Pathogens

BackgroundThe study of retroviruses dates back to the early 1900s during investigations on neoplastic diseases in chickens. Subsequently, Robert Gallo reported the first human retrovirus HLTV in 1980. What we report here is not an archetypal retrovirus, but the discovery of an oncogenic giant microbial agent with a mega-genome, where the transforming retroviral nature co-exists with multiple archaeal oncogenes. MethodsAfter their isolation from human T cells Leukaemia, these organisms were examined at electron microscopy, tested for reverse transcriptase activity, fully sequenced, used for transformation tests on NIH-3T3 cells in vitro and tumours formation in mice. Same type of particles were also isolated from Canine Transmissible Venereal Tumour (CTVT), the oldest contagious cancer in nature. ResultsEM showed the presence of giant viral particles displaying retroviral antigens. These microbial entities harbour in their mega-genome a transforming retroviral kinase, cell-based oncogenes and have reverse transcriptase activity. The purified viral particles transformed NIH-3T3 cells and induced metastatic tumours in nude mice, three weeks post infection. Ruling out the possible presence of filterable retroviruses, a filtered supernatant did not display RT activity and did not transforms. ConclusionsWe discovered an ancestral microbial agent, acutely transforming. For its giant dimensions, the ability to retain the Gram stain, the presence of a mega-genome and its retroviral nature, we tentatively named the agent Retro-Giant-Virus (RGV). However, distinct from amoeba giant Mimiviruses, this transforming human agent has a different nature and does not require for its isolation amoeba co-culture, since amoeba is not its natural host. The morphology, biology and genetic features allocate this mammalian giant microbe halfway in between a classic oncogenic virus and an infectious cancer cell. Its transforming nature goes with its constant ability to induce tumours formation in mice.

Acanthamoebae, which are free-living amoebae, cause corneal inflammation (keratitis) and blindness, if not diagnosed and effectively treated. While trophozoites adhere to and damage the cornea, Acanthamoeba cysts, the walls of which contain cellulose and have two layers connected by conical ostioles, are the diagnostic form by microscopy of the eye or of corneal scrapings. We recently used structural and experimental methods to characterize cellulose-binding domains of Luke and Leo lectins, which are abundant in the inner layer and ostioles. However, no antibodies have been made to these lectins or to a Jonah lectin and a laccase, which are abundant in the outer layer. Here we used confocal microscopy to show that rabbit antibodies to recombinant Luke, Leo, Jonah, and laccase generally support localizations of GFP-tagged proteins in walls of transfected Acanthamoebae. Rabbit antibodies to all four wall proteins efficiently detected calcofluor white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC, including five T4 genotypes that cause most cases of keratitis. Laccase shed into the medium during encystation was detected by an enzyme-linked immunoassay. We also used structural and experimental methods to characterize the mannose-binding domain of an Acanthamoeba mannose-binding protein and showed that rabbit antibodies to the mannose-binding domain efficiently detected trophozoites of all 11 Acanthamoeba isolates. We conclude that four wall proteins are all excellent targets for diagnosing Acanthamoeba cysts in the eye or corneal scrapings, while the mannose-binding domain is an excellent target for identifying trophozoites in cultures of corneal scrapings. ImportanceFree-living amoeba in the soil or water cause Acanthamoeba keratitis, which is diagnosed by identification of cysts by microscopy of the eye or of corneal scrapings, using calcofluor-white that unfortunately cross-reacts with fungi and plants. Alternatively, Acanthamoeba infections are diagnosed by identification of trophozoites in cultures of scrapings. Here we showed that rabbit antibodies to four abundant cyst wall proteins (Jonah, Luke, Leo, and laccase) each efficiently detect calcofluor-white-labeled cysts of 10 of 11 Acanthamoeba isolates obtained from the ATCC. Further, laccase released into the medium by encysting Acanthamoebae was detected by an enzyme-linked immunoassay. We also showed that rabbit antibodies to the mannose-binding domain of the Acanthamoeba mannose-binding protein, which mediates adherence of trophozoites to keratinocytes, efficiently identifies trophozoites of all 11 ATCC isolates. In summary, four wall proteins and the ManBD appear to be excellent targets for diagnosis of Acanthamoeba cysts and trophozoites, respectively.

