Cpb Gene Research Articles

Shaping Drosophila eggs: unveiling the roles of Arpc1 and cpb in morphogenesis.

The Drosophila egg chamber (EC) starts as a spherical tissue at the beginning. With maturation, the outer follicle cells of EC collectively migrate in a direction perpendicular to the anterior-posterior axis, to shape EC from spherical to ellipsoidal. Filamentous actin (F-actin) plays a significant role in shaping individual migratory cells to the overall EC shape, like in every cell migration. The primary focus of this article is to unveil the function of different Actin Binding Proteins (ABPs) in regulating mature Drosophila egg shape. We have screened 66 ABPs, and the genetic screening data revealed that individual knockdown of Arp2/3 complex genes and the "capping protein β" (cpb) gene have severely altered the egg phenotype. Arpc1 and cpb RNAi mediated knockdown resulted in the formation of spherical eggs which are devoid of dorsal appendages. Studies also showed the role of Arpc1 and cpb on the number of laid eggs and follicle cell morphology. Furthermore, the depletion of Arpc1 and cpb resulted in a change in F-actin quantity. Together, the data indicate that Arpc1 and cpb regulate Drosophila egg shape, F-actin management, egg-laying characteristics and dorsal appendages formation.

Infection of Leishmania donovani in Phlebotomus orientalis Sand Flies at Different Microhabitats of a Kala-Azar Endemic Village in Eastern Sudan.

A study was carried out to compare the infection rates of Leishmania donovani in Phlebotomus orientalis sandflies at different microhabitats of a VL endemic village in Gedarif state, Sudan. DNA extracts of 1078 P. orientalis sand fly females sampled by CDC light traps from indoor, outdoor, peri-domestic, and sylvatic sites, in three transmission seasons, March-June 2016-18, in Helat-Belo village, were subjected to independent PCR amplifications targeting Leishmania kDNA and the cpb gene followed by ITS1 region sequencing. Leishmania kDNA was detected in 1.4% of the 1078 P. orientalis females captured in the area. Two of these specimens showed a characteristic 741 bp band of L. donovani after cpb gene amplification. The DNA sequence of the ITS1 region of the parasites matched the ITS1 L. donovani genotype F. There were no signficant differences between rates of infection of L. donovani in P. orientalis captured at different sites. Blood meals found in infected flies origninated from human (5 specimens), cattle (4 specimens) and donkey (2 specimens). The finding of fresh cow and donkey blood in the infected flies suggests the possible role of these animals in the zoopotentiation and/or zooprophylaxis against VL. The study provides important information for VL transmission models and control programs in East Africa.

Toxin-producing Clostridium perfringens in cooked cereal food in restaurants in Bangladesh

Clostridium perfringens causes food poisoning in humans worldwide and C. perfringens beta toxin is associated with improperly heated or reheated cooked food. A study was undertaken to determine the prevalence of beta toxin-producing C. perfringens in cooked Plain rice, Pulao, and Biryani in different types of restaurants (general, well furnished) of four districts [Dhaka (north and south city corporation area), Cumilla, Narayangonj, Gazipur)] of Bangladesh. A total of 200 food samples were examined for the presence of C. perfringens and its beta toxin. The positive samples were further tested for the CPB gene (236 bp) of beta toxin-producing C. perfringens using PCR assay. Three samples had C. perfringens (1.5%), in food samples of the restaurants in Dhaka South City Corporation (DSCC). Beta toxin-producing C. perfringens was in one sample (0.5%) in a sample of pulao in the same area during the winter. It is suggested that the prevalence of beta toxin-producing C. perfringens was low but, further studies are required in other cities in Bangladesh.
 Bangl. vet. 2023. Vol. 40, No. 1-2, 8–15

ITS1 and cpb genetic polymorphisms in Algerian and Tunisian Leishmania infantum isolates from humans and dogs.

