Development of an experimental model allowing discrimination between virulent and avirulent isolates of Serpulina (Treponema) hyodysenteriae.

Variation in virulence among different strains of Serpulina hyodysenteriae was studied by oral inoculation of specific pathogen free piglets and CD-1 mice. Piglets infected with serotype 2 reference strain B204 and an untypable field strain LHV-90-9-I had severe diarrhea tainted intermittently with mucus and fresh blood. The piglets inoculated with B169, B8044, B6933, and ACK300-8 reference strains representing serotypes 3, 5, 6, and 7 respectively developed moderate diarrhea. However, reference strains B234 and A-1 of serotypes 1 and 4, respectively, failed to cause any diarrhea. None of the S. hyodysenteriae strains caused diarrhea in mice. The results indicate a great variation in virulence among strains of different serotypes of S. hyodysenteriae. Mice were less susceptible to infection with S. hyodysenteriae.     

Comparison of six commercially available transport media for maintenance of Serpulina (Treponema) hyodysenteriae.

Two anaerobic (A1 and A2), 1 selective (S1), and 3 conventional (C1, C2, and C3) transport media formulations were compared for their capacity to maintain the viability of Serpulina (Treponema) hyodysenteriae. Initial experiments compared the recovery of S. hyodysenteriae from pure cultures held in each transport medium for 0.5, 1, 2, 3, 5, and 7 days at -40 C, 4 C, 25 C, and 36 C. Subsequent experiments compared each transport medium for maintenance of S. hyodysenteriae in fecal specimens obtained from experimentally infected pigs after holding for up to 7 days at 25 C. In each experiment, the viability of S. hyodysenteriae in each transport medium incubated at each temperature and for each period was determined by inoculating the transport medium onto either trypticase soy agar with 5% sheep blood or selective BJ agar and incubating at 42 C anaerobically. Viability and fecal flora contamination were evaluated blindly after 2-, 4-, and 6-day incubation periods. At -40 C, recovery of viable S. hyodysenteriae from pure culture did not differ among the transport media from 0.5 to 7 days, and all of the transport media consistently maintained the viability of the spirochetes for 7 days. At 4 C, the anaerobic and selective transport media maintained the viability of pure cultures of S. hyodysenteriae significantly better than did the conventional transport media group at day 7 (P = 0.019). At the same temperature, the anaerobic media maintained viability better than did the conventional media at 5 days (P less than 0.042).(ABSTRACT TRUNCATED AT 250 WORDS)     

Teat skin normal flora and colonization with mastitis pathogen inhibitors

Isolates of bacteria from normal teats were used to attempt colonization of teats of dry cows or neonatal calves. Isolates for inoculation were chosen on the basis of ability to inhibit mastitis pathogens in vitro, with the ultimate goal of in vivo inhibition of mastitis pathogens at the teat surface. Three bacterial normal flora isolates (Corynebacterium xerosis, Bacillus sp. and Aerococcus viridans) persisted less than 10 days on the teats of dry cows. The fourth isolate, Staphylococcus hominis 1, was studied in greatest detail because studies characterizing the normal teat flora showed staphylococci to be the predominant flora. The S. hominis 1 isolated used for inoculation was an inhibitor of Gram-positive mastitis pathogens. It was a biotype not found on these teats prior to inoculation, thus facilitating identification of the inoculated isolate on sequential sampling. Colonization of newborn calves, before other bacterial floral became established, resulted in recovery of inoculated S. hominis 1 for an average of 51 days or longer. On dry cow teats it was detected for up to 28 days. On several occasions the inoculated S. hominis 1 was found in pure culture. Since many new infections occur during the dry period, the colonization of dry cow tests with S. hominis 1 organisms inhibitory for Gram-positive pathogens should be tested as an adjunct to other methods of mastitis prevention.     

Swine dysentery: studies of gnotobiotic pigs inoculated with Treponema hyodysenteriae, Bacteroides vulgatus, and Fusobacterium necrophorum

Transmission experiments were carried out in gnotobiotic pigs to determine whether lesions typical of swine dysentery could be produced by oral inoculation of Treponema hyodysenteriae in combination with Bacteroides vulgatus or Fusobacterium necrophorum, or both. Each of the organisms had been isolated from swine with early lesions of the disease. Lesions were not found in 6 pigs inoculated with T hyodysenteriae alone, in 4 pigs given F necrophorum and T hyodysenteriae, or in 4 pigs given B vulgatus and F necrophorum. Lesions typical of swine dysentery developed in 8 pigs given B vulgatus, F necrophorum, and T hyodysenteriae as well as in 3 of 4 pigs given B vulgatus and T hyodysenteriae. In both of these groups, the inoculated bacteria were recovered from the colon, and T hyodysenteriae was demonstrated in the colonic crypts, epithelium, and lamina propria. The pathogenicity of the T hyodysenteriae was shown by the development of characteristic signs and lesions of swine dysentery in 12 of 14 naturally farrowed pigs inoculated with T hyodysenteriae alone.     

