Non-specific immune responses in juveniles of Indian major carps

The non‐specific immune parameters are useful to determine the health status of fish and to evaluate the immunomodulatory substances for fish farming as markers of pollution and disease resistance. Some of the important parameters, viz. superoxide production by neutrophils through nitroblue tetrazolium (NBT) assay; haemagglutination (HA), haemolysin (HLY) and bacterial agglutination titres; myeloperoxidase (MPO), and lysozyme activities, and alternative complement levels in serum of the juveniles of three Indian major carp species (Cirrhinus mrigala, Catla catla and Labeo rohita) were measured to establish their physiological normal range. A wide variation among the individuals within a species in the ranges of the most of the immune parameters was recorded. Significantly higher levels in the mean values of HA, HLY, bacterial agglutination titres; superoxide production by neutrophils in nitroblue tetrazolium assay; serum MPO and lysozyme activities, i.e. 371.20, 4.60, 18.80, 0.40, 0.62 and 6.55 μg ml^?1, respectively, were obtained in L. rohita except a much lower alternative haemolytic complement activity (29.06 units ml^?1) compared with the other two species. In most of the parameters, L. rohita showed the highest value, possibly indicating its more natural resistance compared with the other two species.     

Comparative and evolutionary analysis of mitochondrial genes in Indian major carps

Direct sequencing of mitochondrial DNA regions such as cytochrome b, ATPase 6/8 and control region was performed to study comparative and evolutionary status of the three mitochondrial genes in Labeo rohita, Catla catla and Cirrhinus mrigala. DNA sequence alignment among species using specific software revealed comparative rates of divergence with considerably faster and more heterogeneous substitution rate for control region as compared to cytochrome b and ATPase 6/8. Despite the relatively high variability of control region, the overall levels of sequence divergence were low in coding regions. Two protein coding genes and the control region with varying degree of sequence divergence established two distinct groups which are genetically distant from each other exhibiting identical phylogenetic structure in IMCs. Closest relationship was between Labeo rohita and Catla catla indicating that they might have diverged from a common ancestral stock in genealogical lineage whereas Cirrhinus mrigala showed greater divergence with all the three DNA regions studied. Findings of this study will help to understand evolution of mitochondrial DNA genes in carps and facilitate future investigations on phylogeographic structure of Indian carps.     

Density-dependent growth performance of Indian major carps in rainwater reservoirs

The study was carried out to evaluate growth performance of Indian major carps at different stocking densities in rainwater reservoirs. In this experiment, the absolute growth performance of Catla catla, Labeo rohita and Cirrhinus mrigala at stocking densities of 5000, 8000 and 11 000 fingerlings per hectare was 377.7, 215.4 and 241.9; 370.2, 186.7 and 219.1; and 306.7, 163.4 and 180.6 g, respectively. The recorded biomass yield at 5000, 8000 and 11 000 fingerlings per hectare was 1035, 1572 and 1573 kg ha^?1 per 180 days of culture. The effect of stocking density on production performance (performance index, PI) was highly significant (P < 0.05) at the higher stocking density of 11 000 ha^?1, while there was no significant variation between PI at stocking densities of 5000 and 8000 ha^?1. This indicates optimum production performance at 8000 ha^?1, where yield is significantly higher (P < 0.05) than yield at 5000 ha^?1 and almost equal to the yield at 11 000 ha^?1. An increase in stocking density from 8000 to 11 000 ha^?1, however, showed a sharp decline in average mean body weight of each species, even with supplemental feeding. With an increase in stocking density, the biomass yield increased to an optimum (1572 kg ha^?1), with no substantial increase thereafter. Reductions in growth, which occurred at high density, did not appear to be due to poor water quality as the water quality did not differ significantly among various treatments. Thus, the reduced survival and growth at high density appears to be a behavioural interaction or physiological response to density itself.     