Leishmania (L.) infantum strains, isolated from varying hosts and clinical manifestations (cutaneous, visceral and canine leishmaniasis), were investigated in order to understand the genetic polymorphisms within this species in Algeria and Tunisia. Two DNA-based typing methods were tested in order to evaluate their effectiveness against Multilocus enzyme electrophoresis (MLEE), widely considered as the reference method for Leishmania parasite typing. On the other hand, MLEE is cumbersome, high-cost, time consuming and frequently does not detect intra-species genetic polymorphisms. In this work, we used two molecular target regions to discriminate L. infantum strains, Internal transcribed spacer 1 (ITS1) and the cysteine proteinase B (cpb). The ITS1 region offers good resolution for Leishmania discrimination but does not spotlight intra-species polymorphisms. In contrast, cpbE and cpbF PCR-Sequencing demonstrated a certain variability within CL and VL Algerian and Tunisian L. infantum isolates. Following phylogenetic analyses of 44 L.infantum isolates, two main groups were identified, a group with 39 bp deletion in the cpb sequence, composed of cutaneous, visceral and canine isolates from both countries with no significant clinical or geographic distribution; these samples were typed as MON-1, MON-24, and MON-80 zymodemes. A second group which presents a clear clusterization of Tunisian cutaneous strains belonging to the L. infantum MON-24. This group, with no deletion in the mature domain of the cpb gene sequence, should be further explored with a higher number of samples.

Assessment of pan-Leishmania detection by recombinase polymerase amplification assay

The spread of vector habitats along with increasing human mobility can introduce atypical Leishmania species and hence can challenge existing diagnostic practices for rapid detection of active infection with species outside the narrow target range. Here we assessed the pan-Leishmania detection ability of isothermal recombinase polymerase amplification (RPA) assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions. While the lowest limit of detection of the 18S rRNA-RPA and Cpb-RPA assays were estimated as 12 and 17 standard DNA molecules, respectively, both assays could amplify genomic DNA of 7 pathogenic Leishmania species. Evaluation of 18S rRNA-RPA and our previously developed kDNA-RPA assays on 70 real-time PCR-positive leishmaniasis samples of varying pathologies resulted in sensitivity rates of 35.71% and 88.57%, respectively, while the combined sensitivity was 98.57%. Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays can be recommended for further diagnostic assessments.

Molecular epidemiology and genetic characterization of Clostridium perfringens infections in lambs

Clostridium perfringens is gram positive bacterium, wide spread in environment causing various diseases in animals and human. The current study was conducted to indentify the genetic identity of C. perfringens isolates from lambs from Egypt. Using specific primers amplifying genes associated to the toxins produced by C. perfringens, multiplex PCR was used to confirm C. perfringens in 87 out of 140 samples were collected from diseased and suspected lambs. The isolates were classified as type A in 49.4%, type B in 31.1% and type D in 19.5% of isolates. The phylogenetic analysis for the partial sequences of C. perfringens strains based on plc gene, cpb gene and etx gene obtained in the present study showed high degree of similarity with other sequences of C. perfringens strains in GenBank, isolating from sheep from Egypt and other countries. According to the findings, lambs with enterotoxaemia more frequently have C. perfringens type A and an efficient hygienic control program is necessary to reduce the infection spreading among susceptible animals.

Prevalence and genotypic characterization of Clostridium perfringens associated with goat (Capra hircus) enterotoxemia in Southeast Iran

Goat enterotoxaemia, a worldwide disease with a high case-fatality rate (up to 100%), is mainly caused by Clostridium perfringens (C. perfringens) toxinotypes D, C, and B, respectively. Here, the prevalence and genotypic characteristics of C. perfringens in goats were investigated in southeast Iran during 2016–2020. In total, 5300 goats from 57 herds were examined, and 112 strains of C. perfringens were isolated from 142 goats (79 dysenteric and 63 healthy goats with a history of enterotoxemia). PCR tests were conducted on isolates using the 16SrRNA, cpa, cpb, etx, iap, cpe, and netb gene primers. Toxinotyping of C. perfringens isolates revealed that the existing toxinotypes were type D (26.8%), followed by types A and F (25.9% for each), then type B (15.2%), and type C (6.2%). The overall prevalence of C. perfringens at the animal and farm levels was 19.0% and 36.8%, respectively. The isolation rate of C. perfringens from dysenteric goats (30.4%) was significantly higher than that of the healthy goats (p = 0.000). Among the isolates, 44.6% (50 of 112) were PCR positive for the cpe gene. The majority of cpe-positive isolates were characterized as type F (58.0%), followed by types D (28.0%), B (8.0%), and C (6.0%). Dysenteric goats were most likely to yield cpe-positive isolates (94.1%). Our results show the high prevalence of Clostridium perfringens toxinotypes in goats and, for the first time, indicate the presence of type F and cpe-positive type B strains in goat enterotoxemia.