Swine dysentery: inoculation of gnotobiotic pigs with Treponema hyodysenteriae and Vibrio coli and a Peptostreptococcus.

Pure cultures of Treponema hyodysenteriae given orally to conventional pigs resulted in the development of swine dysentery, whereas identical cultures given to gnotobiotic pigs did not produce the disease. Oral inoculation of gnotobiotic pigs with Vibrio coli and/or a peptostreptococcus in addition to T. hyodysenteriae did not result in dysentery. Neutralization of gastric secretions with NaHCO3 immediately prior to inoculation with T. hyodysenteriae increased the period during which treponemes were evident in the feces, as did the inoculation of this organism via the intracecal route. None of the gnotobiotic pigs with a persistent fecal Treponema population developed signs of dysentery. Factors other than those investigated in this work must play a part in the etiology of swine dysentery.     

Swine dysentery: pathogenicity of Treponema hyodysenteriae.

When pure cultures of Treponema hyodysenteriae were orally inoculated into pigs, severe disease characteristic of swine dysentery developed. Less severe lesions were produced by oral inoculation of infective minced colon. Noninoculated pigs were used as controls. Inoculations of surgically isolated porcine colonic loops with either pure cultures or infective minced colon produced lesions only in the injected loop; the adjacent noninjected colon remained normal. Pigs and other experimental animals, including rabbits, guinea pigs, and mice, inoculated with washed cultures by various parenteral routes remained normal.     

FAD3基因编辑小鼠内脏重量、肌肉中脂肪酸组成与含量以及盲肠菌群分布

为了解析外源基因的转入对动物生长特性、组织中脂肪酸含量和肠道菌群分布的影响,该研究以FAD3基因编辑小鼠为试验组,以BDF1小鼠为对照组,在相同条件下饲养至45日龄处死,检测两组小鼠体重、各器官重量、肌肉中不饱和脂肪酸含量,并利用PCR-DGGE方法在16S rDNA的V₃领域检测小鼠盲肠菌群分布。结果显示,FAD3组小鼠肝脏重量显著（P<0.05）低于对照组,其他各内脏器官重量和体重两组均无显著（P>0.05）差异。FAD3组小鼠肌肉中n-6多不饱和脂肪酸（PUFAs）含量低于对照组,但无显著（P>0.05）差异,而n-3 PUFAs含量显著（P<0.05）高于对照组。两组小鼠盲肠菌群分布个体差异大,未观测到明显的组间差异,Anaerotaenia torta为共同的优势菌,而Flavonifractor plautii可能为FAD3组小鼠特有菌。该试验结果提示,外源基因FAD3的引入对小鼠生长性能和盲肠菌群的影响较小,可提升肌肉中n-3多不饱和脂肪酸含量。     

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

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

Pathogenesis of canine parvovirus enteritis: sequential virus distribution and passive immunization studies

After oral inoculation, the sequential distribution of canine parvovirus was studied in 14 nine-week-old seronegative beagle dogs. Two or three dogs were necropsied on days 1 through 6 after inoculation. Tissues were collected for virus isolation, immunofluorescence testing, and light microscopy. Virus was isolated from, and fluorescent cells were seen in the tonsil, retropharyngeal and mesenteric lymph nodes one and two days after inoculation. Virus infection of systemic and intestinal lymphoid tissues occurred as early as three days after inoculation and was associated with viremia. Intestinal epithelial infection was first seen four days after oral inoculation. All dogs were viremic before intestinal epithelial infection was found. Fecal virus excretion first occurred four days after oral virus inoculation. Intestinal virus infection and lesions became progressively more severe between four and six days after inoculation. The severity of intestinal lesions was variable and related to the severity of systemic lymphoid tissue lesions and the magnitude and duration of viremia. Four littermates of virus-infected dogs were passively immunized against canine parvovirus with convalescent canine serum 24 hours after oral virus inoculation. Neither clinical signs, lymphopenia, nor fecal virus excretion occurred in passively immunized dogs. Intestinal epithelial infection was not demonstrable by immunofluorescence testing when passively immunized dogs were necropsied four, five, and six days after virus inoculation.     

