Experimental Infection Of Piglets Research Articles

The Impact of SsPI-1 Deletion on Streptococcus suis Virulence.

(1) Background: Streptococcus suis is an important zoonotic pathogen that infects pigs and can occasionally cause life-threatening systemic infections in humans. Two large-scale outbreaks of streptococcal toxic shock-like syndrome in China suggest that the pathogenicity of S. suis has been changing in recent years. Genetic analysis revealed the presence of a chromosomal pathogenicity island (PAI) designated SsPI-1 in Chinese epidemic S. suis strains. The purpose of this study is to define the role of SsPI-1 in the virulence of S. suis. (2) Methods: A SsPI-1 deletion mutant was compared to the wild-type strain regarding the ability to attach to epithelial cells, to cause host disease and mortality, and to stimulate host immune response in experimental infection of piglets. (3) Results: Deletion of SsPI-1 significantly reduces adherence of S. suis to epithelial cells and abolishes the lethality of the wild-type strain in piglets. The SsPI-1 mutant causes no significant pathological lesions and exhibits an impaired ability to induce proinflammatory cytokine production. (4) Conclusions: Deletion of the SsPI-1 PAI attenuates the virulence of this pathogen. We conclude that SsPI-1 is a critical contributor to the evolution of virulence in epidemic S. suis.

Limited shedding of an S-InDel strain of porcine epidemic diarrhea virus (PEDV) in semen and questions regarding the infectivity of the detected virus

PEDV is mainly transmitted by the oro-fecal route although PEDV shedding in semen has already been shown for an S-non-InDel PEDV strain infection. The aim of this study was to determine if PEDV can be shed in semen from SPF (specific pathogens free) boars infected by a French S-InDel PEDV strain (PEDV/FR/001/2014) and in case of positive semen to determine the infectivity of that semen. Both infected boars had diarrhea after inoculation and shed virus in feces. PEDV genome was also detected by RT-qPCR in the sperm-rich fraction of semen (6.94 × 103 and 4.73 × 103 genomic copies/mL) from the two boars infected with the S-InDel PEDV strain but only once at 7DPI. In addition, PEDV RNA in Peyer’s patches and in mesenteric lymph nodes was also present for the two inoculated boars. The PEDV positive semen (S-non-InDel and S-InDel) sampled during a previous trial and in this boar trial were inoculated to six SPF weaned pigs. The inoculated piglets did not seroconvert and did not shed virus throughout the duration of the study except for one pig at 18 DPI. But, PEDV could be detected in intestinal tissues such as duodenum, jejunum and jejunum Peyer’s patches by RT-qPCR except for one pig. Even if PEDV genome has been detected in semen, experimental infection of piglets with positive semen failed to conclude to the infectivity of the detected PEDV.

A new porcine reproductive and respiratory syndrome virus strain with highly conserved molecular characteristics in its parental and attenuated strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes substantial economic losses to many swine-producing regions. In this study, PRRSV strain NT0801-F80 was derived from its parental isolate NT0801 by 80 passages in Marc-145 cells. Experimental infection of piglets clearly demonstrated that strain NT0801-F80 is less virulent than NT0801. However, whole genome sequencing showed that the genomes of the parental and attenuated strains are highly conserved compared with those of four other pairs of virulent parental/attenuated vaccine strains (VR2332 and RespPRRS MLV, JA142 and Ingelvac(®) ATP MLV, CH-1a and CH-1R, and JXA1 and JXAR). The attenuated strain NT0801-F80 has only 21 nucleotide changes, producing only 14 amino acid changes in NSP2, GP2, GP3, and GP5, compared with those aa sequences of the virulent parental strain. These mutated aa in the attenuated virus may be involved in virulence. These data provide valuable information on the attenuation mechanism of PRRSV that should be useful in future research.

Comparative analysis of apoptotic changes in peripheral immune organs and lungs following experimental infection of piglets with highly pathogenic and classical porcine reproductive and respiratory syndrome virus

BackgroundOur previous studies have demonstrated that piglets infected with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) may develop significant thymus atrophy, which related to thymocytes apoptosis. However, apart from that detected in the thymus, there are no reports describing cell apoptosis induced by HP-PRRSV infection. In this study, we analyzed comparatively the pathological changes, cell apoptosis and viral load in peripheral immune organs including tonsil, inguinal lymph nodes (ILNs) and spleen and lungs following experimental infection of piglets with HP-PRRSV HuN4 and classical PRRSV CH-1a.FindingsHP-PRRSV HuN4 exhibited much stronger cell tropism than CH-1a in immune organs and lungs of piglets. HuN4 infection led to the serious injuries in tonsils, ILNs, spleens and lungs, especially apoptosis in these organs was significant.ConclusionsHuN4 infection induced severe lesions (gross pathology, histopathology and cell apoptosis) in the peripheral immune organs and lungs of infected piglets. Large numbers of apoptotic cells in immune organs and lung induced by HuN4 may play a role in the pathogenesis of the HP-PRRS and the distinct injuries caused by HuN4 infection may be associated with the high mortality rate of HP-PRRS in pigs.