Malaria Box Compounds against Anopheles gambiae (Diptera: Culicidae) Carboxypeptidase B Activity to Block Malaria Transmission.

Carboxypeptidase B (CPB) plays an important role in blood digestion in mosquitos, aiding the release of free amino acids. Anopheles CPB is a target to block malaria transmission because it facilitates Plasmodium invasion of the mosquito midgut. Our study aimed to discover inhibitors of Anopheles CPB to prevent Plasmodium development in the mosquito. The Anopheles gambiae cpb (Agcpb) gene without a signal sequence was cloned into the pET28b expression vector. The recombinant AgCPB protein was expressed in E. coli BL21(DE3) within inclusion bodies after induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside at 37°C for 4 h. The protein pellet was dissolved in 6 M urea, purified by affinity chromatography, and dialyzed in reaction buffer. The refolded recombinant AgCPB could digest the hippuryl-arginine substrate similarly to that of the commercial porcine pancreas CPB. The 20 top-scoring malaria box compounds from the virtual-screening results were then chosen for an in vitro inhibition assay against AgCPB. Four of the 20 malaria box compounds could inhibit AgCPB activity. The compound MMV007591 was the most potent inhibitor with an IC50 at 0.066 µM. The results indicate that these candidate compounds may be utilized in drug development against mosquito CPB activity to curb malaria transmission.

Immunogenicity and potential protection of DNA vaccine of Leishmania martiniquensis against Leishmania infection in mice.

In Thailand, Leishmania martiniquensis is the predominant species causing cutaneous and visceral leishmaniasis. Its incidence has been increasing among immunocompetent and immunocompromised hosts. We developed a prototype DNA vaccine using a partial consensus sequence of the cysteine protease B (cpb) gene derived from L. martiniquensis from Thai patients. The laboratory inbred strain of albino BALB/c mice were immunized intramuscularly three times at 2-week intervals (weeks 0, 2, and 4) with cpb plasmid DNA (pcDNA_cpb) with or without the adjuvant, monoolein (pcDNA_cpb-MO). Mice were challenged at week 6 with L. martiniquensis promastigotes. Sera were analysed for IgG1, IgG2a, interferon gamma and interleukin 10 (IFN-γ and IL-10, respectively) levels at weeks 0, 4, and 9. Additionally, livers and spleens were also analysed for parasite burden using immunohistochemistry and real-time polymerase chain (qPCR) assays. Three weeks after promastigote challenge, vaccinated mice showed significantly increased levels of IgG2a and IFN-γ while IL-10 level was significantly reduced when compared with those in the control group (p < 0.01). Parasite burden in the livers and spleens of vaccinated mice significantly decreased. In addition, a significant increase in mature granuloma formation in the livers when compared with those of the control group (p < 0.05) was found, indicating increased T-helper cells (Th1)-induced inflammation and destruction of amastigotes. Monoolein produced a booster effect to enhance the mouse Th1 protective immunity. The prototype DNA vaccine could induce a Th1 immune response that conferred potential protection to the L. martiniquensis promastigote challenge in BALB/c mice.

Identifying the Basis for VirS/VirR Two-Component Regulatory System Control of Clostridium perfringens Beta-Toxin Production.

