Abortion Storms Research Articles

Intranasal treatment with CpG-B oligodeoxynucleotides protects CBA mice from lethal equine herpesvirus 1 challenge by an innate immune response

Equine herpesvirus 1 (EHV-1) is the causative agent of a number of equine disease manifestations, including severe disease of the central nervous system, respiratory infections, and abortion storms. Our results showed that intranasal treatment with CpG-B oligodeoxynucleotides (ODN 1826) protected CBA mice from pathogenic EHV-1 RacL11 challenge. The IFN-γ gene and seven interferon-stimulated genes (ISGs) were upregulated 39.4- to 260.3-fold at 8 h postchallenge in the lungs of RacL11-challenged mice that had been treated with CpG-B ODN. Interestingly, IFN-γ gene expression was upregulated by 26-fold upon RacL11 challenge in CpG-B ODN-treated mice lungs as compared to that of CpG-A ODN (ODN 1585)-treated mice lungs; however, the seven ISGs were upregulated by 2.4–5.0-fold, suggesting that IFN-γ is a major factor in the protection of CBA mice from the lethal challenge. Pre-treatment with IFN-γ significantly reduced EHV-1 yield in murine alveolar macrophage MH-S cells, but not in mouse lung epithelial MLE12 cells. These results suggest that CpG-B ODN may be used as a prophylactic agent in horses and provide a basis for more effective treatment of EHV-1 infection.

In Situ Hybridization for Detection of Rift Valley Fever Virus

Rift Valley fever virus (RVFV) is a mosquito‐borne zoonotic arbovirus with a tripartite genome consisting of a small, a medium (M), and a large (L) segment. This RNA virus causes frequent epidemics in Africa and the Arabian Peninsula. Outbreaks of Rift Valley fever typically result in abortion storms, the deaths of young ruminants and less often the deaths of adult ruminants. As a zoonotic virus, RVFV can also cause symptoms ranging from a mild flu‐like illness to encephalitis and death in humans. As such, RVFV‐infected tissues must be handled with caution. Formalin‐fixed, paraffin‐embedded (FFPE) tissues offer a safer format for researchers and diagnosticians testing for the presence of this virus. Currently, immunohistochemistry (IHC) serves as a confirmatory diagnostic test in South Africa alongside RT‐qPCR on fresh frozen tissue. However, PCR does not enable the ability to visualize the virus within a histological context and IHC has limitations in its ability to detect viral antigen in suboptimally preserved samples. These limitations create a need for more powerful confirmatory diagnostic tools. Here we demonstrate the use of RNAscope® in situ hybridization (ISH) technology for the detection of RVFV viral RNA (vRNA) in FFPE tissues.Samples tested included a broad set of previously published, confirmed positive field samples from sheep naturally infected during the 2010 South African outbreak. Prior, a DNA probe was designed to target the nucleotides between 20‐997 of the RVFV L segment (NC_014397.1) and the assay was validated on experimentally infected tissues. Here we tested this pan‐RVFV ISH assay on naturally infected liver, spleen, kidney, lung, thyroid, brain, skeletal and cardiac muscle, lymph node, salivary gland, adrenal gland, gastrointestinal tract, and male and female reproductive organs. We also compared ISH results to other tests, a previously established triplex FFPE RT‐qPCR targeting all three gene segments as well as IHC targeting the RVFV Gn glycoprotein. Finally, we were able to dual label samples with pan‐RVFV ISH and IHC for analysis of both RVFV RNA and protein distribution as well as RVFV RNA and cell type markers, providing finer grained detail regarding cellular tropism.Pan‐RVFV ISH appropriately labeled vRNA and this signal correlated with visible lesions in the various organs tested. Additionally, vRNA was detected in macrophages in the lymph nodes, a result previously seen by IHC. The positive ISH results mirrored those obtained by FFPE RT‐qPCR at the tissue block level. In comparison to IHC, ISH subjectively appears to produce a stronger and more reliable signal. This was observed in multiple organs where histopathology was present but RVFV antigen IHC was negative. We believe that this highly sensitive and specific ISH assay has potential as a RVFV confirmatory diagnostic test and research applications.Support or Funding InformationThis work was funded by the Department of Diagnostic Medicine, College of Veterinary Medicine, Kansas State University; the Department of Paraclinical Sciences, University of Pretoria, USDA Agricultural Research Services, and APHIS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Rift Valley Fever Viral RNA Detection by In Situ Hybridization in Formalin-Fixed, Paraffin-Embedded Tissues.

