Investigation of transplacental transmission of Caryospora bigenetica (Apicomplexa: Eimeriidae) in mice.

Five pairs of female Swiss-Webster mice were caged with 5 males (2 females/1 male). Eight females were inoculated orally with 2.6 x 10(5) Caryospora bigenetica oocysts either 3 days before mating, 3 days after mating (PMD), 9 PMD, or 16 PMD. The remaining 2 females were inoculated orally with Hanks' balanced salt solution and served as controls. One female from each cage delivered naturally at full term and the second female delivered by cesarean section on postmating day 18. The number of offspring per litter ranged from 7 to 12. One female produced a litter of 3 stillborn and 5 liveborn offspring. Seven of 8 female mice exhibited swollen muzzles and footpads 8 days after inoculation. Caryospora bigenetica was identified in tissues of muzzle, tongue, footpad, uterus, and placenta at necropsy. This is the first report of C. bigenetica in uterus and placenta. Clinical signs and tissue infections were not observed in control mice or in any offspring of the 10 female mice. This study presents evidence that C. bigenetica is not transmitted transplacentally.     

Fatal Caryospora bigenetica (Apicomplexa: Eimeriidae) infections in cotton rats, Sigmodon hispidus

Four groups of cotton rats, Sigmodon hispidus, were shown to be suitable secondary hosts for the viperid coccidium, Caryospora bigenetica, following oral inoculation of a mixture of oocysts and sporocysts. Swelling of the face, ears, and scrota and hemorrhagic ears were the predominant clinical signs and some cotton rats died in 3 of 4 experiments. Developmental stages of C. bigenetica were found in connective tissue components of the ear, nose, cheeks, anal skin, scrotum, and penile sheath of all cotton rats in which these tissues were examined. Additionally, developmental stages of C. bigenetica were found in connective tissue components of the following tissues examined from some cotton rats: tongue, lung, testicle, epididymis, rectum, base of the tail, footpad, and bone marrow. The present study shows that C. bigenetica can be pathogenic for cotton rats and demonstrates many new anatomic sites for developmental stages of this parasite in the secondary host.     

Effects of route of inoculation on the site of development of Caryospora bigenetica (Apicomplexa: Eimeriidae)

The sites of infection by Caryospora bigenetica in Swiss-Webster mice (Mus musculus) were demonstrated after 7 routes of inoculation: oral, intraperitoneal, intravenous, intramuscular, subcutaneous, dermal, and intraocular. All mice exhibited clinical signs of dermal coccidiosis 9 days after inoculation regardless of the inoculation route. Signs included swelling of the facial tissue, footpads, and scrota (male mice). Developmental stages of the parasite were found in the muzzle, tongue, footpad, lumbar subcutaneous tissue, biceps femoris muscle, conjunctiva, and eye; the latter 3 sites represent new sites of development. The site of development of the parasite in the host tissue was independent of experimental inoculation route.     

Serial transmission of Caryospora bigenetica Wacha and Christiansen, 1982 (Apicomplexa: Eimeriidae) between different species of rodents

Transmission of Caryospora bigenetica by cannibalism between cotton rats, between cotton rats and mice, and between mice was demonstrated. All experimental animals developed swollen muzzles and scrota 8 days after ingestion of infected tissues. Infections were confirmed by light microscopy of fresh tissue smears. Tissues of cannibalized animals contained caryocysts that, after ingestion by the next host, released sporozoites that underwent merogony, gamogony, sporogony, and caryocyst formation in dermal tissues. This study demonstrates that C. bigenetica can be transmitted by predation between species of rodents and that, in the recipient host, asexual and sexual reproduction occur before caryocysts appear.     

White spots of the liver in pigs experimentally infected with Ascaris suum

To make clear the relationship between Ascaris suum infection and the appearance of white spot lesions on the surface of the liver in pigs, three groups of pigs were inoculated orally with embryonated A. suum eggs and observed clinicopathologically. Group A of three pigs were inoculated 21 times with 100 eggs each of the nematode, group B of three pigs 4 times with 50,000 eggs each for 10 weeks, and group C of two pigs 2 times with 50,000 eggs each at a one-week interval. All the pigs were sacrificed at the same time 1 week after the final inoculation. Such signs of the nematode infection as dyspnea, coughing and fever appeared in all the pigs of groups B and C seven days after inoculation to continue for several days. In addition, peripheral blood eosinophilia was recorded in these animals 7 or 14 days after inoculation. At autopsy, mesh-worked white spots, some compact and others lymphonodular, were observed on the surface of the liver in all the pigs of the three groups. Main white spots were mesh-worked and lymphonodular in the pigs of group A. They were severe and compact in group B. Therefore, they were rough to the touch. In group C mesh-worked white spots fused with one another and covered the surface of the liver. These white spot lesions observed were morphologically very similar to those found in the field conditions. Complement-fixating antibodies reacting to adult A. suum antigen were detected only in sera from the pigs of group B. Moreover, antibodies involved in the intradermal reaction of immediate type were found in the pigs of groups A and B.     

