Immunofluorescent Antibody Technique Research Articles

Detection of Main Causative Agents among Young Children Suffering from Epiglottitis in Hilla City, Iraq.

Epiglottitis is a rapidly progressive epiglottis infection leading to upper airway edema. This study aimed to detect the main causative agent, viral infection, by immunofluorescence antibody technique and PCR technique and bacterial infection detection by specific gene among young children suffering from epiglottitis. This study included 85 young children aged 10-15 years. The virus was identified on 85 blood samples using the CER test Human simplex virus Card test; the results revealed that 12 (14.1%) specimens were related to virus infection, and the sera of patients showed anti-IgM to HSV-1 antibodies. HSV-1 was detected in blood samples by qPCR technique. Eighty-five saliva samples were collected from young children suffering from epiglottitis. The samples were cultured for 18-24 hours at 37°C. They were then cultivated for 18-24 hours on various selective media at 37°C. The colony morphology, microscopically, and biochemical testing were used to identify Haemophilus influenzae as a first Identification. Out of 85 clinical specimens, 63 (74.1%) were positive culture, while 22 (25.9%) had no growth on culture media; out of 63 specimens, only 22 (34.9%) isolates belonged to Haemophilus influenzae by biochemical tests, while 41 (65.1%) related to other types of microorganisms. VITEK 2 was used to validate bacteria isolates from young children suffering from epiglottitis. The findings indicate that 22 (34.9%) isolates related to Haemophilus influenzae have been confirmed with an excellent ID message confidence level (94 to 99.8% likelihood percentage). This method is characterized by quick bacterial detection. DNA was taken from all suspected isolates previously identified as Haemophilus influenzae using the vitek2 technology, and traditional PCR was used to amplify specific hel gene for Haemophilus influenzae primers utilizing these DNA samples. After that, when compared to an allelic ladder, gel electrophoresis revealed that all 22 (100%) samples of Haemophilus influenzae produced 101 bp DNA fragments. For isolates previously identified as Haemophilus influenzae, molecular identification of the ompP gene was performed. The results showed that 12 (or 54.5 percent) of the 22 isolates tested positive for this virulence gene. When compared to an allelic ladder, the presence of (459 bp) bands indicated positive results. In addition, the bexA gene was molecularly detected in 22 Haemophilus influenzae isolates, showing that only 8 (36.3 percent) of the isolates had this gene. When compared to an allelic ladder, the presence of a (343 bp) band indicated positive results for bexA gene pathogenicity; in conclusion, HSV (1) and Hib were considered almost causative agents of epiglottitis in young children.

Immunofluorescent Antibody Techniques in the Diagnosis of SARS-CoV-2 Infection in Humans.

Immunofluorescence (IF) is an important technique used in the diagnosis of many infectious diseases. In virology, it has proven to be particularly suited to detecting antibody directed against newly emerging viruses able to be cultivated in cell culture. It permits visualization of antibody and allows for antibody class to be determined which is critical to understanding the timing of infection. The procedure used to determine IgG, IgA, and IgM antibody directed against SARS-CoV-2 in humans is described in this chapter. These methods were developed for routine diagnosis of SARS-CoV-2 infection in Australia at the start of the global pandemic in 2020.

Pathological study and detection of Bovine parainfluenza 3 virus in pneumonic sheep lungs using direct immunofluorescence antibody technique.

Bovine parainfluenza 3 virus is a common virus that causes respiratory tract infection in cattle, sheep, and goats worldwide. The objective of this study is to identify macroscopic and histopathological lung lesions in slaughtered sheep during the period from December 2018 to December 2019 and to determine the presence of Bovine parainfluenza 3 virus (BPI3V) in frozen sheep pneumonic lung using a direct immunofluorescence antibody technique (DFAT). The overall prevalence of lung affection was 11% (1440/13084). The gross lesions were acute bronchopneumonia (58.12%), interstitial pneumonia (07.15%), fibrinous bronchopneumonia (10.70%), suppurative bronchopneumonia (03.47%), verminous pneumonia (13.75%), and ovine pulmonary adenomatosis (06.81%). There was a significant difference in the rate of pulmonary lesions according to the seasons of the study. The lesions were more frequently observed in autumn and winter with a rate of 34.17% and 28.05%, respectively. The DFAT was carried out only on 107 pneumonic samples with interstitial pneumonia, fibrinous bronchopneumonia, and acute bronchopneumonia. The BPI3V antigens were detected in 12 samples (11.21%). This is the first study that revealed the presence of the BPI3V in pneumonic sheep lungs in Batna region using the direct immunofluorescence antibody technique. The latter may be used for definite diagnosis when histopathological modifications in pneumonic sheep caused by this virus are difficult to distinguish from those caused by other respiratory viruses.

