Direct Immunofluorescent Antibody Test Research Articles

Prevalence of Cryptosporidium spp. in humans and dromedaries (Camelus dromedarius) in Algeria

Cryptosporidium is one of the most important gastrointestinal parasites of a wide range of animals and humans. A study was conducted between January and March 2011 to investigate the prevalence of Cryptosporidium infection in dromedaries and humans in Oued Souf in southern Algeria. Stool samples of humans (n=110) and dromedaries (n=40) were collected. Cryptosporidium infection was diagnosed in human samples by the Heine staining method and by amplification of the 18S rRNA gene using polymerase chain reaction, and in dromedaries by the direct immunofluorescent antibody test. The overall prevalence of Cryptosporidium infection in dromedaries was 10% and none of these Cryptosporidium-infected animals showed diarrhoea. No significant difference was observed between males (10.52%) and females (9.52%). The number of oocysts calculated per gram of faeces varied between 100 and 450 oocysts/g. In humans, no positive cases were detected using the Heine staining method or 18S rRNA gene amplification. This finding highlighted the presence of Cryptosporidium in dromedaries in Oued Souf, Algeria for the first time. Further molecular epidemiology studies including a higher number of dromedaries and different parts of the country are recommended to establish the distribution and national impact of the disease. More efforts are required to isolate and to characterise by PCR the Cryptosporidium species in humans in Algeria.

Performance of bovine genital campylobacteriosis diagnostic tests in bulls from Uruguay: a Bayesian latent class model approach.

The sensitivity (Se) and specificity (Sp) of three diagnostic tests for the detection of Campylobacter fetus venerealis (Cfv) using field samples were estimated using a Bayesian latent class model (BLCM), accounting for the absence of a gold standard. The tests included in this study were direct immunofluorescence antibody test (IFAT), polymerase chain reaction (PCR), and real-time PCR (RT-PCR). Twelve farms from two different populations were selected and bull prepuce samples were collected. The IFAT was performed according to the OIE Manual. The conventional PCR was performed as multiplex, targeting the gene nahE for C. fetus species identification and insertion element ISCfe1 for Cfv identification. The RT-PCR was performed as uniplex: one targeting the gene nahE for C. fetus and the other targeting the insertion ISCfe1 (ISC2) for Cfv. Results from the BLCM showed a median Se of 11.7% (Bayesian credibility interval (BCI): 1.93–29.79%), 53.7% (BCI: 23.1–95.0%), and 36.1% (BCI: 14.5–71.7%) for IFAT, PCR, and RT-PCR respectively. The Sp were 94.5% (BCI: 90.1–97.9%), 97.0% (BCI: 92.9–99.3%), and 98.4% (BCI: 95.3–99.7%) for IFAT, PCR, and RT-PCR respectively. The correlation between PCR and RT-PCR was positive and low in samples from both sampled population (0.63% vs 8.47%). These results suggest that diagnostic sensitivity of the studied tests is lower using field samples than using pure Cfv strains.

Occurrence of faecal endoparasites in reindeer (Rangifer tarandus) in two grazing areas in northern Norway

