Pathogenicity of Rhodococcus equi in mice, isolated from environment, human and horse clinical samples

Rhodococcus equi is a facultative intracellular pathogen associated with bronchopneumonia, mesenteric lymphadenitis and enterocolitis in foals. Although R. equi is likely to be found in every horse-breeding farm, the clinical disease is unrecognized in most of them. Capsule components, equi factor, micolic acid and some products encoded by the large 85-90Kb plasmid were described as virulence factors. However, the pathogenesis of R. equi infections and the sensibility of foals are not completely understood. The aim of this study was evaluate the virulence of R. equi isolated from human, horses and environment for mices. Nine strains carrying the 85-90Kb plasmid isolated from foal clinical specimens, one from immunodeficient human patient and six plasmidless strains (four isolated from feces, one from pasture and one from immunodeficient human patient) were inoculated in cyclophosphamide immunossuppressed mice. The pathological changes and viability of R. equi cells in the liver of mice was verified after the 3rd, 6th an 10th day after inoculation for horse and environmental isolates and for R. equi isolates from human patients on the 1st, 3rd and 6th day. During the necropsy procedures, infiltrate of macrophages and pyogranulomatous lesions were detected after the sixth pos-inoculation day in the liver and spleen. In horse isolates, only plasmid positive strains were virulent, but in human isolates both strains (plasmid positive e plasmid negative) were virulent. Both groups of the immunossupressed mice inoculated with R. equi isolated from environment showed pathological changes. All R. equi strains were unable to kill non imunossuppressed mice.

Animal infections by vaccinia-like viruses in the state of Rio de Janeiro: an expanding disease

In the present study we investigated the presence of infections by vaccinia-like viruses in dairy cattle from 12 counties in the state of Rio de Janeiro in the last 9 years. Clinical specimens were collected from adult animals with vesicular/pustular lesions mainly in the udder and teats, and from calves with lesions around the nose and mouth. A plaque reduction neutralization test (PRNT) was applied to search for antibodies to Orthopoxvirus; the vesicular/pustular fluids and scabs were examined by PCR, electron microscopy (EM) and by inoculation in VERO cells for virus isolation. Antibodies to Orthopoxvirus were detected in most cases. The PCR test indicated a high nucleotide homology among the isolates and the vaccinia viruses (VACV) used as controls. By EM, typical orthopoxvirus particles were observed in some specimens. The agents isolated in tissue culture were confirmed as vaccinia-like viruses by EM and PCR. The HA gene of the vaccinia-like Cantagalo/IOC virus isolated in our laboratory was sequenced and compared with other vaccinia-like isolates, showing high homology with the original Cantagalo strain, both strains isolated in 1999 from dairy cattle. Antibodies to Orthopoxvirus were detected in one wild rodent (genus Akodon sp.) collected in the northwestern region of the state, indicating the circulation of poxvirus in this area. Nonetheless, PCR applied to tissue samples collected from the wild rodents were negative. Vesicular/pustular lesions in people in close contact with animals have been also recorded. Thus, the vaccinia-like virus infections in cattle and humans in the state seem to be an expanding condition, resulting in economic losses to dairy herds and leading to transient incapacitating human disease. Therefore, a possible immunization of the dairy cattle in the state should be carefully evaluated.

Infection by Toxoplasma gondii in Neotropical non-human primates

Toxoplasma gondii (Nicolle et Manceaux, 1909) is an obligatory intracellular protozoan parasite of warm animals, including human and non-human primates. Domestic and wild felids are considered definitive hosts. Several authors have already identified lesions in New World primates caused by T. gondii. Nevertheless, little is known about serological studies on those animals. With this reason, New World non-human primates of the genera Cebus and Callithrix that were apprehended by governmental authorities and sent to the Wildlife Screening Center (Cetas)/IBAMA, at the municipality of Seropédica, state of Rio Janeiro, were bled and sera were submitted to the indirect hemagglutination test for detection of anti-T. gondii antibodies. From 21 sera of Cebus primates, 76.19% (16/21) had anti-T. gondii antibodies. Titles varied from 16 to 2048. In samples from 21 Callithrix, only 4.5% (1/22) had anti-T. gondii antibodies. Only one animal had a title of 32. During all the time those animals were clinical evaluated until sample was collected; none of them had any clinical sign or sequel related to infection by T. gondii. The fact that the origin of these primates is unknown and that there is no information about their feeding habits before captivity makes it difficult to determine the source of T. gondii infection.