Influence of euthanasia, the intensity of inflammatory lesion and viral load in the laboratory diagnosis of rabies in cattle

ABSTRACT: This research reports the use of different diagnostic tests in cattle, naturally infected by Rabies lyssavirus (RABV), and correlates the positivity of the tests with the clinical moment of euthanasia, the intensity of the inflammatory lesion and viral load. It also highlights the possibility of euthanasia in early stages of the disease as a way to improve animal welfare. For that, samples of 34 bovine brains were collected for analysis, preserved in 10% buffered formaline and refrigerated with subsequent freezing. The samples were subjected to direct immunofluorescence antibody technique (DFAT) tests, viral isolation in cell culture (VICC), histopathology with hematoxylin and eosin staining (HE), immunohistochemistry (IHC), Shorr stainied neural tissue smears (DSS), Reverse transcription polymerase chain reaction (RT-PCR) and polymerase chain reaction by quantitative reverse transcriptase (qRT-PCR). The areas used for analysis were the cerebellum, parietal telencephalon and thalamus. Samples with Negri bodies (NBs) or immunostaining in at least one of the analyzed areas were considered positive. For the study of the intensity of histological lesions, the lesions were classified into grades 0, 1, 2 and 3 and the positivity of the test in the presence or absence of NBs in one of the three areas analyzed. To verify the influence of the disease clinical evolution, 4-four groups of analysis were created according to the animal’s clinical status at moment of the euthanasia, being: M1 = animal euthanized while standing, M2 = euthanized when in sternal recumbence, M3 = euthanized when in lateral recumbence, M4 = animal with natural death. Of the 34 brains evaluated, IHC was positive in 100% of cases, DFAT was positive in 97.05% of them, and in this negative sample the presence of RABV was confirmed by VICC. NBs ere seen in 88.23% of the cases, and the DSS test was positive in 82.35% of them. All diagnostic techniques showed positive cases in all groups analyzed. Each case was positive in at least two diagnostic methods. All cases that contained NBs were positive for rabies in the other tests. In this study, it was observed that the variables analyzed (intensity of injury and clinical evolution at the moment of euthanasia) had an influence only on HE and DSS techniques, which are based on NB research to form the diagnosis, but did not interfere with the effectiveness of the diagnosis performed by detecting the viral antigen performed by DFAT and IHC. All isolated RABV samples included in the present study have a genetic lineage characteristic of hematophagous bats Desmodus rotundus. The evaluation of qRT-PCR showed that the amount of virus did not interfere in the positivity of the tests. This work shows that IHC and DFAT are safe diagnostic techniques. They are capable of detecting RABV even in euthanized animals in the early stages of clinical evolution with mild intensities of histological lesions.

Anti-nuclear antibodies: A practical approach to testing and interpretation

Anti-nuclear antibodies (ANAs) are a group of antibodies that are characteristically associated with connective tissue diseases (CTDs). Indirect immunofluorescence antibody technique, having a high sensitivity, is the most common technique used for detection, results of which are expressed in terms of the pattern of fluorescence, substrate used, and the titer of a positive test. Other methods include solid-phase assays. ANA test must be performed only when there is a clinical suspicion of an autoimmune CTD. ANA should not be used as a screening tool for asymptomatic individuals. It is essential in clinical practice to be aware of when to order ANA testing, and how to correctly interpret the test results.

Development and characterization of ORF68 negative equine herpes virus type–1, Ab4p strain

