The segmented genome of Borrelia burgdorferi, the tick-borne agent of Lyme disease, encodes numerous chromosomal and plasmid-borne proteins and small RNAs (sRNAs) of unknown function that are critical for infectivity. Two recent examples are the linear plasmid (lp)17-encoded protein BBD18 and sRNA SR0736 (also termed as ittA), which promote spirochete survival in ticks and mammals, respectively. Using targeted mutagenesis of the bbd18 locus, complementation, and phenotypic analysis of isogenic mutants, we herein confirm and extend the regulatory roles of BBD18 and SR0736 (ittA). A mutant lacking BBD18 and SR0736 (ittA) persisted in ticks, yet failed to infect immunocompetent or immunodeficient mice. Although bbd18 complementation selectively restored bbd18 expression, it did not rescue murine infectivity, supporting an essential role for SR0736 (ittA) during mammalian infection. Transcriptomic and proteomic analyses revealed widespread alterations in expression profiles that were only partially rescued by bbd18 complementation, suggesting distinct regulatory functions for BBD18 and SR0736 (ittA). Because an additional sRNA (SR0735) lies immediately downstream relative to bbd18, we generated an isogenic SR0735 inactivation mutant, which was likewise largely noninfectious in mice and exhibited dysregulation of multiple gene products, including the induction of several lp17 genes, such as bbd18, and the downregulation of multiple proteins, such as OspC, BamA, and DbpA. Together, these data indicate that the bbd18 locus is surrounded by two essential sRNA elements, SR0735 and SR0736 (ittA), all three of which independently regulate genes, including ones impacting mammalian infectivity. Further characterization of such atypical regulatory elements in B. burgdorferi may inform new control strategies.IMPORTANCEBorrelia burgdorferi, the tick-borne agent of Lyme disease, is the causative agent of one of the most prevalent vector-borne infections in many regions worldwide. Despite extensive study, the biological functions of many of its protein and small RNA (sRNA) products remain poorly defined. Here, we confirm and extend the regulatory roles of the linear plasmid (lp)17-encoded protein BBD18 and the sRNA SR0736 (ittA) in spirochete infectivity. Importantly, we identify a previously unrecognized regulatory function for an adjacent sRNA, SR0735, underscoring lp17 as a key regulatory region in B. burgdorferi. Together, our findings highlight the bbd18 locus and its surrounding sRNA elements as an independent, multilayered regulatory module that controls gene products, including those required for mammalian infection. Defining how these three regulators shape gene expression and virulence will reveal new mechanisms underlying Lyme disease pathogenesis and may inform the development of new strategies to prevent this widespread illness.

Trigger Factor (TF) is an essential ribosome-associated molecular chaperone that plays a critical role in protein folding, stabilization, and proteostasis maintenance across various bacterial systems. The multifunctional roles of TF, particularly its involvement in virulence modulation, stress response, and adhesion, are well established across multiple organisms, including Leptospira interrogans. However, its precise function has yet to be fully explored. Transcriptomic studies of Leptospira have unequivocally demonstrated that the genes tig and clpP1 are co-transcribed, indicating a significant regulatory interplay with the Clp protease complex. To further elucidate this relationship, this study engineered three LinTF mutant variants, targeting the N-terminal (LinTFΔNTD) and C-terminal regions (LinTFΔCTD and LinTFΔpCTD). Functional assays confirmed that LinTFΔpCTD and LinTFΔCTD markedly enhanced LinClpP activity by 49% and 34%. No increase in LinClpP activity was observed with LinTFΔNTD. Transmission electron microscopy and immunofluorescence techniques decisively revealed that LinTF is localized on the spirochete surface, a conclusion reinforced by subcellular fractionation. While LinTF is predominantly cytoplasmic, a small membrane-associated fraction indicates surface-associated moonlighting function. Full-length LinTF complemented the E. coli ΔtigΔdnaK strain, fully restoring growth at 37 °C. Conversely, LinTFΔpCTD complementation permitted only moderate recovery, while neither LinTFΔCTD nor LinTFΔNTD exhibited any rescuing effects on the E. coli ΔtigΔdnaK strain. Moreover, LinTF significantly promoted the refolding of chemically denatured GAPDH, as evidenced by a fluorescence emission shift and dynamic light scattering. Overall, these findings decisively highlight the domain-specific contributions of LinTF to LinClp protease regulation and protein folding, reaffirming its critical role in maintaining proteostasis in L.interrogans.

