Abortion In Ruminants Research Articles

Blood-feeding, susceptibility to infection with Schmallenberg virus and phylogenetics of Culicoides (Diptera: Ceratopogonidae) from the United Kingdom

BackgroundCulicoides biting midges (Diptera: Ceratopogonidae) are responsible for the biological transmission of internationally important arboviruses of livestock. In 2011, a novel Orthobunyavirus was discovered in northern Europe causing congenital malformations and abortions in ruminants. From field studies, Culicoides were implicated in the transmission of this virus which was subsequently named Schmallenberg virus (SBV), but to date no assessment of susceptibility to infection of field populations under standardised laboratory conditions has been carried out. We assessed the influence of membrane type (chick skin, collagen, Parafilm M®) when offered in conjunction with an artificial blood-feeding system (Hemotek, UK) on field-collected Culicoides blood-feeding rates. Susceptibility to infection with SBV following blood-feeding on an SBV-blood suspension provided via either (i) the Hemotek system or via (ii) a saturated cotton wool pledglet was then compared. Schmallenberg virus susceptibility was defined by RT-qPCR of RNA extractions of head homogenates and related to Culicoides species and haplotype identifications based on the DNA barcode region of the mitochondrial cytochrome c oxidase 1 (cox1) gene.ResultsCulicoides blood-feeding rates were low across all membrane types tested (7.5% chick skin, 0.0% for collagen, 4.4% Parafilm M®, with 6029 female Culicoides being offered a blood meal in total). Susceptibility to infection with SBV through membrane blood-feeding (8 of 109 individuals tested) and pledglet blood-feeding (1 of 94 individuals tested) was demonstrated for the Obsoletus complex, with both C. obsoletus (Meigen) and C. scoticus Downes & Kettle susceptible to infection with SBV through oral feeding. Potential evidence of cryptic species within UK populations was found for the Obsoletus complex in phylogenetic analyses of cox1 DNA barcodes of 74 individuals assessed from a single field-site.ConclusionsMethods described in this study provide the means to blood-feed Palaearctic Culicoides for vector competence studies and colonisation attempts. Susceptibility to SBV infection was 7.3% for membrane-fed members of the subgenus Avaritia and 1.1% for pledglet-fed. Both C. obsoletus and C. scoticus were confirmed as being susceptible to infection with SBV, with potential evidence of cryptic species within UK Obsoletus complex specimens, however the implications of cryptic diversity in the Obsoletus complex on arbovirus transmission remains unknown.

Genetic Separation of Listeria monocytogenes Causing Central Nervous System Infections in Animals.

Listeria monocytogenes is a foodborne pathogen that causes abortion, septicemia, gastroenteritis and central nervous system (CNS) infections in ruminants and humans. L. monocytogenes strains mainly belong to two distinct phylogenetic groups, named lineages I and II. In general, clinical cases in humans and animals, in particular CNS infections, are caused by lineage I strains, while most of the environmental and food strains belong to lineage II. Little is known about why lineage I is more virulent than lineage II, even though various molecular factors and mechanisms associated with pathogenesis are known. In this study, we have used a variety of whole genome sequence analyses and comparative genomic tools in order to find characteristics that distinguish lineage I from lineage II strains and CNS infection strains from non-CNS strains. We analyzed 225 strains and identified single nucleotide variants between lineages I and II, as well as differences in the gene content. Using a novel approach based on Reads Per Kilobase per Million Mapped (RPKM), we identified 167 genes predominantly absent in lineage II but present in lineage I. These genes are mostly encoding for membrane-associated proteins. Additionally, we found 77 genes that are largely absent in the non-CNS associated strains, while 39 genes are especially lacking in our defined “non-clinical” group. Based on the RPKM analysis and the metadata linked to the L. monocytogenes strains, we identified 6 genes potentially associated with CNS cases, which include a transcriptional regulator, an ABC transporter and a non-coding RNA. Although there is not a clear separation between pathogenic and non-pathogenic strains based on phylogenetic lineages, the presence of the genes identified in our study reveals potential pathogenesis traits in ruminant L. monocytogenes strains. Ultimately, the differences that we have found in our study will help steer future studies in understanding the virulence mechanisms of the most pathogenic L. monocytogenes strains.