Immunisation of Indian major carps against Aeromonas hydrophila by intraperitoneal injection

The Indian major carps Catla catla, Labeo rohita and Cirrhinus mrigala were immunised against the potent bacterial fish pathogen, Aeromonas hydrophila by the intraperitoneal route, in field conditions. Two different polyvalent antigen preparations namely, whole cell and extracellular products (ECP) were used. Immunisation schedule consisting of a single booster dose given 28 days after priming resulted in a good agglutinating antibody response in the carps. Kinetics of the response were similar in the three carp species with both whole cell and ECP. Upon challenge with virulent strains, relative percent survival as high as 80-90% was recorded. Indian major carps are the most important freshwater species cultured in India and it is essential that they are protected against infections of A. hydrophila, a scourge of freshwater fish worldwide.     

Serologic cross-reactivity of human IgM and IgG antibodies to five species of Ebola virus

Five species of Ebola virus (EBOV) have been identified, with nucleotide differences of 30-45% between species. Four of these species have been shown to cause Ebola hemorrhagic fever (EHF) in humans and a fifth species (Reston ebolavirus) is capable of causing a similar disease in non-human primates. While examining potential serologic cross-reactivity between EBOV species is important for diagnostic assays as well as putative vaccines, the nature of cross-reactive antibodies following EBOV infection has not been thoroughly characterized. In order to examine cross-reactivity of human serologic responses to EBOV, we developed antigen preparations for all five EBOV species, and compared serologic responses by IgM capture and IgG enzyme-linked immunosorbent assay (ELISA) in groups of convalescent diagnostic sera from outbreaks in Kikwit, Democratic Republic of Congo (n=24), Gulu, Uganda (n=20), Bundibugyo, Uganda (n=33), and the Philippines (n=18), which represent outbreaks due to four different EBOV species. For groups of samples from Kikwit, Gulu, and Bundibugyo, some limited IgM cross-reactivity was noted between heterologous sera-antigen pairs, however, IgM responses were largely stronger against autologous antigen. In some instances IgG responses were higher to autologous antigen than heterologous antigen, however, in contrast to IgM responses, we observed strong cross-reactive IgG antibody responses to heterologous antigens among all sets of samples. Finally, we examined autologous IgM and IgG antibody levels, relative to time following EHF onset, and observed early peaking and declining IgM antibody levels (by 80 days) and early development and persistence of IgG antibodies among all samples, implying a consistent pattern of antibody kinetics, regardless of EBOV species. Our findings demonstrate limited cross-reactivity of IgM antibodies to EBOV, however, the stronger tendency for cross-reactive IgG antibody responses can largely circumvent limitations in the utility of heterologous antigen for diagnostic assays and may assist in the development of antibody-mediated vaccines to EBOV.     

印度北方河流中主要鲤科鱼类卡拉鲃的年龄和生长

为了获得印度北部赣达（Ganga）盆地河流中野生卡特拉鲃种群的年龄结构和重要生长参数,对该鱼的年龄和生长进行了研究。鳞片取自商业捕捞和实验室饲养的样品。根据研究分析,该鱼最大年龄可达8龄;巴吉拉蒂河（Bhagirathi R.）的种群平均体长为521.51 mm,退算体长为288.9 -1132.3 mm;旁遮普邦（Punjab）Satluj河种群平均体长为641.6 mm,退算体长为335.4 -1096.08 mm。2龄时,种群线性生长率（Cl）和体重增加率（Cw）表现出迅速下降的趋势。其它生长参数值（Clt）也呈现快速下降。退算体长差异（ANOVA）分析显示,生活在赣达盆地不同流域中的种群, 1^＋-4^＋龄组的长度差异较明显（P〈0.05） ,高龄组（5^＋-8^＋）差异不显著。根据本项研究结果,提出了对印度北部赣达盆地相关河流中生活的野生卡特拉鲃种群资源持续利用的对策。     

Production of superoxide anion by head-kidney leucocytes of Indian major carps immunised with bacterins of Aeromonas hydrophila

