ABSTRACT
Abstract Introduction: Wildlife hematological patterns are fundamental for health monitoring, and allows elucidating variations both within and between populations. Among these, hematological parameters are particularly valuable to evaluate the health status of neotropical primate species in the wild. Objective: To define hematological reference values for two species of monkeys in Costa Rica. Methods: During 2014, we collected blood samples from free-ranging mantled howler monkeys, Alouatta palliata (17 females, 18 males) and white-faced capuchin monkeys, Cebus imitator (5 females, 7 males) in seven localities of the Costa Rican Pacific coast. Results: For both species, the hematological values were higher in males, and howler monkey populations differed significantly except for platelets. Conclusions: These hematological values, which differ by sex and locality, will help evaluate the health status of these neotropical primate populations.
Resumen Introducción: Los patrones hematológicos de la vida silvestre son fundamentales para el monitoreo de la salud y permiten dilucidar las variaciones tanto dentro como entre poblaciones. Entre estos, los parámetros hematológicos son particularmente valiosos para evaluar el estado de salud de las especies de primates neotropicales en la naturaleza. Objetivo: Definir valores de referencia hematológicos para dos especies de monos en Costa Rica. Métodos: Durante el 2014 recolectamos muestras de sangre de monos aulladores de manto, Alouatta palliata (17 hembras, 18 machos) y monos capuchinos cariblancos, Cebus imitador (5 hembras, 7 machos) en siete localidades de la costa Pacífica de Costa Rica. Resultados: Para ambas especies, los valores hematológicos fueron mayores en los machos, y las poblaciones de monos aulladores difirieron significativamente con excepción de las plaquetas. Conclusiones: Estos valores hematológicos, que difieren según el sexo y la localidad, ayudarán a evaluar el estado de salud de estas poblaciones de primates neotropicales.
Subject(s)
Animals , Haplorhini/microbiology , Hematologic Tests/veterinary , Costa RicaABSTRACT
En los momentos actuales se viene observando con incertidumbre el aumento de casos por transmisión persona a persona de la viruela del mono, también conocida como viruela símica, viruela de los monos o monkeypox (en inglés) en países no endémicos. Del 13 de mayo al 2 de junio del 2022 se han registrado 780 casos confirmados en 27 países de cuatro regiones de la Organización Mundial de la salud (OMS), siendo Europa la región con el mayor número de casos y que representa el 88% (688 casos) en 20 países, seguido de la Región de las Américas con el 10% (80 casos), Región del Mediterráneo Oriental con el 1% (9 casos) y la Región del Pacífico Occidental con menos del 1 % (3 casos). Los casos reportados al 02 de junio representan un aumento en +203% de lo comunicado al 29 de mayo del 2022. (1) La mayoría de los casos confirmados con antecedentes de viaje informaron que estos fueron a países de Europa y América del Norte, en lugar de África occidental o central que son las áreas endémicas. Ninguno de los casos estuvo asociado a muerte a diferencia de las 66 defunciones ocurridas en países endémicos en el presente año.
Subject(s)
Smallpox , Epidemiologic Studies , Epidemiologic Measurements , Haplorhini , Monkeypox , Epidemiological MonitoringABSTRACT
Captive animals, despite the constant care provided, are susceptible to infections from different sources. We herein report the natural trypanosome infection of 11 (28.2% positive) out of 39 non-human primates from 13 different species, in a Brazilian zoological park. Immunofluorescent antibody test (IFAT) and conventional polymerase chain reaction (cPCR) ruled out Trypanosoma cruzi, the etiological agent of Chagas disease. However, sequencing performed with positive samples employing hsp70 primers revealed similarities from 86% to 88% to diverse trypanosomes, including T. cruzi, Trypanosoma grayi, Trypanosoma lewisi, Trypanosoma rangeli and Trypanosoma vivax. We believe that the low similarity values obtained by sequencing reflect the difficulties in the molecular identification of trypanosomes, which share a large portion of their genetic material; this similarity may also preclude the diagnosis of co-infection by more than one trypanosome species. Thus, our study demonstrates the presence of diverse trypanosomes in primates, which are susceptible to infection by these parasites. Mechanical devices such as windows and bed nets, etc., are required to avoid vector insects in these environments, in addition to preventive quarantining of animals recently introduced into zoos. Therefore, investigation of the parasites in both the animals already residing in the zoo and those being introduced is of paramount importance, although no easy task.