Abortion Caused by Coxiella burnetii in a Cow and Goat in Korea

Q fever is a zoonosis caused by Coxiella burnetii, an obligate intracellular Gram-negative bacterium. Q fever is usually subclinical, but is occasionally associated with abortion in ruminants. Two cases of abortion, one in a goat and the other in a cow, were reported to the Animal and Plant Quarantine Agency for diagnostic testing. Histologically, suppurative placentitis was apparent in both cases and more severe in the goat than in the cow. C. burnetii antigens were detected by polymerase chain reaction of DNA from various tissue extracts and antibodies were detected in the sera in both cases. To our knowledge, this is the first report of bovine and caprine abortion caused by C. burnetii in South Korea.

Serological prevalence of Coxiella burnetii in dairy goats and ewes diagnosed with adverse pregnancy outcomes in Greece.

Coxiella burnetii is an obligatory intracellular bacterial pathogen causing the zoonotic disease Q fever. The most common reservoirs of C. burnetiiare wild mammals, birds and ticks. Pregnant domestic ruminants infected with this bacterium are also a major source of human infection. The serological prevalence of C. burnetii in goats and sheep diagnosed with adverse pregnancy outcomes was assessed by undertaking a survey on 800 dairy goats and 800 dairy ewes reared in four different regions of Greece (Macedonia, Thrace, Thessaly, and Peloponnese). A stratified sampling was carried out, taking also as a criterion the age of the animals. Serum antibodies were analyzed by a commercial ELISA according to the manufacturer's recommendations. Generally, there was a statistically significantly higher serological prevalence of C. burnetii (14.4%) in goats compared to sheep (8%). Serological prevalence was higher in adults (15.5% in goats and 8.5% in sheep) compared to yearlings (7.4% in goats and 4.6% in sheep). The prevalence increased significantly with age only in goats. Finally, all animals reared in Peloponnese had a prevalence significantly higher (21% in goats and 18% in sheep) than animals reared in the other three regions. To the best of the authors' knowledge, this is the first report that associates C. burnetii with reproductive disturbances of domestic ruminants in Greece. However, considering the importance of coxiellosis for public health, further investigations are required on its epidemiology regarding abortion, premature delivery, stillbirth and weak offspring in small ruminants, as well as in other domestic and wild animal species.

Molecular detection of Chlamydophila abortus, Coxiella burnetii, and Mycoplasma agalactiae in small ruminants' aborted fetuses in southern Iran.

Abortion in sheep and goats has become increasingly important worldwide because of the significant economic losses and potential zoonotic implication of commonly involved pathogens. Therefore, this cross-sectional study was conducted in southern Iran to detect the Chlamydophila abortus and Coxiella burnetii, as zoonotic pathogens, and Mycoplasma agalactiae, as a neglected abortifacient agent in small ruminants' aborted fetuses, by using polymerase chain reaction (PCR). From a total of 300 aborted fetuses (183 sheep and 117 goats), 46 samples (15.5%) were positive by PCR, 11% for C. abortus, 2% for C. burnetii, and 3% for M. agalactiae. Also, the association of suggested risk factors with abortion due to these bacterial agents was investigated using univariable and multivariable logistic regression. Results of the statistical analysis showed significant association of C. abortus with flock size (OR = 2.82, P = 0.014), season (P < 0.05), and the number of pregnancy in the aborted dam (OR = 2.5, P = 0.05). Our results indicated that C. abortus has a relatively substantial role in small ruminant abortions, and C. burnetii and M. agalactiae are likely important abortifacient agents in our region, too. Regarding veterinary and/or public health importance of these bacterial agents, more attention from veterinary and/or human health services and, maybe, a surveillance system for control and prevention of them are recommended.

Bacterial, fungal, parasitological and pathological analyses of abortions in small ruminants from 2012-2016.