Species of Chaetomiaceae family are ubiquitous filamentous fungi, that responsible for a wide range of opportunistic human infections. To date, it encompasses more than 300 species have been described in genus Chaetomium. It have been globally reported as being are capable of colonizing various substrates. Due to the lack of genetic studies on the species belonging to this genus in Sudan, This work aimed to investigate the environmental fungal occurrence within the family Chaetomiaceae by using morphological characters and molecular sequencing. A total of 260 environmental samples from soil, animal dung and water were collected from six different states in Sudan in two ecozones: .desert or semis desert ecozones (Dongola in Northern sudan, El-Obeid in western sudan); a low rainfall woodland savanna ecozone (Gazira, El Geteina and Khartoum from central Sudan and AlQadarif in eastern Sudan). During a study of environmental fungi in Sudan, 119 isolates were identified as members of Chaetomiaceae after the ITS sequencing combined with an examination of the macro- and micromorphology. Out of 63 Chaetomium strains obtained from soil, animal dung and water samples, 25 were obtained from soil, 22 from animal dung and 16 from water. 56 additional strains isolated from other genus within the Chaetomiaceae family, such as (Amesia, Collariella, Ovatospora, Subramaniula and Thielavia) were recorded for the first time in Sudan. In conclusion: Sequence-based identification of fungal isolates is often considered to be the most reliable and accurate identification method.

A mouse monoclonal antibody (Moab 4B8) cross-reacting with cilia/flagella was obtained by immunization with Pneumocystis-infected human lung tissue. A key observation was that Moab 4B8 reacted with the ventral flagella of Giardia intestinalis, but not with the three other flagellar pairs of this protozoan. To further identify the 4B8 target, its distribution was studied by immunofluorescence staining of cells and tissues of various origin. The target epitope recognized by Moab 4B8 was found to be associated with structures rich in microtubules; e.g. the mitotic spindle of cultured cells, ciliated airway epithelia, Sertoli cells of the testis and ependymal cells lining brain ventricles. The conserved nature of the 4B8 target was further shown by its presence in cilia of metazoan Schistosome larva and the green alga Chlamydomonas reinhardtii. Absence of the 4B8 target from Trypanosomes and Leishmania flagella suggested that it is involved in some function not primarily related to motility. Its presence in only the ventral flagella of Giardia therefore provides a unique opportunity to elucidate the relationship between ciliary structure and function in the same organism. The observed locations of the 4B8 target in tissues and cells of various origin, suggest a similarity to annexins - and specifically to -19-giardin. This raises the possibility that it is involved in intra-flagellar transport and provides a basis for further studies aiming at its identification. Author SummaryPneumocystis is a ubiquitous fungal organism apparently colonizing the lung at an early age to cause pneumonia only in individuals with an impaired immune system. In the alveolar spaces of such individuals, extensive and frequently fatal proliferation of the pathogen occurs. Pneumocystis has no known reservoir in nature and apparently is transmitted directly from infected individuals via an airborne route. Adaptation of this Ascomycotic fungus to a parasitic lifestyle during its evolution apparently resulted in dependence upon host nutrients, but little is known about this presumed adaptation process. In this report, a previously unrecognized constituent of human Pneumocystis is detected using a monoclonal anti-Pneumocystis jiroveci antibody (Moab 4B8) which was obtained as a by-product in the search for reagents useful in diagnostics. The Moab 4B8 was shown to react with Pneumocystis but also with cytoskeletal microtubules, e.g. in ciliated epithelia, but not ubiquitously a constituent of the conserved cilia/flagella axonemal structure. A striking example of the discriminating capacity of antibody 4B8 was seen in immunofluorescent staining of the protozoan Giardia intestinalis, where only one out of four flagellar pairs expresses the target epitope. This observation of flagellar heterogenicity provoked the question raised in the title of this report. It also provides the basis for the discussion, which arrives at suggestive evidence for the involvement of the described evolutionarily conserved target in host-pathogen interactions related to membrane transport.

