Increased pulmonary clearance of Serratia marcescens in calves given intravenous Freund's complete adjuvant

Lung and alveolar macrophage studies were conducted following experimental immunostimulation of calves with Freund's complete adjuvant (FCA). Intravenous administration of FCA to calves 35 days before aerosol exposure to serratia marcescens resulted in a 53.1% reduction in the 2-hour pulmonary mean percent retention of the organism when compared to control calves. FCA treatment increased alveolar macrophage concentration (cells/g lung) by 30.9%. Lymphoid and granulomatous responses were increased in the lungs of treated calves.     

Effect of Pasteurella multocida and Haemophilus pleuropneumoniae toxins on swine alveolar macrophages

Supernatants were obtained from 18 hr. broth cultures of Pasteurella multocida strains D82 and D62 (serotype D, toxigenic), Kobe 6 (type D, non-toxigenic), A50 and X73 (type A, non-toxigenic), Haemophilus pleuropneumoniae serotypes 1, 5 and 6, Haemophilus sp. taxon "minor group" (2 strains) and an avirulent serotype 1 strain of H. pleuropneumoniae. The supernates were filtered, pH-neutralized and tested for cytotoxicity after incubation for 18 hours in the presence of swine alveolar macrophage monolayers. Supernatants from H. pleuropneumoniae serotypes 1, 5 and 6 were cytocidal.     

Detection of the nucleolar activity in alveolar macrophages of swine]

With the help of the toluidine blue-staining-technique different characteristic shapes of alveolar macrophage nucleoli were detected, indicating differences in the rRNA synthesis activity of these cells. After application of a Pasteurella multocida aerosol the percentage of alveolar macrophages with a high rRNA synthesis increased. No correlation between the nucleolar activity and the efficacy of the clearance of aerogenously administered Pasteurella could be measured.     

Significance of Freund's adjuvant/antigen injection granuloma in the maintenance of serum antibody response

An experimental system involving injections of ovalbumin (OVA) and ferritin (FER) in Freund's incomplete adjuvant (FIA) into the right and left flank skin folds of sheep was used to study the influence of the FIA/antigen depot and the draining lymph node in maintaining an antibody response. Excision of the injection granuloma and draining lymph node from one side 2-3 months after injections resulted in a profound decrease in serum antibody titres. This response was observed in all eight sheep in the experimental group. In five of eight animals in another experiment, excision of the injection sites had no appreciable effect on antigen-specific antibody titres when compared with antibody specific for antigen on the intact side of the sheep. In the remaining three animals, excision of the injection site did cause some fall in titre. Radiotracer studies revealed that about one-third of the original [125I]OVA/FIA injected was present in the granuloma 20 weeks after injection. Lymphatic cannulation approaches were used to study the responsiveness of the lymph node draining an FIA/antigen granuloma established 12 weeks earlier and showed that increments of 1-2 mg OVA in saline administered adjacent to the granuloma at 6-7 day intervals gave rise to strong anti-OVA containing cell (AOCC) responses in lymph. There were 2-6-fold increases in serum antibody titre in response to 3-5 doses of OVA or FER (1-2 mg) in saline injected adjacent to the FIA/antigen injection site (which had been administered 14-16 weeks previously). It is concluded that the release rate of antigen from a FIA/antigen depot is insufficient to sustain maximal antibody levels in blood serum.     

Bacterial lipopolysaccharide induces porcine circovirus type 2 replication in swine alveolar macrophages

Monocyte/macrophage lineage cells are major target cells of porcine circovirus type 2 (PCV2). From tissues of field pigs suffering from postweaning multisystemic wasting syndrome (PMWS), both intracytoplasmic and intranuclear PCV2 signals, including antigens and nucleic acid, were easily detected in the monocyte/macrophage lineage cells. However, there was a high incidence of intracytoplasmic PCV2-positive signals, but lack of intranuclear signals and PCV2 replication in these cells in vitro. Concurrent infection with bacteria and activation of immune system are suggested to promote viral replication. In this study, lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA) were used to stimulate PCV2-inoculated alveolar macrophages (AMs). A decrease in intracytoplasmic but increase in intranuclear PCV2-positive signals, including antigens and nucleic acid, were detected in LPS-treated PCV2-inoculated AMs, but not in PMA-treated cells. Additionally, the replication product corresponding to PCV2 spliced major capsid protein (Cap) mRNA and a significant elevation in PCV2 titer were demonstrated in the LPS-treated PCV2-inoculated AMs. The results imply that Gram-negative bacterial co-infection in PCV2-infected pigs may be an important factor in promoting PCV2 replication and contributing, at least partially, to the full development of PMWS.     

