Comparative study of cellulose waste versus organic waste as substrate in a sulfate reducing bioreactor.

The biodegradability and comparative effectiveness in treatment of acid mine drainage of ten locally available organic waste materials were examined. pH of AMD increased from 2.70 to 6.25, 7.10 and 7.50 with buffalo, cow and goat manures whereas cellulosic wastes increased the pH within the range of 4.83-5.32 in laboratory scale single substrate bioreactors. Significant reduction was observed in Eh, acidity and sulfate with manures in treated AMD. Maximum metal removal efficiency was 99.3%, 99.9%, 99.8%, 99.1%, 99.1%, and 73.8% for Fe, Cu, Zn, Ni, Co and Mn in maximum retention period of 10 days. The highest efficiency of metal removal was observed in bioreactors with manures as single substrate. The effectiveness of substrate depends on its biodegradation ability, the results with cellulosic waste indicates it may need more than 10 days to biodegrade. Biodegradability of organic waste was evaluated according to COD/SO(4)(2-) and C/N ratio and the ratios of 0.48-0.57 and 22.22-23.00 respectively were adequate parameters for activity of sulfate reducing bacteria and pollutant removal efficiency.

Cryptosporidium parvum DNA replication in cell-free culture.

The lack of robust methods for culturing Cryptosporidium parasites remains a major challenge and is hampering efforts to screen for anti-cryptosporidial drugs. In existing culture methods, monolayers of mammalian epithelial cells are inoculated with oocysts. The system supports an initial phase of asexual proliferation of the parasite. For reasons that are not clear, development rapidly declines within 2-3 days. The unexpected report of Cryptosporidium parvum culture in the absence of host cells, and the failure of others to reproduce the method, prompted us to apply quantitative PCR to measure changes in C. parvum DNA levels in cell-free cultures, and parasite-specific antibodies to identify different life cycle stages. Based on this approach, which has not been applied previously to analyze C. parvum growth in cell-free culture, we found that the concentration of C. parvum DNA increased by about 5-fold over 5 days of culture. Immuno-labeling of cultured organisms revealed morphologically distinct stages, only some of which reacted with Cryptosporidium-specific monoclonal antibodies. These observations are indicative of a modest proliferation of C. parvum in cell-free culture.

Characterization of a human monoclonal antibody against Shiga toxin 2 expressed in Chinese hamster ovary cells.

Shiga toxin-producing Escherichia coli infections can often lead to the development of hemolytic-uremic syndrome (HUS) in a small percentage of infected humans. Patients with HUS receive only supportive treatment as the benefit of antibiotic therapy remains uncertain. We have previously reported the generation and preclinical evaluation of neutralizing human monoclonal antibodies (HuMAbs) against the Shiga toxins (Stx). In this paper, we describe the expression in Chinese hamster ovary (CHO) cells of 5C12 HuMAb, which is directed against the A subunit of Stx2. The cDNAs of the light and heavy chain immunoglobulin (Ig) variable regions of 5C12 HuMAb were isolated and cloned into an expression vector containing human IgG1 constant regions. The vector was transfected into CHO cells, and transfectants secreting Stx2-specific antibody were screened by an Stx2-specific enzyme-linked immunosorbent assay. The CHO-produced recombinant 5C12 (r5C12) showed similar specificity and binding affinity to Stx2 as the parent hybridoma-produced 5C12. More significantly, the r5C12 displayed the same neutralizing activity as the parent 5C12 in vitro and in vivo. In the mouse toxicity model, both antibodies significantly and equally prolonged survival at a dose of 0.312 microg/mouse. The data showed that since r5C12, produced in CHO cells, was equally effective as the parent 5C12, it is our choice candidate as a potential prophylactic or therapeutic agent against hemolytic-uremic syndrome.

Nasal mucosal immunogenicity for the horse of a SeM peptide of Streptococcus equi genetically coupled to cholera toxin.

