Cervical lymphadenitis in guinea pigs: infection via intact ocular and nasal mucosa by Streptococcus zooepidemicus.

The traditional view regarding the pathogenesis of cervical lymphadenitis in guinea pigs is that Lancefield Group C Streptococcus gains access to cervical lymph nodes via an abraded oral mucosa. In this study, it is established that inoculation of intact nasal and conjunctival mucous membranes with Streptococcus zooepidemicus (Lancefield Group C) also can produce the disease. Weanling (SPF) guinea pigs (Cavia porcellus) were divided into two experimental groups of 10 and two control groups of four each. Guinea pigs from each group were individually housed in separate cubicles. Group I was inoculated with 0.05 ml of culture containing 2.8 x 10(7) CFU/ml of S. zooepidemicus into the conjunctiva of the left eye. Group II received a similar inoculum into the left nares. Control groups received 0.05 ml of TSB broth in the same sites. Five of ten guinea pigs in Group II died four to nine days postinoculation. Surviving guinea pigs were euthanatized at intervals between days 4-13 postinoculation. All guinea pigs were necropsied, cultured and examined for evidence of infection. S. zooepidemicus was recovered from 30/50 and 39/46 sites cultured from Groups I and II, respectively. Lymphadenitis was found in cervical lymph nodes from 8/10 guinea pigs in Group I and 10/10 in Group II. The conjunctival and nasal mucosa, therefore, represent potential sites of entry resulting in cervical lymphadenitis in guinea pigs.     

Role of the Salmonella abortusovis virulence plasmid in the infection of BALB/c mice

Following oral inoculation of BALB/c mice, Salmonella abortusovis strain SS44 was recovered in lower numbers from the Peyer's patches and mesenteric lymph nodes compared with S. typhimurium strain SL1344, whereas splenic infections were equivalent between the two serovars. SS44 was cured of its virulence plasmid or subjected to mutagenesis of the spv genes, and the Spv(-) derivatives were tested for virulence in mice. Plasmid-cured S. abortusovis SU40 retained virulence in BALB/c mice when inoculated intraperitoneally. On the other hand, mice infected orally with SU40 had greatly reduced splenic infection compared to those infected with wild-type SS44. Similar results were obtained after Tn5 insertion mutagenesis of the spvR gene or deletion of the spvABCD locus. These results suggest that in the gut-associated lymphoid tissues S. abortusovis may replicate less than S. typhimurium and that the S. abortusovis virulence plasmid primarily affects systemic infection after oral inoculation but not after intraperitoneal administration in the mouse model.     

Enhanced multiplication and transmission of Streptococcus pneumoniae in guinea pigs by Sendai virus infection

It was demonstrated that the transmission S. pneumoniae in guinea pigs was remarkably promoted by the combined infection with Sendai virus in the following experiments. When guinea pigs infected with S. pneumoniae alone (infector) were cagemated with non-treated guinea pigs (contact) for 2 and 4 weeks, only 2 of 30 contacts were infected with the organism. On the contrary, when the contact guinea pigs were infected with Sendai virus and immediately cage-mated with the infectors, the pneumococcal infection occurred in 25 of 30 contacts during 2 to 4 weeks period. In the experiment in which 30 non-treated contacts were cage-mated with pneumococcal infectors for 4 weeks and then infected with Sendai virus, no pneumococcal infection was demonstrated in the contacts, suggesting no presence of latent infection of the organism in the contact guinea pigs. Twenty-five of 30 contacts suffered from pneumococcal infection when they were exposed to Sendai virus for 2 weeks and then cage-mated with infectors. The multiplication of S. pneumoniae in the respiratory tract of the guinea pigs was remarkably enhanced by combined infection with Sendai virus. Namely, a 1000-fold increase in the number of organism resulted in the guinea pigs suffered from combined infection as compared with that in the animals received pneumococcal single infection.     

Humoral and cellular immunity factors in peroral vaccination with an S. typhimurium antigenic complex]

The role of individual immunity factors in protection from S. typhimurium infection was studied on mice immunized orally in a single administration with an antigenic complex obtained by the treatment of bacterial cells with hydroxylamine. The oral administration of this preparation induced the systemic and local transformation of the organism, which was manifested by the accumulation of antibody-producing cells in the immunocompetent organs (spleen, mesenteric lymph nodes) and the increase of the phagocytic activity of macrophages in peritoneal fluid. Cellular immunity factors were found to be important for the development of resistance to salmonellosis caused by S. typhimurium after oral immunization with the antigenic complex.     

Immunity status of guinea pigs infected with Brucella L forms

B. abortus L-forms injected subcutaneously into guinea pigs adapt in the lymph nodes of the animals in the absence of reversion to normal cells. Complete and incomplete antibodies belonging to macro- and microglobulins (IgM and IgG) were synthetized. The allergic transformation of the organism is faintly pronounced. After this form of infection guinea pigs become resistant to B. melitensis infection for 6 months (the term of observation).     

