Application of a pig ligated intestinal loop model for early Lawsonia intracellularis infection

Background

Porcine proliferative enteropathy in pigs is caused by the obligate, intracellular bacterium Lawsonia intracellularis. In vitro studies have shown close bacterium-cell interaction followed by cellular uptake of the bacterium within 3 h post inoculation (PI). However, knowledge of the initial in vivo interaction between porcine intestinal epithelium and the bacterium is limited. The aims of the present study were to evaluate the usefulness of a ligated small intestinal loop model to study L. intracellularis infections and to obtain information on the very early L. intracellularis-enterocyte interactions.

Methods

A ligated small intestinal loop model using three different L. intracellularis inocula was applied to 10-11-week-old pigs. The inocula were 1) wild type bacteria derived from overnight incubation of L. intracellularis bacteria from spontaneous disease, 2) crude vaccine bacteria (Enterisol^® Ileitis Vet), and 3) vaccine bacteria propagated in cell culture. The bacteria-enterocyte interaction was visualised using immunohistochemistry on specimens derived 1, 3 and 6 h PI respectively.

Results

Although at a low level, close contact between bacteria and the enterocyte brush border including intracellular uptake of bacteria in mature enterocytes was seen at 3 and 6 h PI for the vaccine and the propagated vaccine inocula. Interaction between the wild-type bacteria and villus enterocytes was scarce and only seen at 6 h PI, where a few bacteria were found in close contact with the brush border.

Conclusions

The ligated intestinal loop model was useful with respect to maintaining an intact intestinal morphology for up to 6 h. Furthermore, the study demonstrated that L. intracellularis interacts with villus enterocytes within 3 to 6 h after inoculation into intestinal loops and that the bacterium, as shown for the vaccine bacteria, propagated as well as non-propagated, was able to invade mature enterocytes. Thus, the study demonstrates the early intestinal invasion of L. intracellularis in vivo.     

Arthroderma benhamiae infection in a rabbit

The isolate from the rabbit with dermatophytosis which was transmitted to the owners was proved to be Arthroderma benhamiae (-) by mating experiments as well as by chitin synthase 1 (CHSI) gene analysis.     

Development of a laboratory animal model of postoperative small intestinal adhesion formation in the rabbit.

In order to establish a model of postoperative intestinal adhesions that would simulate the problem experienced in horses, New Zealand White rabbits were utilized to compare two models of adhesion formation that had been successful in the horse, an ischemic strangulating obstruction (ISO) model and a serosal scarification model. An untreated control group was compared with animals subjected to 1, 2, 3 and 4 h periods of ISO, and to serosal scarification. At postmortem examination 14 d postoperatively, the number of rabbits in each group with adhesions was recorded. Serosal scarification was significantly more consistent at producing adhesions than ISO (Fisher's exact test, P = 0.0022). The 3 h of ISO group was significantly different from the control group: however, compared to the serosal scarification group, fewer animals had adhesions and one animal died of complications associated with the experimental procedure. Based on these results, serosal scarification was selected as the best model for utilization in further studies of adhesion prevention.     

Evaluation of the rabbit as a laboratory model for infectious bovine rhinotracheitis virus infection

Experimental infection of rabbits with infectious bovine rhinotracheitis virus (IBRV) produced diverse manifestations of disease which included abortion, conjunctivitis, dermatitis, vulvovaginitis, systemic infection, neonatal death and respiratory tract infection. Each disease syndrome was studied using virus isolation, fluorescent antibody examination and histologic examination. Conjunctivitis, dermatitis and vulvovaginitis lesions were characterized by edema, infiltration of mucosa and submucosa with inflammatory cells and ulceration of epithelium. Systemic infection resulted in severe necrosis of liver and adrenal glands with large numbers of cells containing intranuclear inculsions. Pregnant rabbits aborted within 48 hours following inoculation of IBRV. Virus infection and viral lesions were not demonstrated in aborted fetuses.     