Bacterial caseinolytic protease (Clp) chaperone-protease complexes are essential for the degradation of misfolded and aggregated protein substrates. The spirochaete Leptospira interrogans possesses a set of Clp adaptor proteins (ClpS1 and ClpS2) and chaperones (ClpX, ClpA and ClpC), which are believed to associate with two distinct isoforms of ClpP (ClpP1 and ClpP2). This study explores the structural and functional properties of LinClpA, LinClpS1 and LinClpS2 derived from L. interrogans. LinClpA, a 740-amino acid protein, features an N-terminal domain and two AAA+ ATPase domains (D-I and D-II), containing conserved motifs critical for ATP binding and hydrolysis. LinClpS1 and LinClpS2 exhibit similar structures, yet they possess distinct binding pockets for N-degron substrates. Biochemical assays indicate that the N-domain-deleted variant of LinClpA (LinClpAΔN) exhibits a nucleotide-induced oligomerization tendency similar to LinClpA's but demonstrates higher ATPase activity. Interaction studies have shown that LinClpA's ATPase activity is enhanced in the presence of LinClpP isoforms and inhibited by LinClpS isoforms. In contrast, the activity of LinClpAΔN remained unaffected by LinClpS1 and LinClpS2, highlighting the significance of the N-domain of LinClpA in adaptor protein interactions. Furthermore, the study predicted and evaluated the role of the C-degron tag called small stable RNA A in facilitating protein degradation by the L. interrogans ClpAP1P2 machinery.

The functionally active ClpP (LinClpP) of Leptospira interrogans is composed of two different isoforms (LinClpP1 and LinClpP2). In this study, five mutants of LinClpP (LinClpP1E170D, LinClpP1N172D, LinClpP2IG_del, LinClpP2S40AK41N, LinClpP2Y62A) targeting its critical hotspot residues were generated. The functional activity of pure LinClpP mutant variants or its heterocomplex and its effect when associated with a chaperone (LinClpX)/antibiotic acyldepsipeptide (ADEP1)/trigger factor (LinTF) was examined. The two mutants (LinClpP2S40AK41N and LinClpP2Y62A) displayed gain-of-function (GOF) in peptidase activity. The ADEP1-bound heterocomplex (LinClpP1P2S40AK41N and LinClpP1P2Y62A) measured 1.7 and 1.5-fold higher protease activity than ADEP-bound LinClpP1P2. The dynamic light scattering analysis of ADEP1-bound GOF mutants displayed increased hydrodynamic diameter. In the presence of LinTF, the heterocomplex (LinClpP1P2S40AK41N and LinClpP1P2Y62A) exhibited a 3-fold surge in peptidase activity. The deletion mutant (LinClpP2IG_del) or its heterocomplex (LinClpP1P2IG_del) displayed no activity. Similarly, the pure LinClpP1E170D and LinClpP1N172D could not cleave a model dipeptide. However, its heterocomplex (LinClpP1E170DP2 and LinClpP1N172DP2) showed 0.5-fold lower peptidase activity than the LinClpP1P2. Collectively, two mutants (LinClpP2S40AK41N and LinClpP2Y62A) have GOF and can degrade model dipeptide substrate without the aid of LinClpP1 isoform and thus provide new insights into unprecedented LinClpP activation.