The intranasal immunogenicity of cholera toxin (CT) genetically coupled to peptide sequence aa236-334 (F3) of the SeM protein of Streptococcus equi was studied in five young adult Welsh ponies. All ponies made rapid CTB- and SeMF3-specific serum antibody responses following the first immunization. Specific nasal IgA responses were detected in two ponies 14 days after the first immunization, in another two 14 days after a second immunization on day 14, and in all ponies 28 days after a third immunization on day 42. SeMF3-specific antibody responses in sera and nasal washes were dominated by IgGb and IgA, respectively, and remained elevated for at least 140 days. Strong serum IgGa and IgG(T) responses were also observed. These antibody responses were qualitatively similar to those induced during recovery from equine strangles. Antibody responses in mucosal secretions were boosted in some ponies by immunizations subsequent to the first immunization, but antibodies in serum were never boosted. In vitro survival of S. equi was significantly reduced by SeMF3-specific antibodies in sera obtained 14 days after the second immunization but survival increased in sera collected following subsequent immunizations, possibly due to absence of synthesis of high affinity antibodies. Finally, the susceptibility of all immunized ponies to commingling challenge by S. equi indicated either that SeMF3 lacks protective epitopes or that the antibodies induced by the chimera were not at effective levels.

Characterization and immunogenicity of pyrogenic mitogens SePE-H and SePE-I of Streptococcus equi.

Two pyrogenic mitogens, SePE-H and SePE-I, were characterized in Streptococcus equi, the cause of equine strangles. SePE-H and SePE-I have molecular masses of 27.5 and 29.5 kDa, respectively, and each is almost identical to its counterpart in Streptococcus pyogenes M1. Both genes are adjacent to a gene encoding a phage muramidase of 49.7 kDa and are located immediately downstream from a phage genomic sequence almost identical to a similar phage sequence in S. pyogenes M1. Strong mitogenic responses were elicited by both proteins from horse peripheral blood mononuclear cells. However, although both were pyrogenic for rabbits, only SePE-I was pyrogenic in ponies. Convalescent sera contained antibody to each mitogen and horses recovered from strangles or immunized with SePE-I were resistant to the pyrogenic effect of SePE-I. The immunogenicity of SePE-I suggests that it should be included in new generation strangles vaccines. In isolates of S. equi sepe-I and sepe-H were consistently present but they were absent from the closely related Streptococcus zooepidemicus, suggesting that phage mediated transfer was an important event in the formation of the clonal, more virulent, S. equi from its putative S. zooepidemicus ancestor.

Intranasal immunogenicity of a Delta cya Delta crp-pabA mutant of Salmonella enterica serotype Typhimurium for the horse.

The aim of this study was to investigate the intranasal immunogenicity for the horse of a Deltacya Deltacrp-pabA mutant (MGN-707) of Salmonella enterica serotype Typhimurium (S. typhimurium). MGN-707 caused no sign of disease, was not detected in feces and a single administration induced strong Salmonella-specific serum and nasal mucosal antibody responses. All ponies had made strong salmonella specific serum IgGa, IgGb, IgA and IgM antibody responses by day 25 after the first immunization. IgM responses to salmonella lipopolysaccharide (LPS) were short lived whereas salmonella specific serum IgGa and IgGb persisted at high levels in all ponies until 83 and 140 days, respectively. Specific nasal mucosal antibody responses dominated by IgA and IgM were evident by day 25 in all ponies except one in which only specific IgGa and IgGb were evident. Specific nasal mucosal IgA persisted in most ponies until day 69. A second immunization on day 140 boosted antibody responses, and stimulated a strong nasal mucosal IgA response in the pony that failed to make an IgA response after primary immunization. At the termination of the experiment, IgA and IgGb dominated jejunal antibody responses whereas vaginal responses were mainly IgA. The latter response unequivocally confirms the existence of a common mucosal immune system in equids. The results indicate that a S. typhimurium Deltacya Deltacrp-pabA mutant has potential as an intranasal vaccine against salmonellosis in the horse.

M cells and associated lymphoid tissue of the equine nasopharyngeal tonsil.