On contact with micro-organisms or any other antigenic particles which are recognised as phagocytosable, the leucocytes of vertebrates raise their oxygen consumption suddenly (respiratory burst). The consumed oxygen is then converted into reactive oxygen species (ROS). An effort has been made in the present study to estimate the production of superoxide anion, one of the ROS, by the resident as well as activated head-kidney leucocytes of Indian major carps. Activation was accomplished by immunisation with formalin killed (FK) and heat killed (HK) whole cell bacterins of Aeromonas hydrophila. In the ex vivo experiment conducted, activated leucocytes yielded a significant increase in O.D. values for nitroblue tetrazolium (NBT) reduction reaction, reflecting an increase in superoxide anion production, from day 1 post immunisation. Of the three species of Indian major carps immunised, Catla catla showed the greatest production of superoxide anion, followed by Labeo rohita and then by Cirrhinus mrigala. The enhancement of superoxide anion production of leucocytes by immunisation justifies the role of immunisation in the microbicidal defence mechanism of fish.     

印度北方河流中主要鲤科鱼类卡拉(鱾)的年龄和生长

为了获得印度北部赣达(Ganga)盆地河流中野生卡特拉(鱾)种群的年龄结构和重要生长参数,对该鱼的年龄和生长进行了研究.鳞片取自商业捕捞和实验室饲养的样品.根据研究分析,该鱼最大年龄可达8龄;巴吉拉蒂河(Bhagirathi R.)的种群平均体长为521.51 mm,退算体长为288.9-1132.3 mm;旁遮普邦(Punjab)Satluj河种群平均体长为641.6 mm,退算体长为335.4-1096.08 mm.2龄时,种群线性生长率(Cl)和体重增加率(Cw)表现出迅速下降的趋势.其它生长参数值(Clt)也呈现快速下降.退算体长差异(ANOVA)分析显示,生活在赣达盆地不同流域中的种群,1 -4 龄组的长度差异较明显(P<0.05),高龄组(5 -8 )差异不显著.根据本项研究结果,提出了对印度北部赣达盆地相关河流中生活的野生卡特拉(鱾)种群资源持续利用的对策.     

Characterization of digitalis-like factors in human plasma. Interactions with NaK-ATPase and cross-reactivity with cardiac glycoside-specific antibodies

Much of the evidence for a physiologically important endogenous inhibitor of the sodium pump has been either contradictory or indirect. We have identified three discrete fractions in desalted deproteinized plasma from normal humans that resemble the digitalis glycosides in that they: are of low molecular weight; are resistant to acid and enzymatic proteolysis; inhibit NaK-ATPase activity; inhibit Na+ pump activity in human erythrocytes; displace [3H]ouabain bound to the enzyme; and cross-react with high-affinity polyclonal and monoclonal digoxin-specific antibodies but not with anti-ouabain or anti-digitoxin antibodies. An additional fraction cross-reacted with digoxin-specific antibodies but had no detectable activity against NaK-ATPase. The three inhibitory fractions differed from cardiac glycosides in that their concentration-effect curves in a NaK-ATPase inhibition and [3H]ouabain radioreceptor assays were steeper than unlabeled ouabain. This suggests that these inhibitors are not simple competitive ligands for binding to NaK-ATPase. In the presence of sodium, no fraction required ATP for binding to NaK-ATPase, and in the presence of potassium, only one fraction had the reduced affinity for the enzyme that is characteristic of cardiac glycosides. Unlike digitalis, all three NaK-ATPase inhibitory fractions stimulated the activity of skeletal muscle sarcoplasmic reticulum Ca-ATPase. The presence of at least three fractions in human plasma that inhibit NaK-ATPase and cross-react to a variable degree with different digoxin-specific antibody populations could explain much of the conflicting evidence for the existence of endogenous digitalis-like compounds in plasma.     

