Joseph S. Haynes

Lymphoid Hyperplasia Resulting in Immune Dysregulation Is Caused by Porcine Reproductive and Respiratory Syndrome Virus Infection in Neonatal Pigs

Amid growing evidence that numerous viral infections can produce immunopathology, including nonspecific polyclonal lymphocyte activation, the need to test the direct impact of an infecting virus on the immune system of the host is crucial. This can best be tested in the isolator piglet model in which maternal and other extrinsic influences can be excluded. Therefore, neonatal isolator piglets were colonized with a benign Escherichia coli, or kept germfree, and then inoculated with wild-type porcine reproductive and respiratory syndrome virus (PRRSV) or sham medium. Two weeks after inoculation, serum IgM, IgG, and IgA levels were 30- to 50-, 20- to 80-, and 10- to 20-fold higher, respectively, in animals receiving virus vs sham controls, although <1% was virus specific. PRRSV-infected piglets also had bronchial tree-associated lymph nodes and submandibular lymph nodes that were 5–10 times larger than colonized, sham-inoculated animals. Size-exclusion fast performance liquid chromatography revealed that PRRSV-infected sera contained high-molecular-mass fractions that contained IgG, suggesting the presence of immune complexes. Lesions, inflammatory cell infiltration, glomerular deposits of IgG, IgM, and IgA, and Abs of all three isotypes to basement membrane and vascular endothelium were observed in the kidneys of PRRSV-infected piglets. Furthermore, autoantibodies specific for Golgi Ags and dsDNA could be detected 3–4 wk after viral inoculation. These data demonstrate that PRRSV induces B cell hyperplasia in isolator piglets that leads to immunologic injury and suggests that the isolator piglet model could serve as a useful model to determine the mechanisms of virus-induced immunopathology in this species.

Experimental reproduction of Haemophilus parasuis infection in swine: clinical, bacteriologic, and morphologic findings

Haemophilus parasuis is a common cause of polyserositis and polyarthritis in swine. Little is known about the mucosal and systemic sites of replication and lesions which follow an aerosol exposure to H. parasuis. In this experiment 5–week-old cesarean-derived, colostrum-deprived (CDCD) pigs were inoculated intranasally with an inoculum containing 2 × 109 colony-forming units of H. parasuis. Two principals and one control pig were necropsied at 12, 36, 84, and 108 hours postinoculation (PI) and samples obtained for bacteriologic culture and microscopic examination. Inoculated pigs developed clinical signs of inappetence, reluctance to move, lameness, and a serous nasal discharge. Macroscopic findings included a fibrinous polyserositis and polyarthritis 36 hours PI which became progressively more severe at 84 and 108 hours PI. No lung lesions were grossly visible. Microscopic lesions included a mild purulent rhinitis at each post inoculation interval and fibrinous to fibrinopurulent synovitis and serositis at 36, 84, and 108 hours PI. A focal suppurative bronchopneumonia was observed in one pig examined at 36 hours PI. The nasal cavity and trachea were the only mucosal sites from which H. parasuis was reisolated. Haemophilus parasuis was isolated from the blood and systemic sites at 36, 84, and 108 hours PI. Findings presented indicate that intranasal inoculation of 5-week-old CDCD pigs with H. parasuis results in clinical signs and lesions of polyserositis and polyarthritis typical of field cases and is a useful model for the study of H. parasuis pathogenesis. The results also suggest that H. parasuis initially colonizes the nasal mucosa.

Phagosomal maturation and intracellular survival of Mycobacterium avium subspecies paratuberculosis in J774 cells.

