Herp Hematopoietic, Vascular and Immune Systems

Question 1

Describe the vascular anatomy and physiology of reptiles.

Where are their chemoreceptors located? What do they sense that regulates respiration and shunting?

Following a meal in snakes, blood flow increases by how much in what regions?

Answer 1

Vascular anatomy and physiology:

• Chemoreceptors
  
  • Identified in aortae, common carotid artery, and pulmonary artery of turtles
  • Oxygen-sensing cells innervated by 10th cranial nerve
    
    • Sense changes in blood oxygenation and regulate respiration and blood cardiovascular shunting
• Vascular shunting
  
  • Can shunt blood through heart and between great vessels depending on lung function and blood gas levels
  • Renal portal system - caudal blood flows through kidneys via the caudal, hypogastric, or iliac veins
    
    • Drugs injected caudally may be excreted via tubules or cause nephrotoxicity
  • Can shunt blood to extremities or core dependent of temp - decreased or increased vasomotor tone
  • Snakes
    
    • Post prandial blood flow to intestines and liver may increase up to 30% and portal venous flow up to 300%

Question 2

What causes metastatic mineralization in herps?

What bacterial disease may predispose reptiles to thromboembolic disease?

What congenital vascular diseases have been documented?

Aneurysms are common in which species?

Answer 2

Vascular diseases:

• Metabolic
  
  • Metastatic mineralization from hypervitaminosis D or Ca/P imbalance (primary or secondary hyperparathyroidism)
    
    • Vascular mineralization
  • Dystrophic vascular mineralization – may be associated with stress, high lipid diets, hypercholesterolemia, hepatic lipidosis, and low daily activity levels
• Bacterial thromboembolic disease
  
  • Embolic septicemia
    
    • Salmonella in green iguana
  • Severe periodontal disease – lizards with acrodont dentition
• Congenital and developmental
  
  • Congenital aortic stenosis in green iguana
  • Aneurysms – bearded dragons and Burmese pythons
    
    • Etiology unknown

Question 3

Describe the cellular immunity of reptiles - what cells do they have?

What are the immunoglobulins of reptiles and what are their mammalian equivalents?

Answer 3

Hematopoietic system and immunology:

• Cellular immunity
  
  • Specific
    
    • Reptiles have B and T lymphocyte equivalents and subsets (T-helper lymphocytes)
  • Nonspecific
    
    • Mononuclear leucocytes – monocytes, azurophils
    • Granulocytes - heterophils, basophils, eosinophils
• Humeral Immunology and Acute Phase Proteins
  
  • Specific humeral immunity - immunoglobulins
    
    • IgY - equivalent of mammalian IgG but has facets of IgE activity
    • IgM - functions in reptiles as combo of IgM and IgA antibodies in mammals
    • IgE and IgD - not proven to exist in reptiles, subsets of IgY may cover some of their spectrum of functions
  • Nonspecific humeral immunity - interferons, transferrins, lysozymes, complement, and other acute phase proteins
    
    • Plasma protein electrophoresis – accurate measurement of albumin and globulin levels in reptiles
      
      • Fibrinogen – major acute phase protein
      • Plasma preferred sample for PPE
      • Pre-albumin – mostly chelonians
        
        • Many are carrier proteins – thyroxine

Question 4

Describe the hematopoietic diseases of reptiles.

What nematode causes aneurysms and fibrous nodules in the aorta? What species does it affect?

What microfiliarial parasites affect reptiles.

What fluke affects turtles, what is its intermediate host?

What parasites (3) and viruses (1) may cause anemia?

What is the most common causes of neoplasia in reptiles?

What toxicity may result in anemia?

Answer 4

Hematopoietic diseases:

• Ophidascaris papuanus
  
  • Nematode
  • Aneurysms and fibrous nodules in aorta, nodules in peritoneum in Papuan python
• microfilaria
  
  • Family Onchocercidae  Subfamily Dirofilariinae
    
    • Foleyella, Oswaldofilaria
  • Common in lizards but seen in other reptiles
  • Intermediate host - ticks, mites, and mosquitoes
  • Treatment – maintain temp at 95°–99°F for 24-48 hours to kill adult worms
• Trematodes
  
  • Family Spirorchidae
    
    • Digenetic Spirorchid Flukes - Spirorchis, Henotosoma, Vasotrema
  • Common in chelonians
  • Intermediate host – aquatic snail
  • Diagnosis – pulmonary biopsy and squash prep, eggs rarely seen in peripheral blood
  • Lesions - endothelial hyperplasia in major arteries, adults rarely cause clinical disease
    
