Immunocapture ELISA Research Articles

Prevalence of peste des petits ruminants virus antibodies in sheep and goats sera from Central-Western Sudan.

Clinical signs suggestive of peste des petits ruminants (PPR) involved herds of small ruminants, which were described elsewhere in Sudan. Peste des petits ruminants was confirmed using an Immunocapture ELISA (IC-ELISA) assay in samples of infected and dead animals in areas of outbreaks. Therefore, to update information regarding the current situation and for assessment of the serological prevalence of PPR in small ruminants mingled at Central and Western Sudan during 2018-2019, 368 sera were collected from sheep (325 sera) and goats (43 sera) with different ages and breeds. These sera included 186 sera (173 sheep and 13 goats) from White Nile State and 182 sera (152 sheep and 30 goats) from Kordofan States. Competitive ELISA demonstrated higher prevalence of PPRV antibodies of 88.9%, 90.7% and 88.6% in both sheep and goats, goats, and sheep sera, respectively. Moreover, 100%, 94.7% and 78.5% seroprevalence values were demonstrated in South Kordofan, North Kordofan and White Nile States. The higher seroprevalence values detected in sera of unvaccinated sheep and goats indicated the wide exposure of these animals to PPRV and presence of protection following PPR viral infection. The findings of the study indicated that PPR is endemic in the surveyed areas of Sudan.Contribution:The study will contribute effectively to the global eradication programme of PPR organised by the World Organization for Animal Health (WOAH, formerly OIE) and Food and Agriculture Organization (FAO). To completely eliminate PPR from Sudan by 2030, local efforts should be directed towards effectively and wholly vaccinating small ruminants using PPRV vaccine especially in routes of seasonal animal's movement and shared grazing areas.

Diagnosis and Control of Peste des Petits Ruminants Disease in Small Ruminants: A Review

Peste des Petits Ruminants (PPR) is an acute highly contagious febrile disease of sheep and goats characterized by erosive and necrotizing stomatitis and associated with severe pneumo-enteritis and bronchopneumonia ended by recovery or death. The aim of the present study was to throw light on the diagnosis and control of PPR. Diagnosis of PPR depends on clinical signs, pathological lesions, and specific detection of the viral antigen, viral genome, or specific antibodies by serological tests and nucleic acid-based assays. The most commonly used diagnostic techniques are cell culture isolation, agar gel immunodiffusion, hemagglutination tests, immunocapture ELISA, and competitive ELISA. In addition to the abovementioned techniques, virus neutralization tests and reverse transcriptase PCR are used. Peste des Petits Ruminants is characterized by high fever associated with watery nasal and ocular discharges, mucopurulent stomatitis, and broncho-pneumonia. Moreover, severe bloody diarrhea and the disease associated with high levels of mortality reached up to 90%. The diagnosis of viral diseases is important in determining the control strategies. Therefore, it can be concluded that recent diagnostic tools are urgently needed not only for the diagnosis but also for following-up combating programs and control of viral diseases. Early and rapid complete identification of infectious viral agents in small ruminants as well as in the surrounding environment is recommended for effective control of PPR. The control program depends mainly on vaccination, hygiene and sanitation measures, and effective quarantine measures.

Proteomic and mitochondrial adaptations to early-life stress are distinct in juveniles and adults

