Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi

A new immunogenic outer membrane protein, Omp-28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp-28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp-28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N-terminal amino acid sequence of Omp-28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c-DNA. ELISA and Western blotting identified Omp-28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp-28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp-28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp-28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.     

Developing a new clinical tool for diagnosing chronic Q fever: the Coxiella ELISPOT

Definitively establishing a clinical diagnosis of chronic Q fever remains challenging, as the diagnostic performance of both conventional serological tests and PCR is limited. Given the importance of an early diagnosis of chronic Q fever, there is a need for a reliable diagnostic test. We developed an enzyme-linked immunospot assay to measure Coxiella burnetii (C.?burnetii)-specific T-cell responses (Coxiella ELISPOT) to both phase I and phase II antigens and tested convalescent Q fever patients (without chronic disease, n?=?9) and patients with an established diagnosis of chronic Q fever (n?=?3). The Coxiella ELISPOT adequately identified convalescent Q fever patients from healthy controls by demonstrating C.?burnetii-specific T-cell interferon-γ production to both phase I and phase II antigens. Compared to convalescent Q fever patients, chronic Q fever patients showed a distinct Coxiella ELISPOT profile characterized by a much higher spot count for both phase I and phase II (18-fold for phase II, 8-fold higher for phase I) and a consistent shift towards more phase I reactivity. The diagnostic potential of the Coxiella ELISPOT is promising and warrants further investigation.     

Hyper-IgD and periodic fever syndrome: a new MVK mutation (p.R277G) associated with a severe phenotype

Hyperimmunoglobulinemia D and periodic fever syndrome (HIDS; MIM# 260920) is a rare recessively-inherited autoinflammatory condition caused by mutations in the MVK gene, which encodes for mevalonate kinase, an essential enzyme in the isoprenoid pathway. HIDS is clinically characterized by recurrent episodes of fever and inflammation. Here we report on the case of a 2 year-old Portuguese boy with recurrent episodes of fever, malaise, massive cervical lymphadenopathy and hepatosplenomegaly since the age of 12 months. Rash, arthralgia, abdominal pain and diarrhea were also seen occasionally. During attacks a vigorous acute-phase response was detected, including elevated erythrocyte sedimentation rate, C-reactive protein, serum amyloid A and leukocytosis. Clinical and laboratory improvement was seen between attacks. Despite normal serum IgD level, HIDS was clinically suspected. Mutational MVK analysis revealed the homozygous genotype with the novel p.Arg277Gly (p.R277G) mutation, while the healthy non-consanguineous parents were heterozygous. Short nonsteroidal anti-inflammatory drugs and corticosteroid courses were given during attacks with poor benefits, whereas anakinra showed positive responses only at high doses. The p.R277G mutation here described is a novel missense MVK mutation, and it has been detected in this case with a severe HIDS phenotype. Further studies are needed to evaluate a co-relation genotype, enzyme activity and phenotype, and to define the best therapeutic strategies.     

Bone Marrow Colony-stimulating Activity of Sera in Infectious Mononucleosis

From 44 to 100% of sera from patients with infectious mononucleosis exhibited the capacity to stimulate colony formation in vitro by mouse bone marrow cells. The proportion of sera with colony-stimulating activity was highest in patients with a short fever period and developing low Paul-Bunnell titres. Patients with a more severe course of the disease generally displayed no, or only weak, colony-stimulating activity in their sera, and also had higher Paul-Bunnell titres. The level of serum colony-stimulating activity tended to fall in the convalescent stages of the disease.     

Diverse and atypical manifestations of Q fever in a metropolitan city hospital: Emerging role of next-generation sequencing for laboratory diagnosis of Coxiella burnetii

Although Q fever has been widely reported in the rural areas of China, there is a paucity of data on the epidemiology and clinical characteristics of this disease in large metropolitan cities. In this study, we profile the epidemiology and clinical manifestations of Q fever from a tertiary hospital in Shenzhen, a Southern Chinese metropolitan city with a large immigrant population from other parts of China. A total of 14 patients were confirmed to have Q fever during a nine-year-and-six-month period, five of whom were retrospectively diagnosed during case review or incidentally picked up because of another research project on unexplained fever without localizing features. Some patients had the typical exposure histories and clinical features, while a few other patients had rare manifestations of Q fever, including one with heart failure and diffuse intracapillary proliferative glomerulonephritis, a patient presenting with a spontaneous bacterial peritonitis-like syndrome, and another one with concomitant Q fever and brucellosis. Using a combination of clinical manifestation, inflammatory marker levels, echocardiographic findings and serological or molecular test results, nine, three and two patients were diagnosed to have acute, chronic and convalescent Q fever, respectively. Seven, five and two patients were diagnosed to have Q fever by serological test, nested real-time PCR and next-generation sequencing respectively. Diverse and atypical manifestations are associated with Q fever. The incidence of Q fever is likely to be underestimated. Next-generation sequencing is becoming an important diagnostic modality for culture-negative infections, particularly those that the physicians fail to recognize clinically, such as Q fever.     

