Indirect Fluorescent Antibody Assay Research Articles

High seroprevalence of Babesia antibodies among Borrelia burgdorferi-infected humans in Sweden

In northern Europe, tick-borne diseases such as Lyme borreliosis (LB) and tick-borne encephalitis (TBE) are well known. The actual incidence of Babesia infections, however, has remained elusive. In this study, the prevalence of antibodies against two Babesia spp. was investigated in a cohort of patients that were seropositive for Borrelia (B.) burgdorferi sensu lato (s.l.). Data were compared to a control group of healthy individuals. Sera were collected from 283 individuals residing in the southernmost region of Sweden, Skåne County. Almost one third of the sera were from patients with a confirmed seropositive reaction against B. burgdorferi s.l. All sera samples were assessed for IgG antibodies against Babesia (Ba.) microti and Ba. divergens by indirect fluorescent antibody (IFA) assays. Seropositive IgG titers for at least one of the Babesia spp. was significantly more common (p < 0.05) in individuals seropositive for Borrelia (16.3%) compared to the healthy control group (2.5%). Our findings suggest that Babesia infections may indeed be quite common among individuals who have been exposed to tick bites. Furthermore, the results indicate that human babesiosis should be considered in patients that show relevant symptoms; particularly for splenectomized and other immunocompromised individuals. Finally, the data challenges current blood transfusion procedures and highlights the current lack of awareness of the parasite in northern Europe.

THE DYNAMICS OF ORTHOHANTAVIRUS HANTAAN STRAINS REPLICATION ON THE MODEL OF MOUSE PERITONEAL MACROPHAGES

Orthohantaviruses (Orthohantavirus genus, Hantaviridae family) are the causative agents of a widespread natural focal infection in the Russian Federation, hemorrhagic fever with renal syndrome (HFRS). An important role in the persistence of orthohantavirus in reservoir hosts among other immunological responses, as well as in the spread of the virus in the infected organism, is played by infected macrophages, which, along with the vascular endothelium, are the main targets for orthohantaviruses.The aim of our study was to investigate the characteristics of replication of orthohantavirus Hantaan strains isolated from Apodemus mice and detect the influence of different values of multiplicity of infection (MOI) on replication dynamics of orthohantaviruses on cell culture.Materials and methods. We used 4 strains of Hantaan virus, isolated from A. agrarius (n = 2) and A. peninsulae (n = 2), captured in the different areas of Primorsky Krai of Russia. The modeling of infection was performed on the primary cell culture of mouse peritoneal macrophages with different MOI (from 10 to 0.1). The assessing of infection was conducted via indirect fluorescent antibody assay, and results were expressed as rate of antigen- positive cells per all cells in the field of vision. Results. Common dynamics of orthohantavirus infection on this in vitro model was characterized by periodically increased rates of infected cells after 2, 4, 6 и 8 hours post infection (p.i.). Replication of A. agrarius-borne strains was more intensive compare with viruses, isolated from A. peninsulae, in the time point 4 hour p.i. on the background the same MOI the statistically significant difference of rate of antigen-positive cell 24.9 ± 2.38 % vs 15.2 ± 1.87 % (t = 3.20; p = 0.001414) was observed. Additionally, the decrease of MOI was followed by determined decrease of replication effectivity.Conclusion. The results of our study showed the significant phenotyping heterogeneity of orthohantavirus Hantaan strains, isolated from Apodemus mice, resulting in different rates of replication in the culture of mouse peritoneal macrophages.