Abortion in small ruminants presents a clinical and economic problem with legal implications regarding animal health and zoonotic risk by some of the abortive pathogens. Several bacteria, fungi and parasites can cause abortion, but cost-orientated routine diagnostics only cover the most relevant epizootic agents. To cover a broad-range of common as well as underdiagnosed abortifacients, we studied 41 ovine and 36 caprine abortions by Stamp's modification of the Ziehl-Neelsen stain, culture for classical and opportunistic abortive agents, real-time PCR for C. burnetii, C. abortus, pathogenic Leptospira spp., Toxoplasma gondii and Neospora caninum. When the dam's serum was available detection of antibodies against B. melitensis, C. burnetii, C. abortus and Leptospira spp. was performed. In 37 cases sufficient placental tissue was available for pathological and histopathological examination. From the 77 cases 11 (14.3%) were positive by staining whereas real-time PCR detected C. burnetii and C. abortus in 49.3% and 32.5% of the cases. Antibodies against C. abortus and Leptospira spp. (33.3 and 26.7%) were detected. In 23.4% a bacterial culturable pathogen was isolated. Fungal abortion was confirmed in 1.3% of cases. A single abortive agent was identified in 44.2% of the cases and in 31.2% multiple possible abortifacients were present. Our study shows that the highest clarification rate can only be achieved by a combination of methods and evidences the role that multi-infections play as cause of abortion.

Investigation of infectious reproductive pathogens of large ruminants: Are neosporosis, brucellosis, leptospirosis and BVDV of relevance in Lao PDR?

N. caninum, bovine viral diarrhoea virus, Brucella abortus and Leptospira interrogans serovar Hardjo are globally significant reproductive pathogens that cause abortion and reproductive loss in large ruminants. Prevalence information is lacking in Lao People’s Democratic Republic (Laos) despite the poor reproductive performance of cattle and buffalo. Serological examination of frozen cattle (n=90) and buffalo (n=61) sera by commercially available enzyme-linked immunosorbent assays provided the first reported screening of some of these pathogens in Laos. Seroprevalence differed amongst these large ruminant species, with N. caninum, BVDV and L. interrogans serovar Hardjo antibodies found in 68.9% (95% CI±11.6), 4.9% (95% CI±5.4) and 3.3% (95% CI±4.5) of buffalo sera, respectively, and in 7.8% (95% CI±5.5), 10.0% (95% CI±6.2) and 22.2% (95% CI±8.6) of cattle sera, respectively. Buffalo sera had a significantly higher seroprevalence of N. caninum compared to cattle (p<0.001) and cattle sera had a significantly higher seroprevalence of L. interrogans serovar Hardjo compared to buffalo (p=0.003). Variability was also observed across provinces for N. caninum in buffalo (p=0.007) and for L. interrogans serovar Hardjo in cattle (p=0.071), suggesting provincial risk factors conducive to pathogen transmission. BVDV and N. caninum seropositivity were negatively associated in buffalo (p=0.018) and cattle (p=0.003). In buffalo, L. interrogans serovar Hardjo and BVDV seropositivity were associated (p=0.035, p=0.039). The identification of antibodies against three major abortifacient pathogens in Laos prompts further research to determine if infection is associated with low reproductive efficiency and the risk factors for infection. This is needed for the development of evidence based prevention strategies for improved large ruminant reproductive management among smallholders in Laos.

Risk analysis of inter-species reassortment through a Rift Valley fever phlebovirus MP-12 vaccine strain.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Arabian Peninsula. The causative agent, Rift Valley fever phlebovirus (RVFV), belongs to the genus Phlebovirus in the family Phenuiviridae and causes high rates of abortions in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral maintenance by mosquito vectors has led to sporadic RVF outbreaks in ruminants and humans in endemic countries, and effective vaccination of animals and humans may minimize the impact of this disease. A live-attenuated MP-12 vaccine strain is one of the best characterized RVFV strains, and was conditionally approved as a veterinary vaccine in the U.S. Live-attenuated RVF vaccines including MP-12 strain may form reassortant strains with other bunyavirus species. This study thus aimed to characterize the occurrence of genetic reassortment between the MP-12 strain and bunyavirus species closely related to RVFV. The Arumowot virus (AMTV) and Gouleako goukovirus (GOLV), are transmitted by mosquitoes in Africa. The results of this study showed that GOLV does not form detectable reassortant strains with the MP-12 strain in co-infected C6/36 cells. The AMTV also did not form any reassortant strains with MP-12 strain in co-infected C6/36 cells, due to the incompatibility among N, L, and Gn/Gc proteins. A lack of reassortant formation could be due to a functional incompatibility of N and L proteins derived from heterologous species, and due to a lack of packaging via heterologous Gn/Gc proteins. The MP-12 strain did, however, randomly exchange L-, M-, and S-segments with a genetic variant strain, rMP12-GM50, in culture cells. The MP-12 strain is thus unlikely to form any reassortant strains with AMTV or GOLV in nature.