Equine herpesvirus–1 (EHV–1) is an important pathogen, which infects horses worldwide with high morbidity but low mortality rates. The respiratory disorders and abortions are the most common indicators. Ab4p (an abortigenic and paralytic virus) is one of the most important and virulent strains. The development and functional characterization of the open reading frame-68 (ORF68) negative EHV–1 Ab4p mutants and an assessment of their roles in the infection at the cellular level were the main targets of the current study. Escherichia coli DH10β containing the Ab4p bacterial artificial chromosome (pAb4pBAC) and Red/ET expression vector were used to develop different ORF68 mutants. Multi-step growth kinetic experiments were conducted in order to evaluate the growth properties of the constructed mutant viruses. Growth of the Ab4pΔORF68 showed the lowest titer, compared to the Ab4pΔORF68R, Ab4pΔORF68R non-sense, and the parent Ab4p viruses without any significant difference (P > 0.05). The growth of the mutant viruses was almost similar across the cell types, but viruses growth was more efficient in FHK cells as judged by the number of the obtained virus particles. The plaque size of Ab4pΔORF68 was significantly (40%) smaller than those of Ab4p (P < 0.01), Ab4pΔORF68R, and Ab4pΔORF68R non-sense viruses which confirmed the importance of ORF68 protein in the cell-to-cell transmission of EHV–1. Subcellular localization of the green fluorescent protein (GFP) ORF68 gene fusion product showed late expression with intranuclear localization of the transfected cells while immunofluorescent antibody technique (IFAT) localized it at the nucleus and nuclear membranes of the infected cells. Hence, it could be concluded that ORF68 protein may not be essential for EHV–1 Ab4p growth but plays a crucial role in virus penetration and transmission at the cellular level. Therefore, the generated EHV–1 ORF68 negative mutant could be a prospective candidate for the development of a vaccine marker.

Recent Approaches To Optimize Laboratory Assessment of Antinuclear Antibodies.

The presence of antinuclear antibodies (ANAs) is a hallmark of a number of systemic autoimmune rheumatic diseases, and testing is usually performed as part of the initial diagnostic workup when suspicion of an underlying autoimmune disorder is high. The indirect immunofluorescence antibody (IFA) technique is the preferred method for detecting ANAs, as it demonstrates binding to specific intracellular structures within the cells, resulting in a number of staining patterns that are usually categorized based on the cellular components recognized and the degree of binding, as reflected by the fluorescence intensity or titer. As a screening tool, the ANA patterns can guide confirmatory testing useful in elucidating a specific clinical diagnosis or prognosis. However, routine use of ANA IFA testing as a global screening test is hampered by its labor-intensiveness, subjectivity, and limited diagnostic specificity, among other factors. This review focuses on current efforts to standardize the nomenclature of ANA patterns and on alternative methods for ANA determination, as well as on recent advances in image-based computer algorithms to automate IFA testing in clinical laboratories.

Seropositivity for West Nile Virus Antibodies in Patients Affected by Myasthenia Gravis.

BackgroundMyasthenia gravis (MG) is an autoimmune neuromuscular disease characterized by varying degrees of weakness of the skeletal muscles. Specific auto-antibodies against acetylcholine receptor (AChR) are present in the majority of MG patients, although the main cause behind its development still remains unclear. Recently MG development following West Nile virus (WNV) infection has been described in patients without any earlier evidence of MG. It is known that infectious agents trigger immune response and occasionally initiate autoimmune disease. WNV, the causative agent of both benign illness and neuroinvasive disease, has become endemic in many countries in all continents.MethodsIn the present study, 29 patients (15 males and 14 females, 19 - 78 years old) with confirmed diagnosis of MG and elevated levels of AChR autoantibodies were screened for the presence of serum anti-WNV antibodies and compared to a similar population affected by different autoimmune diseases. Indirect immunofluorescent antibody technique was used to evaluate the reaction of patients’ sera on cells infected by WNV.ResultsPositive fluorescent signals for anti-WNV IgG were obtained in 17% of MG patients, although no clinical manifestations related to WNV infection were reported. These results are in agreement with previous data and appear of great interest in the understanding of the pathogenic autoimmune mechanisms at the bases of MG development.ConclusionAs already observed in other human autoimmune diseases, pathogen-triggered autoimmunity could be involved in MG by breaking immunological self-tolerance through possible mechanisms of molecular mimicry between virus proteins and AChR subunits. In predisposed individuals, WNV infection could also represent an additional risk factor to initiate MG.