Exposure to early-life stress (ELS) increases risk for poor mental and physical health outcomes that emerge at different stages across the lifespan. Yet, how age interacts with ELS to impact the expression of specific phenotypes remains largely unknown. An established limited-bedding paradigm was used to induce ELS in mouse pups over the early postnatal period. Initial analyses focused on the hippocampus, based on documented sensitivity to ELS in humans and various animal models, and the large body of data reporting anatomical and physiological outcomes in this structure using this ELS paradigm. An unbiased discovery proteomics approach revealed distinct adaptations in the non-nuclear hippocampal proteome in male versus female offspring at two distinct developmental stages: juvenile and adult. Gene ontology and KEGG pathway analyses revealed significant enrichment in proteins associated with mitochondria and the oxidative phosphorylation (OXPHOS) pathway in response to ELS in female hippocampus only. To determine whether the protein adaptations to ELS reflected altered function, mitochondrial respiration (driven through complexes II-IV) and complex I activity were measured in isolated hippocampal mitochondria using a Seahorse X96 Flux analyzer and immunocapture ELISA, respectively. ELS had no effect on basal respiration in either sex at either age. In contrast, ELS increased OXPHOS capacity in juvenile males and females, and reduced OXPHOS capacity in adult females but not adult males. A similar pattern of ELS-induced changes was observed for complex I activity. These data suggest that initial adaptations in juvenile hippocampus due to ELS were not sustained in adults. Mitochondrial adaptations to ELS were also exhibited peripherally by liver. Overall, the temporal distinctions in mitochondrial responses to ELS show that ELS-generated adaptations and outcomes are complex over the lifespan. This may contribute to differences in the timing of appearance of mental and physical disturbances, as well as potential sex differences that influence only select outcomes.

Development and Evaluation of an Immuno-Capture Enzyme-Linked Immunosorbent Assay to Quantify the Mycoplasma capricolum subsp. capripneumoniae (Mccp) Protein in Contagious Caprine Pleuropneumonia (CCPP) Vaccine

An effective contagious caprine pleuropneumonia (CCPP) vaccine is essential for the increased production of healthy goats in a cost-effective manner and the prevention of animal-to-animal transmission for both domestic animals and wildlife. Quality control of this vaccine ensures that a reliable supply of pure, safe, and potent batches is obtained. As part of this control, in vitro quantification of Mycoplasma capricolum subsp. capripneumoniae (Mccp) protein in the final vaccines is required before the CCPP vaccine undergoes batch release and certification. The current method used for quantification is based on the measurement of total protein using the bicinchonic acid (BCA) test. This method quantifies the total amount of protein in the vaccine including contaminant protein from media, which can lead to overestimation of the quantity of Mccp protein, resulting in reduced vaccine immunogenicity. An immuno-capture ELISA (ICE) was developed for specific detection and quantification of the Mccp antigen in the CCPP vaccine. As the ICE detects and measures the amount of antigen between two layers of antibodies, capture and detecting antibodies are required. Mouse monoclonal antibodies (mAbs) that detect the Mccp antigen were produced and characterized. One of these mAbs, Mccp-25, was used to develop the ICE as an unlabelled capture antibody and horseradish peroxidase conjugated detecting antibody. The ICE was standardized and evaluated using an internal reference sample, experimental CCPP vaccines and commercial CCPP vaccines. A comparison between the polymerase chain reaction (PCR) and ICE showed good correlation between the two assays. Also, an in vitro ICE method correlated well with an in vivo sero-conversion in goats that were vaccinated with selected test vaccines. The sensitivity of the ICE was estimated at 30 ng/ml.

An investigation on the prevalence of peste des petits ruminants in the camels of Sindh, Pakistan.

