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1.  The differentiated airway epithelium infected by influenza viruses maintains the barrier function despite a dramatic loss of ciliated cells 
Scientific Reports  2016;6:39668.
Virus-host interactions in the respiratory epithelium during long term influenza virus infection are not well characterized. Therefore, we developed an air-liquid interface culture system for differentiated porcine respiratory epithelial cells to study the effect of virus-induced cellular damage. In our well-differentiated cells, α2,6-linked sialic acid is predominantly expressed on the apical surface and the basal cells mainly express α2,3-linked sialic acid. During the whole infection period, release of infectious virus was maintained at a high titre for more than seven days. The infected epithelial cells were subject to apoptosis resulting in the loss of ciliated cells together with a thinner thickness. Nevertheless, the airway epithelium maintained trans-epithelial electrical resistance and retained its barrier function. The loss of ciliated cells was compensated by the cells which contained the KRT5 basal cell marker but were not yet differentiated into ciliated cells. These specialized cells showed an increase of α2,3-linked sialic acid on the apical surface. In sum, our results help to explain the localized infection of the airway epithelium by influenza viruses. The impairment of mucociliary clearance in the epithelial cells provides an explanation why prior viral infection renders the host more susceptible to secondary co-infection by another pathogen.
PMCID: PMC5177954  PMID: 28004801
2.  Viral Infection of the Central Nervous System Exacerbates Interleukin-10 Receptor Deficiency-Mediated Colitis in SJL Mice 
PLoS ONE  2016;11(9):e0161883.
Theiler´s murine encephalomyelitis virus (TMEV)-infection is a widely used animal model for studying demyelinating disorders, including multiple sclerosis (MS). The immunosuppressive cytokine Interleukin (IL)-10 counteracts hyperactive immune responses and critically controls immune homeostasis in infectious and autoimmune disorders. In order to investigate the effect of signaling via Interleukin-10 receptor (IL-10R) in infectious neurological diseases, TMEV-infected SJL mice were treated with IL-10R blocking antibody (Ab) in the acute and chronic phase of the disease. The findings demonstrate that (i) Ab-mediated IL-10 neutralization leads to progressive colitis with a reduction in Foxp3+ regulatory T cells and increased numbers of CD8+CD44+ memory T cells as well as activated CD4+CD69+ and CD8+CD69+ T cells in uninfected mice. (ii) Concurrent acute TMEV-infection worsened enteric disease-mediated by IL-10R neutralization. Virus-triggered effects were associated with an enhanced activation of CD4+ T helper cells and CD8+ cytotoxic T lymphocytes and augmented cytokine expression. By contrast, (iii) IL-10R neutralization during chronic TMEV-infection was not associated with enhanced peripheral immunopathology but an increased CD3+ T cell influx in the spinal cord. IL-10R neutralization causes a breakdown in peripheral immune tolerance in genetically predisposed mice, which leads to immune-mediated colitis, resembling inflammatory bowel disease. Hyperactive immune state following IL-10R blockade is enhanced by central nervous system-restricted viral infection in a disease phase-dependent manner.
PMCID: PMC5017624  PMID: 27611574
3.  FlpS, the FNR-Like Protein of Streptococcus suis Is an Essential, Oxygen-Sensing Activator of the Arginine Deiminase System 
Pathogens  2016;5(3):51.
Streptococcus (S.) suis is a zoonotic pathogen causing septicemia and meningitis in pigs and humans. During infection S. suis must metabolically adapt to extremely diverse environments of the host. CcpA and the FNR family of bacterial transcriptional regulators are important for metabolic gene regulation in various bacteria. The role of CcpA in S. suis is well defined, but the function of the FNR-like protein of S. suis, FlpS, is yet unknown. Transcriptome analyses of wild-type S. suis and a flpS mutant strain suggested that FlpS is involved in the regulation of the central carbon, arginine degradation and nucleotide metabolism. However, isotopologue profiling revealed no substantial changes in the core carbon and amino acid de novo biosynthesis. FlpS was essential for the induction of the arcABC operon of the arginine degrading pathway under aerobic and anaerobic conditions. The arcABC-inducing activity of FlpS could be associated with the level of free oxygen in the culture medium. FlpS was necessary for arcABC-dependent intracellular bacterial survival but redundant in a mice infection model. Based on these results, we propose that the core function of S. suis FlpS is the oxygen-dependent activation of the arginine deiminase system.