    • Main lesion - blockage of end arterioles with eggs causing ischemic necrosis
  • Tx – praziquantel, does not prevent clinical disease due to emboli
  • Reduce exposure and infection by controlling aquatic snails
• Other parasites causing anemia
  
  • Ophionyssus natricis (snake mite)
  • Kalicephalus spp (hookworm) – mainly affects snakes
  • Hirudinae (leeches) – anemia with high burden, also vectors for hemogregarines and trypanosomes causing hemolytic anemia
• Viruses causing anemia
  
  • Iridoviridae - erythrocytic metachromicatically staining inclusions, hemolytic anemia
• Neoplasia
  
  • Up to 30.8% of cancers in reptile are lymphoid or other hemopoietic origin
  • Lymphoblasts - rarely found in bloodstream of healthy reptiles
    
    • Moderate to large numbers indicative of leukemia
  • Reported neoplasia
    
    • Leukemia - many species of reptiles
    • Tissue-associated lymphomas/lymphosarcomas
    • Plasma cell tumors
    • Hemangioma – corn snake, RE slider
      
      • May rupture causing massive hemorrhage
  • Treatment
    
    • Lymphoma in green iguana
      
      • Radiation therapy at 10 Gy for cervical mass
      • Chemo - vincristine, cyclophosphamide, and prednisolone, switched to doxorubicin and pred after relapse
      • Remission by day 1008
    • Lymphoma in king cobra
      
      • Week 1 - prednisolone 40 mg/m2 PO q 48 hrs w/ L-Aspargine aminohydrolase 10,000 IU/m2 SC
      • Week 2 - prednisolone (40 mg/m2) PO q 48 hrs w/ vincristine (0.5 mg/m2) IV once
      • Week 3 - prednisolone 40 mg/m2 PO q 48 hrs w/ L-Aspargine aminohydrolase 10,000 IU/m2 SC
      • Regrowth occurred
      • Rescue - prednisolone 40 mg/m2 PO q 48 hrs w/ chlorambucil 2 mg/m2 SC q 24 hrs for 30 days
      • Success with side effects (poor sloughing, anorexia, hypoalbuminemia, hyperglycemia)
• Immune-mediated hematopoietic disease
  
  • Immune-mediated hemolytic anemia
    
    • Reported in Parson’s chameleon
    • Tx with pred and blood transfusion
• Toxic anemia
  
  • Lead or heavy metal toxicity – nonregenerative anemia
    
    • Tx – CaEDTA
      
      • May cause hemolysis or renal injury

Question 5

Describe hemoparasitism in reptiles.

What demographics are most commonly affected?

What is the most serious hemoparasite of reptiles?

What other hemoparasites can cause disease?

Answer 5

MARMS – 152. Hemoparasites

Hemoparasites

• Symbiotes
• Most infections chronic and subclinical
• Indirect life cycle
• Clinical disease of captive-bred indoor animals rare
• Stress may cause clinical disease
• Identification in blood smears frequently an incidental finding
• Immunocompromised, geriatric, and young animals - can cause lethargy, open-mouth breathing, weight loss, hemolytic anemia, splenomegaly, and dehydration
• Diagnosis - microscopic examination of quickly air-dried samples fixed with absolute methanol and stained with Romanowsky-type stain or Giemsa/Wright-Giemsa
• Sporozoites - found in blood cells (erythrocytes, leukocytes, and thrombocytes)
• No effective treatment known for reptiles
  
  • Treatment with iron-dextran and quinidine may be helpful if anemia
• Prognosis – good
• Prevention - quarantine combined with blood smear examinations, control of potential vectors, treatment against ectoparasite transmission hosts
• Plasmodium spp.
  
