Polyclonal Antiserum Research Articles

Effect of Oral Streptococci Expressing Pneumococcus-like Cross-Reactive Capsule Types on World Health Organization Recommended Pneumococcal Carriage Detection Procedure.

Carriage studies are fundamental to assessing the effects of pneumococcal vaccines. Because a large proportion of oral streptococci carry homologues of pneumococcal genes, non-culture-based detection and serotyping of upper respiratory tract (URT) samples can be problematic. In the current study, we investigated whether culture-free molecular methods could differentiate pneumococci from oral streptococci carried by adults in the URT. Paired nasopharyngeal (NP) and oropharyngeal (OP) samples were collected from 100 older adults twice a month for 1 year. Extracts from the combined NP + OP samples (n = 2400) were subjected to lytA real-time polymerase chain reaction (PCR). Positive samples were subjected to pure culture isolation, followed by species confirmation using multiple approaches. Multibead assays and whole-genome sequencing were used for serotyping. In 20 of 301 combined NP + OP extracts with positive lytA PCR results, probable pneumococcus-like colonies grew, based on colony morphology and biochemical tests. Multiple approaches confirmed that 4 isolates were Streptococcus pneumoniae, 3 were Streptococcus pseudopneumoniae, 12 were Streptococcus mitis, and 1 were Streptococcus oralis. Eight nonpneumococcal strains carried pneumococcus-like cps loci (approximate size, 18-25kb) that showed >70% nucleotide identity with their pneumococcal counterparts. While investigating the antigenic profile, we found that some S. mitis strains (P066 and P107) reacted with both serotype-specific polyclonal (type 39 and FS17b) and monoclonal (Hyp10AG1 and Hyp17FM1) antisera, whereas some strains (P063 and P074) reacted only with polyclonal antisera (type 5 and FS35a). The extensive capsular overlap suggests that pneumococcal vaccines could reduce carriage of oral streptococci expressing cross-reactive capsules. Furthermore, direct use of culture-free PCR-based methods in URT samples has limited usefulness for carriage studies.

A sequel study on the occurrence of Tomato spotted wilt virus (TSWV) in cut-chrysanthemum by DAS-ELISA using recombinant nucleocapsid protein to produce polyclonal antiserum

The tomato spotted wilt virus (TSWV) belonging to the genus Orthotospovirus, family Tospoviridae, causes severe necrotic disease in field crops and horticultural crops, resulting in considerable yield loss worldwide. The development of protein-based diagnostics is essential to track the virus transmission and prevent its spread in vegetatively propagated crops such as ornamentals. In this study, nucleocapsid (N) gene of TSWV was cloned in pET 28 a (+) expression vector. Expression of the 32 kDa recombinant TSWV-N protein was induced in BL21 (DE3) cells using 1 mM of Isopropyl β-d-1-thiogalactopyranoside (IPTG), and was confirmed through SDS-PAGE and Western blot by fluorescent-labeled secondary antibody. The bacterial cells expressed recombinant TSWV-N protein up to a concentration of 9.48 μg/mL. The purified protein was used for immunization of a rabbit to produce specific polyclonal antiserum. The TSWV antiserum was conjugated with the enzyme alkaline phosphatase (ALP). Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA) was developed and validated against TSWV infected hosts. This antiserum specifically reacted with recombinant N protein as well as TSWV infected hosts, but not with groundnut bud necrosis orthotospovirus (GBNV) as well as capsicum chlorosis orthotospovirus (CaCV) infecting tomato and chilli plants. The coating antibody at 1 μg/mL concentration and 1:500 dilution of enzyme conjugate were found to be effective and economical in the detection of recombinant N protein of TSWV and the virus present naturally in the infected hosts. Using standardized DAS-ELISA protocol, the TSWV titer also was quantified in artificially inoculated assay hosts. Among 11 hosts tested, higher virus titer was recorded in Nicotiana tabacum (0.270 μg/100 μL), followed by Impatiens balsamiana (0.185 μg/100 μL) and Dahlia pinnata at a low virus tire of 0.083 μg/100 μL. The diagnostic reagents and protocol (DAS-ELISA) are further validated by detecting the infection of TSWV in chrysanthemum stem cuttings from six different nurseries in the hill stations of Tamil Nadu, India. The DAS-ELISA assay experimented on six varieties from four different nurseries revealed that the Mum Yellow variety had a higher percentage of TSWV infection (36 %), which was followed by the Mum White variety (33 %); both collected from Kotagiri Nursery. The same variety exhibited a higher virus titer by DAS-ELISA, an A405 value range of 0.733 (̴ 0.115 μg) and 0.711 (̴ 0.111 μg) respectively, and a total of 27 % of TSWV infection was confirmed by screening 800 stem cuttings by DAS-ELISA. The presence of TSWV was also detected in 54 (6.75 %) asymptomatic stem cuttings from different locations, and the A405 value ranged from 0.325 to 0.468.(̴ 0.044−0.069 μg/100 μL); this is the first reported development of immune-based diagnostics for TSWV in India. This protocol and diagnostics will be highly useful for quarantine purposes while trading large quantities of planting materials.