The present study investigated the status of peste des petits ruminants (PPR) for the first time in the camels of Pakistan. The samples were collected from the camel residing area of Sindh, Pakistan, and analyzed for breeds (Dhatti and Larri), districts (Tharparkar and Umerkot), age (young, adult, and old), and sexes (male and female). The sera samples (n = 200) were analyzed for the detection of antibodies using a competitive enzyme-linked immunosorbent assay (cELISA). Moreover, the nasal and fecal samples were screened for the PPR virus. Finally, the positive nasal and fecal samples were validated using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocapture enzyme-linked immunosorbent assay (Ic-ELISA). The cELISA results showed an overall prevalence of 8.5% PPR in the study area. The camels of Tharparkar (10.9%; 95% confidence interval (CI) 9.2-12.9) showed higher seroprevalence of PPR antibodies than those of Umerkot (5.5%; 95% CI 4.1-7.2). Moreover, the Larri breed exhibited slightly greater resistance against the disease, because the camels of Dhatti breed (9.0%; 95% CI 7.5-11.0) exhibited a numerically higher (p >0.05) seroprevalence of PPR in comparison with those of Larri breed (7.9%; 95% CI 6.4-9.9). Furthermore, the young and old camels were more susceptible to the disease attack, because the adults (6.3%; 95% CI 5.0-7.8) exhibited significantly (p < 0.05) lower prevalence rate than the young (9.2%; 95% CI 7.6-11.1) and old (10.3%; 95% CI 8.9-11.9) camels. Finally, the results of the Ic-ELISA and HA test established the 8.3 and 8.2% prevalence of PPR antigen in nasal and fecal material samples, respectively, while the RT-PCR results validated the seropositive animals. These findings confirmed that the prevalence of PPRV infection in the camels of the Sindh province of Pakistan hence urged the need to take effective measures for prevention and control of the disease.

First detection and genetic characterization of peste des petits ruminants virus from dorcas gazelles "Gazella dorcas" in theSudan, 2016-2017.

In May 2017, many free-ranging dorcas gazelles (Gazella dorcas) with suspected signs of peste des petits ruminants (PPR) were reported in Dinder National Park, South-Eastern Sudan. Peste des petits ruminants virus (PPRV) antigen and nucleic acid were detected in specimens from these gazelles using an immunocapture ELISA and a reverse transcription polymerase chain reaction (RT-PCR) assays. PPRV was also detected in four healthy semi-captive dorcas gazelles from two areas of Khartoum State. Phylogenetic analysis showed that these PPRV strains belonged to the lineage IV genotype. The present study demonstrates that gazelles are a potential wild small ruminant host for PPRV and may influence the epidemiology of PPR in theSudan.

Peste des Petits Ruminants: Aetiology, Pathology, Immunology, Disease Status in Africa, Diagnosis, Control, Prevention and Treatment: A Review

Peste des petits ruminants (PPR) is a disease of economic and veterinary importance leading to considerable economic losses. PPR affects small domestic and wild ruminants. Sheep and goats are the natural hosts of PPR but cattle, pigs, African buffaloes and camels are also affected by the PPR virus. Clinical signs seen are mainly fever, mucopurulent nasal and ocular discharges, cough, dyspnoea, gastroenteritis leading to severe diarrhoea. The post mortem lesions seen are congested lungs, congestion of gastrointestinal tract, especially the discontinuous streaks of congestion, which is referred to as Zebra stripes or Zebra markings, oedematous and congested retropharyngeal and mesenteric lymph nodes, linear haemorrhages in the intestinal mucosa and splenomegaly. PPR infection is characterized by a rise in packed cell volume (PCV), increase in haemoglobin concentration (HbC), leukopenia with lymphopenia. The serum alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase activities are elevated, blood urea nitrogen and creatinine concentrations are also elevated. The disease is also characterized by disseminated intravascular coagulopathy (DIC) evidenced by prolonged prothrombin time (PT), prolonged activated thromboplastin time (APTT), thrombocytopenia, and hypofibrinogenemia. In PPR infection, serum biomarkers of oxidative stress such as vitamins A, C, E and glutathione activity decreases while serum catalase, superoxide dismutase, glutathione reductase and xanthine oxidase increase. Diagnostic techniques include histopathology, virus isolation, immunocapture enzyme-linked immunosorbent assay (icELISA), Competitive Enzyme Linked Imunnosorbent Assay (cELISA) and Combined Indirect ELISA (CI-ELISA) agar gel immunodiffusion, real-time polymerase chain reaction (RT-PCR), reverse transcription-loop mediated isothermal amplification assay (RT-LAMP), Luciferase immunoprecipitation system (LIPS) and immunohistochemistry. Therefore, this review focused on the aetiology, epidemiology, pathology, immunology, and disease status in Africa, diagnosis, control, prevention, treatment and control of this disease.