  • Most serious pathogen
  • Chelonians and lizards
  • Anemia, erythroblastosis, reduced tissue oxygen supply in lizards, severe hemolytic anemia in chelonians
  • Tx - primaquine and chloroquine have been used in chelonians
• Saurocytozoon
  
  • Reported in lizards
  • Considered harmless
• Haemoproteus
  
  • Reported in lizards, turtles, and snakes
  • Generally considered nonpathogenic
  • May cause hemolytic anemia in geckos
• Haemogregarina
  
  • Observed in freshwater turtles, tortoises, the tuatara, some lizards, most snakes, and crocodilians
  • Signs - lethargy, open-mouth breathing, weight loss, and dehydration are uncommon but may be seen in unnatural or aberrant host species or in immune-compromised
  • Anemia in lizards and snakes possible
  • Decrease in parasitemia, but not clearance, noted with atovaquone-proguanil
• Haemolivia
  
  • Encountered in turtles and lizards
  • Usually nonpathogenic
• Hepatozoon
  
  • Found primarily in snakes but also in geckos
  • Snakes - severe consequences on growth and reproduction reported, liver necrosis, inflammation, neurologic signs
• Karyolysus
  
  • Primarily described in squamates
  • Rarely pathogenic
• Hemococcidia, Lainsonia, and Schellackia
  
  • Reported mostly in lizards
  • Schellackia may cause anemia
• Piroplasmids, Sauroplasma, and Serpentoplasma
  
  • Reported in lizards, chelonians, snakes, and chameleons
  • Not associated with clinical signs
• Trypanosoma and Leishmania
  
  • Can infect almost all reptile species
  • Can cause severe parasitemia
  • Infection often subclinical and lifelong
  • Leishmania - found in macrophages, usually detected by culture techniques
  • Trypanosoma - found free in the blood film
• Sauroleishmania
  
  • Found primarily in lizards without pathogenicity
• Microfilaria
  
  • Detected in snakes, lizards, chameleons, and crocodilians
  • Incidental findings without clinical signs even with occlusion of terminal vessels
  • Found free in the blood film

Question 6

Describe the anatomy and physiology of the reptilian immune system.

What is the function of the bone marrow?

Where is the thymus in various species?

Describe the seasonal changes of the thymus and spleen in winter? What effect does this have on the animals?

Describe the GALT. Why are the esophageal tonsils of boids important clinically?

Answer 6

MARMS – 37. Immunopathology

• Cells and Tissues of the Immune System
  
  • Tissue components of the reptile immune system:
    
    • Bone marrow, thymus, spleen (critical)
    • GI, respiratory, urogenital associated lymphoid tissue
    • In some cases, lymph node-like tissues
      
      • Do not have LN
      • Do not form germinal centers (B cell/helper T cell interaction, B cell proliferation, isotype switching, affinity maturation with adaptive immune system).
    • No bursa, but a bursalike lymphoid organ in cloacal wall of some spp.
  • Bone marrow:
    
    • Produces red and white cells
    • Medullary bone
    • Includes carapace and plastron
  • Thymus:
    
    • Bilateral, variable lobulated or nonlobulated gland-like nodules or lobes.
    • Cervical region, extends to base of heart.
    • Adjacent to parathyroid glands, next to ultimobranchial bodies.
    • Lizards and tuatara – nonlobulated.
    • Chelonians and crocodilians – distinct thymic lobules.
      
      • Sea turtles – elongated.
      • Crocs, alligators have elongated thymus on both sides of trachea.
      • Young crocs – may have posterior enlargement of right and left sides.
    • Snakes – spherical, single-lobed, double-lobed thymus immediately anterior to heart.
    • Histo – delimited by a capsule, parenchyma divided into cortex and medulla.
      
      • Small and medium lymphocytes, epithelium.
      • Hassall’s corpuscles may be present.
  • Spleen:
    
    • Produces red and white cells.
    • Histologically similar to other vertebrates.
    • Some reptiles, contiguous with pancreas.
      
      • Aka splenopancreas, normal in many chelonians and some snakes.
  • White pulp of thymus and spleen exhibit seasonal changes.
    
    • Involute during winter.
    • Well developed by spring.
    • Snakes – cortex and medulla of thumus poorly defined during winter.
    • Splenic lymphocytolysis may lead to impaired immune function in winter.
    • Also seasonal variation in GALT.
  • GALT:
    
    • Throughout intestine.
      
      • Most importantly esophagus, ileum, ileo-cecal junction, colon, cloaca.
      • Nonencapsulated aggregates in lamina propria and submucosa.
      • Lymphoid cloaca complex in some reptiles.
    • Lymphoid aggregates also in lung, pancreas, urinary bladder, testes, axillary and inguinal regions.
    • Boid snake esophagus – raised ovoid structures with central cleft = esophageal tonsils.
      
      • Important for detection of viral inclusions in arenavirus inclusion body disease.
      • Also seen in some other snake spp.

Question 7

Describe the innate immunity of reptiles.

What are the three major components?

