Polyclonal Antiserum Research Articles

Purification of Anti-OSH1 IgG and Immunohistochemical Staining of Rice Shoot Apical Meristem.

Immunohistochemical staining (IHC) enables the visualization of protein localization within tissues and organs. Here, we describe IHC to detect the ORYZA SATIVA HOMEOBOX1 (OSH1) protein in sections of rice shoot apical meristems (SAMs). First, anti-OSH1 IgG is purified from OSH1 polyclonal rabbit antisera. For subsequent IHC, rice shoot apices are fixed and embedded in paraffin and sections are prepared for IHC. In this IHC procedure, two different labeling methods, fluorescein isothiocyanate and alkaline phosphatase, are utilized for secondary antibody detection.

Prokaryotic Expression of Coat Protein Gene of Grapevine Berry Inner Necrosis Virus and Preparation of Its Polyclonal Antibody

Grapevine berry inner necrosis virus (GINV) and grapevine Pinot gris virus (GPGV) are prevalent viral diseases affecting viticulture, posing significant threats in grape-producing regions of China. Previous studies have emphasized the harmful effects of grape viruses on the grape industry all over the world. However, few reports have focused specifically on GINV. In wild grapevines, GINV infection frequently leads to grapevine fanleaf degeneration disease (GFDD). GINV often co-occurs with other grape viruses, exacerbating its harmful effects on the grapevine industry in China. In this study, we collected grapevine samples from Taizhou city, Jiangsu Province, where GINV infection was confirmed. Based on the GINV coat protein (CP) gene, we developed a high-throughput and high-sensitivity direct antigen-coated ELISA and Dot blot assay for field diagnosis of GINV CP in grape samples. The CP gene was cloned from GINV-infected grape samples, and the GINV CP was expressed using the pET30(a) vector. Specific polyclonal antiserum CPGINV was generated by immunizing rabbits with the purified protein, and its sensitivity was determined to be satisfactory. Leveraging the high accuracy and sensitivity of the CPGINV antiserum, we developed a rapid, precise, and scalable diagnostic method for GINV in the grapevine industry. The established ELISA and Dot blot assays successfully detected GINV-infected grapevine samples. The occurrence of GINV is relatively common in China, which poses a risk of transmission and threatens the healthy development of the grape industry. Therefore, this study prepared CPGINV antiserum and established an efficient, rapid, sensitive, accurate, and high-throughput diagnostic method, providing a foundational approach for the prevention and control of vitis viral diseases.

C1QTNF Related protein 8 (CTRP8) is a marker of myeloid derived innate immune cell populations in the human breast cancer microenvironment.

Innate immune cells in the tumor microenvironment (TME) play an important role in breast cancer (BC) metastatic spread and influence patient survival. Macrophages differentiate along a proinflammatory M1 to protumorigenic M2 phenotype spectrum which affects distinct functions, like angiogenesis and cytokine production, and modulates BC aggressiveness and affects patient survival. Mast cells (MCs) are myeloid derived cells that serve as the first line of innate immune defense but their role in the TME of BC is not well understood. In this study, we have identified a subpopulation of innate immune cells that shows strong immunopositivity for the least studied adipokine CTRP8. Using a new and highly specific polyclonal antiserum on patient BC tissues, we identify a subset of tryptase + MCs and CD68 + macrophages co-expressing immunoreactive CTRP8. In M1 polarized THP-1 myeloid cells, this adipokine stimulated increased secretion of pro-inflammatory cytokines and elevated expression of the relaxin/ CTRP8 receptor RXFP1. Comparative analysis of secreted cytokine profiles in THP-1 M1 macrophages exposed to either CTRP8, relaxin-2 (RLN2), or the small molecule RXFP1 agonist ML-290 revealed ligand-specific cytokine signatures. Our study identified novel subsets of CTRP8 + myeloid derived innate immune cells and links this adipokine to pro-inflammatory events in the TME of BC.