Development of a polymerase chain reaction-based in vivo method in the diagnosis of subclinical pigeon circovirus infection

This paper describes a polymerase chain reaction (PCR)-based method performed on blood samples and intestinal content to detect subclinical pigeon circovirus (PiCV) infection in live pigeons. In addition, two sets of primers (primer set 1 and 2), designed in two different regions of the viral genome, were used to provide evidence of possible differences in PCR responses. Blood and intestinal content samples were randomly collected from a total of 50 apparently healthy meat pigeons, aged 1 to 5 wk, which came from central Italy. Samples of primary lymphoid organs were also collected. Results showed a high level of PiCV infection, although clinical signs were not present. The results obtained with the two sets of primers showed that primer set 2 was able to detect a higher number of PCR-positive pigeons (45 of 50 pigeons) than primer set 1 (11 of 50 pigeons). In both cases an increase in positive results with pigeon age indicates that the major direction of transmission is likely horizontal. In these circumstances feces can play an important epidemiologic role, as supported by the consistent circovirus detection in intestinal content. The high sensitivity of this PCR test, which is able to detect very low amounts of viral DNA (5.5 x 10(-3) fg of plasmid containing the cloned PiCV genome), makes it suitable for possible application as an epidemiologic tool for identifying virus carriers for subsequent removal from lofts.     

Nicarbazin stimulates intestinal transport of L-leucine in rabbit

Nicarbazin is an anticoccidial drug used mainly in birds, but also in rabbits. Besides the anticoccidial activity, riicarbazin has shown several other effects such as inhibition of growth and feed efficiency in poultries, and stimulation o f sugar intestinal absorption in rabbits. The present work has been performed in order to check whether nicarbazin also affects L-leucine intestinal absorption. The results obtained show that nicarbazin decreases L-leucine accumulation in the jejunal tissue, and increases mucosal to serosal transepithelial fluxes o f this amino acid in a dose-dependent way, without modifying its diffusion across the intestinal epithelium. The drug stimulates the amino acid uptake in brush-border and basolateral membrane vesicles, thus suggesting that nicarlxizin increases the absorption of L-leucine mediated by carriers.     

Experimental infection of young rabbits with a rabbit enteric coronavirus.

The clinical signs and lesions caused by the rabbit enteric coronavirus (RECV) were studied in young rabbits orally inoculated with a suspension containing RECV particles. The inoculated animals were observed daily for evidence of diarrhea. Fecal samples and specimens from the small intestine and from the gut associated lymphoid tissue (GALT) were collected from 2 h to 29 days postinoculation (PI) and processed for immune electron microscopy (IEM) and light microscopy. Coronavirus particles were detected in the cecal contents of most inoculated animals from 6 h to 29 days PI. Lesions were first observed 6 h PI and were characterized by a loss of the brush border of mature enterocytes located at the tips of intestinal villi and by necrosis of these cells. At 48 h PI, short intestinal villi and hypertrophic crypts were noted. In the GALT, complete necrosis of the M cells as well as necrosis of the enterocytes lining the villi above the lymphoid follicules with hypertrophy of the corresponding crypts were observed in all the animals. Five inoculated rabbits had diarrhea three days PI. The presence of RECV particles in the feces of the sick animals and the microscopic lesions observed in the small intestine suggested that the virus was responsible for the clinical signs. A few inoculated rabbits remained free of diarrhea. Fecal material collected at postmortem examination contained RECV particles. The results suggest that the virus could also produce a subclinical infection.     