Peste des petits ruminants infection in domestic ruminants in Sudan

The existence of peste des petits ruminants (PPR) in domestic ruminants and camels in Sudan during 2008-2012 was investigated. Lung tissues and serum samples were randomly collected from sheep, goats, cattle, and camels at different areas of Sudan. A total of 12,384 serum samples were collected from clinically healthy 7413 sheep, 1988 camels, 1501 cattle, 1459 goats, and 23 gazelles at different areas in the Sudan. They were examined for PPR antibodies using competitive ELISA (cELISA). The overall detected seroprevalence of PPR in tested sera was 49.4%; seroprevalence values within species were 67.1, 48.2, 25.8, 2.1, and 21.7% in sheep, goat, cattle, camels, and gazelles, respectively. The highest seroprevalence (68.1%) was observed in sera collected from Darfur states, then the central states (54.3%). A total of 1276 lung tissue samples (623 sheep, 324 cattle, 220 camels, and 109 goats) were collected. The majority of lung samples were collected from clinically healthy animals that showed lesions on PM in slaughterhouses (95%) and during PPR outbreaks; samples were tested for PPR antigen using immunocapture ELISA (IcELISA). PPR antigen was detected in 233 out of the 1276 tested samples (18.3%). Positive results were observed in samples collected from clinically healthy and diseased animals. The observed prevalence values in each species were 33.6, 21.1, 15.4, and 12.3% in camel, goat, sheep, and cattle, respectively. PPR antigen was detected in samples from different areas; however, the highest prevalence (63.9%) was found in samples collected from the eastern states, then Khartoum state (28%). Trials for virus isolation were done in different cell cultures. Out of 30 IcELISA-positive samples inoculated in primary bovine and ovine kidney cells, Vero cells, the PPR virus was successfully isolated from 15 (eight sheep, five camels, and two goats) samples in the three cell culture types. Using RT-PCR, PPRV nucleic acid was detected in all 25 IcELISA-positive tested samples.

Outbreak of foot and mouth disease and peste des petits ruminants in sheep flock imported for immediate slaughter in Riyadh.

Aim:To detect and identify the causative agent or agents of the following clinical symptoms which were fever, lack of appetite, salivation, vesiculation, erosions of the buccal mucosa, nose, and feet. The signs vary from mild to severe. The mortality rate of the disease is high. The morbidity rate reaches up to 100%. Sheep also show bloody diarrhea and rapid respiration. Sheep flock resident in El-Kharje Governorate.Materials and Methods:A total of 50 serum samples and 50 buffy coat samples were collected from Marino sheep flock suffered from high mortalities, fever, lameness, diarrhea, stomatitis, and respiratory distress. PrioCHECK® foot and mouth disease virus (FMDV) nonstructural (NS) (marketable enzyme-linked immunosorbent assay [ELISA] kit) was used for revealing of the NS antibodies and liquid phase blocking enzyme immunoassay (LPBE) for identifying the FMD serotype and examined by competitive ELISA (cELISA) for detection of peste des petits ruminants (PPR) antibodies. The buffy coat samples were examined by immunocapture ELISA (Ic ELISA) for detection of PPR antigen.Results:Using PrioCHECK® FMDV NS: Commercial ELISA kit: 38/50 (76%) of the serum samples were positive for the presence of FMD NS viral proteins. In addition, using LPBE the positive samples were identified as FMD serotype O. Examination of the serum sample by cELISA for detection of PPR antibodies gave positive results in 32/50 (64%). While the Ic ELISA identified 32 (64%) positive reactors for PPR antigen.Conclusion:This study reflected high susceptibility of the imported sheep flocks to the infection with FMD and PPR viruses, which are endemic in the Kingdom of Saudi Arabia (KSA). Hence, the imported flocks that prepared for slaughter must be vaccinated with the used vaccine in KSA in the quarantine for the control of FMD especially when importation occurs from counters that are free from these diseases.