Characterization of a localized basophil hypersensitivity lesion in guinea pig conjunctiva

Basophils accumulate in response to antigen challenge in cutaneous basophil hypersensitivity (CBH) reactions. Two ocular diseases, vernal conjunctivitis and contact-lens-associated conjunctivitis, are also characterized by this histopathology. We have refined a model previously developed in guinea pig conjunctiva by precisely defining the site of antigen injection and correlating the site with the clinical and histologic changes. Guinea pigs were primed by an intradermal injection of keyhole limpet hemocyanin (KLH) in the flank and challenged (Day 6) by injection of a small bolus of KLH just under the conjunctival epithelium. Twenty-four hours later histologic examination showed a perivascular infiltrate of inflammatory cells containing large numbers of basophils. Eosinophils, neutrophils, macrophages, and mast cells were also seen. Serial sections of the reaction site showed discrete boundaries. At all sites examined in antigen-challenged tissues, there were significantly more basophils than in control-injected conjunctiva. Insertion of a sterile needle or injection of PBS or KLH into normal conjunctiva induced a significant increase in neutrophils and some macrophages. Injection of graded doses of antigen into the conjunctiva of primed animals, resulted in a dose-dependent increase in basophils up to 50 micrograms KLH (optimal dose).     

Protective effect of prior immunization on ocular paracoccidioidomycosis in guinea pigs

The present study reproduced the experimental model of ocular paracoccidioidomycosis in guinea pigs, by the intracardiac inoculation of yeast-forms of P. brasiliensis. Ocular involvement was observed in 80% of the infected animals. The uvea, ciliary body, choroid, iris, lids and the conjunctiva were the structures most commonly affected. To protect the animals against the infection, an immunization protocol was standardized utilizing a P. brasiliensis soluble antigen in Freund's complete adjuvant, administered weekly, during 3 weeks, by the subcutaneous route. Two weeks later, previously immunized guinea pigs were challenged by the intracardiac route with yeast-forms of P. brasiliensis (vaccinated group). When compared with a control group (infection in the absence of prior immunization), the vaccinated animals developed higher levels of anti-P. brasiliensis cellular and humoral immune response and a three times lower frequency of ocular involvement (85.7% vs 28.5%). In addition, the ocular lesions were significantly more localized and contained less fungal cells. The data demonstrated that the subcutaneous immunization was effective in decreasing the frequency and extent of ocular lesions, as well as in blocking fungal multiplication.     

Reaction and response of newborn guinea pigs to experimental Salmonella typhi infection

Newborn guinea pigs, orally infected with Salmonella typhi were examined at various intervals of time in order to determine bacterial distribution in tissues and to establish possible correlation with the clinical aspects manifested. Histopathological examination evidenced typical lesions in jejunum, ileum, caecum and especially in regional lymphatic tissues. Spleen, liver and mesenteric lymph nodes presented granulomatous lesions similar to those observed in in human typhoid fever. After oral administration, the animals reacted with anorexia, febrile reactions, bacteremia, diarrhoea, positive stool cultures, dehydration, lethargy and antibodies too were produced. Our results indicate that typhoid infection may be induced in newborn guinea pigs; the model may be used for an assessment of attenuated live typhoid vaccine control.     

Proteomic analysis of porcine mesenteric lymph-nodes after Salmonella typhimurium infection

In this study we employed for the first time an in vivo approach coupled to DIGE-based proteomics to explore the response of porcine mesenteric lymph nodes (MLN) to Salmonella typhimurium infection. MLN samples were collected from four control and twelve infected pigs (at 1, 2 and 6 days post infection) for histological analysis, protein and RNA purification. Afterwards, expressed proteins were screened by differential in gel analysis and data were analyzed by bioinformatic tools to generate interaction networks, and identify enriched signaling pathways and biological annotations. S. typhimurium labeling in tissue and phagocyte infiltration were analyzed by immunohistochemistry and RNA was employed to determine the relative expression of immune-related genes by quantitative RNA analysis. The proteome response of porcine MLN to infection was associated to the induction of processes such as phagocyte infiltration, cytoskeleton remodeling and pyroptosis. Moreover, our results suggest that S. typhimurium antigens are cross-presented via MHC-I in a proteasome-dependent manner in porcine MLN. Since pathogen burden in tissue was noticeably reduced at the end of the time course, we infer that host innate and adaptive immunity act in association in MLN to control S. typhimurium dissemination in swine infections.     