Visceral leishmaniasis: clinical and therapeutic aspects

Introduction : visceral leishmaniasis (VL) is endemic in areas bordering the Mediterranean Sea, including Tunisia. It represents a major public health problem not only in developing countries but also in developed ones. It is caused by Leishmania infantum and it is transmitted by the bite of hematophagous sand fly belonging to phlebotomus spp; dog constitutes the main reservoir of the infection. Methods : systemic review of the national and international medical literature concerning this disease. Results : clinical presentation of VL ranges from asymptomatic or subclinical infection to severe and complicated symptomatic disease. Classical manifestations of visceral leishmaniasis include chronicfever, pallor and splenomegaly. Pancytopenia is present very often. Rarely, it may be complicated by hemophagocytic lymphohistiocytosis (HLH) that has varied presentation and course. Diagnosis is established by serological tests (indirect fluorescent-antibody assay, immunoassay test, indirect hemagglutination assay) and by demonstration of Leishmania parasites by microscopy or polymerase chain reaction (PCR) in the bone marrow aspirates. Visceral Leishmaniasis is fatal if left untreated. Although pentavalent antimonial drugs have been used for many decades as standard treatment for VL, newer therapeutic options are nowadays considered the first-line treatment in many countries. They include conventional or liposomal amphotericin B, paromomycin and miltefosine. Liposomal amphotericin B seems to be the most tolerated drug. Unfortunately, it is not available in Tunisia. Recently, an international scientific seminar has been held in Pasteur institute of Tunis from the 2nd-4th April 2015, about visceral leishmaniasis in Maghreb. Three recommendations have been considered by the experts attending this international scientific seminar. Firstly, the necessity to decrease the delay between onset of symptoms and getting a proper diagnosis This aim can be reached by: The training of medical and nursing staff and education of high risk population, the use of rapid diagnostic tests in area with a high incidence of VL, the use of real time PCR on blood samples for diagnosis of VL to avoid marrow aspiration. Secondly, the usefulness of liposomal amphotericin as first-line therapeutic option in management of visceral leishmaniasis. The experts participating in the international scientific seminar agreed on the need to replace the meglumine antimoniate by liposomal amphotericin as first-line therapeutic option in Maghreb countries. This new drug will shorten considerably the duration of therapeutic cures and improve the effectiveness of the treatment and its safety. For immunocompromised patients (HIV(+), Infection and Solid Organ Transplant Recipients), it was strongly recommended to implement a post treatment long term follow-up because of the risk of relapse. And thirdly, the urgent need of fighting against the zoonosis by the control of reservoir host (dog) and the vector (sandflies). The actions include: The eradication of stray and semi-stray dogs, the early detection of canine leishmaniasis, the prevention of canine leishmaniasis (insecticide-impregnated dog collars), the setting up of clean environment (garbage control, whitewashing the walls, nets). Conclusion : despite many previous studies realized on Leishmaniases in Maghreb, they are still considered as neglected diseases because of absence of efficient control strategies. Poor vector control strategies, limited diagnostic services, drugs, treatments and lack of community awareness are the most important challenges in VL control and elimination especially in endemic areas such as Tunisia. Absence of highly sensitive and specific tests, lack of trained man power, and community awareness are the major challenges in VL control. Therefore, proper case diagnosis, community mobilization and launching of

Preparation of a Monoclonal Antibody Against gD Protein of Bovine Herpesvirus I.

The two DNA fragments encoding predicted main antigenic regions, aa 20-160 and aa 257-344 on gD protein of bovine herpesvirus-1 (BoHV-1) were tandem-cloned into the vector pET28a. The recombinant His-tagged Δ gD1-Δ gD2 was expressed in Escherichia coli BL21(DE3)pLysS by induction with 0.5 mM isopropyl-1-thio-β-D-galactoside (IPTG) and Western blot analysis demonstrated that the recombinant His-tagged Δ gD1-Δ gD2 reacted with the positive serum against BoHV-1. A monoclonal antibody (MAb), designated as 2B6, was prepared by fusion of SP2/0 myeloma cells with splenocytes from a female 8-week-old BALB/c mouse immunized with purified His-tagged Δ gD1-Δ gD2 protein with the addition of Freund's adjuvant. The titer of the ascitic fluid triggered by hybridoma cells secreting MAb 2B6 was 1:2.5 × 108 and the subtype of MAb 2B6 was IgG 2a/κ. MAb 2B6 positively recognized with BoHV-1 infected Madin-Darby bovine kidney cells by an indirect fluorescent antibody assay. Moreover, MAb 2B6 showed 1:160 viral neutralizing activity using 60% plaque reduction assay. Therefore, this work suggested that MAb 2B6 has potential in the diagnosis of bovine respiratory disease complex associated with BoHV-1 and function analysis.