A case of Ebola virus infection.

In November 1976 an investigator at the Microbiological Research Establishment accidentally inoculated himself while processing material from patients in Africa who had been suffering from a haemorrhagic fever of unknown cause. He developed an illness closely resembling Marburg disease, and a virus was isolated from his blood that resembled Marburg virus but was distinct serologically. The course of the illness was mild and may have been modified by treatment with human interferon and convalescent serum. Convalescence was protracted; there was evidence of bone-marrow depression and virus was excreted in low titre for some weeks. Recovery was complete. Infection was contained by barrier-nursing techniques using a negative-pressure plastic isolator and infection did not spread to attendant staff or to the community.     

Bovine Epizootic Fever

A virus, the Yamaguchi strain, was serially propagated in suckling hamsters, mice and rats, and hamster kidney BHK21-WI2 cells from a natural case in the 1966 outbreak of bovine epizootic fever, an acute febrile disease of cattle, resembling ephemeral fever, known in Japan since 1949. An acute phase blood from the natural case was first passaged in calves by intravenous inoculation, and a blood specimen at the second passage was used to initiate serial hamster passage. Infected hamsters died with nervous symptoms, and serial passage was readily accomplished by intracerebral inoculation with brain emulsions. The hamster passage line of virus was serially passaged by intracerebral inoculation in 1- or 2-day-old mice, and then in rats 1 or 2 days after birth, which developed fatal encephalitis. The hamster passage virus was also serially propagated with cytopathic effect in cultures of BHK21-WI2 cell cultures. These viral lines were shown to be neutralized by, and to produce specific complement-fixing antigen reactive with, convalescent sera of calves infected with the original Yamaguchi strain, confirming the identity of these lines as the Yamaguchi strain. The hamster passage line, when inoculated intravenously in calves, induced an acute febrile illness which was similar to bovine epizootic fever; all the inoculated calves had viremia and developed neutralizing and complement-fixing antibodies against the virus. Serological evidence for infection with this virus was obtained in natural cases in the 1966 outbreak. These findings seem to justify this virus to be the causative agent of bovine epizootic fever.     

A Novel Rhabdovirus Associated with Acute Hemorrhagic Fever in Central Africa

Deep sequencing was used to discover a novel rhabdovirus (Bas-Congo virus, or BASV) associated with a 2009 outbreak of 3 human cases of acute hemorrhagic fever in Mangala village, Democratic Republic of Congo (DRC), Africa. The cases, presenting over a 3-week period, were characterized by abrupt disease onset, high fever, mucosal hemorrhage, and, in two patients, death within 3 days. BASV was detected in an acute serum sample from the lone survivor at a concentration of 1.09×10⁶ RNA copies/mL, and 98.2% of the genome was subsequently de novo assembled from ∼140 million sequence reads. Phylogenetic analysis revealed that BASV is highly divergent and shares less than 34% amino acid identity with any other rhabdovirus. High convalescent neutralizing antibody titers of >1∶1000 were detected in the survivor and an asymptomatic nurse directly caring for him, both of whom were health care workers, suggesting the potential for human-to-human transmission of BASV. The natural animal reservoir host or arthropod vector and precise mode of transmission for the virus remain unclear. BASV is an emerging human pathogen associated with acute hemorrhagic fever in Africa.     

