Bovine respiratory and enteric diseases have a profound negative impact on animal, health, welfare, and productivity. A vast number of viruses and bacteria are associated with the diseases. Pathogen ...detection using real-time PCR (rtPCR) assays performed on traditional rtPCR platforms are costly and time consuming and by that limit the use of diagnostics in bovine medicine. To diminish these limitations, we have developed a high-throughput rtPCR system (BioMark HD; Fluidigm) for simultaneous detection of the 11 most important respiratory and enteric viral and bacterial pathogens. The sensitivity and specificity of the rtPCR assays on the high-throughput platform was comparable with that of the traditional rtPCR platform. Pools consisting of positive and negative individual field samples were tested in the high-throughput rtPCR system in order to investigate the effect of an individual sample in a pool. The pool tests showed that irrespective of the size of the pool, a high-range positive individual sample had a high influence on the cycle quantification value of the pool compared with the influence of a low-range positive individual sample. To validate the test on field samples, 2,393 nasal swab and 2,379 fecal samples were tested on the high-throughput rtPCR system as pools in order to determine the occurrence of the 11 pathogens in 100 Danish herds (83 dairy and 17 veal herds). In the dairy calves,
Pasteurella multocida
(38.4%), rotavirus A (27.4%),
Mycoplasma
spp. (26.2%), and
Trueperella pyogenes
(25.5%) were the most prevalent pathogens, while
P. multocida
(71.4%),
Mycoplasma
spp. (58.9%),
Mannheimia haemolytica
(53.6%), and
Mycoplasma bovis
(42.9%) were the most often detected pathogens in the veal calves. The established high-throughput system provides new possibilities for analysis of bovine samples, since the system enables testing of multiple samples for the presence of different pathogens in the same analysis test even with reduced costs and turnover time.
Abstract
In 2021 and 2022, clade 2.3.4.4b H5Nx high pathogenicity avian influenza viruses were detected in one harbor seal and in one adult and three fox cubs in Denmark. The viruses were closely ...related to contemporary viruses found in Europe, and some had obtained amino acid substitutions related to mammalian adaptation. Notably, the virus distribution appeared to have been different in the infected fox cubs, as one exclusively tested positive for the presence of HPAIV in the brain and the other two only in the lung. Collectively, these findings stress the need for increased disease surveillance of wild and farmed mammals.
In this study, the pathogenic behavior of PRRSV 13V091 and 13V117, isolated in 2013 from two different Belgian farms with enzootic respiratory problems shortly after weaning in the nursery, were ...compared with the Belgian strain 07V063 isolated in 2007. Full-length genome sequencing was performed to identify their origin. Twelve weeks-old pigs were inoculated intranasally (IN) with 13V091, 13V117 or 07V063 (9 pigs/group). At 10 days post inoculation (dpi), 4 animals from each group were euthanized and tissues were collected for pathology, virological and serological analysis. 13V091 infection resulted in the highest respiratory disease scores and longest period of fever. Gross lung lesions were more pronounced for 13V091 (13%), than for 13V117 (7%) and 07V063 (11%). The nasal shedding and viremia was also most extensive with 13V091. The 13V091 group showed the highest virus replication in conchae, tonsils and retropharyngeal lymph nodes. 13V117 infection resulted in the lowest virus replication in lymphoid tissues. 13V091 showed higher numbers of sialoadhesin⁻infected cells/mm²in conchae, tonsils and spleen than 13V117 and 07V063. Neutralizing antibody response with 07V063 was stronger than with 13V091 and 13V117. It can be concluded that (i) 13V091 is a highly pathogenic type 1 subtype 1 PRRSV strain that replicates better than 07V063 and 13V117 and has a strong tropism for sialoadhesin⁻cells and (ii) despite the close genetic relationship between 13V117 and 07V063, 13V117 has an increased nasal replication and shedding, but a decreased replication in lymphoid tissues compared to 07V063.