Clostridium perfringens toxin production is often regulated by the Agr-like quorum sensing (QS) system signaling the VirS/VirR two-component regulatory system (TCRS), which consists of the VirS membrane sensor histidine kinase and the VirR response regulator. VirS/VirR is known to directly control expression of some genes by binding to a DNA binding motif consisting of two VirR boxes located within 500 bp of the target gene start codon. Alternatively, the VirS/VirR system can indirectly regulate production levels of other proteins by increasing expression of a small regulatory RNA, VR-RNA. Previous studies demonstrated that C. perfringens beta-toxin (CPB) production by C. perfringens type B and C strains is positively regulated by both the Agr-like QS and the VirS/VirR TCRS, but the mechanism has been unclear. The current study first inactivated the vrr gene encoding VR-RNA to show that VirS/VirR regulation of cpb expression does not involve VR-RNA. Subsequently, bioinformatic analyses identified a potential VirR binding motif, along with a predicted strong promoter, ∼1.4 kb upstream of the cpb open reading frame (ORF). Two insertion sequences were present between this VirR binding motif/promoter region and the cpb ORF. PCR screening of a collection of strains carrying cpb showed that the presence and sequence of this VirR binding motif/promoter is highly conserved among CPB-producing strains. Reverse transcription-PCR (RT-PCR) and a GusA reporter assay showed this VirR binding motif is important for regulating CPB production. These findings indicate that VirS/VirR directly regulates cpb expression via VirS binding to a VirR binding motif located unusually distant from the cpb start codon. IMPORTANCE Clostridium perfringens beta-toxin (CPB) is only produced by type B and C strains. Production of CPB is essential for the pathogenesis of type C-associated infections, which include hemorrhagic necrotizing enteritis and enterotoxemia in both humans and animals. In addition, CPB can synergize with other toxins during C. perfringens gastrointestinal diseases. CPB toxin production is cooperatively regulated by the Agr-like quorum sensing (QS) system and the VirS/VirR two-component regulatory system. This study now reports that the VirS/VirR regulatory cascade directly controls expression of the cpb gene via a process involving a VirR box binding motif located unusually far (∼1.4 kb) upstream of the cpb ORF. This study provides a better understanding of the regulatory mechanisms for CPB production by the VirS/VirR regulatory cascade.

Characterisation of the &lt;i&gt;Leishmania donovani/L. infantum&lt;/i&gt; Hybrid Isolated from an Autochothonous Kala-Azar Patient: Preliminary Results of an In Vivo Model

In the present study, preliminary outcomes of the in vivo assessment of a Leishmania donovani/L. infantum hybrid isolated from a hospitalised patient with visceral leishmaniasis in Manisa and identified through analysis of the Leishmania-specific ITS-1, hsp70 and cpb gene regions are presented in comparison with reference strains of L. donovani and L. infantum. Three different study groups [(SG); n=16 mice each] and a control group (n=8 mice) were established with female Balb/C mice weighing 25-30 g. Reference L. donovani (MHOM/IN/1980/DD8), reference L. infantum (MHOM/TN/1980/IP1) and a L. donovani/L. infantum hybrid (MHOM/TR/2014/CBVL-LI/ LD), stored in liquid nitrogen, were thawed, cultured and incubated at 25 °C. A 15-μL dose of 1x108/mL promastigotes of three strains was applied to the tail veins of mice in the SG. After the mice were sacrificed, the liver and spleen tissues were removed and stored for immunological, immunohistochemical and pathological analyses. The presence of infection in the liver and spleen tissues of mice was detected both by a specific enzyme-linked immunosorbent assay test and from the recovery of Leishmania promastigotes from liver and spleen tissues in NNN medium. However, Leishmania amastigotes were not observed in the touch biopsy smears of livers or spleens in either of the SGs. In addition, no evidence of tissue damage was identified in the SGs after immunohistochemical staining (with antibodies against IL-9, CD-117, MBP, CD163, CD4, CD8 and CD31). The obtained results show that hybrid Leishmania and reference L. donovani and L. infantum strains reached the liver and spleens of Balb/C mice in SGs but were of no pathological consequence. Yet, these three Leishmania isolates caused skin lesions when applied subcutaneously in Balb/C mice in another study. The findings presented in this study will be reassessed upon completion of the project, once the final results are obtained.

