Experimental Colibacillosis in Gnotobiotic Baby Pigs II. Pathology

Sixty-two neonatal gnotobiotic pigs were used in three experiments to determine the lesions produced by two closely related strains of Escherichia coli O138:K81:NM (of Michigan origin) and O138:K81 (of Minnesota origin). Exposure was by subcutaneous injection of bacterial culture into the umbilical stump or by oral inoculation.

Gross signs common to monocontaminated pigs included distention of the flaccid small and large intestines with fluid contents. Edema was prominent in various tissues of most pigs exposed via the umbilical stump but not in those exposed orally.

Histological lesions were predominantly in the gastrointestinal tract and were variable. At one extreme acute hemorrhagic enteritis was present in two pigs, while at the other extreme in a few pigs it was difficult to distinguish tissues of infected pigs from those of noninfected germfree pigs. Significant histological lesions common to monocontaminates included mild inflammatory reaction, hydropic degeneration of the intestinal epithelium, evidence of interference with normal function of the villus-draining mechanisms, and vascular changes generally indicated by edema.

The findings suggest that interference with normal absorption of nutrients plays at least some role in the pathogenesis of colibacillosis in young gnotobiotic pigs.

     

Toxoplasmosis II. Studies of Animal Sera Using Complement-Fixation Tests

Serological studies were performed in guinea pigs, a sheep, calf, goat and two pigs experimentally infected with toxoplasmosis. The direct complement-fixation method was effective in detecting antibodies in guinea-pig, goat and sheep sera. The modified complement-fixation technique supplementing complement with normal bovine serum fraction, was required when testing bovine serum. With swine sera best reactions occurred in the indirect complement-fixation test and definite but low grade reactions were produced in the direct test after pro-complementary activity was removed by pH treatment of the sera.

Allergic skin reactions were produced in the experimental animals but improvement in the antigen is necessary before the test could be used generally in the field as a diagnostic method for animal toxoplasmosis.

     

Glasser's Disease of Swine Produced by the Intratracheal Inoculation of Haemophilus suis

The intracheal inoculation of pigs with Haemophilus suis led to the production of Glasser's disease at every attempt without significant pulmonary involvement. Isolation of this organism from the experimental animals was possible only in the acute phase of the disease.

The indirect fluorescent antibody technique when applied to frozen sections of tissues obtained from the experimentally infected pigs at autopsy, revealed a few rod forms but mostly “round bodies” of H. suis in animals from which the organism was isolated, and “round bodies” only in the pigs from which the organism was not isolated.

Attention is drawn to the similarities between the lesions caused by H. suis and Mycoplasma hyorhinis, and to the confusion which may result therefrom. It is stressed that the laboratory diagnosis of these two diseases is complicated by the fact that both agents may not be isolated on the media commonly used in diagnostic laboratories. Both organisms necessitate the use of special media where the clinical and autopsy results indicate polyserositis and arthritis.

     

A sequential study of experimental infection of pigs with porcine circovirus and porcine parvovirus: immunostaining of cryostat sections and virus isolation

The sequential tissue distribution of virus was investigated using virus isolation and immunofluorescence tests in 1-day-old piglets inoculated with porcine circovirus 2 (PCV2) and/or porcine parvovirus (PPV). Enlarged mesenteric lymph nodes were seen in the pig inoculated with PCV2 alone and killed at 26 days post-inoculation (PI). One of the pigs inoculated with PCV2 and PPV and killed at 21 days PI had an enlarged liver. The pig killed at 26 days PI in this group had enlarged liver, kidneys and heart. Histopathological changes were seen in lymphoid tissues of the pigs inoculated with PCV2 alone and killed at 14 and 26 days PI. Similar, but more severe, lesions were observed in the pigs infected with PCV2 and PPV and killed from 10 days PI onwards. Histological lesions of nephritis, pneumonia and hepatitis were also apparent in these animals. Mild nephritis was also seen in the pigs infected with PPV alone and killed at 14 and 26 days PI. Moderate amounts of PPV antigen were detected in tissues from the pigs inoculated with PPV alone and killed at 14 days PI. Low levels of PCV antigen were detected, mainly in lymphoid tissues, in the pigs inoculated with PCV alone and killed at 14 days PI. Low to moderate amounts of PCV antigen were detected in a wider range of tissues in the pig in this group killed at 26 days PI. In the pigs inoculated with both viruses, PPV antigen was detected in tissues of pigs killed from 3 to 26 days PI with maximal amounts detected between 6 and 14 days PI. PCV2 antigen was detected in low to moderate amounts in the tissues of pigs killed at 14 days PI. Large amounts of PCV2 antigen were detected in most of the tissues from pigs in this group killed between 17 and 26 days PI. Virus isolation results for PCV2 generally correlated well with the results for immunofluorescent staining. PPV was isolated from almost all tissues from pigs inoculated with PCV2 and PPV, a much higher incidence of positive tissues than observed for immunofluorescent staining.     