Secondary bacterial and fungal infections are associated with respiratory viral infections and invasive mechanical ventilation. In Coronavirus disease 2019 (COVID-19), lung injury by SARS-CoV-2 and impaired immune response can provide a favorable environment for microorganism growth and colonization in hospitalized individuals. Recent studies suggest that secondary bacterial pneumonia is a risk factor associated with COVID-19. In Brazil, knowledge about microbiota present in COVID-19 patients is incipient. This work describes the microbiota of 21 COVID-19 patients admitted to intensive care units from two Brazilian centers. We identified respiratory, nosocomial and bacterial pathogens as prevalent microorganisms. Other bacterial opportunistic and commensal species are also represented. Virulence factors of these pathogenic species, metabolic pathways used to evade and modulate immunological processes and the interconnection between bacterial presence and virulence in COVID-19 progression are discussed. Article Summary LineWe identified respiratory, nosocomial and bacterial pathogens as prevalent microorganisms in 21 Brazilian COVID-19 patients admitted to Intensive Care Units. Pathogen virulence factors and immune response evasion metabolic pathways are correlated to COVID-19 severity.

IntroductionUrinary tract infections are common to pregnant and nonpregnant women estimated to 150 million new cases annually. The incidence increases with pregnancy due changes that take place. Causative microbes are E.coli, Klebsiella pneumoniae and Staphylococci species. The disease presents symptomatically or asymptomatically, early investigation, detection and treatment to pregnant mothers are crucial to avoid maternal and foetal complications. Several effective antimicrobials are contraindicated using ineffective agents jeopardizes treatment outcome leading to multidrug resistance. We assessed UTI causative microbes and susceptibility patterns to common antibiotics. MethodsWe conducted a hospital based cross sectional study at Kairuki hospital involving 262 pregnant mothers attending ante-natal clinics. Mid-stream urine was collected and inoculated on Cysteine-lactose-electrolyte-deficient agar, MacConkey and blood agar. Eleven microbes were isolated and tested for susceptibility against antibiotics using Kirby-Bauer disk diffusion technique on Mueller-Hinton agar. Data analysed using SPSS package version 23. ResultsThe prevalence of UTI in pregnant mothers was 31.2% (82/262). The gram positive isolates were more prevalent than gram nmoste (59.3% versus 40.7%) Staphylococcus aureus 22/82 (26.2%) and S. saprophyticus 15/82 (17.9%) were the mostly isolated. Nitrofurantoin, Piperacillin/tazobactam have lowest resistant rate to both gram negative and gram positive isolates ranging from (0-26%) while Erythromycin and Ampicillin have the highest resistant rate ranging from (60-100%) therefore associated with multidrug resistant. ConclusionAsymptomatic UTI is prevalent to pregnant women at this hospital. We recommend culture and sensitivity results to guide treatment and usage of nitrofurantoin, piperacillin/tazobactam as first line treatment of UTI in pregnancy.

AbstractsO_ST_ABSBackgroundC_ST_ABSAcinetobacter baumannii is a multidrug-resistant bacterium responsible for severe infections, particularly in hospital settings. Its resistance is driven by enzymatic genes such as those encoding beta-lactamases and carbapenemases, which degrade antibiotics, and non-enzymatic genes that modify mechanisms like efflux pumps and membrane permeability, further enhancing its defence against treatments. Together, these factors allow A. baumannii to thrive in clinical environments, complicating infection management. ObjectiveThis study aimed to explore the relationships between beta-lactamases, carbapenemases, efflux pumps, and membrane permeability changes, to understand their collective contribution to A. baumanniis multidrug resistance. Materials and MethodsAmong 300 clinical isolates from urine, blood, wounds, and burns, 25 (8.33%) were identified as A. baumannii. These included 8% from urine, 12% from blood, and 40% each from wound and burn swabs. all specimens were taken from patients who have different symptoms in hospital of Al-Hilla Teaching Hospital/ Babylon. The research was carried out through the period January and June 2024. Bacterial identification was conducted using the VITEK-2 system and HI-Chromoagar(R) A. baumannii. Enzymatic genes were detected using conventional PCR, while non-enzymatic genes were analyzed via RT-qPCR. ResultsMolecular analysis revealed the presence of beta-lactamase (blaOXA-51, blaOXA-23) and metallo-beta-lactamase genes (blaVIM, blaIMP), with high antibiotic resistance rates. Gene expression analysis highlighted efflux pump upregulation (adeB) and altered permeability (CarO), reinforcing multidrug resistance mechanisms. ConclusionThe combined action of enzymatic and non-enzymatic resistance genes in A. baumannii presents a significant treatment challenge, necessitating multi-target therapeutic approaches.