Immunohistochemical detection of enteroviruses in pancreatic tissues of patients with type 1 diabetes using a polyclonal antibody against 2A protease of Coxsackievirus.

ABSTRACTAims/IntroductionThe need for antiserum for immunohistochemical (IHC) detection of enterovirus (EV) in formaldehyde‐fixed and paraffin‐embedded samples is increasing. The gold standard monoclonal antibody (clone 5D8/1) against EV‐envelope protein (VP1) was proven to cross‐react with other proteins. Another candidate marker of EV proteins is 2A protease (2Apro), which is encoded by the EV gene and translated by the host cells during EV replication, and participates processing proproteins to viral capsid proteins.Materials and MethodsWe raised polyclonal antiserum by immunizing a rabbit with an 18‐mer peptide of Coxsackievirus B1 (CVB1)‐2Apro, and examined the specificity and sensitivity for EV on formaldehyde‐fixed and paraffin‐embedded tissue samples.ResultsEnzyme‐linked immunosorbent assay study showed a high titer of antibody for 18‐mer peptide of CVB1‐2Apro, cross‐reacting with CVB3‐2Apro peptide. IHC showed that antiserum against 2Apro reacted with CVB1‐infected and VP1‐positive Vero cells. Confocal laser scanning microscopy showed that antigen stained by the 2Apro antibody located in the same cell with VP1 stained by 5D8/1. IHC using 2Apro antiserum showed dense staining in the islets of EV‐associated fulminant type 1 diabetes pancreas and that located in the same cell stained positive for VP1 (5D8/1). Specificity of 2Apro antiserum by IHC staining was confirmed by negative 2Apro in 14 VP1‐negative non‐diabetes control pancreases.ConclusionsOur study provides a new polyclonal antiserum against CVB1‐2Apro, which might be useful for IHC of EV‐infected human tissues stored as archive of formaldehyde‐fixed and paraffin‐embedded tissue samples.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

First report of Xanthomonas campestris pv. campestris as the causal agent of necrotic leaf spot in Phaseolus vulgaris at Puebla, Mexico.