Sporadic outbreaks of Rift Valley fever virus (RVFV), a zoonotic, mosquito-borne Phlebovirus, cause abortion storms and death in sheep and cattle resulting in catastrophic economic impacts in endemic regions of Africa. More recently, with changes in competent vector distribution, growing international trade, and its potential use for bioterrorism, RVFV has become a transboundary animal disease of significant concern. New and sensitive techniques that determine RVFV presence, while lessening the potential for environmental contamination and human risk, through the use of inactivated, noninfectious samples such as formalin-fixed, paraffin-embedded (FFPE) tissues are needed. FFPE tissue in situ hybridization (ISH) enables the detection of nucleic acid sequences within the visual context of cellular and tissue morphology. Here, we present a chromogenic pan-RVFV ISH assay based on RNAscope® technology, which is able to detect multiple RVFV strains in FFPE tissues, enabling visual correlation of RVFV RNA presence with histopathologic lesions.

Ovine abortion — causes and diagnosis

Annually it is estimated that 2–3% of the national flock will produce premature or stillborn lambs. However, infectious causes can also result in much higher abortion storms in individual flocks. An accurate diagnosis of infectious causes is important to manage the current abortion outbreak, safeguard farm workers from zoonotic infections and to allow control plans to be put into place for future years. This article will cover the diagnosis of ovine abortion and some of the main infectious causes in the UK flock.

Chimaeric Rift Valley Fever Virus-Like Particle Vaccine Candidate Production in Nicotiana benthamiana.

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus and hemorrhagic fever agent, which causes abortion storms in farmed small ruminants and potentially causes miscarriages in humans. Although live-attenuated vaccines are available for animals, they can only be used in endemic areas and there are currently no commercially available vaccines for humans. Here the authors describe the production of chimaeric RVFV virus-like particles transiently expressed in Nicotiana benthamiana by Agrobacterium tumefaciens-mediated gene transfer. The glycoprotein (Gn) gene is modified by removing its ectodomain (Gne) and fusing it to the transmembrane domain and cytosolic tail-encoding region of avian influenza H5N1 hemagglutinin. This is expressed transiently in N. benthamiana with purified protein yields calculated to be ≈57 mg kg-1 fresh weight. Transmission electron microscopy shows putative chimaeric RVFV Gne-HA particles of 49-60 nm which are immunogenic, eliciting Gn-specific antibody responses in vaccinated mice without the use of adjuvant. To our knowledge, this is the first demonstration of the synthesis of Gne-HA chimaeric RVFV VLPs and the first demonstration of a detectable yield of RVFV Gn in plants.

Synergistic Activation of Bovine Herpesvirus 1 Productive Infection and Viral Regulatory Promoters by the Progesterone Receptor and Krüppel-Like Transcription Factor 15.

Bovine herpesvirus 1 (BoHV-1), including modified live vaccines, readily infects the fetus and ovaries, which can lead to reproductive failure. The BoHV-1 latency reactivation cycle in sensory neurons may further complicate reproductive failure in pregnant cows. The immediate early transcription unit 1 (IEtu1) promoter drives expression of important viral transcriptional regulators (bICP0 and bICP4). This promoter contains two functional glucocorticoid receptor (GR) response elements (GREs) that have the potential to stimulate productive infection following stressful stimuli. Since progesterone and the progesterone receptor (PR) can activate many GREs, we hypothesized that the PR and/or progesterone regulates productive infection and viral transcription. New studies demonstrated that progesterone stimulated productive infection. Additional studies revealed the PR and Krüppel-like transcription factor 15 (KLF15) cooperated to stimulate productive infection and IEtu1 promoter activity. IEtu1 promoter activation required both GREs, which correlated with the ability of the PR to interact with wild-type (wt) GREs but not mutant GREs. KLF15 also cooperated with the PR to transactivate the bICP0 early promoter, a promoter that maintains bICP0 protein expression during productive infection. Intergenic viral DNA fragments (less than 400 bp) containing two GREs and putative KLF binding sites present within genes encoding unique long 52 (UL-52; component of DNA primase/helicase complex), Circ, bICP4, and IEtu2 were stimulated by KLF15 and the PR more than 10-fold, suggesting that additional viral promoters are activated by these transcription factors. Collectively, these studies suggest progesterone and the PR promote BoHV-1 spread to reproductive tissues, thus increasing the incidence of reproductive failure.IMPORTANCE Bovine herpesvirus 1 (BoHV-1) is the most frequently diagnosed cause of abortions in pregnant cows and can cause "abortion storms" in susceptible herds. Virulent field strains and even commercially available modified live vaccines can induce abortion, in part because BoHV-1 replicates efficiently in the ovary and corpus luteum. We now demonstrate that progesterone and the progesterone receptor (PR) stimulate productive infection. The BoHV-1 genome contains approximately 100 glucocorticoid receptor (GR) response elements (GREs). Interestingly, the PR can bind and activate many promoters that contain GREs. The PR and Krüppel-like transcription factor 15 (KLF15), which regulate key steps during embryo implantation, cooperate to stimulate productive infection and two viral promoters that drive expression of key viral transcriptional regulators. These studies suggest that the ability of progesterone and the PR to stimulate productive infection has the potential to promote virus spread in reproductive tissue and induce reproductive failure.