Diagnostic value of dengue virus-specific IgA and IgM serum antibody detection

The diagnostic value of dengue virus (DV)-specific immunoglobulin A (IgA) serum antibody detection, by an indirect immunofluorescence assay (IFA) was evaluated. For this study, the kinetics of DV-specific IgA serum antibodies was analysed in two experimentally immunised macaques, paired samples from 35 patients suspected of a primary or secondary DV infection, paired sera from patients with high levels of IgA specific antibodies against influenza virus (n = 15), sera from patients with other viral infections (n = 40) and healthy blood donors (n = 10), which served as controls. The presence of DV-specific IgA serum antibodies in humans and in monkeys was compared with that of DV-specific IgM demonstrated in a capture enzyme-linked immunosorbent assay (ELISA). The development of DV-specific IgA and IgM antibodies in macaques proved to be similar to that observed in humans with a DV infection. In sera obtained from suspected primary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 1/6 (17%) and 6/6 (100%), whereas IgM was detected in 4/6 (67%) and 5/6 (83%), respectively. In sera from suspected secondary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 18/29 (62%) and 28/29 (97%), whereas IgM was detected in 20/29 (69%) and 28/29 (97%), respectively. The control group consisted of five paired serum samples from yellow fever vaccinated individuals and a patient with acute tick-borne encephalitis, 15 paired serum samples from patients with high levels of IgA antibodies specific for influenza virus and 40 serum samples from patients with specific IgM antibodies against other viruses. Ten serum samples from healthy blood donors were included. Among the control serum samples, in one patient, both DV-specific IgA and IgM antibodies were present, and in three sera DV-specific IgM antibodies could be demonstrated. These data suggest that detection of DV-specific IgA serum antibodies by IFA may have additional value for the diagnosis of DV infection.     

汉滩病毒感染体外培养神经元损伤及抗体对其保护作用

用体外培养的昆明小鼠胚胎脑皮质神经元,感染汉滩病毒后,　检测原癌基因ＦＯＳ的表达,加入ＨＦＲＳ病人恢复期血清,观察其对神经元的保护作用。将病毒　感染的神经元经免疫组化染色,检测原癌基因ＦＯＳ的快速表达;在病毒感染的神经元内加入Ｈ　ＦＲＳ病人恢复期血清,使用ＭＴＴ比色试验检测神经元存活情况。病毒感染的神经元ＦＯＳ表达明　显　增高,与对照组相比有显著性差异（Ｐ＜０．００１）;　ＨＦＲＳ病人恢复期血清保护病毒感染的神经元　组　与感染对照组神经元活性明显不同,有显著性差异（Ｐ＜０．００１）。　ＨＴＮＶ感染体外培养的神经元　,会使细胞内产生ＦＯＳ的快速表达,ＨＦＲＳ病人恢复期血清对病毒感染的神经元有一定的保护　作用。     

Haemorrhagic fever with renal syndrome: evaluation of ELISA for detection of Puumala-virus-specific IgG and IgM

IgM and IgG ELISA to Puumala virus were evaluated using sera from patients with haemorrhagic fever with renal syndrome (HFRS) from different geographical regions: Sweden, Denmark, Norway, Belgium and the European USSR.IgM ELISA proved useful in the diagnosis of HFRS in patients from all the regions mentioned above. Specific IgM could be detected as early as day 1 post onset of disease, and patients remained IgM-positive for several months. Specific IgG ELISA antibodies were also frequently detected in acute sera, and acute-convalescent serum pairs often failed to show a significant titre rise or increase in optical density (OD) values. This limits the use of IgG ELISA in patient diagnosis. Sera collected 2 years after infection revealed higher IgG ELISA OD readings than convalescent sera, and very high values were still detectable 10 to 20 years postinfection. IgG ELISA is therefore useful for the testing of immunity and in seroepidemiological studies.Acute and convalescent sera from HFRS patients in Korea and the Asian USSR showed no or only very weak reactivity in the Puumala virus IgG and IgM ELISA. These results are consistent with the “one-way” crossing described earlier.     

Immunological responses of the rat to Mycoplasma arthritidis

Arthritis was produced in rats by the intravenous injection of Mycoplasma arthritidis. Metabolic inhibiting antibody and indirect hemagglutinating antibody could not be detected in the sera of arthritic or convalescent animals. Nonmurine species of mycoplasma were capable of inducing metabolic inhibiting antibody in the rat. A hypothesis based upon the possible occurrence of heterogenetic antigens common to M. arthritidis and rat tissue was brought forward to explain these findings. Complement-fixing antibody to M. arthritidis was detected 3 to 4 days after injection and subsequently rose to high levels, depending upon the severity of arthritis and number of organisms injected. Animals that had recovered from intravenous or subcutaneous inoculation with M. arthritidis were resistant to subsequent infections by the organism. Immunity could be passively transferred by the intravenous injection of convalescent serum. Adsorption of the convalescent serum with antigen greatly reduced the complement fixation titer but did not significantly alter the protective properties of the serum. The presence of complement-fixing antibody could not be related to the development of immunity. An avirulent strain of M. arthritidis and a strain previously classified as M. hominis type 2 were capable of inducing resistance to subsequent injection by virulent M. arthritidis.     