Beans are the most cultivated legume in the world. In Mexico, it is the second most important crop after corn (FAO 2020; SIAP 2020). Bean plants "Flor de Mayo M38" variety were affected by a foliar disease during the agricultural cycle 2019 in Puebla-Mexico (19°02'46.6" LN and 98°05'15.6" LO). Necrotic V- shaped lesions were observed on the margins of the leaves surrounded by yellow halos followed by foliar necrosis, affecting 40% of the crop. In Mexico this variety of cultivars is in great demand for local consumption and generates income in foreign currency (Castellanos et al. 1997). Sampling was carried out on 50 plants "Flor de Mayo M38" variety, with necrotic leaf symptoms from ten plots of one hectare. Samples were cut into pieces (5 mm), disinfested with 1% hypochlorite 3 min, and washed with sterile distilled water. Subsequently, samples were dried on sterile paper and placed on Petri plates containing yeast extract calcium carbonate dextrose agar (YDC) medium and kept at 36°C for 3 days. Colonies of ten typical bacteria isolated from all symptomatic plants were Gram (-), small and uniform in size with rounded edges, yellow, convex with entire borders and mucoid appearance on YDC. Bacteria did not grow on 0.1% triphenyl tetrazolium chloride amended casamino acid, peptone, and glucose medium (CPG). Biochemical tests showed that isolates did not reduce nitrate to nitrites, had positive catalase and starch hydrolysis, while the Kovac oxidase test was negative (Schaad and White 1974). Genus identity of the representative isolate Xcf1-APJR, was confirmed by 16S rRNA encoding gene partial sequencing, using universal primers 518F (5'-CCAGCAGCCGCGGTAATACG-3') and 800R (5'-TACCAGGGTATCTAATCC-3') (Halim et al. 2020). BLASTn alignments against the nucleotide collection were 100% identical to Xanthomonas sequences including Xanthomonas campestris pv. campestris strains NZ_AP019684.1, CP025750.1, and MN108237.1. The 1,418 bp sequence was deposited in the GenBank database under accession number MT645246. The identification of species/pathovar was accomplished by serological methods using a polyclonal antiserum specific for X. campestris pv. campestris (Popovic ́ et al. 2013) with the DAS-ELISA commercial kit (catalog number 07122C/096, LOEWE Biochemica GmbH, Germany). The pathogenicity test was carried out on 50 healthy bean plants from the "Flor de Mayo M38" variety. Bacterial culture incubated at 28°C for 48 h in YDC medium was used to prepare the bacterial suspension (108 CFU mL-1). The first two lower leaves of 30-day-old plants were inoculated by sprinkling. Ten plants sprayed with sterile distilled water were used as negative control. All plants were kept for 20 days in greenhouse at 18-26°C and relative humidity of 60%. After seven days, chlorotic lesions developed on all inoculated plants that became necrotic from 14 days after inoculation (dai). Necrotic leaf spots merged at 14 dai to form necrotic areas of more than 20 mm in diameter, reaching total necrosis of the leaf tissue at 20 dai and were similar to the symptoms observed in the field. Koch's postulates were confirmed by the reisolation of Xcf1-APJR strain, which presented the same colony morphology, partial sequence, and polyclonal specific detection. This is the first report of this pathogen causing necrotic leaf spot in beans from the "Flor de Mayo M38" variety in Puebla-Mexico. The author(s) declare no conflict of interest.

Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1) is an immunogenic antigen found in EVs released from pre-acetabular glands of invading cercariae.

Extracellular Vesicles (EVs) are an integral component of cellular/organismal communication and have been found in the excreted/secreted (ES) products of both protozoan and metazoan parasites. Within the blood fluke schistosomes, EVs have been isolated from egg, schistosomula, and adult lifecycle stages. However, the role(s) that EVs have in shaping aspects of parasite biology and/or manipulating host interactions is poorly defined. Herein, we characterise the most abundant EV-enriched protein in Schistosoma mansoni tissue-migrating schistosomula (Schistosoma mansoni Larval Extracellular Vesicle protein 1 (SmLEV1)). Comparative sequence analysis demonstrates that lev1 orthologs are found in all published Schistosoma genomes, yet homologs are not found outside of the Schistosomatidae. Lifecycle expression analyses collectively reveal that smlev1 transcription peaks in cercariae, is male biased in adults, and is processed by alternative splicing in intra-mammalian lifecycle stages. Immunohistochemistry of cercariae using a polyclonal anti-recombinant SmLEV1 antiserum localises this protein to the pre-acetabular gland, with some disperse localisation to the surface of the parasite. S. mansoni-infected Ugandan fishermen exhibit a strong IgG1 response against SmLEV1 (dropping significantly after praziquantel treatment), with 11% of the cohort exhibiting an IgE response and minimal levels of detectable antigen-specific IgG4. Furthermore, mice vaccinated with rSmLEV1 show a slightly reduced parasite burden upon challenge infection and significantly reduced granuloma volumes, compared with control animals. Collectively, these results describe SmLEV1 as a Schistosomatidae-specific, EV-enriched immunogen. Further investigations are now necessary to uncover the full extent of SmLEV1's role in shaping schistosome EV function and definitive host relationships.

Identification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches.