Evaluation of Peripheral Blood Lymphocytes as Cell Line for the Propagation of Human Herpes Simplex 1

Objectives: This study was planned to evaluate the use of peripheral blood lymphocytes as cell line for propagation of human herpes simplex 1, by the using of modern diagnostic techniques. Methodology: Primarily, 40 samples were collected from dermal lesions, investigated by RT-PCR techniquedirected to certify human herpes simplex1 infections Bosphore® HSV 1-2 Genotyping Kit v1(Anatoliagene works, Turkey) was used for the detection protocol. Results: the results revealed that HSV1 was correlated with 23(57.5%) of the total cases investigated. Five of HSV1positive samples were selected and applied to the assay of in vitro studying of specific cytopathic effects(CPE) viacell culture technique. Peripheral blood lymphocytes were isolated and propagated as a cell line. The demonstrationof specific HSV1 cytopathic effect was demonstrated by indirect immunofluorescent antibody technique. Thisapproach was revealed different degrees of sensitivity for supporting the growth of human herpes simplex1 virus;these cells were sensitive enough to support the growth of HSV1 virus. Conclusions: We concluded that Bosphore® HSV 1 Genotyping Kit v1allows very rapid detection of HSV DNA indermal leisions. peripheral blood lymphocytes were efficient enough for the studying of CPE of HSV1, while PCRassay was more efficient and more précised as a diagnostic technique. Recommendations: Real-time polymerase chain reaction is complementary to cell culture technique in diagnosis ofHSV, and it`s preferred to use specific primers of viral virulence factors and monitoring their pathogenicity.

Toxoplasma gondii and Neospora caninum seroprevalences in domestic South American camelids of the Peruvian Andes.

The objective of this study was to investigate the presence of Toxoplasma gondii- and Neospora caninum-specific antibodies in domestic South American camelids (SAC) (llamas and alpacas) from the Peruvian Andes through a cross-sectional study. A wide panel of serum samples collected from 1,845 llamas and 2,874 alpacas from the two main SAC production areas of Peru was selected. Immunofluorescence antibody technique was employed to detect and titrate specific anti-T. gondii and anti-N. caninum immunoglobulins G in serum samples. The association between T. gondii and N. caninum seroprevalence and the geographical origin (Central and South Peruvian Andes) was evaluated. Anti-T. gondii antibodies were found in 460 (24.9%) llamas and 706 (24.6%) alpacas, whereas anti-N. caninum antibodies were detected in 153 (8.3%) llamas and 425 (14.8%) alpacas. Toxoplasma gondii infection was strongly associated with the South Peruvian Andes where moderate climate conditions, larger human population, compared to the Central region, and the presence of wildlife definitive hosts could favor horizontal transmission to SAC. In contrast, N. caninum infection was not associated with the geographical region. These results indicate that T. gondii and N. caninum infections are highly and moderately widespread, respectively, in both species of domestic SAC studied in the sampled areas and appropriate control measures should be undertaken to reduce the prevalence of both parasitic infections.

Prevalence of &lt;i&gt;Besnoitia besnoiti&lt;/i&gt; antibodies in bovine sera and milk in Northern Nigeria

Besnoitia besnoiti , a re-emergent parasite of cattle in Europe, occurs in many countries of Africa and other parts of the world. Clinical observations and incidental findings of B. besnoiti in cattle have been reported in the Southern and Northern regions of Nigeria, but the prevalence of antibodies against this parasite is not yet known. This investigation was designed to determine the seroprevalence of bovine besnoitiosis in Northern Nigeria. A total of 400 cattle were selected at random through cluster sampling of herds from two Local Government Areas (LGA) each, of 5 States in the region (Kano, Kaduna, Katsina, Sokoto and Borno States), between May, 2008 and November, 2009. Sera samples obtained from cows, bulls and calves, and milk from lactating cows with suckling calves were screened with indirect immunofluorescent antibody technique (IFAT) for antibodies to B. besnoiti. Out of the 400 samples 321 (80.3%) were positive for antibodies to B. besnoiti . Cattle sampled in Borno had the highest (87.5%) prevalence of antibodies to B. besnoiti, while those sampled from Katsina State had the least prevalence (62.3%). Wamako LGA of Sokoto State had the highest prevalence of the antibodies (100.0%), while Dan Musa LGA in Katsina had the least prevalence (53.0%) among the ten LGA sampled, however, these differences were not statistically significant (p > 0.05). Similarly, the overall prevalence of antibodies to B. besnoiti did not vary significantly between bulls (84.0%) and cows (79.0%), or in the dry (83.6%) and wet (77.1%) seasons (p > 0.05). The high prevalence of antibodies to B. besnoiti in cattle in Northern Nigeria indicates an endemic state of the disease in this region. Keywords : Antibodies, Besnoitia , Cattle, Northern Nigeria, Prevalence

Diagnostic value of Immunoblotting assay for determination of anti nuclear antibody (ANA) concentration in rheumatologic diseases