Influenza A viruses are major pathogens for humans, domestic animals, and wildlife, and these viruses occasionally cross the species barrier. In spring 2014, increased mortality of harbor seals ...(Phoca vitulina), associated with infection with an influenza A(H10N7) virus, was reported in Sweden and Denmark. Within a few months, this virus spread to seals of the coastal waters of Germany and the Netherlands, causing the death of thousands of animals. Genetic analysis of the hemagglutinin (HA) and neuraminidase (NA) genes of this seal influenza A(H10N7) virus revealed that it was most closely related to various avian influenza A(H10N7) viruses. The collection of samples from infected seals during the course of the outbreak provided a unique opportunity to follow the adaptation of the avian virus to its new seal host. Sequence data for samples collected from 41 different seals from four different countries between April 2014 and January 2015 were obtained by Sanger sequencing and next-generation sequencing to describe the molecular epidemiology of the seal influenza A(H10N7) virus. The majority of sequence variation occurred in the HA gene, and some mutations corresponded to amino acid changes not found in H10 viruses isolated from Eurasian birds. Also, sequence variation in the HA gene was greater at the beginning than at the end of the epidemic, when a number of the mutations observed earlier had been fixed. These results imply that when an avian influenza virus jumps the species barrier from birds to seals, amino acid changes in HA may occur rapidly and are important for virus adaptation to its new mammalian host.
Influenza A viruses are major pathogens for humans, domestic animals, and wildlife. In addition to the continuous circulation of influenza A viruses among various host species, cross-species transmission of influenza A viruses occurs occasionally. Wild waterfowl and shorebirds are the main reservoir for most influenza A virus subtypes, and spillover of influenza A viruses from birds to humans or other mammalian species may result in major outbreaks. In the present study, various sequencing methods were used to elucidate the genetic changes that occurred after the introduction and subsequent spread of an avian influenza A(H10N7) virus among harbor seals of northwestern Europe by use of various samples collected during the outbreak. Such detailed knowledge of genetic changes necessary for introduction and adaptation of avian influenza A viruses to mammalian hosts is important for a rapid risk assessment of such viruses soon after they cross the species barrier.
Pigs are considered the main reservoir of genotypes 3 and 4 of the human pathogen hepatitis E virus (HEV). These viruses are prevalent at a high level in swine herds globally, meaning that consumers ...may be exposed to HEV from the food chain if the virus is present in pigs at slaughter. The aim of this study was to determine the HEV infection dynamics from birth to slaughter using 104 pigs from 11 sows in a single production system. Serum was collected from sows at 2 weeks prior to farrowing, in addition feces and serum samples were collected from the pigs every second week, from week 1 to week 17. Feces and selected organs were also sampled from 10 pigs following slaughter at week 20. All the samples were tested for HEV RNA by real-time RT-PCR and the serum samples were tested for HEV-specific antibodies using a commercial ELISA. Maternal antibodies (MAbs) were only present in pigs from sows with high levels of antibodies and all pigs, except one, seroconverted to HEV during weeks 13-17. In total, 65.5% of the pigs tested positive for HEV RNA at least once during the study (during weeks 13, 15, and/or 17) and significantly fewer pigs with a high level of MAbs became shedders. In contrast, the level of MAbs had no impact on the time of onset and duration of virus shedding. HEV was detected in feces and organs, but not in muscle, in 3 out of 10 pigs at slaughter, indicating that detection of HEV in feces is indicative of an HEV positivity in organs. In conclusion, a high proportion of pigs in a HEV positive herd were infected and shed virus during the finisher stage and some of the pigs also contained HEV RNA in feces and organs at slaughter. The presence of MAbs reduced the prevalence of HEV shedding animals, therefore, sow vaccination may be an option to decrease the prevalence of HEV positive animals at slaughter.
Nasal mucosal explant (NEs) cultured at an air–liquid interface mimics in vivo conditions more accurately than monolayer cultures of respiratory cell linesor primary cells cultured in flat-bottom ...microtiter wells. NEs might be relevant for studies of host-pathogen interactions and antiviral immune responses after infection with respiratory viruses, including influenza and corona viruses.