The mechanisms by which Mycobacterium avium subspecies paratuberculosis (M. a. ptb) survives within macrophages are not well characterized. One strategy for intracellular survival developed by Mycobacterium tuberculosis is inhibition of phagosomal maturation. In this study it was hypothesized that M. a. ptb is capable of survival within macrophages by residing within a phagosomal compartment that does not mature into a functional phagolysosome. To test this hypothesis the following objectives were determined. Phagosomal maturation was evaluated by comparison of stage specific markers on the membranes of phagosomes containing live M. a. ptb with those containing killed M. a. ptb, Mycobacterium smegmatis, and zymosan A using immunofluorescent labeling and confocal microscopy. Intracellular survival of live M. a. ptb within J774 macrophages was compared to that of M. smegmatis by direct determination of bacterial viability by differential live/dead staining. The results of this study show that the phagosomes containing live M. a. ptb had increased levels of an early marker (transferrin receptor [TFR]) and decreased levels of a late maturation marker (lysosome associated membrane protein one [Lamp-1]), relative to those containing killed M. a. ptb, M. smegmatis, and zymosan A. Additionally, compared to M. smegmatis, M. a. ptb has enhanced ability to survive within cultured macrophages. These data indicate that M. a. ptb resists intracellular killing by residing within a phagosomal compartment that retains the characteristics of early phagosomes and resists maturation into functional phagolysosome.

Fluorescence In Situ Hybridization Confirms Clearance of Visible Helicobacter spp. Associated with Gastritis in Dogs and Cats

BACKGROUND The results of studies examining the role of Helicobacter spp. in the pathogenesis of canine and feline gastritis are inconclusive. Furthermore, data evaluating the effectiveness of medical therapy for eradication of Helicobacter infection are limited. AIM To detect Helicobacter spp. in mucosal biopsies of dogs and cats diagnosed with gastritis, with fluorescence in situ hybridization (FISH). ANIMALS Three dogs and 2 cats with signs of chronic gastrointestinal disease. METHODS Dogs and cats infected with Helicobacter spp. were treated with triple antimicrobial therapy and fed an elimination diet for 21 days. Helicobacter spp. status in endoscopic (3 dogs, 1 cat) or surgical biopsies (1 cat) of gastric mucosa was compared pre- and posttreatment in each animal by histology, FISH analysis, and polymerase chain reaction (PCR). RESULTS Gastritis of varying severity with intraglandular spiral bacteria was observed in all animals. Pretreatment diagnostic tests confirmed the presence of mucosal Helicobacter spp. in all animals by FISH and histopathology and in 4/5 animals by PCR. Rapid resolution of vomiting episodes was observed in all animals. Gastric biopsies performed after triple therapy revealed clearance of visible Helicobacter spp. by histopathology and negative FISH analysis, as well as PCR in all animals. CONCLUSIONS AND CLINICAL IMPORTANCE Application of FISH to routine biopsy specimens enabled rapid and specific identification of Helicobacter spp. within the gastric mucosa of dogs and cats. Although medical therapy was useful in resolution of clinical signs and clearance of visible Helicobacter spp. in gastric biopsies, gastric inflammation persisted.

Cryptosporidium parvum Initiates Inflammatory Bowel Disease in Germfree T Cell Receptor-α-Deficient Mice

Flora-bearing mice with targeted disruption of T cell receptor (TCR)-alpha or -beta genes spontaneously develop intestinal inflammation with features similar to ulcerative colitis in humans. TCR-alpha-deficient mice maintained germfree or colonized with a limited number of intestinal bacteria failed to develop inflammatory bowel disease (IBD)-like lesions. Evidently, inflammation in these mice does not develop spontaneously or result from a generalized antigenic stimulation, but rather requires induction by a heretofore unidentified specific stimulus. We describe the development of IBD-like lesions in germfree TCR-alpha-deficient mice monoassociated with the protozoan Cryptosporidium parvum. Lesions were seen in distal ileum, cecum, and colon and were most severe in the cecum. A prominent leukocytic infiltrate within the lamina propria was a common characteristic of the lesions observed in the C. parvum-infected germfree TCR-alpha-deficient mice. The leukocytic infiltrate was composed of aggregates of B220+ cells, the majority of which expressed surface IgD (ie, conventional B lymphocytes). It has been proposed that antigenic stimulation by a microorganism(s) is needed to initiate intestinal inflammation in TCR-alpha-deficient mice. Our results indicate that a single microbial species, C. parvum, is capable of triggering the development of IBD-like lesions in germfree TCR-alpha-deficient mice.

