Detection of Anaplasma phagocytophilum in fetal and placental tissue of bovine abortions and perinatal mortalities.

BACKGROUND: Anaplasma phagocytophilum is a tick-borne zoonotic bacterium that is the aetiologic pathogen of tick-borne fever (TBF) in ruminants. In clinical bovine cases of TBF, abortion and stillbirth may be observed. However, in this regard, the pathophysiology of TBF has not yet been completely elucidated, and no clear guidelines to diagnose A. phagocytophilum-related abortions and perinatal mortalities (APM) are available. METHODS: This exploratory study aimed to investigate the presence of A. phagocytophilum in bovine cases of APM and determine whether placental or fetal spleen tissue has the greatest sensitivity for A. phagocytophilum identification. The placenta and fetal spleen of 150 late-term bovine APM cases were analysed using real-time PCR to detect A. phagocytophilum. RESULTS: A total of 2.7% of sampled placentas were positive for A. phagocytophilum, while none of the fetal spleen samples was. LIMITATIONS: No histopathology to detect associated lesions was performed. Consequently, no evidence of causality between the detection of A. phagocytophilum and APM events could be achieved. CONCLUSION: The detection of A. phagocytophilum suggests a potential role of this pathogen in bovine APM, and placental tissue seems to be the most suitable tissue for its identification.

Genetic Markers Associated with Field PRRSV-Induced Abortion Rates.

In gilts and sows, the more severe clinical manifestation of porcine reproductive and respiratory syndrome virus (PRRSV) occurs in late gestation and can result in up to a 40% abortion incidence. Despite the known genetic component in resilience to PRRSV, there is scarce information regarding the abortive outcome of this disease. We tested the relationship between eight molecular markers (six from published studies and two identified in the present study in the HDAC6 gene) and the probability of abortion during a PRRSV outbreak, using data from two commercial Landrace x Large White sow farms with an incidence of abortion of 35% and 17%. From the markers tested, USP18_-1533G>A did not segregate in these populations, and CD163_c.3534C>T and HDAC6_g.2360C>T did not affect the abortion rate. In contrast, the minor allele of two markers in SSC4 (WUR1000125 in GBP1 and rs340943904 in GBP5), which lower viremia in growing pigs, and the major alleles of CD163_rs1107556229 and HDAC6_rs325981825 were associated with a lower probability of abortion during PRRSV outbreaks. The more striking result was for the MX1 gene, where the odds ratio of aborting versus not aborting was nine times lower in the sows homozygous for a 275-bp insertion than in the other genotypes. Interactions between markers were not relevant. All together, we bring here the first evidence that mutations in the host genome can predispose or protect from complete reproductive failure in sows infected with PRRSV.

Actin from the apicomplexan Neospora caninum (NcACT) has different isoforms in 2D electrophoresis.

Apicomplexan parasites have unconventional actins that play a central role in important cellular processes such as apicoplast replication, motility of dense granules, endocytic trafficking and force generation for motility and host cell invasion. In this study, we investigated the actin of the apicomplexan Neospora caninum - a parasite associated with infectious abortion and neonatal mortality in livestock. Neospora caninum actin was detected and identified in two bands by one-dimensional (1D) western blot and in nine spots by the 2D technique. The mass spectrometry data indicated that N. caninum has at least nine different actin isoforms, possibly caused by post-translational modifications. In addition, the C4 pan-actin antibody detected specifically actin in N. caninum cellular extract. Extracellular N. caninum tachyzoites were treated with toxins that act on actin, jasplakinolide and cytochalasin D. Both substances altered the peripheric cytoplasmic localization of actin on tachyzoites. Our findings add complexity to the study of the apicomplexan actin in cellular processes, since the multiple functions of this important protein might be regulated by mechanisms involving post-translational modifications.

Molecular and serological data supporting the role of Q fever in abortions of sheep and goats in northern Egypt.