Experimental salmonellosis conjunctivitis and keratitis in guinea pigs]

The authors present the results of morphological study of experimentally-induced conjunctivitis and keratitis induced in guinea pigs by salmonellae. It was revealed that injuries of the conjunctiva and cornea in salmonellae infection were different by development and morphological manifestations from keratoconjunctivitis caused by shigellae. This difference was due to specific peculiarities of the pathogenic properties of salmonellae and shigellae clearly expressed on the given experimental model.     

Colonization kinetics of different methicillin-resistant Staphylococcus aureus sequence types in pigs and host susceptibilities

In this study we investigated the kinetics of colonization, the host susceptibility and transmissibility of methicillin-resistant Staphylococcus aureus (MRSA) after nasal treatment of pigs with three different MRSA strains of distinctive clonal lineages (sequence type 398 [ST398], ST8, and ST9), and origin in weaning piglets. The colonization dose of 5.0 × 10(8) CFU/animal was determined in preliminary animal studies. A total of 57 piglets were randomly divided into four test groups and one control group. Each of three test groups was inoculated intranasally with either MRSA ST8, MRSA ST9, or MRSA ST398. The fourth group was a mixture of animals inoculated with MRSA ST398 and noninoculated "sentinel" animals. Clinical signs, the nasal, conjunctival, and skin colonization of MRSA, fecal excretion, and organ distribution of MRSA, as well as different environmental samples were examined. After nasal inoculation with MRSA piglets of all four test groups showed no clinical signs of an MRSA infection. MRSA was present on the nasal mucosa, skin, and conjunctiva in all four test groups, including sentinel animals. Likewise, fecal excretion and internal colonization of MRSA ST8, ST9, and ST398 could be shown in each group. However, fecal excretion and the colonization rate of the nasal mucosa with MRSA ST9 were significantly lower in the first days after infection than in test groups infected with ST8 and ST398. The results of this study suggest differences in colonization potential of the different MRSA types in pigs. Furthermore, colonization of lymph nodes (e.g., the ileocecal lymph node) with MRSA of the clonal lineage ST398 was demonstrated.     

A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells

The attenuated Salmonella typhimurium PhoP^c strain is avirulent but immunogenic via the oral route in mice and is attenuated in survival in macrophage cell lines. In this study, the fate of PhoP^c bacteria expressing green fluorescent protein was investigated in murine Peyer's patches. The survival of PhoP^c was monitored after orogastric inoculation of BALB/c mice. Bacteria persisted for several weeks in the Peyer's patches and were also recovered from the mesenteric lymph nodes and spleen. Confocal microscopy analysis identified dendritic cells as the Peyer's patch cell type that internalized PhoP^c expressing green fluorescent protein at early time points. In addition, live PhoP^c were found in Peyer's patch dendritic cells and not in B cells 3 days after orogastric inoculation. Taken together, these results provide strong evidence that PhoP^c is internalized and survives within Peyer's patch dendritic cells. As these cells are potent antigen-presenting cells, these data could explain the immunogenicity of S. typhimurium vaccine strains in vivo .     

Development of a model of natural infection with Mycobacterium bovis in white-tailed deer.

The objective of this study was to develop a suitable experimental model of natural Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus), describe the distribution and character of tuberculous lesions, and to examine possible routes of disease transmission. In October 1997, 10 mature female white-tailed deer were inoculated by intratonsilar instillation of 2 x 10(3) (low dose) or 2 x 10(5) (high dose) colony forming units (CFU) of M. bovis. In January 1998, deer were euthanatized, examined, and tissues were collected 84 to 87 days post inoculation. Possible routes of disease transmission were evaluated by culture of nasal, oral, tonsilar, and rectal swabs at various times during the study. Gross and microscopic lesions consistent with tuberculosis were most commonly seen in medial retropharyngeal lymph nodes and lung in both dosage groups. Other tissues containing tuberculous lesions included tonsil, trachea, liver, and kidney as well as lateral retropharyngeal, mandibular, parotid, tracheobronchial, mediastinal, hepatic, mesenteric, superficial cervical, and iliac lymph nodes. Mycobacterium bovis was isolated from tonsilar swabs from 8 of 9 deer from both dosage groups at least once 14 to 87 days after inoculation. Mycobacterium bovis was isolated from oral swabs 63 and 80 days after inoculation from one of three deer in the low dose group and none of four deer in the high dose group. Similarly, M. bovis was isolated from nasal swabs 80 and 85 days after inoculation in one of three deer from the low dose group and 63 and 80 days after inoculation from two of four deer in the high dose group. Intratonsilar inoculation with M. bovis results in lesions similar to those seen in naturally infected white-tailed deer; therefore, it represents a suitable model of natural infection. These results also indicate that M. bovis persists in tonsilar crypts for prolonged periods and can be shed in saliva and nasal secretions. These infected fluids represent a likely route of disease transmission to other animals or humans.     