BackgroundSemi-domesticated reindeer represent an important livestock industry and livelihood for a proportion of the human population in northern Fennoscandia. Reindeer husbandry is considered an extensive animal husbandry, where the animals are kept mostly on natural pastures, although sometimes kept in fenced areas for shorter periods. These reindeer may harbour a variety of parasites that may affect animal health and production. The relatively limited close contact between herds and owners gives limited opportunities for diagnosis and treatment of diseases in general. Furthermore, the effects of subclinical parasitism in livestock are commonly expressed as a reduction in productivity rather than clinical disease and mortality. Thus, specific knowledge of endoparasites and parasitic infections in these herds is scarce. This study investigated the occurrence of various endoparasites in reindeer by analysis of a total of 114 faecal samples from winter-slaughtered reindeer from two different grazing areas in Troms and Finnmark, Norway.ResultsUsing a McMaster method, a Baermann technique, and a direct immunofluorescent antibody test, the following parasites were identified in the faecal samples with the occurrence data given as percentages: Strongylid eggs (62%), Nematodirinae spp. eggs (24%), Capillaria sp. eggs (42%) and Moniezia spp. eggs (17%), Dictyocaulus spp. larvae (14%) protostrongylid larvae (40%), Eimera spp. oocysts (23%), and Giardia duodenalis cysts (5%). Cryptosporidium oocysts were not detected. Parasite eggs, oocysts, and cysts were quantified.Molecular analysis revealed G. duodenalis sub-assemblage AI, a potentially zoonotic genotype not previously reported in reindeer from this region. Morphological analyses of Eimeria oocysts identified two species, Eimeria mayeri and Eimeria rangiferis, and molecular analyses of the cytochrome C oxidase I (coi) gene and 18 s rRNA (18SSU) gene of Eimeria confirmed the presence of Eimeria species that are specific to reindeer.ConclusionsA high prevalence, but low burden, of endoparasites was detected in samples from these semi-domesticated reindeer. The samples were collected during winter, when adult gastrointestinal parasites usually produce only low numbers of transmission stages. Therefore, together with the low number of samples, detailed and definitive conclusions regarding parasite status of semi-domesticated reindeer are avoided. Nevertheless, these data provide a snapshot overview of parasite occurrence in a semi-domesticated animal group vulnerable to the various environmental changes to which they are exposed.

Study of vesicobullous lesions of the skin clinical and histopathology correlation

Introduction: Vesiculobullous diseases are a group of disorders in which primary lesion is a vesicle or a bulla on the skin or mucous membrane or both. Though some of the veisculobullous lesions are characteristics in their appearance and distribution, clinically many a times a definitive diagnosis cannot be made by physical examination alone. Then the dermatologists are forced to resort to histopathology for a definite diagnosis and classification. In the present scenario, even though histopathological study is sufficient in most of the cases, it is often accompanied by immunofluorescence antibody tests for confirmation of the diagnosis and monitoring of the disease. Direct immunofluorescence antibody test is a gold standard to confirm the disease and the indirect test helps to monitor the disease. Objectives: This study was carried out to find out clinical and histopathological correlation in diagnosing the disease. Materials and Methods: In the present study, histopathological evaluated vesiculobullous lesions over the period of 2 years. A total number of 35 cases were encountered. Staining procedures done are haematoxylin – eosin staining, leishmann staining and direct immunofluorescence stain. Results: A total number of 35 cases were encountered which constituted 0.70% of overall biopsy specimen and 12.32% of skin biopsy specimen. The pemphigus vulgaris formed commonest disease encountered accounting for 34.30% of all skin lesions. Bullous pemphigoid formed the second most common disease observed (31.34%) affecting both sexes equally. Conclusions: All though the ‘primary’ vesiculobullous lesions of skin are seen in a small group of people, they have been associated with significant a morbidity and mortality. It is important to distinguish each of these entities and separate them appropriate for management and treatment. Even though clinical examination has its own importance, both histopathological and DIF testing is must, as it confirms the diagnosis and

Improvement of Leptospira spp. diagnosis in aborted bovine fetuses by qPCR

Leptospirosis is a disease with major economic impact on livestock industry. The objective of this work was to determine the presence of Leptospira spp. DNA by qPCR in bovine fetuses with presumptive diagnosis of leptospirosis as the cause of abortion. Leptospira spp. DNA was detected by qPCR in 11 out of 34 fetuses. These specimens (10/11) had histopathological findings in hepatic and/or renal tissues compatible with leptospirosis. qPCR detection rate (32.4 %) was higher compared with direct immuno-fluorescence antibody test (DFAT) (11.8 %). The concordance coefficient between both techniques was 0.44. qPCR is a rapid and sensitive technique for the diagnosis of leptospirosis and improved the detection rate in fetal tissues compared with DFAT. Implementation of molecular techniques may increase the accurate detection of leptospirosis as a cause of bovine abortion allowing the application of rapid therapeutic and prophylactics measures in order to reduce the impact of this zoonotic disease.