Association of clinical signs after acute Schmallenberg virus infection with milk production and fertility in Swiss dairy cows

Since its first occurrence in August 2011 in Germany and the Netherlands, the Schmallenberg virus (SBV) spread rapidly across Europe, where it caused production losses and abortions in ruminants as well as congenital malformations in the offspring of affected animals. Several studies have investigated the impact of SBV on fertility and production parameters in dairy cows at herd level. However, the impact of clinical disease at the animal level remained undetermined.This study aimed at estimating the impact of clinical disease during and after an infection with SBV on production and fertility parameters in individual Swiss dairy cows. Sixty-seven case and twenty-four control herds were selected according to whether cows had been showing clinical signs indicative of SBV during the epidemic from July to December 2012 in Switzerland. Of these 91 farms, production and fertility data from 388 cows with clinical signs from case herds were collected over a time period of four years, and compared to data from 932 cows without clinical signs originating from case or control herds. Milk yield, somatic cell count, number of inseminations and non-return at day 56 were analysed by means of hierarchical multivariable regression analysis.A significant drop in milk yield was observed in all groups during the SBV epidemic compared to the time before the infection, which amounted to 1.9kg per test day for clinical animals, 1.1kg for non-clinical animals from case herds and 0.6kg for non-clinical animals from control herds. A prolonged effect on milk yield was observed in clinical cows for about one year, suggesting that animals with clinical disease might not return to their previous milk production level in the current lactation after an acute infection with SBV. Clinical animals showed a significantly higher somatic cell count during the epidemic compared to the time before the infection. The number of inseminations per cow and production cycle was higher for clinical animals during the epidemic compared to the time periods before and after, but not significantly higher than for non-clinical animals from case and control herds. No difference regarding non-return at day 56 was found. Although the overall impact of the SBV epidemic in Switzerland was limited, the consequences could be substantial in farms with a high prevalence of clinical disease.

Pathogenic outcome following experimental infection of sheep with Chlamydia abortus variant strains LLG and POS.

This study investigated the pathogenesis of two variant strains (LLG and POS) of Chlamydia abortus, in comparison to a typical wild-type strain (S26/3) which is known to be responsible for late term abortion in small ruminants. Challenge with the three strains at mid-gestation resulted in similar pregnancy outcomes, with abortion occurring in approximately 50–60% of ewes with the mean gestational lengths also being similar. However, differences were observed in the severity of placental pathology, with infection appearing milder for strain LLG, which was reflected in the lower number of organisms shed in vaginal swabs post-partum and less gross pathology and organisms present in placental smears. Results for strain POS were somewhat different than LLG with a more focal restriction of infection observed. Post-abortion antibody responses revealed prominent differences in seropositivity to the major outer membrane protein (MOMP) present in elementary body (EB) preparations under denaturing conditions, most notably with anti-LLG and anti-POS convalescent sera where there was no or reduced detection of MOMP present in EBs derived from the three strains. These results and additional analysis of whole EB and chlamydial outer membrane complex preparations suggest that there are conformational differences in MOMP for the three strains. Overall, the results suggest that gross placental pathology and clinical outcome is not indicative of bacterial colonization and the severity of infection. The results also highlight potential conformational differences in MOMP epitopes that perhaps impact on disease diagnosis and the development of new vaccines.

Reverse Transcriptase Real Time PCR Detection of Rift Valley Fever Virus RNA in Formalin‐Fixed, Paraffin‐Embedded Tissues

