Recoding anaerobic regulator fnr of Salmonella Typhimurium attenuates it's pathogenicity.

AIMS: How recoding of fnr, an anaerobic regulatory gene, affects pathogenicity related parameters of Salmonella Typhimurium (STM). METHODS AND RESULTS: The fnr gene was recoded by substituting all of it's codons with synonymous rare codons of STM. Recoding fnr gene severely reduced the ability of the recoded mutant to compete with wild strain under nutrient depletion condition. Mutants were also less motile than the wild strain and their biofilm forming ability was significantly decreased as compared to wild strain. The recoded strain showed significant reduced survival within murine macrophages (RAW264.7) and monocyte derived macrophage of poultry origin. The colonisation ability of recoded mutant in liver and spleen of mice on day 5 of post infection was significantly reduced. The recoded strain exhibited significant reduction in faecal shedding on day 1 and 5 after infection. CONCLUSIONS: Our study showed that recoding the anaerobic regulator fnr of STM significantly compromised its growth, decreased motility, biofilm forming ability and survival within macrophages. Further, the recoded fnr strain showed reduced colonisation ability and faecal shedding in mice. Thus, these findings highlight that recoding the global anaerobic regulator fnr of Salmonella Typhimurium attenuates its pathogenicity.

Naturally occurring Anaplasma marginale infection in cattle: Molecular prevalence and associated risk factors, haemato-biochemical alterations, oxidant/antioxidant status and serum trace mineral levels.

The present investigation was undertaken to map the distribution of Anapalsma species infection in cattle from the Aizawl region of Mizoram, India, in relation to various risk factors, and to study the haemato-biochemical alterations, oxidant/antioxidant status and serum trace mineral levels in cattle with naturally occurring Anapalsma marginale infection. The study was carried out over 31 months from June 2019 to December 2021. A total of 401 cattle blood samples were collected and screened for the presence of Anaplasma spp. by microscopic examination and polymerase chain reaction (PCR). Non-infected clinically healthy cattle (n = 21) served as control. Blood samples were collected to study the haemogram and serum samples were used for the evaluation of biochemical parameters, oxidative stress indices and trace minerals. During the study period, an overall prevalence of 15.71% was recorded for A. marginale infection in cattle. The prevalence of A. marginale infection was highly associated with age, sex, breed and tick infestation status of animals, floor system and management of farms, and season. The mean values of total erythrocyte count (TEC), haemoglobin (Hb), packed cell volume (PCV), total platelet count, total protein, albumin, superoxide dismutase (SOD), glutathione (GSH), total antioxidant capacity (TAC), copper (Cu) and zinc (Zn) were significantly (P < 0.05) lower, whereas the mean values of mean corpuscular volume (MCV), aspartate aminotransferase (AST), total bilirubin, indirect bilirubin, blood urea nitrogen (BUN), creatinine and lipid hydroperoxide (LPO) were significantly (P < 0.05) higher in cattle infected with A. marginale. A negative correlation of TEC with LPO, and a positive correlation with SOD, GSH, TAC, Cu and Zn suggest a possible link between oxidative stress and the haemolytic crisis noticed in bovine anaplasmosis. Incorporation of antioxidants and organ protective drugs as an adjunct therapy may result in better prognosis.

Isolation and molecular characterization of GP5 glycoprotein gene of Betaarterivirus suid 2 from Mizoram, India.

Porcine reproductive and respiratory syndrome (PRRS) is a serious swine disease causing great economic impact worldwide. The emergence of highly pathogenic strains in Asian countries is associated with large scale mortality in all age groups of pigs besides the classical presentation of severe respiratory distress, pneumonia, and a series of reproductive disorders in sows, like late-term abortion, premature farrowing, and an increased number of stillborn piglets. The present study was designed with the aim of isolation and characterization of the Betaarterivirus suid 2 from outbreaks in Mizoram in primary porcine alveolar macrophage and subsequently characterized the GP5 gene sequence of the isolate in terms of phylogenetic analysis and deduce amino acid sequence comparison. Virus propagation was performed in the porcine alveolar macrophage (PAM) primary cell culture and confirmed by immunoperoxidase test, FAT, and nested RT-PCR. The full-length GP5 gene (603nt) was amplified from the isolate and subsequently cloned and sequenced (MN928985). Phylogenetic analysis and sequence comparison of the present isolate was found to have similarity 98.7-98.8% with Myanmar HP-PRRS strains, 98-98.5% with Vietnam strains, 98.2-98.3% with China strains, indicating a close lineage with highly pathogenic PRRS strains. In deduced amino acid sequence analysis, one mutation was found in the primary neutralizing epitope (PNE) at position 39L → I39 and one more mutation was also found in the decoy epitope (DCE) at position 30 N → D30. The amino acid at this position is an N-linked glycosylation site, and mutation of the N-linked glycosylation is an immune escaped strategy adopted by this virus causing a persistent infection in the natural host.