Subject(s)
Humans , Animals , Primates , Trypanosoma , Haplorhini , Chagas DiseaseABSTRACT
Whether direct manipulation of Parkinson's disease (PD) risk genes in the adult monkey brain can elicit a Parkinsonian phenotype remains an unsolved issue. Here, we used an adeno-associated virus serotype 9 (AAV9)-delivered CRISPR/Cas9 system to directly co-edit PINK1 and DJ-1 genes in the substantia nigras (SNs) of two monkey groups: an old group and a middle-aged group. After the operation, the old group exhibited all the classic PD symptoms, including bradykinesia, tremor, and postural instability, accompanied by key pathological hallmarks of PD, such as severe nigral dopaminergic neuron loss (>64%) and evident α-synuclein pathology in the gene-edited SN. In contrast, the phenotype of their middle-aged counterparts, which also showed clear PD symptoms and pathological hallmarks, were less severe. In addition to the higher final total PD scores and more severe pathological changes, the old group were also more susceptible to gene editing by showing a faster process of PD progression. These results suggested that both genetic and aging factors played important roles in the development of PD in the monkeys. Taken together, this system can effectively develop a large number of genetically-edited PD monkeys in a short time (6-10 months), and thus provides a practical transgenic monkey model for future PD studies.
Subject(s)
Animals , Brain , CRISPR-Cas Systems/genetics , Dependovirus/genetics , Haplorhini , Phenotype , Protein Kinases/geneticsABSTRACT
Abstract Objective: Myofibroblasts have been associated with the development of several pathologic fibrotic conditions. This longitudinal study aims to assess the proliferative and antiapoptotic effects of cyclosporin, nifedipine and phenytoin on gingival connective tissue cells of nonhuman primate, as well as to analyze a possible role of myofibroblasts in gingival overgrowth. Materials and Methods: Gingival samples from the right superior canine area were obtained from 12 male monkeys ( Sapajus spp ) to comprise the control group. After one week, the animals were randomly assigned to three groups, which received daily oral doses of cyclosporin, nifedipine or phenytoin for 120 days. Gingival samples were collected from the left superior canine area of two animals of each group at 52 and 120 days. Histological sections were stained with hematoxylin and eosin, and immunoreacted against α-SMA, Ki- 67 and bcl-2. Results: α-SMA immunoreaction was negative in the control and experimental groups. Similarly, no difference between groups concerning immunostaining against Ki-67 and bcl-2 was observed in connective tissue cells. Conclusion: Based on this methodology, it may be concluded that gingival overgrowths induced by cyclosporin, nifedipine and phenytoin are not associated with neither myofibroblast transdifferentiation, proliferation nor apoptosis of gingival connective cells in monkeys.
Subject(s)
Animals , Male , Phenytoin/pharmacology , Nifedipine/pharmacology , Cyclosporine/pharmacology , Cell Transdifferentiation/drug effects , Myofibroblasts/drug effects , Gingiva/cytology , Biopsy , Immunohistochemistry , Random Allocation , Longitudinal Studies , Actins/analysis , Haplorhini , Apoptosis/drug effects , Gingival Overgrowth/chemically induced , Gingival Overgrowth/pathology , Ki-67 Antigen/analysis , Ki-67 Antigen/drug effects , Genes, bcl-2/drug effects , Cell Proliferation/drug effects , Myofibroblasts/cytology , Gingiva/drug effectsABSTRACT
Human infections due to the monkey malaria parasite Plasmodium knowlesi is increasingly being reported from most Southeast Asian countries specifically Malaysia. The parasite causes severe and fatal malaria thus there is a need for urgent measures for its control. In this study, the level of polymorphisms, haplotypes and natural selection of full-length pkmsp8 in 37 clinical samples from Malaysian Borneo along with 6 lab-adapted strains were investigated. Low levels of polymorphism were observed across the full-length gene, the double epidermal growth factor (EGF) domains were mostly conserved, and non-synonymous substitutions were absent. Evidence of strong negative selection pressure in the non-EGF regions were found indicating functional constrains acting at different domains. Phylogenetic haplotype network analysis identified shared haplotypes and indicated geographical clustering of samples originating from Peninsular Malaysia and Malaysian Borneo. This is the first study to genetically characterize the full-length msp8 gene from clinical isolates of P. knowlesi from Malaysia; however, further functional characterization would be useful for future rational vaccine design.
