Shigella infection as observed in the experimentally inoculated domestic pig, Sus scrofa domestica.

The domestic pig, Sus scrofa domestica, was investigated as a potential animal model for shigellosis. We examined the effects of pig age, pig breed and antibiotic pretreatment upon Shigella infection. Shigella dysenteriae, and Shigella flexneri (both virulent and avirulent strains) were utilized. Our results indicated that young (4-week-old), conventionally re ared, domestic pigs were routinely, but briefly, colonized (average=3.5+/-2.5 days) following oral or gavage administration ofS. flexneri, as determined by direct rectal cultures. The duration of S. dysenteriae colonization was significantly shorter. Inoculation of younger (2 days) or older (9 weeks) pigs with S. flexneri had no significant effect on infection duration. Similarly, infection of 4-week-old pigs with virulent and avirulent strains of S. flexneri had no effect upon the duration of infection, nor did the use of a swine-passaged S. flexneri isolate. Marked clinical, histopathological (gross and microscopic) and immunoIhistopathological signs of disease were absent in all infections. However, in instances where microscopic histopathological evidence was used to correctly identify infected pigs, tonsillar lesions were the consistently noted criteria. The tonsils are believed to be an important portal of entry for Salmonella choleraesuis, another member of the Enterobacteriaceae and a prevalent pig pathogen. Taken altogether, our results indicate that the domestic pig is unsuitable as a model for shigellosis.

Salt toxicosis in commercial turkeys.

Salt toxicosis was confirmed in a flock of 20,000 thirteen-week-old tom turkeys experiencing an increase in mortality. Clinical signs included polydipsia, diarrhea, ataxia, incoordination, tremors that progressed to depression, sternal and lateral recumbency accompanied by torticollis, and death. Mortality over a 5-day period was 6.7%. Necropsy lesions included pallor and dehydration of pectoral muscles, hepatic congestion, and fluid-filled small and large intestines. Microscopic lesions consisted of bilaterally symmetrical areas of necrosis within the cerebral hemispheres accompanied by vascular congestion and edema, as well as hyalinization of the glomerular capillary walls of the kidney and eosinophilic granular casts in the renal tubules. Average salt concentration in the feed from affected houses with 8.04%.

Decreased egg production in turkeys experimentally infected with eastern equine encephalitis virus or Highlands J virus.

Turkey breeder hens were experimentally infected with strains of eastern equine encephalitis (EEE) virus or Highlands J (HJ) virus previously isolated from turkey hens experiencing decreased egg production. Depression and inappetance were observed on day 1 postexposure (PE) in hens inoculated with either EEE virus or HJ virus, and egg production fell in each virus-inoculated group from approximately 75% to less than 20% within 2-3 days PE. Egg production remained depressed (less than 20%) for 15 days in EEE-virus-inoculated hens and for 7 days in HJ-virus-inoculated hens. EEE virus and HJ virus were recovered from various tissues on days 1-5 PE, and virus was detected in eggs laid on days 2-5 PE. The findings of this study confirm that EEE virus and HJ virus are potential causes of decreased egg production in turkey breeder hens.

Effect of protein restriction during brooding on spontaneous turkey cardiomyopathy.

The effect of early protein restriction on poult performance and mortality due to spontaneous turkey cardiomyopathy were examined in a facility that historically had a high incidence of the condition. Two thousand male turkey poults were divided into two equal subgroups for the first 4 weeks of life: one received standard commercial rations for the first 4 weeks (high-protein subgroup), and the other received rations with a protein content approximately 70% of the first subgroup (low-protein subgroup). Rations were the same after 4 weeks of age (standard commercial rations). At 16 weeks of age, turkeys in the low-protein subgroup weighed an average of 12.32 kilograms (27.1 pounds), whereas turkeys in the high-protein subgroup weighed an average of 12.73 kilograms (28.0 pounds). Total mortality for the low-protein subgroup was 10.1%, whereas total mortality for the high-protein subgroup was 15.7%. Total mortality due to spontaneous turkey cardiomyopathy in the high-protein subgroup was greater than twice that in the low-protein subgroup (10.4% versus 4.6%). These results show that lowering the protein content of the feed in the first 4 weeks significantly reduces mortality due to spontaneous turkey cardiomyopathy, but body weight gain is also reduced.