Pathogenicity of selected Toxoplasma gondii isolates in young pigs

The pathogenicity in 7-week-old pigs to five different Toxoplasma gondii strains of various host species origin was compared after i.v. inoculation of 10(4) tachyzoites. Additionally, one group of pigs was inoculated i.v. with 10(6) tachyzoites of the reference strain, SSI 119. In response to the infection a significant effect of T. gondii tachyzoite inoculation dose as well as differences among strains could be observed in several parameters. The 10(6)-dose inoculated pigs showed variable degrees of clinical illness and recurrent episodes of fever 4-17 days p.i., while pigs of four of the 10(4) tachyzoite inoculated groups experienced a short-lived rise in body temperature from day 6-8 p.i. without any apparent illness or inappetence. Control pigs and pigs infected with the least pathogenic strain had normal body temperature throughout the experiment. In all inoculated pigs, T. gondii-specific IgM and IgG antibodies appeared from day 8-10 and 10-17 p.i., respectively. Serum levels of alkaline phosphatase and the acute phase protein haptoglobin were decreased or increased, respectively, in response to the infection. Differential leukocyte count on peripheral blood revealed a significant lymphocytopenia on day 6 p.i. equal to both CD4+ and CD8+ T-cells, but shifting towards a reduced ratio of CD4+/CD8+ T-cells from day 8-14 p.i. In the 10(6)-dose inoculated pigs a considerable increase in zymosan induced and spontaneous oxidative burst capacity of peripheral blood leukocytes was observed from 6 days p.i. compared with control pigs. Oxidative burst capacity was not examined for other pigs. In conclusion, several useful parameters to identify differences in T. gondii pathogenicity other than mortality were identified. Furthermore, even at low doses, significant differences between recently collected Danish T. gondii field isolates were demonstrated after i.v. inoculation in young pigs.     

Rodents are not a source of endogenously-produced, fecally-transmitted Caryospora bigenetica oocysts.

Fifteen Swiss-Webster mice (Mus musculus) and eight cotton rats (Sigmodon hispidus) were inoculated orally with Caryospora bigenetica oocysts. Feces from these animals were collected from 0 to 180 days postinoculation (DPI) and examined for endogenously-produced oocysts using Nomarski microscopy. Oocysts were recovered from mouse feces at 0, 1, 2, 3, 5, 7, 8, 10, and 14 DPI, and from cotton rat feces at 1, 2, and 9 DPI. The recovered oocysts were determined to be from the original inocula due to the presence of thick walls, polar granules, and Stieda and substieda bodies. All animals exhibited clinical signs at 8 DPI. Developmental stages of C. bigenetica were identified in various tissues of seven cotton rats found dead at 9, 10, 11, 12, and 13 DPI. Caryocysts were found in muzzle, tongue, footpad, scrotum, and rectum of mice and cotton rats at 30 DPI. Fecal samples collected from mice on 0, 8, 10, 12, 14, 16, and 18 DPI, and from cotton rats on 0, 9, 11, 13, 15, and 17 DPI were injected subcutaneously into 13 mice. Of the 13 mice, a Caryospora infection was observed only in the mouse inoculated with 0 DPI mouse feces. We propose that endogenously-produced C. bigenetica oocysts are not fecally-transmitted by Swiss-Webster mice or cotton rats.     

Tumor infiltrating leukocytes (tils) during progressive tumor growth and BCG-mediated tumor regression