Subject(s)
Humans , Asian People , Borneo , Epidermal Growth Factor , Genetic Variation , Haplorhini , Haplotypes , Malaria , Malaysia , Merozoites , Parasites , Plasmodium knowlesi , Selection, GeneticABSTRACT
A rapid diagnostic test (RDT) kit was developed to detect non-structural protein 1 (NS1) of yellow fever virus (YFV) using monoclonal antibody. NS1 protein was purified from the cultured YFV and used to immunize mice. Monoclonal antibody to NS1 was selected and conjugated with colloidal gold to produce the YFV NS1 RDT kit. The YFV RDTs were evaluated for sensitivity and specificity using positive and negative samples of monkeys from Brazil and negative human blood samples from Korea. Among monoclonal antibodies, clones 3A11 and 3B7 proved most sensitive, and used for YFV RDT kit. Diagnostic accuracy of YFV RDT was fairly high; Sensitivity was 0.0% and specificity was 100% against Dengue viruses type 2 and 3, Zika, Chikungunya and Mayaro viruses. This YFV RDT kit could be employed as a test of choice for point-of-care diagnosis and large scale surveys of YFV infection under clinical or field conditions in endemic areas and on the globe.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , Brazil , Clone Cells , Dengue Virus , Diagnosis , Diagnostic Tests, Routine , Gold Colloid , Haplorhini , Korea , Point-of-Care Systems , Sensitivity and Specificity , Yellow fever virus , Yellow FeverABSTRACT
The plasticizer di(2-ethylhexyl) phthalate (DEHP) has been widely used in the manufacture of polyvinyl chloride-containing products such as medical and consumer goods. Humans can easily be exposed to it because DEHP is ubiquitous in the environment. Recent research on the adverse effects of DEHP has focused on reproductive and developmental toxicity in rodents and/or humans. DEHP is a representative of the peroxisome proliferators. Therefore, peroxisome proliferator-activated receptor alpha (PPARα)-dependent pathways are the expected mode of action of several kinds of DEHP-induced toxicities. In this review, we summarize DEHP kinetics and its mechanisms of carcinogenicity and reproductive and developmental toxicity in relation to PPARα. Additionally, we give an overview of the impacts of science policy on exposure sources.
Subject(s)
Animals , Humans , Mice , Rats , Diethylhexyl Phthalate , Toxicity , Environmental Pollutants , Toxicity , Haplorhini , PPAR alpha , Genetics , Metabolism , Plasticizers , ToxicityABSTRACT
BACKGROUND: Gross anatomy and sectional anatomy of a monkey should be known by students and researchers of veterinary medicine and medical research. However, materials to learn the anatomy of a monkey are scarce. Thus, the objective of this study was to produce a Visible Monkey data set containing cross sectional images, computed tomographs (CTs), and magnetic resonance images (MRIs) of a monkey whole body. METHODS: Before and after sacrifice, a female rhesus monkey was used for 3 Tesla MRI and CT scanning. The monkey was frozen and sectioned at 0.05 mm intervals for the head region and at 0.5 mm intervals for the rest of the body using a cryomacrotome. Each sectioned surface was photographed using a digital camera to obtain horizontal sectioned images. Segmentation of sectioned images was performed to elaborate three-dimensional (3D) models of the skin and brain. RESULTS: A total of 1,612 horizontal sectioned images of the head and 1,355 images of the remaining region were obtained. The small pixel size (0.024 mm × 0.024 mm) and real color (48 bits color) of these images enabled observations of minute structures. CONCLUSION: Due to small intervals of these images, continuous structures could be traced completely. Moreover, 3D models of the skin and brain could be used for virtual dissections. Sectioned images of this study will enhance the understanding of monkey anatomy and foster further studies. These images will be provided to any requesting researcher free of charge.