Pigs are natural hosts for swine influenza A virus (IAV) but are also susceptible to IAV from humans, emphasizing the relevance of porcine NEs in the study of IAV infection. Therefore, we performed fundamental characterization and study of innate antiviral responses in porcine NEs using microfluidic high-throughput quantitative real-time PCR (qPCR) to generate expression profiles of host genes involved in inflammation, apoptosis, and antiviral immune responses in mock inoculated and IAV infected porcine NEs.
Handling and culturing of the explants ex vivo had a significant impact on gene expression compared to freshly harvested tissue. Upregulation (2–43 fold) of genes involved in inflammation, including IL1A and IL6, and apoptosis, including FAS and CASP3, and downregulation of genes involved in viral recognition (MDA5 (IFIH1)), interferon response (IFNA), and response to virus (OAS1, IFIT1, MX1) was observed. However, by comparing time-matched mock and virus infected NEs, transcription of viral pattern recognition receptors (RIG-I (DDX58), MDA5 (IFIH1), TLR3) and type I and III interferons (IFNB1, IL28B (IFNL3)) were upregulated 2–16 fold in IAV-infected NEs. Furthermore, several interferon-stimulated genes including MX1, MX2, OAS, OASL, CXCL10, and ISG15 was observed to increase 2–26 fold in response to IAV inoculation. NE expression levels of key genes involved in antiviral responses including IL28B (IFNL3), CXCL10, and OASL was highly comparable to expression levels found in respiratory tissues including nasal mucosa and lung after infection of pigs with the same influenza virus isolate.
The importance of protein glycosylation in regulating lipid metabolism is becoming increasingly apparent. We set out to further investigate this by studying patients with type I congenital disorders ...of glycosylation (CDGs) with defective N-glycosylation.
We studied 29 patients with the 2 most prevalent types of type I CDG, ALG6 (asparagine-linked glycosylation protein 6)-deficiency CDG and PMM2 (phosphomannomutase 2)-deficiency CDG, and 23 first- and second-degree relatives with a heterozygous mutation and measured plasma cholesterol levels. Low-density lipoprotein (LDL) metabolism was studied in 3 cell models-gene silencing in HepG2 cells, patient fibroblasts, and patient hepatocyte-like cells derived from induced pluripotent stem cells-by measuring apolipoprotein B production and secretion, LDL receptor expression and membrane abundance, and LDL particle uptake. Furthermore, SREBP2 (sterol regulatory element-binding protein 2) protein expression and activation and endoplasmic reticulum stress markers were studied.
We report hypobetalipoproteinemia (LDL cholesterol LDL-C and apolipoprotein B below the fifth percentile) in a large cohort of patients with type I CDG (mean age, 9 years), together with reduced LDL-C and apolipoprotein B in clinically unaffected heterozygous relatives (mean age, 46 years), compared with 2 separate sets of age- and sex-matched control subjects. ALG6 and PMM2 deficiency led to markedly increased LDL uptake as a result of increased cell surface LDL receptor abundance. Mechanistically, this outcome was driven by increased SREBP2 protein expression accompanied by amplified target gene expression, resulting in higher LDL receptor protein levels. Endoplasmic reticulum stress was not found to be a major mediator.
Our study establishes N-glycosylation as an important regulator of LDL metabolism. Given that LDL-C was also reduced in a group of clinically unaffected heterozygotes, we propose that increasing LDL receptor-mediated cholesterol clearance by targeting N-glycosylation in the LDL pathway may represent a novel therapeutic strategy to reduce LDL-C and cardiovascular disease.