The TolC family protein of Leptospira is a type I outer membrane efflux protein. Phylogenetic analysis revealed significant sequence conservation among pathogenic Leptospira species (83%-98% identity) compared with intermediate and saprophytic species. Structural modeling indicated a composition of six beta-strands and 10 alpha-helices arranged in two repeats, resembling bacterial outer membrane efflux proteins. Recombinant TolC (rTolC), expressed in a heterologous host and purified via Ni-NTA chromatography, maintained its secondary structural integrity, as verified by circular dichroism spectroscopy. Polyclonal antibodies against rTolC detected native TolC expression in pathogenic Leptospira but not in nonpathogenic ones. Immunoassays and detergent fractionation assays indicated surface localization of TolC. The rTolC's recognition by sera from leptospirosis-infected hosts across species suggests its utility as a diagnostic marker. Notably, rTolC demonstrated binding affinity for various extracellular matrix components, including collagen and chondroitin sulfate A, as well as plasma proteins such as factor H, C3b, and plasminogen, indicating potential roles in tissue adhesion and immune evasion. Functional assays demonstrated that rTolC-bound FH retained cofactor activity for C3b cleavage, highlighting TolC's role in complement regulation. The rTolC protein inhibited both the alternative and the classical pathway-mediated membrane attack complex (MAC) deposition in vitro. Blocking surface-expressed TolC on leptospires using specific antibodies reduced FH acquisition by Leptospira and increased MAC deposition on the spirochete. These findings indicate that TolC contributes to leptospiral virulence by promoting host tissue colonization and evading the immune response, presenting it as a potential target for diagnostic and therapeutic strategies.

The survival and proliferation of pathogenic Leptospira within a host are complex phenomena that require careful consideration. The ErpY-like lipoprotein, found on the outer membrane surface of Leptospira, plays a crucial role in enhancing the bacterium's pathogenicity. The rErpY-like protein, in its recombinant form, contributes significantly to spirochete virulence by interacting with various host factors, including host complement regulators. This interaction facilitates the bacterium's evasion of the host complement system, thereby augmenting its overall pathogenicity. The rErpY-like protein exhibits a robust binding affinity to soluble fibrinogen, a vital component of the host coagulation system. In this study, we demonstrate that the rErpY-like protein intervenes in the clotting process of the platelet-poor citrated plasma of bovines and humans in a concentration-dependent manner. It significantly reduces clot density, alters the viscoelastic properties of the clot, and diminishes the average clotting rate in plasma. Furthermore, the ErpY-like protein inhibits thrombin-catalyzed fibrin formation in a dose-dependent manner and exhibits saturable binding to thrombin, suggesting its significant role in leptospiral infection. These findings provide compelling evidence for the anticoagulant effect of the ErpY-like lipoprotein and its significant role in leptospiral infection.

Bacterial caseinolytic protease -chaperone complexes participate in the elimination of misfolded and aggregated protein substrates. The spirochete Leptospira interrogans possess a set of Clp-chaperones (ClpX, ClpA, and ClpC), which may associate functionally with two different isoforms of LinClpP (ClpP1 and ClpP2). The L. interrogans ClpC (LinClpC) belongs to class -I chaperone with two active ATPase domains separated by a middle domain. Using the size exclusion chromatography, ANS dye binding, and dynamic light scattering analysis, the LinClpC is suggested to undergo nucleotide -induced oligomerization. LinClpC associates with either pure LinClpP1 or LinClpP2 isoforms non -preferentially and with equal affinity. Regardless, pure LinClpP isoforms cannot constitute an active protease complex with LinClpC. Interestingly, the heterocomplex LinClpP1P2 in association with LinClpC forms a functional proteolytic machinery and degrade beta-casein or FITC-casein in an energy -independent manner. Adding either ATP or ATP gamma S further fosters the LinClpCP1P2 complex protease activity by nurturing the functional oligomerization of LinClpC. The antibiotic, acyldepsipeptides (ADEP1) display a higher activatory role on LinClpP1P2 protease activity than LinClpC. Altogether, this work illustrates an in-depth study of heterotetradecamer LinClpP1P2 association with its cognate ATPase and unveils a new insight into the structural reorganization of LinClpP1P2 in the presence of chaperone, LinClpC to gain protease activity.