Expression of Rift Valley fever virus N-protein in Nicotiana benthamiana for use as a diagnostic antigen

BackgroundRift Valley fever virus (RVFV), the causative agent of Rift Valley fever, is an enveloped single-stranded negative-sense RNA virus in the genus Phlebovirus, family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing abortion storms in pregnant ruminants, high neonatal mortality in animals, and morbidity and occasional fatalities in humans. The disease is endemic in parts of Africa and the Arabian Peninsula, but is described as emerging due to the wide range of mosquitoes that could spread the disease into non-endemic regions.There are different tests for determining whether animals are infected with or have been exposed to RVFV. The most common serological test is antibody ELISA, which detects host immunoglobulins M or G produced specifically in response to infection with RVFV. The presence of antibodies to RVFV nucleocapsid protein (N-protein) is among the best indicators of RVFV exposure in animals. This work describes an investigation of the feasibility of producing a recombinant N-protein in Nicotiana benthamiana and using it in an ELISA.ResultsThe human-codon optimised RVFV N-protein was successfully expressed in N. benthamiana via Agrobacterium-mediated infiltration of leaves. The recombinant protein was detected as monomers and dimers with maximum protein yields calculated to be 500–558 mg/kg of fresh plant leaves. The identity of the protein was confirmed by liquid chromatography-mass spectrometry (LC-MS) resulting in 87.35% coverage, with 264 unique peptides. Transmission electron microscopy revealed that the protein forms ring structures of ~ 10 nm in diameter. Preliminary data revealed that the protein could successfully differentiate between sera of RVFV-infected sheep and from sera of those not infected with the virus.ConclusionsTo the best of our knowledge this is the first study demonstrating the successful production of RVFV N-protein as a diagnostic reagent by Agrobacterium-mediated transient heterologous expression in N. benthamiana. Preliminary testing of the antigen showed its ability to distinguish RVFV-positive animal sera from RVFV negative animal sera when used in an enzyme linked immunosorbent assay (ELISA). The cost-effective, scalable and simple production method has great potential for use in developing countries where rapid diagnosis of RVFV is necessary.

Abortion storm induced by the live C. abortus vaccine 1B strain in a vaccinated sheep flock, mimicking a natural wild-type infection

Chlamydia abortus is responsible for enzootic abortion (known as ovine enzootic abortion (OEA) and enzootic abortion of ewes (EAE)) in both sheep and goats and has major economic implications for the farming industry worldwide. A virulence-attenuated mutant strain of C. abortus (strain 1B) is currently commercially available as a live attenuated vaccine for immunization of sheep and goats in several European countries. Following an abortion storm in a French flock of 200 ewes that occurred two years after vaccination of 36 replacement ewes with the commercial 1B vaccine strain, the vaginal swabs of 3 vaccinated and 7 unvaccinated aborted ewes and 12 of the 13 dead fetuses were found to be positive for C. abortus by real-time PCR. Genotyping of the samples, using vaccine-specific SNP markers, identified all as positive for the vaccine-type strain. The recent vaccination of this flock with the attenuated commercial vaccine strain, the large number of abortion cases observed in ewes irrespective of vaccination status, the high C. abortus load detected in vaginal swabs or abortion tissues and the identification of specific vaccine-type markers in these samples strongly suggest that the 1B strain has been transmitted from vaccinated to naïve animals, thus mimicking a natural wild-type infection.

General evaluation of the economic impact of introduction of Chlamydia abortus to a Scottish sheep flock

The investigation of an outbreak of chlamydial abortion in a Scottish sheep flock in which there was an overall abortion rate of 12.2 per cent was used as an example...