Preparation of polyclonal antiserum to Tomato mosaic virus and its application as a viral diagnostic test

Preparation of polyclonal antiserum to Tomato mosaic virus and its application as a viral diagnostic test

Serotype 15C Streptococcus pneumoniae resistant to classical complement deposition and agglutination by polyclonal rabbit anti-capsular 15B sera

BackgroundS. pneumoniae (SP) serotypes 15B and 15C are frequent causative agents of invasive pneumococcal disease (IPD), otitis media, and nasopharyngeal colonization in the post PCV13 era. The principal difference between serotypes 15B and 15C is the presence of an O-acetyl group on the pentasaccharide repeating unit of 15B polysaccharide. It remains unclear if antibodies against SP15B polysaccharide demonstrate functional cross reaction with SP15C strains. We compared functional activity of polyclonal rabbit anti-capsular 15B sera against SP15B and SP15C isolates. MethodsUsing flow cytometry we measured complement factors C3c and C4d deposition on SP15B and 15C in the presence of polyclonal rabbit anti capsular 15B sera. We measured the binding of C3c, common to all complement pathways, and C4d, specific to classical pathway, on SP serotypes 15B and 15C when co-incubated with polyclonal rabbit anti capsular 15B sera and antibody depleted complement. Both the proportion of bacteria with complement deposition and the fluorescence intensity were measured. We also measured agglutination as the increase in forward and side scatter. ResultsPolyclonal rabbit anti-capsular 15B sera activated classical pathway resulting in deposition of C4d and C3c at high intensity on all SP15B cells but only achieved limited deposition and intensity of C4 with SP15C. Similarly, polyclonal rabbit anti-capsular 15B sera induced agglutination of SP15B strains in a dose dependent manner and limited agglutination of SP15C. ConclusionsAnti-capsular 15B sera induce limited C4d and C3c deposition, and minimal agglutination with SP15C strains, reflecting lower classical pathway activation in contrast to high C4d and C3c deposition and agglutination of SP15B. These observations support limited functional cross reactivity of anti-15B to SP15C strains and are consistent with the reduction in opsonophagocytic killing of SP15C reported following immunization with vaccines containing 15B polysaccharide.

Characterization of Streptococcus agalactiae 1a isolated from farmed Nile tilapia (Oreochromis niloticus) in North America, Central America, and Southeast Asia

Streptococcosis caused by Streptococcus agalactiae 1a in Nile tilapia (Oreochromis niloticus) is a severe disease challenge for the global supply of tilapia. Currently, the extensive use of antibiotics is the primary curative tool used to minimize the impact of the disease. Vaccination is a prophylactic measure that has been shown to reduce antibiotic use in the aquaculture sector substantially. However, no commercially licensed vaccine against Streptococcus agalactiae 1a is currently available.This study aimed to investigate, through molecular and immunological methods, if Streptococcus agalactiae 1a isolates collected from North America (NAM), Central America (CAM), and Southeast Asia (SEA) were similarly suitable for the development of a potentially effective vaccine to serve the global tilapia farming industry. Our comparative data showed that the Streptococcus agalactiae 1a isolates from NAM, CAM and SEA had similar biochemical profiles, and genetic multi-locus sequence typing (MLST) analysis showed that the NAM and CAM isolates belonged to sequence type 7 (ST-7) and clonal complex 1, while isolates from SEA grouped into three sequence types (ST-1650, ST-500, and ST-7) and two distinct clonal complexes (CC1 and CC12). Isolates from NAM, CAM, and SEA displayed similar antigenic profiles determined by western blotting with polyclonal rabbit antisera, which was supported by in vivo cross-protection studies, showing that fish immunized with vaccines based on SEA and CAM isolates with different genetic MLST profiles were highly protected against cross-challenge using the same bacterial strains for challenge. Overall, the data obtained from our investigations provide strong indications that Streptococcus agalactiae 1a distributed in NAM, CAM, and SEA are serologically uniform pathogens, and vaccines based on isolates of Streptococcus agalactiae 1a from these regions may be suited for vaccination of tilapia worldwide.

A human monoclonal antibody based immunoenzymetric assay to measure Fel d 1 concentrations in cat hair and pelt allergenic extracts.

In the United States, 19 allergen extracts of different specificities are standardized, which means that their potencies are determined in comparison to a US reference standard. For cat allergen extracts, potency is determined by measuring Fel d 1 content expressed in in Fel d 1 units, and with a unitage that correlates with skin test reactions (bioequivalent allergy units or BAU). Currently, Fel d 1 content is measured with a radial immunodiffusion (RID) assay that uses polyclonal sheep antisera to detect the allergenic protein by producing a white precipitin line in agar gel. However, the RID is considered cumbersome, and the polyclonal sera may qualitatively vary among animals and may recognize epitopes irrelevant to human allergic disease. In this report, we describe a quantitative two-site immunoenzymetric assay (IEMA) for Fel d 1 that uses immobilized capture and soluble biotin-labeled detection Fel d 1-specific human IgE monoclonal antibodies (mAb) that have been class-switched to IgG4. Together, they sandwich Fel d 1 molecules from extracts. Using purified natural Fel d 1 as a calibrator, the historically reported ∼4 micrograms Fel d 1/Fel d 1 unit assignment was directly measured in this mAb-based IEMA at 3.12 ± 0.24 micrograms of Fel d 1 per Fel d 1 unit. This IEMA appears to be equivalent to RID in the measurement of biological potencies of commercial cat hair and cat pelt extracts marketed in the United States.

