Veterinary Medicine Commons^™

Immunocompromise In Gnotobiotic Pigs Induced By Verotoxin-Producing Escherichia Coli (O111:Nm), Jane Christopher-Hennings, Joann A. Willgohs, David H. Francis, Usha A. K. Raman, Rodney A. Moxley, David J. Hurley

School of Veterinary and Biomedical Sciences: Faculty Publications

A verotoxin-producing Escherichia coli serotype O111:NM strain (strain 10049; verotoxin 1 positive) persistently infected experimentally inoculated gnotobiotic pigs, causing attaching-effacing intestinal lesions and chronic diarrhea. Experiments were performed to determine whether persistent infection might be associated with immunocompromise of the host by this organism. Pigs inoculated with this strain had a significant reduction in peripheral blood lymphocytes and lower antibody titers to sheep erythrocytes compared with control pigs. Compared with pigs given a verotoxin-negative pathogenic strain of the same serotype (O111:NM, strain 2430), pigs inoculated with the verotoxin-positive strain had lower peripheral lymphocyte counts and proliferative responses to concanavalin A, …

Rapid Assessment Of Drug Susceptibilities Of Mycobacterium Tuberculosis By Means Of Luciferase Reporter Phages, William R. Jacobs Jr., Raul G. Barletta, Rupa Udani, John Chan, Gary Kalkut, Gabriel Sosne, Tobias Kieser, Gary J. Sarkis, Graham F. Hatfull, Barry R. Bloom

School of Veterinary and Biomedical Sciences: Faculty Publications

Effective chemotherapy of tuberculosis requires rapid assessment of drug sensitivity because of the emergence of multidrug-resistant Mycobacterium tuberculosis. Drug susceptibility was assessed by a simple method based on the efficient production of photons by viable mycobacteria infected with specific reporter phages expressing the firefly luciferase gene. Light production was dependent on phage infection, expression of the luciferase gene, and the level of cellular adenosine triphosphate. Signals could be detected within minutes after infection of virulent M. tuberculosis with reporter phages. Culture of conventional strains with antituberculosis drugs, including isoniazid or rifampicin, resulted in extinction of light production. In contrast, …

Detection Of Serum Antibody Responses In Cattle With Natural Or Experimental Neospora Infections, Patricia Conrad, Karen Sverlow, Mark Anderson, Joan Rowe, Robert Bondurant, Gwen Tuter, Richard Breitmeyer, California Department Of Food And Agriculture, Animal Health Branch, Mark Thurmond, Alex Ardans, J. P. Dubey, Gerarld Duhamel, Bradd Barr

School of Veterinary and Biomedical Sciences: Faculty Publications

Parasite-specific antibody responses were detected using an indirect fluorescent antibody (IFA) test in cattle that were naturally or experimentally infected with Neospora parasites. The test was developed using Neospora tachyzoites isolated from an aborted bovine fetus and grown in bovine cell cultures (isolate BPA1). In all cases, infections were confirmed by the identification of Neospora tachyzoites and/or bradyzoite cysts in fetal or calf tissues using an immunoperoxidase test procedure. Fifty-five naturally infected cows that aborted Neospora-infected fetuses had titers of 320-5,120 at the time of abortion. The titer of 6 cows that were serologically monitored over a prolonged period decreased …

Localization Of Cis-Acting Sequences In The Latency-Related Promoter Of Bovine Herpesvirus 1 Which Are Regulated By Neuronal Cell Type Factors And Immediate Early Genes, A. C. Bratanich, Clinton J. Jones

School of Veterinary and Biomedical Sciences: Faculty Publications

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in sensory ganglionic neurons of cattle. During a latent infection, a single latency-related (LR) transcript is expressed. This observation suggested that DNA sequences in the LR promoter are positively regulated by neural cell type factors. The regulation of the LR gene was examined in neural cells as well as nonneural cells in transient assays. A 258-bp XbaI-SphI fragment from the LR promoter cis activated the herpes simplex virus type 1 thymidine kinase promoter in rat pheochromocytoma (PC-12) cells and differentiated human (HCNIA) neurons. In contrast, cis activation was not …

Diarrhea Associated With Enterococcus Durans In Calves, Douglas G. Rogers, David H. Zeman, E. Denis Erickson

School of Veterinary and Biomedical Sciences: Faculty Publications

Enterococci in the alimentary tract of animals are believed to be commensal bacteria.3 However, Enterococcus durans has been associated with diarrhea in foals, piglets, and puppies. The possibility that E. durans is associated with diarrhea in calves has not been established, although grampositive cocci adhered to villi, occasionally together with Cryptosporidia, have been seen histologically in the small intestine of diarrheic calves. This report describes the association of E. durans and diarrhea in 4 calves.