Citrus concave gum-associated virus (CCGaV) is a negative-stranded RNA virus, first reported a few years ago in citrus trees from Italy. It has been reported in apple trees in the USA and in Brazil, suggesting a wider host range and geographic distribution. Here, an anti-CCGaV polyclonal antiserum to specifically detect the virus has been developed and used in a standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) that has been validated as a sensitive and reliable method to detect this virus both in citrus and apple trees. In contrast, when the same antiserum was used in direct tissue-blot immunoassay, CCGaV was efficiently detected in citrus but not in apple. Using this antiserum, the first apple trees infected by CCGaV were identified in Italy and the presence of CCGaV in several apple cultivars in southern Italy was confirmed by field surveys. High-throughput sequencing (HTS) allowed for the assembling of the complete genome of one CCGaV Italian apple isolate (CE-c3). Phylogenetic analysis of Italian CCGaV isolates from apple and citrus and those available in the database showed close relationships between the isolates from the same genus (Citrus or Malus), regardless their geographical origin. This finding was further confirmed by the identification of amino acid signatures specific of isolates infecting citrus or apple hosts. Analysis of HTS reads also revealed that the CE-c3 Italian apple tree, besides CCGaV, was simultaneously infected by several viruses and one viroid, including apple rubbery wood virus 2 which is reported for the first time in Italy. The complete or almost complete genomic sequences of the coinfecting agents were determined.

Lambda monoclonal free light chain abnormalities detected by a serum immunofixation electrophoresis assay are underrepresented by quantitative serum free light chain results

Abstract Protein and immunofixation (IFIX) electrophoresis are used to diagnose and monitor monoclonal gammopathies. While IFIX detects clonal production of intact immunoglobulins and free light chains (FLC), the latter can also be quantified using a serum free light chain (SFLC) assay, in which polyclonal antisera detects epitopes specific for free kappa (KFLC) or lambda light chains (LFLC). An abnormal KFLC: LFLC ratio (KLR) serves as a surrogate for clonality. While the SFLC assay is highly sensitive, normal LFLC (<2.63mg/dL) and KLR results (>0.26 & <1.65) were found in samples with distinct lambda monoclonal free light chains visualized by IFIX (X-LMFLC). To investigate this discordance, contemporaneous SFLC or KLR values were evaluated for their ability to accurately classify monoclonal FLCs identified by IFIX. We performed a retrospective analysis of serum and urine IFIX (Sebia Hydrasys) and SFLC (Freelite®, Binding Site) results from our institution between July 2010 through December 2020, using R 4.0.2 and Tidyverse packages. From among 9,594 encounters in which a single monoclonal component was initially identified by IFIX, 157 X-LMFLC and 131 X-KMFLC samples were analyzed. Elevated LFLC with normal KFLC was identified in 105/157 X-LMFLC samples (67%), while both LFLC and KFLC were elevated in 42/157 samples (27%). Concordance between X-KMFLC and KFLC was markedly higher, where 122/131 samples (93%) displayed elevated kappa FLC (>1.94mg/dL) with normal LFLC, and only 7/131 X-KMFLC samples (5%) possessed both elevated KFLC and LFLC. The use of KLR to identify pathogenic monoclonal free light chains improved lambda concordance to 85%; however, 19/157 (12%) of X-LMFLC samples still exhibited normal KLR. High concordance of 98% was again observed for X-KMFLC with abnormal KLR. When samples were segregated according to normal or impaired renal function (eGFR > or ≤60mL/min/1.73m², respectively), this disparate identification of X-LMFLC and X-KMFLC by the SFLC assay persisted, suggesting that renal dysfunction (as measured by eGFR) does not underlie this phenomenon. Lastly, we corroborated the above findings in a larger sample population by examining patients with urine Bence Jones FLC identified by IFIX who had free or intact monoclonal components in serum (N=724), grouped by lambda or kappa light chain involvement. The cause(s) of the discrepant performance by the Freelite® SFLC assay, relative to the Sebia Hydrasys IFIX assay, for identifying lambda FLC components is currently unclear. Possible contributory factors include assay reference range cutoffs, other patient disease parameters, and differences in assay-specific polyclonal antisera. Future analyses of these factors will help to further characterize SFLC assay performance and elucidate how interpretation of composite serum FLC test results can be improved to better guide patient management.

Effective purification of human chorionic gonadotropin and production of highly specific polyclonal anti-βHCG as a component of radioimmunoassay kit

ABSTRACT This study aimed to purify human chorionic gonadotropin (HCG) from the urine of pregnant women with high biological activity (10811 IU/mg) and purity (98.2%), by simple capturing of HCG using DEAE Sepharose FF and polishing using Sephacryl S200 HR. The HCG obtained was characterized by SDS-PAGE and dissociated into alpha and beta subunits using the urea treatment method. The βHCG subunits were injected into rabbits for the production of highly specific polyclonal anti-βHCG antisera. The polyclonal anti-βHCG was locally produced in rabbits and assessed for binding titer (1/10000), displacement (84.8%), and specificity (98.8%). Purified HCG along with locally prepared polyclonal anti-βHCG antisera were used as basic components of the in-house Radioimmunoassay system for quantitative estimation of HCG in human serum.