CTXP, The Major Cobra Toxin Peptide from Naja Naja Oxiana Venom; A Promising Target for Antivenom Agent Development.

Snakebite envenoming is a serious public health issue causing more than 135,000 annual deaths worldwide. Naja Naja Oxiana is one of the most clinically important venomous snakes in Iran and Central Asia. Conventional animal-derived polyclonal antibodies are the major treatment of snakebite envenoming. Characterization of venom components helps to pinpoint the toxic protein responsible for clinical manifestations in victims, which aids us in developing efficient antivenoms with minimal side effects. Therefore, the present study aimed to identify the major lethal protein of Naja Naja Oxiana by top-down proteomics. Venom proteomic profiling was performed using gel filtration (GF), reversed-phase (RP) chromatography, and intact mass spectrometry. The toxicity of GF-, and RP-eluted fractions was analyzed in BALB/c mice. The rabbit polyclonal antisera were produced against crude venom, GF fraction V (FV), and RP peak 1 (CTXP) and applied in neutralization assays. Toxicity studies in BALB/c identified FV as the major toxic fraction of venom. Subsequently, RP separation of FV resulted in eight peaks, of which peak 1, referred to as "CTXP" (cobra toxin peptide), was identified as the major lethal protein. In vivo neutralization assays using rabbit antisera showed that polyclonal antibodies raised against FV and CTXP are capable of neutralizing at least 2-LD50s of crude venom, FV, and CTXP in all tested mice. Surprisingly, the Anti-CTXP antibody could neutralize 8-LD50 of the CTXP peptide. These results identified CTXP (a 7 kDa peptide) as a potential target for the development of novel efficient antivenom agents.

Human antibodies neutralizing the alpha-latrotoxin of the European black widow.

Poisoning by widow-spider (genus Latrodectus) bites occurs worldwide. The illness, termed latrodectism, can cause severe and persistent pain and can lead to muscle rigidity, respiratory complications, and cardiac problems. It is a global health challenge especially in developing countries. Equine serum-derived polyclonal anti-sera are commercially available as a medication for patients with latrodectism, but the use of sera imposes potential inherent risks related to its animal origin. The treatment may cause allergic reactions in humans (serum sickness), including anaphylactic shock. Furthermore, equine-derived antivenom is observed to have batch-to-batch variability and poor specificity, as it is always an undefined mix of antibodies. Because latrodectism can be extremely painful but is rarely fatal, the use of antivenom is controversial and only a small fraction of patients is treated. In this work, recombinant human antibodies were selected against alpha-latrotoxin of the European black widow (Latrodectus tredecimguttatus) by phage display from a naïve antibody gene library. Alpha-Latrotoxin (α-LTX) binding scFv were recloned and produced as fully human IgG. A novel alamarBlue assay for venom neutralization was developed and used to select neutralizing IgGs. The human antibodies showed in vitro neutralization efficacy both as single antibodies and antibody combinations. This was also confirmed by electrophysiological measurements of neuronal activity in cell culture. The best neutralizing antibodies showed nanomolar affinities. Antibody MRU44-4-A1 showed outstanding neutralization efficacy and affinity to L. tredecimguttatus α-LTX. Interestingly, only two of the neutralizing antibodies showed cross-neutralization of the venom of the Southern black widow (Latrodectus mactans). This was unexpected, because in the current literature the alpha-latrotoxins are described as highly conserved. The here-engineered antibodies are candidates for future development as potential therapeutics and diagnostic tools, as they for the first time would provide unlimited supply of a chemically completely defined drug of constant quality and efficacy, which is also made without the use of animals.

Immunization with PfGBP130 generates antibodies that inhibit RBC invasion by P. falciparum parasites.

Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions. To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies. We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.