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer's protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

Immunofluorescence and molecular diagnosis of bovine respiratory syncytial virus and bovine parainfluenza virus in the naturally infected young cattle and buffaloes from India

Pneumonia in bovines is a multifactorial disease manifestation leading to heavy economic losses. Infections of bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus-3 (BPI-3) are among the important contributing factors for the development of pneumonia in young animals. These viral agents either primarily cause pneumonia or predispose animals to the development of pneumonia. Although, the role of BRSV and BPI-3 in the pathogenesis of pneumonia is well established, there are no reports of involvement of BRSV and BPI-3 from Indian cattle and buffaloes suffering from pneumonia. In the present investigation, we performed postmortem examinations of 406 cattle and buffaloes, which were below twelve months of age. Out of 406 cases, twelve (2.95%) cases were positive for BRSV and fifteen (3.69%) cases were positive for BPI-3, screened by reverse transcriptase polymerase chain reaction (RT-PCR). Further, positive cases were confirmed by sequence analysis of RT-PCR amplicons and direct immunofluorescence antibody test (d-FAT) in paraffin-embedded lung tissue sections. BRSV positive cases revealed characteristic findings of bronchiolar epithelial necrosis, thickened alveolar septa by mononuclear cells infiltration and edema; alveolar lumens were filled with mononuclear cells and numerous syncytial cells were seen having intracytoplasmic inclusions. The BRSV antigen distribution was found to be in bronchiolar and alveolar epithelium and syncytial cells in the lung sections. In fifteen cases, where BPI-3 was detected, bronchointerstitial pneumonia in the majority of cases with thickened alveolar septa by mild macrophage infiltration, hyperplasia of type-II pneumocytes and bronchiolar necrosis along with syncytial cells having intracytoplasmic inclusions in the majority of cases were observed. The BPI-3 antigen distribution was found to be in bronchiolar and alveolar epithelium and syncytial cells in the lung sections. RT-PCR amplicons of BRSV and BPI-3 obtained were sequenced and their analysis showed homology with already available sequences in the NCBI database. It is the first report of detection of BRSV and BPI-3 from pneumonic cases by RT-PCR and d-FAT from cattle and buffaloes of India, indicating the need for more epidemiological studies.

First report of zoonotic Cryptosporidium parvum GP60 subtypes IIaA15G2R1 and IIaA16G3R1 in wild ponies from the northern Iberian Peninsula.

Studies on the prevalence and molecular characterization of Cryptosporidium spp. affecting feral horses are scarce. The highland areas of the northern Iberian Peninsula are home to a large population of wild ponies which generally roam free in the ancient natural range and are subjected to a traditional exploitation regime. In the present study, a total of 79 non-diarrhoeal faecal samples from the wild ponies were collected from the ground immediately after defecation. Cryptosporidium was detected in 10 of the samples (12.6%) by a direct immunofluorescence antibody test and DNA amplification and sequencing. Analysis of partial sequences of the small subunit ribosomal RNA (SSU-rRNA) and heat shock protein (hsp70) loci revealed the presence of Cryptosporidium parvum. In addition, amplification and sequencing of a fragment of the 60-kDa glycoprotein (GP60) locus identified C. parvum subtypes IIaA15G2R1 and IIaA16G3R1. This study reports, for the first time, the occurrence of C. parvum in wild ponies in Europe, specifically in the northern Iberian Peninsula. Identification of the common subtype IIaA15G2R1 and also subtype IIaA16G3R1 (first description) indicates that these hosts may play a role in the sylvatic transmission of C. parvum and that they may act as a reservoir of zoonotic cryptosporidiosis.