What proteins are present from the humoral immunity that profide defense against infection?

What are teh acute phase proteins of reptiles?

What are the functions of the various leukocytes of reptiles?

Why are reptile livers dark colored?

What role does melanin play in the innate immune system?

Answer 7

• Innate Immunity:
  
  • First line of defense.
  • Critical in activation and regulation of adaptive immunity.
  • Reptile major components:
    
    • Intact epithelial barriers (skin, GI, resp, urinary).
    • Physiologic parameters (stomach pH, body temp).
    • Humoral and cellular effector components.
  • Humoral innate immunity:
    
    • Lysozyme, antimicrobial peptides, APP, complement, cytokines.
    • Antimicrobial peptides – testudines’, crocodilians, squamata.
      
      • Unlike birds, have cathelicidin, defensins, and hepcidins.
    • Reptile heterophils – granules with cathelicidin like peptides, beta defensing.
      
      • Beta defensing-like peptides and lysozyme also found in reptile eggs.
      • Broad-spectrum antimicrobial/antifungal activity.
      • Also expressed in wounds i.e. when lizards lose tails.
    • Acute phase proteins – early defense, activated by infection, injury, inflammation.
      
      • Produced mostly by liver.
      • SAA, fibrinogen.
    • Complement system – poorly defined in reptiles.
      
      • Only complement protein C3 has been identified.
      • Cytokines also poorly understood – type 2 interferons, certain interleukins, TGF-beta.
  • Cell-mediated immunity:
    
    • Intraepithelial T lymphocytes in intestines, phagocytic ells, basophils, eos, cytotoxic cells, phagocytic B cells.
    • Phagocytosis important role in immune response in reptiles because less influenced by temperature vs adaptive. Also seasonal variations.
    • NK cells – can kill pathogens without being primed and activated. Not ID in reptiles. Believed to have functionally similar cells.
    • Azurophils function similar to mammalian monocytes.
    • Heterophils – functionally similar to birds.
      
      • Primarily phagocytic.
      • Contain granules with cationic proteins, lysozyme, acid phosphatases, acid hydrolases.
        
        • Lack myeloperoxidase, catalase, ALP.
        • Depend on non-oxidative method of killing, do not form pus.
        • Also contain B-defensins.
    • Eosinophils – role in parasitic infection.
    • Basophils degranulate and release histamine when triggered by antigen.
      
      • Release dependent on antigen conc and temperature.
    • Aggregates of melanomacrophages normally in liver.
      
      • With inflammation can proliferate and develop in spleen, other sites.
      • In yellow mud turtle – liver aggregations increase in number and size with age.
        
        • Melanin granules produce free radicals, may be involved in killing bacteria.

Question 8

Describe the adaptive immunity of reptiles.

What is unique about reptilian B cells?

What are some differences of reptilian antibodies?

What immunoglobulins do they have?

Answer 8

• Adaptive Immunity:
  
  • Follows innate immune response.
  • T-cells and B-cells.
    
    • Site of B-cell origin in reptiles unknown.
    • Lymphopoiesis likely in BM, spleen.
    • B-cells have phagocytic activity.
    • T-cells probably arise from lymphocytes in BM that colonize thymus and differentiate.
  • Antibodies.
    
    • Reptile immunoglobulins do not have a hinge, otherwise suspected similar to mammals. Undergo class switch recombination similar to mammals.
    • Both cold and prolonged high temps can affect Ab production (also leukocytes).
    • IgM – probably in all reptiles.
      
      • Variability among spp with which other classes are present, multiple forms.
      • i.e. Sea turtles have 3 forms of IgM and two of IgY.
      • Highest in blood and spleen, also present in lung and intestine.
      • May play role in mucosal immunity.
    • IgY – considered functional equivalent to IgG in mammals.
      
      • Expressed in liver and spleen in a T cell dependent manner.
      • Binds protein G in contrast to IgY of birds (dose not).
    • IgA – IgA-like gene in some spp but not in others.
      
      • Gecko and crocs expressed in high levels in intestines.
        
        • Mucosal immunity.
    • IgD – may play role in mucosal protection.
  • Lymphocytes are affected by stress, temp, season (decreased in fall and winter).
  • Pregnancy in viviparous lizards may impair the immune response and is assoc with splenic involution.
  • Humoral response in reptiles measured with ELISAs.
  • CMI present in reptiles.
    