Bacterial cancer therapy has gained lots of attention in the past decade and is now considering a reliable option for the future. However, some concerns have limited its application into clinic settings like insufficient colonization of tumors and infectious origin of the currently used bacteria like Clostridium and Salmonella species, especially in cancer patients which exhibit different levels of immunocompromising. In the present study, Veillonella parvula (V. parvula) as a strictly anaerobic bacterium which has rarely identified as a pathogen in human, was administrated into 4T1 breast tumor-bearing mice. At first, 4T1 breast tumor-bearing BALB/c mice were injected with 107 bacteria intravenously, intraperitoneally, orally, or intratumorally. The best administration route according to tumor colonization and safety was selected. Then, the therapeutic effect of V. parvula administration was investigated according to the 4T1 breast tumors growth, metastasis, and tumor-bearing mice survival. Besides, histopathological evaluations were done to estimate microscopic changes at the inner of the tumor. V. parvula exhibited significant tumor-targeting and colonization efficacy, 24 h after intravenous administration and formed clustered colonies at the central region of the tumors. Although a negligible number of the bacteria were localized at normal organs, these organs became clear from the bacteria after 72 h, and no side effects or death were observed at the animals after intravenous administration of V. parvula. Although mean tumor volumes in the V. parvula treated group was lower than the control (~ 25.4%), their difference wasnt statistically significant (P > 0.05). Despite significant tumor colonization (5500000:1 in comparison with normal organs after 72 h), V. parvula didnt cause a significant therapeutic effect on the metastasis or survival time of tumor-bearing mice. Taking together, V. parvula is a completely safe and tumor-specific agent per se, without any genetic manipulation. Also, it exhibits high tumor penetration and colonization at the deep regions of the tumor.

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm, freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2'-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (5'ethynyl-2'-deoxyadenosine), an adenine analog, also was incorporated by N. fowleri, but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

Phylogenetic group B2 Escherichia coli is associated with urinary tract infections and other pathologies, but the basis for this phylogenetic skew is not understood. One aspect of urinary tract infections is binding to and entering uroepithelial cells. To test whether a phylogenetic skew exists for cell invasion, we examined invasion of 10 E. coli strains from three phylogenetic groups into CRL2169 and HTB-9 cells, which are derived from grade 1 and grade 2 bladder carcinomas, respectively. The top four strains that invaded CRL2169 were from group B2: three of these strains had more flagella gene transcripts than the other seven strains. The seven strains that invaded HTB-9 were from different phylogenetic groups. For the model uropathogenic group B2 strain UTI89, which expresses pili over flagella, loss of flagella or pili impacted invasion into CRL2169 to similar extents, but loss of pili had a greater effect on invasion into HTB-9 and a murine infection model than loss of flagella. A hyperflagellated variant of a group A strain did not invade either cell line better than the parental strain. Reported transcript differences, which were confirmed experimentally, showed that CRL2169 was more differentiated. The endocytosis stimulator tanshinone enhanced invasion into HTB-9, but not into CRL2169, which suggests differences in endocytic pathways and is consistent with differences in differentiation states. If the initial or recurring event in urinary tract infection is invasion into differentiated urothelial cells, as opposed to tight junctions, then the role of flagella may have been underestimated.