Dry and liquid formulations of IBT-V02, a novel multi-component toxoid vaccine, are effective against Staphylococcus aureus isolates from low-to-middle income countries.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) in the U.S. as well as more serious invasive diseases, including bacteremia, sepsis, endocarditis, surgical site infections, osteomyelitis, and pneumonia. These infections are exacerbated by the emergence of antibiotic-resistant clinical isolates such as methicillin-resistant S. aureus (MRSA), highlighting the need for alternatives to antibiotics to treat bacterial infections. We have previously developed a multi-component toxoid vaccine (IBT-V02) in a liquid formulation with efficacy against multiple strains of Staphylococcus aureus prevalent in the industrialized world. However, liquid vaccine formulations are not compatible with the paucity of cold chain storage infrastructure in many low-to-middle income countries (LMICs). Furthermore, whether our IBT-V02 vaccine formulations are protective against S. aureus isolates from LMICs is unknown. To overcome these limitations, we developed lyophilized and spray freeze-dried formulations of IBT-V02 vaccine and demonstrated that both formulations had comparable biophysical attributes as the liquid formulation, including similar levels of toxin neutralizing antibodies and protective efficacy against MRSA infections in murine and rabbit models. To enhance the relevancy of our findings, we then performed a multi-dimensional screen of 83 S. aureus clinical isolates from LMICs (e.g., Democratic Republic of Congo, Palestine, and Cambodia) to rationally down-select strains to test in our in vivo models based on broad expression of IBT-V02 targets (i.e., pore-forming toxins and superantigens). IBT-V02 polyclonal antisera effectively neutralized toxins produced by the S. aureus clinical isolates from LMICs. Notably, the lyophilized IBT-V02 formulation exhibited significant in vivo efficacy in various preclinical infection models against the S. aureus clinical isolates from LMICs, which was comparable to our liquid formulation. Collectively, our findings suggested that lyophilization is an effective alternative to liquid vaccine formulations of our IBT-V02 vaccine against S. aureus infections, which has important implications for protection from S. aureus isolates from LMICs.

Development of lateral flow immunoassay strip for rapid detection of acute hepatopancreatic necrosis disease-causing PirAB toxins in shrimp

Acute hepatopancreatic necrosis disease (AHPND) is currently the most affected disease of shrimp farms worlwide As rapid and specific detection methods are essential to improve disease management, a lateral flow immunoassay (LFIA) was designed for the detection of PirAvp and PirBvp toxins in shrimp. In this study, polyclonal antibody based lateral flow immunoassay (LFIA) strip has been developed. The recombinant PirAvp and PirBvp toxin‐like proteins of Vibrio parahaemolyticus (VPAHPND) was used to immunize rabbits with the highest antibody titre after the third booster. This polyclonal rabbit antiserum was purified and used to develop a LFIA strip for the detection of AHPND pathogen. This test strip could detect PirAvp and PirBvp toxins up to 125 ng/mL by naked eye within 15 min. No cross-reactivity was observed with VPnon-AHPND. Furthermore, the sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. This LFIA strip could be used as a handy tool by the farmers and technicians in situ. ® 2023 Journal of Science and Technology - NTTU

Evaluation of species-specific polyclonal antibodies to detect and differentiate between Neospora caninum and Toxoplasma gondii.

Neosporosis and toxoplasmosis are major causes of abortion in livestock worldwide, leading to substantial economic losses. Detection tools are fundamental to the diagnosis and management of those diseases. Current immunohistochemistry (IHC) tests, using sera raised against whole parasite lysates, have not been able to distinguish between Toxoplasma gondii and Neospora caninum. We used T. gondii and N. caninum recombinant proteins, expressed in Escherichia coli and purified using insoluble conditions, to produce specific polyclonal rabbit antisera. We aimed to develop species-specific sera that could be used in IHC on formalin-fixed, paraffin-embedded (FFPE) tissue sections to improve the diagnosis of ruminant abortions caused by protozoa. Two polyclonal rabbit sera, raised against recombinant proteins, anti-Neospora-rNcSRS2 and anti-Toxoplasma-rTgSRS2, had specificity for the parasite they were raised against. We tested the specificity for each polyclonal serum using FFPE tissue sections known to be infected with T. gondii and N. caninum. The anti-Neospora-rNcSRS2 serum labeled specifically only N. caninum-infected tissue blocks, and the anti-Toxoplasma-rTgSRS2 serum was specific to only T. gondii-infected tissues. Moreover, tissues from 52 cattle and 19 sheep previously diagnosed by lesion profiles were tested using IHC with our polyclonal sera and PCR. The overall agreement between IHC and PCR was 90.1% for both polyclonal anti-rNcSRS2 and anti-rTgSRS2 sera. The polyclonal antisera were specific and allowed visual confirmation of protozoan parasites by IHC, but they were not as sensitive as PCR testing.