Spatialization of climate, physical and socioeconomic factors that affect the dairy goat production in Brazil and their impact on animal breeding decisions

Brazil has high climate, soil and environmental diversity, as well as distinct socioeconomic and political realities, what results in differences among the political administrative regions of the country. The objective of this study was to determine spatial distribution of the physical, climatic and socioeconomic aspects that best characterize the production of dairy goats in Brazil. Production indices of milk per goat, goat production, milk production, as well as temperature range, mean temperature, precipitation, normalized difference vegetation index, relative humidity, altitude, agricultural farms; farms with native pasture, farms with good quality pasture, farms with water resources, farms that receive technical guidance, family farming properties, non-familiar farms and the human development index were evaluated. The multivariate analyses were carried out to spatialize climatic, physical and socioeconomic variables and so differenciate the Brazilian States and Regions. The highest yields of milk and goat production were observed in the Northeast. The Southeast Region had the second highest production of milk, followed by the South, Midwest and North. Multivariate analysis revealed distinctions between clusters of political-administrative regions of Brazil. The climatic variables were most important to discriminate between regions of Brazil. Therefore, it is necessary to implement animal breeding programs to meet the needs of each region.

Emerging animal viruses: real threats or simple bystanders?

The list of animal viruses has been frequently added of new members raising permanent concerns to virologists and veterinarians. The pathogenic potential and association with disease have been clearly demonstrated for some, but not for all of these emerging viruses. This review describes recent discoveries of animal viruses and their potential relevance for veterinary practice. Dogs were considered refractory to influenza viruses until 2004, when an influenza A virus subtype H3N8 was transmitted from horses and produced severe respiratory disease in racing greyhounds in Florida/USA. The novel virus, named canine influenza virus (CIV), is considered now a separate virus lineage and has spread among urban canine population in the USA. A new pestivirus (Flaviviridae), tentatively called HoBi-like pestivirus, was identified in 2004 in commercial fetal bovine serum from Brazil. Hobi-like viruses are genetically and antigenically related to bovine viral diarrhea virus (BVDV) and induce similar clinical manifestations. These novel viruses seem to be widespread in Brazilian herds and have also been detected in Southeast Asia and Europe. In 2011, a novel mosquito-borne orthobunyavirus, named Schmallenberg virus (SBV), was associated with fever, drop in milk production, abortion and newborn malformation in cattle and sheep in Germany. Subsequently, the virus disseminated over several European countries and currently represents a real treat for animal health. The origin of SBV is still a matter of debate but it may be a reassortant from previous known bunyaviruses Shamonda and Satuperi. Hepatitis E virus (HEV, family Hepeviridae) is a long known agent of human acute hepatitis and in 1997 was first identified in pigs. Current data indicates that swine HEV is spread worldwide, mainly associated with subclinical infection. Two of the four HEV genotypes are zoonotic and may be transmitted between swine and human by contaminated water and undercooked pork meat. The current distribution and impact of HEV infection in swine production are largely unknown. Avian gyrovirus type 2 (AGV2) is a newly described Gyrovirus, family Circoviridae, which was unexpectedly found in sera of poultry suspected to be infected with chicken anemia virus (CAV). AGV2 is closely related to CAV but displays sufficient genomic differences to be classified as a distinct species. AGV2 seems to be distributed in Brazil and also in other countries but its pathogenic role for chickens is still under investigation. Finally, the long time and intensive search for animal relatives of human hepatitis C virus (HCV) has led to the identification of novel hepaciviruses in dogs (canine hepacivirus [CHV]), horses (non-primate hepaciviruses [NPHV] or Theiler's disease associated virus [TDAV]) and rodents. For these, a clear and definitive association with disease is still lacking and only time and investigation will tell whether they are real disease agents or simple spectators.