Q fever is a worldwide zoonotic disease, caused by Coxiella burnetii (C. burnetii), an obligate intracellular bacterium. The epidemiological data about the Q fever situation in Egypt is limited. The present study investigated the seroprevalence of Q fever among small ruminants in some localities in the northern Egypt and reported the shedders using specific real-time PCR (Rt-PCR). A total of 190 sera and vaginal swabs (110 sheep and 80 goats) were collected from aborted cases. Indirect ELISA was used to detect specific antibodies against C. burnetii, and Rt-PCR was used to detect DNA in the shedder animals. The study revealed that infection was significantly higher in sheep (22.7%) than in goats (12.5%) (p < 0.05). The Menoufia and Gharbia governorates had 20% seropositive animals while Qalubia and Alexandria had 15% and 17.5% seropositive animals, respectively. Using a Rt - PCR assay, C. burnetii was detected in 33.6% and 16.3% of sheep and goats, respectively. The findings of the study demonstrate that Q fever may be enzootic among small ruminants and distributed in the northern Egyptian Governorates. Further studies are needed in different regions to gain better understanding of the epidemiology of Q fever all over the country and to develop an appropriate preventive strategy for human and animals.

A multi-screening Fast qPCR approach to the identification of abortive agents in ruminants.

Abortion in ruminants represents an important economic concern for farmers. Microbial agents, such as Brucella spp., Chlamydia spp., Coxiella burnetii, Leptospira spp., Neospora caninum, Salmonella spp. and Toxoplasma gondii, are among the main infectious causes of abortion and require rapid and reliable diagnosis. This study describes the development of a multi-screening assay using Fast Real-Time PCR (Fast qPCR) that allows, in a single test, the simultaneous identification of the above-mentioned abortive agents. This multi-screening approach is characterized by a mean diagnostic sensitivity and specificity of 100% and 97%, respectively; it has a limit of detection (LOD) ranging from 5 × 103 to 4 × 104 genomic copies/g of tissue and a very good concordance with traditional end-point PCR assays used in routine diagnostic activity. The proposed method represents a rapid approach to the simultaneous detection of the main abortive agents in ruminants that allows to make an accurate diagnosis and to set up appropriate control measures in a short period of time.

Pharmacokinetics of chlortetracycline in maternal plasma and in fetal tissues following oral administration to pregnant ewes.

The purpose of this study was to determine if concentrations of chlortetracycline could be detected in fetal plasma or tissues after administering an oral dose of chlortetracycline (CTC; 500 mg/head/day) reported to be effective in controlling Campylobacter spp. abortions. Five pregnant ewes were administered 250 mg/head twice a day (total dose 500 mg/hd/d) for 7 days. On the beginning of day 7, intravenous catheters were surgically implanted or inserted into the fetus and dam. Plasma samples were collected from the ewe and fetus at various time points before and up to 36 hr after the last dose of CTC. All ewes were then sacrificed, and tissues were harvested from the fetus for drug analysis. Concentrations of CTC in maternal plasma were consistent with our previous study and below the minimum inhibitory concentration of Campylobacter abortion isolates. Concentrations of CTC were below the limit of detection in three of five fetal plasma samples and all of the placenta, amniotic fluid, and fetal stomach contents. Low concentrations were detectable in fetal kidney and liver, suggesting that CTC reaches the fetus, although at a variable and low ratio when compared to maternal concentrations.

Experimental challenge of pregnant cattle with the putative abortifacient Waddlia chondrophila.

Waddlia chondrophila is a Gram-negative intracellular bacterial organism that is related to classical chlamydial species and has been implicated as a cause of abortion in cattle. Despite an increasing number of observational studies linking W. chondrophila infection to cattle abortion, little direct experimental evidence exists. Given this paucity of direct evidence the current study was carried out to investigate whether experimental challenge of pregnant cattle with W. chondrophila would result in infection and abortion. Nine pregnant Friesian-Holstein heifers received 2 × 108 inclusion forming units (IFU) W. chondrophila intravenously on day 105-110 of pregnancy, while four negative-control animals underwent mock challenge. Only one of the challenged animals showed pathogen-associated lesions, with the organism being detected in the diseased placenta. Importantly, the organism was re-isolated and its identity confirmed by whole genome sequencing, confirming Koch's third and fourth postulates. However, while infection of the placenta was observed, the experimental challenge in this study did not confirm the abortifacient potential of the organism.