PMCID: PMC5039431  PMID: 27455333
Streptococcus suis; FNR-like protein; arginine deiminase system
4.  Clearance of Streptococcus suis in Stomach Contents of Differently Fed Growing Pigs 
Pathogens  2016;5(3):56.
Streptococcus (S.) suis translocates across the intestinal barrier of piglets after intraintestinal application. Based on these findings, an oro-gastrointestinal infection route has been proposed. Thus, the objective of this study was to investigate the survival of S. suis in the porcine stomach. Whereas surviving bacteria of S. suis serotypes 2 and 9 were not detectable after 60 min of incubation in stomach contents with a comparatively high gastric pH of 5 due to feeding of fine pellets, the number of Salmonella Derby bacteria increased under these conditions. Further experiments confirmed the clearance of S. suis serotypes 2 and 9 within 30 min in stomach contents with a pH of 4.7 independently of the bacterial growth phase. Finally, an oral infection experiment was conducted, feeding each of 18 piglets a diet mixed with 1010 CFU of S. suis serotype 2 or 9. Thorough bacteriological screenings of various mesenteric-intestinal lymph nodes and internal organs after different times of exposure did not lead to any detection of the orally applied challenge strains. In conclusion, the porcine stomach constitutes a very efficient barrier against oro-gastrointenstinal S. suis infections. Conditions leading to the passage of S. suis through the stomach remain to be identified.
PMCID: PMC5039436  PMID: 27509526
Streptococcus suis; stomach content; pig; oro-gastrointestinal infection
5.  Porcine Bocavirus Infection Associated with Encephalomyelitis in a Pig, Germany1 
Emerging Infectious Diseases  2016;22(7):1310-1312.
PMCID: PMC4918158  PMID: 27315461
central nervous system; encephalomyelitis; next generation sequencing; in situ-hybridization; parvovirus; porcine bocavirus; viruses; pigs; Germany
6.  Identification of a Novel Hepacivirus in Domestic Cattle from Germany 
Journal of Virology  2015;89(14):7007-7015.
Hepatitis C virus (HCV) continues to represent one of the most significant threats to human health. In recent years, HCV-related sequences have been found in bats, rodents, horses, and dogs, indicating a widespread distribution of hepaciviruses among animals. By applying unbiased high-throughput sequencing, a novel virus of the genus Hepacivirus was discovered in a bovine serum sample. De novo assembly yielded a nearly full-length genome coding for a polyprotein of 2,779 amino acids. Phylogenetic analysis confirmed that the virus represents a novel species within the genus Hepacivirus. Viral RNA screening determined that 1.6% (n = 5) of 320 individual animals and 3.2% (n = 5) of 158 investigated cattle herds in Germany were positive for bovine hepacivirus. Repeated reverse transcription-PCR (RT-PCR) analyses of animals from one dairy herd proved that a substantial percentage of cows were infected, with some of them being viremic for over 6 months. Clinical and postmortem examination revealed no signs of disease, including liver damage. Interestingly, quantitative RT-PCR from different organs and tissues, together with the presence of an miR-122 binding site in the viral genome, strongly suggests a liver tropism for bovine hepacivirus, making this novel virus a promising animal model for HCV infections in humans.
IMPORTANCE Livestock animals act as important sources for emerging pathogens. In particular, their large herd size and the existence of multiple ways of direct and food-borne infection routes emphasize their role as virus reservoirs. Apart from the search for novel viruses, detailed characterization of these pathogens is indispensable in the context of risk analysis. Here, we describe the identification of a novel HCV-like virus in cattle. In addition, determination of the prevalence and of the course of infection in cattle herds provides valuable insights into the biology of this novel virus. The results presented here form a basis for future studies targeting viral pathogenesis of bovine hepaciviruses and their potential to establish zoonotic infections.
PMCID: PMC4473572  PMID: 25926652
7.  Differential Contributions of the Complement Anaphylotoxin Receptors C5aR1 and C5aR2 to the Early Innate Immune Response against Staphylococcus aureus Infection 
Pathogens  2015;4(4):722-738.