A Novel Transposon Trap For Mycobacteria: Isolation And Characterization Of Is1096, Jeffrey D. Cirillo, Raul G. Barletta, Barry R. Bloom, William R. Jacobs Jr.

School of Veterinary and Biomedical Sciences: Faculty Publications

In the course of developing strategies to obtain a mutation in the aspartate semialdehyde dehydrogenase (asd) gene of Mycobacterium smegmatis, an efficient transposon trap was constructed which may be generally useful for the identification of transposable elements in mycobacteria. A DNA fragment containing the asd gene was replaced with an aminoglycoside phosphotransferase gene (aph) to generate a Δasd::aph allele. Attempts to replace the wild-type asd gene with the Δasd::aph allele were unsuccessful, suggesting that this deletion was lethal to the growth of M. smegmatis. The plasmid, pYUB215, which …

Actinobacillus Pleuropneumoniae-Induced Thymic Lesions In Mice And Pigs, D.L. Stine, M.J. Huether, Rodney A. Moxley, Subramaniam Srikumaran

School of Veterinary and Biomedical Sciences: Faculty Publications

Actinobacillus pkuropneumoniae produces several hemolysins/cytotoxins that may be important in the pathogenesis of acute lesions. Little is known, however, about the role of these virulence factors in chronic disease or the carrier state. We investigated the effects of live bacterial infection and transthoracic injection of a sterile culture supernatant on primary lymphoid organs and lymphocyte populations. Transthoracic inoculation of mice or intranasal inoculation of pigs with virulent A. pleuropneumoniae serotypes 1 and 7 induced thymic cortical lymphoid necrosis. These lesions were reproduced in mice by transthoracic injection of a concentrated sterile culture supernatant. The cytotoxic effect of this culture supernatant …

Cells That Express All Five Proteins Of Vesicular Stomatitis Virus From Cloned Cdnas Support Replication, Assembly, And Budding Of Defective Interfering Particles, Asit K. Pattnaik, Gail W. Wertz

School of Veterinary and Biomedical Sciences: Faculty Publications

An alternative approach to structurefunction analysis of vesicular stomatitis virus (VSV) gene products and their interactions with one another during each phase of the viral life cycle is described. We showed previously by using the vaccinia viruslT7 RNA polymerase expression system that when cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with defective interfering (DI) particles, rapid and efflicient replication and amplification of DI particle RNA occurred. Here, we demonstrate that all five VSV proteins can be expressed simultaneously when cells are cotransfected with plasmids containing the matrix protein …

Monoclonal Antibodies To The Fusion Protein Of Bovine Respiratory Syncytial Virus, Kent M. Mulkey, Gary A. Anderson

School of Veterinary and Biomedical Sciences: Faculty Publications

Five monoclonal antibodies specific for bovine respiratory syncytial virus were characterized by Western immunoblotting, radioimmunoprecipitation, and epitope mapping assays. The monoclonal antibodies were found to be specific for the fusion protein, and there were at least two antigen binding sites, one of which was neutralizing.

Effects Of Ethanol Extraction And Heat Treatment Of Soybean Flakes On Function And Morphology Of Pig Intestine, J. D. Hancock, E. R. Peo Jr., A. J. Lewis, Rodney A. Moxley