Murine Typhus-Related Acute Febrile Hepatitis: A Case Series

Murine typhus is a flea-borne zoonosis caused by Rickettsia typhi (R. typhi). Acute hepatitis accompanying this infection has not been well emphasized in the literature. We describe two patients with febrile hepatitis due to rickettsial infection. Case 1 A 62 year old (y.o.) male with an unremarkable past medical history presented with high grade fever. A thorough review of systems was unremarkable for rash, arthralgias, headache, abdominal pain, back pain or cough. Physical examination was notable for a temperature of 102.2 degrees Fahrenheit (F). Laboratory findings are noted in the attached table. Computed tomography of the abdomen revealed no intra-abdominal pathology. Serological assays for hepatitis A, B,C, Cytomegalovirus, Epstein Barr virus and Chikungunya virus were negative. An indirect fluorescent antibody (IFA) assay showed immunoglobulin M (IgM) antibodies against R. typhi with a titer of 1:64. A liver biopsy demonstrated mild hepatitis with portal and lobular infiltration by mixed inflammatory cells, apoptotic hepatocytes, edematous portal tracts, and Kupffer cell hyperplasia. He was treated with Doxycycline for 2 weeks and is due for follow-up.Figure: Case 1.Figure: Case 2.Figure: Laboratory values of patients on admission to our institution. WBC= White Blood Cells; AST= Aspartate Transaminase; ALT=Alanine Transaminase; ALP= Alkaline Phosphatase; T.BR= Total Bilirubin; PT=Prothrombin time.Case 2: A 50 y.o. male state trooper with no prior history of liver disease presented with high grade fever, headache, myalgias and polyarthralgias. On admission, he was febrile with a temperature of 104 F. Scleral icterus and a diffuse faint erythematous macular rash on the upper torso were present. Abdominal examination was remarkable for splenomegaly. Laboratory data are provided in the attached table. Ultrasound of the abdomen was notable for splenomegaly measuring 14 cm in largest dimension. An extensive infectious workup was negative except for a positive IgM antibody titer against R.typhi of 1:512. A liver biopsy showed acute mild hepatitis with an inflammatory infiltrate comprised of histiocytes, lymphocytes, plasma cells, and eosinophils. He was treated with Doxycyline for 2 weeks and lost to follow-up. Discussion: Rickettsial infections may have liver involvement and should be considered in the differential diagnosis of acute hepatitis. These cases highlight the role of a liver biopsy in the evaluation of acute febrile hepatitis. To our knowledge, this is the first case series reported in the United States with serologic diagnosis of murine typhus and accompanying acute hepatitis confirmed on liver biopsy.

Unreliability of three commercial Coxiella burnetii phase II IgM ELISA kits for the seroscreening of acute Q fever in human cases.

Background & objectives:Seroprevalence of Q fever (QF) caused by Coxiella burnetii has been reported from different parts of India. Usually serological/molecular tests are employed for detection of infection. The present study was undertaken to verify the validity of three different QF phase II IgM ELISA kits for acute QF diagnosis by comparing with the gold standard indirect fluorescent antibody assay (IFA).Methods:Fifty eight serum samples collected from 42 patients (26 patients provided acute sample only and 16 both acute and convalescent samples) which were examined by all three commercial kits, were cross-checked with QF Phase II IgM IFA for confirmation.Results:Eleven patients were positive for C. burnetii antibodies by IFA in acute and/or convalescent serum samples. Taking IFA as a reference, percentages of sensitivity, specificity, positive predictive value and negative predictive value for Virion-Serion/Vircell/NovaTec were 36.36, 61.29, 25.00, 73.08; 81.82, 35.48, 31.03, 84.62 and 100, 25.81, 32.35, 100 per cent, respectively.Interpretation & conclusions:The three different ELISA kits exhibited poor agreement amongst them and unacceptable level of false positivity. IFA remains to be the only option for diagnosing acute QF. Discrepancy between the clinical findings and IFA/ELISA results needs confirmation by C. burnetii DNA detection in real-time polymerase chain reaction.

Indirect ELISA (iELISA) for routine detection of antibodies against Minute Virus of Mice (MVM) in mice colonies

In this study we developed an indirect ELISA to detect antibodies against Minute Virus of Mice (MVM) using an antigen produced from BHK-21 cells infected with a prototype strain of the virus. The optimal antigen concentration and serum dilutions were established. In order to analyze variability in the laboratory, reproducibility and repeatability within and between plates were determined. Then, a panel of 460 sera from conventional facilities and previously classified as positive or negative by the indirect fluorescent antibody assay was analyzed. The cutoff value was determined by a receiver operating characteristic (ROC) curve. The results of the indirect ELISA were compared with those of the indirect fluorescent antibody assay. The ELISA assay showed 100% sensitivity and 99% specificity. ELISA is a useful tool to be developed in standard virology laboratories and can be used for screening animals faster than the traditional indirect fluorescent antibody assay.