Infection of rhesus monkeys with topical instillation of simian immunodeficiency virus (SIV)B670 into the conjunctival sac

We tested the ability of SIV to cause local and systemic infection in three rhesus monkeys after topical instillation of cell-free virus into the conjunctival cul-de-sac. Conjunctivitis or other signs of infection were monitored after inoculation. Conjunctiva were swabbed for virus culture and biopsied for PCR. Changes in lymphocyte subsets, seroconversion, antigenemia, and virus isolation from PBL were assessed systemically postinoculation. Viral DNA was detected in conjunctival biopsy by PCR in one of three animals that later developed systemic infection. The other two animals remained uninfected. These data demonstrate that the conjunctiva is a route by which SIV (and perhaps HIV) may cause systemic infection.     

Establishment of a salmonella-free Guinea pig colony

Salmonellosis due to Salmonella typhimurium was enzootic in a guinea pig breeding colony for over 25 years. A Salmonella-free auxiliary colony was established by removing weanlings from the infected colony to a clean area, and preventing infection. Examination of agglutinin titers and necropsy specimens indicated that the auxiliary colony was still free from Salmonella 18 months after its establishment while 24% of the guinea pigs dying in the infected colony yielded Salmonella typhimurium.     

Experimental Haemonchus contortus infections in guinea pigs

Approximately 40% of exsheathed Haemonchus contortus larvae administered to guinea pigs established in the stomach and developed into fourth stage larvae. Most worms were then lost between 5 and 7 days after infection and the guinea pigs were resistant to a second infection. Haemorrhage, oedema and infiltration with inflammatory cells, especially eosinophils, developed in the stomach wall of infected guinea pigs and reactive hyperplastic changes occurred in the gastric lymph node. H. contortus infection of guinea pigs has some potential as a model for study of the pathology, immunology and chemotherapy of gastric nematodiasis.     

Enterohepatitis in Mongolian gerbils (Meriones unguiculatus) inoculated perorally with Tyzzer's organism (Bacillus piliformis)

Enterohepatitis was produced in Mongolian gerbils by intragastric inoculation with Tyzzer's organism from natural infection of a gerbil. Death occurred in 50 to 60% animals 5 to 7 days postinoculation (p.i.). On day 3 p.i., when a few necrotic foci appeared in the liver, a large amount of bacterial antigen was present within ileocecal enterocytes and reticuloendothelial cells of the Peyer's patches. Neutrophil and monocyte infiltration was found in the lamina propria. On day 5 or 6 p.i. there was severe necrotizing and hemorrhagic ileotyphlocolitis. Bacterial antigen was abundant within not only enterocytes, but also smooth muscle cells of the ileum and jejunum as well as reticular cells of the mesenteric lymph nodes. On day 7 p.i. the intestinal lesions subsided in the presence of fewer bacteria, while necrotizing hepatitis became well developed. The results indicated that Mongolian gerbils were highly susceptible to the oral route of infection with the Tyzzer's organism.     

Protection against experimental toxoplasmosis by adoptive immunotherapy

The role of humoral and cell-mediated immunity against toxoplasmosis in experimentally infected guinea pigs was examined by using a syngeneic passive transfer system. Serum or spleen and lymph node cells from guinea pigs immune to infection with the RH strain of Toxoplasma gondii conferred partial protection against symptomatic disease in recipient guinea pigs. This result was based on the reduced dissemination or growth of T. gondii parasites from the primary inoculation site to various selected organ sites of the recipients of immune serum or cells. Similar levels of partial protection against disseminated toxoplasmosis occurred in animals infused with cell suspensions enriched for immune T cells, whereas treatment of immune cells with a monoclonal anti-guinea pig T cell antibody plus complement abolished their ability to transfer resistance. These findings provide substantial direct evidence implicating both cellular and humoral components of the immune response as important effector mechanisms in host resistance to toxoplasmosis.     

Systemic dissemination of H5N1 influenza A viruses in ferrets and hamsters after direct intragastric inoculation

Although oral exposure to H5N1 highly pathogenic avian influenza viruses is a risk factor for infection in humans, it is unclear how oral exposure to these virus results in lethal respiratory infections. To address this issue, we inoculated ferrets and hamsters with two highly pathogenic H5N1 strains. These viruses, inoculated directly into the stomach, were isolated from the large intestine and the mesenteric lymph nodes within 1 day of inoculation and subsequently spread to multiple tissues, including lung, liver, and brain. Histopathologic analysis of ferrets infected with virus via direct intragastric inoculation revealed lymph folliculitis in the digestive tract and mesenteric lymph nodes and focal interstitial pneumonia. Comparable results were obtained with the hamster model. We conclude that, in mammals, ingested H5N1 influenza viruses can disseminate to nondigestive organs, possibly through the lymphatic system of the gastrointestinal tract.