The first report of peste des petits ruminants (PPR) in camels (Camelus dromedarius) in Iran.

In mid-July 2013, an outbreak of peste des petits ruminants (PPR) was observed in a herd of camels after they were imported from Kuwait to the Khuzestan province in southwest of Iran. The clinical signs of the affected animals included sudden death, fever, oral erosion, and ecthyma like lesions, yellowish diarrhea, pneumonia and respiratory distress, enlargement of lymph node, severe dehydration, dermatitis, ulcerative keratitis, and conjunctivitis. Necropsy findings included keratoconjunctivitis, congestion and consolidation of the lung, paleness of the liver, and enlargement and edema of lymph nodes. Histopathological exam revealed degeneration and acute hyperemia of the lungs, fatty change and necrotic foci in the liver, tubular necrosis in the kidneys, and necrotic dermatitis. We used immunocapture enzyme linked immunosorbent assay (ELISA) to confirm peste des petits ruminants virus (PPRV) and differentiate it from rinderpest virus. Then virus genome was studied by molecular analysis for detecting of strain and substrain of the virus. Immunocapture ELISA of all specimens reacted positively against PPRV antigens. Also, reverse transcription polymerase chain reaction (RT-PCR) results in the lung and lymph nodes of the dead camels consolidated the cause of disease to be PPRV. The present study is the first report of the PPRV outbreak in camels in Iran.

Mixed infection of peste des petits ruminants virus (PPRV) and other respiratory viruses in dromedary camels in Sudan, an abattoir study.

This study was intended to determine the role played by peste des petits ruminants (PPR) in causing respiratory infections in camels and its association with other respiratory viruses. A total of 474 lung specimens showing pneumonia were collected from clinically healthy camels in slaughterhouses at five different areas in Sudan. Using immunocapture ELISA (IcELISA), 214 specimens (45.1%) were found to be positive for PPR antigen. The highest prevalence was found in central Sudan (59.9%) then northern Sudan (56.6%) and eastern Sudan (26.6%). Parainfluenza virus 3 (PIV 3), respiratory syncytial virus (RSV), bovine herpes virus-1 (BHV-1), bovine viral diarrhea (BVD), and adenovirus were detected in 4.4, 2.9, 2.0, 9.0, and 1.3% of the specimens, respectively. PPR antigen was found in about 50% of specimens that showed positive result for other viral antigens. Twenty-five of 28 BVD, 15 of 16 PIV3, 8 of 12 RSV, 4 of 4 adenovirus, and 4 of 5 BHV-1 were found in association with other respiratory antigens. Results revealed the existence of PPRV infection in dromedary camels in Sudan and present evidence for mixed virus infection, suggesting that respiratory infections in camels might be exacerbated by PPRV.

Peste des petits ruminants outbreaks in White Nile State, Sudan.

Eight outbreaks of peste des petits ruminants in sheep and goats were reported in White Nile State, Sudan, between 2008 and 2009. A mortality rate of 4.2% was reported across the different outbreaks. Clinically the disease was characterised by high fever, ocular and nasal discharge, pneumonia, ulceration of the mucous membranes, diarrhoea and death. The post-mortem findings included necrotic lesions in the mouth and gastrointestinal tract, and swollen, oedematous lymph nodes associated with the lungs and intestine. Of the 209 serum samples tested by competitive enzyme-linked immunosorbent assay, 113 (54%) were found positive. Peste des petits ruminants virus was confirmed in tissues, nasal swabs and blood samples by immunocapture enzyme-linked immunosorbent assay, reverse-transcription polymerase chain reaction and isolation of the virus in culture of lamb testicle cells.