Cultivation of Anaplasma ovis in the HL-60 human promyelocytic leukemia cell line

The tick-borne bacterium Anaplasma ovis is a widely distributed pathogen affecting sheep, goats and wild ruminants. Here, the HL-60 human promyelocytic leukemia cell line was used to isolate A. ovis from PCR-positive sheep and goats in Heilongjiang Province, China. Two weeks after inoculation, morulae were observed in cytoplasmic vacuoles in four different HL-60 cultures. Confocal microscopy using a Cy3-labeled A. ovis-specific probe confirmed that the HL-60 cells were infected with A. ovis. Cells from the 6th HL-60 subculture displayed positive fluorescence when incubated with A. ovis antiserum in the indirect fluorescent antibody assay. PCR amplification and sequencing of 16S rRNA, groEL, gltA, msp2 and msp4 Anaplasma genes revealed that the four A. ovis culture isolates were identical. Phylogenetic analysis showed that the sequences clustered with other A. ovis strains but could clearly be distinguished from other Anaplasma species. When the 18th subculture of infected HL-60 cells was examined by electron microscopy, lysosomes were often observed near the vacuoles. After the 24th subculture, Giemsa staining and PCR indicated that the HL-60 cells were negative for A. ovis. Although A. ovis can infect HL-60 cells for only four months, the ability of the organism to infect and multiply in HL-60 cells provides a tool to study intra-erythrocytic Anaplasma and host cell interactions.Emerging Microbes & Infections (2017) 6, e83; doi:10.1038/emi.2017.70; published online 20 September 2017

The utility of a country-specific Bartonella henselae antigen in an IgM-indirect fluorescent antibody assay for the improved diagnosis of cat scratch disease

Bartonella henselae strains genetically differ among nations. The utility of Japanese-specific YH-01 strain was investigated in developing indirect fluorescence antibody assay (IFA) for IgM in comparison with conventional IFA employing Houston-1 strain by testing 100 Japanese patients suspected of cat scratch disease. The country-specific IFA greatly improved the accuracy of diagnosis.

Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens.

The African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study.

Feasibility of Using Convalescent Plasma Immunotherapy for MERS-CoV Infection, Saudi Arabia.

We explored the feasibility of collecting convalescent plasma for passive immunotherapy of Middle East respiratory syndrome coronavirus (MERS-CoV) infection by using ELISA to screen serum samples from 443 potential plasma donors: 196 patients with suspected or laboratory-confirmed MERS-CoV infection, 230 healthcare workers, and 17 household contacts exposed to MERS-CoV. ELISA-reactive samples were further tested by indirect fluorescent antibody and microneutralization assays. Of the 443 tested samples, 12 (2.7%) had a reactive ELISA result, and 9 of the 12 had reactive indirect fluorescent antibody and microneutralization assay titers. Undertaking clinical trials of convalescent plasma for passive immunotherapy of MERS-CoV infection may be feasible, but such trials would be challenging because of the small pool of potential donors with sufficiently high antibody titers. Alternative strategies to identify convalescent plasma donors with adequate antibody titers should be explored, including the sampling of serum from patients with more severe disease and sampling at earlier points during illness.

Production and characterization of VP6-specific monoclonal antibodies against bovine group C rotavirus

Rotaviruses are enteric pathogens causing acute watery dehydrating diarrhea in humans and animals. The importance of group C rotavirus (GpC-RV) infections has not been established as the studies on the GpC-RV have been hampered by the lack of an in vitro culture system. However, diarrheal diseases associated with GpC-RV have been gradually increasing worldwide. In this study, VP6 gene of bovine GpC-RV Korean isolate was expressed, and monoclonal antibodies (mAbs) against VP6 were produced and characterized. The VP6 gene was cloned and expressed based on a baculovirus expression system. Indirect fluorescence antibody (IFA), polymer chain reaction (PCR), and Western blot assays were used to confirm expression of VP6 gene synthesized by the recombinant baculovirus. Eleven mAbs against VP6 were produced using expressed VP6. Cross-reactivity of the mAbs was assessed with recombinant VP6 proteins from porcine GpC-RV and human GpA-RV, or different serotypes of group A rotavirus strains by IFA test. Some mAbs reacted with intact porcine GpC-RV Cowden strain as well as bovine GpC-RV VP6 recombinant baculoviruses, but not with human and animal GpA-RV strains. The VP6-specific mAbs might be useful to develop immunodiagnostic tests such as rapid diagnostic kit, IFA and enzyme-linked immunosorbent assay (ELISA) for detection of GpC-RV.