Pathogenic species of Leptospira are recalcitrant for genetic manipulation using conventional tools, and therefore there is a need to explore techniques of higher efficiency. Application of endogenous CRISPR-Cas tool is emerging and efficient; nevertheless, it is limited by a poor understanding of interference machinery in the bacterial genome and its associated protospacer adjacent motif (PAM). In this study, interference machinery of CRISPR-Cas subtype I-B (Lin_I-B) from L. interrogans was experimentally validated in E. coli using the various identified PAM (TGA, ATG, ATA). The overexpression of the Lin_I-B interference machinery in E. coli demonstrated that LinCas5, LinCas6, LinCas7, and LinCas8b can self-assemble on cognate CRISPR RNA to form an interference complex (LinCascade). Moreover, a robust interference of target plasmids containing a protospacer with a PAM suggested a functional LinCascade. We also recognized a small open reading frame within lincas8b that independently co-translates into LinCas11b. A mutant variant LinCascade-Cas11b that lacks LinCas11b co-expression erred to mount target plasmid interference. At the same time, LinCas11b complementation in LinCascade-Cas11b rescued target plasmid interference. Thus, the present study establishes Leptospira subtype I-B interference machinery to be functional and, soon, may pave the way for scientists to harness it as a programmable endogenous genetic manipulation tool.

A corkscrew-shaped spirochaete named Leptospira interrogans causes the infectious disease called leptospirosis. Leptospirosis, a growing public health concern worldwide, from subclinical infections to potentially lethal pulmonary hemorrhage. The disease caused by the pathogenic Leptospira, poses a threat to both humans and animals, and its transmission occurs through contact with infected animals, contaminated water, and soil. The prevalence of leptospirosis is influenced by various factors, including climate, urban development, and animal-rearing practices. It can manifest with severe symptoms in humans, making early diagnosis crucial. Diagnostic methods like microscopic agglutination test (MAT) and enzyme-linked immunoassay (ELISA) are widely used for the screening of leptospira infection. Molecular technique like PCR and qPCR offering higher sensitivity and rapidity. This paper meta-analyses the incidence of leptospirosis in various animals based on the literature published from 2005 to 2023 and provides prevalence of the disease in various animal including humans. Results suggest the significance prevalence of the disease in humans and various animal species, namely buffalo, rodents, and dogs. Coastal regions in India were particularly vulnerable to the disease. Efforts to control leptospirosis include surveillance programs and public health initiatives. Understanding the epidemiology and prevalence of leptospirosis, as highlighted in this paper, is essential for implementing effective preventive measures. Finally, a continued research, diagnostic advancements, public awareness campaigns and addressing research gaps in epidemiology of the disease are critical in mitigating the impact on human and animal health. This review provides important data for public health authorities, veterinarians, scientists and for the public, in general.

Leptospira interrogans serovar Copenhageni's genome harbors two CRISPR-Cas systems belonging to subtypes I-B and I-C. However, in L. interrogans, the subtype I-C locus lacks an array component essential for assembling an interference complex. Thus, the reason for sustaining the expense of a cluster of cas genes (I-C) is obscure. Type I-C (previously Dvulg) is the only CRISPR subtype that engages Cas5c, a Cas5 variant, to process precursor CRISPR-RNA (pre-crRNA). In this study, thus, the recombinant Cas5c (rLinCas5c) of L.interrogans and its mutant variants were cloned, expressed, and purified. The purified rLinCas5c is illustrated as metal-independent, sequence, and size-specific cleavage on repeat RNA and pre-crRNA of subtype I-B or orphan CRISPR array. However, the Cas6-bound mature crRNA of subtype I-B fends off from the rLinCas5c activity. In addition, rLinCas5c holds metal and size-dependent DNase activity. The bioinformatics analysis of LinCas5c inferred that it belongs to the subgroup Cas5c-B. Substitution of Phe141 with a more conserved His residue and deletion of unique (β1'-β2') insertions usher a gain of rLinCas5c activity over nucleic acid. Overall, our results uncover the functional diversity of Cas5c ribonucleases and infer an incognito auxiliary role in the absence of a cognate CRISPR array.