A or AB? Resolving the dilemma of weaker subgroup through serological evaluation: Reporting two cases from Uttarakhand

Abstract Blood group discrepancies and their resolution can sometimes become tricky when it comes to weaker subgroups. A number of weaker B subgroups, which are very infrequently encountered, have been described, including B3, Bx, Bm, and Bel. The varied serologic reactivity of these subgroups, using routine human polyclonal antisera, usually startles the personnel involved in such blood group discrepancies and interpretation is either delayed or incorrect. Here, we present two cases of AB Rh (D) positive with weak B subgroups, detected in two whole blood donors. Their red cells showed strong agglutination with anti-A, anti-AB, and anti-D, no reaction with anti-B, and serum showed no reaction with A1, A2, B, and O cells. Adsorption-elution procedure and salivary testing suggested the presence of a weaker B subgroup (Bel). Hence, ABel subgroup was reported for both the donors. Resolution of blood group discrepancies is crucial, as it would aid to prevent hemolytic transfusion reactions due to inaccurate blood group interpretation.

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies.

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.

Development of lateral flow immunoassay strip for rapid detection of acute hepatopancreatic necrosis disease-causing PirAB toxins in shrimp

Acute hepatopancreatic necrosis disease (AHPND) is currently the most affected disease of shrimp farms worlwide As rapid and specific detection methods are essential to improve disease management, a lateral flow immunoassay (LFIA) was designed for the detection of PirAvp and PirBvp toxins in shrimp. In this study, polyclonal antibody based lateral flow immunoassay (LFIA) strip has been developed. The recombinant PirAvp and PirBvp toxin‐like proteins of Vibrio parahaemolyticus (VPAHPND) was used to immunize rabbits with the highest antibody titre after the third booster. This polyclonal rabbit antiserum was purified and used to develop a LFIA strip for the detection of AHPND pathogen. This test strip could detect PirAvp and PirBvp toxins up to 125 ng/mL by naked eye within 15 min. No cross-reactivity was observed with VPnon-AHPND. Furthermore, the sensitivity and specificity of LFIA were 94.0% and 98.0%, respectively. This LFIA strip could be used as a handy tool by the farmers and technicians in situ.
 ® 2023 Journal of Science and Technology - NTTU

Non-invasive assessment of hormonal fluctuations during pregnancy in guanacos (Lama guanicoe) and its application in a wild population.

Obtaining endocrinological profiles using non-invasive methodologies by the measurement of hormone fecal metabolites is a widely used method to monitor ovarian activity and pregnancy in wild species. These tools allow the obtention of physiological information without causing capture-related stress on the individuals. In this research, we aimed to 1) biologically validate a non-invasive method to assess fecal progestagens and estrogens fluctuations during gestation in guanacos (Lama guanicoe) and 2) apply this technique to assess pregnancy in a wild free-ranging population. Fecal samples were collected through the gestation period (~12months) of female guanacos in a 6.5-ha paddock. An increase in fecal metabolites of both hormones was detected. Progestagens increased gradually, in contrast to estrogens, which remained at basal values for most of the gestation period and peaked only a few days before calving. To assess pregnancy in wild free-ranging animals, fecal samples were collected from a population of La Payunia provincial reserve (Mendoza, Argentina) during the beginning of gestation and at the end of gestation. Through the first months of possible gestation, pregnant females represented between 40 and 80% of the population; at the end of gestation, only 20-40% of the females had confirmed pregnancies. Our results demonstrated that the polyclonal antisera and sexual hormone metabolite assays used here detect variations in the metabolites excreted through feces in guanacos and provide the possibility of non-invasive hormone monitoring of female reproductive status. Also, the findings in wild conditions suggest that natural abortions could have occurred during the first months of gestation. Although some abortions may be natural, the harsh environmental conditions that challenge the support of such a long gestational process may be another relevant factor to consider. The results obtained here enhance our understanding of the reproductive physiology of one of the most emblematic ungulates in South America.

Use of Reductive Amination to Produce Capsular Polysaccharide-Based Glycoconjugates.