The Orphan Response Regulator CovR: a Globally Negative Modulator of Virulence inStreptococcus suisSerotype 2

Streptococcus suis serotype 2 is an emerging zoonotic pathogen responsible for a wide range of life-threatening diseases in pigs and humans. However, the pathogenesis of S. suis serotype 2 infection is not well understood. In this study, we report that an orphan response regulator, CovR, globally regulates gene expression and negatively controls the virulence of S. suis 05ZYH33, a streptococcal toxic shock syndrome (STSS)-causing strain. A covR-defective (DeltacovR) mutant of 05ZYH33 displayed dramatic phenotypic changes, such as formation of longer chains, production of thicker capsules, and increased hemolytic activity. Adherence of the DeltacovR mutant to epithelial cells was greatly increased, and its resistance to phagocytosis and killing by neutrophils and monocytes was also significantly enhanced. More importantly, inactivation of covR increased the lethality of S. suis serotype 2 in experimental infection of piglets, and this phenotype was restored by covR complementation. Colonization experiments also showed that the DeltacovR mutant exhibited an increased ability to colonize susceptible tissues of piglets. The pleiotropic phenotype of the DeltacovR mutant is in full agreement with the large number of genes controlled by CovR as revealed by transcription profile analysis: 2 genes are positively regulated, and 193 are repressed, including many that encode known or putative virulence factors. These findings suggested that CovR is a global repressor in virulence regulation of STSS-causing S. suis serotype 2.

SalK/SalR, a Two-Component Signal Transduction System, Is Essential for Full Virulence of Highly Invasive Streptococcus suis Serotype 2

Background Streptococcus suis serotype 2 (S. suis 2, SS2) has evolved into a highly infectious entity, which caused the two recent large-scale outbreaks of human SS2 epidemic in China, and is characterized by a toxic shock-like syndrome. However, the molecular pathogenesis of this new emerging pathogen is still poorly understood.Methodology/Principal Findings89K is a newly predicted pathogenicity island (PAI) which is specific to Chinese epidemic strains isolated from these two SS2 outbreaks. Further bioinformatics analysis revealed a unique two-component signal transduction system (TCSTS) located in the candidate 89K PAI, which is orthologous to the SalK/SalR regulatory system of Streptococcus salivarius. Knockout of salKR eliminated the lethality of SS2 in experimental infection of piglets. Functional complementation of salKR into the isogenic mutant ΔsalKR restored its soaring pathogenicity. Colonization experiments showed that the ΔsalKR mutant could not colonize any susceptible tissue of piglets when administered alone. Bactericidal assays demonstrated that resistance of the mutant to polymorphonuclear leukocyte (PMN)-mediated killing was greatly decreased. Expression microarray analysis exhibited a transcription profile alteration of 26 various genes down-regulated in the ΔsalKR mutant.Conclusions/SignificanceThese findings suggest that SalK/SalR is requisite for the full virulence of ethnic Chinese isolates of highly pathogenic SS2, thus providing experimental evidence for the validity of this bioinformatically predicted PAI.

Investigations of influence of colostral antibodies on development of pathomorphological changes following experimental infection of piglets with classical swine plague virus

The pathomorphological changes established following infection with the virus of classical swine plague in non-immune individuals are well known. However, piglets present a problem from the clinical-pathological aspect, in which this virus can be multiplied in spite of established colostral antibodies, but without the exhibiting of the clinical symptoms characteristic for the disease. The question of the characteristics of the pathomorphological finding is raised in the event of the breakdown of the colostral immunity of the piglets. With the objective of determining the influence of colostral antibodies on the development of pathomorphological changes in classical swine plague, piglets aged 28, 35, 44, and 54 days, originating from sows that had received a vaccine of the K-strain of the classical swine plague virus, were experimentally infected with a virulent variety of this virus (Becker strain). The control group comprised non-vaccinated animals originating from non-vaccinated sows. Following the death and/or sacrificing of the piglets in the experiment, a pathomorphological examination was performed of all organic systems and the presence of classical swine plague virus antigens was established in organs and tissues of piglets using the immunoenzyme (ELISA) test. Even though clinical signs characteristic for this disease were not found in all animals following artificial infection, the pathomorphological findings following death and/or sacrificing indicated a successful experimental infection and was typical for the acute course of classical swine plague. Bleeding was established in most organs and serous membranes (haemorrhagic diathesis). However, certain variations were also established regarding the expression and distribution of the pathomorphological changes in certain animals.