Evaluation of serological cross-reactivity and cross-neutralization between the United States porcine epidemic diarrhea virus prototype and S-INDEL-variant strains.

BackgroundAt least two genetically different porcine epidemic diarrhea virus (PEDV) strains have been identified in the United States (U.S. PEDV prototype and S-INDEL-variant strains). The current serological assays offered at veterinary diagnostic laboratories for detection of PEDV-specific antibody are based on the U.S. PEDV prototype strain. The objectives of this study were: 1) isolate the U.S. PEDV S-INDEL-variant strain in cell culture; 2) generate antisera against the U.S. PEDV prototype and S-INDEL-variant strains by experimentally infecting weaned pigs; 3) determine if the various PEDV serological assays could detect antibodies against the U.S. PEDV S-INDEL-variant strain and vice versa.ResultsA U.S. PEDV S-INDEL-variant strain was isolated in cell culture in this study. Three groups of PEDV-negative, 3-week-old pigs (five pigs per group) were inoculated orally with a U.S. PEDV prototype isolate (previously isolated in our lab), an S-INDEL-variant isolate or virus-negative culture medium. Serum samples collected at 0, 7, 14, 21 and 28 days post inoculation were evaluated by the following PEDV serological assays: 1) indirect fluorescent antibody (IFA) assays using the prototype and S-INDEL-variant strains as indicator viruses; 2) virus neutralization (VN) tests against the prototype and S-INDEL-variant viruses; 3) PEDV prototype strain whole virus based ELISA; 4) PEDV prototype strain S1-based ELISA; and 5) PEDV S-INDEL-variant strain S1-based ELISA. The positive antisera against the prototype strain reacted to and neutralized both prototype and S-INDEL-variant viruses, and the positive antisera against the S-INDEL-variant strain also reacted to and neutralized both prototype and S-INDEL-variant viruses, as examined by IFA antibody assays and VN tests. Antibodies against the two PEDV strains could be detected by all three ELISAs although detection rates varied to some degree.ConclusionsThese data indicate that the antibodies against U.S. PEDV prototype and S-INDEL-variant strains cross-reacted and cross-neutralized both strains in vitro. The current serological assays based on U.S. PEDV prototype strain can detect antibodies against both U.S. PEDV strains.

Development of a Highly Specific IgM Enzyme-Linked Immunosorbent Assay for Bartonella henselae Using Refined N-Lauroyl-Sarcosine-Insoluble Proteins for Serodiagnosis of Cat Scratch Disease.

The conventional anti-Bartonella henselaeIgM enzyme-linked immunosorbent assay (IgM-ELISA) methods for diagnosing cat scratch disease (CSD) remain poor in both sensitivity and specificity. We sought to develop an IgM-ELISA with improved accuracy in the serodiagnosis of CSD by exploring the antigens that are most suitable for an ELISA. We prepared 5 different protein antigens: antigen I (sonicatedB. henselaewhole-cell antigen), antigen II (N-lauroyl-sarcosine-insoluble antigen), antigen III (processed sarcosine-soluble antigen), and antigen IV and antigen V (sarcosine-insoluble and sarcosine-soluble antigens refined by DEAE-Sepharose Fast Flow ion-exchange chromatography). The IgM antibodies in the sera of 47 patients with clinically suspected CSD (24 definite, 23 suspected) and of 85 healthy individuals were examined by ELISAs using the 5 antigens, and the results were compared with those of an IgM indirect fluorescent antibody assay (IgM-IFA). In a reference panel, which consisted of 5 positive and 5 negative sera, antigen I and antigen III failed to distinguish between the two statuses, whereas the other three antigens succeeded in distinguishing between them. When the cutoff value was set at the 98th percentile of the ELISA index for healthy individuals, the sensitivity of IgM-IFA for the 24 cases of definite CSD was 54%, whereas the sensitivities of the IgM-ELISAs with antigen II, IV, and V were 75%, 83%, and 75%, respectively. The sensitivities of these three IgM-ELISAs for all 47 of the clinically suspected cases were 49%, 64%, and 51%, respectively. In contrast, the sensitivity of IgM-IFA was 28%. These results indicate that the refined sarcosine-insoluble proteins (antigen IV), which possessed the highest specificity among the 5 antigens, are the most appropriate for developing an IgM-ELISA for the highly specific serodiagnosis of CSD.