Tumor regression was induced by intralesional injection with BCG, 7 days after inoculation of line 10 hepatocellular carcinoma cells into strain 2 guinea pigs. Tumor-infiltrating leukocytes (TILS) were characterized immunohistochemically with 11 monoclonal antibodies (MoAbs) during the induction phase of line 10-immunity, and during immune-mediated regression of the tumor, at days 12 and 28 after tumor cell inoculation, respectively. At day 5 after BCG-injection (day 12 after tumor cell inoculation), there were no major differences between the TIL subpopulations of the BCG-treated and untreated tumors. The TILS were mainly T-cells, as identified by MoAbs against Pan T-cells (CT5), T-cytotoxic/suppressor cells (CT6) and T-helper/inducer cells (H155). A limited number of macrophages was also present. However, at day 21 after BCG-treatment (28 days after tumor cell inoculation), the fibrous stroma was increased dramatically in most of the BCG-treated tumors, and as a result, the tumor cell islets were smaller than in control tumors. In the BCG treated tumors, the numbers of T-cells and macrophages were increased. In growing and regressing tumors, MHC class I and II antigens were strongly expressed in TILS and in the tumor stroma. Line 10 tumor cells prior to inoculation expressed no MHC class I or II antigens. In the centers of the tumor islets at days 12 and 28, expression of these antigens was not found. However, MHC class I and II antigens were expressed on tumor cells at sites where they lay close to the fibrous stroma or TILS. This observation was made in progressively growing tumors and was most apparent in BCG-treated tumors.     

Experimental Infection with Mycobacterium Avium,Serotype 2, in Pigs

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).     

Methicillin-resistant Staphylococcus aureus ST398 contamination and transmission in pigs after a low dose inoculation

Aims: Methicillin‐resistant Staphylococcus aureus (MRSA) ST398 has recently been described as a zoonotic agent. Its transmission between animals seems to be a pivotal factor in its emergence and dissemination. This experimental trial was performed to describe MRSA ST398 contamination and transmission in pigs after a low dose inoculation. Methods and results: Twelve specific pathogen‐free (SPF) pigs were randomly divided between two separate pens. Three pigs in each pen received a nasal inoculation of 2 × 10⁴ colony‐forming units per animal, and three naïve pigs were left in contact with them. Every 2 days and at necropsy, different samples were screened for MRSA. It was detected in nasal swabs from five inoculated and three naïve contact pigs, as early as 1 day after inoculation. MRSA was also found in environmental wipes but never in faecal samples. At necropsy, MRSA was detected in the lymph nodes of two contact pigs and in the tonsils and lymph nodes of three inoculated pigs. Twelve other SPF pigs were included as negative control in a separate room. Conclusion: This experiment showed that inoculation of a low dose of MRSA ST398 could lead to the horizontal transmission of the bacterium between pigs, the contamination of mandibular lymph nodes and the contamination of the environment without faecal carriage. Significance and Impact of the Study: The minimal inoculated dose via nasal route to observe transmission of MRSA ST398 between pigs is equal or lower to 2 × 10⁴ colony‐forming units per animal, and faecal excretion seems not to be a necessary condition for horizontal transmission.     

Fascioloides magna: development in selected nonruminant mammalian hosts.

Metacercariae of Fascioloides magna were inoculated per os into selected nonruminant animals to evaluate them as laboratory hosts for this trematode. Immature F. magna were recovered from 36 of 52 guinea pigs, 6 of 15 mice, 2 of 3 rabbits, and 16 of 25 domestic swine. The parasite was not recovered from two beavers (Castor canadensis), two ferrets (Mustela putorius), one red fox (Vulpes fulva), one Shetland pony (Equus caballus) and two Rhesus monkeys (Macaca mulatta). Mortality in guinea pigs inoculated with 10 metacercariae each, occurred 56 to 116 (mean of 80) days after inoculation. Trematodes were in the liver up to 69 days after inoculation and usually free in the peritoneal cavity after 69 days after inoculation. Swine 2 weeks of age when inoculated were more susceptible to infection (5.6% trematode recovery rate) than were those inoculated at 4 weeks of age (1.9% trematode recovery rate) or 10 weeks of age (0.4% trematode recovery rate), indicating an age resistance to infection.     

非洲钝缘蜱感染Q热立克次体敏感度的测定

Q热立克次体以10^-1—10^-12不同稀释度,用血腔注射法感染非洲钝缘蜱,感染35天,取蜱血淋巴液,分别用Gimenez染色法及免疫荧光技术检查,在10^-1—10^-5的稀释度,两种方法检出率基本相似。10^-6以下各组,免疫荧光尚可检出少数阳性标本,而Gimcnez法则无阳性,前者总阳性率为48.28%,后者为40.68%。感染的蜱悬液接种豚鼠,12组动物血清做Q热补体结合试验全部阳性。动物发病情况如潜伏期长短、发烧天数等似与蜱内立克次体的含量有关。     

Intrauterine inoculation of minipigs with Chlamydia trachomatis during diestrus establishes a longer lasting infection compared to vaginal inoculation during estrus