Seroprevalence of Toxoplasma gondii and Neospora caninum infections in cattle in Mongolia.

Toxoplasma gondii and Neospora caninum are protozoan parasites that cause huge economic losses in animal industries worldwide. N. caninum can cause abortion storms and high culling rates in cattle, whereas T. gondii infection is a significant concern in both human and animals because it can induce abortion and clinical symptoms in immunocompromised hosts. The aim of this study was to determine the seroprevalence of T. gondii and N. caninum in cattle in Mongolia. Specific antibodies to T. gondii and N. caninum were detected by using an indirect enzyme-linked immunosorbent assay (iELISA) based on recombinant antigens of dense granule protein 7 of Toxoplasma gondii and surface antigen 1 of Neospora caninum, respectively. A total of 1438 cattle sera from 20 of 21 provinces of Mongolia and the capital city of Ulaanbaatar were tested. Overall, 18.7% and 26.2% of cattle were positive for specific antibodies to T. gondii and N. caninum, respectively. Prevalence rates were higher (T. gondii infection: P < .0001, N. caninum infection: P = .002) in the central region of Mongolia (T. gondii infection: 27.1%, N. caninum infection: 30.8%) compared with western region, suggesting that prevalence rates might be influenced by geographical condition, particularly warmer temperatures around this area in Mongolia. The lowest prevalence rates were observed in the western region of Mongolia (T. gondii: 9%, N. caninum: 20.8%). In addition, the seroprevalence of N. caninum in female animals (27.5%) was significantly higher than that in male animals (20.4%) (P = .018), suggesting an important risk factor of abortion and stillbirth in cattle. The present results showed that T. gondii and N. caninum infections might be a risk for public health and economy of the livestock industry in Mongolia. In conclusion, this study demonstrates high seroprevalences of T. gondii and N. caninum in Mongolia and provides valuable new data for development of control measures against these infections in Mongolia.

Coagulation parameters following equine herpesvirus type 1 infection in horses.

Equine herpesvirus type 1 (EHV-1) is the cause of respiratory disease, abortion storms, and outbreaks of herpesvirus myeloencephalopathy (EHM). Infection of the spinal cord is characterised by multifocal regions of virally infected vascular endothelium, associated with vasculitis, thrombosis and haemorrhage that result in ischaemia and organ dysfunction. However, the mechanism of thrombosis in affected horses is unknown. To evaluate tissue factor (TF) procoagulant activity and thrombin-antithrombin complex (TAT) levels in horses following infection with EHV-1. In vitro and in vivo studies following experimental EHV-1 infection. Horses were infected with EHV-1 and levels of peripheral blood mononuclear cell (PBMC)-associated TF activity; plasma and cerebrospinal fluid (CSF)-derived microvesicle (MV)-associated TF activity and TAT complexes in plasma were examined. EHV-1 infection increased PBMC TF procoagulant activity in vitro and in vivo. In infected horses, this increase was observed during the acute infection and was most marked at the onset and end of viraemia. However, no significant differences were observed between the horses that showed signs of EHM and the horses that did not develop EHM. Significant changes in MV-associated TF procoagulant activity and TAT complexes were not observed in infected horses. A small number of horses typically exhibit clinical EHM following experimental infection. The results indicate that EHV-1 infection increases PBMC-associated TF procoagulant activity in vivo and in vitro. Additional in vivo studies are needed to better understand the role of TF-dependent coagulation during EHM pathogenesis in horses.

Porcine macrophage-like cells permit viral replication, produce inflammatory mediators, and undergo apoptosis following infection with Rift Valley fever virus MP-12

Abstract Rift Valley fever virus (RVFV) is a mosquito-borne virus that can cause severe disease in a variety of domestic animal species as well as in humans. Outbreaks occur primarily in Sub-Saharan Africa with ruminant livestock most affected and outcomes that include abortion storms and high mortality among young animals. Clinical disease is not known to occur in domestic pigs (Sus scrofa); however, a limited number of studies have detected circulating antibodies to RVFV among wild and domestic members of the pig family (Suidae). Experimentally, RVFV has been shown to replicate in some porcine epithelial and neuronal cells in vitro, and induce a viremia in a fraction of pigs inoculated in vivo. No studies to date have investigated the behavior of RVFV in porcine cells of immune origin, whereas in humans and mice, macrophages and dendritic cells have been implicated as having roles in RVFV pathogenicity. Here, we use a porcine monocyte-derived macrophage-like cell line, CΔ2+, to investigate porcine innate immune cell permissiveness to MP-12, an attenuated form of RVFV. MP-12 caused observable cytopathic effects in CΔ2+ cells and reduced cell viability in dose- and time-dependent manners. At 24 hours, the reduced viability was coincident with apoptosis, with necrosis also occurring at a higher multiplicity of infection. At early time points, MP-12 replicated at levels comparable to cells derived from sheep kidney—a target organ of a highly susceptible host species. Infected CΔ2+ cells also upregulated transcription of inflammatory mediators. This work provides the first evidence that porcine immune cells are permissive to RVFV, and indicates that additional studies are warranted to better understand the dynamics of RVFV in suids.