Egg-production drop in turkeys associated with alphaviruses: eastern equine encephalitis virus and Highlands J virus.

Alphaviruses were isolated from tracheas of turkey breeders in two North Carolina flocks experiencing a severe drop in egg production. Highlands J virus was isolated from one of the breeder flocks, in which production decreased by as much as 72.6% in selected houses over a 48-to-96-hour period. Eastern equine encephalitis virus was isolated from the second breeder flock, which experienced an egg-production drop of 44.5%. Clinical signs in both flocks were similar, with inactivity and the egg-production drop being the only clinical signs observed. Eggs from affected breeders were small and white, and a few were soft-shelled. Sera collected from the flocks 2 to 3 weeks after production began dropping confirmed the presence of antibodies to the viruses recovered. In the first flock, egg production failed to return to above 50%, although heat stress may have played a role in production recovery. The second flock was taken out of production and recycled.

Cerebral encephalomalacia in commercial turkeys.

A flock of 9 1/2-week-old commercial tom turkeys experienced high mortality after consuming a complete feed containing an unidentified toxic substance. Initially, turkeys were found dead. Clinically, the birds were calm and still but became hyperexcitable with noise. A small percentage of birds exhibited torticollis, opisthotonos, circling, ataxia, and blindness. Findings at necropsy and upon microscopic examination were bilaterally symmetrical areas of necrosis of the cerebral hemispheres in the area of the neostriatum that were well demarcated from the surrounding normal neuropil. A feeding trial with the suspect feed in twelve 4-week-old turkey hens induced clinical disease and gross and microscopic brain changes similar to those observed in the field case. Analyses for the following substances in the suspect feed were either negative or within acceptable limits: salt, selenium, furazolidone, monensin, amprolium, 3-nitro-4-hydroxyphenylarsonic acid, aflatoxin, deoxynivalenol, zearalenone, T-2 toxin, ochratoxin, fumonisin, organophosphates, chlorinated hydrocarbons, and carbamates. The toxic component of the feed remains unidentified.

Experimental infection of young turkeys with eastern equine encephalitis virus and highlands J virus.

Depression, somnolence, and increased mortality were observed in 2-week-old turkeys inoculated intramuscularly with either eastern equine encephalitis (EEE) virus or Highlands J (HJ) virus. Mortality rates in EEE virus- and HJ virus-inoculated turkeys were 7/30 (23%) and 9/30 (27%), respectively; no sham-inoculated controls died. Both EEE virus- and HJ virus-inoculated turkeys developed viremia that lasted 2 days; peak mean titers were 5.5 and 3.2 log10 plaque-forming units per ml of blood, respectively. Pathologic changes in both EEE virus- and HJ virus-inoculated turkeys consisted primarily of multifocal necrosis in the heart, kidney, and pancreas, and lymphoid necrosis and depletion in the thymus, spleen, and bursa of Fabricius. The findings indicate that EEE virus and HJ virus are pathogenic for young turkeys.

High mortality of domestic turkeys associated with Highlands J virus and eastern equine encephalitis virus infections.

High mortality occurred in two flocks of commercial turkey hens placed in southern North Carolina in fall 1991. Daily mortality peaked at 3.19% in Flock 1 and 3.79% in Flock 2. Clinical signs included restlessness, somnolence, vocalization, and acute death. Gross lesions included atrophy of the bursa of Fabricius, thymus, and spleen, and watery intestinal contents. Microscopic changes included moderate to marked lymphocyte necrosis and depletion in the bursa, thymus, and spleen, widely scattered necrosis of pancreatic acinar cells, and mild villous atrophy and fusion in the jejunum and ileum with cuboidal to low columnar epithelial cells covering the villous tips. In Flock 1, at 27 days of age, reovirus and picornavirus particles were detected in the feces. One week later, togavirus-like particles were observed in fecal contents, and two of seven serum samples showed seroconversion to Highlands J virus. Eleven days later, five of six serum samples were positive for antibodies against Highlands J virus, with a fourfold increase in the geometric mean titer. In Flock 2, seroconversion to eastern equine encephalitis virus was observed in four of 10 serum samples 11 days after the onset of clinical signs. Based on the above observations, it is suspected that these alphaviruses were the cause of the clinical syndrome.