A comparison of the morphologic effects of Serpulina hyodysenteriae or its beta-hemolysin on the murine cecal mucosa.

Studies were carried out to compare the early morphologic changes in the cecal mucosa of mice either infected with Serpulina hyodysenteriae or exposed to the beta-hemolysin of S. hyodysenteriae. Sixty-five 12-24-week-old C3H/HeOuJ mice were infected with S. hyodysenteriae by gastric intubation. Two mice were necropsied every hour for 30 hours following infection. S. hyodysenteriae was isolated from the cecal contents of each mouse at all time points. Macroscopic lesions were first apparent at 14 hours postinfection (PI), and light microscopic lesions were first apparent at 10 hours PI, earlier than has been previously reported. Ultrastructural changes, first evident at 6 hours PI, included disarray and loss of microvilli and terminal web, with dilatation of intercellular spaces. Luminal bacteria were translocated through epithelial cells to the lamina propria, where capillaries exhibited changes indicative of increased permeability. In another experiment, solutions containing between 2,500 and 25,000 hemolytic units of purified S. hyodysenteriae hemolysin were placed within the lumen of surgically closed murine ceca (n = 10); ceca were collected for examination 3 hours following treatment. Ultrastructural changes consisted of loss of microvilli and terminal web and marked vacuolation and exfoliation of epithelial cells. Significant numbers of necrotic and apoptotic epithelial cells were present, and epithelial cells internalized moderate numbers of bacteria. The hemolysin of S. hyodysenteriae induces some of the same early ultrastructural changes in the cecal epithelium of mice as occur following infection with S. hyodysenteriae. Based on the observed bacterial translocation, luminal bacteria also appear to play a unique role in lesion development in this model.     

Experimentally induced coronavirus infections in calves: viral replication in the respiratory and intestinal tracts

Eleven 3- to 50-day-old colostrum-deprived gnotobiotic calves and seven 25- to 63-day-old colostrum-deprived conventional calves were allotted into 3 groups. Each group was inoculated with a fecal isolate of bovine coronavirus via different routes: orally/intranasally OR/IN, No. 1 through 8, group 1 calves; OR, No. 9 through 13, group 2 calves; IN, No. 14 through 18, group 3 calves. Nasal swab specimens and fecal specimens were collected daily and were examined for coronavirus antigen by use of direct immunofluorescent staining (nasal epithelial cells) or by use of immune electron microscopy (fecal specimens). All but 4 calves (No. 11, 13, 17, and 18) were euthanatized on postinoculation days (PID) 3 to 7. Calves 11 and 17 became severely dehydrated and died at PID 5. Calves 13 and 18 were evaluated for nasal and fecal shedding of coronavirus through PID 14. Distribution of coronavirus antigen in the respiratory and intestinal tracts of the 14 euthanatized calves was evaluated by use of direct immunofluorescent staining. All calves developed profuse diarrhea by PID 2 to 4; however, calves did not develop clinical signs of respiratory tract disease before euthanasia or death. Inoculated calves shed coronavirus in their feces as detected by use of immune electron microscopy. Infected nasal epithelial cells were detected in all but 2 orally inoculated calves (No. 9 and 10). Route of inoculation influenced the sequence of initial detection of coronavirus antigen from fecal specimens or nasal swab specimens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Salmonella-associated conjunctivitis in a cat.

Salmonella typhimurium-associated conjunctivitis in an adult female cat was characterized by epiphora and blepharospasm. The cat also harbored intestinal S typhimurium, as did one other cat in the same shipment of 7 cats obtained from a commercial cat dealer. The 3 isolates were resistant to ampicillin, cephaloridine, cephalothin, streptomycin, triple sulfonamides, and tetracycline. All 3 isolates were capable of transferring a part of the antibiotic resistance pattern to an Escherichia coli K-12 recipient. Salmonella were not recovered from the conjunctiva or fecal flora of either cat after 10 days of therapy with chloramphenicol.     