School of Veterinary and Biomedical Sciences: Faculty Publications

Digesta and tissue samples were collected from the intestinal tracts of 27 pigs to determine the relationship between intestinal morphology and the utilization of nutrients from soybean flakes. Soybean flake treatments were under-, intermediate- and overprocessed (Le., 5, 20 and 60 min of autoclaving) either without extraction or with heating before or after extraction with a 55% ethanol-water mixture. Final BW was greatest (P< .001) for pigs fed soybean flakes given 20 min of heat treatment. There was a trend (P < .09) for plasma lysine concentrations to be reduced when the unextracted soybean flakes were over-processed (60 min of heat). Differences in the flow rate of DM and N through the ileum and colon reflected differences in DM and N intake, rather than differences in intestinal function. The soybean flake treatments had no effect (P > .OS) on pH of the contents of the stomach, duodenum, ileum or colon. The ethanol extraction process increased (P < .001) N digestibility of the soybean flakes, especially when the soybean flakes were underprocessed (interaction, P < .02). Villus size (area, height and perimeter length) tended to be greater in pigs fed the soybean flakes heated after extraction and(or) exposed to the intermediate level of heat treatment. Indicators of villus shape (villus area/ villus height) and proliferative activity (crypt depth and villus heigh/crypt depth) were not affected by soybean flake treatment (P > .OS). Ethanol extraction and heat treatment affected the utilization of nutrients …

Replication And Amplification Of Defective Interfering Particle Rn As Of Vesicular Stomatitis Virus In Cells Expressing Viral Proteins From Vectors Containing Cloned Cdnas, Asit K. Pattnaik, Gail W. Wertz

School of Veterinary and Biomedical Sciences: Faculty Publications

Replication and amplification of RNA genomes of defective interfering (DI) particles of vesicular stomatitis virus (VSV) depend on the expression of viral proteins and have untD now been attained only in ceUs coinfected with helper VSV. In the work described in this report, we used a recombinant vaccinia virus-T7 RNA polymerase expression system to synthesize individual VSV proteins in cells transfected with plasmid DNAs that contain cDNA copies of the VSV genes downstream of the T7 RNA polymerase promoter. In this way, we were able to examine the ability of VSV proteins, individually and in combination, to support DI particle …

Analysis Of The Transcriptional Promoter Which Regulates The Latency-Related Transcript Of Bovine Herpesvirus 1, Clinton J. Jones, G. Delhon, A. Bratanich, G. Kutish, D. Rock

School of Veterinary and Biomedical Sciences: Faculty Publications

As a transcriptional promoter in primary cultures of sensory ganglionic neurons, DNA sequences near the 5' terminus of the latency-related (LR) gene of bovine herpesvirus 1 were at least 10 times more efficient than the simian virus 40 early promoter-enhancer. In contrast, as a promoter in bovine, rodent, or monkey cells, the LR promoter was approximately six times less efficient than the simian virus 40 early promoter-enhancer. The LR promoter had strict orientation preferences in neurons and all other mammalian cell lines tested. Removal of a 146-base-pair XhoI fragment from the LR promoter resulted in stimulation of LR promoter …

Efficacy Of A Cell Extract From Actinobacillus (Haemophilus) Pleuropneumoniae Serotype 1 Against Disease In Swine, P. J. Fedorka-Cray, M.J. Huether, D.L. Stine, G. A. Anderson

School of Veterinary and Biomedical Sciences: Faculty Publications

We partially characterized a cell extract (CE) from Actinobacillus pleuropneumoniae serotype 1 and used the CE to test the efficacy of secreted proteins against disease. Secreted products from 4-h culture supernatants were precipitated with 20% polyethylene glycol. Analysis of the CE indicated the presence of protein, endotoxin, and carbohydrate. Hemolytic activity to bovine erythrocytes and cytotoxic activity to porcine mononuclear leukocytes was also demonstrated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the CE from a 4-h culture showed a major band at 110 kilodaltons (kDa), while a CE of a 26-h culture indicated the presence of a number of additional …

Edema Disease-Like Brain Lesions In Gnotobiotic Piglets Infected With Escherichia Coli Serotype 0157:H7, David H. Francis, Rodney A. Moxley, Cecile Y. Andraos

School of Veterinary and Biomedical Sciences: Faculty Publications

Gnotobiotic piglets inoculated with Escherichia coli serotype 0157:H7 strains that produced Shiga-like toxin II developed brain lesions similar to those observed in edema disease of swine, including arteriolar necrosis and malacia. Loss of ability to produce Shiga-like toxin II resulted in loss of ability to cause brain lesions.

