Infections In Laboratory Animals Research Articles

Animal models of human cytomegalovirus congenital infection

Human cytomegalovirus (HCMV) infection is highly species-specific, which means that it is unable to productively infect laboratory animals. Despite this caveat, studies of animal CMV counterparts in their natural hosts have revealed significant correlations with observed neuropathological effects of congenital HCMV infection and have improved our understanding of host responses to vaccination. The biological relatedness between human and animal CMVs has been confirmed by phylogenetic analyses; the conservation of ‘core' genes that are essential for virus replication as well as genes that contribute similar mechanisms for virus persistence in their respective host species. The common animal models of HCMV congenital infection include Rhesus CMV (RhCMV), guinea-pig CMV (GPCMV) and mouse CMV (MCMV). Whilst animal models of CMV do not fully recapitulate HCMV infection, they each offer specific advantages in understanding HCMV congenital/perinatal infection (summarised in Table 1).

Ribonucleases as antiviral agents.

Many ribonucleases (RNases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but the molecular mechanisms of their antiviral activity remain unclear. The review discusses the well-known RNases that possess established antiviral effects, including both intracellular RNases (RNase L, MCPIP1 protein, and eosinophil-associated RNases) and exogenous RNases (RNase A, BS-RNase, onconase, binase, and synthetic RNases). Attention is paid to two important, but not always obligatory, aspects of molecules of RNases that have antiviral properties, i.e., catalytic activity and ability to dimerize. The hypothetic scheme of virus elimination by exogenous RNases that reflects possible types of interaction of viruses and RNases with a cell is proposed. The evidence for RNases as classical components of immune defense and thus perspective agents for the development of new antiviral therapeutics is proposed.

A Water-Soluble Fenbendazole (FBZ) Formulation for Treating Pinworm Infections in Laboratory Animals

Fenbendazole (FBZ) is an oral veterinary medication given daily for 3 consecutive days to treat oxyurid (Syphacia, pinworm) infections (oxyuriasis) and other parasites. A species-dependent oral dose of 5-20 mg/kg/day completely eliminates the parasite. FBZ is usually administered in food rations, but costs associated with FBZ-treated food are high. The low water solubility of FBZ (

Determination the therapeutiction of Datura metel leaves extract for some urinary system bacteria in rabbits (in vitro and in vivo )

This study was designed to evaluate the effects the watery and alcoholic extracts from leaves of Datura metel in vitro to ten of pathogenic bacteria and study the watery extract toward Candida on the urinary system in rabbits. The results showed the watery and alcoholic extracts from leaves of Datura metel have antibacterial activity against both Gram-positive (Staphylococcus. aureus – Streptococcus .agylactiae ) And Gram-negative bacteria (Klebsiella. pneumonia –Proteus. vulgarities – E.coli – Pseudomonas – Vibrio – Salmonella – Enterobacter ) and Candida. albicans, however, the watery extract of Datura metel were more potent than alcoholic extract against pathogenic bacteria (80%) . According to results , Candida pathogen that more sensitive toward Datura metel watery extract was choosing to injected intraperitoneally as experimental infection in laboratory animals (vivo) which cause morphological and histopathological degenerative lesion of kidney cortex and medulla tissue in addition to change of renal profile test that include blood urea nitrogen, creatinine, creatinine kinase, uric acid, in addition to Potassium. but after watery extract of Datura metel injected in these laboratory animals cause significant improvement (p≤0.01) in the value of blood urea nitrogen, creatinine, creatinine kinase, and uric acid. Potassium concentration, histopathological studies confirm these results which include regeneration of degenerative lesion for medulla and kidney cortex with convoluted tubules tissue.

Interaction between Bacillus anthracis and Pattern-Recognizing Receptors of Innate and Adaptive Immunity

According to modern views, protection of an organism from different pathogens is achieved through functioning of the two chains of immune system - innate and acquired ones. Initially, receptors of non-specific immunity, including Toll-like receptors, identify conservative pathogen-associated molecular structures. The subsequent activation of signaling pathways leads to rapid neutralization and elimination of foreign agent. Concurrently, initiation of the adaptive immunity, and realization of pro-inflammatory reactions' cascade take place. The review summarizes literature data concerning peculiarities of innate and adaptive types of immunity in case of interaction between macro-organism and Bacillus anthracis. Protective antigen, lipoprotein, cell wall components, anthrolysine O, CpG DNA sequence and others are the potential ligands of Toll-like receptors in B. anthracis . Demonstrated are the results of experiments that bear evidence of the fact, that synthetic agonists of Toll-like receptors influence realization of B. anthracis cytotoxicity and course of infection in laboratory animals. Modificators of Toll-like receptor functions can be used as immunostimulators for designing of effective means of anti-infectious protection. Innate and adaptive components of the immunity, having their own unique mechanisms of the specific B. anthracis identification, interact with each other, strengthen and complement each other.