The genome of pathogenic Leptospira interrogans serovars (Copenhageni and Lai) are predicted to have CRISPR-Cas of subtypes I-B and I-C. Cas2, one of the core Cas proteins, has a crucial role in adaptive defense against foreign nucleic acids. However, subtype I-C lacks the CRISPR element at its loci essential for RNA-mediated adaptive immunity against foreign nucleic acids. The reason for sustaining the expense of cas genes are unknown in the absence of a CRISPR array. Thus, Cas2C was chosen as a representative Cas protein from two well-studied serovars of Leptospira to address whether it is functional. In this study, the recombinant Cas2C of Leptospira serovars Copenhageni (rLinCas2C, 12 kDa) and Lai (rLinCas2C_Lai, 8.6 kDa) were overexpressed and purified. Due to natural frameshift mutation in the cas2c gene of serovar Lai, rLinCas2C_Lai was overexpressed and purified as a partially translated protein. Nevertheless, the recombinant Cas2C from each serovar exhibited metal-dependent DNase and metal-independent RNase activities. The crystal structure of rLinCas2C obtained at the resolution of 2.60 Å revealed the protein is in apostate conformation and contains N- (1–71 amino acids) and C-terminal (72–90 amino acids) regions, with the former possessing a ferredoxin fold. Substitution of the conserved residues (Tyr7, Asp8, Arg33, and Phe39) with alanine and deletion of Loop L2 resulted in compromised DNase activity. On the other hand, a moderate reduction in RNase activity was evident only in selective rLinCas2C mutants. Overall, in the absence of an array, the observed catalytic activity of Cas2C may be required for biological processes distinct from the CRISPR-Cas-associated function.

In the genome of various Leptospira interrogans serovars , the subtype I-B locus of CRISPR-Cas possesses either one or multiple CRISPR arrays. In silico database (CRISPRCasdb) for predicting CRISPR-Cas reveals seven CRISPR arrays in L. interrogans serovar Lai positioned between the two independent cas -operons. Here, we present the redefined repeat-spacer boundaries of the CRISPR subtype I-B locus of serovar Lai. Such refinement of boundaries of arrays in serovar Lai was done after comparison with the characterized array of another serovar Copenhageni and the manual analysis of CRISPR flanking sequences. Using the reverse transcription-PCR (RT-PCR), we account that the seven CRISPR are transcriptionally active in serovar Lai. Our RT-PCR and quantitative real-time PCR analysis of transcripts in serovar Lai indicated that seven CRISPR of subtype I-B transcribe together as a single precursor unit. Moreover, the cleavage of the two miniature pre-crRNA of the subtype I-B by Cas6 demonstrates the biogenesis of the expected size of mature crRNA essential for the guided interference of foreign DNA. This study features insight into transcription direction and the crRNA biogenesis in serovar Lai essential for RNA-mediated interference of invading nucleic acids.

The survival of pathogenic Leptospira in the host depends on its proficiency to circumvent the immune response. These pathogens evade the complement system in serum by enticing and amassing the serum complement regulators onto their surface. ErpY-like lipoprotein, a surface-exposed protein of Leptospira spp., is conserved in the pathogenic Leptospira serovars. The recombinant form of this protein interacts with multiple extracellular matrix (ECM) components and serum proteins such as soluble complement regulators factor H (FH) and factor I (FI). Here, we document that the supplementation of rErpY-like protein (10 μg/mL) in human serum inhibits complement-mediated bacterial cell lysis and augments the viability of Escherichia coli and saprophytic Leptospira biflexa by more than two-fold. Complement regulators FH and FI, when bound to rErpY-like protein, preserve their respective cofactor and protease activity and cleave the complement component C3b. The supplementation of rErpY-like protein (40 μg/mL) in serum ensued in an ∼90% reduction of membrane attack complex (C5b-9/MAC) deposition through the alternative pathway (AP) of complement activation. However, rErpY-like protein could moderately reduce (∼16%) MAC deposition in serum through the classical pathway (CP). In addition, the rErpY-like protein solely initiated the AP, suggesting its role in the rapid consumption and depletion of the complement components. Blocking the pathogenic Leptospira interrogans surface with anti-rErpY-like antibodies resulted in an increase in MAC formation on the bacterial surface, indicating a specific role of the ErpY-like lipoprotein in complement-mediated immune evasion. This study underscores the role of the ErpY-like lipoprotein of Leptospira in complement evasion.