    • Cutaneous delayed hypersensitivity reaction to tuberculin and alloantigens, allogeneic and xenogeneic graft rejection, phagocytosis, two-way mixed lymphocyte reaction, and graft vs host phenomena.
    • Can be measured by in vitro cell proliferation assays using whole blood or peripheral blood mononuclear cells cultured with phytohemagglutinin, concanavalin A, or soluble chicken egg white lysozyme.

Question 9

Describe reptilian immune responses and disorders.

How does exposure to exogenous estrogens or androgens affect immune function?

How does exposure to endocrine-disrupting chemicals (DDT, PCBs) affect immune function?

How does the reptilian immune system respond to most infectious agents? What about mycobacteria? What about mycoplasma? What about fungi? What about parasites?

Are hypersensitivities reported in reptiles?

Answer 9

• Responses and Disorders:
  
  • Sex Hormone-Induced Immunopathology
    
    • Estrogens, androgens.
    • Lizards and turtles demonstrate immune-related pathologies after estrogen administration.
      
      • E2 exposure induced thymic atrophy and inhibited thymocyte proliferation in Hemidactylus flaviviridis.
      • 17 alpha ethinylestradiol (sewage tx effluent) decreased blood leukocyte and total splenocyte levels in Eceloporus occidentalis.
    • Testosterone induces lymphopenia in turtles.
      
      • High levels disrupt immune function, lead to thymic degeneration.
  • Endocrine-Disrupting Chemicals
    
    • Estrogenic endocrine-disrupting chemicals
      
      • DDT, PCBs.
      • May overstimulate immune response, induce autoimmunity.
      • Alteration of testosterone levels or E:T ratio.
        
        • May increase thymic hematopoiesis, shift to proinflammatory response.
        • Decreased thymic ratio (medulla:cortex), decrease size of Malpighian bodies and lymphocyte sheaths in spleen.
  • Response to Infectious Agents
    
    • Granulomatous
      
      • Heterophilic – heterophils degranulate, elicit MP response.
      • Histiocytic – composed mostly of MP.
    • Mycobacteriosis
      
      • Multinucleated giant cells
      • Heterophils, lymphocytes, plasma cell
      • Central core of necrosis
      • Become walled off with fibrous tissue
    • Mycoplasma
      
      • Typically colonize ciliated epithelial mucosa
      • Epithelial hyperplasia, infiltration of heterophils and histiocytes
      • Lymphoid hyperplasia often found
    • Non-mycobacteria, fungi
      
      • Heterophilic granuloma.
      • O. ophidoiicola – central necrosis surrounded by degenerate heterophils, MP, MGC.
    • Parasites
      
      • Rarely eosinophilic response
      • Granulomatous
  • Immune Complex-Associated Glomerulonephritis
    
    • Acute to chronic
    • Glomerular tuft sclerosis
    • Thickening of capillary membranes due to deposition of hyaline PAS reactive-positive material.
  • Splendore-Hoeppli Reaction
    
    • Eosinophilic clublike material radiating around an infectious agent
    • Common in mammals
    • Reported in Neisseria spp in two iguana spp
  • Hypersensitivity
    
    • Except type 4 HST to mycobacteria, not much otherwise reported.
    • Delated type HST has been used to assess immune function.
    • Acute HST reaction in lungs of a Terrapene Carolina has been reported.
  • Amyloidosis
    
    • Most commonly assoc with chronic infection, deposits from SAA.
    • Amyloid deposition is common in mammals and birds, not reptiles.
    • Amyloid-like plaques have been reported in brains of reptiles with severe neurologic signs.
  • Vaccination
    
    • Humeral response is slow
    • Antigen-specific Ab may not peak until 6-9 weeks following vx
    • Prolonged IgM response before switching to another isotype reported
  • Immunohistochemistry
    
    • Can be helpful in determining tumor type in reptiles
    • Ability of commercially available antibodies to cross-react in reptiles is often unknown, may vary between reptile spp.
    • Vimentin expression in snapping turtle fibroma, did not cross-react in other species.
      
      • Anti-actin antibodies seem more likely to cross-react in reptiles.
      • For gastric neuroendocrine carcinomas in bearded dragons – variety of antibodies have been used.
      • Melan A also shown to cross-react in crocodile lizards, may be useful for melanomas.
      • T-cell markers work well for lymphoid tumors, but B-cell markers do not cross-react.
    • Antibody and species are both important considerations in using IHC for tumor ID in reptiles, and a positive control of the same spp should always be used.