In recent years nidoviruses have emerged as an important respiratory pathogen of reptiles, affecting especially captive python populations. In pythons, nidovirus infection induces an inflammation of the upper respiratory and alimentary tract which can develop into a severe and often fatal proliferative pneumonia. We observed pyogranulomatous and fibrinonecrotic lesions in organ systems other than the respiratory tract during full post mortem examinations on 30 nidovirus RT-PCR positive pythons of varying species originating from Switzerland and Spain. The observations prompted us to study whether the atypical tissue tropism associates with previously unknown nidoviruses or changes in the nidovirus genome. RT-PCR and inoculation of Morelia viridis cell cultures served to recruit the cases and to obtain virus isolates. Immunohistochemistry and immunofluorescence staining against nidovirus nucleoprotein demonstrated that the virus not only infects a broad spectrum of epithelial (respiratory and alimentary epithelium, hepatocytes, renal tubules, pancreatic ducts etc.), but also intravascular monocytes, intralesional macrophages and endothelial cells. By next-generation sequencing we obtained full length genome for a novel nidovirus species circulating in Switzerland. Analysis of viral genomes recovered from pythons showing nidovirus infection-associated respiratory or systemic disease did not explain the observed phenotypes. The results indicate that python nidoviruses have a broad cell and tissue tropism, further suggesting that the course of infection could vary and involve lesions in a broad spectrum of tissues and organ systems as a consequence of monocyte-mediated systemic spread of the virus. IMPORTANCEDuring the last years, python nidoviruses have become a primary cause of fatal disease in pythons. Nidoviruses represent a threat to captive snake collections, as they spread rapidly and can be associated with high morbidity and mortality. Our study indicates that, different from previously evidence, the viruses do not only affect the respiratory tract, but can spread in the entire body with blood monocytes, have a broad spectrum of target cells, and can induce a variety of lesions. Nidovirales is an order of animal and human viruses that compromise important zoonotic pathogens such as MERS-CoV and SARS-CoV, as well as the recently emerged SARS-CoV-2. Python nidoviruses belong to the same subfamily as the mentioned human viruses and show similar characteristics (rapid spread, respiratory and gastrointestinal tropism, etc.). The present study confirms the relevance of natural animal diseases to better understand the complexity of viruses of the order nidovirales.

BackgroundViral Hemorrhagic Septicemia Virus (VHSV) is a rhabdovirus that causes high mortalities linked to high economic losses in aquaculture. It has been grouped in four genotypes of which some do not easily propagate on continuous cell lines. As an alternative, the objectives of this study was to develop a primary gill epithelial cell (GEC) model from olive flounder (Paralichthys olivaceus) as an in vitro model for the propagation of VHSV.\n\nResultsOur findings show that the primary GECs developed herein are highly permissive to replication of the JF-09 genotype IVa strain leading to high cytopathic effect observed within 96 hours post virus inoculation. Our findings also show that the viability GECs produced herein corresponded with increase in the concentration fetal bovine serum in growth medium. We envision that GECs produced herein will heighten our understanding of immune mechanisms associated with virus entry on gill mucosal surfaces in flounder.

In 2023, a new yellow fever virus (YFV) lineage was introduced in Sao Paulo State, Brazil. From July 2024 to June 2025, nine Callithrix penicillata tested positive, showing low YFV viral load but characteristic organ lesions, suggesting this genus may replace Alouatta sp. as sentinel to detect YFV circulation.

EsxA has long been recognized as an important virulence factor of Mycobacterium tuberculosis (Mtb) that plays an essential role in Mtb cytosolic translocation presumably by penetrating phagosomal membranes with its acidic pH-dependent membrane permeabilizing activity (MPA). However, current data suggest that the observed cytolytic activity of EsxA at neutral pH is due to contamination of ASB-14, a detergent used in EsxA protein purification, and the role of EsxA MPA in Mtb cytosolic translocation is also questionable. Here, we have obtained evidence that it is ASB-14, not EsxA that causes cytolysis at neutral pH. Quantitative liquid chromatography and mass spectrometry showed that even after gel filtration, dialysis, or passing through detergent removal column, the remaining ASB-14 in the EsxA protein solution was still at a concentration enough to kill cultured lung epithelial cells. When treated with trypsin or proteinase K, the digested EsxA protein solution with ASB-14 was still cytotoxic. Interestingly, however, we have found that the exogenously added EsxA is endocytosed into lung epithelial cells and inserts into the host membranes within acidic subcellular compartments, which can be blocked by cytochalasin D and bafilomycin A. It is for the first time EsxA is found to insert into the host membranes within acidic subcellular compartments. ImportanceEsxA has long been recognized as an important virulence factor of Mycobacterium tuberculosis (Mtb) that plays an essential role in Mtb virulence. However, current data regarding to its role in Mtb virulence are controversial. Here, we have obtained evidence showing that the cytolytic activity of EsxA at neutral pH is due to contamination of ASB-14, a detergent used in EsxA preparation. Moreover, it is for the first time we have found that EsxA protein is endocytosed into lung epithelial cells and inserts into the host membranes within acidic subcellular compartments, implicating an important role of the acidic pH-dependent membrane permeabilizing activity of EsxA in Mtb virulence.