The complement anaphylatoxin C5a contributes to host defense against Staphylococcus aureus. In this study, we investigated the functional role of the two known C5a receptors, C5aR1 and C5aR2, in the host response to S. aureus. We found that C5aR1−/− mice exhibited greater susceptibility to S. aureus bloodstream infection than wild type and C5aR2−/− mice, as demonstrated by the significantly higher bacterial loads in the kidneys and heart at 24 h of infection, and by the higher levels of inflammatory IL-6 in serum. Histological and immunohistochemistry investigation of infected kidneys at 24 h after bacterial inoculation revealed a discrete infiltration of neutrophils in wild type mice but already well-developed abscesses consisting of bacterial clusters surrounded by a large number of neutrophils in both C5aR1−/− and C5aR2−/− mice. Furthermore, blood neutrophils from C5aR1−/− mice were less efficient than those from wild type or C5aR2−/− mice at killing S. aureus. The requirement of C5aR1 for efficient killing of S. aureus was also demonstrated in human blood after disrupting C5a-C5aR1 signaling using specific inhibitors. These results demonstrated a role for C5aR1 in S. aureus clearance as well as a role for both C5aR1 and C5aR2 in the orchestration of the inflammatory response during infection.
PMCID: PMC4693161  PMID: 26512700
Staphylococcus aureus; C5a; C5aR1; C5aR2; complement system; neutrophils
8.  Effect of a single injection of autologous conditioned serum (ACS) on tendon healing in equine naturally occurring tendinopathies 
Autologous blood-derived biologicals, including autologous conditioned serum (ACS), are frequently used to treat tendinopathies in horses despite limited evidence for their efficacy. The purpose of this study was to describe the effect of a single intralesional injection of ACS in naturally occurring tendinopathies of the equine superficial digital flexor tendon (SDFT) on clinical, ultrasonographic, and histological parameters.
Fifteen horses with 17 naturally occurring tendinopathies of forelimb SDFTs were examined clinically and ultrasonographically (day 0). Injured tendons were randomly assigned to the ACS-treated group (n = 10) receiving a single intralesional ACS injection or included as controls (n = 7) which were either untreated or injected with saline on day 1. All horses participated in a gradually increasing exercise programme and were re-examined nine times at regular intervals until day 190. Needle biopsies were taken from the SDFTs on days 0, 36 and 190 and examined histologically and for the expression of collagen types I and III by immunohistochemistry.
In ACS-treated limbs lameness decreased significantly until day 10 after treatment. Swelling (scores) of the SDFT region decreased within the ACS group between 50 and 78 days after treatment. Ultrasonographically, the percentage of the lesion in the tendon was significantly lower and the echogenicity of the lesion (total echo score) was significantly higher 78 and 106 days after intralesional ACS injection compared to controls. Histology revealed that, compared to controls, tenocyte nuclei were more spindle-shaped 36 days after ACS injection. Immunohistochemistry showed that collagen type I expression significantly increased between days 36 and 190 after ACS injection.
Single intralesional ACS injection of equine SDFTs with clinical signs of acute tendinopathy contributes to an early significant reduction of lameness and leads to temporary improvement of ultrasonographic parameters of repair tissue. Intralesional ACS treatment might decrease proliferation of tenocytes 5 weeks after treatment and increase their differentiation as demonstrated by elevated collagen type I expression in the remodelling phase. Potential enhancement of these effects by repeated injections should be tested in future controlled clinical investigations.
PMCID: PMC4513386  PMID: 26113022
Horse; Tendon; Ultrasonography; Biopsy; Histology; Collagen; Autologous conditioned serum; ACS; Irap
9.  The immunoglobulin M-degrading enzyme of Streptococcus suis, IdeSsuis, is involved in complement evasion 
Veterinary Research  2015;46(1):45.