Persistence of porcine parvovirus in swine infected in utero and followed through maturity

The potential of porcine parvovirus (PPV) to persistently infect swine exposed in utero was studied. Forty eight 80- to 95-day-old fetuses from 5 PPV seropositive sows were inoculated intramusculary with a virulent strain of PPV or with cell culture medium (controls). Blood samples were collected at birth prior to nursing and at monthly intervals thereafter and tested for antibodies to PPV. Virus-inoculated and control pigs were euthanized at either 1 week before birth (-1), at birth (0) and at weeks 2, 4, 6, 8, 10, 22, and 28 after birth. Presence of viral DNA and antigen was evaluated using slot blot DNA hybridization and indirect FA techniques, respectively. All inoculated fetuses (n = 26) and 7 control fetuses (n = 22) seroconverted in utero, and these pigs maintained antibody titers greater than log10 2 for the period of testing (0-38 weeks after birth). After passive antibody titers had reached subdetectable levels in control animals, animals remained seronegative through an additional 14 weeks of testing in spite of close contact with infected pigs. Virus antigen was not detected in any tissues examined from pigs euthanized at term. In contrast, PPV DNA was detected consistently from pigs at birth from various tissues, and from the lung of one pig at 6 weeks of age and from the lymph nodes of one pig euthanized at 28 weeks of age. The results indicate that pigs infected with PPV in utero may be persistently infected, however the likelihood of shedding to contact animals is minimal.     

V. Detection of the Virus in Infected Materials by Immunofluorescence

The fluorescent-antibody technique was employed for the detection of bluetongue virus in bovine foetal kidney cell cultures inoculated with tissues and blood from experimentally infected animals.

In the first series, a total number of 79 inoculated suckling-mouse brains were examined, 36 as frozen sections alone and 43 as impression slides in conjunction with tissue culture inoculation of the same material. With the combined tissue culture immunofluorescent methods, 36 suspicious were detected by impression smears and 37 positives by the tissue culture out of 43 brains examined. Twenty-two were suspicious out of the 36 examined as frozen sections.

Results obtained with the second series, using sheep tissues, showed that the combined tissue culture-fluorescent antibody method was satisfactory for demonstrating the virus in blood of infected animals 1 to 9 days postinfection, and in some organs after death. No false positive reactions were obtained.

     

Diagnosis of swine influenza with an immunofluorescence technique using monoclonal antibodies

Summary

Direct diagnosis of swine influenza infection by an indirect immunofluorescence technique using anti‐nucleoproteine monoclonal antibody was compared with virus isolation. Five 8‐week‐old pigs were inoculated with 2 × 107 EID₅₀ of strain A H₁N₁ Sww//4115/85. Clinical signs developed in only three pigs. Antigen was detected in nasal epithelial cells obtained from all animals the first day after inoculation; the antigen was detected in one pig 6 days after the infection. Fluorescence was present in the nucleus, nucleolus and cytoplasm of infected cells. The indirect immunofluorescence test was specific and as sensitive as virus isolation in embryonated eggs, allowing a rapid diagnosis that could be achieved within hours.     

Studies on the Susceptibility of Water Buffaloes to Leptospira

Eight mature farming type, Taiwan, water buffaloes were inoculate with L. australis A while six received L. canicola. Before inoculation all animals were negative to the microscopic-agglutination test (agglutinationlysis test) using the above species as antigen.

No sign of clinical leptospirosis was observed although four animals developed temperatures.