Effects of Porcine Circovirus Type 2 (PCV2) Maternal Antibodies on Experimental Infection of Piglets with PCV2

To determine the effects of porcine circovirus type 2 (PCV2) maternal antibodies on and response to experimental PCV2 infection, 24 piglets were divided into four groups on the basis of the enzyme-linked immunosorbent assay titers of PCV2 maternal antibodies: group A (n = 6; sample/positive [S/P] ratio, <0.2), group B (n = 5; S/P ratio, >0.2 to <0.5), and groups C (n = 8) and D (n = 5) (S/P ratio, >0.5). Piglets in groups A, B, and C were inoculated with PCV2 at day 0 and challenged with PCV2 at day 42. Group D piglets were not exposed to PCV2 at day 0 but were challenged at day 42. Before challenge, seroconversion to PCV2 antibodies occurred in five of six group A piglets, and the antibody level rose above the cutoff level in one of five group B piglets. Viremia was detected in five of six, four of five, and two of eight pigs in groups A, B, and C, respectively. After challenge, PCV2 DNA was detectable from 7 to 21 days postchallenge in the sera from six of six, four of five, three of eight, and five of five pigs in groups A, B, C, and D, respectively. The results indicated that protection against PCV2 infection conferred by maternal antibodies is titer dependent: higher titers are generally protective, but low titers are not.

Effect of diet composition on postweaning colibacillosis in piglets.

The weaning of piglets is often associated with digestive disorders, particularly diarrhea--postweaning colibacillosis (PWC)--which is caused by infection with enterotoxigenic strains of Escherichia coli. It has been shown previously that a diet for newly weaned pigs based on cooked white rice and animal protein decreases the occurrence of PWC, whereas the addition of carboxymethylcellulose (CMC) to this diet enhances PWC. The aims of the current work were to 1) determine whether substitution of animal protein with plant proteins in the cooked-white-rice diet influenced its protective effects on PWC and 2) confirm that an increase in viscosity of the digesta by adding CMC to the diet favors the development of PWC--with (Exp. 1) or without (Exp. 2) experimental infection of piglets with E. coli. The diets were 1) cooked white rice and animal protein sources (RAP), 2) RAP + CMC added at 40 g of CMC/kg (air-dry basis) of diet, 3) cooked white rice and plant protein sources (RPP), and 4) wheat and plant protein sources (WPP). Experiments 1 and 2 were conducted using 32 and 24 piglets (eight and six per treatment), respectively. Piglets were weaned at 21 d (d 1), and fed ad libitum until slaughter on d 9. In Exp. 1, piglets were orally infected with enterotoxigenic E. coli on d 4, 5, 6, and 7. On d 8 of Exp. 1, the E. coli scores in feces of pigs fed RAP + CMC were higher than with RAP (P < 0.01). On d 9 after weaning, feces from pigs fed diet RAP were normal or moist, whereas feces from pigs fed RAP + CMC were wet to diarrheic. On d 7 of Exp. 2, pigs fed diets RAP + CMC and WPP had wetter feces than pigs fed diets RAP or RPP (P < 0.05). On d 8, the E. coli scores in feces were higher (P < 0.01) with pigs fed RAP + CMC than with all other diets. The E. coli scores in the digesta were also higher with pigs fed RAP + CMC, and to a lesser extent with diet WPP, than with pigs fed RAP or RPP (P < 0.01). The large intestine was heavier in pigs fed diets RPP and WPP, and the digesta were more acidic (P < 0.05). This study confirmed that diet RAP was protective against PWC, and that substitution of animal proteins with plant protein in a rice-based diet did not diminish its protective effects. The addition of CMC to cooked white rice increased digesta viscosity and enhanced PWC. Consequently, this diet represents a useful model for studying this condition.

