Experimental infection with Mycobacterium avium, serotype 2, in pigs. 1. Intravenous inoculations

A porcine strain of Mycobacterium avium, Serotype 2, was used for intravenous inoculation of pigs in doses 5, 1, 10⁻¹, 10⁻² and 10⁻³ mg (1 mg = 78 × 10⁶ viable units), 2 pigs per dose.Dose 5 mg proved fatal for both of the inoculated pigs, which were killed in extremis 64 and 69 days, respectively, after inoculation. Dose 1 mg caused clinical disease in 1 of 2 pigs, but was not lethal. Post mortem, the clinically affected pigs showed a generalized granulomatous tuberculosis. The other pig given 1 mg and the pigs given smaller doses, showed no clinical signs, and lesions and presence of acid-fasts were mostly limited to the lymph nodes of the lung, liver and digestive tract.All the pigs showed delayed hypersensitivity to avian PPD tuberculin (1000 t.u.) and some of them cross-reacted with human PPD tuberculin (1000 t.u.). The clinically affected pigs gave a very weak response to tuberculin, the others a strong response.The smallest dose capable of establishing an infection and producing tuberculous lesions was not determined, but seems to be less than 10⁻³ mg (78000 viable organisms).     

The ability to enter into an avirulent viable but non-culturable (VBNC) form is widespread among Listeria monocytogenes isolates from salmon, patients and environment

Media-based bacteriological testing will fail to detect non-culturable organisms and the risk of consuming viable but non-culturable (VBNC) Listeria monocytogenes is unknown. We have here studied whether L. monocytogenes obtained from seafoods, processing environment and clinical cases enter the VBNC state and assessed the virulence of the non-culturable forms of the bacteria. A number of 16 L. monocytogenes strains were starved in microcosm water at 4 °C until loss of culturability. Metabolic activity in the VBNC form was measured as ATP generation using a luciferase assay and membrane integrity was examined using the LIVE/DEAD BacLight assay. All tested L. monocytogenes strains entered the VBNC state after starvation in microcosm water. Ongoing mRNA synthesis of hly in VBNC L. monocytogenes cells re-incubated in culture medium indicated a potential virulence of these forms. Sodium pyruvate and replenishment of nutrient were used in attempts to resuscitate VBNC cells. However, VBNC L. monocytogenes were not resuscitated under these conditions. VBNC L. monocytogenes were tested for virulence in a cell plaque assay and by intraperitoneally inoculation in immunodeficient RAG1^−/− mice. Inoculation of VBNC L. monocytogenes in immunodeficient mice did not cause morbidity, and plaque assay on HT-29 cells in culture indicated that the VBNC cells were avirulent. The results indicate that the risk of non-culturable L. monocytogenes in foods, when the VBNC state is induced by starvation, is negligible.     

Experimental infections of mice and pigs with Streptococcus suis type 2.

Five inbred strains of mice were tested for their susceptibility to Streptococcus suis type 2 including the type strain, two isolates from meningitis in pigs and two isolates from tonsils of clinically healthy pigs. C57BL/6, ICR and ddY strain mice showed lower susceptibility to all strains of S. suis type 2 than BALB/c and SS strain mice. The type strain and the isolates from diseased pigs produced septicaemia and meningitis in BALB/c and SS mice inoculated with 10(8) colony forming unit of the bacteria and 60 to 100% of these infected mice died. On the other hand, mice inoculated with the isolates from healthy pigs showed mild clinical signs but none of them died. In BALB/c mice which died or developed nervous signs, the purulent meningo-encephalitis, myocarditis, ophthalmitis, labyrinthitis and otitis media were observed. S. suis type 2 antigen was demonstrated in these lesions by immunoperoxidase staining using rabbit S. suis type 2 antiserum. These results were similar to those in the experimentally infected pigs with these virulent and avirulent strains against mice. These results indicate that BALB/c and SS strains of mice are useful as an experimental model of S. suis type 2 infections in pigs, and that there are virulent and avirulent strains against mice and pigs among the strains of S. suis type 2.     