Opportunistic feeding behaviour and Leishmania infantum detection in Phlebotomus perniciosus females collected in the human leishmaniasis focus of Madrid, Spain (2012-2018).

BackgroundAn outbreak of human leishmaniasis due to Leishmania infantum has been registered in an urban area of southwestern Madrid, Spain, since 2010. Entomological surveys carried out in the municipalities of Fuenlabrada, Leganés, Getafe and Humanes de Madrid showed that Phlebotomus perniciosus is the only potential vector. In this work, an intensive molecular surveillance was performed in P. perniciosus females captured in the region between 2012 and 2018.Methodology/Principal findingsA total of 1805 P. perniciosus females were analyzed for Leishmania infection, and 1189 of them also for bloodmeal identification. Eleven different species of vertebrate were detected by amplification and subsequent sequencing of the 359 bp cytb fragment. The most prevalent blood source identified was hare (n = 553, 46.51%), followed by rabbit (n = 262, 21.95%). Less frequent were cat (n = 45, 3.80%), human (n = 34, 2.90%), pig (n = 14, 1.20%), horse (n = 11, 0.93%), sheep (n = 3, 0.25%), rhea (n = 3, 0.25%), partridge (n = 1, 0.09%) and chicken (n = 1, 0.09%). The distribution of the blood meal sources varied between the different locations. Regarding L. infantum detection, PCR amplification of a fragment of kDNA, cpb gene and ITS1 region showed 162 positive specimens (8.97%). The highest infection rate was found in the municipality of Leganés (15.17%).ConclusionsThe results of this molecular survey in P. perniciosus, the only leishmaniasis vector in the outbreak occurred in southwestern Madrid region, showed its opportunistic blood-feeding behaviour, high infection rates and the differences between the different points. This study was an essential part of the intensive surveillance plan in the area and the results obtained have supported the implementation of control measures in the outbreak.

Cropland connectivity affects genetic divergence of Colorado potato beetle along an invasion front.

The population genetic structure of invasive species can be strongly affected by environmental and landscape barriers to dispersal. Disentangling the relative contributions of these factors to genetic divergence among invading populations is a fundamental goal of landscape genetics with important implications for invasion management. Here, we relate patterns of genetic divergence in a global invasive agricultural pest, Colorado potato beetle (CPB; Leptinotarsa decemlineata), to environmental and landscape factors along an invasion front in Northwestern China. We first used microsatellite markers and spatial‐temporal samples to assess broad patterns of genetic diversity as well as fine‐scale changes in patterns of genetic divergence. We then distinguished the relative contributions of five factors to genetic divergence among front populations: geographic distance (isolation by distance), climate dissimilarity (isolation by environment), and least‐cost distances (isolation by resistance) modeled with three factors: climate suitability, cropland cover, and road networks. Genetic diversity broadly decreased from West to East, with the exception being Eastern China. Low levels of genetic diversity and varying degrees of divergence were observed in Northwestern China, reflecting the potential effect of landscape heterogeneity. Least‐cost distance across cropland cover was most positively correlated with genetic divergence, suggesting a role of croplands in facilitating gene flow. The contribution of climate to genetic divergence was secondary, whether modeled in terms of local adaptability or connectivity of the climatic landscape, suggesting that constraints to CPB gene flow imposed by a harsh climate may be ameliorated in agricultural landscapes. No evidence was found for an obvious effect of road networks on genetic divergence and population structuring. Our study provides an example of how agricultural landscape connectivity can facilitate the spread of invasive pests, even across a broad climatic gradient. More broadly, our findings can guide decisions about future land management for mitigating further spread.