Streptococcus (S.) suis is one of the most important pathogens in pigs causing meningitis, arthritis, endocarditis and serositis. Furthermore, it is also an emerging zoonotic agent. In our previous work we identified a highly specific IgM protease in S. suis, designated IdeSsuis. The objective of this study was to characterize the function of IdeSsuis in the host-pathogen interaction. Edman-sequencing revealed that IdeSsuis cleaves the heavy chain of the IgM molecule between constant domain 2 and 3. As the C1q binding motif is located in the C3 domain, we hypothesized that IdeSsuis is involved in complement evasion. Complement-mediated hemolysis induced by porcine hyperimmune sera containing erythrocyte-specific IgM was abrogated by treatment of these sera with recombinant IdeSsuis. Furthermore, expression of IdeSsuis reduced IgM-triggered complement deposition on the bacterial surface. An infection experiment of prime-vaccinated growing piglets suggested attenuation in the virulence of the mutant 10ΔideSsuis. Bactericidal assays confirmed a positive effect of IdeSsuis expression on bacterial survival in porcine blood in the presence of high titers of specific IgM. In conclusion, this study demonstrates that IdeSsuis is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.
Electronic supplementary material
The online version of this article (doi:10.1186/s13567-015-0171-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4404118  PMID: 25928761
10.  Role of Capsule and Suilysin in Mucosal Infection of Complement-Deficient Mice with Streptococcus suis 
Infection and Immunity  2014;82(6):2460-2471.
Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis. Experimental infection of wild-type (WT) and C3−/− mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3−/− mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3−/− mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant. Ex vivo multiplication in murine WT and C3−/− blood depended on capsule but not suilysin expression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR−/− mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity.
PMCID: PMC4019146  PMID: 24686060
11.  Limited role of regulatory T cells during acute Theiler virus-induced encephalitis in resistant C57BL/6 mice 
Theiler’s murine encephalomyelitis virus (TMEV) infection represents a commonly used infectious animal model to study various aspects of the pathogenesis of multiple sclerosis (MS). In susceptible SJL mice, dominant activity of Foxp3+ CD4+ regulatory T cells (Tregs) in the CNS partly contributes to viral persistence and progressive demyelination. On the other hand, resistant C57BL/6 mice rapidly clear the virus by mounting a strong antiviral immune response. However, very little is known about the role of Tregs in regulating antiviral responses during acute encephalitis in resistant mouse strains.
In this study, we used DEREG mice that express the diphtheria toxin (DT) receptor under control of the foxp3 locus to selectively deplete Foxp3+ Tregs by injection of DT prior to infection and studied the effect of Treg depletion on the course of acute Theiler’s murine encephalomyelitis (TME).
As expected, DEREG mice that are on a C57BL/6 background were resistant to TMEV infection and cleared the virus within days of infection, regardless of the presence or absence of Tregs. Nevertheless, in the absence of Tregs we observed priming of stronger effector T cell responses in the periphery, which subsequently resulted in a transient increase in the frequency of IFNγ-producing T cells in the brain at an early stage of infection. Histological and flow cytometric analysis revealed that this transiently increased frequency of brain-infiltrating IFNγ-producing T cells in Treg-depleted mice neither led to an augmented antiviral response nor enhanced inflammation-mediated tissue damage. Intriguingly, Treg depletion did not change the expression of IL-10 in the infected brain, which might play a role for dampening the inflammatory damage caused by the increased number of effector T cells.
We therefore propose that unlike susceptible mice strains, interfering with the Treg compartment of resistant mice only has negligible effects on virus-induced pathologies in the CNS. Furthermore, in the absence of Tregs, local anti-inflammatory mechanisms might limit the extent of damage caused by strong anti-viral response in the CNS.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-014-0180-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4236492  PMID: 25391297
Regulatory T cells; Theiler’s virus; Interleukin-10
12.  Canine Distemper Virus Infection Leads to an Inhibitory Phenotype of Monocyte-Derived Dendritic Cells In Vitro with Reduced Expression of Co-Stimulatory Molecules and Increased Interleukin-10 Transcription 
PLoS ONE  2014;9(4):e96121.