The aim of this study was to characterise the morphological and histochemical features of equine nasopharyngeal tonsillar tissue. Nasal and oropharyngeal tonsillar tissue has been described as the gatekeeper to mucosal immunity because of its strategic location at the entrance to the respiratory and alimentary tracts. A combination of light, scanning and transmission electron microscopy has revealed the presence of follicle-associated epithelium (FAE) overlying lymphoid tissue of the equine nasopharyngeal tonsil caudal to the pharyngeal opening of the guttural pouch. Membranous microvillus (M) cells were identified in the FAE on the basis of short microvilli, an intimate association with lymphocytes, cytoplasmic vimentin filaments and epitopes on the apical surface reactive with lectin GS I-B4 specific for alpha-linked galactose. CD4-positive lymphocytes were scattered throughout the lamina propria mucosae as well as forming dense aggregates in the subepithelial part. The central follicular area was heavily populated with B lymphocytes and the dome and parafollicular areas contained both CD4- and CD8-positive lymphocytes. CD8-positive lymphocytes were also present in the epithelium and, together with B lymphocytes, in small numbers in the lamina propria mucosae. These observations indicate that the nasopharyngeal tonsil is potentially an important mucosal immune induction site in the horse and an appropriate target for intranasally administered vaccines.

Intranasal immunogenicity of a Deltacya Deltacrp-pabA mutant of Salmonella enterica serotype Typhimurium for the horse.

The aim of this study was to investigate the intranasal immunogenicity for the horse of a Deltacya Deltacrp-pabA mutant (MGN-707) of Salmonella enterica serotype Typhimurium (S. typhimurium). MGN-707 caused no sign of disease, was not detected in feces and a single administration induced strong Salmonella-specific serum and nasal mucosal antibody responses. All ponies had made strong salmonella specific serum IgGa, IgGb, IgA and IgM antibody responses by day 25 after the first immunization. IgM responses to salmonella lipopolysaccharide (LPS) were short lived whereas salmonella specific serum IgGa and IgGb persisted at high levels in all ponies until 83 and 140 days, respectively. Specific nasal mucosal antibody responses dominated by IgA and IgM were evident by day 25 in all ponies except one in which only specific IgGa and IgGb were evident. Specific nasal mucosal IgA persisted in most ponies until day 69. A second immunization on day 140 boosted antibody responses, and stimulated a strong nasal mucosal IgA response in the pony that failed to make an IgA response after primary immunization. At the termination of the experiment, IgA and IgGb dominated jejunal antibody responses whereas vaginal responses were mainly IgA. The latter response unequivocally confirms the existence of a common mucosal immune system in equids. The results indicate that a S. typhimurium Deltacya Deltacrp-pabA mutant has potential as an intranasal vaccine against salmonellosis in the horse.

The mucosal humoral immune response of the horse to infective challenge and vaccination with equine herpesvirus-1 antigens.

Equine herpesvirus-1 (EHV-1) remains a frequent cause of upper respiratory tract infection and abortion in horses worldwide. However, little is known about the local antibody response elicited in the upper airways of horses following exposure to EHV-1. This study analysed the mucosal humoral immune response of weanling foals following experimental infection with virulent EHV-1, or vaccination with either of 2 commercial vaccines. Twenty weanlings were assigned to 5 groups and were inoculated with, or vaccinated against, EHV-1 following different regimens. Finally, all weanlings were simultaneously challenged intranasally with virulent EHV-1 Army 183 (A183). Nasal wash and serum samples were collected at regular intervals until 13 weeks after final challenge. Nasal washes were assayed for EHV-1-specific equine IgGa, IgGb, IgG(T), IgA, IgM and total virus-specific antibody using an indirect, quantitative ELISA. Total serum antibody responses were also monitored, and clinical signs of EHV-disease were recorded for each individual. Virus-specific IgA dominated the mucosal antibody response elicited in weanlings inoculated with A183, being detectable at up to 3.1 microg/mg total IgA 13 weeks after challenge. Neither inactivated EHV-1 administered i.m., nor attenuated EHV-1 administered intranasally induced detectable mucosal antibodies. EHV-1-specific mucosal antibodies impeded EHV-1 plaque formation in vitro. Such virus-neutralising antibody probably contributes to a reduction of shedding of EHV-1 from the respiratory tract of virus-infected horses.