Induction of swine dysentery in swine by the intravenous injection of filtered Treponema hyodysenteriae.

Swine dysentery was induced in 18 swine exposed by intravenous injection of a filtrate which contained Treponema hyodysenteriae and was obtained from macerated colonic scrapings of swine dysentery. However, swine dysentery did not develop in swine injected intravenously with a pure culture of T. hyodysenteriae or when combined with a colonic filtrate from normal swine. Diarrheal feces from the swine injected intravenously with the filtered T. hyodysenteriae contained more mucus, and fecal smears contained more T. hyodysenteriae and fewer other bacteria than did swine exposed orally to colon infected with swine dysentery or filtered T. hyodysenteriae. In the colons of the 12 swine injected intravenously with filtered T. hyodysenteriae that died, there was a minimum amount of croupous membrane and, microscopically, the T. hyodysenteriae were located deep in the colonic crypts. Five of the six surviving swine injected intravenously with filtered T. hyodysenteriae developed serum anti-T. hyodysenteriae antibodies using the indirect fluorescent antibody test and four of these swine developed diarrhea when reexposed with swine dysentery infected colon six weeks after initial exposure. None of the swine injected intravenously with cultured T. hyodysenteriae developed serum anti-T. hyodysenteriae antibodies and all were highly susceptible to swine dysentery.     

Immunization of rats against Fasciola hepatica using crude antigens conjugated with Freund's adjuvant or oligodeoxynucleotides

Chronic Fasciola hepatica infection is correlated with the development of a T helper (Th2)-predominant immune response. To determine whether immunostimulatory CpG-containing oligodeoxynucleotides (CpG-ODN) or Freund's complete adjuvant (FCA), known to promote a Th1 (T helper 1) immune responses, could provide protection from F. hepatica infection, total homogenate (TH) of F. hepatica mixed with CpG-ODN or FCA were injected subcutaneously (s.c.) into Wistar rats. A F. hepatica-specific Th1-predominant immune response was induced with CpG-ODN or FCA in lymph nodes of immunized animals. Lymph node cells from TH-CpG-ODN or TH-FCA immunized rats showed increased antigen-specific proliferation with high levels of INFgamma, compared to lymphocytes from rats injected with TH alone. In contrast, these two groups of immunized animals did not modify IL-4 release by draining lymph node cells, when they were subsequently stimulated with TH in vitro. However, a significant reduction in the burden of flukes (76.7%) was only observed in rats immunized with TH-FCA. Conversely, immunization of rats with TH-CpG-ODN did not promote protection against the parasite. Therefore, even though CpG-ODNs and FCA induced Th1 type responses, only FCA provided a significant protection to rats infected with F. hepatica.     

Lesions in sheep following administration of a vaccine of a Freund's complete adjuvant nature used in the control of ovine paratuberculosis

CASE HISTORIES: Occurrences of adverse reactions in seven sheep flocks in Australia following vaccination against paratuberculosis where veterinary attention was requested are reviewed. All cases occurred within the 3-year period following commencement of use of a vaccine of a Freund's complete adjuvant nature, at a time when approximately six million doses of vaccine had been administered.

CLINICAL FINDINGS: In the first case, 26/58 (45%) Merino sheep vaccinated as adults had palpable tissue reactions at or near the site of vaccination; enlarged prescapular lymph nodes were palpated in 17 (29%), and nine (16%) sheep had both palpable lesions at the site of vaccination and enlarged prescapular lymph nodes. The reactions included caseous nodules up to 5.5 cm in diameter. In the other cases, fistulating or granulomatous wounds were occasionally found at the recommended site of injection behind the ear, and myiasis was rare. Occurrences of inappropriate choice of injection site were recorded, including injection into the axilla of two Merino rams, and lesions in the tissues of the maxilla and nose of almost 50% of 350 Border Leicester lambs. Four outbreaks of progressive paralysis due to injection into cervical musculature were reported, described as ‘OJD staggers’ by producers.

DIAGNOSIS: Granulomatous cellulitis and lymphadenitis associated with oil droplets typical of ‘oil granulomata’. Injection of vaccine into the dorsal cervical area resulted in progressive paralysis due to myonecrosis and suspected granulomatous leptomeningitis.