Subject(s)
Female , Humans , Anatomy, Cross-Sectional , Brain , Dataset , Haplorhini , Head , Macaca mulatta , Magnetic Resonance Imaging , Primates , Skin , Tomography, X-Ray Computed , Veterinary MedicineABSTRACT
Microorganisms play important roles in obesity; however, the role of the gut microbiomes in obesity is controversial because of the inconsistent findings. This study investigated the gut microbiome communities in obese and lean groups of captive healthy cynomolgus monkeys reared under strict identical environmental conditions, including their diet. No significant differences in the relative abundance of Firmicutes, Bacteroidetes and Prevotella were observed between the obese and lean groups, but a significant difference in Spirochetes (p < 0.05) was noted. Microbial diversity and richness were similar, but highly variable results in microbial composition, diversity, and richness were observed in individuals, irrespective of their state of obesity. Distinct clustering between the groups was not observed by principal coordinate analysis using an unweighted pair group method. Higher sharedness values (95.81% ± 2.28% at the genus level, and 79.54% ± 5.88% at the species level) were identified among individual monkeys. This paper reports the association between the gut microbiome and obesity in captive non-human primate models reared under controlled environments. The relative proportion of Firmicutes and Bacteroidetes as well as the microbial diversity known to affect obesity were similar in the obese and lean groups of monkeys reared under identical conditions. Therefore, obesity-associated microbial changes reported previously appear to be associated directly with environmental factors, particularly diet, rather than obesity.
Subject(s)
Bacteroidetes , Diet , Environment, Controlled , Firmicutes , Gastrointestinal Microbiome , Haplorhini , Macaca fascicularis , Methods , Microbiota , Obesity , Prevotella , Primates , SpirochaetalesABSTRACT
Quantitative susceptibility mapping (QSM) can provide tissue susceptibility information and has been adapted for clinical research and diagnosis. QSM of monkey brain at 9.4 T has not been demonstrated so far. In this study 9.4 T monkey brain QSM was performed with 200 μm isotropic high-resolution. It was found that the inherent singularity problem for QSM diverged significantly at ultra-high image resolution during regularization process and resulted in severe image artifacts. The K-space division (TKD) was applied to eliminate the artifacts, with an optimal threshold level between 0.2 and 0.3. High resolution QSM of monkey brain can thus provide a novel tool for brain research.
Subject(s)
Animals , Algorithms , Brain , Diagnostic Imaging , Brain Mapping , Haplorhini , Magnetic Resonance ImagingABSTRACT
The function of microglia/macrophages after ischemic stroke is poorly understood. This study examines the role of microglia/macrophages in the focal infarct area after transient middle cerebral artery occlusion (MCAO) in rhesus monkeys. We measured infarct volume and neurological function by magnetic resonance imaging (MRI) and non-human primate stroke scale (NHPSS), respectively, to assess temporal changes following MCAO. Activated phagocytic microglia/macrophages were examined by immunohistochemistry in post-mortem brains (n=6 MCAO, n=2 controls) at 3 and 24 hours (acute stage), 2 and 4 weeks (subacute stage), and 4, and 20 months (chronic stage) following MCAO. We found that the infarct volume progressively decreased between 1 and 4 weeks following MCAO, in parallel with the neurological recovery. Greater presence of cluster of differentiation 68 (CD68)-expressing microglia/macrophages was detected in the infarct lesion in the subacute and chronic stage, compared to the acute stage. Surprisingly, 98~99% of transforming growth factor beta (TGFβ) was found colocalized with CD68-expressing cells. CD68-expressing microglia/macrophages, rather than CD206⁺ cells, may exert anti-inflammatory effects by secreting TGFβ after the subacute stage of ischemic stroke. CD68⁺ microglia/macrophages can therefore be used as a potential therapeutic target.
Subject(s)
Brain , Haplorhini , Immunohistochemistry , Infarction, Middle Cerebral Artery , Inflammation , Macaca mulatta , Magnetic Resonance Imaging , Microglia , Middle Cerebral Artery , Primates , Stroke , Transforming Growth Factor betaABSTRACT
Spinal cord contusion injury is one of the most serious nervous system disorders, characterized by high morbidity and disability. To mimic spinal cord contusion in humans, various animal models of spinal contusion injury have been developed. These models have been developed in rats, mice, and monkeys. However, most of these models are developed using rats. Two types of animal models, i.e. bilateral contusion injury and unilateral contusion injury models, are developed using either a weight drop method or impactor method. In the weight drop method, a specific weight or a rod, having a specific weight and diameter, is dropped from a specific height on to the exposed spinal cord. Low intensity injury is produced by dropping a 5 g weight from a height of 8 cm, moderate injury by dropping 10 g weight from a height of 12.5–25 mm, and high intensity injury by dropping a 25 g weight from a height of 50 mm. In the impactor method, injury is produced through an impactor by delivering a specific force to the exposed spinal cord area. Mild injury is produced by delivering 100 ± 5 kdyn of force, moderate injury by delivering 200 ± 10 kdyn of force, and severe injury by delivering 300 ± 10 kdyn of force. The contusion injury produces a significant development of locomotor dysfunction, which is generally evident from the 0–14(th) day of surgery and is at its peak after the 28–56th day. The present review discusses different animal models of spinal contusion injury.