Immunohistochemical demonstration of influenza A nucleoprotein in lungs of turkeys with natural and experimental influenza respiratory disease.

Influenza A virus (H1N1) and several bacteria were recovered from lungs of turkey breeder hens during a respiratory disease outbreak. Influenza A nucleoprotein was detected in the pneumonic lung tissue within macrophages and, rarely, in atrial-lining epithelium. Inconsistent recovery of pathogenic bacteria suggested that death in some turkeys resulted from acute primary viral pneumonia. In an experimental study, the gross and histologic lesions confirmed the respiratory pathogenicity of the influenza virus. The presence of intranuclear and intracytoplasmic influenza A nucleoprotein within macrophages and atrial lining epithelium of the lung, respiratory epithelium of the trachea and hypertrophied epithelial cells of the airsacs verified influenza virus replication in the respiratory system. However, the absence of mortality may suggest that secondary factors, such as bacteria, may modify the disease in natural outbreaks.

Vaccination of turkeys with cell-free culture filtrate of Pasteurella multocida: effects of dilution, iron chelation, and heterologous challenge.

Two experiments were done to further define cell-free culture filtrate (CCF) from Pasteurella multocida and its endotoxin content in protecting turkeys against challenge. In the first experiment, the greater-than-30,000-molecular-weight fraction of P. multocida strain R44/6 (serotype 3/4/9/12) CCF was used in 10-fold dilutions given by air-sac inoculation or aerosol to vaccinate turkeys, which were subsequently challenged with either homologous (P-1059, serotype 3) or heterologous (X-73, serotype 1) strains. Endotoxin content of the CCF fraction was high. Compared with positive controls given either live Clemson University vaccine or a commercial bacterin, homologous protection was provided by undiluted CCF and 1:10 dilutions of CCF, but there was no heterologous protection. In the second experiment, CCF of strain R44/6 in regular and iron-limiting media and CCF of strain FC127B (serotype 1/4) were used alone or in combination to vaccinate turkeys, which were challenged as in the first experiment. Homologous but not heterologous protection occurred, even though growth of strain R44/6 in iron-limiting media reduced endotoxin content of CCF by approximately 93%. These results indicate that endotoxin levels of less than 10% but greater than 1% of those in CCF from regular media are sufficient to induce protection in turkeys against homologous challenge but that CCF from either regular or iron-limiting medium does not provide protection against heterologous challenge.

Breeder turkey hens seropositive and culture-negative for Mycoplasma synoviae.

Four flocks of clinically normal turkey breeder hens were shown to have suspect and positive Mycoplasma synoviae (MS) hemagglutination-inhibition (HI), enzyme-linked immunosorbent assay, and, in some cases, serum plate agglutination serology in the absence of MS isolation. In all cases, HI serology for Mycoplasma gallisepticum (MG) and M. meleagridis was negative. Acholeplasma laidlawii was isolated from some hens in each of these MS-seropositive culture-negative flocks. Immunoblotting was used to help determine if this positive MS serology was a result of cross-reactive antibodies to A. laidlawii or to some other Mycoplasma species. When sera from two of the flocks were reacted with MS antigen in immunoblotting, a strong and characteristic MS immunoblot profile was seen. Immunoblotting gave no evidence of a strong antibody response to A. laidlawii, M. iowae, or MG. This suggests the presence (or earlier presence) of MS in these flocks that is difficult to isolate by routine methods. Furthermore, this work shows that immunoblotting can be an important tool in the diagnosis of poultry diseases.

Vaccination of turkeys with cell-free culture filtrate of Pasteurella multocida: effects of ultrafiltration and endotoxin removal.