Cultures made from buffalo blood, kidneys and urine and from blood of guinea pigs inoculated with kidney emulsion and urine from the inoculated buffalo were all negative for leptospiral organisms.

Blood samples drawn from the water buffalo 2, 3 and 4 weeks post inoculation were negative to the microscopic-agglutination test except for one animal. Blood from the animal taken two weeks post-inoculation was positive at 1:100 dilution with L. australis A antigen but that taken at 3 and 4 weeks was negative.

     

Swine brucellosis in Indonesia

Summary

Brucella suis biotype 1 was isolated from 13.1% of the pigs slaughtered in Kapuk Jakarta, West Java and from 15.09% of the pigs slaughtered in Surabaya, East Java. The prevalence of B. suis by means of the Rose Bengal Plate Test, was 22.3% for West Java and 14.9% for East Java. The Rose Bengal Plate Test detected more B. suis infected animals (73% of the infected animals) than did the Complement Fixation Test (41%) and the Serum Agglutination Test (54.5%).

The high infection rate is a potential health danger for the abattoir workers.     

Detection of Actinobacillus pleuropneumoniae Infection in Pigs

It is difficult to control the spread of porcine haemophilus pleuropneumonia caused by Actinobacillus pleuropneumoniae because there is no sensitive and specific way to accurately determine whether or not a pig herd is infected. This paper reports bacteriological and serological techniques used to detect A. pleuropneumoniae infection in pigs from a herd with endemic disease.

The bacteria were isolated from the anterior nasal mucosa of grower pigs, but not from younger or older pigs. Bacteriological culture of several tissues from the respiratory tract showed that nine of ten young finishing pigs were infected, but culture of lung tissue from slaughtered hogs detected infection in only 39 of 288 (13.5%). Both cooler storage temperature and use of selective medium prolonged the time that lung tissue could be stored and the organism still recovered. An enzyme-linked immunosorbent assay detected serotype-specific antibodies in serum of infected pigs.

     

Leptospirosis in pigs: The effectiveness of streptomycin in stopping leptospiruria

A trial was conducted to assess the efficacy of a single intra-muscular injection of 25mg per kg of streptomycin in stopping leptospiruria in growing pigs exposed to a natural infection.

The investigation involved 54 pigs, 10–12 weeks old, that were free of serological evidence of infection with serotypes pomona or tarassovi at the start of the trial. The animals were kept in pens in a finishing house through which flowed effluent from other pens containing infected pigs. Urine samples were collected from each pig three times weekly until it was slaughtered for bacon at 20–24 weeks old.

Leptospiruria was first detected between 16 and 74 days after exposure. Twenty-one of 37 pigs which showed leptospiruria were injected with streptomycin. Leptospirae were detected in 11 of 185 (5.9%) subsequent urine samples from these 21 treated animals and serotype pomona was cultured from the kidney of 1 of the 10 animals that were examined from this group at slaughter.

Following the initial detection of leptospiruria in the 16 un-treated pigs, 179 of 211 (84.8%) subsequent urine samples con-tained leptospirae, which were also cultured from the kidneys of 4 out of 11 of these animals.     

Diagnostic tests for Trichinella spiralis infection in pigs. A comparative study of ELISA for specific antibody and histamine release from blood cells in experimental infections.

A study on the histamine release test (HR) for the demonstration of infections with Trichinella spiralis in pigs was carried out on 18 pigs, six infected with 200 larvae, six infected with 5000 larvae and six non-infected (control group). The results obtained by HR during a 7 week infection were compared with those of the enzyme-linked immunosorbent assay (ELISA). All inoculated pigs were found to be positive on Day 40 post-inoculation (p.i.) by necropsy examination of selected muscle groups, with mean recoveries of 7.9 and 225 larvae g-1 of tissue in the low- and high-dose group, respectively. At this time, all animals of the high-dose group and five out of six animals of the low-dose group were antibody positive in ELISA with any of three coating antigens employed (a crude muscle larva extract, an excretory/secretory (ES) antigen and a purified 45 kDa antigen). HR performed on whole blood was positive in four out of six pigs of the high-dose group and one out of six pigs of the low-dose group. The earliest ELISA seroconversions took place at Day 15 p.i. with crude and ES antigens. The earliest measurable reaction in HR performed on whole blood was found on Day 19 p.i. There was considerable individual variation regarding which test was the most sensitive for the early detection of infection. Washing of the blood cells prior to antigen provocation led to a markedly improved sensitivity of HR, all animals of the high-dose and three out of six animals of the low-dose group being positive by Day 40 p.i. The time course of the development of ELISA titres and HR reactivity indicated that this effect is due to the removal of blocking antibodies.     