Development of polyclonal antibodies-based serological methods and a DIG-labelled DNA probe-based molecular method for detection of the Vicia cryptic virus-M in field plants

Vicia cryptic virus M (VCV-M), a member of the genus Amalgavirus of the family Amalgaviridae, was first identified in 2009 in a Vicia faba Linn. planting in Hangzhou, Zhejiang Province, China. However, there has been no further research on the biological features of VCV-M to date and the viral particles and coat protein (CP) have not been identified. The putative CP of VCV-M was predicted from the viral genomic RNA. In this study, a recombinant version of the putative CP of VCV-M (His-CPVCV−M) was produced and used to prepare a polyclonal antiserum against the His-CPVCV−M. Using this antiserum, a Western blot, an immuno-dot-blot and an enzyme-linked immunosorbent assay were developed for testing field samples of V. faba for the presence of VCV-M. Additionally, a digoxigenin (DIG)-labelled DNA probe-based Northern blot assay was established for VCV-M genome detection in field samples. The results showed that both the serological and nucleic acid assays could accurately and sensitively detect VCV-M in V. faba. This research represented the first confirmed expression of the putative CP of VCV-M in infected V. faba tissues. The serological and nucleic acid assays provided two complementary methods for VCV-M detection which could contribute to seed quality control and production increases of V. faba crops.

Making Weak Antigens Strong: Preparing Immune Complexes for Injection.

If antibodies against a particular antigen are available, that antigen can be purified and used for further immunizations, and antigens thus purified can show enhanced immunogenicity. Purified immune complexes can be injected directly, or while coupled to beads; the presence of antibodies and/or beads stimulates phagocytosis and usually will not influence the response. This method provides a useful means of antigen enrichment for a variety of applications, such as using antibodies raised against a denatured antigen to harvest a native protein for further immunizations, or when using a monoclonal antibody as an intermediate to the preparation of polyclonal antisera. Injecting antibody-coated antigens has also been used to mask a particularly immunodominant epitope on an antigen, and thereby develop a response against other epitopes. The amount of antigen needed to elicit a strong response using immune complexes will vary from one compound to another. Doses as low as 50 ng of antigen have been used successfully when delivered this way.

Histopathological, Immunohistochemical and In-Situ Hybridization Findings in Suckling Rats Experimentally Infected With Akabane Genogroups Ⅰ and Ⅱ, Aino and Peaton Viruses

Akabane, Aino and Peaton viruses are closely related arthropod-borne viruses in the genus Orthobunyavirus of the family Peribunyaviridae that can cause congenital abnormalities in cattle, sheep and goats. East Asian Akabane virus strains are subdivided into genogroups Ⅰ and Ⅱ, and the former can also cause non-suppurative encephalomyelitis in post-natal animals. Specific detection of the infecting virus in tissues is essential for accurate diagnosis. Immunohistochemistry (IHC) has been used to identify viral antigen but cannot always detect specific viruses due to potential cross-reactivity of the primary antisera. We compared in-situ hybridization (ISH), based on the use of cocktail probe sets targeted at the RNA of each virus, with IHC for the detection of the specific viruses in tissues of suckling rats inoculated intracerebrally with Akabane (KM-1 or OBE-1 strains), Aino or Peaton viruses at 3 or 7 days of age. Most inoculated rats developed severe neurological signs and histopathological brain lesions including necrosis, spongy degeneration and non-suppurative inflammation. A rabbit polyclonal antiserum immunolabelled antigen of all three viruses within the lesions, whereas ISH specifically detected RNA of each individual virus. The distribution of viral RNA was comparable to that of viral antigens, but tended to be more widespread, especially in immature nervous tissue. Viral antigen and RNA were detected in skeletal muscle and heart of the rats infected with the KM-1 strain of Akabane virus but not with any of the other viruses. This study demonstrates the value of ISH detection of these viruses in a rat model and may prove useful for clarification of the pathogenesis of post-natal arbovirus infection.

Characterization of taro reovirus and its status in taro (Colocasia esculenta) germplasm from the Pacific.