Cell-free culture filtrate (CCF) of Pasteurella multocida strain R44/6 (serotype 3/4/9/12) was fractionated by ultrafiltration into fractions of less than 10,000, greater than 10,000, greater than 30,000, and 10,000 to 30,000 molecular weight (MW). The less-than-10,000-MW fraction contained little endotoxin comparable to bacteriologic medium; the 10,000-to-30,000-MW fraction had a moderate amount of endotoxin, whereas the greater-than-10,000- and greater-than-30,000-MW fractions contained high levels of endotoxin. Following ultrafiltration, each fraction, except the less-than-10,000-MW fraction, was divided into two equal parts, and endotoxin was removed from one part. Turkeys were vaccinated with the various MW fractions of CCF, with and without endotoxin, via the air sacs at 6 and 9 weeks of age and compared with negative controls given bacteriologic medium and positive controls vaccinated with a commercial bacterin. Before oral challenge with strain P-1059 (serotype 3) at 12 weeks of age, antibody titers were detected only in positive control turkeys. Protection against challenge, as measured by post-challenge mortality and body-weight gain, was provided by the greater-than-10,000-, greater-than-30,000-, and 10,000-to-30,000-MW fractions containing endotoxin and the commercial bacterin. Turkeys that had been vaccinated with bacteriologic medium and the four different fractions without endotoxin were not protected. Results indicated that endotoxin in CCF of P. multocida is critical in protecting turkeys from pasteurellosis.

Acute airsacculitis in untreated and cyclophosphamide-pretreated broiler chickens inoculated with Escherichia coli or Escherichia coli cell-free culture filtrate.

Ninety commercial broiler chickens were divided into three equal groups; 30 were injected with brain-heart-infusion broth into the cranial thoracic air sacs (controls), 30 were similarly inoculated with a culture of Escherichia coli, and 30 were similarly inoculated with E. coli cell-free culture filtrate. Birds were examined from 0 to 6 hours post-inoculation. E. coli-inoculated and cell-free culture filtrate-inoculated chickens reacted similarly, with exudation of heterophils into the air sac. Microscopically, heterophils were present in low numbers perivascularly 0.5 hour after inoculation and became more numerous by 3 hours post-inoculation. By 6 hours post-inoculation, there was severe swelling of air sac epithelial cells and thickening of the air sac by proteinaceous fluid and heterophils. Ultrastructurally, air sac epithelial cells were swollen and vacuolated, and interdigitating processes were separated. Histologically and ultrastructurally, all features in control chickens were normal, with only rare heterophils in the air sac interstitium. In E. coli-inoculated and cell-free culture filtrate-inoculated chickens, cell counts (predominantly heterophils) in air sac lavage fluids increased markedly at 3 and 6 hours, with only slight increases in counts from lavages of controls. Heteropenia was observed in E. coli-inoculated chickens, whereas heterophilia was observed in cell-free filtrate chickens and controls. Ninety additional chickens were pretreated with cyclophosphamide, subdivided into three equal groups, and inoculated and examined similarly as above. Cyclophosphamide pretreatment reduced inflammatory changes in air sacs, lowered cell numbers in lavage fluids, and abolished hematologic changes; however, it did not prevent epithelial cell changes. These results indicate that cell-free culture filtrate of E. coli induces changes similar to those induced by cultures of E. coli.

Marek's disease virus isolates with unusual tropism and virulence for ocular tissues: clinical findings, challenge studies and pathological features.

Outbreaks of Marek's disease (MD) were diagnosed in two flocks from the same company. Clinical signs, mainly blindness (>90%), but also depression, mild paralysis, and 11 to 12% mortality by 20 weeks of age were observed. MD virus, serotype 1 was isolated. The isolates were designated NC-1 (flock 1) and NC-2 (flock 2). Challenge experiments were conducted with these isolates and with two reference MD virus strains (JM/102W and Md5) in unvaccinated, turkey herpesvirus- (HVT) vaccinated and bivalent- (HVT and SB-1) vaccinated chickens. Blindness, gross ocular lesions and tumour formation were observed in a high proportion of all groups challenged with NC-1 and NC-2 when compared with chickens challenged with JM/102W and Md5. In chickens challenged with isolates NC-1 and NC-2, corneal changes included oedema, midstromal cellular infiltration consisting of macrophages, lymphocytes, plasma cells and lesser numbers of heterophils, collagen degeneration and keratic precipitates consisting primarily of macrophages covering the central endothelium. Eosinophilic intranuclear inclusion bodies were present in mononuclear cells infiltrating the cornea. Changes in the uveal tract consisted of inflammatory cell infiltrates similar to those present in the cornea. Retinal lesions included disruption of the retinal pigmented epithelium, inflammatory cell infiltration in the subretinal space, photoreceptor degeneration and in severely affected eyes, necrosis of retinal cellular elements. Pecten changes consisted of necrosis and mononuclear cell infiltration. Intranuclear inclusion bodies were abundantly present in cells of the retina's ganglion and inner nuclear cell layers. The unusual clinical manifestation of MD, the unusual tropism and virulence of NC-1 and NC-2 for ocular tissues and the incomplete protection afforded by conventional vaccination suggest that these isolates may be new pathotypes.