Canine distemper virus (CDV) exhibits a profound lymphotropism that causes immunosuppression and increased susceptibility of affected dogs to opportunistic infections. Similar to human measles virus, CDV is supposed to inhibit terminal differentiation of dendritic cells (DCs), responsible for disturbed repopulation of lymphoid tissues and diminished antigen presenting function in dogs. In order to testify the hypothesis that CDV-infection leads to an impairment of professional antigen presenting cells, canine DCs have been generated from peripheral blood monocytes in vitro and infected with CDV. Virus infection was confirmed and quantified by transmission electron microscopy, CDV-specific immunofluorescence, and virus titration. Flow cytometric analyses revealed a significant down-regulation of the major histocompatibility complex class II and co-stimulatory molecules CD80 and CD86 in CDV-infected DCs, indicative of disturbed antigen presenting capacity. Molecular analyses revealed an increased expression of the immune inhibitory cytokine interleukin-10 in DCs following infection. Results of the present study demonstrate that CDV causes phenotypical changes and altered cytokine expression of DCs, which represent potential mechanisms to evade host immune responses and might contribute to immune dysfunction and virus persistence in canine distemper.
PMCID: PMC4000198  PMID: 24769532
13.  Neurotoxocarosis: marked preference of Toxocara canis for the cerebrum and T. cati for the cerebellum in the paratenic model host mouse 
Parasites & Vectors  2014;7:194.
Infective larvae of the worldwide occurring zoonotic roundworm T. canis exhibit a marked affinity to the nervous tissues of paratenic hosts. In humans, most cases of neurotoxocarosis are considered to be caused by larvae of T. canis as T. cati larvae have rarely been found in the CNS in previous studies. However, direct comparison of studies is difficult as larval migration depends on a variety of factors including mouse strains and inoculation doses. Therefore, the present study aims to provide a direct comparison of both roundworm species in mice as a model for paratenic hosts with specific focus on the CNS during the acute and chronic phase of disease to provide a basis for further studies dealing with neurotoxocarosis.
C57Bl/6J mice were infected with 2000 embryonated T. canis and T. cati eggs, respectively as well as Balb/c mice infected with T. cati eggs only. On 8 time points post infection, organs were removed and microscopically examined for respective larvae. Special focus was put on the CNS, including analysis of larval distribution in the cerebrum and cerebellum, right and left hemisphere as well as eyes and spinal cord. Additionally, brains of all infection groups as well as uninfected controls were examined histopathologically to characterize neurostructural damage.
Significant differences in larval distribution were observed between and within the infection groups during the course of infection. As expected, significantly higher recovery rates of T. canis than T. cati larvae were determined in the brain. Surprisingly, significantly more T. canis larvae could be found in cerebra of infected mice whereas T. cati larvae were mainly located in the cerebellum. Structural damage in brain tissue could be observed in all infection groups, being more severe in brains of T. canis infected mice.
The data obtained provides an extensive characterization of migrational routes of T. canis and T. cati in the paratenic host mouse in direct comparison. Even though to a lesser extent, structural damage in the brain was also caused by T. cati larvae and therefore, the potential as pathogenic agents should not be underestimated.
PMCID: PMC4017833  PMID: 24754900
Toxocara canis; Toxocara cati; Roundworms; Larval migration; Neurotoxocarosis; Zoonotic helminths
14.  Morphometric magnetic resonance imaging and genetic testing in cerebellar abiotrophy in Arabian horses 
Cerebellar abiotrophy (CA) is a rare but significant disease in Arabian horses caused by progressive death of the Purkinje cells resulting in cerebellar ataxia characterized by a typical head tremor, jerky head movements and lack of menace response. The specific role of magnetic resonance imaging (MRI) to support clinical diagnosis has been discussed. However, as yet MR imaging has only been described in one equine CA case. The role of MR morphometry in this regard is currently unknown. Due to the hereditary nature of the disease, genetic testing can support the diagnosis of CA.
Therefore, the objective of this study was to perform MR morphometric analysis and genetic testing in four CA-affected Arabian horses and one German Riding Pony with purebred Arabian bloodlines in the third generation.
CA was diagnosed pathohistologically in the five affected horses (2 months - 3 years) supported by clinical signs, necropsy, and genetic testing which confirmed the TOE1:g.2171G>A SNP genotype A/A in all CA-affected horses.