Reductive amination is a relatively simple and convenient strategy for coupling purified polysaccharides to carrier proteins. Following their synthesis, glycoconjugates can be used to assess the protective capacity of specific microbial polysaccharides in animal models of infection and/or to produce polyclonal antiserum and monoclonal antibodies for a variety of immune assays. Here, we describe a reproducible method for chemically activating the 6-deoxyheptan capsular polysaccharide (CPS) from Burkholderia pseudomallei and covalently linking it to recombinant CRM197 diphtheria toxin mutant (CRM197) to produce the glycoconjugate, CPS-CRM197. Similar approaches can also be used to couple other types of polysaccharides to CRM197 with little to no modification of the protocol.

Distribution of Papaya Ring Spot Virus Infecting Papaya in Kerala, India

In the midst of the rising interest of papaya cultivation in Kerala, it is inevitable to study the current status of the Papaya ring spot disease in the state. Knowing the severity and distribution of PRSV isolates, causing ring spot disease, helps in the better formulation of effective management strategies against it. Purposive sampling surveys carried out in 2021-22 across five agro ecological units (AEUs) of Kerala, recorded papaya ring spot disease incidence (PRSD) ranging from 50 to 100 per cent in the cultivated areas with vulnerability index of 33.54 to 98.22. Highest disease incidence was recorded from AEU 8- Southern Laterites: Thiruvananthapuram (100.0%) and highest vulnerability index was recorded from Kalliyoor (98.22). Twenty symptomatic samples tested positive for PRSV in double antibody sandwich- ELISA (DAS-ELISA) using PRSV polyclonal antiserum. RT-PCR using coat protein gene specific primers RKJ 52 and 3 yielded amplicons of size approximately ~890bp in all the samples. Virulence of the Kerala isolates was evaluated based on the symptom expression, disease incidence and vulnerability index, upon mechanical transmission of PRSV on two months old papaya plants (variety- Red Lady). PRSV isolate from Kalliyoor (TVM1) inoculated on papaya plants expressed chlorosis, mottling, malformation of leaves and stunting with 96.80 vulnerability index. Maximum vulnerability index and severe symptoms including stunting in the inoculated plants were observed in isolates from southern and central Kerala, which include Thiruvananthapuram, Alappuzha and Thrissur had the most virulent PRSV isolates compared to north Kerala.

Concurrent colonization of rodent kidneys with multiple species and serogroups of pathogenic Leptospira.

Rodents are important reservoir hosts of pathogenic leptospires in the US Virgin Islands. Our previous work determined that trapped rodents were colonized with Leptospira borgpetersenii serogroup Ballum (n = 48) and/or Leptospira kirschneri serogroup Icterohaemorrhagiae (n = 3). In addition, nine rodents appeared to be colonized with a mixed population comprising more than one species/serogroup. The aim of this study was to validate this finding by characterizing clonal isolates derived from cultures of mixed species. Cultures of presumptive mixed species (designated LR1, LR5, LR37, LR57, LR60, LR61, LR68, LR70, and LR72) were propagated in different media including Hornsby-Alt-Nally (HAN) media, incubated at both 29℃ and 37℃, and T80/40/LH incubated at 29℃. Polyclonal reference antisera specific for serogroup Ballum and Icterohaemorrhagiae were used to enrich for different serogroups followed by subculture on agar plates. Individual colonies were then selected for genotyping and serotyping. Of the nine cultures of mixed species/serogroups, a single clonal isolate was separated in five of them: L. borgpetersenii serogroup Ballum in LR1, LR5, and LR37, and L. kirschneri serogroup Icterohaemorrhagiae in LR60 and LR72. In four of the cultures with mixed species (LR57, LR61, LR68, and LR70), clonal isolates of both L. borgpetersenii serogroup Ballum and L. kirschneri serogroup Icterohaemorrhagiae were recovered. Our results definitively establish that rodents can be colonized with more than one species/serogroup of Leptospira concurrently. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations. IMPORTANCE Pathogenic Leptospira, the causative agent of human and animal leptospirosis, comprise a diverse genus of species/serogroups which are inherently difficult to isolate from mammalian hosts due to fastidious growth requirements. Molecular evidence has indicated that reservoir hosts of Leptospira may shed multiple species concurrently. However, evidence of this phenomena by culture has been lacking. Culture is definitive and is essential for comprehensive characterization of recovered isolates by high-resolution genome sequencing and serotyping. In this work, a protocol using recently developed novel media formulations, in conjunction with reference antisera, was developed and validated to demonstrate the recovery of multiple species/serogroups of pathogenic Leptospira from the same host. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.