Campylobacter jejuni infections in gnotobiotic pigs

At 3 days of age, 12 gnotobiotic pigs were inoculated orally with broth cultures of Campylobacter jejuni. One pig was euthanatized and evaluated each day for 12 days. In the cecum and colon, there was diffuse edema, neutrophilic infiltration, and sloughing of epithelial cells from the mucosa on postinoculation days (PID) 2 through 5. Dysplastic colonic crypt epithelial cells were observed in the submucosa of the colon on PID 5 through 12. Curved, rod-shaped bacteria were detected on the surface of ileal, cecal, and colic absorptive and glandular epithelial cells. Bacteria also were found around small submucosal vessels on PID 3 and 4 and were associated with numerous perivascular neutrophils. The gnotobiotic pig appears to provide a simple, well-controlled in vivo model for the study of the pathogenesis of C jejuni infections in human beings, pigs, and other mammals.     

Campylobacter jejuni infection in the ferret: an animal model of human campylobacteriosis

Campylobacter infection in weanling ferrets (Mustela putorius furo) was studied as an animal model for enteric campylobacteriosis in persons. The screening of fecal cultures on selective campylobacter media showed that Campylobacter jejuni/coli was not present in the normal enteric flora. Intragastric feeding of a mixture of cat feed and 2.5 X 10(8) C jejuni isolated from ferrets with naturally occurring proliferative colitis was accomplished. All ferrets (n = 8) became infected on 3 days after they were inoculated, and at 5 to 7 days, they had bile-tinged, liquid feces with excessive mucus and blood. Ferrets gradually recovered from the diarrhea, and feces were normal 10 to 14 days after inoculation was done. Feces contained C jejuni at 14, 23, 28, 39, 46, 60, 91, 101, 109 and 144 days. In the second experiment, weanling ferrets initially were treated with 10% sodium bicarbonate, and 1 X 10(10) C jejuni organisms were administered in the cat feed. Diarrhea with fecal leukocytes and occult blood with occasional mucus appeared in almost all of the 21 ferrets from days 4 through 7. Campylobacter jejuni was isolated from the blood of 11 ferrets between 3 hours and 14 days after they were inoculated. Campylobacter jejuni bactericidal antibodies were present in serum samples at 14 days, with titers of 1:16 to 1:32. Intestinal lesions including cellular infiltration with mononuclear and polymorphonuclear leukocytes were in the lamina propria of the pyloric mucosa and small intestine of infected and control ferrets. The colon of 3 infected ferrets had small focal infiltrates of neutrophils on the lamina propria; one ferret had perivascular cuffing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Campylobacter jejuni and Campylobacter coli inoculation of neonatal calves

Three groups of neonatal calves were inoculated orally with pathogenic strains of Campylobacter jejuni or C coli. The calves developed a mild, self-limiting enteritis characterized by thick mucoid feces. Bacteremia and fecal shedding of Campylobacter were sporadic in all inoculated calves. Two groups of calves were killed 1 to 3 weeks after inoculation to study the pathogenesis of infection. Postmortem culture of tissues revealed C jejuni or C coli most frequently in the ileum, cecum, colon, and blood. Clinical or pathologic differences between C jejuni-inoculated and C coli-inoculated calves were minimal.     

Effect of anaerobic cecal microflora and dietary lactose on colonization resistance of layer chicks to invasive Salmonella enteritidis.

The effect of oral inoculation with anaerobic cultures of cecal microflora and providing lactose in the feed on colonization resistance to invasive Salmonella enteritidis was evaluated in newly hatched leghorn chicks. Salmonella colonization of the ceca, tissue invasion and organ colonization, horizontal transmission, and seroconversion were significantly decreased (P less than 0.01) in chicks inoculated with cecal flora. The addition of lactose to the feed, in the absence of cecal microflora, failed to provide protection. Dietary lactose enhanced colonization resistance in chicks that were inoculated with anaerobic cultures of cecal flora. The results indicated that establishment of normal cecal flora in layer chicks together with the addition of lactose to the diet markedly increases resistance to cecal colonization and organ invasion, and decreases horizontal transmission of S. enteritidis.     

Experimental infection of cats with Tritrichomonas foetus.