Molecular detection of Babesia microti in laboratory mice from India.

BACKGROUND & OBJECTIVES: For detection and molecular characterization of Babesia microti in laboratory mice from India. METHODS: A total of 625 mice were screened by peripheral blood smear examination and subsequently was confirmed by PCR using a piroplasm conserved primer set (Piro A/B). Nested PCR was done using a species-specific primer targeting the gene encoding the small subunit ribosomal RNA (18S rRNA). The PCR products were cloned, purified and sequenced. A total of 12 isolates were obtained. The sequences were aligned and phylogenetic trees were prepared with other published Babesia spp. sequences. RESULTS: B. microti was detected with a total infection rate of 8.80%. The higher rate of infection was observed by species specific PCR (8.80%) than examined by blood smear (7.20%). Sequence and phylogenetic analysis showed that Babesia species detected in mice were genetically identical to the genotypes of B. microti and can be easily distinguished from other genotypes of Babesia parasites by neighbour joining and maximum likelihood method. Intra-species analysis indicated that all the twelve isolates from six North-Eastern states of India have a close identity but inter-species showed genetic reservoir host for transmission of babesial infection to humans. INTERPRETATION & CONCLUSION: The detection of Babesia microti may suggest that laboratory mice may serve as potential reservoir host for human infection and possibility of innovative way of diagnosing and control of human babesiosis.

Comparative proteomic analysis of Salmonella Typhimurium wild type and its isogenic fnr null mutant during anaerobiosis reveals new insight into bacterial metabolism and virulence.

AIM: The aim of this study was to understand the role of anaerobic regulator FNR (Fumarate Nitrate Reduction) in Salmonella Typhimurium through proteomic approach. METHODS AND RESULTS: We did label free quantitative proteomic analysis of Salmonella Typhimurium PM45 wild type and the fnr null mutant cultured under anaerobic conditions. The data revealed 153 significantly differentially expressed proteins (DEPs) in the mutant out of 1798 total proteins identified. Out of 153 DEPs, 94 proteins were up-regulated (repressed by FNR) and 59 proteins were down-regulated (activated by FNR) in the mutant. The network analysis indicated up-regulation of TCA cycle, electron transport chain and ethanolamine metabolism and down regulation of pyruvate metabolism and glycerol and glycerophospholipid metabolism. CONCLUSIONS: Our study showed that FNR represses ethanolamine utilization. The different metabolic pathways such as pyruvate metabolism, glycerol metabolism and glycerophospholipid metabolism were activated by FNR. Further, FNR positively regulated the DNA binding protein Fis, one of the global regulators of virulence in Salmonella Typhimurium. Thus, our finding highlights the pivotal role of FNR in regulating bacterial metabolism and virulence during anaerobiosis for systemic infection of the host.

Systematic evaluation of species-independent serum pre-fractionation strategies revealed cost-effective methods to reduce proteome complexity.

In this study, the efficiency of one commercial (ProteoMiner™ -PM) and five simple and cost-effective laboratory chemicals (Acetone, TCA/acetone, DTT, ACN and DTT-ACN) based serum protein pre-fractionation strategies was compared in pig model by label-free quantitation based mass spectrometric approach to find out the most suitable strategy for reducing the complexity of serum proteome for subsequent proteomic studies. The highest serum protein depletion percentage and highest depletion of albumin, the most abundant serum protein, was observed in DTT-ACN method. The maximum number of serum proteins was identified in ACN followed by DTT-ACN method and importantly, detection of more number of low-abundant proteins (LAPs) could also be achieved by these two methods. Although PM method resulted into lowest dynamic range of protein abundance, quite a less number of proteins were identified by this method. Overall, sequential depletion using DTT-ACN and ACN methods provided advantage of simultaneous detection of more number of proteins along with LAPs with a reasonably high dynamic range of protein abundances over other methods and thus emerged as cheaper and effective alternatives to the commercial methods. Further, these methods are species-independent and hence can be applied in human and in any livestock species to simplify the serum proteome.

Molecular Characterization of Chitin Synthase Gene of Demodex canis from Mizoram, India.

AIMS: Canine demodicosis is a parasitic condition affecting the skin of dogs. The present study was designed to characterize chitin synthase gene of Demodex canis. The molecular technique was used for better understanding of this gene. METHODS: A total of 75 dogs which are reared as pets with or without showing any skin lesions were examined during the study period. Skin scrapings were examined by indirect method using 10% potassium hydroxide solution under 10 × microscope. DNA samples were extracted from positive skin samples and were subjected to PCR for molecular identification. RESULTS: A total of 25 dogs irrespective of age, sex, breed or coat showed positive result for D. canis. The PCR revealed a single amplified product of 339 bp length which exactly matched with D. canis. The chitin synthase gene was amplified by PCR, subsequently cloned, sequenced, and compared with available data in GenBank for the particular gene of D. canis. Only one single nucleotide polymorphism (SNP) was noticed at 231 position of the chitin synthase gene sequence when compared to other isolates. CONCLUSION: The molecular technique confirms with the morphological identity of D. canis. This report signifies the value of peculiar tool to identify 'follicular mite' even from apparently healthy skin.