Experimental production of congenital persistent swine fever infections: I. Clinical,pathological and virological observations

Twelve sows were inoculated with a low-virulent field strain of swine fever (SF) virus at 40, 65 and 90 days of pregnancy. Transplacental infection occurred in eight sows and these farrowed 88 piglets.Prenatal mortality was highest in litters from sows infected at 40 days after service, and postnatal death was most frequent in litters from sows infected at 65 days. Three sows gave birth to completely infected litters, whereas the five others farrowed litters in which uninfected piglets were found. The later the sows were infected during pregnancy, the more uninfected piglets were born. Twelve piglets recovered from the infection and the percentage of recoveries increased with the stage of pregnancy at which inoculation took place. Twenty-three piglets developed a persistent infection. The earlier infection occurred during pregnancy, the more persistent infections were produced. The persistently infected pigs developed a runting syndrome from about one week after weaning. The clinical signs were more severe in pigs from sows infected at 65 days of pregnancy than in pigs from sows infected at 40 days. A persistent viraemia was present in these pigs with titres ranging between 10^5.7 and 10^7.0 plaque forming units/ml of plasma. At autopsy the most pronounced lesions were an atrophy of the thymus, and swollen and pale lymph nodes. Virus antigen was present in lymphoidal, reticulo-endothelial and epithelial tissues.One persistently infected pig survived superinfection with a virulent strain of SF virus for 45 days.     

Experimental infection of the possum (Trichosurus vulpecula) with Leptospira interrogans serovar balcanica I. Characteristics of infection

The serological, bacteriological and histopathological characteristics of experimental infection with serovar balcanica in possums (Trichosurus vulpecula) are described, and the possum is shown to be a potential maintenance host for this organism. Serum agglutination titres were maintained at almost constant levels for longer than a year, and leptospiruria was present in 50% of animals for a similar period. A paradoxical reaction to hardjo antigen was found in sera from all possums infected with balcanica.

Comparative studies were conducted using recently isolated field strains of serovars hardjo and ballum. Young possums seronegative to Hebdomadis group titres were insusceptible to challenge with hardjo, and the pathogenesis of ballum infection was characteristic of leptospiral infection in an accidental host.

The occurrence of antibodies in the urine of possums infected with balcanica is also described.     

Immune response to haemophilus parahaemolyticus (HP) infection in the pig I. In vitro tests to detect sensitized cells in the blood of infected and vaccinated animals

The in vitro immune response of pigs in herds infected or non-infected with H. parahaemolyticus (HP) was investigated by lymphocyte stimulation and leucocyte migration inhibition tests. The stimulation of pig lymphocytes with HP bacteria has been measured by ³H-thymidine incorporation. The response of the cells seems to be specific, lymphocytes from chronically HP-infected animals showed positive reactions with high frequency, while on the contrary, cells from SPF (specific pathogen free) animals reacted only occasionally to the HP antigens. However, in the leucocyte migration inhibition test the reactions were less specific; the widely occurring H. parasuis bacteria seem to possess antigen(s) which causes cross reactions with HP in this test.Vaccination with HP bacteria induced sensitive lymphocytes in the blood of SPF pigs and an increase of the in vitro lymphocyte reactions in already positive animals.     

III. The Use of the Agar Double-Diffusion Precipitation Test For the Detection of the Virus in Swine Tissue

The agar double-diffusion precipitation test was applied successfully in the demonstration of ASF viral antigen in spleen and liver from swine experimentally infected by the oral route.

Positive reactions were obtained with tissues collected as early as 24 hours after the onset of pyrexia and before other clinical manifestation of the disease. Cross-reactions were observed between the various ASF strains used in the study, making the test practical for routine diagnosis in which different strains may be encountered.