Bath immunisation of spawn,fry and fingerlings of Indian major carps using a particulate bacterial antigen

Larval mortality in Indian major carps is one of the major problems encountered in the pond culture system. The present investigation was carried out to investigate the proper age, duration of exposure, and optimum bacterin concentration for vaccinating rohu (Labeo rohita) and catla (Catla catla) at their early stages with a formalin killed Edwardsiella tarda bacterin suspension. The development of immunological competence was recorded with spawn of rohu and catla of 3 weeks of age exposed to a bacterin at a concentration 10(9) cfu ml(-1) for 15 min, where it persisted up to 4 weeks post vaccination. They showed significant resistance against challenge with virulent E. tarda bacteria. Significant antibody titre could be recorded in advanced fries and fingerlings exposed to 10(9) cfu/ml(-1) bacterin concentration for 45 and 60 min, respectively.     

Ingestion of bacteria in suspension Indian major carps (Catla catla,Labeo rohita) and Chinese carps (Hypophthalmichthys molitrix,Aristichthys nobilis)

Single-species groups of fry of the planktivorous, filter-feeding carps (silver carp Hypophthalmichthys molitrix, bighead carp Aristichthys nobilis, rohu Labeo rohita and catla Catla catla) were exposed to the bacterium Chromobacterium violaceum to assess if they could detect or ingest unattached bacteria. The results indicate that planktivorous carp fry are able to ingest unattached bacteria and that the numbers ingested increase with time and with bacterial concentration in the media, although probably by passive means. It is also suggested that unattached bacteria are of little nutritional significance to carps.     

Widespread hybridization among species of Indian major carps in hatcheries, but not in the wild

Twenty‐one allozyme loci in samples of wild‐caught and hatchery‐reared Indian major carps from Bangladesh were analysed. Bayesian model‐based clustering analysis revealed the presence of four taxa, corresponding to the three known species along with a fourth unknown taxon present in two hatchery samples. Individual admixture coefficients showed that 24% of all hatchery‐reared fishes were hybrids, whereas a single hybrid was observed in the wild‐caught samples. Only catla Catla catla × rohu Labeo rohita and mrigal Cirrhinus cirrhosus × rohu hybrids were observed, the vast majority of which were F₁ hybrids, though five individuals represented putative backcrosses. Mitochondrial DNA analysis revealed that catla × rohu hybridization primarily involved catla males and rohu females, whereas mrigal × rohu hybrids primarily resulted from rohu males and mrigal females. Despite the high percentage of F₁‐hybrids in hatchery samples, reproductive barriers among species have so far precluded widespread introgression. Continued hybridization may eventually lead to a breakdown of species barriers, thereby compromising the genetic integrity of the species in the wild, and leading to production losses in aquaculture.     

Comparative sensitivity of different serological tests for seromonitoring and surveillance of Edwardsiella tarda infection of Indian major carps

Different serological tests viz. indirect ELISA, indirect blocking ELISA, competitive ELISA and serum agglutination tests were evaluated to detect antibodies against Edwardsiella tarda in naturally infected fish sera for seromonitoring and epizootiological studies. Approximately 66.6, 62.5, 57.6 and 16.6% of the field sera samples were found to be positive by indirect ELISA, competitive ELISA, indirect blocking ELISA and serum agglutination test, respectively. The percentage of serum samples positive for E. tarda antibodies in serum agglutination, competitive ELISA and indirect blocking ELISA, when compared with indirect ELISA, were 33.3, 83.6 and 66.6%, respectively, but its use was restricted due to the requirement of several conjugates against different fish species and the difficulty in assaying large numbers of serum samples from different fish species in a limited time to enable seromonitoring of the disease prevalence. No significant difference (P<0.05) in the mean optical density value was found in indirect and competitive ELISA. Although the competitive ELISA was slightly less sensitive than the indirect ELISA, it could accommodate a large number of serum samples with one anti-rabbit conjugate, and the need for different fish conjugates as required in indirect ELISA was eliminated. As in medical and veterinary practices, these tests can now be used in aquaculture practices for seromonitoring and study of pre-exposure of Indian major carps to pathogens in enzootic areas.     