Analysis of Humoral Immune Responses to Surface and Virulence-Associated Chlamydia abortus Proteins in Ovine and Human Abortions by Use of a Newly Developed Line Immunoassay.

The obligate intracellular bacterium Chlamydia abortus is the causative agent of enzootic abortion of ewes and poses a significant zoonotic risk for pregnant women. Using proteomic analysis and gene expression library screening in a previous project, we identified potential virulence factors and candidates for serodiagnosis, of which nine were scrutinized here with a strip immunoassay. We have shown that aborting sheep exhibited a strong antibody response to surface (MOMP, MIP, Pmp13G) and virulence-associated (CPAF, TARP, SINC) antigens. While the latter disappeared within 18 weeks following abortion in a majority of the animals, antibodies to surface proteins persisted beyond the duration of the study. In contrast, nonaborting experimentally infected sheep developed mainly antibodies to surface antigens (MOMP, MIP, Pmp13G), all of which did not persist. We were also able to detect antibodies to these surface antigens in C abortus-infected women who had undergone septic abortion, whereas a group of shepherds and veterinarians with occupational exposure to C abortus-infected sheep revealed only sporadic immune responses to the antigens selected. The most specific antigen for the serodiagnosis of human C abortus infections was Pmp13G, which showed no cross-reactivity with other chlamydiae infecting humans. We suggest that Pmp13G-based serodiagnosis accomplished by the detection of antibodies to virulence-associated antigens such as CPAF, TARP, and SINC may improve the laboratory diagnosis of human and animal C abortus infections.

Seroprevalence study of the main causes of abortion in dairy cattle in Morocco.

Sera from 221 cattle were collected in 25 farms in Morocco to investigate the evidence and circulation of some of the main bovine abortive agents in the dairy cattle farming, where abortions are often reported. All sera were examined for brucellosis, 176 for neosporosis, 88 for leptospirosis, and 42 for Bovine viral diarrhoea (BVD/MD), Bovine Herpesvirus 1 (BHV-1) (Infectious bovine rhinotracheitis, IBR/IPV), and Bovine Herpesvirus 4 (BHV-4) infections (at least 1 sample per herd). Abortions were reported in 23 (10.4%) of the 221 tested cattle. Antibodies against the investigated pathogens were detected in all samples tested, with an overall seroprevalence of 33.48% for Brucella, 9.09% for Leptospira, 8.52% for Neospora, 37.71% for BVDV, 50% for BHV-1, 9.52% for BHV-4. As for Leptospira antibodies against serovars Hardjo, Pomona, and Tarassovi were identified. Mixed infections were common. The lack of evidence of non-infectious factors epidemiologically related to abortions suggested that the investigated agents are to be considered important risk factors in the dynamic of the abortion syndrome, even if further investigations are necessary to identify the abortion cause. Particular attention should be paid on brucellosis, considering the high seroprevalence and its zoonotic relevance.

Insights into potential pathogenesis mechanisms associated with Campylobacter jejuni-induced abortion in ewes.