Event Abstract Back to Event Diagnostic value of Immunoblotting assay for determination of anti nuclear antibody (ANA) concentration in rheumatologic diseases Mohammad Naghavi-Behzad1* 1 Tabriz University of Medical Sciences, Students' Research Committee, Iran Abstract Background: Antinuclear antibodies (ANAs) are common features of autoimmune connective tissue diseases. Various detection methods are used and there are newer techniques that are continuously put forward to facilitate diagnosis and therapeutic monitoring in connective tissue disease (CTD) patients. Immunofluorescence (IF) or Fluorescent Antibody Technique the most used and “gold standard” test for diagnosis. Enzyme-Linked Immunosorbent Assay (ELISA) is another routine test. For Immunoblotting (IB) assay, autoimmune ANA profiles are used which provide a qualitative in vitro assay for human autoantibodies to 15 different antigens. This study was conducted to compare three techniques (IF, IB, ELISA) in detection of ANA. If the sensitivity and specificity of IB superior to other methods, we can replace IF and ELISA with IB. Materials and Methods: An analytical cross-sectional study of 85 sera from patients with Systemic lupus erythematous (SLE), Systemic sclerosis (SSc) and Dermatomyosities(DM) was undertaken at rheumatology and nephrology department and clinic of Emam Reza hospital from 89/11/1 to 90/10/30. Sera collected and stored at -80˚c. Then they were used to detection of ANA with three techniques. Results: Of all sera 63 (74.1%) were ELISA positive and 22 (25.9%) had negative ELISA.74 (87.1 %) IF and IB positive and 11 (12.9%) IF and IB negative were observed. The sensitivity and specificity of IB in comparison with IF was 98.65% and 90.91% respectively. In comparison with ELISA we found 93.65% and 31.82% of sensitivity and specificity. Conclusion: Immunoblotting, has high sensitivity and specificity, can be used in screening of ANA.

Prevalence of cryptosporidiosis in dairy cattle, cattle-keeping families, their non-cattle-keeping neighbours and HIV-positive individuals in Dagoretti Division, Nairobi, Kenya

This paper reports a study estimating the prevalence of cryptosporidiosis, an emerging zoonosis, in people and cattle in Dagoretti, Nairobi. A repeated cross-sectional survey was carried out among randomly selected cattle keepers in Dagoretti, their dairy cattle and their non-cattle-keeping neighbours in the dry and wet seasons of 2006. A survey was also carried out among a group of people living with human immunodeficiency virus (HIV). Faecal samples were examined for Cryptosporidium oocysts using the modified Ziehl-Neelsen method; 16 % of the samples were also examined using immunofluorescence antibody (IFA) technique. Quality control consisted of blind reviews of slides, examining split samples and confirming slide results with IFA. We found that members of dairy households had a dry season cryptosporidiosis prevalence of 4 % and wet season prevalence of 0.3 %, and non-dairy households, a prevalence of 5 and 0 %, respectively. The cattle dry season prevalence was 15 %, and the wet season prevalence, 11 %. The prevalence in people living with HIV was 5 %. The laboratory quality control system showed some inconsistency within and between different tests, indicating challenges in obtaining consistent results under difficult field and working conditions. In conclusion, this is the first reported study to simultaneously survey livestock, livestock keepers and their neighbours for cryptosporidiosis. We failed to find evidence that zoonotic cryptosporidiosis is important overall in this community. This study also draws attention to the importance of quality control and its reporting in surveys in developing countries.

Acanthamoeba spp. as vehicle and reservoir of adenoviruses

Adenoviruses are important pathogens which are responsible for human enteritic, respiratory and eye infections. These viruses have been found to be prevalent in several natural and artificial water reservoirs worldwide. Free-living amoebae (FLA) have been recovered from similar water reservoirs, and it has been shown that FLA may act as reservoirs or vehicles of various microorganisms living in the same environment. To examine the ability of FLA to harbour adenoviruses, an in vitro study was conducted. Several Acanthamoeba strains were ‘co-cultivated’ with adenoviruses (adenoviruses 11 and 41), grown on A549 cells, using a proven test protocol. After phagocytosis and ingestion, the adenoviruses could be found within the cytoplasm of the Acanthamoeba trophozoites. The intake of the viruses into the cytoplasm of the trophozoites was demonstrated in an Acanthamoeba castellanii strain with the help of fluorescence microscopy and electron microscopy. An adenovirus DFA kit, which utilizes a direct immunofluorescent antibody technique for identifying adenovirus in infected tissue cultures, was used. In our study, it was demonstrated that adenoviruses were incorporated into the host amoebae (Acanthamoeba sp. Grp. II, three strains). So far, there were only a few publications concerning the relationship of free-living amoebae and viruses; only one of these described the detection of adenoviruses within acanthamoebae with molecular biological methods. We conducted this descriptive study to further examine the association between viable adenoviruses and FLA. To our knowledge, this is the first study to demonstrate directly the adenoviruses within FLA as vectors and vehicles. Therefore, we concluded that free-living amoebae appear able to act as carriers or vectors of the adenoviruses and thus may play a certain role in the dispersal of adenoviruses.