Taro reovirus (TaRV) has been reported infecting taro (Colocasia esculenta) in the South Pacific, but information on the virus is limited. Here, we report the genome sequence of a reovirus infecting taro in Papua New Guinea that had 10 genomic segments ranging from 1.1 to 3.9 kilobase pairs (kbp) in length with a total genome length of 26.3 kbp. TaRV was most closely related to rice ragged stunt virus (RRSV) but did not cross-react with RRSV polyclonal antisera. TaRV was not detected in 82 germplasm accessions of taro in Hawaii, or samples collected in American Samoa, Fiji, Guam, Palau, or Vanuatu.

Erythrocyte Adhesion of Merozoite Surface Antigen 2c1 Expressed During Extracellular Stages of Babesia orientalis.

Babesia orientalis, a major infectious agent of water buffalo hemolytic babesiosis, is transmitted by Rhipicephalus haemaphysaloides. However, no effective vaccine is available. Essential antigens that are involved in parasite invasion of host red blood cells (RBCs) are potential vaccine candidates. Therefore, the identification and the conduction of functional studies of essential antigens are highly desirable. Here, we evaluated the function of B. orientalis merozoite surface antigen 2c1 (BoMSA-2c1), which belongs to the variable merozoite surface antigen (VMSA) family in B. orientalis. We developed a polyclonal antiserum against the purified recombinant (r)BoMSA-2c1 protein. Immunofluorescence staining results showed that BoMSA-2c1 was expressed only on extracellular merozoites, whereas the antigen was undetectable in intracellular parasites. RBC binding assays suggested that BoMSA-2c1 specifically bound to buffalo erythrocytes. Cytoadherence assays using a eukaryotic expression system in vitro further verified the binding and inhibitory ability of BoMSA-2c1. We found that BoMSA-2c1 with a GPI domain was expressed on the surface of HEK293T cells that bound to water buffalo RBCs, and that the anti-rBoMSA2c1 antibody inhibited this binding. These results indicated that BoMSA-2c1 was involved in mediating initial binding to host erythrocytes of B. orientalis. Identification of the occurrence of binding early in the invasion process may facilitate understanding of the growth characteristics, and may help in formulating strategies for the prevention and control of this parasite.

C1Q‐TNF‐related peptide 8 (CTRP8) in human prostate cancer

Introduction C1Q Tumor Necrosis Factor related Peptide 8 (CTRP8) is the one of the least characterized members of the C1Q/TNF family of proteins which is engaged in diverse functions ranging from metabolism to immunity and is one of only two CTRP family members absent in the mouse genome. CTRP8 was identified in our lab as a novel ligand of the RXFP1 receptor and was found to promote invasiveness and chemoresistance to temozolomide in glioblastoma (GB)(Glogowska et al., 2013; Thanasupawat et al., 2018). We have generated high-affinity rabbit antisera to human CTRP8 to identify a selective mast cell subpopulation in human prostate cancer tissues (Hempel Sullivan et al., 2020). Mast cells are innate immune cells which are primarily known for their role in the mediation of allergic responses. They have been detected within prostate cancer tissues and serve as potential prognostic marker in prostate cancer (Johansson et al., 2010; Hempel Sullivan et al., 2020). Methods Affinity-purified rabbit polyclonal antisera directed against an epitope unique to human CTRP8 were characterized by Western blot, immunofluorescence, and immunohistochemistry in prostate cancer tissue sections and human prostate cancer tissue microarrays (TMAs). Results Of several Flag-tagged human and mouse CTRP family members transiently expressed in HEK293T cells, the CTRP8 antisera exclusively detected human CTRP8 as assessed by Western blot, demonstrating the specificity of the antisera. We studied the expression of CTRP8 in human prostate tissues. CTRP8 was expressed in a subpopulation of human mast cells distributed in prostate cancer tissues, as demonstrated by co-localization of CTRP8 immunoreactivity and toluidine blue positive mast cells in prostate cancer tissues. This was further validated by dual immunofluorescence of CTRP8 with Tryptase, a marker for mature mast cells. Initial analysis of prostate cancer tissue microarrays (TMAs) (n= 360) revealed a comparative increase in the proportion of Tryptase+/ CTRP8+ cells versus total number of mast cells (Tryptase+/ CTRP8+ and Tryptase+/ CTRP8- cells) in the extra-tumoral compartment as compared to intra-tumoral regions for Gleason grades 6 and 7 prostate cancer samples. Weak staining of CTRP8 was detected in prostate epithelial cells. The RXFP1+ human prostate cancer cell line PC3 responded to CTRP8 treatment with increased protein kinase C delta signaling and enhanced motility as determined by real-time migration assays. Conclusions Here we have identified the expression of adipokine family member CTRP8 in prostate epithelial cells and in a subpopulation of mature mast cells in patient prostate cancer tissues, thus, identifying CTRP8 as a novel player in prostate cancer and associated mast cell compartment. References Glogowska, A., et al. J. Pathol., 231, 466–479, 2013 Thanasupawat, T., et al. Mol. Oncol. 12, 1464–1479, 2018 Johansson, A., Am. J. Pathol, 177, 1031–1041, 2010 Hempel Sullivan H, et al. J. Pathol., 2020 Hempel Sullivan H, et al. Cancer Epidemiol Biomarkers Prev., 2020