ELISA and some biochemical tests of heterophyidae infection in laboratory animals

Heterophyiasis is an important food-borne parasitic zoonosis in Egypt, among the inhabitants living around brackish-water lakes especially fishermen, and it is a common human parasite in the Nile Delta. The experiment was done on two laboratory animals (rats and dogs), and the time of sample collection was done periodically at 6, 9, 15, 21, and 28 days post-infection to evaluate different tests required. Whole blood was collected with heparin or ethylenediamine tetra-acetic acid as anticoagulant to help in the hematological studies such as red blood cells count (RBCs), white blood cells count, packed cell volume (PCV), and hemoglobin (Hb). Only marked increase in the total leuckocytic count was recorded while RBCs, PCV, and Hb were decreased in most of the results obtained. Total protein and globulin decreased while albumin and A/G ratio increased. Liver enzymes showing marked increase in aspartate aminotransferase and increase in alanine aminotransferase in dogs and rats denoting that liver has a role in the response to that infection. Kidney-function tests, urea, and creatinine showed slight increase at 6 days post-infection (d.p.i.). After preparation of different Ag (antigen) from different collected helminthes, the protein content of each was determined. The sera of infected animals were collected to find antibodies in their blood against the parasite using enzyme-linked immunosorbent assay and using crude heterophyid antigen collected from their intestines after scarification. The worms washed, homogenized, and then centrifuged to collect supernatant fluid as antigens. The results indicated that antibody starts to appear at 9 d.p.i. and increases till 21 and 28 d.p.i. and detection depends on antigen concentration.

Sensitive and Specific Detection ofMycoplasma speciesby Consensus Polymerase Chain Reaction and Dot Blot Hybridization

Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Many species of mycoplasmas are important pathogens that cause respiratory infection in laboratory animals and that are known to affect experimental results obtained with contaminated animals. The aim of the present study was to develop a sensitive and specific assay for the detection of mycoplasma species. To this end, we developed a polymerase chain reaction and dot blot hybridization assay (PCR/DBH) for detecting mycoplasma DNA and evaluated it for its sensitivity and specificity. Mycoplasma consensus primer pairs were used for the amplification of target DNA. When PCR product was visually detected, the limit of detection of the PCR test was 102 pg of mycoplasma purified DNA. For DBH, the amplified DNA was labeled by incorporation of digoxigenin (DIG). This DIG-labeled probe was capable of detecting 104 pg of purified mycoplasma DNA by DBH. PCR/DBH was more sensitive than PCR or DBH alone and was also very specific. Our PCR/DBH assay can be applied efficiently to confirm the presence of mycoplasma species on clinical samples and to differentiate between mycoplasma species infection and other bacterial infections.

Lipidomics in biomarker development

Research into the cell biology of intracellular pathogens has provided information on host-pathogen interactions and on the importance of lipids at various stages in the interactions. Lipids are “gatekeepers” in important chemical reactions involving cell signaling during pathogen docking, invasion and traffic in and out of cells. Working with the Novartis Institute for Tropical Diseases, our team is exploring how TB takes advantage of normal cellular processes by using its own lipids to subvert the host’s lipid signaling - allowing it to enter the host cell and survive there. Our focus is on developing host and pathogen biomarkers that correlate with TB infection in laboratory animals and in humans. We are performing mass spectrometry-based multi-marker quantification of lipids in tissue and body fluids (blood, urine, sputum), specifically host lipids (e.g. phospholipids and ceramides) and pathogen-derived lipids (e.g. mycolates, LAM, etc.). Our research is focused on laboratory animals and TB patients in clinical trials, to be used both for drug efficacy studies as well as case detection and monitoring of treatment response. For analysis of the host response, lipids are extracted from infected tissues and cells, isolated and analyzed by mass spectrometry to develop a lipid profile, which is used to identify the metabolic pathways involved in the infection and the enzyme/protein that facilitates the process. This technique is very sensitive - a drop of blood is potentially enough to perform the analysis. For pathogen biomarker discovery, mycobacterial lipids are isolated directly from small volumes of infected body fluids such as sputum and subsequently quantified via mass spectrometry. We expect this research will be (i) useful in providing a better description of mycobacterial biosynthetic activity, which will be helpful in developing new diagnostics, drugs and vaccines, and (ii) for the development of lipid biomarker for TB case detection.