FRECUENCIA DE SERORREACTORES A Lawsonia intracellularis EN GRANJAS PORCINAS TECNIFICADAS

The objective of this study was to detect the presence of Lawsonia intracellularis antibodies in commercial swine herds in the Peruvian departments of Ica, Arequipa, La Libertad and Lambayeque. The antibodies were detected using the Indirect Immunofluorescence Antibody Technique (IFAT) in serum samples of 293 animals in five different age groups. La Libertad department was the most affected (14.6%) and female breeders were the most affected age group (23.1%). Positive results were found in 5.5 ± 0.6% of the samples. The results showed that L. intracellularis is an enteric ethiologic agent that affects the commercial swine production system of Peru.

Plasma sanguíneo desidratado na recuperação de leitões leves ao desmame: desempenho zootécnico, perfil hematológico, freqüência de diarréia e viabilidade econômica

Water buffaloes ( Bubalus bubalis), are capable of becoming infected with Neospora caninum and Toxoplasma gondii, contributing to disease dissemination. The incidence of these antibodies was assessed in sera of water buffaloes, using an immunofluorescent antibody technique (IFAT). Any possible influence of breed or age of the buffaloes was investigated, besides assessment of sera antibody titres of any dogs and humans that were in close contact with the tested animals. Serum samples were taken from water buffaloes (n=411), dogs (n=8) and humans (n=14), from buffalo dairy farms in the São Paulo State, and tested. Of the samples of buffalo sera tested, 56.0% were reactive ( >1:200) to N. caninum and 49.9% were reactive ( >1:64) to T. gondii. Of all, 33.9% of the buffaloes presented antibody titres for both protozoon parasites. Of the canine serum samples, 25.0% presented titres ( >1:50) for N. caninum and 62.5% for T. gondii ( >1:16). The human serum samples did not show detectable antibodies for N. caninum when using a >100 limit titre for positive findings. The obtained data allows the conclusion that the protozoan parasite N. caninum, as well as T. gondii, are widely disseminated in the water buffalo herds assessed and, apparently, the factors breed and age do not interfere with frequency of N. caninum positives among adult female buffaloes. When concerning T. gondii, female buffaloes of 5 to 6 years of age showed more frequent presence of specific antibodies.

Study on the transmission of Hantaan virus and Orientia tsutsugamushi by naturally dual infected Leptotrombidium scutellare through stinging

To investigate whether Leptotrombidium scutellare could be naturally infected by both Hantaan virus (HV) and Orientia tsutsugamushi (OT) and transmission status by stinging. 3459 Leptotrombidium scutellares from mice bodies and 3265 which were free were collected in the epidemic area of hemorrhagic fever with renal syndrome (HFRS) and tsutsugamushi disease.15 days later, the suspensions of lung and spleen of mice with 6 in a group stung by 1, 5 or 10 infected mites were injected intra-cerebrally into other mice for the detection of HV and OT in the next 6 generations of the mice, with immunofluorescent antibody technique (IFAT) and Giemsa staining technique. The passages of Vero-E6 cells inoculated on the aseptic filtrations from different number of infected mites were used to detect HV and OT pathogens. HV-RNA and OT-DNA were detected by PCR. After passage, HV positive mouse body mite group out of both 5 and 10 mites in the sixth generation, OT positive mouse body mite group out of the 10 mites in the sixth generation, both HV and OT positive mouse body mite group out of 1 mite in the fifth and sixth generation, both HV and OT positive mouse body mite group out of 5 and 10 mites in the sixth generation, and free mites group out of 1, 5 and 10 mites in the sixth generation, were found one mouse infected by both HV and OT, respectively. Out of the fourth generation of Vero-E6 cells, one sample was found both HV and OT positive out of 5 and 10 HV and OT mouse body mite group, respectively. In the sixth generation, both HV and OT positive cells were detected in one mouse mite group and the 1, 5, 10 free mite groups, respectively. HV-RNA and OT-DNA were all detected by PCR. Both HV and OT could be coexisted in wild Leptotrombidium scutellare and transmitted by stinging.