Subject(s)
Animals , Female , Humans , Mice , Rats , Body Weight , Cervical Vertebrae , Contusions , Haplorhini , Locomotion , Methods , Models, Animal , Nervous System Diseases , Spinal Cord Injuries , Spinal CordABSTRACT
Simian malaria is a zoonotic disease caused by Plasmodium knowlesi infection. The common natural reservoir of the parasite is the macaque monkey and the vector is the Anopheles mosquito. Human cases of P. knowlesi infection has been reported in all South East Asian countries in the last decade, and it is currently the most common type of malaria seen in Malaysia and Brunei. Between 2007–2017, 73 cases of P. knowlesi infection were notified and confirmed to the Ministry of Health in Brunei. Of these, 15 cases (21%) were documented as work-related, and 28 other cases (38%) were classified as probably related to work (due to incomplete history). The occupations of those with probable and confirmed work related infections were border patrol officers, Armed Forces and security personnel, Department of Forestry officers, boatmen and researchers. The remaining cases classified as most likely not related to work were possibly acquired via peri-domestic transmission. The risk of this zoonotic infection extends to tourists and overseas visitors who have to travel to the jungle in the course of their work. It can be minimised with the recommended use of prophylaxis for those going on duty into the jungles, application of mosquito/insect repellants, and use of repellant impregnated uniforms and bed nets in jungle camp sites.
Subject(s)
Humans , Anopheles , Arm , Asian People , Brunei , Culicidae , Forestry , Haplorhini , Macaca , Malaria , Malaysia , Occupations , Parasites , Plasmodium knowlesi , Plasmodium , ZoonosesABSTRACT
RESUMO O conhecimento sobre modelos animais para estudo metabólico representa a base da pesquisa nessa área. Este trabalho tem por objetivo revisar os principais modelos animais a serem utilizados no estudo da obesidade e da síndrome metabólica. Para isso, pesquisa no banco de dados Pubmed foi realizada usando as palavras-chave "animal models", "obesity", "metabolic syndrome", e "bariatric surgery". Várias espécies de animais podem ser usadas para o estudo de distúrbios metabólicos, no entanto, os roedores, tanto modelos monogênicos quanto modelos de obesidade induzida por dieta (DIO), são os animais mais utilizados nessa área. Animais monogênicos são a melhor escolha se apenas um aspecto estiver sendo avaliado. Animais DIO tendem a demonstrar melhor a interação entre doença, ambiente e gene. No entanto, eles ainda não são totalmente eficazes para a compreensão de todos os mecanismos dessa doença.
ABSTRACT Knowledge about animal models for metabolic study is the basis of research in this area. This work aims to review the main animal models used in the study of obesity and metabolic syndrome. For this, we performed a search in the Pubmed database using the terms "animal models", "obesity", "metabolic syndrome" and "bariatric surgery". Several species of animals can be used for the study of metabolic disorders. However, rodents are the most commonly used, both as monogenic models and as diet-induced obesity (DIO) ones. Monogenic animals are the best choice if only one aspect is being evaluated. DIO animals tend to better demonstrate the interaction between disease, environment and genetics. However, they are still not fully effective in providing understanding of all disease mechanisms.