Multiple PCR analyzes were performed using 19 different primer sets to open and broaden the search spectrum for shrimp pathogens. In addition, multiple primer pairs for 10 pathogens were compared to see if there were differences in selectivity or sensitivity among them. Some pathogens that did not present histological lesions were detected. The most important outcome was that selection of appropriate primers was the most critical factor in obtaining reliable results. We found high variability in results among primers and we learned it was prudent to seasonally assess among them for the best set selection. It is important to understand that a PCR positive test result alone does not confirm the presence of a viable pathogen or a disease state. Nor, as might be expected, does it mean that the positive PCR test results will be necessarily accompanied by histological lesions characteristic of the targeted pathogen. However, the use of appropriately selected primers sets can reveal whether there is an evolution in the result spectrum over time and if some pathogens disappear or reappear or new ones emerge. In general, most shrimp presented coinfections that consisted of the presence of WzSV8, DHPV, chronic midgut inflammation and tubule distension/epithelial atrophy consistent with Pir A/B toxicity. Also included were RLB/NHPB, microsporidia, striated muscle necrosis, gregarines in the hindgut caecum (gametocyte stage, and not associated with tegumental glands but glands that line the mouth and anus) and encysted, presumed nematode larvae. WzSV8 was newly discovered in gonads. Histological changes and the presence of spheroids in the lymphoid organ were considered as healthy host responses of often unidentified cause.

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and the leading cause of mortality from foodborne and waterborne gastrointestinal disease. In neonates of domestic animals it is associated with consistent diarrhoea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy, antigen trap ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA based duplex TaqMan(R) probe PCR (dRT-qPCR) was developed to target the Cryptosporidium oocyst wall protein (COWP) gene and 18ssu rRNA gene in a single tube that can detect metabolically active Cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and it will help decipher the active stages of its lifecycle in host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection. For COWP and 188ssu rRNA genes the limit of detection was 7.08x1004 and 5.95x1005 respectively. During active infections the oocyst wall protein will be active and so its COWP gene transcripts will act as marker for active infection. While transcripts for 18SSU rRNA are constitutively expressing in Cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among the healthy animals. ImportanceCryptosporidiosis is an important neonatal disease affecting goats causing diarrhoea, dehydration and stunted growth. For diagnosing this condition, many diagnostic tests are available including microscopy, immunological tests, but none of the diagnostic tests available currently can differentiate between active and passive infection in the host. The mRNA transcripts are the direct indicator of any actively replicating cell and especially in intracellular parasites it will help decipher the infective stages of a lifecycle in the host, and hence the test was developed in a reverse transcriptional format in a duplex mode. The currently developed diagnostic assay for cryptosporidiosis was evaluated for sensitivity using Limit of detection (LOD). This diagnostic test will act as a quantitative marker to aid in detecting active stages of Cryptosporidium infection in neonatal goats and will eventually lead to better control strategies for managing cryptosporidial infections in the future.

This novel study detected persistent low level infections of Elephant Endotheliotropic Herpesviruses (EEHV), that can cause highly pathogenic Elephant Hemorrhagic Disease (EHD) in Loxodonta and Elephas, and co-infection of presumed less pathogenic Elephant Gammaherpesviruses (EGHV), in skin nodule biopsies, saliva and tissues collected from 43 wild L. africana (savannah elephant) in Botswana, Kenya, South Africa and Zimbabwe; in saliva from 25 wild L. cyclotis (forest elephant) in Gabon; and in saliva collected over seven years from 7 wild-born L.africana at Six Flags Safari Park, USA; and in saliva, blood and tissues from an additional 200 L. africana in USA zoos. DNA from these samples was extracted in our USA laboratories and amplified by conventional polymerase chain reaction using three-round nested primer sets designed specifically to screen for known EEHV and EGHV genes loci and to discover new species and subtypes. Sanger sequencing of purified DNA from nearly all samples yielded unambiguous positive genetic matches to previously known Loxodonta-associated EEHV2, EEHV3A, EEHV3B, EEHV6, EEHV7A, and EGHV1B, EGHV2, EGHV3B, EGHV4B, EGHV5B and discovered novel types EEHV3C-H and EEHV7B and the prototype EGHV1B. Many of the primer sets used could also have detected known Elephas-associated EEHV1A, EEHV1B, EEHV4, and EEHV5 if present in these samples, but they did not. Our extensive library of EEHV and EGHV sequences from wild and zoo Loxodonta, (as well as from 100 zoo Elephas maximus not discussed in this review), is a significant contribution to the elephant virology community, particularly for comparing subtypes types of EEHV found in pathogenic cases of EHD in zoos as well as determining and comparing species and subtypes of EEHV present in existing zoo herds, and in individual elephants being transported between zoos, and for importation of wild elephants into existing zoo herds.