CLINICAL RELEVANCE AND CONCLUSIONS: Although lesions at and near the site of injection are common, adverse reactions to vaccination were rare and included mortality from cervical spinal injection, production losses from injection in the maxilla or axilla or if myiasis resulted, and potential marketing losses if animals or carcasses are discounted as a result of the lesions. Risk factors for adverse reactions included inadequate restraint of sheep, breed of sheep, experience of the operator, poor injection technique, and inappropriate placement of vaccine. Increasing attention to the proper restraint of animals, restricting vaccination to the recommended site behind the ear, careful placement of the vaccine into subcutaneous tissue to avoid drainage of vaccine material into tissues such as the spinal cord, and post-vaccination supervision to address welfare concerns should adverse reactions occur are recommended.     

Lesions in sheep following administration of a vaccine of a Freund's complete adjuvant nature used in the control of ovine paratuberculosis

CASE HISTORIES: Occurrences of adverse reactions in seven sheep flocks in Australia following vaccination against paratuberculosis where veterinary attention was requested are reviewed. All cases occurred within the 3-year period following commencement of use of a vaccine of a Freund's complete adjuvant nature, at a time when approximately six million doses of vaccine had been administered. CLINICAL FINDINGS: In the first case, 26/58 (45%) Merino sheep vaccinated as adults had palpable tissue reactions at or near the site of vaccination; enlarged prescapular lymph nodes were palpated in 17 (29%), and nine (16%) sheep had both palpable lesions at the site of vaccination and enlarged prescapular lymph nodes. The reactions included caseous nodules up to 5.5 cm in diameter. In the other cases, fistulating or granulomatous wounds were occasionally found at the recommended site of injection behind the ear, and myiasis was rare. Occurrences of inappropriate choice of injection site were recorded, including injection into the axilla of two Merino rams, and lesions in the tissues of the maxilla and nose of almost 50% of 350 Border Leicester lambs. Four outbreaks of progressive paralysis due to injection into cervical musculature were reported, described as "OJD staggers" by producers. DIAGNOSIS: Granulomatous cellulitis and lymphadenitis associated with oil droplets typical of "oil granulomata". Injection of vaccine into the dorsal cervical area resulted in progressive paralysis due to myonecrosis and suspected granulomatous leptomeningitis. CLINICAL RELEVANCE AND CONCLUSIONS: Although lesions at and near the site of injection are common, adverse reactions to vaccination were rare and included mortality from cervical spinal injection, production losses from injection in the maxilla or axilla or if myiasis resulted, and potential marketing losses if animals or carcasses are discounted as a result of the lesions. Risk factors for adverse reactions included inadequate restraint of sheep, breed of sheep, experience of the operator, poor injection technique, and inappropriate placement of vaccine. Increasing attention to the proper restraint of animals, restricting vaccination to the recommended site behind the ear, careful placement of the vaccine into subcutaneous tissue to avoid drainage of vaccine material into tissues such as the spinal cord, and post-vaccination supervision to address welfare concerns should adverse reactions occur are recommended.     

Actinobacillus pleuropneumoniae culture supernatants interfere with killing of Pasteurella multocida by swine pulmonary alveolar macrophages.

The effect of Actinobacillus pleuropneumoniae culture supernatant on swine pulmonary alveolar macrophage (PAM) functions was studied. The A. pleuropneumoniae culture supernatant was toxic to PAMs when tested by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and lactate dehydrogenase (LDH) release assays. Biological activity of the supernatant was ascribed to cytotoxins. Both the LDH and MTT assays were used for measurement of crude A. pleuropneumoniae cytotoxin concentration with good reproducibility. A preparation containing 6,800 toxic units/mL (determined by MTT assay) was used for subsequent experiments. The objective was to study the effect of crude cytotoxin on the ability of swine PAMs to kill Pasteurella multocida. Phagocytosis of opsonized P. multocida type A by PAMs was not efficient. Only 8% of incubated organisms were ingested by noncytotoxin-treated PAMs after 30 min phagocytosis. The bactericidal effect of noncytotoxin-treated PAMs only last for 60 min, after which, the rate of growth of surviving P. multocida exceeded the rate of bacterial killing by PAMs. Complete elimination of P. multocida by PAMs was not observed in this study. A total loss of ability to kill P. multocida by PAMs was seen when the PAMs were pretreated with a high concentration (340 toxic units/mL) of A. pleuropneumoniae cytotoxin. If the PAMs were pretreated with a low concentration (3.4 toxic units/mL) of cytotoxin, a significant reduction in the killing of P. multocida was still observed. The reductions in phagocytosis, phagosome-lysosome fusion (demonstrated using yeast particles of Candida albicans), and oxidative burst (demonstrated by nitro blue tetrazolium reduction (NBT) assay) may have contributed to the impaired killing of P. multocida by PAMs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of intravenous lidocaine on the minimum alveolar concentration of isoflurane in cats