Giardia sp. and Cryptosporidium sp. in Iberian Wolf

A subsample consisting of fifty fecal samples from wild Iberian Wolf (Canis lupus signatus), from the northwest of Spain were collected in the field. The samples were analyzed for cysts of Giardia sp. and oocysts of Cryptosporidium sp. using a direct immunofluorescence antibody test (IFA). Giardia sp. and Cryptosporidium sp. were found in 20.0 % of the samples examined. Simple infections were more frequent (90.0 %) with seven (14.0 %) positive for Giardia sp. and two (4.0 %) positive for Cryptosporidium sp. To the authors’ knowledge, this is the first report of occurrence of Cryptosporidium sp. in Iberian Wolf.

Immunologic detection of Giardia duodenalis in a specific pathogen-free captive olive baboon ( Papio cynocephalus anubis) colony.

Several commercial Giardia immunoassays were evaluated in baboons for sensitivity and specificity as well as ease of use in a large specific pathogen-free (SPF) colony. An additional objective was to identify the assemblage(s) of Giardia duodenalis present in this baboon colony. A direct immunofluorescent antibody test (IFAT) was used as the reference test. Tests evaluated were a patient-side rapid test for dogs and cats, a human rapid test, and a well-plate ELISA designed for use with humans. Test sensitivities and specificities were compared using the McNemar paired t-test and were further evaluated for agreement using an unweighted Cohen kappa statistic. When compared to the IFAT reference, both human tests were more sensitive than the veterinary test. Based on PCR and sequencing of the G. duodenalis small-subunit ribosomal RNA and glutamate dehydrogenase loci, assemblage AI was present in this baboon colony. We found that 10 of the 110 (9%) baboons in this SPF colony were infected with a zoonotic strain of G. duodenalis.

Occurrence of Giardia in Swedish Red Foxes ( Vulpes vulpes ).

Giardia duodenalis is an intestinal protozoan capable of causing gastrointestinal disease in a range of vertebrate hosts. It is transmitted via the fecal-oral route. Understanding the epidemiology of G. duodenalis in animals is important, both for public health and for the health of the animals it infects. We investigated the occurrence of G. duodenalis in wild Swedish red foxes ( Vulpes vulpes ), with the aim of providing preliminary information on how this abundant predator might be involved in the transmission and epidemiology of G. duodenalis . Fecal samples (n=104) were analysed for G. duodenalis using a commercially available direct immunofluorescent antibody test. Giardia duodenalis cysts were found in 44% (46/104) of samples, with foxes excreting 100 to 140,500 cysts per gram of feces (mean, 4,930; median, 600). Molecular analysis, using PCR with sequencing of PCR amplicons, was performed on 14 samples, all containing over 2,000 cysts per gram feces. Amplification only occurred in four samples at the tpi gene, sequencing of which revealed assemblage B in all four samples. This study provides baseline information on the role of red foxes in the transmission dynamics of G. duodenalis in Sweden.

Occurrence of Giardia, Cryptosporidium, and Entamoeba in wild rhesusmacaques (Macaca mulatta) living in urban and semi-rural North-West India.

Giardia duodenalis, Cryptosporidium spp., and Entamoeba spp. are intestinal protozoa capable of infecting a range of host species, and are important causes of human morbidity and mortality. Understanding their epidemiology is important, both for public health and for the health of the animals they infect. This study investigated the occurrence of these protozoans in rhesus macaques (Macaca mulatta) in India, with the aim of providing preliminary information on the potential for transmission of these pathogens between macaques and humans. Faecal samples (n = 170) were collected from rhesus macaques from four districts of North-West India. Samples were analysed for Giardia/Cryptosporidium using a commercially available direct immunofluorescent antibody test after purification via immunomagnetic separation. Positive samples were characterised by sequencing of PCR products. Occurrence of Entamoeba was investigated first by using a genus-specific PCR, and positive samples further investigated via species-specific PCRs for Entamoeba coli, Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii. Giardia cysts were found in 31% of macaque samples, with all isolates belonging to Assemblage B. Cryptosporidium oocysts were found in 1 sample, however this sample did not result in amplification by PCR. Entamoeba spp. were found in 79% of samples, 49% of which were positive for E. coli. Multiplex PCR for E. histolytica, E. dispar and E. moshkovskii, did not result in amplification in any of the samples. Thus in 51% of the samples positive at the genus specific PCR, the Entamoeba species was not identified. This study provides baseline information on the potential for transmission of these zoonotic parasites at the wildlife-human interface.