     

Ascaris suum in Saskatchewan Pigs: An Abattoir Survey of Prevalence and Intensity of Infection

In a survey of 2500 market weight pigs in a Saskatchewan abattoir, 37% were infected with adult Ascaris suum and in 46% there were milkspot hepatic lesions. A total of 60% of the pigs examined had some evidence of A. suum infection. Most infected pigs contained less than 50 adult ascarids.

In a second abattoir survey of a further 2500 market weight pigs, 44% of the animals had milk-spot hepatic lesions. In approximately 12% of the affected pigs these lesions were very severe and a very similar proportion of pigs' livers were condemned or sent for use as animal feed.

The results of these surveys are discussed with particular reference to the significance of A. suum infection in growing pigs in Saskatchewan.

     

Comparative immunohistopathology in pigs infected with highly virulent or less virulent strains of hog cholera virus

Eight pigs were inoculated subcutaneously with a highly virulent hog cholera virus (HCV) strain ALD. The infected pigs developed severe illness and became moribund on postinoculation day (PID) 7 or PID 10. Histologic lesions were characterized by severe generalized vasculitis, necrosis of lymphocytes, and encephalitis. HCV antigen was detected in crypt tonsilar epithelial cells, macrophages, and reticular endothelial cells of lymphoid tissues. Antigen localization corresponded well with histologic lesions. Five pigs were inoculated with less virulent HCV Kanagawa/74 strain and were euthanatized on PID 30. All five infected pigs recovered from the illness but became stunted. They also had a slight follicular depletion of lymphocytes, histiocytic hyperplasia, and hematopoiesis in the spleen. Less virulent HCV antigen was observed in the tonsils, kidneys, pancreas, adrenal glands, and lungs. Although antigen localization was less associated with histologic lesions, immunoreactivity was stronger than that in the pigs infected with the ALD strain of HCV. An almost complete loss of B lymphocytes was recognized in pigs infected with the ALD strain and was correlated with follicular necrosis in lymphoid tissues. Loss of B lymphocytes was not prominent in the pigs infected with Kanagawa/74 strain. The number of CD4+ and CD8+ T lymphocytes was significantly higher than that in the noninfected control pigs.     

Localization of classical swine fever virus from chronically infected pigs by in situ hybridization and immunohistochemistry

Classical swine fever (CSF) virus (CSFV) nucleic acid and antigen were detected in 15 pigs with naturally occurring chronic CSF by in situ hybridization and immunohistochemistry. The most consistent and prominent microscopic lesions were perivascular mononuclear cell infiltration and gliosis in the central nervous system of pigs with chronic CSF. Positive cells typically exhibited a dark brown (in situ hybridization) or red (immunohistochemistry) reaction product in the cytoplasm without background staining. A positive signal for both in situ hybridization and immunohistochemistry was detected in mononuclear cells and lymphocytes of lymphoid tissues. Viral nucleic acid was detected in some tissue sections in the absence of viral antigen. The in situ hybridization technique developed in this study was useful for the detection of CSFV RNA in tissues taken from chronically infected pigs and may be a valuable technique for studying the pathogenesis of chronic CSFV infection.     

Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors.

Congenital tremors (CT) type A2 is associated with porcine circovirus (PCV) and deficient and abnormal myelin. The aim of this study was to determine the tissue distribution and genetic type of PCV in 1-2-day-old pigs with naturally occurring CT type A2 using in situ hybridization, polymerase chain reaction (PCR), and indirect fluorescent antibody tests on frozen tissue sections. CT-affected and clinically normal pigs were selected from 4 farms in the midwestern USA that were undergoing outbreaks of CT type A2. All CT and most normal pigs were infected with PCV. PCV was widely distributed in tissues of infected pigs and was most common in tissues of the central nervous system and liver. In all infected pigs, there were more PCV-infected cells in brain and spinal cord than in nonneural tissues. CT pigs had many more PCV-infected cells in the brain and spinal cord than did clinically normal pigs because of a more diffuse distribution and a larger proportion of infected cells. The cells most commonly infected with PCV in brain and spinal cord were large neurons. In nonneural tissues, macrophages were the most frequent cell type infected. PCR analysis demonstrated only PCV type 2 and not PCV type 1 in all PCV-infected pigs on all 4 farms.