Avirulent ts and thymidine kinase-deficient mutant of Aujeszky's disease virus.

The temperature-sensitive (ts), thymidine kinase-deficient (TK-) mutant designated ZHtsTK- strain, of Aujeszky's disease virus (ADV) was isolated from a virulent strain with the treatments using 5-bromodeoxyuridine and arabinosylthymine. The ZHtsTK- strain was easily distinguished from the other virulent ADV strains by plaque size on HmLu-1 and chicken embryo fibroblast cells and by restriction endonuclease analyses using Bam HI, Sal I and Kpn I. The ZHtsTK- strain was avirulent for mice, guinea pigs and rabbits, and produced neutralizing antibodies to ADV in these animals. The rabbits inoculated with the ZHtsTK- strain did not shed detectable amounts of virus after dexamethasone treatment. The ZHtsTK- strain was also avirulent for 5-day-old piglets and did not cause disease. No virus was detected from the piglets inoculated intramuscularly in the nasal swabs or the tissues examined on postinoculation day 9. These findings presented here suggested that there is a significant correlation between pathogenicity and properties such as ts and TK-, and the combination of ts and TK- properties plays a much larger role in reducing virulence for animals.     

Immunization of mice against Streptococcus suis serotype 2 infections using a live avirulent strain.

In this study, the IgG response of mice injected with two virulent strains and one avirulent Streptococcus suis capsular type 2 strain was compared by Western blotting. The serum from mice immunized against the avirulent strain could recognize most proteins of the various strains tested and similar results were obtained with serum from mice injected with virulent strains. The live avirulent strain was injected twice (days 0 and 10) to groups of five mice, and four virulent strains from different geographical origins were used to challenge the animals. All mice, except one in one group, survived the challenge. These results suggest that a live avirulent strain could be used for immunization of swine, the natural host.     

Serum resistance and virulence of Escherichia coli isolated from turkeys

Twenty-five strains of Escherichia coli isolated from turkeys were characterized for their serum resistance and virulence. An in vitro bactericidal assay was used to determine the serum resistance of E coli. Virulence was determined by survival time after IV inoculation of each strain into 3-week-old turkeys. Serum-resistant E coli strains were generally found to be virulent for turkeys, whereas serum-sensitive E coli strains were avirulent. Of the 25 strains, 18 strains were placed in the 2 categories of serum-resistant/virulent and serum-sensitive/avirulent. Five strains were serum-resistant and avirulent, and 2 strains were serum-sensitive and virulent. Serum resistance appears to be an important determinant of virulence for E coli in turkeys; however, the requirement for other virulence factors, in addition to serum resistance, was suggested by the finding that 5 serum-resistant strains were avirulent in turkeys.     

猪多杀性巴氏杆菌对HeLa细胞附着能力的研究

本研究通过猪肺疫的活菌疫苗和死菌疫苗多杀性巴氏杆菌菌株(Pasteurella multocida,Pm)对小鼠的毒力试验测定它们的毒力性。结果表明,死菌疫苗Pm的毒力性比活菌疫苗Pm的强,即死亡率分别为10 0 %和0 %。通过两菌株对He L a细胞的附着试验测定它们的附着能力,结果证明强毒菌的附着能力明显地比弱毒菌强(P<0 .0 1) ,平均附着数分别为11.96和2 .4 4 ;从上述菌株细胞荚膜中分别提取荚膜蛋白,用SDS- PAGE分离测定两菌株荚膜蛋白质结构,结果表明39k Da荚膜蛋白是强毒菌的特异性蛋白。以上研究结果证明Pm的毒力与He L a细胞的附着能力是密切相关的,同时暗示本菌39k Da荚膜蛋白可能与它们的毒力和He L a细胞的附着能力有关     

Pathogenicity of field isolates of Erysipelothrix rhusiopathiae in mice, rats and pigs