Alterations in the host transcriptome in vitro following Rift Valley fever virus infection

Rift Valley fever virus (RVFV) causes major outbreaks among livestock, characterized by “abortion storms” in which spontaneous abortion occurs in almost 100% of pregnant ruminants. Humans can also become infected with mild symptoms that can progress to more severe symptoms, such as hepatitis, encephalitis, and hemorrhagic fever. The goal of this study was to use RNA-sequencing (RNA-seq) to analyze the host transcriptome in response to RVFV infection. G2/M DNA damage checkpoint, ATM signaling, mitochondrial dysfunction, regulation of the antiviral response, and integrin-linked kinase (ILK) signaling were among the top altered canonical pathways with both the attenuated MP12 strain and the fully virulent ZH548 strain. Although several mRNA transcripts were highly upregulated, an increase at the protein level was not observed for the selected genes, which was at least partially due to the NSs dependent block in mRNA export. Inhibition of ILK signaling, which is involved in cell motility and cytoskeletal reorganization, resulted in reduced RVFV replication, indicating that this pathway is important for viral replication. Overall, this is the first global transcriptomic analysis of the human host response following RVFV infection, which could give insight into novel host responses that have not yet been explored.

Economic impact of a Salmonella outbreak on a Welsh dairy farm and an estimation of the breakeven point for vaccination

Salmonellosis is a disease present in the cattle herds of England with clinical presentations such as abortion storms that can have devastating farm-level economic consequences. Using farm-specific costs and revenues,...

Unfractionated and Low-Molecular-Weight Heparin and the Phosphodiesterase Inhibitors, IBMX and Cilostazol, Block Ex Vivo Equid Herpesvirus Type-1-Induced Platelet Activation.

Equid herpes virus type-1 (EHV-1) is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins) or platelet inhibitors that impede post-receptor thrombin signaling [phosphodiesterase (PDE) antagonists] would inhibit EHV-1-induced platelet activation ex vivo. We exposed platelet-rich plasma (PRP) collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque-forming unit/cell in the presence or absence of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX), and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1–0.2 U/mL anti-factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM, but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX) or isoenzyme-3 selective (cilostazol) PDE antagonists inhibit ex vivo EHV-1-induced platelet activation. These drugs have potential as adjunctive therapy to reduce the serious complications associated with EHV-1-induced thrombosis. Treatment trials are warranted to determine whether these drugs yield clinical benefit when administered to horses infected with EHV-1.

Are all bovine herpesvirus-1 reproductive disease events vaccine-induced?

Modified-live virus (MLV) bovine herpesvirus 1 (BHV-1) vaccines have been a major part of the program to the prevention of reproductive disease in cattle. However, their use (or misuse) continues to result in major abortion storms. The results of 20 additional cases with reproductive losses are described where the single nucleotide polymorphism (SNP) analysis was done to identify the source.

HoBi-Like Pestivirus and Its Impact on Cattle Productivity.

The clinical features and economic impact of the infection caused by an emerging group of pestiviruses, namely HoBi-like pestivirus, in a cattle herd of southern Italy are reported. In 2011, the virus was first associated with respiratory disease, causing an abortion storm after 1year and apparently disappearing for the following 3years after persistently infected calves were slaughtered. However, in 2014, reproductive failures and acute gastroenteritis were observed in the same herd, leading to a marked decrease of productivity. A HoBi-like strain closely related to that responsible for previous outbreaks was detected in several animals. Application of an intensive eradication programme, based on the detection and slaughtering of HoBi-like pestivirus persistently infected animals, resulted in a marked improvement of the productive performances.