Activation of Autophagy and Nucleotide-Binding Domain Leucine-Rich Repeat–Containing-Like Receptor Family, Pyrin Domain–Containing 3 Inflammasome during Leishmania infantum–Associated Glomerulonephritis

Chronic kidney disease is a major contributor to human and companion animal morbidity and mortality. Renal complications are sequelae of canine and human visceral leishmaniasis (VL). Despite the high incidence of infection-mediated glomerulonephritis, little is known about pathogenesis of VL-associated renal disease. Leishmania infantum-infected dogs are a naturally occurring model of VL-associated glomerulonephritis. Membranoproliferative glomerulonephritis type I [24 of 25 (96%)], with interstitial lymphoplasmacytic nephritis [23 of 25 (92%)], and glomerular and interstitial fibrosis [12 of 25 (48%)] were predominant lesions. An ultrastructural evaluation of glomeruli from animals with VL identified mesangial cell proliferation and interposition. Immunohistochemistry demonstrated significant Leishmania antigen, IgG, and C3b deposition in VL dog glomeruli. Asymptomatic and symptomatic dogs had increased glomerular nucleotide-binding domain leucine-rich repeat-containing-like receptor family, pyrin domain containing 3 and autophagosome-associated microtubule-associated protein 1 light chain 3 associated with glomerular lesion severity. Transcriptional analyses from symptomatic dogs confirmed induction of autophagy and inflammasome genes within glomeruli and tubules. On the basis of temporal VL staging, glomerulonephritis was initiated by IgG and complement deposition. This deposition preceded presence of nucleotide-binding domain leucine-rich repeat-containing-like receptor family, pyrin domain containing 3-associated inflammasomes and increased light chain 3 puncta indicative of autophagosomes in glomeruli from dogs with clinical VL and renal failure. These findings indicate potential roles for inflammasome complexes in glomerular damage during VL and autophagy in ensuing cellular responses.

Ischemic myelomalacia due to cartilage emboli associated with chronic diskospondylitis in a group of swine

Rear limb paralysis in market age swine occurs sporadically and can be attributed to a variety of causes, including fractures, spinal abscesses, and ischemic myelomalacia due to cartilage (fibrocartilage) emboli from the nucleus pulposus.11,16 Cartilage emboli resulting in ischemic myelomalacia have been reported in several species, most commonly in adult largeand giant-breed dogs and rarely in swine.4– 6,9,10,14–16 In a recent report, there was a high incidence of rear limb paresis/paralysis in a large group of finishing pigs caused by ischemic myelomalacia due to fibrocartilage emboli in the spinal cord.3 However, no factors were identified in the affected pigs that predisposed them to development of cartilage emboli. In this follow-up report to that case, we describe an additional group of finishing pigs with ischemic myelomalacia caused by cartilage emboli. These pigs also had chronic diskospondylitis. Market weight swine in a production unit with approximately 1,800 grow–finish swine developed a high incidence of acute rear limb paralysis following transportation to market (25/1,800 swine affected).3 These swine were from a high-lean, heavily muscled genetic line that had a rapid growth rate, and they were extremely large (127 kg) when transported. Nine of these paralyzed swine were submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL) for necropsy. All 9 of these swine had ischemic myelomalacia associated with cartilage emboli in spinal cord vessels. However, abnormalities in the spine were not detected, and a predisposing cause for the cartilage emboli was not discovered. Several weeks later, acute rear limb paralysis developed in several market weight swine in additional groups (500 swine/group) from the same production unit. The circumstances of this episode were similar to those of the previous episode of paralysis, occurring after transportation to market. Five paralyzed swine were submitted to the ISU-VDL for necropsy and complete examination of the spinal cord and spine. The spinal cords were removed following dorsal laminectomy, and the spines were sectioned longitudinally with a bandsaw in a dorsoventral vertical plane. Sections of vertebrae, intervertebral disks, and the entire spinal cords from all 5 pigs were fixed in 10% neutral buffered formalin. Vertebrae were demineralized in a 25% aqueous solution of formic acid, and all tissues were embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin. Replicate sections were stained with Alcian blue at pH 2.5 to identify fragments of nucleus pulposus.