BACKGROUND: Campylobacter jejuni is commonly found in the gastrointestinal tract of many food-animals including sheep without causing visible clinical symptoms of disease. However, C. jejuni has been implicated in ovine abortion cases worldwide. Specifically, in the USA, the C. jejuni sheep abortion (SA) clone has been increasingly associated with sheep abortion. In vivo studies in sheep (the natural host) are needed to better characterize the virulence potential and pathogenesis of this clone. RESULTS: Pregnant ewes intravenously (IV) or orally inoculated with ovine or bovine abortion-associated C. jejuni SA clones exhibited partial or complete uterine prolapse with retained placenta, and abortion or stillbirth, whereas delivery of healthy lambs occurred in pregnant ewes inoculated with C. jejuni 81-176 or in the uninfected group. In sheep inoculated with the SA clone, histopathological lesions including suppurative necrotizing placentitis and/or endometritis coincided with: 1) increased apoptotic death of trophoblasts, 2) increased expression of the host genes (e.g. genes encoding interleukin IL-6 and IL-15) related to cellular necrosis and pro-inflammatory responses in uterus, and 3) decreased expression of the genes encoding GATA binding protein 6, chordin, and insulin-like 3 (INSL3) that account for embryonic development in uterus. Immunohistochemistry revealed localization of bacterial antigens in trophoblasts lining the chorioallantoic membrane of ewes inoculated with the C. jejuni SA clone. CONCLUSIONS: The results showed that C. jejuni SA clones are capable of causing abortion or stillbirth in experimentally infected sheep. Furthermore, down- or up-regulation of specific genes in the uterus of infected pregnant ewes might implicate host genes in facilitating the disease progression. Since the C. jejuni SA strains share genotypic similarities with clones that have been isolated from human clinical cases of gastroenteritis, these strains might represent a potential public health risk.

Caracterização morfológica e imuno-histoquímica de lesões em casos de aborto bovino bacteriano e viral no sul de Minas Gerais / Morphological and immunohistochemical characterization of the lesions in cases of bacterial and viral bovine abortion in southern Minas Gerais, Brazil

O presente estudo avaliou a participação de agentes bacterianos e virais em abortos em bovinos de propriedades rurais do sul de Minas Gerais. Foi realizada análise histopatológica e imuno-histoquímica dos casos de aborto recebidos pelo Setor de Patologia Veterinária da Universidade Federal de Lavras no período de 1999 a 2013. De 60 fetos analisados, em 30 (50%) foram observadas lesões microscópicas. Destes, oito apresentavam lesões compatíveis com infecção por agentes bacterianos e três apresentaram lesões sugestivas de agentes virais. Dos abortos bacterianos, um feto tinha lesões compatíveis com leptospirose, caracterizadas por icterícia e colestase, nefrite intersticial linfoplasmocítica e nefrose tubular. Sete fetos apresentaram pneumonia ou broncopneumonia purulenta; num deles havia também pleurite e peritonite fibrinosas; e em dois desses fetos houve imunomarcação para Brucella abortus. Dos três fetos com lesões sugestivas de aborto viral ocorreu imunomarcação anti-Herpesvírus bovino em um. Os resultados demonstram a ocorrência de abortos de origem bacteriana e viral na Região do estudo e que medidas profiláticas devem ser adotadas nas propriedades. O trabalho demonstra também que a imuno-histoquímica (IHQ); associada à histopatologia; é uma ferramenta útil e viável para o diagnóstico, especialmente quando provas microbiológicas e/ou sorológicas não estão disponíveis.

The paper evaluates the participation of bacterial and viral agents in bovine abortions in farms of southern Minas Gerais state, Brazil. Histopathological and immunohistochemical analysis was performed in aborted fetuses of cattle received by the Setor de Patologia Veterinária, Universidade Federal de Lavras, from 1999 to 2013. From 60 fetuses studied, 30 (50%) had microscopic lesions. From these, eight had lesions consistent with bacterial agents and three had lesions suggestive of viral agents. In the bacterial abortions, one fetus presented lesions compatible with leptospirosis, characterized by jaundice, cholestasis, lymphoplasmacytic intersticial nephritis, and tubular nephrosis. Seven fetuses had purulent pneumonia or bronchopneumonia and one of them had also fibrinous pleuritis and peritonitis; two of them presented positive immunostaining for Brucella abortus. One of the three fetuses with lesions of viral infection revealed positive imunostaining for bovine herpesvirus. The results showed that abortions of bacterial and viral origin occur in the Region of this study and prophylactic measures should be adopted on the farms. This study also demonstrates that immunohistochemistry associated with histopathology is a useful and viable tool for the diagnosis, especially when microbiological and/or serological tests are not available.

Pharmacokinetics of oral chlortetracycline in nonpregnant adult ewes.