Fatal Human herpesvirus 1 (HHV-1) infection in captive marmosets (Callithrix jacchus and Callithrix penicillata) in Brazil: clinical and pathological characterization

Fatal Human herpesvirus 1 (HHV-1) was diagnosed in 12 captive marmosets (Callithrix jacchus and Callithrix penicillata) from metropolitan region of São Paulo, São Paulo State. Clinical signs were variable among the cases, but most affected marmosets presented signs associated with viral epithelial replication: oral, lingual and facial skin ulcers and hypersalivation, and viral replication in the central nervous system: prostration, seizure and aggressive behavior. Consistent microscopic findings were diffuse mild to severe nonsuppurative necrotizing meningoencephalitis with gliosis, vasculitis and neuronal necrosis. Additionally, in the brain, oral cavity, skin, adrenal gland and myoenteric plexus intranuclear inclusion bodies were present. Immunohistochemistry confirmed the presence of the HHV-1 antigen in association with lesions in the brain, oral and lingual mucosa, facial skin, adrenal gland and myoenteric plexus. HHV-1-specific polymerase chain reaction (PCR) analysis of the brain was carried out and the virus was detected in 7/8 infected marmosets. It is concluded that HHV-1 causes widespread fatal infection in marmosets.

Brains and guts in human evolution: The Expensive Tissue Hypothesis

The brain is a very expensive organ in metabolic terms. Each unit of brain tissue requires over 22 times the amount of metabolic energy as an equivalent unit of muscle tissue. There is no correlation across mammals, however, between the relative size of the brain and the relative basal metabolic rate. The Expensive Tissue Hypothesis explains this apparent paradox by looking at the metabolic cost of the brain in the context of the costs of other metabolically expensive organs in the body. The results show that the increase in brain size in humans is balanced by an equivalent reduction in the size of the gastro-intestinal tract. In other words, the increased energetic demands of a relatively large brain are balanced by the reduced energy demands of a relatively small gastro-intestinal tract. This relationship also seems to be true in non-human primates. The size of the gastro-intestinal tract is dependent on both body size and the quality of the diet. It is argued that humans (and other primates) could not have developed a relatively large brain without also adopting a high quality diet that would have permitted a reduction in the relative size of the gastro-intestinal tract. Dietary change is therefore viewed as a 'prime releaser' in brain evolution. It is argued that a high quality diet is necessary for the evolution of a relatively large brain. However, the change to such a high quality diet, which involved an increased proportion of animal based products, need not have been one of the 'prime movers' in brain evolution. In this context, and based on the archaeological and palaeoanthropological record, the factors most probably surrounding the evolution of the human brain are discussed.

Aluminum induces lipid peroxidation and aggregation of human blood platelets

Aluminum (Al3+) intoxication is thought to play a major role in the development of Alzheimer's disease and in certain pathologic manifestations arising from long-term hemodialysis. Although the metal does not present redox capacity, it can stimulate tissue lipid peroxidation in animal models. Furthermore, in vitro studies have revealed that the fluoroaluminate complex induces diacylglycerol formation, 43-kDa protein phosphorylation and aggregation. Based on these observations, we postulated that Al3+-induced blood platelet aggregation was mediated by lipid peroxidation. Using chemiluminescence (CL) of luminol as an index of total lipid peroxidation capacity, we established a correlation between lipid peroxidation capacity and platelet aggregation. Al3+ (20-100 µM) stimulated CL production by human blood platelets as well as their aggregation. Incubation of the platelets with the antioxidants nor-dihydroguaiaretic acid (NDGA) (100 µM) and n-propyl gallate (NPG) (100 µM), inhibitors of the lipoxygenase pathway, completely prevented CL and platelet aggregation. Acetyl salicylic acid (ASA) (100 µM), an inhibitor of the cyclooxygenase pathway, was a weaker inhibitor of both events. These findings suggest that Al3+ stimulates lipid peroxidation and the lipoxygenase pathway in human blood platelets thereby causing their aggregation

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.