Induction of mucosal and systemic antibody specific for SeMF3 of Streptococcus equi by intranasal vaccination using a sucrose acetate isobutyrate based delivery system.

Streptococcus equi causes equine strangles, a highly contagious disease of the upper respiratory tract. The antiphagocytic surface protein SeM is strongly immunogenic and evokes mucosal and systemic antibodies during convalescence. The present study investigated the potential of sucrose acetate isobutyrate (SAIB); a high viscosity excipient that provides controlled release of biologically active substances, to enhance antibody responses following intranasal immunization of horses with a 108 a.a. peptide of SeM (SeMF3). SeMF3-SAIB was administered intranasally to each of the 11 adult horses on days 0 and 28. A second group of seven horses was vaccinated with SeMF3 alone. SAIB enhanced the mucosal and systemic immunogenicity of SeMF3, whereas SeMF3 by itself stimulated only a shortlived mucosal IgA and no systemic response. Moreover, nasal mucosal responses of horses immunized with SeMF3-SAIB were qualitatively and quantitatively similar to those observed in convalescent horses and involved similar linear epitopes of SeM. Epitope analysis also suggested that the nasal response was different from that observed in serum. A booster response was obtained after the second vaccination. These results suggest that SAIB has potential as a vehicle for intranasal immunization of horses with antigenic peptides.

Immunoglobulin isotypes in sera and nasal mucosal secretions and their neonatal transfer and distribution in horses.

OBJECTIVE: To determine concentrations of IgA and IgG subclasses in serum, colostrum, milk, and nasal wash samples of adult horses and foals. ANIMALS: Seven 2-year-old Welsh ponies, 27 adult mixed-breed horses, and 5 Quarter Horse mares and their foals. PROCEDURE: Serum was obtained from ponies and adult horses. Colostrum and milk were obtained from mares and serum and nasal wash samples from their foals immediately after parturition and on days 1, 7, 14, 28, 42, and 63. Nasal wash samples were also obtained from 23 adult horses. Concentrations of immunoglobulins were determined by use of inhibition ELISA. To determine transfer of maternal isotypes to foals, concentrations in colostrum and milk were compared with those in foal serum. Serum half-lives of isotypes in foals were also determined. RESULTS: IgGb was the most abundant isotype in serum and colostrum from adult horses, whereas IgA was the predominant isotype in milk. The major isotype in nasal secretions of adult horses and foals > or = 28 days old was IgA, but IgGa and IgGb were the major isotypes in nasal secretions of foals < or = 14 days old. Serum half lives of IgGa, IgGb, IgG(T), and IgA in foals were 176, 32, 21, and 3.4 days, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The early immunoglobulin repertoire of neonatal foals comprised IgGa, IgG(T), and IgA; endogenous synthesis of IgGb could not be detected until 63 days after birth. The restricted repertoire of immunoglobulins in foals may influence humoral immune responses to vaccination.

Light and scanning electron microscopic studies of the nasal turbinates of the horse.

The nasal turbinates of 5 young horses were studied by light and scanning electron-microscopy. Stratified cuboidal epithelium lined the rostral part of the dorsal and ventral nasal turbinates of the vestibular region. The polyangular microvillus cells of this region were separated by linear depressions. The mid and caudal parts of the dorsal and ventral nasal turbinates and the rostral part of the ethmoturbinates were lined by pseudostratified columnar ciliated respiratory epithelium. Numerous cilia with dilated blebs on the ciliated cells concealed adjacent non-ciliated supporting cells and goblet cells. The olfactory zone consisting of the olfactory vesicle and a dense network of olfactory cilia localized to the caudal part of the ethmoturbinates. The three regions were delineated from each other by transitional zones.

Antibody isotypes in sera of equine fetuses aborted due to Leptospira interrogans serovar pomona-type kennewicki infection.