Eight field isolates of Erysipelothrix rhusiopathiae serotypes 1 and 2, from different sources, were examined for their pathogenicities for mice and pigs. Arthritogenicity for pigs correlated with virulence for mice at the highest and lowest levels, but not with strains of intermediate virulence. The most virulent strain was also arthritogenic in rats. In pigs, after repeated intravenous challenge the number of affected joints ranged from 0 to 11 of 12 examined. For the 8 strains, the mean number of affected joints ranged from 1 to 7.7 per pig. Clinical course and pathological findings were correlated, but the onset, severity and duration of lameness was variable both within and between groups. Clinical lameness, joint swelling and urticariae were of limited use as indicators of joint changes. The more virulent strains caused lameness as early as 2 days, whereas strains of low virulence took up to 8 weeks.     

The physiological and biochemical response of standardbred horses to exercise of varying speed and duration

LINDHOLM, ARNE and BENGT SALTIN: The physiological and biochemical response of standardbred horses to exercise of varying speed and duration. Acta vet. scand. 1974, 15, 310–324. — Welltrained standardbred horses were studied to examine the metabolic response to excercise of various speeds and duration. Comparisons between interval (400, 700, 1,000 and 2,000 m) and continuous trotting (1 hr., 2 hrs.) and racing were made.Muscle and rectal temperatures were recorded before and immediately after each work bout. Heart rate was linearly related to trotting speed, and maximal heart rate (240 beats × min.⁻¹) was achieved when trotting at least 700 m at close to maximal speed (12.0–12.5 m×sec.⁻¹).Biopsy specimens from the gluteus medius muscle and venous blood were obtained before and after each work bout.Muscle and blood lactate values were markedly increased first at speeds close to maximal speed (11.4–12.5 m×sec.⁻¹). Trotting 6×700 m at 12.5 m×sec.⁻¹ produced as high muscle and blood lactate values as 23.7 and 19.0 mmol×kg⁻¹ wet weight and l⁻¹, respectively. Corresponding values after a race were about 15 mmol×kg⁻¹ (muscle) and l⁻¹ (blood).Glycogen utilization was related to work intensity and was most pronounced during the first work bouts. At a speed of 12 m×sec.⁻¹ and trotting 2000 m, there was a glycogen utilization of near 12 mmol glucose units × kg⁻¹ × min.⁻¹ wet muscle.It is concluded that interval training over a distance of 700–1000 m repeated 4–6 times with a trotting speed close to maximal speed (11.4–12.5 m×sec.⁻¹) appears to be optimal.ATP; CP; blood lactate; glycogen utilization; heart rate; horse skeletal muscle; muscle lactate; racing training.     

Virulence genes and plasmid profiles in Rhodococcus equi isolates from domestic pigs and wild boars (Sus scrofa) in Brazil

The virulence genes and plasmid profiles of 23 Rhodococcus equi isolates from 258 lymph nodes from domestic pigs (129 nodes with lesions and 129 without lesions) and 120 lymph nodes from slaughtered wild boars (60 nodes with lesions and 60 without) were characterized. R. equi was obtained from 19 lymph nodes of domestic pigs, 17 with, and two without lesions, and from four lymph nodes with lesions, from wild boars. The 23 isolates were tested for the presence of vapA and vapB genes, responsible for the 15–17 and 20 kDa virulence-associated proteins, respectively, by PCR in order to characterize as virulent (VapA), intermediately virulent (VapB) and avirulent. Plasmid DNAs were isolated and analyzed by digestion with restriction endonucleases to estimate size and compare their polymorphisms. Of the 19 domestic pigs strains, seven (36.8%) were avirulent and 12 (63.2%) were intermediately virulent, with the intermediately virulent isolates being plasmid types 8 (8 isolates), 10 (2 isolates), 1 (1 isolate) and 29 (1 isolate). The plasmid type of four strains isolated from wild boars was also intermediately virulent type 8. None of the domestic pigs and wild boar isolates showed the vapA gene. These findings demonstrate a high occurrence of plasmid type 8 in isolates from pigs and wild boars, and the similarity of plasmid types in the domestic pigs, wild boars and human isolates in Brazil.     