Efficacy of gallium maltolate against Lawsonia intracellularis infection in a rabbit model

Antimicrobial efficacy against Lawsonia intracellularis is difficult to evaluate in vitro, thus, the effects of gallium maltolate's (GaM) were investigated in a rabbit model for equine proliferative enteropathy (EPE). Juvenile (5–6‐week‐old) does were infected with 3.0 × 10⁸ L. intracellularis/rabbit and allocated into three groups (n = 8). One week postinfection, one group was treated with GaM, 50 mg/kg; one, with doxycycline, 5 mg/kg; and one with a sham‐treatment (control). Feces and blood were collected daily and weekly, respectively, to verify presence of L. intracellularis fecal shedding using qPCR, and seroconversion using immunoperoxidase monolayer assay. Rabbits were sacrificed after 1 week of treatment to collect intestinal tissues focusing on EPE‐affected sections. Intestinal lesions were confirmed via immunohistochemistry. No difference was noted between treatments regarding EPE‐lesions in jejunum (P = 0.51), ileum (P = 0.74), and cecum (P = 0.35), or in L. intracellularis fecal shedding (P = 0.64). GaM and doxycycline appear to have similar efficacy against EPE in infected rabbits.     

Fluid accumulation in the ligated intestinal loop and histopathological changes of the intestinal mucosa caused by Clostridium botulinum C2 toxin in the pheasant and chicken

Fluid accumulation was induced in ligated intestinal loops of both pheasants and chickens injected with crude Clostridium botulinum C2 toxin. Necrosis of the epithelium and lamina propria of the duodenum and jejunum occurred in both species of bird after intraduodenal administration of crude C2 toxin. The severity of such reactions depended upon the dose and the period after administration of C2 toxin up to eight hours, and such reactions were suppressed specifically with rabbit anti-C2 toxin. These results confirm that C2 toxin is one of the causes of diarrhoea in avian botulism.     

Virulence of wild and mutant strains of Salmonella typhimurium in ligated intestinal segments of calves, pigs, and rabbits

A ligated intestine model in calves, pigs, and rabbits was tested for its value as an indicator of virulence of potential vaccine strains of Salmonella typhimurium. A wild virulent strain (3860C), a laboratory strain LT2, and mutants of these 2 strains were evaluated. Inoculation of calf intestinal segments with strain 3860C revealed that fluid responses were greatest in the proximal portion of the small intestine and that doses greater than 10(7) organisms were required to produce fluid responses and mucosal damage. Immunoperoxidase-stained sections of intestine revealed that a large dose of Salmonella organisms was required before mucosal invasion could be detected. Aromatic (aroA), galactose epimerase (galE), and diaminopimelic acid (dap) mutants of strain 3860C all resulted in much less fluid response, mucosal invasion, and mucosal damage compared with those by the parent organism. Strain LT2 induced such weak responses that it was not possible to evaluate reductions in virulence of its mutants. In 6-week-old pigs, there was no fluid response to any strains; however, in 1-week-old pigs, there was fluid response to the wild strain and some of its mutants. In adult rabbits, fluid responses were not observed, except when the wild strain was inoculated in the proximal portion of the small intestine. The calf and 1-week-old pig models appeared to be best suited for assessment of virulence of mutant strains of S typhimurium.     

Development of a technique for the in vivo assessment of flatulence in dogs.

OBJECTIVE: To develop a noninvasive method for the in vivo assessment of flatulence in dogs. ANIMALS: 8 adult dogs. PROCEDURE: Rectal gases were collected via a perforated tube held close to each dog's anus and attached to a monitoring pump fitted with a sensor that recorded hydrogen sulfide concentrations every 20 seconds. Patterns of flatulence were monitored for 14 hours after feeding on 4 days, and within- and between-dog variation was assessed over 4 hours on 4 consecutive days. Rate of hydrogen sulfide production (flatulence index) and frequency and number of emissions were evaluated as potential indicators of flatus characteristics. An odor judge assigned an odor rating to each flatulence episode, and the relationship between that rating and hydrogen sulfide concentration was determined. RESULTS: Flatulence patterns varied within and between dogs. Variation was most pronounced for flatulence index; mean coefficients of variance within dogs over time and between dogs on each day were 75 and 103%, respectively. Flatus with hydrogen sulfide concentrations > 1 parts per million could be detected by the odor judge, and severity of malodor was highly correlated with hydrogen sulfide concentration. Odor ratings were accurately predicted by use of the equation 1.51 X hydrogen sulfide concentration(0.28). CONCLUSIONS AND CLINICAL RELEVANCE: The technique described in this report appears to provide sensitive, reliable, and relevant data and will enable further studies of the factors that influence flatulence in dogs. Use of this technique also has the potential to aid in investigations of colonic physiology and pathology.     