Impairment Of Melibiose Utilization In Streptococcus Mutans Serotype C Gtfa Mutants, Raul G. Barletta, Roy Curtiss Iii

School of Veterinary and Biomedical Sciences: Faculty Publications

The Streptococcus mutans serotype c gtfA gene encodes a 55-kilodalton sucrose-hydrolyzing enzyme. Analysis of S. mutans gtfA mutants revealed that the mutant strains were specifically impaired in the ability to use melibiose as a sole carbon source. S. mutans gafA mutant strains synthesized less α-galactosidase activity inducible by raffinose than wild-type strains. Melibiose (an inducer in wild-type strains) failed to induce significant levels of a-galactosidase in the mutant strains. We hypothesize that melibiose use by . mutans requires the interaction of the GtfA enzyme, or another gene product under the control of the gtfA promoter, with other gene product(s) involved …

Analysis Of The Virulence Of Streptococcus Mutans Serotype C Gtfa Mutants In The Rat Model System, Raul G. Barletta, Suzanne M. Michalek, Roy Curtis Iii

School of Veterinary and Biomedical Sciences: Faculty Publications

The Streptococcus mutans serotype c gtfA gene encodes a 55-kilodalton protein which catalyzes the synthesis of a small glucan (1.5 kilodaltons) from sucrose (J. P. Robeson, R. G. Barletta, and R. Curtiss III, J. Bacteriol. 153:211-221, 1983). To investigate the role of the GtfA enzyme in virulence, we constructed S. mutans gtfA mutants from three cariogenic serotype c strains. A plasmid that carried an erythromycin resistance determinant and an internal fragment of the gtfA gene but that was unable to replicate in streptococci was used to transform S. mutans. The erythromycin-resistant transformants carried a partial duplication of the internal …

Improved Selective Medium For The Isolation Of Treponema Hyodysenteriae, Robert A. Kunkle, J. M. Kinyon

School of Veterinary and Biomedical Sciences: Faculty Publications

An agar medium with improved selection for Treponema hyodysenteriae was developed. Cultures of T. hyodysenteriae and T. innocens, feces from 11 clinically normal pigs, and colonic contents from 6 pigs with gross lesions consistent with swine dysentery were diluted in phosphate-buffered saline and plated on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) with 5% citrated bovine blood (TSA), TSA with 400 ,ug of spectinomycin per ml (TSA-S400), TSA-S400 with 25 ,ug each of colistin and vancomycin per ml, and TSA with 5% pig feces extract and five antimicrobial agents (spiramycin, rifampin, vancomycin, colistin, and spectinomycin) (BJ). Viable numbers …

Purification Of Maturation-Promoting Factor, An Intracellular Regulator Of Early Mitotic Events, Manfred J. Lohka, Marianne K. Hayes, James L. Maller

School of Veterinary and Biomedical Sciences: Faculty Publications

Maturation-promoting factor causes germinal vesicle breakdown when injected into Xenopus oocytes and can induce metaphase in a cell-free system. The cell-free assay was used to monitor maturation-promoting factor during its purification from unfertilized Xenopus eggs. Ammonium sulfate precipitation and six chromatographic procedures resulted in a preparation purified >3000-fold that could induce germinal vesicle breakdown within 2 hr when injected into cycloheximide- treated oocytes. Proteins of 45 kDa and 32 kDa were correlated with fractions of highest activity in both assays. These fractions contained a protein kinase activity able to phosphorylate the endogenous 45-kDa protein, as well as histone Hi, phosphatase …

Natural And Experimental Infection With An Attaching And Effacing Strain Of Escherichia Coli In Calvest, Rodney A. Moxley, David H. Francis

School of Veterinary and Biomedical Sciences: Faculty Publications

Gnotobiotic calves were inoculated with an 05:K4:H^-, urease-positive strain of Escherichia coli isolated from a 2-day-old calf with diarrhea. The calves developed elevated temperatures and passed loose mucoid feces, with or without blood. The E. coli strain was negative for heat-stable and heat-labile enterotoxins but produced high levels of Shiga-like toxin. Bacteria attached diffusely to the epithelium of the large intestine and multifocally to the epithelium of the ileum. The duodenum and jejunum were not affected. At the sites of bacterial attachment, microvilli were effaced, enterocytes were degenerate, and necrosis and exfoliation had occurred. These results confirm a …

