Levels of feline infectious peritonitis virus in blood, effusions, and various tissues and the role of lymphopenia in disease outcome following experimental infection.

Twenty specific pathogen free cats were experimentally infected with a virulent cat-passaged type I field strain of FIPV. Eighteen cats succumbed within 2-4 weeks to effusive abdominal FIP, one survived for 6 weeks, and one seroconverted without outward signs of disease. A profound drop in the absolute count of blood lymphocytes occurred around 2 weeks post-infection (p.i.) in cats with rapid disease, while the decrease was delayed in the one cat that survived for 6 weeks. The absolute lymphocyte count of the surviving cat remained within normal range. Serum antibodies as measured by indirect immunofluorescence appeared after 2 weeks p.i. and correlated with the onset of disease signs. Viral genomic RNA was either not detectable by reverse transcription quantitative real-time PCR (RT-qPCR) or detectable only at very low levels in terminal tissues not involved directly in the infection, including hepatic and renal parenchyma, cardiac muscle, lung or popliteal lymph node. High tissue virus loads were measured in severely affected tissues such as the omentum, mesenteric lymph nodes and spleen. High levels of viral genomic RNA were also detected in whole ascitic fluid, with the cellular fraction containing 10-1000 times more viral RNA than the supernatant. Replicating virus was strongly associated with macrophages by immunohistochemistry. Virus was usually detected at relatively low levels in feces and there was no evidence of enterocyte infection. Viral genomic RNA was not detected at the level of test sensitivity in whole blood, plasma, or the white cell fraction in terminal samples from the 19 cats that succumbed or in the single survivor. These studies reconfirmed the effect of lymphopenia on disease outcome. FIPV genomic RNA was also found to be highly macrophage associated within diseased tissues and effusions as determined by RT-qPCR and immunohistochemistry but was not present in blood

Feline infectious peritonitis: role of the feline coronavirus 3c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats.

Feline infectious peritonitis virus (FIPV) was presumed to arise from mutations in the 3c of a ubiquitous and largely nonpathogenic feline enteric coronavirus (FECV). However, a recent study found that one-third of FIPV isolates have an intact 3c and suggested that it is not solely involved in FIP but is essential for intestinal replication. In order to confirm these assumptions, 27 fecal and 32 FIP coronavirus isolates were obtained from resident or adopted cats from a large metropolitan shelter during 2008-2009 and their 3a-c, E, and M genes sequenced. Forty percent of coronavirus isolates from FIP tissues had an intact 3c gene, while 60% had mutations that truncated the gene product. The 3c genes of fecal isolates from healthy cats were always intact. Coronavirus from FIP diseased tissues consistently induced FIP when given either oronasally or intraperitoneally (i.p.), regardless of the functional status of their 3c genes, thus confirming them to be FIPVs. In contrast, fecal isolates from healthy cats were infectious following oronasal infection and shed at high levels in feces without causing disease, as expected for FECVs. Only one in three cats shed FECV in the feces following i.p. infection, indicating that FECVs can replicate systemically, but with difficulty. FIPVs having a mutated 3c were not shed in the feces following either oronasal or i.p. inoculation, while FIPVs with intact 3c genes were shed in the feces following oronasal but not i.p. inoculation. Therefore, an intact 3c appears to be essential for intestinal replication. Although FIPVs with an intact 3c were shed in the feces following oronasal inoculation, fecal virus from these cats was not infectious for other cats. Attempts to identify potential FIP mutations in the 3a, 3b, E, and M were negative. However, the 3c gene of FIPVs, even though appearing intact, contained many more non-synonymous amino acid changes in the 3' one-third of the 3c protein than FECVs. An attempt to trace FIPV isolates back to enteric strains existing in the shelter was only partially successful due to the large region over which shelter cats and kittens originated, housing conditions prior to acquisition, and rapid movement through the shelter. No evidence could be found to support a recent theory that FIPVs and FECVs are genetically distinct

Migrating leukocytes are the source of Peroxiredoxin V during inflammation in the airways

BACKGROUND: We characterized changes in expression of the antioxidant protein Peroxiredoxin V (PRXV) during airway inflammation. METHODS: Studies in anesthetized rats and mice; PRXV expression determined by Western blot analyses and immunohistochemistry; PRXV m-RNA expression determined by Taq-Man RT-PCR. RESULTS: Bacterial lung inflammation did not change expression of PRXV in murine epithelia but produced massive influx of leukocytes highly expressing PRXV. Endotoxin and f-MLP induced leukocyte migration in rat trachea but did not change mRNA levels and PRXV protein expression in tracheal epithelial cells. In primary airway cell culture (cow), alveolar epithelial cells A549, or co-culture of A549 with murine macrophages RAW264.7, exposure to live bacteria increased expression of PRXV, which required serum. PRXV was secreted in vitro by epithelial and immune cells. CONCLUSION: Inflammation increased expression of PRXV in airways by at least 2 mechanisms: cell population shift by massive influx of leukocytes expressing PRXV, and moderate post-transcriptional up-regulation of PRXV in epithelial cells