Some characteristics of four attenuated vaccine virus strains and a virulent strain of Aujeszky's disease virus

Summary

In the present study four attenuated virus strains, used as vaccines, and a virulent strain of Aujeszky's disease virus (AD V) were compared with respect to their virulence in mice, their ability to induce virus‐specified thymidine kinase (TK) in infected cells, and their cleavage profiles of viral DNA's after treatment with the restriction endonuclease Kpnl.

The survival time of mice inoculated with the B‐KAL or the virulent NIA‐3 strain was comparable. whereas the Hanha and BUK strains required significantly loniser periods to kill mice. Mice were resistant to the MK‐25 strain of ADV.

The strains were assayed for TK phenotype by plaque autoradiography after ³H‐thymidine labelling of infected cells. MK‐25 proved to be the only strain defective in induction of TK in pig kidney cells. Restriction endonuclease analysis of viral DNA's revealed that each vaccine strain showed a characteristic fragment pattern that could easily be differentiated from that of other vaccine and field strains of ADV. The present results demonstrate that the mouse virulence lest and the TK assay detect differences in biological properties of ADV strains, but that restriction endonuclease analysis is required for unambiguous identification of vaccine and field strains of ADV.     

Evidence that the 36 kb plasmid of Brachyspira hyodysenteriae contributes to virulence

Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently the genome of virulent Australian B. hyodysenteriae strain WA1 was sequenced, and a 36 kilobase (kb) circular plasmid was identified. The plasmid contained 31 genes including six rfb genes that were predicted to be involved with rhamnose biosynthesis, and others associated with glycosylation. In the current study a set of PCRs was developed to amplify portions of nine of the plasmid genes. When used with DNA extracted from virulent strain B204, PCR products were generated, but no products were generated with DNA from avirulent strain A1. Analysis of the DNA using pulsed field gel electrophoresis (PFGE) identified a plasmid band in strains WA1 and B204, but not in strain A1. These results demonstrate that strain A1 does not contain the plasmid, and suggests that lack of the plasmid may explain why this strain is avirulent. To determine how commonly strains lacking plasmids occur, DNA was extracted from 264 Australian field isolates of B. hyodysenteriae and subjected to PCRs for three of the plasmid genes. Only one isolate (WA400) that lacked the plasmid was identified, and this absence was confirmed by PFGE analysis of DNA from the isolate and further PCR testing. To assess its virulence, 24 pigs were experimentally challenged with cultures of WA400, and 12 control pigs were challenged with virulent strain WA1 under the same conditions. Significantly fewer (P=0.03) of the pigs challenged with WA400 became colonised and developed SD (13/24; 54%) compared to the pigs infected with WA1 (11/12; 92%). Gross lesions in the pigs colonised with WA400 tended to be less extensive than those in pigs colonised with WA1, although there were no obvious differences at the microscopic level. The results support the likelihood that plasmid-encoded genes of B. hyodysenteriae are involved in colonisation and/or disease expression.     

Evaluation of the live vaccine efficacy of virulence plasmid-cured,and phoP- or aroA-deficient Salmonella enterica serovar Typhimurium in mice

We evaluated the protective efficacy of 94-kb virulence plasmid-cured, and phoP- or aroA-deficient strains of Salmonella enterica serovar Typhimurium (ΔphoP or ΔaroA S. Typhimurium) as oral vaccine candidates in BALB/c mice. Two weeks after the completion of 3 oral immunizations with 1 × 10⁸ colony-forming units (CFU) of virulence plasmid-cured, and ΔphoP or ΔaroA S. Typhimurium at 10-day intervals, S. Typhimurium lipopolysaccharide (LPS)-specific mucosal secretory immunoglobulin A (s-IgA) antibody titers were detected in the cecal homogenate, bile and lung lavage fluid, but not in the intestinal lavage fluid. In addition, the increases in S. Typhimurium LPS-specific immunoglobulin G (IgG) and IgA antibody titers in the serum were also observed 2 weeks after completing 3 oral immunizations with virulence plasmid-cured, and ΔphoP or ΔaroA S. Typhimurium. The series of 3 oral immunizations protected the mice against an oral challenge with 5 × 10⁸ CFU of the virulent strain of S. Typhimurium, suggesting that both the virulence plasmid-cured, and ΔphoP and ΔaroA S. Typhimurium strains are promising candidates for safe and effective live S. Typhimurium vaccines.     