High and low virulence Staphylococcus aureus strains in a rabbit skin infection model

In the present study, an in vivo rabbit skin infection model was developed to reproduce the lesions caused by high and low virulence Staphylococcus aureus strains from rabbits. "O"-shaped dermal skin lesions were induced on the shaved flanks of anaesthetised rabbits using a tattoo pin and pincers. The induced lesions on the flanks of four groups of 10 rabbits were then inoculated by topical application of 0.1 ml of 10(8)cfu S. aureus bacteria. One group was inoculated with a typical high virulence (HV) S. aureus strain from rabbits, one group received an atypical HV strain and two groups were inoculated with low virulence (LV) strains. Five animals were kept as negative controls. The development, appearance and size of abscesses were scored daily for a period of 2 weeks. The infection model showed reproducible results for the different S. aureus inoculation groups. Inoculation of the skin with the typical HV strain resulted in significantly larger abscesses than those caused by the LV strains. The atypical HV strain caused abscesses of a size intermediate to that obtained with the HV and LV strains. In rabbits infected with LV strains, most of the lesions had healed by day 14 post-inoculation. The devised infection model is able to reliably reproduce the virulence properties of HV and LV S. aureus strains.     

Development of a control strategy for Leptospira hardjo infection in a closed beef herd.

Serological evidence of infection with a leptospire belonging to the Sejroe serogroup was identified in a closed population of Luing cattle in the west of Scotland, and the geographical isolation of the population presented an opportunity to control and possibly eradicate the infection in a large beef herd farmed under extensive conditions. Serological and bacteriological studies revealed that infection was present at a high level throughout the herd, and that the infecting serovar was hardjo. Unlike endemic hardjo infection in dairy herds, new infections were still occurring in older age-groups. Investigations of other domestic and free-living species sharing the habitat demonstrated that the maintenance of an endemic focus of hardjo was restricted to the cattle. Changes in management to prevent the transmission of infection to successive cohorts of young animals were impractical and risky, and antibiotic treatment followed by removal to clean pasture failed to prevent new cases. Thus vaccination offered the only means of control and possible eradication, and the epidemiological characteristics of the infection dictated that the programme be applied to the whole herd.     

Development of a hamster model of Chlamydophila pneumoniae infection

The aim of this study was to develop a new experimental model of Chlamydophila pneumoniae infection in the hamster. Intraperitoneal injection of C. pneumoniae purified elementary bodies (EBs) in the hamsters caused a systemic infection, since it was possible to isolate viable chlamydiae from several organs up to 14 days after infection. In particular, spleen infection was detectable up to 7 days post infection in 100% of animals. In contrast, cultures of the organs obtained from intranasally infected animals were far less frequently positive. Systemic infection probably occurred via macrophages, as demonstrated by the presence of intracellular chlamydial inclusions in peritoneal macrophages of peritoneally inoculated animals four days after infection. Furthermore, by infecting LLC-MK2 cells with supernatant preparations obtained from these macrophages, it was possible to observe the development of chlamydial intra-cytoplasmic inclusions after 96 h. Immunization of 18 hamsters with heat-inactivated purified EBs completely protected 16 animals and substantially reduced infection levels in the remaining two. Sera obtained from immunized hamsters prior to challenge reacted mainly against two C. pneumoniae proteins of about 60 kDa, when tested by immunoblot.