A Primer on the Differential Calculus of 3D Orientations

The proper handling of 3D orientations is a central element in many optimization problems in engineering. Unfortunately many researchers and engineers struggle with the formulation of such problems and often fall back to suboptimal solutions. The existence of many different conventions further complicates this issue, especially when interfacing multiple differing implementations. This document discusses an alternative approach which makes use of a more abstract notion of 3D orientations. The relative orientation between two coordinate systems is primarily identified by the coordinate mapping it induces. This is combined with the standard exponential map in order to introduce representation-independent and minimal differentials, which are very convenient in optimization based methods

Infection of Mice with the Agent of Human Granulocytic Ehrlichiosis after Different Routes of Inoculation

Population kinetics of the agent of human granulocytic ehrlichiosis (aoHGE) were examined after needle and tickborne inoculation of C3H mice. Blood, skin, lung, spleen, liver, kidney, brain, lymph node, and bone marrow samples were analyzed by using real-time polymerase chain reaction (PCR) at various intervals after inoculation, using a p44 gene target. The highest number of copies of the p44 gene target occurred in blood and bone marrow samples, emphasizing aoHGE leukocytotropism. Numbers of copies of the p44 gene target in other tissues reflected vascular perfusion rather than replication. Needle-inoculated infected mice had earlier dissemination, but kinetics of infection in both groups were parallel, with declining rates of infection by day 20 and recovery in some mice on days 20-60 after inoculation. On the basis of an aoHGE lysate ELISA, mice seroconverted by day 10 after inoculation. Therefore, real-time PCR is useful for quantitative studies with the aoHGE in experimental infections, and results showed that needle inoculation can be used to study the aoHGE infection because of its similarity to tickborne inoculatio

In vivo gene expression profiling of human intestinal epithelial cells: analysis by laser microdissection of formalin fixed tissues

<p>Abstract</p> <p>Background</p> <p>The small intestinal epithelium mediates vital functions of nutrient absorption and host defense. The spatial organization of the epithelial cells along the crypt-villus axis segregates them into regions of specialized function. However, the differences in transcriptional programming and the molecular machinery that governs the migration, adhesion, and differentiation of intestinal epithelial cell lineages in humans remain under-explored. To increase our understanding of these mechanisms, we have evaluated gene expression patterns of ileal epithelial cells isolated by laser capture microdissection from either the villus epithelial or crypt cell regions of healthy human small intestinal mucosa. Expression profiles in villus and crypt epithelium were determined by DNA microarray, quantitative real-time PCR, and immunohistochemistry based methods. The expression levels of selected epithelial biomarkers were also compared between gastrointestinal tissues.</p> <p>Results</p> <p>Previously established biomarkers as well as a novel and distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation were found to be enriched in each of the two corresponding cell populations (GEO accession: GSE10629). Additionally, high baseline expression levels of innate antimicrobials, alpha defensin 5 (HD5) and regenerating islet-derived 3 alpha (Reg3A), were detected exclusively within the small bowel crypt, most notably in the ileum in comparison to other sites along the gastrointestinal tract.</p> <p>Conclusion</p> <p>The elucidation of differential gene expression patterns between crypt and villus epithelial cell lineages in human ileal tissue provides novel insights into the molecular machinery that mediates their functions and spatial organization. Moreover, our findings establish an important framework of knowledge for future investigations of human gastrointestinal diseases.</p

Anaplasma spp. in dogs and owners in north-western Morocco

Background: Anaplasma phagocytophilum is an emerging tick-borne zoonotic pathogen of increased interest worldwide which has been detected in northern Africa. Anaplasma platys is also present in this region and could possibly have a zoonotic potential. However, only one recent article reports on the human esposure to A. phagocytophilum in Morocco and no data are available on canine exposure to both bacteria. Therefore, we conducted a cross-sectional epidemiological study aiming to assess both canine and human exposure to Anaplasma spp. in Morocco. A total of 425 dogs (95 urban, 160 rural and 175 working dogs) and 11 dog owners were sampled from four cities of Morocco. Canine blood samples were screened for Anaplasma spp. antibodies by an enzyme-linked immunosorbent assay (ELISA) and for A. phagocytophilum and A. platys DNA by a real-time polymerase chain reaction (RT-PCR) targeting the msp2 gene. Human sera were tested for specific A. phagocytophilum immunoglobulin G (IgG) using a commercial immunofluorescence assay (IFA) kit. Results: Anaplasma spp. antibodies and A. platys DNA were detected in 21.9 and 7.5% of the dogs, respectively. Anaplasma phagocytophilum DNA was not amplified. Anaplasma platys DNA was significantly more frequently amplified for working dogs. No statistically significant differences in the prevalence of Anaplasma spp. antibodies or A. platys DNA detection were observed between sexes, age classes or in relation to exposure to ticks. A total of 348 Rhipicephalus sanguineus (sensu lato) ticks were removed from 35 urban and working dogs. The majority of dog owners (7/10) were seroreactive to A. phagoyctophilum IgG (one sample was excluded because of hemolysis). Conclusions: This study demonstrates the occurrence of Anaplasma spp. exposure and A. platys infection in dogs, and A. phagocytophilum exposure in humans in Morocco