A novel combination of fipronil and permethrin (Frontline Tri-Act®/Frontect®) reduces risk of transmission of Babesia canis by Dermacentor reticulatus and of Ehrlichia canis by Rhipicephalus sanguineus ticks to dogs.

BackgroundThe ability of Frontline Tri-Act®/Frontect®, a topical ectoparasiticide containing fipronil and permethrin for dogs, to prevent the transmission of Babesia canis as well as Ehrlichia canis was evaluated by infesting dogs with infected vector ticks.MethodsFor the Babesia canis study, 16 dogs were randomly allocated to two groups. Eight dogs were treated on day 0 with a topical spot-on formulation containing 6.76 % w/v fipronil plus 50.48 % w/v permethrin and eight dogs served as the untreated control group. Dermacentor reticulatus ticks, with a B. canis infection rate ranging between 2 and 10 %, were placed onto dogs on days 7, 14, 21 and 28. In situ tick counts were performed on Days 9, 16 and 23. Ticks were counted and removed on Day 30. Infection of the dogs with B. canis was monitored by rectal temperature readings, clinical examinations and blood smears as well as PCR and IFA (indirect fluorescent antibody assay).For the Ehrlichia canis study, another 16 dogs were allocated to two groups. Eight dogs were treated with the fipronil and permethrin combination on days 0 and 28 and eight dogs served as untreated controls. Rhipicephalus sanguineus ticks, carrying an infection rate of 13 % for E. canis, were released in the sleeping kennels of the dogs on days 7, 14, 21, 28, 35, 42, 49 and 56. Ticks were counted in situ on the dogs on a weekly basis. All ticks were removed and counted on the final assessment day 58. Infection of the dogs with E. canis was monitored by rectal temperature, clinical examinations, and testing of blood samples by PCR, IFA and platelet counts.ResultsB. canis was transmitted by D. reticulatus ticks to all eight untreated control dogs and to one treated dog, which was confirmed by blood smears, PCR and IFA. E.canis was transmitted by R. sanguineus ticks to all eight untreated control dogs. Two of the dogs in the treated group were found positive based on PCR and/or IFA.ConclusionsFrontline Tri-Act®/Frontect® significantly lowered the risk for dogs to acquire a B. canis infection by 87.5 % over a challenge period of 28 days. The risk for dogs to acquire E. canis was reduced by 75 % over a period of 56 days.

The health status of a village population, 7 years after a major Q fever outbreak.

From 2007 to 2010, The Netherlands experienced a major Q fever outbreak with more than 4000 notifications. Previous studies suggested that Q fever patients could suffer long-term post-infection health impairments, especially fatigue. Our objective was to assess the Coxiella burnetii antibody prevalence and health status including fatigue, and assess their interrelationship in Herpen, a high-incidence village, 7 years after the outbreak began. In 2014, we invited all 2161 adult inhabitants for a questionnaire and a C. burnetii indirect fluorescence antibody assay (IFA). The health status was measured with the Nijmegen Clinical Screening Instrument (NCSI), consisting of eight subdomains including fatigue. Of the 70·1% (1517/2161) participants, 33·8% (513/1517) were IFA positive. Of 147 participants who were IFA positive in 2007, 25 (17%) seroreverted and were now IFA negative. Not positive IFA status, but age <50 years, smoking and co-morbidity, were independent risk factors for fatigue. Notified participants reported significantly more often fatigue (31/49, 63%) than non-notified IFA-positive participants (150/451, 33%). Although fatigue is a common sequel after acute Q fever, in this community-based survey we found no difference in fatigue levels between participants with and without C. burnetii antibodies.