Leptospira-specific antibody isotypes in sera of late term equine fetuses aborted due to Leptospira interrogans serovar pomona-type kennewicki infection were characterized and compared with those of their dams. IgM was the dominant Leptospira-Specific isotype in both fetuses and mares. However, IgGa was the isotype in highest concentration in petal sera and strong Leptospira-specific IgGa but no IgGb and little or no IgG(T) were detected. In contrast, although IgGb was quantitatively the dominant isotype in mares serum, Leptospira-specific serum IgG in aborting mares was dominated by IgG(T) but also included large amounts of IgGa and IgGb. IgGa and IgGb were quantitatively the dominant isotypes in sera of fetuses and mares, respectively. Affinity purified IgGa from fetuses did not agglutinate leptospires but serum devoid of IgGa did, suggesting that IgM is the principal agglutinating antibody. It is concluded that the equine fetus is deficient in IgGb and IgG(T) synthesis.

Streptococcus equi but not Streptococcus zooepidemicus produces potent mitogenic responses from equine peripheral blood mononuclear cells.

Streptococcus equi causes equine strangles. The acute disease has many of the hallmarks of an acute response including high fever, elevated plasma fibrinogen and neutrophilia, affects known to be mediated by proinflammatory cytokines. The objective of this study was to screen-culture supernatants from equine clinical isolates of S. equi and S. zooepidemicus for stimulation of mitogenic responses by horse peripheral blood mononuclear cells. Mitogenicity comparable to that of concanavalin A was detected in culture supernatants of S. equi strains but not in those of S. zooepidemicus. Mitogenicity was neutralised by Proteinase K and a post-strangles convalescent serum, and evidence for the presence of both thermo-resistant and thermo-labile mitogenic factors was obtained. Release of proteinaceous immunogenic mitogens in combination with the antiphagocytic protein SeM unique to S. equi may therefore contribute to some of the severe clinical manifestations of acute strangles in the horse.

Organization of the equine immunoglobulin heavy chain constant region genes; III. Alignment of c mu, c gamma, c epsilon and c alpha genes.

Previous restriction analysis of cloned equine DNA and genomic DNA of equine peripheral blood mononuclear cells had indicated the existence of one c epsilon, one c alpha and up to six c gamma genes in the haploid equine genome. The c epsilon and c alpha genes have been aligned on a 30 kb DNA fragment in the order 5' c epsilon-c alpha 3'. Here we describe the alignment of the equine c mu and c gamma genes by deletion analysis of one IgM, four IgG and two equine light chain expressing heterohybridomas. This analysis establishes the existence of six c gamma genes per haploid genome. The genomic alignment of the cH-genes is 5' c mu/(/) c gamma 1/(/) c gamma 2/(/) c gamma 3/(/) c gamma 4/(/) c gamma 5/(/) c gamma 6/(/) c epsilon-c alpha 3', naming the c gamma genes according to their position relative to c mu. For three of the c gamma genes the corresponding IgG isotypes could be identified as IgGa for c gamma 1, IgG(T) for c gamma 3 and IgGb for c gamma 4.

Local and systemic isotype-specific antibody responses to equine influenza virus infection versus conventional vaccination.

Inactivated alum-adjuvanted conventional equine influenza virus vaccines are of poor efficacy and offer limited short-term protection against infection. In sharp contrast, natural infection with equine influenza virus confers long-term protective immunity. In order to identify the protective immune responses to equine influenza virus, the influenza virus-specific IgA, IgGa, IgGb, IgGc and IgG(T) antibody responses in nasal secretions and serum induced by natural infection and a commercial vaccine were studied by ELISA. Two groups of four influenza-naive ponies were established. In the natural infection group, ponies received 10(8.5) EID50 of A/equine/Ky/1/81 by intranasal instillation, were allowed to recover, and then were rechallenged 100 days later. All four ponies exhibited clinical signs of influenza virus infection and viral shedding following primary infection, but were completely protected from challenge infection. Antibody responses to primary infection were characterized by nasal IgA and serum IgGa and IgGb responses. Ponies in the conventional vaccine group received a commercially available vaccine by intramuscular injection followed by a booster injection 3 weeks later. Challenge infection 100 days after vaccination resulted in clinical signs of infection and viral shedding. Antibody responses to vaccination were restricted to serum IgG(T) responses only. These results demonstrate that the protective immunity generated by natural equine influenza virus infection is associated with a mucosal IgA immune response and humoral IgGa and IgGb sub-isotype responses, and that this pattern of response is not generated by conventional vaccines.