Non-surgical cannulation of the vena cava for chronic blood collection in mature swine.

A nonsurgical cannulation technique for blood collections from mature swine was evaluated. Primiparous Yorkshire-Landrace sows (n = 6) received an indwelling jugular vein cannulae for 7 days duration. Recannulation was performed at monthly intervals for a total of 14 months. During cannulation, sows were restrained in a standing position using a rope snout snare. A 12-gauge by 10 cm needle was inserted into the jugular vein. Sterilized polyvinyl chloride tubing was advanced through the needle into the vein and a blunted 18-gauge needle and attached intermittent injection hub was inserted into the free end of the tubing. Surgical tape was used to form a butterfly on the tubing by suturing the tape to the animals' skin. Foam padding, livestock cement, and elastic tape helped to keep the tubing in position. Problems with cannulae patency and maintenance were few. No behavioral problems or systemic signs of illness were noted and necropsy examinations performed after the final cannulation revealed few abnormalities associated with chronic intermittent cannulation. This technique provides a safe, quick, effective means for multiple and repeated cannulae placement for blood collection from mature swine with minimal effects on the animal and without the risks associated with surgical techniques.

Vaccination of turkeys with cell-free culture filtrate of Pasteurella multocida.

Turkeys given cell-free culture filtrate (CCF) of Pasteurella multocida strain R44/6 orally, via air sacs, or subcutaneously mixed 1:1 with incomplete Freund's adjuvant (IFA) at 6 and 9.5 weeks of age were compared with negative controls given bacteriologic medium and positive controls vaccinated with a commercial bacterin. At 13 weeks of age, serum antibody titers to P. multocida were detectable only in turkeys given CCF in IFA (low titers) and positive control turkeys (high titers), at which time turkeys were challenged orally with either the homologous strain or strain P-1059. Protection against challenge with strain R44/6 was provided by the commercial bacterin, CCF in IFA, and CCF given via air sacs. When turkeys were challenged with strain P-1059, protection was superior in turkeys given CCF via air sacs, intermediate in turkeys given commercial bacterin or CCF in IFA, and absent in negative control turkeys and turkeys given CCF orally. These results indicate CCF is an effective immunogen when administered via the lower respiratory tract for protecting turkeys against pasteurellosis.

Acute airsacculitis in turkeys inoculated with cell-free culture filtrate of Pasteurella multocida.

Twenty-six female and 26 male turkeys, inoculated into the caudal thoracic air sacs with cell-free culture filtrate of Pasteurella multocida strain R44/6, were examined from 0 to 6 hours post-inoculation and compared with 26 female and 26 male sham-inoculated control turkeys given brain-heart-infusion broth. The air sac reacted rapidly with exudation of heterophils. Microscopically, low numbers of heterophils were present within air sac blood vessels and also perivascularly by 0.5 hour after inoculation. These became more numerous by 1.5 and 3 hours post-inoculation. By 6 hours post-inoculation, there was severe swelling of air sac epithelial and mesothelial cells and thickening of the air sac by proteinaceous fluid and heterophils. Ultrastructurally, mesothelial and air sac epithelial cells were vacuolated, and interdigitating processes of epithelial cells were separated. Microscopically, in control turkeys, rare heterophils were present perivascularly at 1.5, 3, and 6 hours after inoculation. Ultrastructurally, all features were normal. In turkeys given cell-free culture filtrate, total cell counts in air sac lavage fluids increased markedly by 3 hours post-inoculation in which heterophils predominated (greater than 97%). There were only slight increases in cell counts of air sac lavages from control turkeys. The circulating blood heterophil cell count dropped transiently at 1.5 hours post-inoculation, followed by a return to normal 3 hours after inoculation, and by heterophilia by 6 hours post-inoculation in turkeys given either cell-free culture filtrate or brain-heart-infusion broth. These results indicate cell-free culture filtrate of P. multocida induces hematologic, cytologic, and morphologic changes indistinguishable from those induced by cultures of P. multocida.