Development of an indirect ELISA, blocking ELISA, fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of Porcine Epidemic Diarrhea Virus.

BackgroundRecent, severe outbreaks of porcine epidemic diarrhea virus (PEDV) in Asia and North America highlight the need for well-validated diagnostic tests for the identification of PEDV infected animals and evaluation of their immune status to this virus. PEDV was first detected in the U.S. in May 2013 and spread rapidly across the country. Some serological assays for PEDV have been previously described, but few were readily available in the U.S. Several U.S. laboratories quickly developed indirect fluorescent antibody (IFA) assays for the detection of antibodies to PEDV in swine serum, indicating prior exposure. However, the IFA has several disadvantages, including low throughput and relatively subjective interpretation. Different serologic test formats have advantages and disadvantages, depending on the questions being asked, so a full repertoire of tests is useful. Therefore, the objective of this study was to develop and validate multiple improved serological assays for PEDV, including an indirect ELISA (iELISA); a highly specific monoclonal antibody-based blocking ELISA (bELISA); fluorescent microsphere immunoassays (FMIA) that can be multiplexed to monitor exposure to multiple antigens and pathogens simultaneously; and a fluorescent focus neutralization assay (FFN) to measure functional virus neutralizing antibodies.ResultsA recombinant North American nucleoprotein (NP) based iELISA was developed and validated along with a bELISA using newly developed PEDV-NP specific biotinylated monoclonal antibodies (mAbs) and an FMIA using magnetic beads coupled with expressed NA PEDV-NP. Receiver operating characteristic (ROC) analysis was performed using swine serum samples (iELISA n = 1486, bELISA n = 1186, FMIA n = 1420). The ROC analysis for the FMIA showed estimated sensitivity and specificity of 98.2 and 99.2 %, respectively. The iELISA and bELISA showed a sensitivity and specificity of 97.9 and 97.6 %; and 98.2 and 98.9 %, respectively. Inter-rater (kappa) agreement was calculated to be 0.941 between iELISA and IFA, 0.945 between bELISA and IFA and 0.932 between FMIA and IFA. Similar comparative kappa values were observed between the iELISA, bELISA and FMIA, which demonstrated a significant level of testing agreement among the three assays. No cross-reactivity with the closely related coronaviruses, transmissible gastroenteritis virus (TGEV) or porcine respiratory coronavirus (PRCV) was noted with these assays. All three assays detected seroconversion of naïve animals within 6–9 days post exposure. The FFN assay allows relative quantitation of functional neutralizing antibodies in serum, milk or colostrum samples.ConclusionWell-validated iELISA, bELISA and FMIA assays for the detection of PEDV antibodies were developed and showed good correlation with IFA and each other. Each assay format has advantages that dictate how they will be used in the field. Newly developed mAbs to the PEDV-NP were used in the bELISA and for expediting FFN testing in the detection and quantitation of neutralizing antibodies. In addition, these PEDV mAbs are useful for immunohistochemistry, fluorescent antibody staining and other antigen-based tests. Measurement of neutralizing antibody responses using the FFN assay may provide a valuable tool for assessment of vaccine candidates or protective immunity.

Virus, strain, and epitope specificities of neutralizing bovine monoclonal antibodies to bovine herpesvirus 1 glycoproteins gB, gC, and gD, with sequence and molecular model analysis