Detection of peste des petits ruminants virus in formalin-fixed tissues.

Peste des petits ruminants virus that causes a highly infectious and often fatal disease of sheep and goats is confirmed by various diagnostic techniques among them being isolation of the virus from cell culture systems, viral ribonucleic acid (RNA) detection by molecular assays, and viral antigen detection by immunocapture enzyme-linked immunosorbent assay (IC ELISA), immunohistochemistry (IHC), and AGAR gel test. Whereas most of the confirmatory diagnostic procedures require pathological samples to be stored frozen to preserve integrity of the peste des petits ruminants (PPR) virus RNA, samples for IHC tests are preserved in 10% formalin. In this study, nine formalin-fixed pathological samples from three goats suspected of PPR were processed for extraction of PPR viral RNA and analyzed for detection with real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay. The results showed that five out of the nine tested samples returned positive for presences PPR viral genome. This study has established that field pathological samples of PPR-suspected cases, collected and stored in 10% formalin for up 2 years, could be used for PPR virus RNA extraction for disease virus confirmation.

English

This report describes the details of outbreaks of Peste des petits ruminants (PPR) which occurred during 2008 in Khartoum and River Nile States in Sudan. An outbreak was reported in a sheep flock at the southern part of the State (Soba) in Khartoum, with 100% morbidity and mortality rates. In ElDamer city at River Nile State, the morbidity rate was 31.4% while mortality rate was 17.1% in the total animal population&nbsp;of 3,500. PPR antigen was detected in seven samples from both outbreaks using immunocapture enzyme-linked immunosorbent assay (IcELISA). The PPR ELISA results were confirmed by reverse transcription polymerase chain reaction (RT-PCR). Using cELISA, only one serum was sent from Khartoum and 6 out of 13 sera from ElDamer were positive for PPR antibodies. Key words: Peste des petits ruminants (PPR), outbreaks, sheep, Sudan.

Antemortem and postmortem examinations of the cattle calf naturally infected withMycobacterium aviumsubsp.paratuberculosis

A male cattle calf was detected as subclinically and naturally infected with Mycobacterium avium subspecies paratuberculosis (MAP) by a series of antemortem and postmortem tests. The MAP infection was identified by strong antibody and cell-mediated immune (CMI) response by a commercial ELISA kit and an intradermal Johnin test, respectively, in the initial antemortem examination. The antemortem status of the calf was further confirmed by MAP-specific interferon gamma (IFN-γ) response. For detection of IFN-γ response, MAP-specific IFN-γ release assays (IGRAs): (a) immuno capture ELISA (IC-ELISA) and (b) ELISPOT was employed. In addition, the presence of intracellular cytokine IFN-γ was detected by flow cytometry. For all cytokine assays, MAP-specific recombinant antigens HSP65 and 35 kDa were employed to overcome the poor sensitivity and specificity resulting from the use of Johnin, the crude protein purified derivative of MAP. Postmortem examination of the MAP-infected/suspected cattle calf did not reveal any pathognomonic gross lesions in the gastro-intestinal tract. Histopathological examination of multiple organs showed the presence of epithelioid cells/macrophages and edematous lesions in the mesenteric lymph nodes suggestive of MAP; however, no granulomas were observed in the intestinal tract. The necropsy samples of rectum and mesenteric lymph nodes were positive for isolation of MAP by culture in the BACTEC™ MGIT™ 960 system, and acid fast bacilli were demonstrated by fluorescence microscopy confirming the infection. Due to differential and complex expression patterns of MAP antigens reported in literature, a combination of assays such as those based on IGRAs and antibody detection is essential. Therefore, the current experimental evidence confirms the efficacy of the approach adopted. However, further studies will be needed to understand the optimal combination MAP-specific antigens for use in IGRAs or antibody assays that can be used for detecting MAP infection in every stage of the disease.