Advanced animal models, such as minipigs, are needed for the development of a globally requested human Chlamydia vaccine. Previous studies have shown that vaginal inoculation of sexually mature Göttingen minipigs with Chlamydia trachomatis resulted in an infection lasting only 3–5 days.The aim of this study was to evaluate the effect of targeting the upper porcine genital tract by transcervical and transabdominal intrauterine inoculation, compared to previously performed vaginal inoculation. Furthermore, we investigated the effect of the hormonal cycle, estrus vs. diestrus, on the establishment of a C. trachomatis infection in the minipig.Targeting the upper genital tract (transcervical inoculation) resulted in a longer lasting infection (at least 7 days) compared to vaginal inoculation (3–5 days). When comparing intrauterine inoculation during estrus and diestrus, inoculation during diestrus resulted in a longer lasting infection (at least 10 days) compared to estrus (3–5 days). Furthermore, we found a significant C. trachomatis specific IFN-γ response in pigs inoculated during estrus correlating with the accelerated clearance of infection in these pigs.These findings suggest that for implementation of an optimal model of C. trachomatis in minipigs, inoculation should bypass the cervix and preferable be performed during diestrus.     

Complete development of Caryospora bigenetica (Apicomplexa: Eimeriidae) in vitro

The development of Caryospora bigenetica in vitro is described by light microscopy. Sporozoites from snake-derived oocysts were purified and inoculated onto cultures of primary testicle cells of the cotton rat, cotton rat kidney cells, and human fetal lung cells. Intracellular sporozoites were observed one and two days postinoculation (DPI). Motile, extracellular first-generation merozoites were present 3 DPI, and second-generation merozoites were present 5 DPI. Mature gamonts were observed 9 DPI and developed into unsporulated oocysts by 10 DPI. Oocystes sporulated in vitro, and excystation was observed. Cells that were penetrated by in vitro-produced sporozoites formed caryocysts by 16 DPI. To test infectivity of in vitro-derived stages, merozoites were removed from cultured cells 5 DPI and inoculated intraperitoneally into a mouse; infection resulted. Sporulated oocysts removed from cell cultures 12 DPI produced facial swelling in an orally inoculated cotton rat.     

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.     

Leishmania braziliensis spp. in the nasal mucosa of guinea pigs inoculated in the tarsi.

Two lots of 20 young male guinea pigs were inoculated subcutaneously in the tarsi with 10(4) amastigotes of Leishmania braziliensis braziliensis or L. b. guyanensis to study the susceptibility of this Neotropical hystricomorph rodent the autochthonous parasites. Almost 50% of the animals showed lesions in the inoculation site and had parasitizations that were infective to hamsters, as shown by inoculating homogenates of the dermal lesion, of the spleen, of the liver, and of the nasal mucosa into hamsters at 20, 40, 60, and 120 days after inoculation of the guinea pig. Smears of the above organs showed the presence of amastigotes. Parasites inoculated into the tarsi were detected early in the skin, spleen, and liver of the guinea pig host. Blood cultures made by cardiopuncture on sacrifice of the guinea pigs were uniformly negative. The nasal mucosa of nearly all animals positive in the skin or viscera was invaded early by the parasites, although with greater frequency between 60 and 120 days post-inoculation. The use of this model for the study of mucocutaneous parasitism by L. braziliensis is discussed, together with the phenomena of parasitism at a distance from the inoculation site, the temperature of the body regions affected, and the possible genetic influence on susceptibility of the guinea pig to L. braziliensis.     

Trichinella spiralis: acquired immunity in swine

The ability of domestic pigs to develop protective immunity to Trichinella spiralis in response to inoculation with different doses of muscle larvae was assessed. Adult worms developing from the inoculations of 112, 500, and 10,000 larvae were expelled from the intestine about 6 weeks after inoculation. Inoculation with 25,000 larvae, however, resulted in more rapid intestinal worms expulsion, indicating that gut expulsion is dose dependent. Secondary expulsion also tended to be dependent upon primary infection level. Pigs initially inoculated with 500 to 10,000 larvae expelled the challenge infection of adult worms after 22 to 25 days; in contrast, infection by inoculation of only 112 larvae failed to induce significant enchanced gut expulsion of the challenge infection intestinal worms. However, all primary infection levels, including inoculation with 112 larvae, induced nearly absolute resistance to the muscle establishment of larvae from challenge adult worms. The fecundity of female worms recovered from immune pigs was reduced 75% in comparison to controls. These results show that, in contrast to some host species, very rapid gut expulsion does not occur in domestic swine. Yet, immune responses at the gut level are important, perhaps responsible for much of the inhibition reflected as reduction in the establishment of muscle larvae.     