On MR images morphometric analysis of the relative cerebellar size and relative cerebellar cerebrospinal fluid (CSF) space were compared to control images of 15 unaffected horses. It was demonstrated that in MR morphometric analyses, CA affected horses displayed a relatively smaller cerebellum compared to the entire brain mass than control animals (P = 0.0088). The relative cerebellar CSF space was larger in affected horses (P = 0.0017). Using a cut off value of 11.0% for relative cerebellar CSF space, the parameter differentiated between CA-affected horses and controls with a sensitivity of 100% and a specificity of 93.3%.
In conclusion, morphometric MRI and genetic analysis could be helpful to support the diagnosis of CA in vivo.
PMCID: PMC3671216  PMID: 23702154
Ataxia; Heredity; Horse; Purkinje cells; TOE1
15.  Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation 
Unlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation.
Thirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6) and four days later (group 2, n = 7). The oviduct and uterine horn of one side were flushed separately and the flushing’s were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution.
The total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P<0.05) and a marked reduction of the mean sperm number in uterine horn glands were observed. A concomitant diminution of spermatozoa was indicated in the utero-tubal junction accompanied by a slight increase in sperm numbers in the mid oviduct.
Oocyte maturation to metaphase-II stage is accompanied by a continuous sperm detachment and elimination in the uterine horns. Entrance of spermatozoa into the caudal oviduct seems to be steadily controlled by the utero-tubal junction thus providing a selected sperm population to be shifted towards the site of fertilization when oocyte maturation is completed.
PMCID: PMC3526419  PMID: 22932162
Dog; Sperm storage; Oviduct; Oocyte maturation; Fertilization
16.  A One Base Pair Deletion in the Canine ATP13A2 Gene Causes Exon Skipping and Late-Onset Neuronal Ceroid Lipofuscinosis in the Tibetan Terrier 
PLoS Genetics  2011;7(10):e1002304.
Neuronal ceroid lipofuscinosis (NCL) is a progressive neurodegenerative disease characterized by brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin in neurons and other cells. Tibetan terriers show a late-onset lethal form of NCL manifesting first visible signs at 5–7 years of age. Genome-wide association analyses for 12 Tibetan-terrier-NCL-cases and 7 Tibetan-terrier controls using the 127K canine Affymetrix SNP chip and mixed model analysis mapped NCL to dog chromosome (CFA) 2 at 83.71–84.72 Mb. Multipoint linkage and association analyses in 376 Tibetan terriers confirmed this genomic region on CFA2. A mutation analysis for 14 positional candidate genes in two NCL-cases and one control revealed a strongly associated single nucleotide polymorphism (SNP) in the MAPK PM20/PM21 gene and a perfectly with NCL associated single base pair deletion (c.1620delG) within exon 16 of the ATP13A2 gene. The c.1620delG mutation in ATP13A2 causes skipping of exon 16 presumably due to a broken exonic splicing enhancer motif. As a result of this mutation, ATP13A2 lacks 69 amino acids. All known 24 NCL cases were homozygous for this deletion and all obligate 35 NCL-carriers were heterozygous. In a sample of 144 dogs from eleven other breeds, the c.1620delG mutation could not be found. Knowledge of the causative mutation for late-onset NCL in Tibetan terrier allows genetic testing of these dogs to avoid matings of carrier animals. ATP13A2 mutations have been described in familial Parkinson syndrome (PARK9). Tibetan terriers with these mutations provide a valuable model for a PARK9-linked disease and possibly for manganese toxicity in synucleinopathies.
Author Summary
The neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative storage diseases characterized by psychomotor retardation, blindness, and premature death. NCL has been reported in several dog breeds. NCL is characterized by progressive brain and retinal atrophy and the intracellular accumulation of autofluorescent lysosomal storage bodies resembling lipofuscin. Tibetan terriers show a late-onset and lethal NCL (age of onset 5–7 years) with an autosomal recessive inheritance. The most frequently described first symptom is blindness in twilight. In the disease progress the affected dogs often appear nervous or anxious and the lack of motor coordination becomes more severe. In the final stages of this disease, mild but also severe seizures have been observed by the owner. There are no treatment options for affected dogs. Through a genome-wide association analysis using the 127K canine Affymetrix SNP chip, we found a 1 Mb candidate genomic region and identified ATP13A2 as the most likely candidate for NCL. A 1-base pair deletion mutation within exon 16 of the ATP13A2 gene caused the loss of an exonic splicing enhancer and, consequently, the alternative splicing lead to skipping of exon 16. This study provides a suitable animal model for PARK9 in man to develop therapeutic approaches.