Pharmacology of human experimental anxiety

This review covers the effect of drugs affecting anxiety using four psychological procedures for inducing experimental anxiety applied to healthy volunteers and patients with anxiety disorders. The first is aversive conditioning of the skin conductance responses to tones. The second is simulated public speaking, which consists of speaking in front of a video camera, with anxiety being measured with psychometric scales. The third is the Stroop Color-Word test, in which words naming colors are painted in the same or in a different shade, the incongruence generating a cognitive conflict. The last test is a human version of a thoroughly studied animal model of anxiety, fear-potentiated startle, in which the eye-blink reflex to a loud noise is recorded. The evidence reviewed led to the conclusion that the aversive conditioning and potentiated startle tests are based on classical conditioning of anticipatory anxiety. Their sensitivity to benzodiazepine anxiolytics suggests that these models generate an emotional state related to generalized anxiety disorder. On the other hand, the increase in anxiety determined by simulated public speaking is resistant to benzodiazepines and sensitive to drugs affecting serotonergic neurotransmission. This pharmacological profile, together with epidemiological evidence indicating its widespread prevalence, suggests that the emotional state generated by public speaking represents a species-specific response that may be related to social phobia and panic disorder. Because of scant pharmacological data, the status of the Stroop Color-Word test remains uncertain. In spite of ethical and economic constraints, human experimental anxiety constitutes a valuable tool for the study of the pathophysiology of anxiety disorders.

Random amplification of polymorphic DNA reveals clonal relationships among enteropathogenic Escherichia coli isolated from non-human primates and humans

Enteropathogenic Escherichia coli (EPEC) strains are important agents of infantile diarrhea all over the world, gaining even greater importance in developing countries. EPEC have also been isolated from various animal species, but most isolates belong to serotypes that differ from those recovered from humans. However, it has been demonstrated that several isolates from non-human primates belong to the serogroups and/or serotypes related to those implicated in human disease. The objective of this study was to evaluate the genetic differences between thirteen strains isolated from non-human primates and the same number of strains isolated from human infections. Human isolates belonged to the same serogroup/serotype as the monkey strains and the evaluation was done by analysis of random amplified polymorphic DNA. Dendrogram analysis showed that there was no clustering between human and monkey strains. Human and non-human isolates of the EPEC serotypes O127:H40 and O128:H2 shared 90 and 87% of their bands, respectively, indicating strong genomic similarity between the strains, leading to the speculation that they may have arisen from the same pathogenic clone. To our knowledge, this study is the first one comparing genomic similarity between human and non-human primate strains and the results provide further evidence that monkey EPEC strains correlate with human EPEC, as suggested in a previous investigation.

Efficacy of HUMN criteria for scoring the micronucleus assay in human lymphocytes exposed to a low concentration of p,p'-DDT

