IgG1 to IgG1 in ewe serum was 2:1. 6. The immunoglobulin concentration in milk dropped sharply on the first day of lactation, followed by a continuous, more gradual decrease over the entire course of lactation. A terminal rise, as observed in sows, could not be detected. 7. The ratio of IgG1:IgG2 : IgM : IgA in the whey changed from 85 : 1 : 12 : 2 on day one to 70 : 7 : 12 : 11 on the last day of lactation. 8. While characteristic trends in immunoglobulin patterns in the sera of ewes over the course of lactation are clearly discernible, it is not possible to denote "normal" values.     

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.     

Metabolomic Profile of Allantoic and Amniotic Fluid in Late-term Gestational Mares Characterized by 1H-nuclear Magnetic Resonance Spectroscopy

The amniotic and allantoic fluid compartments in the mare serve essential roles throughout pregnancy and parturition. Although the global metabolomic profile of amniotic fluid in women has been extensively characterized, current data for equine fetal fluids are limited. Therefore, the goal of this study was to characterize the global metabolomic profile of equine allantoic and amniotic fluid through nuclear magnetic resonance spectroscopy. Fetal fluids were collected between 270 and 295 days of gestation from 12 pregnancies through ultrasound-guided transabdominal puncture. A total of 24 samples (n = 10 allantoic fluid; n = 9 amniotic fluid; n = 5 admixed fluid) were analyzed by one-dimensional proton (¹H) and two-dimensional (¹H-¹³ C) nuclear magnetic resonance spectroscopy. Metabolites were integrated and compared between fluid types using a Kruskal–Wallis test at P < .05 significance. A total of 28 distinct metabolites were found in allantoic and admixed fluid, whereas 23 metabolites were identified in amniotic fluid. Allantoic fluid contained significant elevations (P < .05) in the metabolites betaine, creatine, creatinine, citrate, histidine, nitrophenol, tryptophan, π-methylhistidine, and unknown metabolite #1 compared with amniotic fluid, whereas amniotic fluid contained statistically increased concentrations of the metabolite lactate compared with allantoic fluid (P = .003).     

Akabane virus infection in the pregnant ewe. 1. Growth of virus in the foetus and the development of the foetal immune response

Three different pools of the CSIRO 16 strain of Akabane virus differing in their laboratory passage histories were used to inoculate 39 ewes between 32 and 36 days pregnant; 22 pregnant ewes received inocula containing no virus. There was no difference in the development, duration and titre of the viraemia and neutralising antibody response between the three infected groups of ewes. Both infected and control ewes had 141% foetuses when autopsied at 69 to 105 days gestation. Of the 55 foetuses from infected ewes 44 (80%) had gross developmental abnormalities.At autopsy of the dams Akabane virus was isolated only from the uterine caruncle. From foetal samples virus was isolated from a wide range of tissues, from one foetus at 69 days and from the blood of four foetuses at 95 to 106 days gestation. Virus was also isolated from 24 of the choriolllantoic fluid samples and from 37 placentomes of the 44 foetuses with developmental defects, in concentrations ranging from 10² to 10^5.5 TCID₅₀/ml or/g. No virus was isolated from the tissues of the control ewes or their foetuses.Neutralising antibody to Akabane virus was detected in 78% of the foetal sera from the infected group, titres ranging from 2 to 64. IgM and IgG₁ and neutralising antibody were detected in sera of 40 foetuses with developmental abnormalities including three that were of 76 to 78 days gestation. Neutralising antibody was detected only in serum that contained IgG₁ but may also have been associated with IgM in infected foetuses. IgM was detected in the serum of most foetuses including the non-infected controls, but sera from the control foetuses did not contain IgG₁ or neutralising antibody to Akabane virus. No IgG₂ or IgA were detected in any foetal serum.     

Experimental infection of dogs with Brucella abortus: effect of exposure dose on serologic responses and comparison of culture methods

Two studies of experimentally induced Brucella abortus infections in dogs are reported. Twenty dogs in experiment 1 were fed approximately 4.8 X 10(10) colony forming units of B. abortus strain 2308 (BA 2308), and 11 dogs in experiment 2 were fed approximately 3.4 X 10(14) BA 2308. Serum samples from each infected dog and noninfected control were tested on the day of infection and at weekly and biweekly intervals post-infection (PI) for antibodies to B. abortus. The standard tube agglutination (STA) and rivanol (RIV) mean log-titers of the infected dogs in the 2 experiments were compared statistically on the day of infection and on PI days 7, 14, 21, 35, 42 and 49. The STA and RIV titers of the infected dogs in experiment 2 were significantly higher than were those of the dogs in experiment 1. Brucella abortus was isolated from 29 infected dogs in experiments 1 and 2 including 10 dogs, which were seronegative when cultured.     

Biochemical Profile of Amniotic and Allantoic Fluid During Different Gestational Phases in Mares

Fetal fluids have different vital functions that sustain both pregnancy and normal parturition. The biochemical composition of amniotic fluid during gestation is not well established; thus the purpose of the present study was to determine the biochemical profile of both amniotic and allantoic fluids from mares during initial, mid, and latter third phases of pregnancy. Samples were collected after slaughter, using allantocentesis and amniocentesis. Sixty samples of fetal fluids were analyzed. Alkaline phosphatase (AP), glucose, total protein (TP), urea, creatinine, Ca, chloride (Cl), Na, and K concentrations were measured using commercially available kits. The AP concentration in amniotic fluid was higher than that in allantoic fluid during the three gestational phases (P < .05). There were no differences between glucose mean values of allantoic and those of amniotic fluids (P < .05). However, glucose values were higher in the allantoic fluid in the last trimester of pregnancy. TP was higher in the amniotic fluid than in allantoic fluid (P < .05). Urea values varied among the phases; however, there were no differences between the amniotic and allantoic fluid values (P > .05). Creatinine values were higher in allantoic fluid (P < .05). Na and Cl concentrations were higher in amniotic fluid (P < .05). However, Ca and K concentrations were higher in the allantoic fluid.