The objectives of this study were to determine plasma concentrations and pharmacokinetic parameters of feed-grade chlortetracycline (CTC) in sheep after oral administration of 80 or 500 mg/head daily, divided into two equal doses given at 12-h intervals for 8 days. These are the approved, and commonly used but unapproved, feed additive doses, respectively, in the United States for the prevention of ovine infectious abortion. Blood samples were collected just prior to dosing at 0, 12, 24, 72, 96, and 192 h, as well as 4, 8, 12, 24, and 36 h after the last dose, and noncompartmental pharmacokinetic analysis was performed to estimate elimination half-life and area under the plasma concentration-time curve (AUC). Mean observed maximum CTC concentrations (Cmax ) were 20.0 ng/mL (80 mg dose) and 101 ng/mL (500 mg dose). Mean apparent elimination half-life was 18 h (80 mg dose) and 20 h (500 mg dose). Although published data do not exist to estimate plasma CTC concentrations necessary for the prevention of ovine infectious abortion, concentrations reached in our study suggest that either the FDA-approved and FDA-unapproved dosages are not high enough or that the pharmacodynamic parameter relating preventive dose to pathogen minimum inhibitory concentrations is yet to be determined.

Genetic diversity and antimicrobial susceptibility of Campylobacter jejuni isolates associated with sheep abortion in the United States and Great Britain.

Campylobacter infection is a leading cause of ovine abortion worldwide. Historically, genetically diverse Campylobacter fetus and Campylobacter jejuni strains have been implicated in such infections, but since 2003 a highly pathogenic, tetracycline-resistant C. jejuni clone (named SA) has become the predominant cause of sheep abortions in the United States. Whether clone SA was present in earlier U.S. abortion isolates (before 2000) and is associated with sheep abortions outside the United States are unknown. Here, we analyzed 54 C. jejuni isolates collected from U.S. sheep abortions at different time periods and compared them with 42 C. jejuni isolates associated with sheep abortion during 2002 to 2008 in Great Britain, using multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and array-based comparative genomic hybridization (CGH). Although clone SA (ST-8) was present in the early U.S. isolates, it was not as tetracycline resistant (19% versus 100%) or predominant (66% versus 91%) as it was in the late U.S isolates. In contrast, C. jejuni isolates from Great Britain were genetically diverse, comprising 19 STs and lacking ST-8. PFGE and CGH analyses of representative strains further confirmed the population structure of the abortion isolates. Notably, the Great Britain isolates were essentially susceptible to most tested antibiotics, including tetracycline, while the late U.S. isolates were universally resistant to this antibiotic, which could be explained by the common use of tetracyclines for control of sheep abortions in the United States but not in Great Britain. These results suggest that the dominance of clone SA in sheep abortions is unique to the United States, and the use of tetracyclines may have facilitated selection of this highly pathogenic clone.

Proteomic identification of immunodominant membrane-related antigens in Campylobacter jejuni associated with sheep abortion.

Campylobacter jejuni clone SA is the predominant agent inducing sheep abortion and a zoonotic agent causing gastroenteritis in humans in the United States. In an attempt to identify antigens of clone SA that may be useful for vaccine development, immunoproteomic analyses were conducted to characterize the membrane proteome of C. jejuni clone SA. 2-DE of C. jejuni membrane-related proteins was followed by immunoblotting analyses using convalescent sera that were derived from ewes naturally infected by C. jejuni clone SA. Totally 140 immunoreactive spots were identified, 50 of which were shared by all tested convalescent sheep sera. Conserved and immunodominant spots were identified by mass spectrometry. Among the 26 identified immunogenic proteins, there were 8 cytoplasmic proteins, 2 cytoplasmic membrane proteins, 11 periplasmic proteins, 3 outer membrane proteins, and 2 extracellular proteins. Notably, many of the immunodominant antigens were periplasmic proteins including HtrA, ZnuA, CjaA, LivK, CgpA, and others, some of which were previously shown to induce protective immunity. Interestingly, 11 immunoreactive proteins including 9 periplasmic proteins are known N-linked glycosylated proteins. These findings reveal immunogens that may potentially elicit protective immune responses and provide a foundation for developing vaccines against C. jejuni induced sheep abortion. BIOLOGICAL SIGNIFICANCE: Campylobacter jejuni clone SA is the predominant agent inducing sheep abortion and incurs a significant economic loss to sheep producers. This emergent strain is also a zoonotic agent, causing gastroenteritis in humans. However, the immunogens of C. jejuni induced abortion are largely unknown. Considering the significance of C. jejuni clone SA in causing sheep abortion and foodborne illnesses, protective vaccines are needed to control its transmission and spread. Additionally, immunological markers are required for detection and identification of this highly pathogenic clone. To address these needs, we applied an immunoproteomic approach to identify the membrane-associated antigens of this highly virulent C. jejuni clone associated with sheep abortions in the U.S. The findings reveal immunogens that may potentially elicit protective immune responses and provide a foundation for developing vaccines against C. jejuni induced sheep abortion.