Inquérito sorológico e molecular de Leptospira spp. em cavalos carroceiros de área endêmica para leptospirose humana em Curitiba, Sul do Brasil

Introdução: Cavalos carroceiros são uma população reemergente empregada para transportar materiais recicláveis em cidades. Métodos: Em área endêmica para leptospirose humana foram amostrados 62 cavalos. Soroaglutinação microscópica e reação em cadeia da polimerase em tempo real foram empregadas. Resultados: Observou-se soropositividade em 75,8% com sorovar Icterohaemorrhagiae em 80,8% cavalos. Amostras de sangue e urina foram negativas no qPCR. Observou-se correlação positiva entre SAM e pluviosidade (p = 0,02) e alagamentos (p = 0,03). Conclusão: Embora cavalos possam estar constantemente expostos a Leptospira spp. no ambiente, principalmente por chuvas e inundações, leptospiremia e leptospiruria não foram encontradas neste estudo.Introduction: Cart horses are a re-emerging population employed to carry recyclable material in cities. Methods: Sixty-two horses were sampled in an endemic area of human leptospirosis. The microscopic agglutination test (MAT) and real-time polymerase chain reaction (qPCR) were performed. Results: A seropositivity of 75.8% with serovar Icterohaemorrhagiae in 80.8% of the horses was observed. Blood and urine were qPCR negative. MAT showed positive correlations with rainfall (p = 0.02) and flooding (p = 0.03). Conclusions: Although horses may be constantly exposed to Leptospira spp. in the environment mostly because of rainfall and flooding, no leptospiremia or leptospiruria were observed in this study

A novel bocavirus in canine liver

Background: Bocaviruses are classified as a genus within the Parvoviridae family of single-stranded DNA viruses and are pathogenic in some mammalian species. Two species have been previously reported in dogs, minute virus of canines (MVC), associated with neonatal diseases and fertility disorders; and Canine bocavirus (CBoV), associated with respiratory disease. Findings: In this study using deep sequencing of enriched viral particles from the liver of a dog with severe hemorrhagic gastroenteritis, necrotizing vasculitis, granulomatous lymphadenitis and anuric renal failure, we identified and characterized a novel bocavirus we named Canine bocavirus 3 (CnBoV3). The three major ORFs of CnBoV3 (NS1, NP1 and VP1) shared less than 60% aa identity with those of other bocaviruses qualifying it as a novel species based on ICTV criteria. Inverse PCR showed the presence of concatemerized or circular forms of the genome in liver. Conclusions: We genetically characterized a bocavirus in a dog liver that is highly distinct from prior canine bocaviruses found in respiratory and fecal samples. Its role in this animal’s complex disease remains to be determined. Keywords: Canine bocavirus 3; Episome; Coinfectio

Presence of infectious agents and co-infections in diarrheic dogs determined with a real-time polymerase chain reaction-based panel

BACKGROUND: Infectious diarrhea can be caused by bacteria, viruses, or protozoan organisms, or a combination of these. The identification of co-infections in dogs is important to determine the prognosis and to plan strategies for their treatment and prophylaxis. Although many pathogens have been individually detected with real-time polymerase chain reaction (PCR), a comprehensive panel of agents that cause diarrhea in privately owned dogs has not yet been established. The objective of this study was to use a real-time PCR diarrhea panel to survey the frequencies of pathogens and co-infections in owned dogs attended in a veterinary hospital with and without diarrhea, as well the frequency in different countries. Feces samples were tested for canine distemper virus, canine coronavirus, canine parvovirus type 2 (CPV-2), Clostridium perfringens alpha toxin (CPA), Cryptosporidium spp., Giardia spp., and Salmonella spp. using molecular techniques. RESULTS: In total, 104 diarrheic and 43 control dogs that were presented consecutively at a major private veterinary hospital were included in the study. Overall, 71/104 (68.3%) dogs with diarrhea were positive for at least one pathogen: a single infection in 39/71 dogs (54.9%) and co-infections in 32/71 dogs (45.1%), including 21/32 dogs (65.6%) with dual, 5/32 (15.6%) with triple, and 6/32 (18.8%) with quadruple infections. In the control group, 13/43 (30.2%) dogs were positive, all with single infections only. The most prevalent pathogens in the diarrheic dogs were CPA (40/104 dogs, 38.5%), CPV-2 (36/104 dogs, 34.6%), and Giardia spp. (14/104 dogs, 13.5%). CPV-2 was the most prevalent pathogen in the dual co-infections, associated with CPA, Cryptosporidium spp., or Giardia spp. No statistical difference (P = 0.8374) was observed in the duration of diarrhea or the number of deaths (P = 0.5722) in the presence or absence of single or co-infections. CONCLUSIONS: Diarrheic dogs showed a higher prevalence of pathogen infections than the controls. Whereas the healthy dogs had only single infections, about half the diarrheic dogs had co-infections. Therefore, multiple pathogens should be investigated in dogs presenting with diarrhea. The effects of multiple pathogens on the disease outcomes remain unclear because the rate of death and the duration of diarrhea did not seem to be affected by these factors