Assessment of rat polyclonal antisera’s suitability in hemagglutination inhibition assay for influenza surveillance and antigenic mapping

This paper presents comparative hemagglutination inhibition (HI) assay data obtained using ferret or rat antisera to analyze influenza viruses. The results indicate that rat antisera can be successfully applied both for identification and for antigenic analysis of human influenza A and B viruses. Data gained with rat antisera were comparable to those obtained with ferret antisera. In-depth statistical analysis, based on Confusion Matrix analysis and Receiver Operating Characteristic (ROC) analysis, confirmed good coincidence between ferret antisera-based and rat antisera-based results. Two-dimensional antigenic mapping, based on HI assays using rat and ferret antisera, supported these findings. Both antisera types yielded identical antigenic attributions for the viruses analyzed, and both permitted visualization of contemporary human influenza virus evolutionary trends.

Evidence for Unknown Sarcocystis-Like Infection in Stranded Striped Dolphins (Stenella coeruleoalba) from the Ligurian Sea, Italy.

Simple SummaryTwo stranded striped dolphins presented meningoenchepalitic lesions associated with the presence of unknown protozoan tissue cysts. The present study aimed at fully characterizing these previously undescribed parasites. Light microscopy re-examination of affected CNS areas showed high numbers of tissue cysts with morphological features resembling those of Sarcocystis species. Tissue cyst bradyzoites positively stained when labeled with polyclonal antisera but cross-reactivity could not be precluded. Sarcocystis sp. sequences with high homology to species infecting livestock were amplified by means of PCR from myocardial and muscle tissues. This is the first report of Sarcocystis-like tissue cysts in the cerebral tissue of stranded cetaceans with muscular sarcocystosis in Mediterranean dolphins. The obtained results may suggest a land-to-sea cycling of Apicomplexan parasites in this region and the need for further investigations in order to foster marine mammal conservation.Two striped dolphins (SD1, SD2), stranded along the Ligurian coast of Italy, were diagnosed with a nonsuppurative meningoencephalitis associated with previously undescribed protozoan tissue cysts. As tissue cysts were morphologically different from those of Toxoplasma gondii, additional histopathological, immunohistochemical, ultrastructural, and biomolecular investigations were performed, aiming to fully characterize the organism. Histopathology revealed the presence of large Sarcocystis-like tissue cysts, associated with limited inflammatory lesions in all CNS areas studied. IHC was inconclusive, as positive staining with polyclonal antisera did not preclude cross-reaction with other Sarcocystidae coccidia. Applied to each animal, 11 different PCR protocols precluded a neural infection by Sarcocystis neurona, Sarcocystis falcatula, Hammondia hammondi, and Neospora caninum. T. gondii coinfection was confirmed only in dolphin SD2. Sarcocystis sp. sequences, showing the highest homology to species infecting the Bovidae family, were amplified from SD1 myocardium and SD2 skeletal muscle. The present study represents the first report of Sarcocystis-like tissue cysts in the brain of stranded cetaceans along with the first description of Sarcocystis sp. infection in muscle tissue of dolphins from the Mediterranean basin.