Study on impact of habitat degradation on proximate composition and amino acid profile of Indian major carps from different habitats

This investigation is aimed to study an impact of habitat degradation on proximate composition and amino acid (AAs) profile of Catla catla, Labeo rohita and Cirrhinus mrigala collected from polluted, non-polluted area (upstream) and a commercial fish farm. The amino acid profile was estimated by the amino acid analyzer. C. catla collected from the polluted environment had highest lipid, protein and ash contents (12.04?±?0.01, 13.45?±?0.01 and 0.93?±?0.03%, respectively). The high protein content (14.73?±?0.01 and 14.12?±?0. 01%) was recorded in C. catla procured from non-polluted (upstream) wild habitat of River Chenab and controlled commercial fish farm. Farmed fish species showed comparatively higher moisture contents followed by upstream and polluted area fishes. C. mrigala showed significant differences in amino acid and proximate composition collected from a polluted site of the river Chenab. C. catla collected from non-polluted site of the river showed an excellent nutrient profile, followed by L. rohita (wild and farmed) and C. mrigala (polluted area), respectively. All fishes from the polluted areas of the River Chenab indicated a significant decrease in the concentration of some AAs when compared to farmed and wild (upstream) major carps. Omitting of some important AAs was also observed in the meat of fish harvested from polluted habitat of this river. C. mrigala and L. rohita exhibited a significant increase in the concentration of some of non-essential amino acids such as cysteine in their meat. The results indicated that wild fish (upstream) and farmed fish species had highest protein contents and amino acid profile and hence appeared to be the best for human consumption. The proximate composition and AAs profiles of fish harvested from the polluted area of the river clearly indicated that efforts shall be made for the restoration of habitat to continue the requirement of high quality fish meat at a low cost to the human population.     

Monoclonal antibodies to bovine UDP-galactosyltransferase. Characterization, cross-reactivity, and utilization as structural probes

A series of mouse monoclonal antibodies has been developed against a soluble form of bovine UDP-galactose:N-acetylglucosamine galactosyltransferase purified to apparent chemical homogeneity by a combination of affinity and immunoadsorption chromatography. The purified enzyme consists of two molecular mass variants of 42 and 48 kDa. Individual monoclonal antibodies were selected for by their ability to recognize immobilized affinity-purified galactosyltransferase and were not reactive against bovine alpha-lactalbumin and bovine immunoglobulins. Based on competitive binding assays and Western blot analysis with either galactosyltransferase or lactose synthetase (covalently cross-linked alpha-lactalbumin galactosyltransferase), these monoclonal antibodies can be subdivided into four groups. Group A (3 clones) recognize an epitope at or near the alpha-lactalbumin binding site. In addition, this group is cross-reactive with soluble galactosyltransferase from human milk and pleural effusion. Group B (6 clones) and D (1 clone) appear to recognize two different epitopes on the 6-kDa fragment which is released when the 48-kDa galactosyltransferase polypeptide is converted to the 42-kDa form, apparently by proteolysis. Groups A and C (1 clone) recognize epitopes found on both the 48- and 42-kDa polypeptide. Interestingly, immunofluorescence studies indicate that only two monoclonal antibody groups (C and D) are able to decorate membrane-bound galactosyltransferase (Golgi-associated) in formalin-fixed, methanol-, or detergent-permeabilized cells. Thus, these groups of monoclonal antibodies appear to identify four separate structural/functional domains on soluble galactosyltransferase, two of which are not readily accessible for binding in situ.     