Lidocaine has been reported to decrease the minimum alveolar concentration (MAC) of inhalation anesthetics in several species and has been used clinically to reduce the requirements for other anesthetic drugs. This study examined the effects of intravenous lidocaine on isoflurane MAC in cats. Six cats were studied. In experiment 1, the MAC of isoflurane was determined. An intravenous bolus of lidocaine 2 mg kg^–1 was then administrated and venous plasma lidocaine concentrations measured to determine pharmacokinetic values. In experiment 2, lidocaine was administered to achieve target plasma concentrations between 1 and 11 μg mL^–1 and the MAC of isoflurane was determined in triplicate at each lidocaine plasma concentration, using the tail‐clamp method. End‐tidal isoflurane concentration was determined using a calibrated infrared analyzer. Systolic blood pressure (Doppler), SpO2 and end‐tidal PCO2 (calibrated Raman spectrometer) were measured prior to each MAC determination. Body temperature was maintained between 38.5 and 39.5 °C by supplying external heat as needed. MAC values at the different lidocaine plasma concentrations were analyzed by a repeated measures ANOVA , using the Huynh–Feldt correction. The MAC of isoflurane in these cats was 2.21 ± 0.17. For the target concentrations of 1, 3, 5, 7, 9, and 11 μg mL^–1, the actual lidocaine plasma concentrations was 1.06 ± 0.12, 2.83 ±0.39, 4.93 ± 0.64, 6.86 ± 0.97, 8.86 ± 2.10, and 9.84 ± 1.34 μg mL^–1, respectively. At these target concentrations, the MAC of isoflurane was 2.14 ± 0.14, 1.88 ± 0.18, 1.66 ± 0.16, 1.47 ±0.13, 1.33 ± 0.23, and 1.06 ± 0.19%, respectively. Lidocaine, at target plasma concentrations of 1, 3, 5, 7, 9, and 11 μg mL^–1, linearly decreased isoflurane MAC by –6 to 6, 7 to 28, 19 to 35, 28 to 45, 29 to 53, and 44 to 59%, respectively. Lidocaine significantly dose‐dependently and linearly decreases the requirements for isoflurane in cats. No ceiling effect was observed within the range of plasma concentrations studied.     

Effects of inorganic or organic selenium on immunoglobulins in swine

A study was conducted to determine if Se source fed during gestation and lactation affects passive transfer of immunoglobulins. Sixty days prior to breeding (d -60), gilts were randomly assigned to one of three treatments prior to breeding and throughout gestation: control (Control, no supplemental Se; n = 8), inorganic Se (Inorganic Se, 0.3 ppm; n = 4) and organic Se (Organic Se, 0.3 ppm; n = 4). Blood was collected on d -60, 57 and 113 of gestation and on d 21 of lactation and milk was collected at d 0, 1, 7, 14, and 21 of lactation. Blood was collected from piglets at d 0, 1, 7, 14, and 21 of age. Gilts fed organic Se had greater (P < 0.05) circulating concentrations of Se than Inorganic and Control gilts. Regardless of treatment, circulating concentrations of Se were greatest (P < 0.05) at d -60 compared to all other days. Serum concentrations of IgG were greatest (P < 0.05) in gilts at d 57 of gestation compared to d 113. Serum concentrations of IgA were greatest (P < 0.05) on d 113 of gestation and d 21 of lactation compared to d -60 and 57. Serum concentrations of IgM were greater (P < 0.05) at d 57 compared to d -60. Inorganic gilts had greater (P < 0.05) colostral and milk concentrations of IgG and IgM than Organic or Control gilts. Circulating concentrations of Se in piglets were greatest (P < 0.05) at d 14 and 21 of age compared to all other days. Piglets from gilts supplemented with organic Se had greater (P < 0.05) circulating concentrations of Se on d 1 versus piglets from gilts supplemented with no additional Se. The immunoglobulin concentrations of IgG, IgA, and IgM were lowest (P < 0.05) on d 0 and then increased when compared to d 1. The addition of different sources of Se did not affect the immunoglobulin concentrations in the gilt or piglet.     