The spirochete Leptospira interrogans serovar Copenhageni harbors the genetic elements of the CRISPR-Cas type I-B system in its genome. CRISPR-Cas is a CRISPR RNA (crRNA) mediated adaptive immune system in most prokaryotes against mobile genetic elements (MGEs). To eliminate the intruding MGEs, CRISPR-Cas type I systems utilize a Cascade (CRISPR-associated complex for antiviral defense) complex composed of Cas5, Cas6, Cas7, and Cas8 bound with a crRNA. The Cas7 is essentially known to constitute the major component of the Cascade complex. The present study reports the biochemical characterization of the Cas7 (LinCas7) from the CRISPR-Cas type I-B system of L. interrogans serovar Copenhageni. The pure recombinant LinCas7 (rLinCas7) exists as a monomer in the solution by size exclusion chromatography. The rLinCas7 demonstrates an endoDNase activity dependent upon divalent Mg2+ ions, monovalent ions, pH, temperature, and substrate size. Analysis of ribonucleoprotein composite (rLinCas7-crRNA) by electron microscopy and native-PAGE demonstrated that rLinCas7 could oligomerize on the mature CRISPR RNA (crRNA) framework in the presence of Mg2+ ions. The ribonucleoprotein composite attains a helical shape similar to the backbone of the Cascade complex. However, in the absence of Mg2+ ions, rLinCas7 acts as an RNase. The fluorescence spectroscopy disclosed a weak interaction (K-d = 26.81 mM) between rLinCas7 and Mg2+ ions, leading to an overall conformational change in rLinCas7 that modulates the rLinCas7's activity on DNA and RNA substrates. The nuclease activity of LinCas7 characterized in this study aids to the functional divergences among proteins of the Cas7 family from different CRISPR-Cas systems in various organisms.

Background: Water buffaloes are important milch species of tropical and subtropical countries. In general, they are considered to more resistant to diseases compared to crossbred cattle. However, a recent problem of tick-borne diseases in the water buffaloes by the field veterinarians and farmers causing production losses was observed. The study was conducted to explore the spectrum of tick-borne diseases (TBDs) infections in buffaloes and analyze the associated risk factors. Methods: Acute-phase response, cytokine and oxidative stress in infected buffaloes with TBDs were evaluated and compared with the negative buffaloes (control) to elucidate their role in pathogenesis and outcome of infection. The study was undertaken in 107 tick-infested water buffaloes. The conventional Giemsa stained blood smear (GSBS) based confirmation and classification of infection of haemo-parasites were made. The statistical model was used to understand their relevance with TBDs. Conclusion: Tick-borne disease in water buffaloes must be looked upon seriously to maintain good productivity. The buffaloes are often accompanied by low and un-yielding clinical symptoms due to associated co-infections of haemo-parasites. Our study showed that the buffaloes had a high prevalence (47.66%) of haemo-parasites transmitted by the ticks associated with co-infections and a low level of parasitemia. Most buffaloes responded symptomatically with the different modalities administered. Production parameter was not restored post 15 days of treatment. Oxidative stress is one of the important mechanisms of production losses in infected buffaloes.