Molecular Characterization of Peste des Petits Ruminants Viruses From Outbreaks Caused by Unrestricted Movements of Small Ruminants in Pakistan

Peste des petits ruminants (PPR) is an endemic disease of small ruminants, and vaccination has been the method of control but outbreaks are continuously occurring in Pakistan. The following study presents a detailed investigation of an outbreak, suspected to be PPR, probably introduced by PPRV-infected sheep and goats from Sindh Province (north-west) to Punjab Province (central) of Pakistan during the flood relief campaign in 2011. A total of 70 serum samples from 28 different flocks were tested with competitive ELISA (H antibodies), which detected 24 (34.2%) samples positive for PPRV antibodies. Nasal swabs and faeces were tested with immunocapture ELISA (N antigen), which detected 18 (25.7%) samples positive for PPRV antigen. The RNA detected positive (n=28, 40%) using real-time PCR was subjected to conventional PCR for the amplification of the fusion and nucleoprotein genes. Sequencing of both genes and subsequent phylogenetic analysis indicated the grouping of all the sequences to be in lineage IV along with other Asian isolates of PPRV. However, sequences of both genes were divided into two groups within lineage IV. One group of viruses clustered with previously characterized Pakistani isolates, whereas the other group was distinctly clustered with isolates from the Middle East or India. The sequence identity indicated the introduction of at least one population of PPRV from a different source and circulation in the local flocks of small ruminants, which emphasized the need to obtain health clearance certificate before movement of animals. The results of this study provide baseline data for the genetic characterization of different PPRV populations in Pakistan.

Apport de l'immunocapture-Elisa au diagnostic de l'infection à virus grippal A(H1N1)pdm09 : une alternative à la RT-PCR ?

To evaluate immunocapture-Elisa (ICE) sensitivity versus RT-PCR in diagnosis of influenza A(H1N1)pdm09 virus infection. Sixty-seven RT-PCR-negative and 282 RT-PCR-positive nasopharyngeal swabs collected during winter 2009-2010 have been analyzed using ICE. Among all the samples tested, a sensitivity of 31.3% was found for ICE. The sensitivity of ICE was directly correlated to the virus load determined through the number of cycling reactions necessary to reach detection by RT-PCR. ICE can be a suitable method compared to RT-PCR when RT-PCR cannot be used for economical or epidemiological reasons. Its sensitivity is largely dependent of the nasopharyngeal sampling quality.

Abstract 5251: CXCL14 as a functional determinant in a novel model of inflammatory, triple negative breast cancer progression

Abstract CXCL14 (BRAK) is a cytokine implicated in inflammatory responses, but its contribution to cancer is largely unknown. Recent studies in clinical breast cancer indicate an association between CXCL14 expression and shortened time to metastatic progression, as well as the presence of CXCL14 in a subset of breast epithelial cells with a stem-like phenotype. Using global gene expression profiling, we identified CXCL14 as the most prominently upregulated cytokine in a novel progression model of triple-negative inflammatory and metaplastic breast cancer that we generated. The model comprises tumor cells established from a locally aggressive primary tumor obtained from a patient at the time of initial surgery (TES-1), and tumor cells from the same site at the time of local failure in the chest wall after chemo and radiation therapy (TES-2b). In contrast to TES-1, TES-2b cells are tumorigenic in immuno deficient mice and can be followed and quantified by non-invasive bioluminescence imaging after injecting the F-luc tagged cancer cells into the 4th mammary fat pad. Illumina microarray analysis revealed a 66-fold increase in CXCL14 expression in TES-2b cells over TES-1 cells, ranking it as one of the top 25 genes most differentially expressed among over 25,000 identified gene transcripts. We validated CXCL14 overexpression in TES-2b cells by real time PCR (TaqMan) (64 fold), and found a significant increase in CXCL14 protein secretion in cultured TES-2b cells (3.8 fold) by immuno-capture ELISA. To address a functional role in CXCL14 in the progression of these aggressive breast cancer cells, we stably reduced CXCL14 expression in TES-2b cells by lentiviral transduction with small interfering RNA and confirmed an 80% reduction in gene expression. CXCL14 protein production was concomitantly reduced 2.7 fold. Importantly, xenograft studies revealed a critical role of CXCL14 in the in vivo growth behavior of these breast cancer cells, as the tumor growth rate in the mammary fat pad of CXCL14 knock-down cells was significantly reduced compared to scrambled control vector treated cells. After injecting 5×105 F-luc tagged tumor cells, 4/6 mice had tumors measuring 10 mm in diameter after 11 weeks (requiring sacrifice of the animals) compared with 1/6 in the CXCL14 knock-down group. These initial results indicate a critical role of tumor cell derived CXCL14 in the in vivo survival and proliferation of these breast cancer cells. Our findings provide a basis for further analyses of CXCL14 in breast cancer progression in vivo and for detailed studies of the mechanisms through which CXCL14 may promote disease progression of highly aggressive triple-negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5251. doi:10.1158/1538-7445.AM2011-5251