Neglected human Rickettsia felis infection in Taiwan: A retrospective seroepidemiological survey of patients with suspected rickettsioses

BackgroundCurrent knowledge on Rickettsia felis infection in humans is based on sporadic case reports. Here we conducted a retrospective seroepidemiological survey of R. felis infection among febrile patients visiting a medical center in Taipei.Methodology/Principal findingsA total of 122 patients with suspected rickettsioses presenting with fever of unknown origin (FUO) but tested negative for scrub typhus, murine typhus, or Q fever were retrospectively identified during 2009 to 2010. The archived serum samples were examined for the presence of antibodies against R. felis, Rickettsia japonica, and Rickettsia typhi using microimmunofluorescence (MIF) assay. Serological evidence of Rickettsia exposure was found in 23 (19%, 23/122) patients. Eight patients had antibodies reactive to R. felis, including four with current infection (a ≥4-fold increase in IgG titer between acute and convalescent sera). The clinical presentations of these four patients included fever, skin rash, lymphadenopathy, as well as more severe conditions such as pancytopenia, hepatomegaly, elevated liver enzymes/bilirubin, and life-threatening acute respiratory distress syndrome. One of the patients died after doxycycline was stopped after being tested negative for scrub typhus, Q fever, and murine typhus.ConclusionsRickettsia felis is a neglected flea-borne pathogen in Taiwan, and its infection can be life-threatening. Further prospective studies of the prevalence of R. felis among patients with FUO and compatible clinical manifestations are warranted.     

Protection and recovery in influenza virus-infected mice immunosuppressed with anti-IgM

BALB/c mice, immunosuppressed from birth with goat anti-mouse IgM, were able to recover from influenza virus infection in the absence of detectable serum and nasal antibody. Recovery was delayed a few days when compared with control animals. Antibody-deficient mice, that had recovered from an initial influenza virus infection, i.e., convalescent mice, were subsequently rechallenged with homologous influenza virus in order to study the importance of nasal and serum antibody in prevention of infection. Convalescent mice were susceptible to reinfection when nasal and serum antibody were not detectable. The mice were resistant to reinfection when serum and/or nasal antibody was detectable by radioimmunoassay. Normal mice that were passively immunized with high titer mouse anti-influenza virus serum were susceptible to challenge with homologous influenza virus. The serum antibody levels in these mice were higher than most of those found in the immune convalescent mice suppressed with anti-IgM, thereby suggesting that the serum antibody, found in convalescent suppressed mice, is not protective. We conclude that 1) mice can recover from influenza virus infection in the absence of detectable levels of nasal and serum antibody, thus indirectly confirming the role of cell-mediated immunity in recovery; 2) serum IgM, IgG2A, IgG2B, IgG3, and probably IgG1 antibody levels are not responsible for protection against influenza virus infection of the upper respiratory tract; and 3) nasal IgA antibody correlates best with protection against reinfection of the upper respiratory tract, but some other locally protective agent cannot be excluded.     

Experimental study of the immunotherapy of burns

Experiments were conducted on rats. The influence of the serum of a burn convalescent on the toxic properties, the level of proteolytic enzymes activity, and morphological changes following burn were studied. After the burn the blood serum and the extracts of the organs acquired toxic properties; there was an increase in the activity of proteolytic enzymes, and marked morphological changes developed Administration of the serum of aburn convalescent promoted detoxication, diminished the activity of proteolytic enzymes distinctly, and decreased the morphological disturbances.     

Appendiceal abscess revealed by right renal colic and hydronephrosis

Reported is the case of a patient who had an appendiceal abscess revealed by right renal colic with fever and general fatigue. The abdominal computed tomography scan showed hydronephrosis and an appendiceal abscess surrounding and compressing the ureter. The appendix contained a stercolith and was perforated.     