Giardia and Cryptosporidium in cetaceans on the European Atlantic coast.

The occurrence of Giardia and Cryptosporidium was investigated in cetacean specimens stranded on the northwestern coast of Spain (European Atlantic coast) by analysis of 65 samples of large intestine from eight species. The parasites were identified by direct immunofluorescence antibody test (IFAT) and by PCR amplification of the β-giardin gene, the ITS1-5.8S-ITS2 region and the SSU-rDNA gene of Giardia and the SSU-rDNA gene of Cryptosporidium. Giardia and Cryptosporidium were detected in 7 (10.8 %) and 9 samples (13.8 %), respectively. In two samples, co-infection with both parasites was observed. Giardia duodenalis assemblages A, C, D and F, and Cryptosporidium parvum were identified. This is the first report of G. duodenalis in Balaenoptera acutorostrata, Kogia breviceps and Stenella coeruleoalba and also the first report of Cryptosporidium sp. in B. acutorostrata and of C. parvum in S. coeruleoalba and Tursiops truncatus. These results extend the known host range of these waterborne enteroparasites.

High excretion of Cryptosporidium ubiquitum by peri-parturient goats in one flock in western France

Cryptosporidium spp. is an important agent of neonatal diarrhoea in goat kids. Little is known about its molecular characterization in adult goats. A longitudinal study was set up to identify the species excreted by adult goats around parturition. Individual faecal samples were collected from 20 pregnant adult goats between 1 and 5 years old in one flock. Samplings began 3 weeks before the estimated kidding date and were done weekly until kidding and for 2 weeks after kidding. Cryptosporidium oocysts were concentrated from 15g of faeces using a caesium chloride (CsCl) method. Oocyst output was determined using a direct immunofluorescent antibody test (IFAT). Genomic DNA was extracted from each CsCl-concentrated faecal sample positive by IFAT and submitted to a nested PCR-RFLP on the SSU rDNA gene followed by sequencing to identify the isolates at species level. According to their kidding date, goats were sampled between 4 and 8 times. Sixteen goats, out of the eighteen which kidded, were found positive at least at one sampling date. Infection was asymptomatic. Prevalence of excretion was maximal 14 days before kidding with half of the goats excreting oocysts at this date. Excretion was higher before kidding than after kidding. Unexpected levels of excretion were observed with individual oocyst excretion ranging from 6 to 2.5×105 oocysts per gram of faeces. All isolates were identified as Cryptosporidium ubiquitum.

Detection and molecular characterization of Giardia and Cryptosporidium in common dolphins (Delphinus delphis) stranded along the Galician coast (Northwest Spain)

The ubiquitous protozoan parasites Giardia and Cryptosporidium have been detected from many species of captive and free-living wildlife, representing most mammalian orders. Twenty species of marine mammals have been reported to inhabit Galician waters and the region has one of the highest rates of stranding in Europe. Evidence from stranding, reported by-catches and sightings, suggests that the common dolphin (Delphinus delphis) is the most abundant cetacean on the Galician coast (Northwest Spain). The objective of this study was to detect and molecularly characterize isolates of Giardia and Cryptosporidium obtained from common dolphins stranded in this area. Between 2005 and 2012, sections of large intestine from 133 common dolphins stranded along the Galician coast were collected by the personnel of the Galician Stranding Network (Coordinadora para o Estudo dos Mamíferos Mariños, CEMMA). Using direct immunofluorescence antibody test (IFAT) and PCR amplification and sequencing of the SSU-rDNA, β-giardin genes and the ITS1-5.8S-ITS2 region, Giardia and Cryptosporidium were detected in 8 (6.0%) and 12 samples (9.0%), respectively. In two samples, co-infection by both parasites was observed. The molecular characterization revealed the presence of Giardia duodenalis assemblages A (genotypes A1 and A2) and B and Cryptosporidium parvum in these samples. This constitutes the first study in which the presence of Giardia and Cryptosporidium has been investigated in common dolphins on the European Atlantic coast, and it is also the first report of C. parvum in this host. Our findings indicate that these animals could act as reservoir of these waterborne parasites or could be victims of the contamination originated by anthropogenic activities.