MOLECULAR CHARACTERIZATION OF CLOSTRIDIUM PERFRINGENS ISOLATED FROM TURKEYS

A total of 82 C. perfringensisolates (41%) were recovered from 200 samples collected from 100 turkeys (20 apparently healthy, 40 clinically diseased and 40 freshly dead), 4-6 weeks old. The clinical signs of diseased birds were sudden mortality, depression, ruffled feathers, diarrhea, dehydration, emaciation and decrease in feed consumption with increase in water consumption. While, postmortem findings were consistent with necrotic enteritis (NE), the small intestine was severely affected. Small and demarcated lesions were found in the duodenal loop, hepatitis and cholecystitis were also observed. Unopened intestines were gas-filled and enlarged with thin wall. The intestinal contents were dark due to necrotic material. The opened intestines showed necrotic lesions of varying severity of the mucosa. Isolation and biochemical identification of C.perfringenswere done.Five C.Perfringens field isolates were analyzed by PCR assay to determine the presence of some toxin genes.In all tested isolates, the Cpa gene (alpha toxin) was detected (100%) confirming the isolates were C.perfringens. While, Cpb (beta toxin) gene was detected in two samples (40%). But the Etx gene (epsilon toxin) was not detected in any isolate (0%). NetB gene was detected in two isolates (40%). Antimicrobial susceptibility of 40 C.perfringens field isolates recovered from turkeys revealed that C.perfringens isolates were sensitive to Ampicillin (85%), Amoxicillin (85%), Penicillin (82.5%), Florfenicol (72.5%), Enrofloxacin (67.5%), Vancomycin (65%), Bacitracin (60%), Oxytetracyclin (32.5%), Lincomycin (22.5%) and Clindamycin (15%).These five C. perfringens isolates were also screened by PCR which detected the presence of tetracycline resistance gene tet(K) in three isolates (60%) and Lincomycin resistance gene lin(B) in three isolates (60%).

Development and Assessment of Leishmania major and Leishmania tropica Specific Loop-Mediated Isothermal Amplification Assays for the Diagnosis of Cutaneous Leishmaniasis in Tunisia.

Cutaneous leishmaniasis (CL) remains one of the world's most prevalent neglected diseases, particularly in developing countries. Identification of the involved Leishmania species is an important step in the diagnosis and case management process. In this study, we tested simple, rapid, and highly sensitive loop-mediated isothermal amplification (LAMP) assays for Leishmania DNA species-specific detection from cutaneous lesions. Two LAMP assays, targeting cysteine protease B (cpb) gene, were developed to detect and identify Leishmania major and Leishmania tropica species. Loop-mediated isothermal amplification specificity was examined using DNA samples from other Leishmania species and Trypanosoma species. No cross-reactions were detected. The developed LAMP assays exhibited sensitivity with a detection limit of 20 fg and 200 fg for L. major and L. tropica, respectively. Both tests were applied on clinical samples of CL suspected patients living in endemic Tunisian regions and compared with kinetoplast DNA quantitative PCR (qPCR), microscopic, and conventional cpb-based polymerase chain reaction (PCR) assays. Our LAMP tests were able to discriminate between L. major and L. tropica species and showed a sensitivity of 84% and a specificity of 100%. However, when compared with the performance of the diagnostic tests with latent class analysis (LCA), our LAMP assays show a sensitivity of 100%. These assays can be used as a first-line molecular test for early diagnosis and prompt management of CL cases in public health programs.

Identification, Molecular Characterization, and In Silico Structural Analysis of Carboxypeptidase B2 of Anopheles stephensi.

Malaria is a vector-borne infectious disease that is considered a priority of the World Health Organization due to its enormous impacts on global health. Plasmodium spp. (Haemosporida: Plasmodiidae), Anopheles spp. (Diptera: Culicidae), and a suitable host are the key elements for malaria transmission. To disrupt the parasitic life cycle of malaria or prevent its transmission, these three key elements should be targeted by effective control strategies. Development of vaccines that interrupt malaria transmission is one of the solutions that has been recommended to the countries that aim to eliminate malaria. With respect to the important role of Anopheles stephensi in malaria transmission and involvement of Anopheles carboxypeptidase B1 in sexual parasite development, we characterized the second member of cpb gene family (cpbAs2) of An. Stephensi to provide some basic information and evaluate significance of cpbAs2's role in complementing sexual plasmodium development role of cpbAs1. The cpbAs2 mRNA sequence was characterized by 3' and 5' RACE and the structural features of its coded protein were studied by in silico modeling. The coding sequence and gene structure of cpbAs2 were determined empirically and compared with the in silico predictions from the An. stephensi genome sequencing project. Furthermore, homology modeling revealed that its structure is very similar to the structurally important domains of procarboxypeptidase B2 in humans. This study provides basic molecular and structural information about another member of the cpb gene family of An. stephensi. The reported results are informative and necessary for evaluation of the role of this gene in sexual parasite development by future studies.