PMCID: PMC3192819  PMID: 22022275
17.  Immunogenicity of an Autogenous Streptococcus suis Bacterin in Preparturient Sows and Their Piglets in Relation to Protection after Weaning▿ †  
Clinical and Vaccine Immunology : CVI  2010;17(10):1589-1597.
Streptococcus suis is an important porcine pathogen causing meningitis and other invasive diseases in piglets of different ages. Application of S. suis serotype 2 bacterins to specific-pathogen-free (SPF) weaning piglets has been demonstrated to protect against the homologous serotype. However, autogenous S. suis bacterins are also applied to sows and suckling piglets in the field. Therefore, comparative evaluation of different bacterin immunization regimes, including sow vaccination, was performed in this study. The main objectives were to determine the immunogenicity of an S. suis bacterin in sows prepartum and its influence on active immunization of piglets. Experimental infection of 6- and 8-week-old weaning piglets was performed to elucidate protective efficacies. Humoral immune responses were investigated by an enzyme-linked immunosorbent assay (ELISA) measuring muramidase-released protein (MRP)-specific IgG titers and by opsonophagocytosis assays. Bacterin application elicited high MRP-specific IgG titers in the serum and colostrum of sows, as well as opsonizing antibodies. Piglets from vaccinated sows had significantly higher MRP-specific titers than respective piglets from nonvaccinated sows until 6 weeks postpartum. Vaccination of suckling piglets did not result in high MRP-specific titers nor in induction of opsonizing antibodies. Furthermore, neither vaccination of suckling nor of weaning piglets from immunized sows was associated with a prominent active immune response and protection at 8 weeks postpartum. However, protection was observed in respective 6-week-old weaning piglets, most likely because of protective maternal immunity. In conclusion, this study provides the first results suggesting protective passive maternal immunity for S. suis serotype 2 after bacterin vaccination of sows and a strong inhibitory effect on active immunization of suckling and weaning piglets, leading to highly susceptible growers.
PMCID: PMC2953004  PMID: 20739502
18.  Lack of cardiac fibrosis in a new model of high prorenin hyperaldosteronism 
The aim of the present study was to test the hypothesis that elevation of prorenin in plasma is sufficient to induce cardiac fibrosis. Normotensive cyp1a1ren-2 transgenic rats with normal plasma prorenin and aldosterone levels were given 0.125% indole-3-carbinol (I3C) orally for a period of 12 wk. Plasma prorenin and aldosterone levels were determined in 4-wk intervals, and cardiac marker enzymes for hypertrophy, fibrosis, and oxidative stress as well as cardiac pathology were investigated. In I3C-treated cyp1a1 ren-2 transgenic rats, plasma prorenin concentrations were >100-fold elevated (≥7.1 ± 2.6 μg ANG I·ml−1·h−1 vs. ≤0.07 ± 0.1; P < 0.001), whereas active renin levels were suppressed (0.09 ± 0.02 vs. 0.2 ± 0.1; P < 0.05). Aldosterone concentrations were elevated three- to fourfold for a period of >4 wk (574 ± 51 vs. 160 ± 68 pg/ml; P < 0.01). After 12 wk of I3C, rats exhibited moderate cardiac hypertrophy (heart weight/body weight 2.5 ± 0.04 vs. 3.1 ± 0.1 mg/g; P < 0.01). There was a slight increase in mRNA contents of endothelin 1 (1.21 ± 0.08 vs. 0.75 ± 0.007; P < 0.001), NADP oxidase-2 (1.03 ± 0.006 vs. 0.76 ± 0.04; P < 0.001), transforming growth factor-β (0.99 ± 0.06 vs. 0.84 ± 0.04; P < 0.05), collagen type I (1.32 ± 0.32 vs. 0.94 ± 0.18; P < 0.05), and intercellular adhesion molecule-1 (1.12 ± 0.12 vs. 0.84 ± 0.08; P < 0.05). These genes are known to be stimulated by the renin-angiotensin system. There were no histological signs of fibrosis in the heart. We found that prorenin and aldosterone alone are not sufficient to induce considerable cardiac fibrosis in the absence of sodium load.