Comparative serum proteome analysis reveals potential early pregnancy-specific protein biomarkers in pigs.

In this study, the comparative serum proteome profile of Day 5, 12 and 16 of gestation, representing three early embryonic events, namely formation, elongation and implantation of blastocysts, and non-pregnant control were explored by a label-free quantitation-based mass spectrometric approach to identify early pregnancy biomarkers in pigs. A total of 131 proteins were identified with respect to different groups, out of which 105 were found to be differentially expressed proteins (DEPs). Among the DEPs, 54 and 66 proteins were found to be up and downregulated respectively in early pregnancy groups (fold change >2) and the maximum number of upregulated proteins was observed in the Day 12 pregnancy stage. Functional classification and pathway analysis of the DEPs revealed involvement of most of the proteins in complement and coagulation cascades, metabolic processes and immune and inflammatory responses. Proteins such as glutathione peroxidise (GPX), pregnancy zone protein (PZP), thrombospondin-1 (THBS1), α-1-antitrypsin (AAT) and mannose-binding lectin C (MBLC) were differentially expressed during early pregnancy and actively involved in different pregnancy-related activities. To the best of our knowledge, this is the first report on comparative serum protein profiling of different early pregnancy stages in pigs and our results provide a set of proteins that can be used as potential biomarkers for early pregnancy diagnosis in pigs.

Retraction Note to: Therapeutic management of trypanosomosis with ophthalmic involvement in a dog.

[This retracts the article DOI: 10.1007/s12639-017-0953-z.].

Therapeutic management of trypanosomosis with ophthalmic involvement in a dog.

The present report communicates a case of canine trypanosomosis with ophthalmic involvement, its diagnosis, hemato-biochemistry and therapeutic management in a 2 year old dog. The dog had history of bilateral corneal opacity and impaired vision since last 4 weeks. On the basis of history and clinical signs, a presumptive diagnosis of canine trypanosomosis was made followed by confirmation with Giemsa stained buffy coat smear examination. Therapeutic regimen was comprised of Diminazine aceturate @ 3.5 mg/kg deep IM for two occasions 24 h apart along with parenteral fluids, hematinics, NSAID and multivitamins which yielded favourable response by third day post-therapy. Haemato-biochemical parameters took nearly 14 days of time to return to near normal levels. Improvement with respect to corneal opacity took 6 weeks of time and the animal was followed up to 3 months without any recurrence.

Dynamics of cytokine gene expression in peripheral blood mononuclear cells of indigenous and exotic breeds of pigs in India.

To incorporate immune competence traits in swine breeding programs, association between immune responsiveness and susceptibility to specific infectious diseases must be established. In order to understand the differences in immune competence between indigenous (Zovawk) and exotic (Large White Yorkshire: LWY) pigs reared in India, we carried out a time course expression analysis of immune-regulating key cytokine genes (interleukin (IL)-2, interferon (IFN)-γ, IL-4 and IL-10) in the phytohemagglutinin-P stimulated peripheral blood mononuclear cells (PBMCs). The IL-2 transcript levels in PBMCs increased several thousand-fold when compared to unstimulated cells in both the breeds, albeit the response in that of Zovawk was remarkably higher. Higher and earlier IFN-γ and IL-4 expression levels in Zovawk pigs suggest that both TH 1 and TH 2 immune responsiveness of this indigenous breed affords better preparedness for danger signals. Moreover, the low expression levels of IL-10 depict a regulated adaptive immune responsiveness. Remarkable difference between the two breeds of the pigs is evident showing a clear advantage of the Zovawk over LWY in terms of a shorter lag period of adaptive immune response. These findings provide a lead for understanding the genetic differences with respect to immune competence levels of indigenous pigs compared to exotic counterparts.

Changing the Codon Usage of hfq Gene has Profound Effect on Phenotype and Pathogenicity of Salmonella Typhimurium.