OBJECTIVE: To determine whether infection with Tritrichomonas foetus causes diarrhea in specific-pathogen-free or Cryptosporidium coinfected cats. ANIMALS: 4 cats with subclinical cryptosporidiosis (group 1) and 4 specific-pathogen-free cats (group 2). PROCEDURE: Cats were infected orogastrically with an axenic culture of T. foetus isolated from a kitten with diarrhea. Direct microscopy and protozoal culture of feces, fecal character, serial colonic mucosal biopsy specimens, and response to treatment with nitazoxanide (NTZ; group 1) or prednisolone (groups 1 and 2) were assessed. RESULTS: Infection with T. foetus persisted in all cats for the entire 203-day study and resulted in diarrhea that resolved after 7 weeks. Group-1 cats had an earlier onset, more severe diarrhea, and increased number of trichomonads on direct fecal examination, compared with group-2 cats. Use of NTZ eliminated shedding of T. foetus and Cryptosporidium oocysts, but diarrhea consisting of trichomonad-containing feces recurred when treatment was discontinued. Prednisolone did not have an effect on infection with T. foetus but resulted in reappearance of Cryptosporidium oocysts in the feces of 2 of 4 cats. During necropsy, T. foetus was isolated from contents of the ileum, cecum, and colon. Tritrichomonas foetus organisms and antigen were detected on surface epithelia and within superficial detritus of the cecal and colonic mucosa. CONCLUSIONS AND CLINICAL RELEVANCE: After experimental inoculation in cats, T. foetus organisms colonize the ileum, cecum, and colon, reside in close contact with the epithelium, and are associated with transient diarrhea that is exacerbated by coexisting cryptosporidiosis but not treatment with prednisolone.     

The early pathogenesis in chicken inoculated with non-pathogenic serotype 2 Marek's disease virus.

A newly cloned serotype 2 Marek's disease virus (MDV), strain ML-6, was inoculated via the nasal cavity in specific-pathogen-free chicks to examine early virus replication and the expression of Marek's disease (MD)-related antigens. Following inoculation, viral intracellular antigens (VIAs) were detected in lymphoid organs (bursas and spleens) between 5 and 14 days post inoculation (PI), in feather follicles between 14 and 30 days PI, and in lungs at 3 days PI by the immunohistopathological staining of avidin-biotin-peroxidase complex method. But, very few VIAs were expressed in the thymuses between 5 and 14 days PI. However, MD tumor-associated surface antigens were not detected in any organs. Viruses were isolated from separated spleen cells at 14 and 30 days PI. Fluorescent antibodies of convalescent sera were also detected after 10 days PI. As most of the VIAs were detectable in B-cells in bursas and spleens. B-cells were considered to be the main first target cells for the serotype 2 MDV infection.     

Mycobacterium paratuberculosis monoassociated nude mice as a paratuberculosis model

In this study, a paratuberculosis (Johne's disease) model was developed by intragastrically dosing gnotobiotic athymic nude mice with Mycobacterium paratuberculosis. The mice infrequently shed bacilli from their intestinal tracts during the first 4 months after inoculation. Following this time, increasing numbers of M. paratuberculosis (greater than 4.0 log10 bacilli per fecal pellet by 40 weeks) were recovered from the feces of the 12 mice that remained in the isolator. A similar pattern of recovery of M. paratuberculosis was obtained from the ileum, cecum, colon, and liver. Histopathologic lesions and acid-fast bacilli were rare during the first 4 months of infection and then, with time, increased in prevalence and severity. Mice maintained for 7 months or longer exhibited severe granulomatous inflammation and large numbers of acid-fast bacilli in the gastrointestinal tract and liver (up to 10(8) log10 colony forming units per gram wet weight). Five mice maintained for 7 months or more developed clinical signs consistent with those seen in paratuberculosis (weight loss, chronic diarrhea); three of these mice eventually died or became moribund and were euthanatized. M. paratuberculosis monoassociated mice released increased levels of tumor necrosis factor activity into their sera, as compared to uninfected control mice, when they were injected with bacterial lipopolysaccharide. The clinical signs, fecal shedding of M. paratuberculosis, granuloma formation, and progressive bacillary multiplication observed with these mice are consistent with naturally occurring M. paratuberculosis infection of ruminants (Johne's disease). This model will be useful for future studies of immunoregulation and antimicrobial therapy of paratuberculosis.     

Experimental fecal transmission of human cryptosporidia to pigs, and attempted treatment with an ornithine decarboxylase inhibitor

Fecal material collected from an immunologically deficient man with persistent cryptosporidia infection was stored in potassium dichromate for two weeks and then fed (inoculated) to newborn pigs. The six inoculated newborn pigs shed the organism in their feces starting four to five days afer inoculation and continuing for as long as 22 days after inoculation. Pigs which were killed and necropsied while shedding had cryptosporidia infection of ileum, cecum, and colon. Infected pigs had atrophied ileal villi and flattened irregular cecal and colonic epithelium. Uninoculated littermate controls remained free to the infection and had histologically normal intestinal tracts at necropsy. Treatment of three of the six inoculated pigs with the ornithine decarboxylase inhibitor, DL-alpha-difluoromethylornithine, orally for ten days had no apparent effect on the infection.