Dot-blot immunoassay for detection of tomato chlorosis virus and reaction of potato genotypes to virus infection

Tomato chlorosis virus (ToCV) is a whitefly-transmitted crinivirus that causes yield losses, mainly in tomato (Solanum lycopersicum) and potato (S. tuberosum) crops. In this work, a polyclonal antiserum for detecting the virus using a dot-blot immunoassay was developed and the responses of crinivirus-infected potato genotypes were evaluated. The virus was purified using infected tomato leaves and the antiserum was obtained by intramuscular injection of purified viral preparations in rabbit. Tomato and potato tissue samples were used to validate the polyclonal antiserum for detection of ToCV, previously analyzed by RT-PCR. A total of 81 tomato and potato samples were analyzed by RT-PCR and dot-blot immunoassay (DBIA). All samples were positive in RT-PCR, but three of them did not react in DBIA, showing of 96.3% efficiency compared with that in RT-PCR. All potato genotypes inoculated with ToCV by Bemisia tabaci MEAM1 were susceptible to infection. Potato plants of cv. Camila infected by ToCV showed the lowest virus titer and were asymptomatic.

In Vitro and In Vivo Characterization of a Pigeon Paramyxovirus Type 1 Isolated from Domestic Pigeons in Victoria, Australia 2011.

Significant mortalities of racing pigeons occurred in Australia in late 2011 associated with a pigeon paramyxovirus serotype 1 (PPMV-1) infection. The causative agent, designated APMV-1/pigeon/Australia/3/2011 (P/Aus/3/11), was isolated from diagnostic specimens in specific pathogen free (SPF) embryonated eggs and was identified by a Newcastle Disease virus (NDV)-specific RT-PCR and haemagglutination inhibition (HI) test using reference polyclonal antiserum specific for NDV. The P/Aus/3/11 strain was further classified as PPMV-1 using the HI test and monoclonal antibody 617/161 by HI and phylogenetic analysis of the fusion gene sequence. The isolate P/Aus/3/11 had a slow haemagglutin-elution rate and was inactivated within 45 min at 56 °C. Cross HI tests generated an R value of 0.25, indicating a significant antigenic difference between P/Aus/3/11 and NDV V4 isolates. The mean death time (MDT) of SPF eggs infected with the P/Aus/3/11 isolate was 89.2 hr, characteristic of a mesogenic pathotype, consistent with other PPMV-1 strains. The plaque size of the P/Aus/3/11 isolate on chicken embryo fibroblast (CEF) cells was smaller than those of mesogenic and velogenic NDV reference strains, indicating a lower virulence phenotype in vitro and challenge of six-week-old SPF chickens did not induce clinical signs. However, sequence analysis of the fusion protein cleavage site demonstrated an 112RRQKRF117 motif, which is typical of a velogenic NDV pathotype. Phylogenetic analysis indicated that the P/Aus/3/11 isolate belongs to a distinct subgenotype within class II genotype VI of avian paramyxovirus type 1. This is the first time this genotype has been detected in Australia causing disease in domestic pigeons and is the first time since 2002 that an NDV with potential for virulence has been detected in Australia.

Splenectomy in zebrafish: a new model for immune thrombocytopenia

In humans, splenectomy is performed to treat many clinical disorders, including immune thrombocytopenia. However, the incidence of splenectomies for immune thrombocytopenia as a therapeutic has significantly declined over the past decade due to the availability of new therapies. Infection and sepsis as a result of splenectomies are well documented, but other long-term effects are not well characterized. Evidence suggests that persons who have had a prior splenectomy may be at an increased risk of vascular conditions. Also, elevated levels of cell-derived microparticles appear to contribute to an increased risk of thrombosis and cardiovascular disease. However, in vivo studies on the increased levels of microparticles following splenectomy are limited. In order to understand the effects of splenectomies, we developed a protocol for splenectomy in adult zebrafish. After anesthesia, the spleen was removed under a stereomicroscope after making an incision on the ventral side of the fish. The spleen was removed by pulling with forceps. The incision was closed by Vetbond tissue glue. Blood collected from both splenectomized zebrafish and those that underwent sham surgeries was immunolabeled with polyclonal antisera against αIIb, followed by flow cytometry. We observed elevated levels of thrombocytes and their microparticles in splenectomized zebrafish. Finally, by injecting αIIb antibody intravenously into zebrafish, we found the thrombocyte counts decreased, suggesting the fish developed immune thrombocytopenia like conditions, which were then reversed by splenectomy. In summary, the model developed here should be useful to study molecular changes due to splenectomy. Also, the zebrafish will be useful in modeling treatment of immune thrombocytopenia like conditions.