Bubaline herpesvirus 1 associated with abortion in a Mediterranean water buffalo.

During routine analysis of water buffalo foetuses, one sample was positive for herpesvirus and negative to all the other abortive agents investigated. Sequencing of the herpesvirus glycoprotein E gene identified the virus as bubaline herpesvirus 1, showing few differences with the published sequences. This represents the first finding of bubaline herpesvirus in a water buffalo foetus associated with abortion.

Experimental infection of pregnant cows with noncytopathogenic bovine viral diarrhoea virus between days 26 and 50 postbreeding.

The effect of bovine viral diarrhoea virus (BVDV) infection on early pregnant cows between 10 and 24 days after virus inoculation at day 26 of pregnancy was determined. Four cows were inoculated intravenously with either BVDV (treated, n=3) or growth medium (control, n=1). The treated cows were euthanized on either day 10, 17 or 24 post-infection and the control cow was euthanized on day 24 post-infection. The level of serum 2-5A synthetase increased in all of the three treated cows. Progesterone levels decreased to below 1.0 ng/ml between 10 and 22 days after inoculation in two of the three treated cows and the embryos/foetuses of two cows died. Therefore, BVDV may be a cause of early embryonic or feotal loss in early pregnant cows and serum 2-5A synthetase may be useful as an indicator of viral infection in cows.

Salmonellosis in cattle: advantages of being an experimental model.

Salmonellosis is an important disease of cattle caused predominantly by Salmonella enterica serotypes Typhimurium (S. typhimurium) and Dublin (S. dublin). S. typhimurium causes acute enteritis and exudative diarrhea in calves. In addition to enteric disease, S. dublin can cause systemic infections, and may cause abortion in pregnant cows. Calves are considered a relevant model for non-typhoidal salmonellosis in humans. Experimental oral infections or inoculation of ligated ileal loops in calves have been extensively studied recently. This article reviews relevant published results regarding bovine salmonellosis as a natural disease or as an animal model.

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.

Immunological paradigms and the pathogenesis of ovine chlamydial abortion.

Successful mammalian pregnancy involves complex immunological interactions between the mother and foetus that are not yet fully understood. A number of immunological paradigms have been established to explain the failure of the maternal immune system to reject the semi-allogeneic foetus, mainly based on studies in mice and humans. However, as placental structure, gestation periods and number of concepti per pregnancy can vary greatly between mammals, it is not always clear how applicable these immunological paradigms are to reproduction in other species. Here, we discuss the predictions of three important immunological paradigms in relation to the pathogenesis of ovine enzootic abortion (OEA), a common cause of infectious abortion in sheep and other ruminants. OEA is caused by the intracellular Gram-negative bacterium Chlamydophila abortus that exhibits a tropism for placental trophoblast. The paradigms of particular relevance to the pathogenesis of OEA are as follows: (i) intracellular bacterial infections are controlled by T(H)1-type CD4(+ve) T cells; (ii) indoleamine 2,3-dioxygenase is expressed in the placenta to prevent immunological rejection of the semi-allogeneic foetus; and (iii) pregnancy is a maternal T(H)2-type phenomenon. We discuss the relevance and validity of these paradigms for chlamydial abortion and reproductive immunology in sheep.