Osteopenic disease in growing pigs: diagnostic methods using serum and urine calcium and phosphorus values, parathormone assay, and bone analysis.

This research was performed to evaluate the utility of several serum and urine parameters as well as bone ash and plasma parathormone assay to diagnose and monitor diet-related osteopenia in growing pigs. Five diets were tested as follows: calcium-deficient, phosphorus-replete; moderate-deficiency of calcium and phosphorus; marked deficiency of calcium and phosphorus; calcium replete, phosphorus deficient; and vitamin D deficient. Parameters monitored included serum calcium and phosphorus as well as ratios of urine calcium to creatinine, phosphorus to creatinine, calcium to phosphorus, and percent fractional excretions of calcium and phosphorus. Plasma parathormone (PTH) levels were monitored in 2 of 3 experiments. Osteopenic bone differences at necropsy were evaluated by bone density, percent ash, ash per milliliter bone, calcium per milliliter bone, and phosphorus per milliliter bone. Marked change in urine mineral parameters, especially the calcium-to-phosphorus ratio, typically occurred within 1 to 2 days of treatment and preceded significant change in serum mineral or plasma PTH by 2 to 3 weeks. When monitored, plasma PTH levels were elevated following treatment, which confirms the hyperparathyroid state induced by the test diets. Significant differences in bone mineralization between control and treatment diets at necropsy were generally observed. The results of this study indicate that the analysis of urine minerals offers an early, noninvasive technique to investigate diet-associated osteopenic disease in growing pigs, which can be supported further by bone mineral analysis at postmortem using techniques herein described. Several urine mineral reference intervals for application to field investigations are included. Research into application of similar techniques to evaluate calcium and phosphorus homeostasis in pigs of all ages, including gestating and lactating gilts and sows, appears warranted.

Use of Terbinafine in the Treatment Protocol of Intestinal Cryptococcus neoformans in a Dog

A 2.5 yr old sexually intact male vizsla was admitted to the Iowa State University Veterinary Teaching Hospital for persistent diarrhea, weight loss, and panhypoproteinemia. Examination revealed an emaciated condition and melena. Two masses were palpated in the cranial abdomen. Hematology and serum biochemistry exhibited a regenerative anemia and confirmed the presence of panhypoproteinemia, suggestive of a protein-losing eneteropathy. Distinct areas of thickened intestinal wall and enlarged mesenteric lymph nodes were found on abdominal ultrasound. Cytology from those nodes showed the presence of suspected Cryptococcus spp., and infection was confirmed utilizing a cryptococcal antigen titer. Medical therapy with lipid-complexed amphotericin B and fluconazole was unsuccessful. Two surgical procedures were performed to remove the affected areas of intestine and lymph nodes, but the disease persisted as evidenced by a persistently elevated cryptococcal antigen titer. Terbinafine was prescribed, which resulted in complete resolution of clinical signs and a steadily decreasing cryptococcal antigen titer. Very few cases of intestinal cryptococcosis have been reported. In this case, infection resulted in a protein-losing enteropathy. In addition, this article describes the use of terbinafine in the treatment of intestinal cryptococcal infection in the dog, which has not been previously reported.

Idiopathic Eosinophilic Meningoencephalomyelitis in a Rottweiler Dog

A 6-month-old, female, intact Rottweiler dog was presented to the Iowa State University Veterinary Teaching Hospital for a progressive history of abnormal behavior and generalized ataxia. At necropsy, there was eosinophilic infiltration of the brain and spinal cord, most severe in the medulla oblongata, cerebellum, and cervical spinal cord. Infiltrates of eosinophils were also present in the liver and small intestines. The dog was diagnosed with idiopathic eosinophilic meningoencephalomyelitis based on cerebrospinal fluid analysis, histopathology, and special stains to exclude etiologic agents.