American tripanosomiasis: a study on the prevalence of Trypanosoma cruzi and Trypanosoma cruzi-like organisms in wild rodents in San Luis province, Argentina

Chagas disease is caused by Trypanosoma cruzi. Wild and perianthropic mammals maintain the infection/transmission cycle, both in their natural habitat and in the peridomestic area. The aim of this paper was to present the results from a study on wild rodents in the central and northern regions of San Luis province, Argentina, in order to evaluate the prevalence of this infection. Sherman traps were set up in capture areas located between latitudes 32 masculine and 33 masculine S, and longitudes 65 masculine and 66 masculine W. The captured rodents were taxonomically identified and hemoflagellates were isolated. Morphological, biometric and molecular studies and in vitro cultures were performed. Infection of laboratory animals and histological examination of the cardiac muscle and inoculation area were also carried out. Parasites were detected in circulating blood in Calomys musculinus, Graomys griseoflavus, Phyllotis darwini and Akodon molinae. The parasites were identified using biological criteria. Molecular PCR studies were performed on some isolates, which confirmed the characterization of these hemoflagellates as Trypanosoma cruzi. Forty-four percent of the 25 isolates were identified as Trypanosoma cruzi, and the remaining 56% as Trypanosoma cruzi-like. These findings provide evidence that wild rats infected with Trypanosoma cruzi and Trypanosoma cruzi-like organisms are important in areas of low endemicity.

Detection of Trichinella spiralis DNA in mouse faeces during the early stage of infection

Trichinella spiralis is an etiological agent of trichinellosis, a parasitic disease present worldwide, affecting both animals and humans. The infection starts from the intestinal stage which usually lasts a few weeks and develops into the muscle phase when larvae enter the muscle cells and create a nurse cell-larva-complex. In animals, the infection is asymptomatic even if a high number of larvae is ingested. The aim of this work was to develop a sensitive test to monitor the early phase of trichinellosis in laboratory animals. T. spiralis DNA presence was assayed in faeces of mice during the first 21 days of experimental infection. The target gene fragment of the mitochondrial ATP6 synthase F0 subunit was amplified by PCR from faecal samples of mice infected with 50, 250 or 500 larvae per mouse (lpm). Trichinella DNA was detected in 45% (116) of the examined faecal samples from days 1 to 21 post infection (p.i.). The time of appearance of the parasite DNA in the faeces and the percentage of PCR positive results were dose dependent. In collective samples consisting of faeces from 3 mice average 80%, 83% and 90% of PCR positive results were obtained for 50, 250 and 500 lpm doses, respectively, throughout 21 days of the observation period. In individual mice faeces DNA was detected on days 2–19 and 9–18 p.i. for 250 lpm and 50 lpm doses, respectively. For both infective doses the highest probability of DNA detection was on day 11 p.i. Since the developed test is simple and non-invasive, it can be useful for monitoring the intestinal phase of Trichinella sp. infection in laboratory animals.

Immunoblot Analysis as an Alternative Method to Diagnose Enterohepatic Helicobacter Infections

Enterohepatic Helicobacter species have been associated with chronic infections of the hepatobiliary tract and lower bowel in naturally and experimentally infected mice, Helicobacter-infected animals should thus not be used in studies of diseases associated with chronic inflammation. Helicobacter species induce inflammation and modulate host immune responses, thus emphasizing the need to diagnose these infections in laboratory animals. An immunoblot assay was developed to analyze antibodies to enterohepatic Helicobacter species in naturally colonized laboratory mouse colonies. We evaluated the serum antibody responses to cell surface proteins of H. bilis, H. hepaticus, and H. ganmani in 188 mouse sera from four different university animal facilities. Lower bowel tissue specimens from 56 of these animals were available and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and the results compared with matched immunoblot patterns. Specific antibody reactivity to H. bilis was detected in 8 of 186 (4.3%) sera, to H. hepaticus in 45 of 184 (24%) sera, and to H. ganmani in 51 of 188 (27%) of tested sera. These results were compared with PCR-DGGE analyses of tissue samples of corresponding animals, and concordance between the two diagnostic tests was found in 96% for H. bilis, in 91% for H. hepaticus, and in 82% for H. ganmani. The PCR-DGGE also detected DNA of H. typhlonius, H. sp. flexispira, and H. rodentium. Infection with enterohepatic species was common in the laboratory mouse colonies tested, independent of strain and stock. Immunoblot analysis seems to be a promising diagnostic tool to monitor enterohepatic Helicobacter species infections of laboratory rodents.