PMCID: PMC2781377  PMID: 19749160
prorenin; aldosterone; cardiac fibrosis; renin receptor
19.  The renin–angiotensin system as a primary cause of polyarteritis nodosa in rats 
Polyarteritis nodosa is a necrotizing vasculitis of medium-sized arteries of unknown origin. Hypertension is present in 30% of patients with polyarteritis nodosa. In those cases, high renin levels are thought to be secondary to renal involvement. The present study was performed to identify causal factors of polyarteritis nodosa. In cyp1a1ren-2 transgenic rats, vasculitis of medium-sized arteries resembling classical polyarteritis nodosa can be induced. In this model, oral administration of indole-3-carbinol (I3C) activates the liver-specific cyp1a1 promoter, leading to prorenin expression in a dose-dependent manner. After the first 6 weeks of chronic induction with 0.125% I3C, the mean arterial pressure reached a plateau of about 170 mmHg. Ten out of 11 I3C-treated rats, which were chronically instrumented with a telemetric device to measure blood pressure, developed polyarteritis nodosa within 10 weeks of I3C treatment. I3C alone or instrumentation alone did not cause polyarteritis nodosa. The angiotensin-converting enzyme inhibitor captopril completely prevented the development of polyarteritis nodosa, indicating that local angiotensin II generation is a pathogenetic factor in this model. The renin–angiotensin system can play a primary role in the development of polyarteritis nodosa in rats.
PMCID: PMC3828848  PMID: 19432815
polyarteritis nodosa; angiotensin; inflammation; prorenin
20.  Streptococcus suis Bacterin and Subunit Vaccine Immunogenicities and Protective Efficacies against Serotypes 2 and 9▿†  
Streptococcus suis causes numerous diseases in pigs, most importantly, meningitis, arthritis, septicemia, and bronchopneumonia. One of the major problems in modern swine production is the lack of a vaccine protecting against more than one S. suis serotype. The objective of this study was to determine the protective efficacy of a serotype 2 murein-associated protein (MAP) fraction subunit vaccine in comparison to that of a bacterin against experimental challenge with serotype 2 (containing muramidase-released protein [MRP], extracellular factor, and suilysin [SLY]) and serotype 9 (containing MRP variant MRP* and SLY) strains. MAP was shown to include different surface-associated proteins, such as the MRP and surface antigen one (SAO) expressed by both pathotypes used for challenge. The results of this study demonstrated that the serotype 2 bacterin induced protective immunity against homologous challenge. In contrast, the protective efficacy of the MAP subunit vaccine was low, though MAP immunization resulted in high serum immunoglobulin G2 titers against MRP and SAO. Importantly, immunization with bacterin but not with MAP induced opsonizing antibody titers against the serotype 2 strain, and these antibody titers were found to correlate with protection. However, after absorption with a nonencapsulated isogenic mutant, the sera from bacterin-immunized piglets failed to facilitate neutrophil killing, indicating that antibodies directed against capsule may not have been essential for opsonophagocytosis. Furthermore, induction of opsonizing antibodies against serotype 9 was not detectable in the group receiving bacterin or in the group receiving the MAP vaccine. In agreement, protection against the heterologous serotype 9 strain was low in both groups. Thus, identification of an antigen protecting against these two important S. suis pathotypes remains an important goal of future studies.
PMCID: PMC2643536  PMID: 19109449
21.  Phocine Distemper in German Seals, 2002 
Emerging Infectious Diseases  2004;10(4):723-725.
Approximately 21,700 seals died during a morbillivirus epidemic in northwestern Europe in 2002. Phocine distemper virus 1 was isolated from seals in German waters. The sequence of the P gene showed 97% identity with the Dutch virus isolated in 1988. There was 100% identity with the Dutch isolate from 2002 and a single nucleotide mismatch with the Danish isolate.
PMCID: PMC3323098  PMID: 15200869
harbor seal; phocine distemper virus; germany; RT-PCR; immunohistochemistry; serology

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