Detection of Cryptosporidium oocysts in fresh calf faeces: Characteristics of two simple tests and evaluation of a semi-quantitative approach

The objective of the present study was to evaluate the characteristics of two rapid tests, namely, a faecal smear staining method (Heine staining) and a commercially available immunochromatographic (IC) assay, against a direct immunofluorescent antibody test (IFAT) for the diagnosis of Cryptosporidium infections in 917 faecal samples from calves aged <3weeks. These rapid tests were performed on non-concentrated faeces using a semi-quantitative approach using a 6-point scale (0–5) for Heine staining according to the number of oocysts per microscopic field, and a 4-point scale (0–3) for the IC assay reflecting the intensity of the positive line compared to the control line. Direct IFAT was performed following a diethyl ether concentration and results were expressed as oocysts per g of faeces (opg).Heine staining showed a sensitivity of 76.7% and a specificity of 90.7%. For faecal samples with ⩾10,000opg, sensitivity increased to 90.0%. The sensitivity of the IC assay was lower (61.8%) but the specificity was 100%. For faeces with ⩾100,000opg, the sensitivity of the IC assay reached 81%, indicating some limitation for clinical cryptosporidiosis diagnosis. Additional scoring (1–5) of the Heine staining correlated with the corresponding direct IFAT results, particularly for ranges of 1000 to >1,000,000opg. Additional scoring from 1 to 3 according to the thickness of the sample line for the IC test correlated with increasing levels of Cryptosporidium opg measured by IFAT in the range of 10,000 to >1,000,000opg. In conclusion, both Heine staining and the IC test can be reliably used through a simple semi-quantitative scale for grossly quantifying oocyst output in calf faecal samples.

Cryptosporidium in pet snakes from Italy: Molecular characterization and zoonotic implications

To provide information on the occurrence of Cryptosporidium species and genotypes in captive snakes from Italy, faecal specimens from 120 snakes belonging to 13 different genera of the families Boidae, Colubridae and Pythonidae were collected. Faecal samples were taken from the ground of the terrarium when available; otherwise cloacal cotton swabs were used. No clinical signs of cryptosporidiosis were observed in any animal at the time of sampling. Samples were examined for the presence of Cryptosporidium by using a direct immunofluorescence antibody test (IFAT) and two-step nested PCR at the small subunit (SSU) rRNA locus. PCR-positive samples were genotyped by restriction fragment length polymorphism (RFLP) analysis with the endonucleases SspI and VspI.By IFAT, 42 out of 120 snakes (35.0%) were found to be shedding Cryptosporidium oocysts. A significant higher percentage of positive ophidians were detected by using faecal specimens obtained from the terrarium (55.5%) than by cloacal cotton swabs (29.0%).SSU rRNA gene products were obtained from 25 isolates. Twenty samples tested positive to both microscopy and molecular techniques. Our data reveal a wide extent of cryptosporidial infections in snake-food animals since most of the identified isolates belonged to Cryptosporidium species, some of them with zoonotic potential, considered specific for rodents and resulting from ingestion of infected preys. The reptilian-specific species Cryptosporidium serpentis was identified in only one isolate.The common presence of reptile non-specific and, in some cases, zoonotic Cryptosporidium oocysts in snake faeces should to be taken into consideration in order to avoid the misidentification of the protozoan as well as the possible public health implications.