Rift Valley Fever Virus (RVFV) is a mosquito‐borne zoonotic pathogen endemic in parts of Africa and the Arabian Peninsula. RVFV causes acute febrile disease with mass abortion and death in ruminants and occasional encephalitis and death in humans. In the US, RVFV is a BSL‐3Ag Select Agent and a foreign animal disease, requiring special containment laboratories for research work. Research goals include the study of RVFV pathogenesis and the development of novel diagnostic tools and disease prevention methods such as vaccines. Ongoing surveillance plays a critical role in prevention and control of outbreaks and virus spread. Formalin‐fixed, paraffin‐embedded tissues (FFPET) are non‐infectious diagnostic specimens, routinely used for histopathology and immunohistochemistry (IHC), however use of this diagnostic specimen for molecular detection of pathogen RNA is underinvestigated. The objective of this work is to develop and validate a protocol for use of FFPET for the sensitive and specific detection of RVFV RNA.Study samples were fresh‐frozen tissues (FFT) and FFPET (liver, spleen, lung, kidney) collected from sheep and calves experimentally infected with two RVFV strains (Kenya 2006 and Saudi Arabia 2001). Deparaffinization of paraffin whorls (5‐5μm thick), tissue lysis with proteinase K, and reversal of nucleic acid cross linking in heated alkaline buffer was performed in a single tube using commercial buffers (Qiagen). RNA was purified using automated magnetic bead extraction on the Taco mini (GeneReach) for FFPET or the Applied Biosystems reagents on the Kingfisher (FFT). Multiplex Reverse Transcriptase Real Time PCR (RT‐qPCR) for RVFV L, M and S gene segments was used to detect RVFV RNA using Quanta qScript XLT One‐Step RT‐qPCR ToughMix (FFPET) or AgPath RT‐qPCR mix (FFT). IHC for RVFV antigen was done with a mouse monoclonal anti‐RVFV nucleoprotein antibody and avidin‐biotin complex detection.To date, 18 FFPET samples have been tested. For samples that were both IHC and FFT RT‐qPCR positive (n=13), FFPET results were 100% positive. For FFT samples (n=5) that were not tested by RT‐qPCR but were IHC positive, the FFPET RVFV RT‐qPCR results were also positive. Testing of an additional 46 FFPE samples is presently underway.Using this newly developed single tube FFPET technique for nucleic acid recovery, followed by a rapid, low cost magnetic bead nucleic acid extraction allows for highly sensitive detection of RNA specific for RVFV. This novel method could strengthen routine diagnostics, surveillance and molecular epidemiological studies that are currently limited by the strict biosecurity requirements for RVFV samples. Moreover, this methodology could also be expanded for detection of RNA targets for additional high impact pathogens or for transcriptional studies that must relie on archival FFPET.Support or Funding InformationThis work was funded by startup funds provided to Dr. Davis by the Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University and uses archival animal study materials from research funded by grants from the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD), Grant No. 2010‐ST061‐AG0001, USDA Agricultural Services project 3020‐32000‐005‐00D and the Kansas Bioscience Authority.

Molecular detection of Neospora caninum infection in ovine aborted foetuses in the Mashhad area, Iran

N. caninum could cause abortion in small ruminants. The aim of the study was to detect N. caninum infection in ovine aborted foetuses in the Mashhad area by PCR examination. During the period 2009 to 2013, 71 ovine aborted foetuses were collected and their brain samples examined by PCR. Of the 71 brains of the aborted foetuses, N. caninum DNA was detected in seven (9.8%) samples. In conclusion, it seems that N. caninum may act as a causative agent of abortion in sheep in the Mashhad area.

Enterolobium contortisiliquum is a cause of acute ruminal acidosis in sheep

The ingestion of pods of Enterolobium contortisiliquum is associated with digestive disturbances, photosensitivity and abortion in domestic ruminants. This experiment was designed to test the hypothesis that digestive disturbances in this toxicosis are really caused by acute ruminal acidosis. Three sheep fed large doses (10–15 g/kg/body weight [bw]) of E. contortisiliquum pods developed ruminal acidosis and were treated with sodium bicarbonate to try to control this metabolic disturbance, thus providing additional evidence of the involvement of ruminal acidosis in the pathogenesis of toxicosis. Two of the sheep died, and one recovered after treatment. In the two sheep that developed severe signs of ruminal acidosis, the values of blood lactate were 18 mg/dL and 196.88 mg/dL, indicating metabolic acidosis as the cause of death. Additionally, four sheep developed elevated serum levels of aspartate aminotransferase and gamma glutamyl transferase, indicating that the pods had hepatotoxic effects. Necropsy findings included the accentuation of the hepatic lobular pattern and multiple focally extensive red areas in the rumen mucosa and on the surface of the liver. Repeated ingestion of small doses induced tolerance but did not induce cumulative effects. Histopathologically, the epithelial mucosa of the rumen and reticulum exhibited swollen and vacuolated epithelia with intraepithelial pustules. Focal ulceration of the mucosa was also observed. Multifocal vacuolar degeneration of hepatocytes and scattered individual hepatocellular necrosis were evident in the liver. We concluded that the main clinical manifestation of intoxication by E. contortisiliquum pods in sheep was acute ruminal lactic acidosis and metabolic acidosis. Ingestion of repeated sublethal doses could stimulate proliferation of the ruminal fauna that degrades the sugar present in the pods, and thereby prevent the occurrence of ruminal acidosis. The plant is also hepatotoxic, and no abortions were observed.