Serological detection and epidemiology of Neospora caninum and Cryptosporidium parvum antibodies in cattle in southern Egypt

Neospora caninum and Cryptosporidium parvum are intracellular protozoan parasites that are distributed worldwide and of major economical concern in cattle industry. N. caninum can cause abortion storms and high culling rates, whereas C. parvum has zoonotic implications and can cause diarrhea in calves. There are currently no data on the prevalence of neosporosis and cryptosporidiosis in humans or animals in southern Egypt. Prevalence of these two infections was determined in a sample of cattle from two different areas in southern Egypt, Sohag and Qena, using enzyme-linked immunosorbent assay. A total 301 cattle were sampled, of which 18.9% were positive for N. caninum, 35.9% were positive for C. parvum and 10.0% were positive for both. Geographical location and breeding system were considered as potential risk factors for C. parvum infection. A higher prevalence of infection was identified on small scale farms, compared with larger, intensive systems, with a prevalence of 50.2% compared with 37.8%, respectively. Animals in Sohag had a significantly higher prevalence compared with Qena, with a seroprevalence of 46.1% compared with 31.6%, respectively. In brief, marked seroprevalence recorded in this study indicates a high incidence of N. caninum and C. parvum infections in cattle, and this necessitates the application of more effective strategies for combating these types of infections on farms in Egypt.

Tolerogenic dendritic cells (DCs) are induced after seminal fluid priming in allogeneic mating mice

Neospora caninum and Cryptosporidium parvum are intracellular protozoan parasites that are distributed worldwide and of major economical concern in cattle industry. N. caninum can cause abortion storms and high culling rates, whereas C. parvum has zoonotic implications and can cause diarrhea in calves. There are currently no data on the prevalence of neosporosis and cryptosporidiosis in humans or animals in southern Egypt. Prevalence of these two infections was determined in a sample of cattle from two different areas in southern Egypt, Sohag and Qena, using enzyme-linked immunosorbent assay. A total 301 cattle were sampled, of which 18.9% were positive for N. caninum, 35.9% were positive for C. parvum and 10.0% were positive for both. Geographical location and breeding system were considered as potential risk factors for C. parvum infection. A higher prevalence of infection was identified on small scale farms, compared with larger, intensive systems, with a prevalence of 50.2% compared with 37.8%, respectively. Animals in Sohag had a significantly higher prevalence compared with Qena, with a seroprevalence of 46.1% compared with 31.6%, respectively. In brief, marked seroprevalence recorded in this study indicates a high incidence of N. caninum and C. parvum infections in cattle, and this necessitates the application of more effective strategies for combating these types of infections on farms in Egypt.

The L, M, and S Segments of Rift Valley Fever Virus MP-12 Vaccine Independently Contribute to a Temperature-Sensitive Phenotype.

Rift Valley fever (RVF) is endemic to Africa, and the mosquito-borne disease is characterized by "abortion storms" in ruminants and by hemorrhagic fever, encephalitis, and blindness in humans. Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) has a tripartite negative-stranded RNA genome (L, M, and S segments). A live-attenuated vaccine for RVF, the MP-12 vaccine, is conditionally licensed for veterinary use in the United States. MP-12 is fully attenuated by the combination of the partially attenuated L, M, and S segments. Temperature sensitivity (ts) limits viral replication at a restrictive temperature and may be involved with viral attenuation. In this study, we aimed to characterize the ts mutations for MP-12. The MP-12 vaccine showed restricted replication at 38°C and replication shutoff (100-fold or greater reduction in virus titer compared to that at 37°C) at 39°C in Vero and MRC-5 cells. Using rZH501 reassortants with either the MP-12 L, M, or S segment, we found that all three segments encode a temperature-sensitive phenotype. However, the ts phenotype of the S segment was weaker than that of the M or L segment. We identified Gn-Y259H, Gc-R1182G, L-V172A, and L-M1244I as major ts mutations for MP-12. The ts mutations in the L segment decreased viral RNA synthesis, while those in the M segment delayed progeny production from infected cells. We also found that a lack of NSs and/or 78kD/NSm protein expression minimally affected the ts phenotype. Our study revealed that MP-12 is a unique vaccine carrying ts mutations in the L, M, and S segments. Rift Valley fever (RVF) is a mosquito-borne viral disease endemic to Africa, characterized by high rates of abortion in ruminants and severe diseases in humans. Vaccination is important to prevent the spread of disease, and a live-attenuated MP-12 vaccine is currently the only vaccine with a conditional license in the United States. This study determined the temperature sensitivity (ts) of MP-12 vaccine to understand virologic characteristics. Our study revealed that MP-12 vaccine contains ts mutations independently in the L, M, and S segments and that MP-12 displays a restrictive replication at 38°C.