Comparative investigation of the role of the YadA, InvA, and PsaA genes in the pathogenicity of Yersinia pseudotuberculosis

The role of yadA, invA, and psaA genes in virulence was studied using a Yersinia pseudotuberculosis strain isolated from a patient with Far-East scarlatinoid fever as a model. Isogenic single, double, and multiple mutants of the studied genes were constructed, in which wild-type alleles were inactivated by the insertion of various antibiotic-resistance genes. LD50 and body weight dynamics of infected laboratory animals were used as virulence indicators. It has been established that the yadA gene is the primary determinant of bacterial virulence. This gene also causes the loss of body weight in laboratory animals infected by sublethal doses of Y. pseudotuberculosis. The invA gene, rather than yadA or psaA, plays a major role in the penetration of pathogenic microorganisms into eukaryotic cells. The effects of the yadA and invA genes on bacterial virulence and invasion, respectively, do not depend on the expression of other studied genes.

Evaluation of polymerase chain reaction methods for detection of murine Helicobacter in nine diagnostic laboratories

Helicobacter infections of laboratory animals may influence the results of in vivo experiments, necessitating diagnostic methods that are specific, sensitive and rapid. Polymerase chain reaction (PCR) is currently the preferred diagnostic tool for detecting Helicobacter infections in mice; however, detection ability may vary considerably among laboratories. Nine commercial and academic European labs participated in a 3-year ring study that was designed to explore this problem. The authors sought to identify which PCR methods were used for detection of murine Helicobacter spp. in fecal pellets and to compare the sensitivity, specificity and reproducibility of these methods. The study consisted of four rounds in which labs tested mouse fecal samples spiked with H. bilis, H. hepaticus or H. muridarum. The first round showed differences of up to 3 logs in detection sensitivity. Over the course of the study, sensitivity, specificity and reproducibility of PCR results in all labs improved substantially. By the study's conclusion, diagnostic ability in all labs was sufficient to reliably detect Helicobacter in naturally infected mice.

Prion protein allotype profiling by mass spectrometry

Abstract Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative pathologies characterized by the formation in the central nervous system of the amyloid protein PrPSc, which derives from a cellular precursor called PrPc. Epidemiological and laboratory studies have shown that in species where the PrPc gene is polymorphic, the genotype composition is an important factor for the development of the disease. Identification of PrPSc allotypes accumulated in the brain during the disease proved valuable to investigate whether these polymorphisms are critical for the pathological conversion. These analyses are complicated by the heterogeneity and the insolubility of the prion amyloid extracted from affected brains, which have been obviated by extensive digestion of extracted fractions and analysis of peptide fragment composition. We have developed an optimized protocol of liquid chromatography/mass spectrometry (LC/MS) that can reliably map PrP peptides in digested fractions with a low PrPSc/contaminants ratio. This approach has been successfully applied to the analysis of amyloidogenesis in experimentally infected PrP-heterozygous laboratory animals.

Elemental and intravenous total parenteral nutrition diet-induced gut barrier failure is intestinal site specific and can be prevented by feeding nonfermentable fiber.

Parenteral nutrition and elemental diets both cause bacterial translocation, immune dysfunction, and increased infection in laboratory animals, whereas elemental diets, with or without fiber, ameliorate some, but not all gut barrier failure. The purpose of this study is to investigate, in an Ussing chamber system, whether elemental vs. parenteral diets induce gut barrier failure in specific anatomical sites in the intestine and whether fiber can ameliorate this phenomenon. Controlled study in laboratory animals. University laboratory. Male Sprague-Dawley rats. Nutritional support was provided to rats for 7 days by oral total parenteral nutrition (TPN; elemental diet) 307 kcal/kg/day, intravenous TPN (parenteral diet) 307 kcal/kg/day via jugular venous catheters, or rodent chow (controls). Permeability to bacteria in intestinal segments of ileum, jejunum, and colon was evaluated in an Ussing chamber. Results were correlated with bacterial translocation to the mesenteric lymph nodes. Intravenous TPN caused greater bacterial translocation in all small intestinal segments and the cecum when compared with chow (p <.05). Oral TPN caused gut barrier failure only in the ileal segment, but not in the remainder of the small intestine (p <.001). Addition of cellulose provided a greater protection of the ileum to permeability than did pectin (p <.01). TPN causes global intestinal barrier failure, but elemental diet prevents barrier failure in parts of the small intestine other than the ileum. The addition of cellulose fiber to elemental diet can ameliorate further barrier failure in the ileum.

Inadequate management of natural ecosystem in the Brazilian Amazon region results in the emergence and reemergence of arboviruses.