IgG-subclass-specific antibody reactivity to respiratory syncytial virus polypeptides investigated by Western blot

IgG1 and IgG3 subclass-specific antibody reactivity (ABR) in serum samples obtained from infants and children in relation to acute lower respiratory disease caused by respiratory syncytial virus (RSV) infection was investigated by Western blot. IgG1 ABR was directed against the nucleocapsid polypeptides VPN and VPP as well as against the glycoproteins GP48 (F1) and GP90. IgG3 ABR was directed only against VPN and VPP. In infants, a low IgG1 reactivity against glycoproteins was observed. When serum samples obtained in the early convalescent phase were tested, ABR against GP48 and GP90 as well as against VPP differed with respect to RSV subtypes A and B. IgG1 ABR increased in the late convalescent phase, while IgG3 ABR decreased during this phase when serum samples from primary infections were tested.     

Serological Response of the EBV Antibodies in Pediatric Cases of Infectious Mononucleosis and in Their Contacts

The EBV antibodies were measured in 378 sera from a group of 129 pediatric and older cases of infectious mononucleosis and from 117 family and social contacts. Cases of infectious mononucleosis with only one exception were EBV seropositive in acute and/or convalescent sera. Fourteen cases, however, from whom no convalescent serum was available were EBV seronegative. A rise in EBV antibodies of two dilutions or more was demonstrated in 15 of the 129 cases. The prevalence of these antibodies in contacts reached 50 to 70% in each of four age groups. A significant antibody rise was encountered in only four cases, one of whom was found to have infectious mononucleosis simultaneously with the index case and one after an interdisease period of 10 months. The infectivity of the EB virus (and of infectious mononucleosis if causally related) and its horizontal transmission seem to be as low in nature as they appear to be experimentally in the laboratory.     

有关SARS病原冠状病毒合并感染组织胞浆菌的探讨

目的探讨SARS病原学的冠状病毒复合组织胞浆菌感染的问题。方法对象为SARS患者尸检1例和南猴(某猴场病死猴)尸检1例,采用病理组织学和免疫组化荧光检测的方法作检查,观察和比较两者尸检材料肺、脾、淋巴结和肝的病理变化和组织内组织胞浆菌的存在。结果1.人SARS和南猴的病理组织学改变极为相似。2.免疫组化结果表明,用人SARS恢复期血清和兔抗组织胞浆菌血清与组织胞浆菌抗原均呈阳性反应,显示此例SARS患者曾感染组织胞浆菌,其次用此两种抗血清分别与人SARS和南猴肺、脾、淋巴结尸检材料反应,均有组织胞浆菌阳性菌体的存在,说明人SARS和南猴脏器均有组织胞浆菌的感染。结论人SARS病原除冠状病毒外,还有组织胞浆菌的合并感染。     

Serum Metabolome and Lipidome Changes in Adult Patients with Primary Dengue Infection

Background

Dengue virus (DENV) is the most widespread arbovirus with an estimated 100 million infections occurring every year. Endemic in the tropical and subtropical areas of the world, dengue fever/dengue hemorrhagic fever (DF/DHF) is emerging as a major public health concern. The complex array of concurrent host physiologic changes has hampered a complete understanding of underlying molecular mechanisms of dengue pathogenesis.

Methodology/Principle Findings

Systems level characterization of serum metabolome and lipidome of adult DF patients at early febrile, defervescence, and convalescent stages of DENV infection was performed using liquid chromatography- and gas chromatography-mass spectrometry. The tractability of following metabolite and lipid changes in a relatively large sample size (n = 44) across three prominent infection stages allowed the identification of critical physiologic changes that coincided with the different stages. Sixty differential metabolites were identified in our metabolomics analysis and the main metabolite classes were free fatty acids, acylcarnitines, phospholipids, and amino acids. Major perturbed metabolic pathways included fatty acid biosynthesis and β-oxidation, phospholipid catabolism, steroid hormone pathway, etc., suggesting the multifactorial nature of human host responses. Analysis of phospholipids and sphingolipids verified the temporal trends and revealed association with lymphocytes and platelets numbers. These metabolites were significantly perturbed during the early stages, and normalized to control levels at convalescent stage, suggesting their potential utility as prognostic markers.

Conclusions/Significance

DENV infection causes temporally distinct serum metabolome and lipidome changes, and many of the differential metabolites are involved in acute inflammatory responses. Our global analyses revealed early anti-inflammatory responses working in concert to modulate early pro-inflammatory processes, thus preventing the host from development of pathologies by excessive or prolonged inflammation. This study is the first example of how an omic- approach can divulge the extensive, concurrent, and dynamic host responses elicited by DENV and offers plausible physiological insights to why DF is self limiting.