Experimental poisoning by Enterolobium contortisiliquum in sheep

ABSTRACT: Ingestion of Enterolobium contortisiliquum pods causes digestive disturbances, secondary hepatogenous photosensitization and abortions in ruminants. Pods were administered to sheep via a ruminal cannula to characterize acute poisoning. In Experiment 1, a single dose of 12g/kg of body weight (BW) was administered to three sheep in one experiment. One sheep died, and the other two recovered after presenting clinical signs. In Experiment 2, 10g/kg BW were administered daily to 15 sheep until the onset of clinical signs or for three consecutive days. Fourteen sheep showed mild to severe signs after the ingestion of 1-3 doses. Two sheep died, and the others recovered. Clinical signs in both experiments were diarrhea, anorexia, rumen atony, apathy, dehydration and tachypnea. The main macroscopic findings were an orange, frothy ruminal content witch contained pods fragments. The intestinal content was liquid. Detachment of the mucosa from the submucosa and ballooning degeneration of mucosal cells were observed histologically in the forestomachs. Evaluation of ruminal contents revealed acute lactic ruminal acidosis (ALRA). Bromatological analysis of E. contortisiliquum pods revealed 537.8g/kg DM (dry matter) of non-fibrous carbohydrates, which is sufficient to cause ALRA. Only one sheep in Experiment 2 had liver failure, characterized by jaundice, elevated serum activity of liver enzymes and histological lesions in liver biopsies. It is concluded that the administration of E. contortisiliquum pods in forage-fed sheep at doses of 10g/kg BW or higher may cause ALRA. The induction of liver failure in one sheep suggests that liver damage may occur in those sheep that do not develop acidosis.

Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization.

ABSTRACTSchmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death.IMPORTANCE Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family.

Post-epidemic Schmallenberg virus circulation: parallel bovine serological and Culicoides virological surveillance studies in Ireland.

BackgroundSchmallenberg virus (SBV) emerged in northern-Europe in 2011 resulting in an epidemic of ruminant abortions and congenital malformations throughout the continent. In the years following the epidemic there have been reports of SBV overwintering and continued circulation in several European countries. When the population-level of immunity declines in exposed regions, re-introduction of SBV could result in further outbreaks of Schmallenberg disease. The aims of this study were to determine the SBV seroprevalence in previously exposed Irish dairy herds in 2014 and to investigate if SBV continued to circulate in these herds in the three years (2013–2015) following the Irish Schmallenberg epidemic.Whole-herd SBV serosurveillance was conducted in 26 herds before (spring) and following the 2014 vector-season (winter), and following the 2015 vector-season (winter). In spring 2014, 5,531 blood samples were collected from 4,070 cows and 1,461 heifers. In winter 2014, 2,483 blood samples were collected from 1,550 youngstock (8–10 months old) and a subsample (n = 933; 288 cows, 645 heifers) of the seronegative animals identified in the spring. Youngstock were resampled in winter 2015. Culicoides spp. were collected in 10 herds during the 2014 vector-season and analysed for SBV; a total of 138 pools (3,048 Culicoides) from 6 SBV vector species were tested for SBV RNA using real-time PCR.ResultsIn spring 2014, animal-level seroprevalence was 62.5 % (cows = 84.7 %; heifers = 0.6 %). Within-herd seroprevalence ranged widely from 8.5 %–84.1 % in the 26 herds. In winter 2014, 22 animals (0.9 %; 10 cows, 5 heifers, 7 youngstock) originating in 17 herds (range 1–4 animals/herd) tested seropositive. In winter 2015 all youngstock, including the 7 seropositive animals in winter 2014, tested seronegative suggesting their initial positive result was due to persistence of maternal antibodies. All of the Culicoides pools examined tested negative for SBV-RNA.ConclusionsSBV appears to have recirculated at a very low level in these herds during 2013 and 2014, while there was no evidence of SBV infection in naïve youngstock during 2015. A large population of naïve animals was identified and may be at risk of infection in future years should SBV re-emerge and recirculate as it has done in continental Europe.

Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4.

BackgroundBrucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons (breeding and non-breeding season) on superovulation and the imported exogenous gene on growth.ResultsIn total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep. Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05). The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group (P < 0.05). The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.ConclusionsHere we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.

Serological survey of viral diseases relating to reproductive failure among Artiodactyla in Ethiopian Camelus dromedarius.

Serological surveys were performed on Ethiopian camels with a history of abortion to investigate the presence of antibodies against viruses that infect animals classified in the order Artiodactyla. In 2013, 120 serum samples were collected from camels in various parts of Ethiopia. Several viruses related to abortion in ruminants were prevalent. In particular, antibodies against bluetongue virus, were detected at a high rate (76.7% of samples). Additionally, antibodies against Akabane virus and Japanese encephalitis virus were also detected in samples from more than 40% of the camels; however, their antibody titers were relatively low.

Short Report: Identification of a Potential Marker of Anaplasma Phagocytophilum Associated with Cattle Abortion.

Anaplasma phagocytophilum is a tick-borne pathogen that causes tick-borne fever in domestic ruminants. Tick-borne fever is characterized by diverse symptoms and occasionally causes abortions in domestic ruminants, resulting in significant economic impact. However, in France, the potential frequency of A.phagocytophilum-related abortions is unknown, and thus, it remains difficult to estimate the full extent of its economic impact. This gap in our knowledge is likely explained, at least in part, by the absence of suitable and specific tools capable of detecting A.phagocytophilum associated with abortion. Our objective was to identify a genetic marker able to differentiate A.phagocytophilum strains isolated from domestic ruminants that had aborted compared to those that had not. Thus, we typed a total of 123 A.phagocytophilum obtained from cattle, of which 25 originated from cows that had aborted, via multiple-locus variable-number tandem repeat (VNTR) analysis. These included 56 new A.phagocytophilum samples and 67 previously published A.phagocytophilum samples. A multivariate logistic model demonstrated that the triple-repeat allele of the APV-A VNTR was statistically more frequent in A.phagocytophilum from cattle that had aborted, compared to A.phagocytophilum from cattle that had not aborted (P=0.03), while controlling for any regional effects (P<0.0001). For four other VNTR, there were no statistical associations between specific alleles and abortion. The APV-A triple-repeat VNTR allele could thus act as a marker of A.phagocytophilum involved in abortions. If this hypothesis is confirmed in additional samples from other regions, this marker could then be utilized in the development of A.phagocytophilum abortive strain surveillance measures.

The role of herpesviruses (BoHV-1 and BoHV-4) and pestiviruses (BVDV and BDV) in ruminant abortion cases in western Turkey.

The economic impact of abortions in ruminant breeders is one of the biggest problems in livestock. Of the infectious agents, viruses, especially herpesviruses and pestiviruses, are the most important causative agents of abortion in ruminants. In the present study, the role of herpesviruses (bovine herpesvirus-1 (BoHV-1), bovine herpesvirus-4 (BoHV-4)) and pestiviruses (bovine viral diarrhea virus (BVDV), border disease virus (BDV)) was investigated in cases of ruminant abortion between 2007 and 2015 in western Turkey. Out of 81 aborted fetal samples (60 calves, 19 lambs, and 2 kids), 42 were positive, which included 31 calves, 9 lambs, and 2 goats; 39 aborted fetal samples were negative for the pestivirus antigen ELISA. BoHV-1 antigen ELISA was positive in 3 cases which included 2 calves and 1 lamb; the remainder 78 cases were negative. Pestivirus and BoHV-1 were positive in 51.85 and 3.70%, respectively, of the samples. According to PCR analysis, BoHV-4 was not encountered in any of the tested samples. In one of the calf fetus samples, both BVDV and BoHV-1 were positive; in one of the lamb fetus samples, BoHV-1 was positive. There was a much higher level of pestivirus antigen than the other viral agents evaluated in the study (p < 0.0001). The results of this study indicate that pestiviruses are a common viral cause of ruminant abortions in the examined area.