Subject(s)
Animals , Cats , Dogs , Rats , Metabolic Syndrome , Disease Models, Animal , Obesity , Haplorhini , Bariatric Surgery , MiceABSTRACT
This study was carried out to determine the pathogen-causing diarrhoea in sheep Ovis aries in the Qinghai Tibetan Plateau Area, China. A trophozoite was identified as species of ciliate alveolates infecting the sheep based on morphological characteristics examined by microscope. It was mostly spherical, colourless and transparent, with many vesicles. Macronucleus and contractile vacuoles could not be distinguished. Size of the trophozoite was 80–180×70–150 μm and its surface was covered with cilia. Molecular analysis based on sequences of 18S rRNA and ITS genes confirmed the ciliate species as Balantidium coli. According to the literature, there have been many epidemiological investigations of B. coli infection in pigs, monkeys and humans. To our knowledge, this was the first report of B. coli infections in sheep in the Qinghai Tibetan Plateau Area of China, or eleswhere around the world. Importantly, the sheep case was rare but raised our concern that B. coli may spread across species and expand its host range.
Subject(s)
Humans , Balantidium , China , Cilia , Haplorhini , Host Specificity , Macronucleus , Sheep , Sheep, Domestic , Swine , Trophozoites , VacuolesABSTRACT
PURPOSE: A major question remaining in approaches to tissue engineering and organ replacement is the role of native mobilized native cells in the regeneration process of damaged tissues and organs. The goal of this study was to compare the cell mobilizing effects of the chemokine CXCL12 and cell therapy on the urinary sphincter of nonhuman primates (NHP) with chronic intrinsic urinary sphincter dysfunction. METHODS: Either autologous lenti-M-cherry labeled skeletal muscle precursor cells (skMPCs) or CXCL12 were injected directly into the sphincter complex of female NHPs with or without surgery-induced chronic urinary sphincter dysfunction (n=4/treatment condition). All monkeys had partial bone marrow transplantation with autologous lenti-green fluorescent protein (GFP) bone marrow cells prior to treatment. Labeled cells were identified, characterized and quantified using computer-assisted immunohistochemistry 6 months posttreatment. RESULTS: GFP-labeled bone marrow cells (BMCs) were identified in the bone marrow and both BMCs and skMPCs were found in the urinary sphincter at 6-month postinjection. BMCs and skMPCs were present in the striated muscle, smooth muscle, and lamina propria/urothelium of the sphincter tissue. Sphincter injury increased the sphincter content of BMCs when analyzed 6-month postinjection. CXCL12 treatment, but not skMPCs, increased the number of BMCs in all layers of the sphincter complex (P < 0.05). CXCL12 only modestly (P=0.15) increased the number of skMPCs in the sphincter complex. CONCLUSIONS: This dual labeling methodology now provides us with the tools to measure the relative number of locally injected cells versus bone marrow transplanted cells. The results of this study suggest that CXCL12 promotes mobilization of cells to the sphincter, which may contribute more to sphincter regeneration than injected cells.
Subject(s)
Female , Humans , Bone Marrow , Bone Marrow Cells , Bone Marrow Transplantation , Cell- and Tissue-Based Therapy , Chemokine CXCL12 , Chemokines , Haplorhini , Immunohistochemistry , Muscle, Skeletal , Muscle, Smooth , Muscle, Striated , Primates , Regeneration , Stem Cells , Tissue EngineeringABSTRACT
Obesity-related metabolic disorders can affect not only systemic health but also brain function. Recent studies have elucidated that amyloid beta deposition cannot satisfactorily explain the development of Alzheimer's disease (AD) and that dysregulation of glucose metabolism is a critical factor for the sporadic onset of non-genetic AD. Identifying the pathophysiology of AD due to changes in brain metabolism is crucial; however, it is limited in measuring changes in brain cognitive function due to metabolic changes in animal models. The touchscreen-based automated battery system, which is more accurate and less invasive than conventional behavioral test tools, is used to assess the cognition of mice with dysregulated metabolism. This system was introduced in humans to evaluate cognitive function and was recently back-translated in monkeys and rodents. We used outbred ICR mice fed on high-fat diet (HFD) and performed the paired associates learning (PAL) test to detect their visual memory and new learning ability loss as well as to assess memory impairment. The behavioral performance of the HFD mice was weaker than that of normal mice in the training but was not significantly associated with motivation. In the PAL test, the average number of trials completed and proportion of correct touches was significantly lower in HFD mice than in normal diet-fed mice. Our results reveal that HFD-induced metabolic dysregulation has detrimental effects on operant learning according to the percentage of correct responses in PAL. These findings establish that HFD-induced metabolic stress may have an effect in accelerating AD-like pathogenesis.