Localization of Pseudomonas aeruginosa in lung tissue in patients with diffuse panbronchiolitis using an immunofluorescent antibody technique and immunoperoxidase staining procedure

Localization of Pseudomonas aeruginosa in lung tissue in patients with diffuse panbronchiolitis using an immunofluorescent antibody technique and immunoperoxidase staining procedure

Viral haemorrhagic septicaemia virus (VHSV) genotype II isolated from European river lamprey Lampetra fluviatilis in Finland during surveillance from 1999 to 2008

We examined the occurrence of viral haemorrhagic septicaemia virus (VHSV) in the main spawning stocks of wild European river lamprey Lampetra fluviatilis in the rivers of Finland from 1999 to 2008. Pooled samples of internal organs (kidney, liver and heart or brain) from 2621 lampreys were examined for the presence of VHSV by standard virological techniques. VHSV was isolated from 5 samples from the rivers Lestijoki and Kalajoki, which flow from Finland into the Bothnian Bay of the Baltic Sea. The presence of VHSV was confirmed by immunofluorescent antibody technique (IFAT), ELISA and RT-PCR. Phylogenetic analysis based on the full-length VHSV glycoprotein (G) gene sequence revealed that the isolates were most closely related to the VHSV strain isolated in 1996 from herring Clupea harengus and sprat Sprattus sprattus in the Eastern Gotland Basin of the Baltic Sea, and were therefore assigned to VHSV genotype II. The partial G gene sequences obtained (nt 1 to 672-1129) of all 5 lamprey VHSV isolates were identical, and so were the entire G genes (nt 1 to 1524) of 2 isolates sequenced. The virulence of one of the lamprey isolates was evaluated by an experimental infection trial in rainbow trout Oncorhynchus mykiss fry. No mortality was induced postinfection by waterborne and intraperitoneal challenge, respectively, while 2 genotype Id isolates originating from Finnish rainbow trout caused marked mortality under the same conditions. The infection in the European river lamprey is thought to be independent from the epidemic in farmed rainbow trout in Finnish brackish waters, because the isolates from rainbow trout were of a different genotype. This is the first report of VHSV found in the European river lamprey. The role of wild river lampreys in maintaining the infection in the marine environment remains unclear.

Zebra mussels (Dreissena polymorpha) are effective sentinels of water quality irrespective of their size

Zebra mussels (Dreissena polymorpha) are recognised biomonitors in determining the presence and viability of the human waterborne pathogens Cryptosporidium parvum, C. hominis, Giardia intestinalis and microsporidia in surface waters. This study investigated whether the size of zebra mussels is a significant factor in the concentration of protozoan Cryptosporidium oocysts, Giardia cysts and microsporidian spores. Zebra mussels were collected in Lough Arrow, a small Irish lake, which is utilized for drinking water abstraction and is subject to agricultural and human wastewater pollution drivers, both recognised risk factors for human waterborne pathogens. Zebra mussels were cleaned, divided into size (5 mm) interval classes based on their shell length and made up to 150 g samples (wet weight with shell). Combined fluorescence in situ hybridization (FISH) and immunofluorescent antibody (IFA) techniques were utilized as biomolecular techniques to assess the presence and concentration of the pathogens. PCR analysis provided source-tracking information on human and animal pollution sources. There was no significant relationship between the size of D. polymorpha and pathogen loads in similar sized samples, indicating that different sites in the same or different waterbody can be compared in terms of relative concentrations of human waterborne parasites irrespective of the zebra mussels' size. Cryptosporidium was the most abundant species, with lower counts of Giardia and the microsporidian Encephalitozoon hellem, respectively. Cryptosporidium oocysts and Giardia cysts were detected in zebra mussel samples at all three lake water abstraction points. A lake transect showed a decline in Cryptosporidium with increasing distance from a stream discharging sewage. Samples from agricultural sites indicated faecal inputs contaminated with these pathogens. Species identification implicated both human and animal faecal inputs to the lake from treated effluent, septic tanks, and agriculture. The research demonstrates the efficacy of zebra mussels as sentinels of water quality i rrespective of their size.