Experimental Infection of Piglets with Transmissible Gastroenteritis Virus: a Comparison of Three Strains (Korean, Purdue and Miller)

Eighty-four colostrum-deprived piglets aged 1 day were inoculated with either a Korean strain of transmissible gastroenteritis virus (TGEV) or one of two American strains (Purdue and Miller). The purpose was to compare, by morphometric analysis and in-situ hybridization, the Korean strain with the two American strains in respect of pathogenicity and viral distribution over a period of 72 h. The progression of infection in pigs infected with the Korean strain was much slower than that in pigs infected with either of the two American strains. At 6 h post-inoculation (hpi), the mean score of the villous height/crypt depth (VH/CD) ratio of pigs inoculated with the Purdue or Miller strain was significantly less than that of pigs inoculated with the Korean strain or uninfected control pigs. At 12–72 hpi, however, the VH/CD ratio of all infected pigs was significantly less than that of control pigs. In-situ hybridization showed significant differences between the Korean and American strains in terms of the amount of TGEV nucleic acid at 6, 12 and 60 hpi. TGEV nucleic acid was detected in the duodenum, jejunum and ileum of pigs inoculated with the Purdue or Miller strain but only in the jejunum and ileum of those inoculated with the Korean strain. The results suggested that the Korean strain was less virulent than the two American strains.

Environmentally regulated genes of Streptococcus suis: identification by the use of iron-restricted conditions in vitro and by experimental infection of piglets.

The identification of environmentally regulated genes of Streptococcus suis by the use of iron-restricted conditions in vitro and by experimental infection of piglets is described. Eighteen unique iron-restriction-induced (iri) genes and 22 unique in-vivo-selected (ivs) genes of Strep. suis were found. None of the ivs genes was exclusively expressed in vivo. Four iri genes were identical to four clones selected in piglets. Two ivs genes were similar to genes for putative virulence factors. One of these ivs genes was identical to the epf gene of virulent Strep. suis serotype 2 strains and the other showed homology to a gene encoding a fibronectin-binding protein of Streptococcus gordonii. Two additional ivs genes showed homology to environmentally regulated genes previously identified by using an in vivo expression technology (IVET) selection system in other bacterial species. One of these showed similarity to the agrA gene of Staphylococcus aureus, a key locus involved in the regulation of numerous virulence proteins. The promoter selection system described in this paper has been successfully used for the identification of many environmentally regulated genes potentially involved in the pathogenesis of Strep. suis infections in piglets.

Presence of 22-kDa protein reacting with sera in piglets experimentally infected with Brachyspira hyodysenteriae

In order to examine the effect of spectinomycin on outbreaks of swine dysentery, experimental infection of piglets with Brachyspira hyodysenteriae was carried out. Feed with and without spectinomycin (SP) was given to each piglet ad libitum and the susceptibility of the piglets to infection with B. hyodysenteriae was compared between SP-treated and untreated piglets. The results showed that the SP-treated piglets did not display clinical signs of swine dysentery unlike the untreated piglets. The sera obtained from these piglets were examined by the microscopic agglutination test and antibodies to B. hyodysenteriae in both groups of experimentally infected piglets were detected and the reaction was serogroup-specific. The agglutination titers were very high in the untreated piglets with dysentery while the titers in the SP-treated piglets were lower than those in the untreated piglets. In addition, the immunoblotting technique was applied and the results demonstrated that 22- and 17-kDa proteins in strain ATCC 31212 (serogroup B) reacted strongly with the sera from the untreated piglets but not with the sera from the SP-treated piglets. The 22- and 17-kDa proteins also reacted with strain ATCC 27164 (serogroup A) which belongs to a different serogroup. The 22- and 17-kDa proteins were also confirmed in six other strains of B. hyodysenteriae which belong to six different serogroups. These proteins were sensitive to proteinase K. These results indicate that the 22- and 17-kDa proteins are common to eight strains of B. hyodysenteriae which differ serologically from each other.

Presence of 22-kDa protein reacting with sera in piglets experimentally infected with Brachyspira hyodysenteriae.

In order to examine the effect of spectinomycin on outbreaks of swine dysentery, experimental infection of piglets with Brachyspira hyodysenteriae was carried out. Feed with and without spectinomycin (SP) was given to each piglet ad libitum and the susceptibility of the piglets to infection with B. hyodysenteriae was compared between SP-treated and untreated piglets. The results showed that the SP-treated piglets did not display clinical signs of swine dysentery unlike the untreated piglets. The sera obtained from these piglets were examined by the microscopic agglutination test and antibodies to B. hyodysenteriae in both groups of experimentally infected piglets were detected and the reaction was serogroup-specific. The agglutination titers were very high in the untreated piglets with dysentery while the titers in the SP-treated piglets were lower than those in the untreated piglets. In addition, the immunoblotting technique was applied and the results demonstrated that 22- and 17-kDa proteins in strain ATCC 31212 (serogroup B) reacted strongly with the sera from the untreated piglets but not with the sera from the SP-treated piglets. The 22- and 17-kDa proteins also reacted with strain ATCC 27164 (serogroup A) which belongs to a different serogroup. The 22- and 17-kDa proteins were also confirmed in six other strains of B. hyodysenteriae which belong to six different serogroups. These proteins were sensitive to proteinase K. These results indicate that the 22- and 17-kDa proteins are common to eight strains of B. hyodysenteriae which differ serologically from each other.