The vast majority of cystic echinococcosis cases in Southern Brazil are caused by Echinococcus granulosus and Echinococcus ortleppi. Comparative proteomic studies of helminths have increased the knowledge about the molecular survival strategies adopted by parasites. Here, we surveyed the protein contents of the hydatid fluid compartment of E. granulosus and E. ortleppi pulmonary bovine cysts, in an attempt to compare their molecular arsenal in this host-parasite interface. Hydatid fluid samples from three isolates of each species were analyzed by trypsin digestion and mass spectrometry. We identified 280 proteins in E. granulosus and 251 proteins in E. ortleppi, highlighting a core of 52 proteins common to all samples of hydatid fluid. The in silico functional analysis revealed important molecular functions and processes active in pulmonary cystic echinococcosis. Some were more evident in one species, such as apoptosis in E. ortleppi, and cysteine protease activity in E. granulosus, while many molecular activities have been found in fluids of both species, such as proteolysis, development signaling and extracellular structures organization. The similar molecular tools employed by E. granulosus and E. ortleppi for their survival within the host are potential targets for new therapeutic approaches to deal with cystic echinococcosis and other larval cestodiases.

AbatractCarbapenem resistance in Pseudomonas.aeruginosa is particularly worrisome because this class of {beta}-lactam represents the last therapeutic resource for control of bacterial infection.So this study aimed to detect the frequency of bla OXA-48 resistance gene among Pseudomonas aeruginosa clinical isolates during the period from November 2018 to November 2019. Hundred Pseudomonas aeruginosa clinical isolates, 81 carbapenems (imipenem meropenem) resistant and 19 carbapenems sensitive were collected from Omdurman Teaching Hospital, Fedail Hospital and Soba Teaching Hospital in Khartoum State-Sudan. All isolates were re-identified using conventional bacteriological techniques, their susceptibility to carbapenems were tested using Kirby-Bauer method for confirmation and investigated for the presence of the bla OXA-48 gene using conventional PCR technique. 60 (60.0%) out of 100 Pseudomonas aeruginosa clinical isolates were positive for blaOXA-48 gene. Out of 81 carbapenem resistant isolates 54(66.7%) were positive for bla OXA-48 gene, while among the (19) carbapenem sensitive isolates 6 (31.6%) were positive for blaOXA-48 gene. There was statistically significant association between carbapenem resistant isolates and the presence of blaOXA-48 gene (P-value = 0.006). Wound swabs were the predominant clinical samples detected harboring bla OXA-48 gene both among the sensitive 5 (83.3%) and carbapenem resistant isolates 29(53.7) (P.value> 0.05). Our findings revealed high frequency of bla OXA-48 among carbapenem resistant isolates so identification of bla OXA-48 producing strains and taking efforts to reduce the rate of transferring these gene between the different strains is essential for optimization of therapy and improves of patients outcomes.

We designed a multiplex qPCR to differentiate monkeypox virus clades. In evaluations using clinical samples collected in the UK and Nigeria, the assay had an overall sensitivity of 78% (95% CI: 67.67% to 86.14%) and specificity of 94% (95% CI: 80.84% to 99.30%); for samples under Ct35 sensitivity was 98% (95% CI: 91.72% to 99.96%) and specificity was 94% (95% CI: 80.84% to 99.30%).