The Meat Animal Research Center-145 (MARC-145) cell line has been proven to be valuable for viral attenuation regarding vaccine development and production. Cell-adaptation is necessary for the ...efficient replication of porcine reproductive and respiratory syndrome virus (PRRSV) in these cells. Multiple sequence analysis revealed consistent amino acid substitutions in GP2a (V88F, M94I, F95L) of MARC-145 cell-adapted strains. To investigate the putative effect of these substitutions, mutations at either position 88, 94, 95, and their combinations were introduced into two PRRSV1 (13V091 and IVI-1173) infectious clones followed by the recovery of viable recombinants. When comparing the replication kinetics in MARC-145 cells, a strongly positive effect on the growth characteristics of the 13V091 strain (+2.1 log10) and the IVI-1173 strain (+1.7 log10) compared to wild-type (WT) virus was only observed upon triple amino acid substitution at positions 88 (V88F), 94 (M94I), and 95 (F95L) of GP2a, suggesting that the triple mutation is a determining factor in PRRSV1 adaptation to MARC-145 cells.
•First publication of complete genomes of Type 1 PRRSV isolated in Denmark.•Genetic and antigenetic analysis of complete genomes of PRRSV isolated over a period of 10 years.•Two genetic different ...viruses are circulating in Danish pig herds.•A thorough examination of ORF5 diversity.
Porcine Reproductive and Respiratory Syndrome (PRRS) caused by the PRRS virus (PRRSV) is considered one of the most devastating swine diseases worldwide. PRRS viruses are divided into two major genotypes, Type 1 and Type 2, with pronounced diversity between and within the genotypes. In Denmark more than 50% of the herds are infected with Type 1 and/or Type 2 PRRSV. The main objective of this study was to examine the genetic diversity and drift of Type 1 viruses in a population with limited introduction of new animals and semen. A total of 43 ORF5 and 42 ORF7 nucleotide sequences were obtained from viruses collected from 2003 to February 2013. Phylogenetic analysis of ORF5 nucleotide sequences showed that the Danish isolates formed two major clusters within the subtype 1. The nucleotide identity to the subtype 1 protogenotype Lelystad virus (LV) spanned 84.9–98.8% for ORF5 and 90.7–100% for ORF7. Among the Danish viruses the pairwise nucleotide identities in ORF5 and ORF7 were 81.2–100% and 88.9–100%, respectively. Sequencing of the complete genomes, including the 5′- and 3′-end nucleotides, of 8 Danish PRRSV Type 1 showed that the genome lengths differed from 14,876 to 15,098 nucleotides and the pairwise nucleotide identity among the Danish viruses was 86.5–97.3% and the identity to LV was 88.7–97.9%. The study strongly indicated that there have been at least two independent introductions of Type 1 PRRSV in Denmark and analysis of the full genomes revealed a significant drift in several regions of the virus.
The amount of animal manure used in modern agriculture is increasing due to the increase in global animal production. Pig slurry is known to contain zoonotic bacteria such as
E. coli
,
Salmonella
...spp. and
Campylobacter
spp., and viruses such as hepatitis E virus and group A rotavirus. Coliform bacteria, present in manure, have previously been shown to leach into tile drains. This poses a potential threat to aquatic environments and may also influence the quality of drinking water. As knowledge is especially scarce about the fate of viruses when applied to fields in natural settings, this project sets out to investigate the leaching potential of six different microorganisms:
E. coli
and
Enterococcus
spp. (detected by colony assay), somatic coliphages (using plaque assays), and hepatitis E virus, porcine circovirus type 2, and group A rotavirus (by real-time polymerase chain reaction). All six microorganisms leached through the soil entering the tile drains situated at 1-m depth the first day following pig slurry application. The leaching pattern of group A rotavirus differed substantially from the pattern for somatic coliphages, which are otherwise used as indicators for virus contamination. Furthermore, group A rotavirus was detected in monitoring wells at 3.5-m depth up to 2 months after pig slurry application. The detection of viral genomic material in drainage water and shallow groundwater signifies a potential hazard to human health that needs to be investigated further, as water reservoirs used for recreational use and drinking water are potentially contaminated with zoonotic pathogens.
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