Neuronal spread of swine hemagglutinating encephalomyelitis virus (HEV) 67N strain in 4-week-old rats.

The HEV 67N strain causes encephalomyelitis or vomiting and wasting syndrome in piglets 1,2. In experimental infection of piglets, the virus spreads along the nerve pathways to the central nervous system (CNS), and is restricted to the neurons. In our experimental studies of HEV 67N strain, the virus produced encephalitis in mice when inoculated by several routes, and propagated mainly in the neurons in the CNS 4. However, 20-day-old or older mice were resistant to the virus inoculated by intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) route. In contrast, 4-week-old rats died of encephalitis after i.v., i.p. and s.c. as well as intracerebral (i.c.) and intranasal (i.n.) inoculation. However, when rats were inoculated by s.c. route, they died a few days earlier than those by i.p. and i.v. routes, suggesting that the virus might be spread to the CNS by neural routes rather than blood stream. To see the virus spread from the peripheral nerve to the brain, the virus was directly inoculated in to the sciatic nerve of rats. These studies demonstrated that HEV spread from the sciatic nerve to the brain by the neural route, and that persistent infection of HEV was established in rats6.

Experimental infection of piglets with Cryptosporidium

Piglets from five litters were doses orally with cryptosporidium originally derived from diarrhoeic calves. The piglets were either nursed by the sow, artificially reared after sucking colostrum, or weaned on to creep feed. Colostrum-fed, artificially reared piglets obtained from two litters and exposed in the first week of life developed clinical signs of inappetence, vomiting and diarrhoea and shed oocysts in the faeces. Histologically the parasite was observed throughout the small and large intestine attached to epithelial cell surfaces and its presence was associated with extensive mucosal damage, particularly in the posterior small intestine, stunting and fusion of villi, immaturity of villous epithelial cells and oedema with increased cellularity of the lamina propria. Piglets from two other litters, both sucking and colostrum-fed artificially reared, exhibited similar but milder clinical signs. Histological lesions were less severe and cryptosporidium infection less extensive. When weaned piglets were exposed they remained clinically healthy although histologically there was evidence of cryptosporidium attachment in the small intestine and minor mucosal damage. There appears to be a good correlation between the extent of intestinal infection, the degree of mucosal damage and the severity of clinical disease induced by cryptosporidium in piglets.

The experimental infection of piglets with a porcine reovirus

A strain (Quebec) of reovirus isolated from the faeces of a pig with dysentery was neutralised by reovirus type 1 antiserum. Four of eight hysterectomy-produced, colostrum-deprived (HPCD) piglets dosed orally with the third cell culture passage of the virus developed diarrhoea and showed focal areas of villous atrophy in the small intestine. The virus was isolated from the intestinal tract of all eight specific pathogen free piglets, but not from three control animals. Nine germ-free piglets dosed orally with the eight cell culture passage of the virus showed neither clinical signs nor lesions, but virus was recovered from their intestinal tracts for 14 days after infection. No virus was isolated from four control germ-free piglets.

The Effect of Colostrum Derived Antibody on Mortality and Virus Excretion Following Experimental Infection of Piglets with Aujeszky’s Disease Virus

Sows experimentally infected with Aujeszky’s disease virus (ADV) and allowed to recover were mated. Antibody to ADV was detected 11 days following infection, reached a peak by day 34 and remained steady throughout pregnancy and parturition. Antibody in the colostrum had a titre 11–16 fold greater than circulating antibody but this fell rapidly within 24 h. The piglets were experimentally infected with ADV on the third day of life and, in spite of the maternal antibody, all excreted virus for up to 20 days. Antibody protected the piglets from mortality, all litters with a geometric mean neutralizing antibody titre (GMT) of » 1/380 survived, those with a lower GMT had some deaths and all piglets without antibody died. The half life of the maternal antibody was 8.5 days and no production of antibody was detected in the piglets during the course of the experiment. However 4 sows developed a marked antibody boost 64 and 80 days after parturition and one litter in contact with these sows also developed antibody around the same period.

Experimental infection of piglets and pregnant gilts with a parvovirus.

Experimental infection of piglets and pregnant gilts with a parvovirus.