Genome recoding with bias codons (synonymous rare codons) or codon pair bias is being used as a method to attenuate virulence mostly in viruses. The target gene chosen for attenuation in general in bacteria is mostly toxin or virulence gene. We have used RNA chaperone hfq, a global post-transcriptional regulator of bacterial gene expression that regulates about 20 % genes in Salmonella, as the target of recoding. The hfq gene was recoded by replacing the codons of hfq gene with synonymous rare codons. Recoding decreased the expression of Hfq protein about two-fold in the mutant as compared to the parent strain. Recoding did not affect growth kinetics, but in growth competition the mutant strain was outcompeted by the parent strain. There was significant decrease in survivability of mutant strain in macrophage as compared to the parent strain. The biofilm formation was significantly impaired in case of recoded mutant. The mutants were also less motile as compared to the parent strain. Intraperitoneal infection of mice with the mutant strain had shown better survival as compared to parent strain. The results show that recoding is an effective method of reducing virulence.

Effect of age and season on the thyroid hormone activity of Mizoram strain female mithun (Bos frontalis).

AIM: The aim of the present study was to generate baseline data on the normal values of the thyroidhormone (TH) activity as well as their correlation with age and season. MATERIALS AND METHODS: Blood samples (10 ml) were collected from jugular vein of 30 female mithun's of three different age groups viz. Calves (6 months to 1 year), heifer (1-3 years) and adult (above 3 years) during the three season's viz. Monsoon, winter and spring of a year. The serum was analyzed for thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4) activity. RESULT: The result showed a significantly (p<0.05) a higher T3 level in heifers followed by adults and calves and higher T4 level in adults followed by heifers and calves in all the seasons. The TSH level was higher in heifers in all the seasons. The winter season recorded higher level of T3, T4, and TSH as compared to the other seasons of a year. CONCLUSION: The TSH and T3 level were the highest for aheifer, whereas T4 level was the highest for adults inall the season. Furthermore, the higher level of TH was observed in winter season. The increased level of the TH during the winter season signifies their calorigenic effect. Similarly in heifers, the increased T3 concentrations show its importance in reproductive physiology and its association with ovarian activity. This indicates that age and season have aprofound effect on TH activity of Mizoram strain female mithun.

Cloning and sequencing of hfq (host factor required for synthesis of bacteriophage Q beta RNA) gene of Salmonella Typhimurium isolated from poultry.

AIM: The aim was to clone and sequence hfq gene of Salmonella Typhimurium strain PM-45 and compare its sequence with hfq gene of other serovar of Salmonella. MATERIALS AND METHODS: Salmonella Typhimurium strain PM-45 was procured from the G. B. Pant University of Agriculture and Technology, Pantnagar, India. The genomic DNA was isolated from Salmonella Typhimurium. Hfq gene was polymerase chain reaction (PCR) amplified from the DNA using specific primers, which was subsequently cloned into pET32a vector and transformed into Escherichia coli BL21 pLys cells. The recombinant plasmid was isolated and subjected to restriction enzyme digestion as well as PCR. The clone was then sequenced. The sequence was analyzed and submitted in GenBank. RESULTS: PCR produced an amplicon of 309 bp. Restriction digestion of the recombinant plasmid released the desired insert. The hfq sequence shows 100% homology with similar sequences from other Salmonella Typhimurium isolates. Both nucleotide and amino acid sequences are highly conserved. The submitted sequence is having Genbank accession no KM998764. CONCLUSION: Hfq, the hexameric RNA binding protein is one of the most important post-transcriptional regulator of bacteria. The sequence of hfq gene of Salmonella Typhimurium is highly conserved within and between Salmonella enterica serovars. This gene sequence is probably under heavy selection pressure to maintain the conformational integrity of its product in spite of its being not a survival gene.

Trypanosoma evansi infection in a German shepherd dog - Apparent successful treatment using serial low dose of diminazene aceturate.

The present study reports a new case of Trypanosoma evansi infection in a dog possibly due to oral transmission from a wild antelope, Nilgai. A four year old male German shepherd dog presented to the Referral Veterinary Polyclinic with history of inappetance, persistent fever, ocular discharge, standing for prolong periods and head pressing. Physical examination revealed corneal opacity, absence of menace reflex and partial blindness. The blood smear examination revealed the presence of Trypanosoma species. T. evansi infection was confirmed by RoTat 1.2 T. evansi specific Polymerase Chain Reaction (PCR). Haemato-biochemical examination showed anaemia, leukopenia, neutrophilia, a mild increase in concentration of alanine aminotransferase, blood urea nitrogen, and creatinine, and a decrease in concentration of blood glucose level. The dog was treated with diminazene aceturate at 3.5mg/kg body weight by deep intramuscular for 5days along with supportive therapy. Marked recovery in clinical signs as well as restoration of normal organ function and oxidant/antioxidant equilibrium was observed after two weeks of treatment. The dog was followed up to 6month and was negative for T. evansi by microscopy and PCR. The present treatment of a consecutive five dose regime of diminazene aceturate along with supportive therapy may help clinicians to overcome the hurdle of relapsing parasitaemia due to T. evansi and successful recovery in clinical cases.