Synergistic effects of active specific immunotherapy and chemotherapy in guinea pigs with disseminated cancer

Sewall Wright strain 2 guinea pigs bearing pulmonary metastases of the syngeneic line 10 (L10) hepatocarcinoma were treated with a vaccine composed of 10(7) bacillus Calmette-Guérin admixed with 10(7) x-irradiated L10 tumor cells beginning 10 days after tumor inoculation. Although this treatment failed to cure most of the guinea pigs of their metastatic disease, histologic examination of the pulmonary tumors in the vaccinated guinea pigs provided evidence of a cell-mediated hypersensitivity response that disrupted the normally compact architecture seen in control tumors. When a monoclonal antibody against the L10 tumor was injected i.v. to evaluate the vascular permeability of the tumors, significantly more antibody localized in tumors of vaccinated guinea pigs than in tumors of untreated controls. These results suggested that blood-borne substances could be delivered more efficiently to L10 metastases after the tumor-bearing guinea pigs had been treated with vaccine. To determine whether such increased vascular permeability would enhance the antitumor effects of chemotherapeutic agents, combined immunotherapy and chemotherapy studies were performed. Although cyclophosphamide treatment by itself did not cure L10-bearing guinea pigs, cyclophosphamide used in conjunction with prior immunotherapy increased the survival rate of animals to more than twice that of animals treated with immunotherapy alone (74 vs 33%). These results suggest that one mechanism by which active specific immunotherapy enhances chemotherapy of disseminated tumors is by rendering tumor foci more permeable to subsequently administered cytotoxic drugs.     

Salmonella fecal shedding and immune responses are dose- and serotype- dependent in pigs

Despite the public health importance of Salmonella infection in pigs, little is known about the associated dynamics of fecal shedding and immunity. In this study, we investigated the transitions of pigs through the states of Salmonella fecal shedding and immune response post-Salmonella inoculation as affected by the challenge dose and serotype. Continuous-time multistate Markov models were developed using published experimental data. The model for shedding had four transient states, of which two were shedding (continuous and intermittent shedding) and two non-shedding (latency and intermittent non-shedding), and one absorbing state representing permanent cessation of shedding. The immune response model had two transient states representing responses below and above the seroconversion level. The effects of two doses [low (0.65×10(6) CFU/pig) and high (0.65×10(9) CFU/pig)] and four serotypes (Salmonella Yoruba, Salmonella Cubana, Salmonella Typhimurium, and Salmonella Derby) on the models' transition intensities were evaluated using a proportional intensities model. Results indicated statistically significant effects of the challenge dose and serotype on the dynamics of shedding and immune response. The time spent in the specific states was also estimated. Continuous shedding was on average 10-26 days longer, while intermittent non-shedding was 2-4 days shorter, in pigs challenged with the high compared to low dose. Interestingly, among pigs challenged with the high dose, the continuous and intermittent shedding states were on average up to 10-17 and 3-4 days longer, respectively, in pigs infected with S. Cubana compared to the other three serotypes. Pigs challenged with the high dose of S. Typhimurium or S. Derby seroconverted on average up to 8-11 days faster compared to the low dose. These findings highlight that Salmonella fecal shedding and immune response following Salmonella challenge are dose- and serotype-dependent and that the detection of specific Salmonella strains and immune responses in pigs are time-sensitive.     

Experimental infection of Indonesia Taenia (Samosir strain) in domestic animals

Three 58-day old Small-Ear-Miniature (SEM) pigs, six 36-day old Landrace-Small-Ear-Miniature (L-SEM) pigs, and two 5-day old Holstein calves were each fed 3000 or 30,000 Indonesia Taenia (Samosir strain) eggs and sacrificed 27-195 days after inoculation. A total of 4922 cysticerci were recovered only from the livers of the three SEM pigs (1977 cysticerci) and six L-SEM pigs (2945 cysticerci). The infection rate in pigs was 100%. Cysticerci recovery rates of SEM and L-SEM pigs were 22.0% and 1.6%, respectively. Calves were not susceptible to Indonesia Taenia. More cysticerci were found in the liver parenchyma (L-SEM, 66.4%; SEM, 76.2%) than on the liver surface (L-SEM, 33.6%; SEM, 23.8%) of the infected animals. Most (99.86%) of the cysticerci recovered from the livers of L-SEM pigs were degenerated or calcified but 77.9% of those in the livers of SEM pigs were mature and only 22.1% were degenerated or calcified. Measurements of length, width, diameters of protoscolex, rostellum, and sucker and hooklet pattern indicated that Indonesia Taenia is very similar to Taiwan Taenia and very different from Taenia saginata and Taenia solium. The present findings indicate that Indonesia Taenia and Taiwan Taenia may be the same new species.