Formation Of Influenza Virus Particles Lacking Hemagglutinin On The Viral Envelope, Asit K. Pattnaik, Donald J. Brown, Debi P. Nayak

School of Veterinary and Biomedical Sciences: Faculty Publications

We investigated the intraceUular block in the transport of hemagglutinin (HA) and the role of HA in virus particle formation by using temperature-sensitive (Is) mutants (1s134 and 1s61S) of inOuenza virus AlWSN/33. We found that at the nonpermissive temperature (39.5°C), the exit of ts HA from the rough endoplasmic reticulum to the Golgi complex was blocked and that no additional block was apparent in either the exit from the Golgi complex or post-Golgi complex transport. When MDBK ceUs were infected with these mutant viruses, they produced noninfectious virus particles at 39.5°C. The efficiency of particle formation at 39.5°C was essentiaUy …

Identification Of Four Complementary Rna Species In Akabane Virus-Infected Cells, Asit K. Pattnaik, G. Abraham

School of Veterinary and Biomedical Sciences: Faculty Publications

The analysis of RNA extracted from purified Akabane virus demonstrated the presence of three size classes of single-stranded RNAs with sedimentation coefficients of 31S (large, L), 26S (medium, M), and 13S (small, S). Molecular weights of these RNA species were estimated to be 2.15 x 10⁶,1.5 X 10⁶, and 0.48 x 10⁶ for the L, M, and S RNAs, respectively. Hybridization analysis involving viral genomic RNA and RNA from virus-infected cells resulted in the identification of four virus-specific cRNA species in infected cells. These cRNAs were found to be nonpolyadenylated by their inability to bind …

Escherichia Coli Strains Producing Streptococcus Mutans Proteins Responsible For Colonization And Virulence, Roy Curtiss Iii, Robert G. Holt, Raul G. Barletta, James P. Robeson, Shigeno Saito

School of Veterinary and Biomedical Sciences: Faculty Publications

Streptococcus mutans is a principal etiologic agent of dental caries and is likely one of the most ubiquitous bacterial infectious disease agents worldwide. The ability of S. mutans to colonize the oral cavity is due to sucroseindependent and sucrose-dependent adherence to the pellicle-coated tooth surface with glucan facilitated aggregation between cells to result in plaque. Cariogenicity is then caused by the ability of S. mutans in plaque to metabolize free sugars and both extra and intracellular complex carbohydrates to yield predominantly lactic acid. The S. mutans gene products that contribute to colonizing ability and thus virulence include glucosyltransferases, glucanbinding proteins, …

Expression Of A Streptococcus Mutans Glucosyltransferase Gene In Escherichia Coli, James P. Robeson, Raul G. Barletta, Roy Curtiss Iii

School of Veterinary and Biomedical Sciences: Faculty Publications

Chromosomal DNA from Streptococcus mutans strain UAB90 (serotype c) was cloned into Escherichia coli K-12. The clone bank was screened for any sucrosehydrolyzing activity by selection for growth on raffinose in the presence of isopropyl- β-D-thiogalactoside. A clone expressing an S. mutans glucosyltransferase was identified. The S. mutans DNA encoding this enzyme is a 1.73-kilobase fragment cloned into the HindIII site of plasmid pBR322. We designated the gene gtfA. The plasmid-encoded gtfA enzyme, a 55,000-molecular-weight protein, is synthesized at 40% the level of pBR322-encoded β-lactamase in E. coli minicells. Using sucrose as substrate, the gtfA enzyme catalyzes …

Antiphosphocholine Antibodies Found In Normal Mouse Serum Are Protective Against Intravenous Infection With Type 3 Streptococcus Pneumoniae, David E. Briles, Moon Nahm, Kenneth Schroer, Joseph Davie, Phillip Baker, John Kearney, Raul G. Barletta

School of Veterinary and Biomedical Sciences: Faculty Publications

Although it has been clearly demonstrated that antibodies to pneumococcal capsules are protective in both mice and men (1, 2), the protective properties of antibodies to other pneumococcal constituents have been less well investigated. In the present report we describe results indicating that naturally occurring antibodies binding with the phosphocholine (PC) determinant of the pneumococcal cell wall C-carbohydrate (3) are important in the protection of mice from experimental pneumococcal infections.