Evidence of peste des petits ruminants virus (PPRV) infection in Sindh Ibex (Capra aegagrus blythi) in Pakistan as confirmed by detection of antigen and antibody

An outbreak resulting in mortality in Sindh Ibex (Capra aegagrus blythi) was investigated. There was a history of about 36 deaths (both young and adult) during the period of 1 month. Disease appeared in a generalized form, affecting the respiratory and digestive systems. Major lesions were respiratory distress, pustules on and in the mouth, ocular-nasal discharges, and severe diarrhea. The most significant lesion was the oculonasal discharges and diarrhea. Deaths were mainly due to blindness, anorexia, diarrhea, and respiratory arrest. Both adult (mortality = 21) and young (mortality = 15) animals were affected with the disease. Peste des petits ruminants virus (PPRV) antigen was detected in the spleen, lung, lymph node, and swab samples by immunocapture enzyme-linked immunosorbent assay. Spleen and lung samples were also tested and found positive for the presence of F-gene of PPRV by polymerase chain reaction. Thirteen of 20 serum samples from nearby sheep and goats were found positive for antibodies to PPRV. The disease threatened the huge population of ibex in the wild life park, which was spread over a large area, but vaccination of the domestic population of sheep and goats in the surrounding villages appeared to control the disease.

Immunocapture enzyme-linked immunosorbent assay for assessment of in vitro potency of recombinant hepatitis B vaccines.

Quantification of hepatitis B surface antigen (HBsAg) or relative in vitro potency in the final vaccines is a prerequisite for hepatitis B vaccine batch release. The commercial kit for automated analysis (AxSYM) is expensive, and an alternative is required for the estimation of HBsAg in hepatitis B vaccines. Mouse monoclonal antibodies (MAbs) specific for HBsAg were developed and characterized. One of the monoclonal antibodies (HBs06) was used in development of an immunocapture ELISA (IC-ELISA) as an unlabeled capture antibody and biotin-labeled detection antibody. The IC-ELISA was standardized and validated using experimental hepatitis B vaccine batches with various HBsAg concentrations per dose and commercial vaccines. The vaccine was treated with an alkaline solubilizer to desorb the HBsAg from Algel-adjuvanted vaccines before testing, and the sensitivity of the test was 5 ng/ml. A good correlation could be observed between the HBsAg estimates derived by both formats, except for the higher HBsAg concentration range, where the IC-ELISA format could estimate closer to the actual values than AxSYM. There was a significant correlation between the estimated relative potencies of the two methods. There was lack of correlation between the in vivo potency and the relative in vitro potency. However, the estimates of IC-ELISA were comparable to the in vivo values when compared with the estimates of AxSYM. The IC-ELISA can therefore be considered to be a reliable test for deriving in vitro relative potency and antigen concentration in vaccine batches for batch control and release.