Establishment and characterization of a chicken mononuclear cell line.

A new chicken mononuclear cell line (MQ-NCSU) has been established. The starting material used to initiate this cell line was a transformed spleen from a female Dekalb XL chicken which had been experimentally challenged with the JM/102W strain of the Marek's disease virus. After homogenization, a single cell suspension of splenic cells was cultured using L.M. Hahn medium supplemented with 10 microM 2-mercaptoethanol. Under these culture conditions, a rapidly proliferating cell was observed and then expanded after performing limiting dilution cultures. These cells were moderately adherent and phagocytic for sheep red blood cells and Salmonella typhimurium. When tested against a panel of monoclonal antibodies (mAb) using the flow cytometry, MQ-NCSU cells stained readily with anti-chicken monocyte specific (K-1) mAb but did not stain with mAb detecting T-helper, T-cytotoxic/suppressor, and NK cells. MQ-NCSU cells expressed very high levels of Ia antigens and transferrin receptors. In addition, cell-free supernatant obtained from MQ-NCSU culture contained a factor which exhibited cytolytic activity against tumor cell targets. Based on their cultural, morphological, and functional characteristics and mAb reactivity profile, we conclude that MQ-NCSU cell line represents a malignantly-transformed cell which shares features characteristic of cells of the mononuclear phagocyte lineage.

Acute airsacculitis in turkeys inoculated with phorbol myristate acetate.

Phorbol myristate acetate (PMA), which induces acute pulmonary injury in mammals, induced acute airsacculitis in turkeys after intra-airsac inoculation of 0.1 mg/kg. Grossly, air sacs contained multifocal to diffuse hemorrhage and edema at postinoculation hours (PIH) 3 and 6. Microscopically, there was multifocal congestion and small thrombocyte aggregates within small blood vessels by PIH 0.5, with a few vessels containing small numbers of marginating heterophils. By PIH 1.5, thrombocyte aggregates were larger and more numerous, and moderate numbers of heterophils were located perivascularly. Erythrocytes and proteinaceous fluid were in air sac interstitium. By PIH 3 and 6, hemorrhage and exudation of proteinaceous fluid had increased, in some instances severely distending the air sac. Ultrastructurally, changes resulting from PMA-induced injury were thrombocyte aggregation and degeneration, air sac epithelial cell vacuolation with separation of interdigitating cell processes, and endothelial cell vacuolar degeneration with loss of vascular integrity. Air sac lavage fluids had mildly increased total cell counts by PIH 1.5, but values returned to baseline by the end of the experiment, indicating lack of cell exudation into the air sac lumen. Circulating leukocyte changes included transient lymphopenia at PIH 3 and marked heterophilia at PIH 6. These results indicate that thrombocytes and/or heterophils are central to the pathogenesis of injury induced in air sacs by PMA and that the air sac responds differently to PMA than to pathogenic bacteria.

Blood clearance of radiolabeled gold colloid by the turkey mononuclear phagocytic system.

Radiolabeled gold colloid (198Au), which has been used to assess particle clearance in mammalian species, was used to assess blood-borne particle clearance in turkeys. When turkeys 16 weeks of age were injected intravenously with these particles, there was greater than a 98% decrease in blood gamma emission from 1 minute to 6 minutes postinjection. Uptake of particles was predominantly hepatic with minor uptake by the spleen and bone marrow. Negligible uptake was observed in lung, kidney, and skeletal muscle. Autoradiography demonstrated particles within Kupffer cells of the liver, periarteriolar macrophages of the spleen, and bone-marrow macrophages. Particles could not be demonstrated within the lung or kidney. The mononuclear phagocyte system responsible for blood particle clearance in turkeys is therefore located predominantly within the liver, spleen, and bone marrow and is similar to that of rats, mice, rabbits, and dogs. Pulmonary intravascular macrophages, which have recently been described in ruminants and pigs, are not apparent in this species.