Three bovine monoclonal antibodies (BomAb) raised to bovine herpesvirus (BoHV) 1.1 and specific for the viral glycoproteins gB, gC, and gD were tested for reactivity to two isolates of BoHV-1.1, one of BoHV-1.2, and two of BoHV-5 in virus neutralization and indirect fluorescent antibody assays. They were also tested with other herpesviruses infecting cattle and other mammalian alphaherpesviruses, and found negative or of negligible reactivity. Their BoHV-1.1 epitope specificity was examined using competitive ELISA with peroxidase-labeled murine monoclonal antibodies (MumAb) that had been previously characterized. To explain the incongruities observed, the amino acid sequences of the epitopes and adjacent regions of BoHV-1.1, 1.2, and 5 were compared, and molecular modeling was performed using human herpesvirus 1 glycoprotein crystals as templates. The anti-gB BomAb reacted strongly with BoHV-1.1 and BoHV-1.2, and poorly or not at all with BoHV-5. It competed with a MumAb specific for a BoHV-1.1 gB epitope previously shown to only partially cross-react between BoHV-1 and BoHV-5. BoHV-5 gB has nearly identical sequence with BoHV-1.1 in the epitope region, but modeling suggested the lack of cross-reactivity of the MumAb was due to masking of the epitope in BoHV-5 by an adjacent region, which has significant sequence differences between BoHV-1.1 and BoHV-5. The BomAb reactivity could also be explained by masking, or by reactivity with the adjacent region. The anti-gC BomAb reacted strongly with one isolate of BoHV-1.1 and BoHV-1.2, less well with a heterologous isolate of BoHV-1.1, and poorly or not at all with BoHV-5. It did not compete with any of the anti-gC MumAb tested, but a target domain was suggested by BoHV-1.1, 1.2, and 5 sequence divergence. The anti-gD BomAb reacted strongly with all BoHV-1.1, 1.2, and 5 isolates tested. However, it competed with two MumAb previously shown to not cross-react between BoHV-1.1 and BoHV-5. Sequence analysis and modeling suggested the cross-reactivity of the anti-gD BomAb was due to it reacting with an epitope-adjacent region or regions conserved between BoHV-1.1 and BoHV-5, but not with other alphaherpesviruses. The results suggest the usefulness of combining in vitro biological data with sequence or structure modeling data to investigate important epitopes involved in immunity to infectious agents.

Investigation of Pneumocystis jirovecii colonization in patients with pulmonary diseases

The detection of Pneumocystis jirovecii or its DNA in respiratory samples from individuals who do not have signs or symptoms of pneumonia has been defined as colonization. In this study, we aimed to investigate the prevalence of P. jirovecii colonization in patients with various lung diseases. Thirty patients who were followed-up and who had undergone bronchoscopy for diagnosis of different underlying diseases or pulmonary signs were included in the study. Bronchoalveolar lavage (BAL) fluids of these patients were analyzed with nPCR amplification of the mt-LSUrRNA gene of P. jirovecii. In addition to nPCR, giemsa, Gomori's methenamine silver (GMS), and indirect fluorescence antibody (IFA) staining assays were applied to all samples. P. jirovecii DNA was detected in 21 of 30 (70%) BAL samples by nPCR. However, P. jirovecii cysts were found in 1 of 21 nPCR-positive samples by giemsa and GMS. IFA assay showed six samples to be positive, but only four of them were found to be positive by nPCR. Result of our study showed that prevalence of P. jirovecii colonization is particularly high in patients with chronic pulmonary diseases, and nPCR was a good assay for evaluation of the colonization of P. jirovecii.

Prevention of Babesia canis in dogs: efficacy of a fixed combination of permethrin and fipronil (Effitix®) using an experimental transmission blocking model with infected Dermacentor reticulatus ticks.

BackgroundTwo experimental studies using a transmission blocking model with Dermacentor reticulatus ticks infected with Babesia canis were performed to test the ability of Effitix® to prevent the transmission of babesiosis in dogs.MethodsFour groups of seven dogs (experiment 1) and one group of eight dogs (experiment 2) were treated topically with a novel combination of fipronil and permethrin in a spot-on formulation (Effitix®, Virbac) respectively 28, 21, 14 and 7 days (experiment 1) and 2 days (experiment 2) prior to tick infestation. In each study, a control group of seven dogs (experiment 1) and eight dogs (experiment 2) remained untreated. On day 0, all dogs were infested with adult D.reticulatus ticks harboring B. canis. An efficacy failure (successfully infected) was regarded as a dog in the treated groups that was tested serologically positive for B.canis antibodies, using an indirect fluorescent antibody (IFA) assay and tested positive for B.canis by DNA-assay using PCR analysis.ResultsB.canis was transmitted by D.reticulatus to all untreated dogs (experiment 1) and six untreated dogs out of eight (experiment 2) as confirmed by IFA and PCR assays. The large majority of treated dogs (92.9% in experiment 1 and 100% in experiment 2) remained sero-negative over the challenge period.ConclusionsThe treatment of dogs with Effitix® applied 2 to 28 days prior to infestation with D. reticulatus harboring B.canis, successfully prevented the transmission of canine babesiosis.