Preliminary experiments on use of zebrafish as a laboratory model for Giardia duodenalis infection

Although Giardia duodenalis is considered a parasite of mammals, different genotypes have been identified as infecting several species of freshwater and marine fish in Australia. Establishment of G. duodenalis infection in common laboratory zebrafish (Danio rerio), could provide an excellent tool for a range of studies on Giardia. We conducted preliminary experiments to investigate this possibility. Zebrafish were inoculated with viable G. duodenalis cysts from two different Assemblages (A and D) using a modified oro-gastric tube. Direct microscopy and immunofluorescent antibody test were used to check for Giardia cysts/trophozoites in the intestine, and histology was performed on intestinal mucosa to evaluate possible pathological changes. Giardia cysts were successfully deposited in the zebrafish alimentary tract using a modified oro-gastric tube, and were maintained in the fish gut for at least 8 days. Although a single trophozoite was observed in one fish three days post-exposure, we were unable to demonstrate established, propagative infection under the conditions tested.

Duration of shedding of respiratory syncytial virus in a community study of Kenyan children

BackgroundOur understanding of the transmission dynamics of respiratory syncytial virus (RSV) infection will be better informed with improved data on the patterns of shedding in cases not limited only to hospital admissions.MethodsIn a household study, children testing RSV positive by direct immunofluorescent antibody test (DFA) were enrolled. Nasal washings were scheduled right away, then every three days until day 14, every 7 days until day 28 and every 2 weeks until a maximum of 16 weeks, or until the first DFA negative RSV specimen. The relationship between host factors, illness severity and viral shedding was investigated using Cox regression methods.ResultsFrom 151 families a total of 193 children were enrolled with a median age of 21 months (range 1-164 months), 10% infants and 46% male. The rate of recovery from infection was 0.22/person/day (95% CI 0.19-0.25) equivalent to a mean duration of shedding of 4.5 days (95%CI 4.0-5.3), with a median duration of shedding of 4 days (IQR 2-6, range 1-14). Children with a history of RSV infection had a 40% increased rate of recovery i.e. shorter duration of viral shedding (hazard ratio 1.4, 95% CI 1.01-1.86). The rate of cessation of shedding did not differ significantly between males and females, by severity of infection or by age.ConclusionWe provide evidence of a relationship between the duration of shedding and history of infection, which may have a bearing on the relative role of primary versus re-infections in RSV transmission in the community.

Virological investigation of four outbreaks of influenza B reassortants in the northern region of Taiwan from October 2006 to February 2007

BackgroundFrom October 2006 to February 2007, clinical specimens from 452 patients with symptoms related to respiratory tract infection in the northern region of Taiwan were collected. Real-time PCR and direct immunofluorescent antibody tests showed that 145 (32%) patients had influenza B virus infections. Subsequently, nucleotide sequence analyses of both hemagglutinin (HA) and neuraminidase (NA) genes of 39 isolates were performed. Isolated viruses were antigenically characterized using hemagglutinin inhibition (HI) test.FindingsPhylogenetic tree analysis showed that all the isolates belonged to the B reassortant lineage with HA gene belonged to the B/Victoria/2/87 lineage and the NA gene belonged to the B/Yamagata/16/88 lineage. In addition, a group of children aged between 6 to 8 years old resided in Yilan county were infected with a variant strain. Hemagglutinin inhibition (HI) tests confirmed that all the reassortant influenza B viruses were B/Malaysia/2506/04-like viruses. Pre- and post-immunized serum samples from 4 normal volunteers inoculated with 2007 influenza vaccine were evaluated for their HI activity on 6 reassortant B isolates including two variants that we found in the Yilan county. The results demonstrated that after vaccination, all four vaccinees had at least 4-fold increases of their HI titers.ConclusionThe results indicate that the 2006–2007 seasonal influenza vaccine was effective in stimulating protective immunity against the influenza B variants identified in Yilan county. Continuous surveillance of emerging influenza B variants in the northern region of Taiwan is important for the selection of proper vaccine candidate in the future.