HEMAGGLUTINATION AND ANTIGENIC PROPERTIES OF AVIBACTERIUM PARAGALLINARUM ISOLATES RECOVERED IN THE RUSSIAN FEDERATION AND REPUBLIC OF BELARUS

The paper demonstrates examination results of antigenic properties ofAvibacterium paragallinarumreference strain and ten isolates thereof recovered in the Russian Federation and Republic of Belarus. Nine isolates and the reference strain demonstrated type L hemagglutinin (thermoliable, trypsin-sensitive, active against fresh and glutaraldehyde-treated RBC). One isolate was marked by type Hl hemagglutinin (thermoliable, trypsin-resistant, active only against glutaraldehydetreated RBC). The reference strain antigen proved to be inagglutinable by homologous antiserum, and unable to agglutinate RBCs due to hyaluronic acid in the capsular substance. Serological and hemagglutination non-reactivity was removed through the cell treatment with hyaluronidase. The agglutination test demonstrated that seven out of ten tested isolates belonged to the same serological group; herewith, the proportion of the bilateral antigenic relatedness amounted to ≥78.4%. HI results demonstrated ≥92.6% antigenic relatedness of the tested isolates being indicative of the fact that they belonged not only to the same serological group but also to the same serotype. No serological relatedness was identifed between the tested isolates and reference strain of serogroup A both using agglutination test (≤23.6%) and HI (≤12.2%). Polymerase chain reaction demonstrated that all the isolates recovered in the Russian Federation and Republic of Belarus in 2014-2016 belonged to serogroup B.

Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain

ABSTRACT Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a “treatment-resistant” and highly potent toxin. However, the toxin’s chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons. Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated “type H,” has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 107 mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a “treatment-resistant” and highly potent toxin. However, the toxin’s chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons.

Host Range, Purification, and Genetic Variability in Sweet potato chlorotic fleck virus

Sweet potato chlorotic fleck virus (SPCFV) has recently been classified as a putative new member of the genus Carlavirus (family Flexiviridae) on the basis of its molecular properties. In this study, SPCFV was characterized in terms of host range, physical and biological characteristics, and genetic variability. In addition to sweet potato, SPCFV infected some plant species in the families Convolvulaceae, Chenopodiaceae, and Solanaceae. Limited numbers of virus particles were observed in the assimilation parenchyma cells of infected plant tissues; some cells had a distorted and enlarged endoplasmic reticulum though without any cytoplasmic and amorphous inclusions. The normal length of SPCFV particles was determined to be approximately 800 nm. In enzyme-linked immunosorbent assays, polyclonal antibodies raised against purified SPCFV virions were able to detect the virus in infected sweet potato and indicator plant tissues. In immunoelectron microscopy, SPCFV particles were all strongly decorated when reacted with homologous antiserum. Comparison of the 3′ terminal part of the genome of a range of geographically diverse isolates revealed a high level of genetic diversity. The amino acid sequence identity in the coat protein and the nucleic acid binding protein ranged from 89 to 99.7% and from 75.9 to 99.2%, respectively. Phylogenetic analysis of both proteins showed a geographically associated clustering into two genogroups.

The use of sorbitol-MacConkey agar in conjunction with a specific antiserum for the detection of Vero cytotoxin-producing strains ofEscherichia coliO 157

SUMMARYUsing DNA probes specific for the genes encoding Vero cytotoxins 1 and 2 in hybridization experiments on faecal samples. Vero cytotoxin-producingEscherichia coli(VTEC) of serogroup 0 157 were detected in 21 of 63 cases of haemorrhagic colitis, 9 of 31 cases of non-bloody diarrhoea and 14 of 68 cases of haemolytic uraemic syndrome. Compared with these results sorbitol-MacConkey agar in conjunction with a specific 0 157 antiserum gave a sensitivity of 62% in haemorrhagic colitis, 56% in non-bloody diarrhoea and 57% in haemolytic uraemic syndrome.The specificity of this method was 100% in all three groups. This demonstrates that sorbitol-MacConkey agar is a useful screening method for the detection of VTEC of serogroup O 157 when used in conjunction with a specific homologous antiserum. However, this method does not detect VTEC belonging to other serogroups and such strains were found, particularly in cases of haemolytic uraemic syndrome.

Absence of relaxin immunostaining in the male reproductive tracts of the rat and mouse.

By use of the biotin-avidin immunohistochemical method and a homologous antiserum as the primary antiserum, relaxin immunostaining was absent in the testes, prostate, seminal vesicles, and epididymides of the rat. Relaxin immunostaining was also lacking when anti-porcine relaxin serum was employed as the primary antiserum. Furthermore, immunohistochemical studies for relaxin localization in the reproductive tract of the male mouse using both anti-rat and anti-porcine relaxin sera also revealed an absence of the hormone in the reproductive system of this species. Although this study suggests that immunoreactive relaxin is absent in the male reproductive tracts of both the rat and mouse, it raises some questions concerning the reports in the literature of the presence of relaxin-like substances in the male reproductive tracts of other species. These reports are discussed in relation to our current results.

Evaluation of Hypocalcemia with a Highly Sensitive, Homologous Radioimmunoassay for the Midregion of Parathyroid Hormone

The diagnosis of hypoparathyroidism by radioimmunoassay of serum parathyroid hormone (PTH) has been hampered by lack of an assay system sensitive enough to allow discrimination between low and normal values. A new assay for human PTH that has improved sensitivity has been developed. It uses an homologous antiserum (against the human hormone) and uses a carboxy-terminal fragment of bovine PTH as tracer to provide an assay monospecific for the midregion of PTH. Immunoreactive PTH (iPTH) was detectable in 25/27 normal children and borderline detectable in the other two. The pediatric normal range was slightly lower than that previously established in adults. Among patients with secondary hyperparathyroidism and normal renal function, iPTH was 3- to 8-fold elevated in those with rickets, and 1.3- to 2.0-fold above normal in those with more acute forms of hypocalcemia. Twelve patients with hypoparathyroidism were studied. iPTH was undetectable in seven with permanent total hypoparathyroidism, and was borderline detectable in five, including four neonates who proved to have transient hypoparathyroidism. In these four patients, iPTH became detectable as the requirements for supplemental calcium decreased. Measurement of iPTH with an adequately sensitive assay may be useful in the diagnosis and management of pediatric hypocalcemia.

INFLUENCE OF SELECTED HERBICIDES IN PHAGES OF SOME SOIL BACTERIA

Various concentrations of paraquat, atrazine, simazine, linuron, diuron, and paraquat in combinations with each including simazine + diuron, and terbacil alone, did not inhibit lytic activity of four bacteriophages of Agrobacterium radiobacter, three bacteriophages of Rhizobium meliloti, three bacteriophages of R. trifolii, or two bacteriophages of Streptomyces chrysomallus. Generally, the herbicides had no effect on the neutralization of radiobacterphage PR-1001 with its homologous antiserum, the length of the latent period, the percent adsorption or the average burst size. In contrast, paraquat concentrations from 20 to 400 μg∙mL−1 gradually reduced the adsorption from 38 to 21% and the average burst size from 67 to 9 in the PR-1001:R-1001 phage: host system. The same concentrations, however, showed no effect on the particle attachment or the length of the latent period.