Background: A novel, rapid and specific multiplex polymerase chain reaction was developed to diagnose hemo-parasitic infection in bovine blood co-infected with three of the most common hemo-parasites. Methods: The diagnostic process relied on the detection of the three different bovine hemoparasites isolated from red blood cells (RBCs) of cattle (N=30) by conventional Giemsa stained blood smear (GSBS) and confirmed by multiplex PCR. The multiplex PCR system was used to diagnose GSBS positive blood samples (N=12) found infected or co-infected with hemoparasites. The designed multiplex primer sets was attempted to amplify 205, 313 and 422 bp fragments of apocytochrome b, sporozoite and macroschizont 2 (spm2) and 16S rRNA gene for Babesia bigemina, Theileria annulata and Anaplasma marginale, respectively. Result: This multiplex PCR was sensitive with the ability to detect the presence of 150 ng of genomic DNA. The primers used in this multiplex PCR also showed highly specific amplification of specific gene fragments of each respective parasite. Comparing the two detection methods revealed that 58.33% of specimens showed concordant diagnoses with both techniques. The specificity, positive predictive value and kappa coefficient of the agreement was highest for diagnosis of B. bigemina and lowest for A. marginale. The overall Kappa coefficient for diagnosis based on GSBS for multiple pathogens compared to multiplex PCR was 0.56, slightly behind the threshold of 0.6 of agreement. Therefore, confirmation should always be based on PCR to rule out false positives due to differences in subjective observations, stain particles and false negatives due to low parasitemia. The simplicity and rapidity of this specific multiplex PCR method make it suitable for large-scale epidemiological studies and follow-up of drug treatments.

ABSTRACT A novel, rapid and specific multiplex polymerase chain reaction has been developed for the diagnosis of hemo-parasitic infection in bovine blood by three of the most common hemo-parasites. The reported method relied on the detection of the three different bovine hemoparasites isolated from red blood cells (RBCs) of cattle by conventional Giemsa stained blood smear (GSBS) and confirmed by multiplex PCR. The designed multiplex primer sets can amplify 205, 313 and 422 bp fragments of apocytochrome b, sporozoite and macroschizont 2 (spm2) and 16S rRNA gene for Babesia bigemina, Theileria annulata and Anaplasma marginale , respectively. This multiplex PCR was sensitive with the ability to detect the presence of 150 ng of genomic DNA. The primers used in this multiplex PCR also showed highly specific amplification of specific gene fragments of each respective parasite DNA without the presence of non-specific and non-target PCR products. This multiplex PCR system was used to diagnose GSBS confirmed blood samples (N=12) found infected or co-infected with hemoparasites. A comparison of the two detection methods revealed that 58.33% of specimens showed concordant diagnoses with both techniques. The specificity, positive predictive value and kappa coefficient of agreement was highest for diagnosis of B. bigemina and lowest for A. marginale . The overall Kappa coefficient for diagnosis based on GSBS for multiple pathogen compared to multiplex PCR was 0.56 slightly behind the threshold of 0.6 of agreement. Therefore, confirmation should always be made based on PCR to rule out false positive due to differences in subjective observations, stain particles and false negative due to low level of parasitaemia. The simplicity and rapidity of this specific multiplex PCR method make it suitable for large-scale epidemiological studies and for follow-up of drug treatments.

Background: Mastitis is the most common and economically important disease of dairy cattle. Subclinical mastitis is a more important form in India than clinical mastitis. Subclinical mastitis (SCM) detection done by periodic examination of the udder health by evaluation of milk at the herd level or the individual cow level by milk somatic cell count (SCC), followed by culture of random milk samples. The presented study was undertaken by survey and sampling of milk from lactating crossbred cattle of unorganized dairy farms and farmer’s dairy of the peri-urban region of middle Indo-Gangetic Plains. The objective of the study was to monitor the status of SCM in crossbred cattle and associated changes in milk constitutes and oxidative stress in milk. Methods: A total of 147 lactating crossbred cattle were screened for SCM using the modified California Mastitis Test (CMT) using detergent based CMT reagent and compared with conventional CMT reagent, followed by SCC, milk constituents, bacterial isolation and antibiotic sensitivity testing (ABST). General information was collected in pre-tested questionnaire. The data obtained were statistically treated to evaluate significance of the study.Conclusion: The overall prevalence of subclinical mastitis in peri-urban cross-bred cattle was 36.74% which varied with parity and stage of milking. Prevalence of subclinical mastitis was highest (55.77%) in cross-bred cattle in mid-lactation and Parity 3-5. The mean SCC was significantly higher (7.21±0.27) in subclinical mastitis compared to CMT negative (3.66±0.06) milk sample. Somatic cell count was positively and significantly correlated with CMT reactions using conventional CMT reagent (rs= 0.86) as well as modified CMT reagent (rs=0.815) attempted using Spearman rank-order correlation coefficient. The mean values of milk pH, fat and lactic acid acidity increased significantly (P£0.01) in SCM compared to the milk of healthy cattle, except lactose which decreased significantly in SCM milk. The common contagious bacteria responsible for SCM isolate were coagulase-positive Staphylococcus spp. (64.82%) isolates from these SCM milk followed by Streptococcus spp. ABST test conducted on random CMT positive milk sample indicated that gentamicin as most sensitive, followed by enrofloxacin. The present finding indicates the suitability of enrofloxacin as the most useful antibiotic for the treatment of subclinical mastitis in cross-bred cattle of the peri-urban area of middle Indo-Gangetic plains.