Deduced Amino Acid Sequences Surrounding the Fusion Glycoprotein Cleavage Site and of the Carboxyl-terminus of Haemagglutinin–Neuraminidase Protein of the Avirulent Thermostable Vaccine Strain I-2 of <Emphasis Type="Italic">Newcastle disease virus</Emphasis>

A single-tube RT-PCR technique generated a 387 bp or 300 bp cDNA amplicon covering the F₀ cleavage site or the carboxyl (C)-terminus of the HN gene, respectively, of Newcastle disease virus (NDV) strain I-2. Sequence analysis was used to deduce the amino acid sequences of the cleavage site of F protein and the C-terminus of HN protein, which were then compared with sequences for other NDV strains. The cleavage site of NDV strain I-2 had a sequence motif of ¹¹² RKQGRLIG¹¹⁹, consistent with an avirulent phenotype. Nucleotide sequencing and deduction of amino acids at the C-terminus of HN revealed that strain I-2 had a 7-amino-acid extension (VEILKDGVREARSSR. This differs from the virulent viruses that caused outbreaks of Newcastle disease in Australia in the 1930s and 1990s, which have HN extensions of 0 and 9 amino acids, respectively. Amino acid sequence analyses of the F and HN genes of strain I-2 confirmed its avirulent nature and its Australian origin.     

Virulence of Salmonella enteritidis phagetypes 4, 8 and 13 and other Salmonella spp. for day-old chicks, hens and mice.

Virulence of three Canadian poultry strains of Salmonella enteritidis, namely phagetypes (PT) 4, 8 and 13, and one Salmonella heidelberg strain was assessed in orally and intraperitoneally inoculated one-day old chickens and compared to the virulence of a human S. enteritidis PT 4 strain from the United Kingdom (UK). The two PT 4 strains were also compared in orally inoculated adult laying hens. In addition, orally inoculated Balb/c mice were used to evaluate virulence of the above strains and two strains of Salmonella typhimurium containing different plasmids. In orally inoculated one-day old chickens, the UK S. enteritidis PT 4 strain was more virulent than the Canadian PT 4 strain. The UK PT 4 strain was also more virulent and invasive in adult laying hens than the Canadian PT 4 strain. The S. enteritidis PT 8 strain and one S. typhimurium strain isolated from a chicken hatchery were the most virulent for orally inoculated Balb/c mice. This strain of S. typhimurium contained the 60 megadalton plasmid associated with virulence for Balb/c mice which was not present in the S. typhimurium strain isolated from a pig with septicemic disease.     

Toxoplasma gondii virulence prediction using hierarchical cluster analysis based on coding sequences (CDS) of sag1, gra7 and rop18

Toxoplasma gondii consists of three genotypes, namely genotype I, II and III. Based on its virulence, T. gondii can be divided into virulent and avirulent strains. This study intends to evaluate an alternative method for predicting T. gondii virulence using hierarchical cluster analysis based on complete coding sequences (CDS) of sag1, gra7 and rop18 genes. Dendrogram was constructed using UPGMA with a Kimura 80 nucleotide distance measurement. The results showed that the prediction errors of T. gondii virulence using sag1, gra7 and rop18 were 7.41%, 6.89% and 9.1%, respectively. Analysis based on CDS of gra7 and rop18 was able to differentiate avirulent strains into genotypes II and III, whereas sag1 failed to differentiate.     

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10⁹ CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID₅₀ of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 10⁹ CFU of p3D-NT56/S. cho were protected in 9 days.     