Effects Of Thiabendazole Treatment On Weight Gains By Nebraska Range Cattle, D. L. Ferguson, D. A. Reynolds, M. J. Twiehaus

School of Veterinary and Biomedical Sciences: Faculty Publications

The results of these studies indicate that a high percentage of Nebraska range cattle are infected with gastrointestinal parasites, but at a subclinical level of infection. However, significant differences were not observed in average daily gain between thiabendazole-treated range cattle and unmedicated controls in three of four field trials. In the three trials the average improvement in daily gain in animals receiving thiabendazole once averaged .01 lb greater than untreated controls. During a 120 day grazing period this improvement would not be sufficient to pay for the wormer or cost of additional labor.

Effects Of Thiabendazole Treatment On Weight Gains By Nebraska Range Cattle, D. L. Ferguson, D. A. Reynolds, M. J. Twiehaus

School of Veterinary and Biomedical Sciences: Faculty Publications

The results of these studies indicate that a high percentage of Nebraska range cattle are infected with gastrointestinal parasites, but at a subclinical level of infection. However, significant differences were not observed in average daily gain between thiabendazole-treated range cattle and unmedicated controls in three of four field trials. In the three trials the average improvement in daily gain in animals receiving thiabendazole once averaged .01 lb greater than untreated controls. During a 120 day grazing period this improvement would not be sufficient to pay for the wormer or cost of additional labor.

Transferable Drug Resistance Among Enterobacteriaceae Isolated From Cases Of Neonatal Diarrhea In Calves And Piglets, David P. Aden, Norman D. Reed, Norman R. Underdaul, Charles A. Mebus

School of Veterinary and Biomedical Sciences: Faculty Publications

Fecal specimens were collected on 22 different Nebraska ranches and at the Department of Veterinary Science from young calves and pigs with neonatal diarrhea. Enterobacteriaceae isolated from these fecal specimens were screened for resistance to tetracycline, streptomycin, sulfamethizole, kanamycin, chloramphenicol, colistin, nitrofurantoin, and nalidixic acid. Of the 92 strains studied, 57 were resistant to one or more of these antimicrobial agents. Resistant strains were obtained from all herds involved in the study. The two most common resistance patterns were tetracycline streptomycin sulfamethizole (22 of 57) and tetracycline (13 of 57). None of the strains were resistant to chloramphenicol, colistin, nitrofurantoin, …

Viral Infections Of Domestic Animals, George A. Young

School of Veterinary and Biomedical Sciences: Faculty Publications

There is a strong tendency in current research on mammalian virus diseases to concentrate on virus activity at a cellular or subcellular level. This trend is summarized adequately by Enders in relation to viral replication as follows: ','The large and ever-increasing volume of published experimental work on viral replication strikingly reveals the central position of this phenomenon in contemporary virological research. There are good reasons, both biological and practical, underlying this intensive effort to understand, i n detail, how a virus particle, without energy transforming apparatus of its own, manages to utilize the metabolic equipment of the cell to produce …

Viral Infections Of Domestic Animals, George A. Young

School of Veterinary and Biomedical Sciences: Faculty Publications

Contents
Health-Defined Animals 270
Immunity in Health-Defined Animals 272
Virus Infections During Gestation 275
Respiratory Infections 279
Influenza 279
Ascariasis enhancement of respiratory infections 280
Virus pneumonia of pigs 281
Infectious atrophic rhinitis of swine 282
Porcine inclusion body rhinitis 283
Infectious bovine rhinotracheitis 283
Bovine myxovirus parainfluenza 284
Infectious bovine keratoconjunctivitis 284
Enteric Infections 285
Swine polio-encephalomyelitis viruses 285
Transmissible gastroenteritis of swine 286
Beran’s swine enterovirus 287
Bovine virus diarrhea 287
Systemic Diseases 288
Hog cholera 288
African swine fever 290
Nebraska University disease 290
Swine edema disease 291
Exudative epidermitis of swine 292
Foot-and-mouth disease and rinderpest …

Worm Eggs: Cost You Money, E.F. Frolik, E.W. Janike

School of Veterinary and Biomedical Sciences: Faculty Publications

Contents

One Million Eggs Daily.......... 3

Damaging Trip......... 4

Worm Remedies......... 5

Modern Worm Remedies......... 6

A Control Program.......... 8