High-titre IgM anti-sulfatide antibodies in individuals with IgM paraproteinaemia and associated peripheral neuropathy

The common association between monoclonal gammopathy and peripheral neuropathy was studied in seven patients with demyelinating polyneuropathy and IgM paraproteinaemia. Plasma samples from these individuals were thoroughly tested for antiperipheral nerve myelin (PNM) antibodies and then screened for glycoprotein and glycolipid reactivity by western immunoblotting and thin-layer chromatography (TLC) immunostaining. Three of the seven samples showed strong IgM anti-PNM and antisulfatide (GalS) antibody reactivity. Two of these three plasma samples showed extraordinarily high antisulfatide IgM antibody titres, ranging from 1 x 104 to 1 x 106 arbitrary units/L. These same samples also showed intense myelin staining of sciatic nerve sections (paraffin and cryostat) and teased nerve fibres. No axonal immunoreactivity was observed. These results suggest that high titre IgM antisulfatide antibodies may play a pathogenetic role in the immune demyelination associated with IgM paraproteinaemia.     

Cross-priming of microsatellite loci in subfamily cyprininae (family Cyprinidae): their utility in finding markers for population genetic analysis in three Indian major carps

This study is aimed to identify polymorphic microsatellite markers and establish their potential for population genetics studies in three carp (family cyprinidae; subfamily cyprininae) species, Labeo rohita, Catla catla and Cirrhinus mrigala through use of cyprinid primers. These species have high commercial value and knowledge of genetic variation is important for management of farmed and wild populations. We tested 108 microsatellite primers from 11 species belonging to three different cyprinid subfamilies, Cyprininae, Barbinae and Leuciscinae out of which 63 primers (58.33 %) successfully amplified orthologous loci in three focal species. Forty-two loci generated from 29 primers were polymorphic in these three carp species. Sequencing of amplified product confirmed the presence of SSRs in these 42 loci and orthologous nature of the loci. To validate potential of these 42 polymorphic loci in determining the genetic variation, we analyzed 486 samples of three focal species collected from Indus, Ganges and Brahmaputra river systems. Results indicated significant genetic variation, with mean number of alleles per locus ranging from 6.80 to 14.40 and observed heterozygosity ranging from 0.50 to 0.74 in the three focal species. Highly significant (P < 0.00001) allelic homogeneity values revealed that the identified loci can be efficiently used in population genetics analysis of these carp species. Further, thirty-two loci from 19 primers were useful for genotyping in more than one species. The data from the present study was compiled with cross-species amplification data from previous results on eight species of subfamily cyprininae to compare cross-transferability of microsatellite loci. It was revealed that out of 226 heterologous loci amplified, 152 loci that originated from 77 loci exhibited polymorphism and 45 primers were of multispecies utility, common for 2–7 species.     

Characterization of the primary IgM response to GAT and GT: conditions required for the detection of IgM antibodies.

Primary IgM antibody responses to synthetic linear copolymers of L-glutamic acid, L-tyrosine, and L-alanine were investigated. The appearance of primary IgM anti-GAT antibodies was detected in BALB/c mice by using a solid phase radioimmunoassay (SPRIA) procedure. The finding was verified for GAT in responder mice and GAT-MBSA and GT-MBSA in nonresponder mice in an indirect plaque forming cell (PFC) assay by using a rabbit antiserum directed against the mulambda myeloma protein, MOPC 104E. Facilitated IgM PFC could be inhibited by a purified muK myeloma protein, TEPC 183. Maximal facilitated IgM plaque response was found to precede the IgG response by several days. A direct plaque assay was developed for the detection of IgM anti-GAT plaques using poly-L-lysine (PLL) to couple GAT to sheep erythrocytes (SRBC). GAT-SRBC coupled by the PLL method optimally couple 4 to 5 times less antigen to the indicator cell surface than does the CrCl3-coupling method routinely employed in our laboratory. These findings were extended to a conventional antigen, chicken gamma globulin (CgammaG). We found that a less dense epitope coat on the indicator cell surface favors detection of direct IgM PFC, whereas a more densely coated indicator cell favors the detection of facilitated IgM and IgG PFC responses.