The cytokinesis-block micronucleus (CBMN) assay is one of the standard cytogenetic tools employed to assess chromosomal damage subsequent to exposure to genotoxic/cytotoxic agents, and is widely applicable to plant, animal and human cells. In the present study, the CBMN assay was used to assess the baseline damage in binuclear human peripheral blood lymphocytes exposed to 25 µg/L p,p'-DDT for 1, 2, 24, and 48 h by measuring the frequency of micronuclei, nucleoplasmic bridges and nuclear buds. These new scoring criteria facilitated the detection of different types of clastogenic and aneugenic effects induced by this type of pollutant. With these criteria, CBMN can also be used to measure nucleoplasmic bridges which are considered to be consequences of chromosome rearrangements and nuclear buds which are biomarkers of altered gene amplification and gene dosage. The total number of micronuclei observed in binuclear human peripheral blood lymphocytes of the exposed samples (ranging from 32 to 47) was significantly greater (P < 0.05) than that detected in the unexposed (0 time) control sample, where the total number of micronuclei was 7. The number of nucleoplasmic bridges and nuclear buds obtained after 24 and 48 h was also significantly (P < 0.05) greater in the samples treated with p,p'-DDT than in the unexposed control samples. Thus, our results confirmed the usefulness of the new criteria applicable for the CBMN assay employed in measuring the DNA damage and its role of a sensitive cytogenetic biomarker.

Effect of the human leukocyte antigen HLA-DRB1 and anti-cyclic citrullinated peptide on the outcome of rheumatoid arthritis patients

Our objective was to determine whether the presence of the human leukocyte antigen HLA-DRB1 locus is associated with production of anti-cyclic citrullinated peptide antibodies (anti-CCP Abs) and to what extent they are associated with increased susceptibility to and severity of rheumatoid arthritis (RA) in Egyptian patients. Twenty-nine RA patients gave informed consent to participate in a case-control study that was approved by the Ain Shams University Medical Ethics Committee. RA disease activity and severity were determined using the simplified disease activity index and Larsen scores, respectively. We used a wide scale national study on the pattern of HLA typing in normal Egyptians as a control study. Anti-CCP Abs and HLA-DRB1 typing were determined for all subjects. The alleles most strongly associated with RA were HLA-DRB1 [*01 , *04 and *06] (41.4%). RA patients with serum anti-CCP Ab titers above 60 U/mL had a significantly higher frequency of HLA-DRB1*01 (58.3%) and HLA-DRB1*04 alleles (83.3%). Significant positive correlations were found between serum and synovial anti-CCP Ab titer, RA disease activity, and severity (r = 0.87, 0.66 and 0.63, respectively; P < 0.05). HLA-DRB1 SE+ alleles [*01 and *04] were highly expressed among Egyptian RA patients. The presence of these alleles was associated with higher anti-CCP Ab titer, active and severe RA disease. Early determination of HLA-DRB1 SE+ alleles and serum anti-CCP Ab could facilitate the prediction of the clinical course and prognosis of RA when first evaluated leading to better disease control.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

A forced running wheel system with a microcontroller that provides high-intensity exercise training in an animal ischemic stroke model

We developed a forced non-electric-shock running wheel (FNESRW) system that provides rats with high-intensity exercise training using automatic exercise training patterns that are controlled by a microcontroller. The proposed system successfully makes a breakthrough in the traditional motorized running wheel to allow rats to perform high-intensity training and to enable comparisons with the treadmill at the same exercise intensity without any electric shock. A polyvinyl chloride runway with a rough rubber surface was coated on the periphery of the wheel so as to permit automatic acceleration training, and which allowed the rats to run consistently at high speeds (30 m/min for 1 h). An animal ischemic stroke model was used to validate the proposed system. FNESRW, treadmill, control, and sham groups were studied. The FNESRW and treadmill groups underwent 3 weeks of endurance running training. After 3 weeks, the experiments of middle cerebral artery occlusion, the modified neurological severity score (mNSS), an inclined plane test, and triphenyltetrazolium chloride were performed to evaluate the effectiveness of the proposed platform. The proposed platform showed that enhancement of motor function, mNSS, and infarct volumes was significantly stronger in the FNESRW group than the control group (P<0.05) and similar to the treadmill group. The experimental data demonstrated that the proposed platform can be applied to test the benefit of exercise-preconditioning-induced neuroprotection using the animal stroke model. Additional advantages of the FNESRW system include stand-alone capability, independence of subjective human adjustment, and ease of use.