Detection of Clostridium perfringens toxinotypes, enteropathogenic E. coli, rota and corona viruses in the intestine of neonatal goat kids by molecular techniques

The role of various pathogenic agents affecting gastro-intestinal tract of neonatal goat kids was studied. Intestinal tissue samples of 142 necropsied kids died due to enteritis, pneumo-enteritis, tapeworm’s infestations and septicemia were collected for detection of toxinotypes of Clostridium perfringens, enteropathogenic E. coli (EPEC), Group-A rotavirus (GARV) and bovine coronavirus (BCV). C. perfringens toxinotyping was done by multiplex PCR using primers for cpa, cpb, cpb2, etx and iap gene. Further, identification of EPEC was done using SYBR green based real-time PCR assay targeting bfpA gene. Detection of GARV and BCV was done by one-step RT-PCR (osRTPCR). The incidence of C. perfringens was 16.20% (47.83% toxinotype A and 52.17% toxinotype D), with b2- toxin present in 30.43% of the samples. Incidence of EPEC in 0-1 and 1–3 month diarrhoeic kids was 36.62% and 25.35%, respectively. For viral aetiology, 11.97% were positive for GARV and 9.86% positive for BCV. Mixed infection of C. perfringens and EPEC was seen in 12.68% cases, while it was 2.11% for C. perfringens + GARV and 1.40% for C. perfringens + BCV. Similarly, the mixed occurrence of EPEC+GARV was 10.56% and that of EPEC+BCV was 7.04%. The combined infection of EPEC+GARV+BCV was 0.70%. The results suggested that C. perfringens type A and type D are the common toxinotypes affecting neonatal goat kids, with β2 toxin being an additional potential virulence factor. SYBR green based real-time PCR assay can be used as quick lab-based technique for efficient screening of EPEC from enteritis affected kids.Group-A rotavirus and bovine coronavirus appeared to be instrumental in causing mixed infection enteritis in kids.

Leishmaniasis in Turkey: Visceral and cutaneous leishmaniasis caused by Leishmania donovani in Turkey

In Turkey, the main causative agents are Leishmania tropica (L. tropica) and Leishmania infantum (L. infantum) for cutaneous leishmaniasis (CL) and L. infantum for visceral leishmaniasis (VL). In this study, we investigated leishmaniasis cases caused by L. donovani and established animal models for understanding its tropism in in vivo conditions. Clinical samples (lesion aspirations and bone marrow) obtained from CL/VL patients were investigated using parasitological (smear/NNN) and DNA-based techniques. For species identification, a real time ITS1-PCR was performed using isolates and results were confirmed by hsp70 PCR-N/sequencing and cpb gene PCR/sequencing in order to reveal Leishmania donovani and Leishmania infantum discrimination. Clinical materials from CL and VL patients were also inoculated into two experimental groups (Group CL and Group VL) of Balb/C mice intraperitoneally for creating clinical picture of Turkish L. donovani strains. After 45days, the samples from visible sores of the skin were taken, and spleens and livers were removed. Measurements of the internal organs were done and touch preparations were prepared for checking the presence of amastigotes. The strains were isolated from all patients and amastigotes were seen in all smears of the patients, and then isolates were immediately stored in liquid nitrogen. In real time ITS1-PCR, the melting temperatures of all samples were out of range of L. infantum, L. tropica and L. major. Sequencing of hsp70 PCR-N showed that all isolates highly identical to previously submitted L. donovani sequences in GenBank, and cpb gene sequencing showed five isolates had longer cpbF allele, whereas one isolate contained a mixed sequence of both cpbF and cpbE. All mice in both experimental groups became infected. Compared to controls, the length and width of both liver and spleen were significantly elevated (p<0.001) in both groups of mice. However, the weight of the liver increased significantly in all mice whereas the weight of spleen increased only in VL group. Amastigotes were also seen in all touch preparations prepared from skin sores, spleen and liver. L. donovani strain was isolated from autocutaneous a VL patient first time in Turkey. Animal models using clinical samples were successfully established and important clinical differences of the isolated strains were observed.