A total of 187 different species of arboviruses and other viruses in vertebrates were identified at the Evandro Chagas Institute (IEC) from 1954 to 1998, among more than 10,000 arbovirus strains isolated from humans, hematophagous insects, and wild and sentinel vertebrates. Despite intensive studies in the Brazilian Amazon region, especially in Pará State, very little is known about most of these viruses, except for information on date, time, source, and method of isolation, as well as their capacity to infect laboratory animals. This paper reviews ecological and epidemiological data and analyzes the impact of vector and host population changes on various viruses as a result of profound changes in the natural environment. Deforestation, mining, dam and highway construction, human colonization, and urbanization were the main manmade environmental changes associated with the emergence and/or reemergence of relevant arboviruses, including some known pathogens for humans.

Attempts to Establish Experimental Cyclospora cayetanensis Infection in Laboratory Animals

Attempts to Establish Experimental Cyclospora cayetanensis Infection in Laboratory Animals

Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system.

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti-Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-kappaB into large complexes in the cytoplasm. NF-kappaB aggregation was dependent on type III secretion and aggregated NF-kappaB did not respond to TNFalpha activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-kappaB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.

Serum Lysozyme Activity Can Differentiate Infection from Rejection in Organ Transplant Recipients

Background. The presentation of infection in immunocompromised patients can be subtle. Patients often undergo extensive testing and empiric treatment for rejection and infection while awaiting the results of the biopsy and culture results. Delay in diagnosis of either entity can result in a worse clinical outcome. Lysozyme activity has been found to be both a sensitive and a specific marker for infection in laboratory animals and trauma patients. Our goal was to determine whether lysozyme activity could serve as a reliable marker for infection in immunocompromised organ transplant recipients.Methods. Transplant recipients from three clinical categories were enrolled in this study. Group 1 (n = 16) were patients hospitalized for sepsis (culture-positive), and Group 2 (n = 13) for biopsy-proven rejection. Group 3 (n = 51) were recipients who had routine blood sampling in the clinic. In Groups 1 and 2, blood was collected within 24 h of hospital admission and centrifuged, and serum samples were stored at 80°C until processing. Lysozyme activity was assayed by turbidimetric measurement on a sample of a standard suspension (0.15 mg/ml) of Micrococcus lysodeikticus in buffer using a spectrophotometer to read the optical density at 450 nm. The lysozyme activity level was then determined by the measuring activity at 1 min subtracted from activity at 4 min. White blood cell count was determined by using a flow cytometer. Statistical comparisons between groups were made using paired Student's t test.Results. Serum lysozyme was found to be significantly elevated in Group 1; the mean level of activity was 60.1 ± 13 (P < 0.05) compared to the level of activity in Group 2, mean 41.1 ± 11, and Group 3, mean 40.8 ± 13. A level of 45 units of activity/ml of serum or above indicated infection and not rejection in the patients admitted to the hospital.Conclusions. These findings indicate that the lysozyme activity assay appears to be useful in differentiating infection from rejection in transplant recipients.

Helicobacter pylori-Induced Chronic Active Gastritis, Intestinal Metaplasia, and Gastric Ulcer in Mongolian Gerbils

The establishment of persisting Helicobacter pylori infection in laboratory animals has been difficult, but in 1996 Hirayama reported the development of a successful Mongolian gerbil model. The present study was undertaken with two aims: to better characterize the normal histological structure and histochemical properties of the gastric mucosa of the Mongolian gerbil; and to evaluate the progression of the histopathological features of H. pylori-induced gastritis in this animal model for one year after the experimental infection. Seventy-five Mongolian gerbils were used. Mongolian gerbils were sacrificed at 2, 4, 8, 12, 26, 38, and 52 weeks after H. pylori inoculation. Sections prepared from stomachs immediately fixed in Carnoy's solution were stained with hematoxylin and eosin and Alcian blue at pH 2.5/periodic acid-Schiff, a dual staining consisting of the galactose oxidase-cold thionin Schiff reaction and paradoxical Concanavalin A staining, and with immunostaining for H. pylori and BrdU. H. pylori infection induced in the Mongolian gerbil a chronic active gastritis, in which a marked mucosal infiltration of neutrophils on a background of chronic inflammation became detectable 4 weeks after inoculation and continued up to 52 weeks. Intestinal metaplasia and gastric ulcers appeared after 26 weeks in some of the animals, whereas others developed multiple hyperplastic polyps. The Mongolian gerbil represents a novel and useful model for the study of H. pylori-induced chronic active gastritis and may lend itself to the investigation of the epithelial alterations that lead to intestinal metaplasia and gastric neoplasia.