Isoenzymes of lactate dehydrogenase in swine. Stability during storage at different temperatures and by heat treatment

The isoenzymes of lactate dehydrogenase (LDH) in serum from normal pigs were studied after separation by agar gel electrophoresis with subsequent staining with a tetrazolium salt.Experiment 1. The stability of isoenzymes was investigated for 5 successive days after storage at room temperature (22°C), in the refrigerator (4°G), and once after storage for 32 days in the deep-freezer (—20°C). Greatest loss of activity was seen after storage in the refrigerator, where LDH₂ and LDH₃ lost most of its activity after 5 days. In LDH₄ and LDH₅ no loss had occurred at this time. Also at room temperature great losses were seen in LDH₂ and LDH₃. After storage in the deep-freezer an increase in LDH₃ activity was recorded.Experiment 2. Serum samples were kept in water baths for 30 min. at 50, 53, and 56°C. A simultaneous and increasing loss in activity of LDH₃, LDH₄, and LDH₅ was seen from 50° to 56°C. At 56° no activity was left in LDH₃, LDH₄, or LDH₅, and only about 15 % of the original activity was present in LDH₂. LDH₁ showed no loss at 56°, but all activity was lost at 65°.A close correlation was found between total lactate dehydrogenase and α-hydroxybutyrate dehydrogenase activity in both experiments.     

Infection of the chicken with a virulent or avirulent strain of Mycoplasma gallisepticum alone and together with Newcastle disease virus or E. coli or both

The influence of the virulence of M. gallisepticum (Mg) was studied in multiple infections of chickens involving Mg, Newcastle disease virus (NDV) and E. coli. Separate groups of 3-week-old chickens were inoculated supra-conjuctivally with a virulent and an avirulent strain of Mg alone and in combination with the La Sota strain of NDV, the 01 serotype of E. coli, and both NDV and E. coli. In addition, chickens were inoculated with NDV alone, E. coli alone, and with NDV and E. coli; one group was left uninfected.Clinical signs, lesions, recovery of the pathogens and the serological response were observed for all groups for 3 weeks after infection and for those involving Mg alone and Mg together with NDV for 18 weeks.No clinical signs were seen in any of the birds; in each infected group some showed mild lesions of the trachea and air sacs without any marked difference among the groups.Although the numbers of birds examined were small, the virulent strain of Mg was more readily recovered than the avirulent, from the respiratory tract, in the first 3 weeks following multiple infections. However, the virulence of Mg had no influence on the recovery of the other pathogens.For the first 6 weeks after infection there was a direct relationship between the virulence of the Mg and the proportion of birds with agglutinins to the Mg rapid serum agglutination (RSA) test in single or multiple infections; multiple infections enhanced the antibody response to both virulent and avirulent mycoplasma. For NDV, multiple infections, particularly involving E. coli, enhanced the peak titre of haemagglutination-inhibition antibodies and accelerated their appearance; the virulence of the Mg had no apparent effect on this.Neither the mycoplasma nor NDV were detected in the trachea by immunofluorescence.     

Comparison of Cellular and Extracellular Products Expressed by Virulent and Attenuated Strains of Edwardsiella ictaluri

Abstract

Cellular and extracellular products of three virulent Edwardsiella ictaluri isolates were compared with corresponding attenuated strains to evaluate potential virulence factors. The characteristics compared included growth kinetics, hemolysin activity, surface structures, outer membrane protein profiles, and lipopolysaccharide profiles. To produce the attenuated strains, we passed three isolates through multiple subcultures in liquid media. Attenuated strains were found to have shorter generation times than virulent strains. They also apparently failed to express a 55-kdalton outer membrane protein that the virulent strains possessed. Scanning electron microscopy found no conclusive differences in surface structure expression. Hemolysin activity was significantly greater in virulent strain R4383 than in its corresponding attenuated strain. Lipopolysaccharide profiles showed no apparent differences in the O polysaccharide portion; however, the composition of core oligosaccharide sugars differed between the two.