Increasing in cysteine proteinase B expression and enzymatic activity during in vitro differentiation of Leishmania (Viannia) braziliensis: First evidence of modulation during morphological transition

Leishmania (Viannia) braziliensis presents adaptive protease-dependent mechanisms, as cysteine proteinases B (CPB). This study investigates the expression of three cpb gene isoforms and CPB enzymatic activity during the parasite differentiation. Relative expression levels of LbrM.08.0810 gene were assessed, exhibiting a higher quantity of transcripts in the logarithmic promastigotes phase than in the stationary promastigotes phase (>1.5 times). The cbp gene tends to decrease during acid pH shock and increases when the temperature rises (>1.3 times). LbrM.08.0820 and LbrM.08.0830 genes exhibited similar expression profiles to LbrM.08.0810 gene, with lower levels being observed overall. The proteolytic activity exhibits a gradual increase during the parasite's differentiation with low levels in samples of logarithmic promastigotes phase (3.2 ± 0.08 mmol min−1 mg protein−1) to a peak of activity after 72 h of incubation at 32 °C (4.2 ± 0.026 mmol min−1 mg protein−1) followed by a subsequent decrease of 68 % of peak activity levels after 96 h of incubation at 32 °C (2.8 ± 0.37 mmol min−1 mg protein−1). These activities were also measured in the presence of selective inhibitors for cysteine proteinases, such as Z-Phe-Phe-fluoromethyl ketone and trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, demonstrating their source as cathepsin-like proteinases. To the best of our knowledge, this report presents the first description of a modulation of cathepsin L-like expression during the L. (V.) braziliensis in vitro differentiation induced by acid pH and high temperature.

Silica nanowire conjugated with loop-shaped oligonucleotides: A new structure to silence cysteine proteinase gene in Leishmania tropica

The main aim of this study was to evaluate the capability of silica nanowire conjugated with loop-shaped oligonucleotides (SNWCLSOs) to silence cysteine proteinase b (Cpb) gene in Leishmania (L) tropica. On the other hand, its toxicity on amastigotes and mouse peritoneal macrophages was evaluated by 5-diphenyl-tetrazolium bromide (MTT) assay. For control, two loop-shaped oligonucleotides (LSO) were considered. LSO1 and LSO2 were 5ʹ-NH2-cccccaaaaaaaaaaaaaaaaaaaaaaaaaggggg-COOH-3ʹ and LSO2: 5ʹ-NH2-cccccttttttttttttttttttttttttttttttttttttttggggg-COOH-3ʹ, respectively. After 72h incubation at 37°C, AMSNW, LSO1, and LSO2 had no remarkable toxicity on L. tropica amastigote (2×105/mL) and mouse peritoneal macrophages (2×105/mL). In case of SNWCLSOs, they had high toxicity on L. tropica amastigote, but they had no effect on mouse peritoneal macrophages. At concentrations of 1, 10, and 25μg/mL, AMSNW, LSO1 and LSO2 had no effect on the gene expression. But, at concentration of 50 and 100μg/mL, decrease of gene expression was observed. In case of SNWCLSOs, they could dramatically decrease the gene expression. It could be concluded that since SNWCLSOs could silence Cpb gene with no remarkable toxicity, they are good choice for treat cutaneous leishmaniasis in future. As a new agent, it must be checked in vivo.