The genomic analysis of Leptospira reveals a trigger factor (TF) encoding gene (tig) to be colocalized along with the clpP1 and clpX. The TF is a crouching dragon-like protein known to be a ribosome-associated chaperone that is involved in cotranslational protein folding in bacteria in an ATP-independent mode. In Leptospira, tig is localized upstream of the clpP1 with a short (4 bp) overlap. In the present study, we document the distinctive role of Leptospira TF (LinTF) in the caseinolytic protease (ClpP) system. The recombinant LinTF (rLinTF) was found to improve the peptidase or protease activity of the ClpP1P2 heterocomplex and ClpXP1P2 complex, respectively, on model substrates. In addition, on supplementation of rLinTF to rClpP1P2 bound to its physiological ATPase chaperone ClpX or the antibiotic analogue acyldepsipeptide (ADEP), an augmentation in the activity of ClpP1P2 was observed. These studies underscore the novel role of LinTF in aiding the caseinolytic protease activity of Leptospira. Supplementation of rLinTF to a peptidase assay of rClpP1P2 conditionally in the presence of a salt (sodium citrate) with high Hofmeister strength led us to speculate that rLinTF may have a role in the assembly of multimeric proteins. The deletion of one of the arms (arm-2) of the LinTF structure from the carboxy terminal domain indicated a reduction in its capacity to stimulate rClpP1P2 activity. Thus, the C-terminal domain of LinTF may have a role in the assembly of multimeric ClpP protein, leading to enhancement of ClpP activity.

Theileria are tick-borne apicomplexan parasites, which mainly infect ruminants in tropical and subtropical regions of the world. The present study was directed to investigate the serological methods for the diagnosis of theileriosis in crossbred cattle. Blood samples (n = 176) were collected from the regional cattle populations of Bihar state situated at the Gangetic plains of India. Microscopic examination of blood smears from the cattle revealed the presence of tick-borne infectious organisms (Theileria and Anaplasma) in the region. PCR-based detection of Tams1 (Theileria annulata merozoite surface antigen) gene and the sequencing of 18S rRNA amplicon from the blood samples confirmed T. annulata as the primary causative agent of theileriosis in cattle of the Bihar region. Similarly, the amplification of the msp5 gene confirmed Anaplasma marginale infection. For the large-scale epidemiological investigation, sporozoite and macroschizont (spm2) partial gene from T. annulata was cloned in pET-28a (+) vector and overexpressed in E. coli BL21 cells. Overexpressed recombinant-Spm2 (43 kDa) was purified by Ni-NTA affinity chromatography and was used for immunodetection of theileriosis in cattle serum samples. Sequence analysis of the cloned partial spm2 gene in this study showed multiple SNPs (single nucleotide polymorphisms) in T. annulata. Recombinant-Spm2 antigen was explicitly recognised by the immunoglobulins (IgG) of the cattle naturally infected with Theileria. Further, an indirect enzyme-linked immunosorbent assay (ELISA) was developed using partial r-Spm2 antigen that exhibited high sensitivity (100 %) and specificity (90.9 %). Thus, this study suggests that partial r-Spm2 can be used as a diagnostic antigen for seroepidemiological studies of T. annulata infection in crossbred cattle.