Effects of inorganic or organic selenium on immunoglobulins in swine

A study was conducted to determine if Se source fed during gestation and lactation affects passive transfer of immunoglobulins. Sixty days prior to breeding （d -60）, gilts were randomly assigned to one of three treatments prior to breeding and throughout gestation： control （Control, no supplemental Se; n = 8）, inorganic Se （Inorganic Se, 0.3 ppm; n = 4） and organic Se （Organic Se, 0.3 ppm; n = 4）. Blood was collected on d -60, 57 and 113 of gestation and on d 21 of lactation and milk was collected at d 0, 1, 7, 14, and 21 of lactation. Blood was collected from piglets at d 0, 1, 7, 14, and 21 of age. Gilts fed organic Se had greater （P 〈 0.05） circulating concentrations of Se than Inorganic and Control gilts. Regardless of treatment, circulating concentrations of Se were greatest （P 〈 0.05） at d -60 compared to all other days. Serum concentrations of IgG were greatest （P 〈 0.05） in gilts at d 57 of gestation compared to d 113. Serum concentrations of IgA were greatest （P 〈 0.05） on d 113 of gestation and d 21 of lactation compared to d -60 and 57. Serum concentrations of IgM were greater （P 〈 0.05） at d 57 compared to d -60. Inorganic gilts had greater （P 〈 0.05） colostral and milk concentrations of IgG and IgM than Organic or Control gilts. Circulating concentrations of Se in piglets were greatest （P 〈 0.05） at d 14 and 21 of age compared to all other days. Piglets from gilts supplemented with organic Se had greater （P 〈 0.05） circulating concentrations of Se on d 1 versus piglets from gilts supplemented with no additional Se. The immunoglobulin concentrations of IgG, IgA, and IgM were lowest （P 〈 0.05） on d 0 and then different sources of Se did not affect the immunoglobulin ncreased when compared to d 1. The addition of concentrations in the gilt or piglet.     

Immunopathological effects of porcine circovirus type 2 (PCV2) on swine alveolar macrophages by in vitro inoculation

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), a multifactorial disease, in pigs. Monocyte/macrophage lineage cells, including alveolar macrophages (AMs), are the major target cells for PCV2. Swine AMs are essential for the pulmonary defense system against various pathogens. Concurrent infection of lung with opportunistic pathogens in pigs suffered from PMWS is speculated as a feature of immunosuppression. The present study was conducted to characterize the effects of PCV2 inoculation on swine AMs in the in vitro system. The parameters selected for evaluation included PCV2 antigen- and nucleic acid-containing rate, viability, TUNEL-positive rate, phagocytosis, microbicidal capability, and capacity for production of reactive oxygen species (superoxide anion, O₂⁻, and hydrogen peroxide, H₂O₂), cytokines, and chemokines. High intracytoplasmic PCV2 antigen- and nucleic acid-containing rate, absence of intranuclear signals for PCV2 antigen and nucleic acid, and lack of noticeable cell death were seen in PCV2-inoculated AMs. The PCV2-inoculated AMs displayed a transient as well as persistent reduction in the up-take and destruction of Candida albicans, respectively, accompanied by decrease in the production of O₂⁻ and H₂O₂. In PCV2-inoculated AMs, the levels of tumor necrosis- (TNF-) and interleukin-8 (IL-8) were significantly increased; the mRNA expression levels of alveolar macrophage-derived neutrophil chemotactic factors-II (AMCF-II), granulocyte colony-stimulating factor (G-CSF), monocyte chemotactic protein-1 (MCP-1), and IL-8 were strongly up-regulated. The reduced phagocytosis and microbicidal capability in conjunction with decreased production of reactive oxygen species in PCV2-inoculated AMs suggest that PCV2-containing AMs may favor the survival and spread of PCV2. It is speculated that the functional alterations observed in PCV2-containing AMs may be potentially harmful to the lung tissue and local pulmonary defense system, especially in those PCV2-infected pigs conditioned by various PMWS development-dependent co-factors.     

Interactions of porcine alveolar macrophages and bone marrow cells with African swine fever virus and virus-infected cells

Virus yields from porcine alveolar macrophages (AM) infected with African swine fever virus (ASFV) were greater and were achieved more rapidly, when inoculated at a high multiplicity of infection (MOI) than at low MOI. The difference was related to a lower percentage of cells becoming infected after low MOI inoculation. The reduced yields after low MOI were not caused by prolongation of the culture time, by bacterial endotoxins or by production of inhibitory substances by infected AM. Virus-infected AM were not susceptible to lysis in antibody-dependent cell mediated cytotoxicity (ADCC) assays and this was apparently due to a paucity of viral antigen expressed on the cell surface. Uninfected AM did not act as effectors in ADCC.Porcine bone marrow (PBM) cells were effective in mediation of ADCC and their activity was reduced after ASFV infection. Cells separated into adherent and non-adherent populations, depleted by carbonyl iron treatment or separated by Ficoll-Hypaque centrifugation, all showed effector activity in ADCC. The effector cells were not mature neutrophils or lymphocytes and were probably granulocytic precursors.