Monoclonal antibodies to subclass-specific antigenic determinants on equine immunoglobulin gamma chains and their characterization.

This paper describes the production of a panel of monoclonal antibodies (mAbs) identifying the four recognised equine IgG subisotypes IgG, IgGa, IgGb, IgGc and IgG(T). Pure preparations of the subisotypes for use in immunisations and testing were produced using a combination of gel filtration, salt precipitation, ion exchange chromatography and protein A and Protein G affinity chromatography. The specificity of mAbs for the IgG subisotypes was confirmed using ELISA assays, by characterisation of affinity purified proteins recognised by the mAbs, and by Western blotting of equine serum proteins. The expression of equine IgG subisotypes by B cells was examined by flow cytometry using the panel of mAbs.

Immunoepidemiology of the equine tapeworm Anoplocephala perfoliata: age-intensity profile and age-dependency of antibody subtype responses.

The equine intestinal cestode Anoplocephala perfoliata has been the subject of recent epidemiological and immunological studies because of its suspected association with intestinal disease in the horse. We have previously shown that the IgG(T) subtype antibody response to the 12/13 kDa component of the parasite excretory/secretory (E/S) antigen is positively correlated with parasite intensity. In this study, we utilize that correlation to examine the changes in natural infection intensity with age. Infection intensity based on IgG(T) responses showed a triphasic age-dependency pattern with peak mean worm burden in the 6 months-2 years age group, falling to a lower plateau level from 3 to 15 years, and rising again in older age groups. Anti-E/S total IgG was found to have a convex age-dependency curve, with maximal response in the 6 months-2 years old age group. IgG(a) showed a triphasic response similar to the age-intensity profile of IgG(T); IgG(c) showed steadily increasing levels of antibody with age. The IgG(b) age-dependency profile was intermediate between IgG(a) and IgG(c). Age-specific correlation coefficients between anti-12/13 kDa IgG(T) (as a measure of infection intensity) and IgG(a) and IgG(b) revealed statistically significant values for many age groups. The relative importance of exposure to infection and the development of acquired immunity as determinants of the observed age-intensity pattern is considered.

Serum and mucosal antibody isotype responses to M-like protein (SeM) of Streptococcus equi in convalescent and vaccinated horses.

Equine strangles, caused by the clonal pathogen Streptococcus equi, is a source of serious economic loss despite the widespread use of commercial vaccines. The anti-phagocytic 58 kDa M-like protein (SeM) is an important protective antigen. The objective of this study was to define differences, if any, between SeM-specific convalescent serum and mucosal IgA and IgG subisotypes and those induced by vaccination with commercial strangles vaccine. SeM-specific opsonophagocytic IgGb was the predominant serum antibody in horses intramuscularly vaccinated or recently recovered from infection. Infection also induced high levels of specific opsonophagocytic serum IgGa during and shortly after S. equi infection whereas vaccination stimulated only low levels of serum IgGa. Specific serum IgGc and opsonophagocytic IgA were present at very low levels following infection or vaccination. A strong specific mucosal antibody response occurred during the acute and convalescent phases of infection whereas vaccinated horses made no mucosal response. Specific IgGb was generally predominant in nasopharyngeal washings during the acute phase but was replaced by specific IgA during convalescence. SeM-specific mucosal IgGa and IgG(T) but not IgGc were detected only during the acute and early convalescent phase. The results therefore indicate that vaccination, although inducing SeM-specific serum isotype responses qualitatively and quantitatively similar to those seen in convalescence, did not induce mucosal responses. This suggests that mucosal immunity may be important in acquired resistance to strangles.