Canine Inflammatory Bowel Disease

Yang Dewei(1) Liu Fuan(2)

(1)State Key Laboratory for Biocontrol and Biopharmaceutical Center, Zhongshan University, Guangzhou, China 510275

(2) College of Veterinary Medicine, South China Agricultural University
Guangzhou, China 510642

Abstract: This paper reports the results of virological and microbiological studies on Canine Inflammatory Bowel Disease cases of undetermined etiology occurring in various parts of mainland China during the period 1997 through 2003. It was found that viruses could act synergistically with the secretion of certain bacteria to produce disease when alone they could not do so.

Keywords: Canine Inflammatory Bowel Disease; virus synergism; multiple antibiotic resistant bacteria

Preface Canine inflammatory bowel disease(CIBD)is a chronic inflammatory disease of the intestines and, although discovered by veterinarians in the late 1970s, the etiological agent has yet to be determined. Two hypotheses have evolved regarding the etiology,

1. Bacterial infection. Since CIBD cases could be healed with antibiotics?bacteria were considered the pathogen. However, up to the present nobody has succeeded in reproducing the disease by introducing isolated organisms into the intestine of experimental dogs, and so there is no proof to verify this hypothesis.

2. Antigen-antibody reaction within the intestinal tract. Scholars in favor of this hypothesis believe that canines ingest food possessing antigenicity(thus eliciting antigen-antibody reaction in the intestinal tract and triggering inflammation)leading to heightening of cell permeability. This in turn would allow protein macromolecules to inter the intestinal mucosa where more antigen-antibody reaction would take place, the exacerbated inflammatory reaction assuming a vicious circle. Owing to the fact that dogs belonging to the Alsatian, Sharpei, Rottweiler and other purebreds appear to be more susceptible to the disease, with a wide age range of 4 months to 15 years, some scholars consider genetic factors to be involved.

The symptoms of CIBD are: vomiting, diarrhea (acute at first then severe later) feces streaked with blood or mucus, loss of weight, abdominal flatulence, anorexia etc. Afflicted canines display one kind or various kinds of symptoms?and although diarrhea is usually present?it is not invariably so.

The severity of the disease depends on the immunocompetence of the sick animal and the extent of damaged intestinal area?there being great disparity between individuals. Inflammation in the large intestine leads to decrease in the amplitude of intestinal peristalsis. Enteritis is accompanied by vomiting, loss of bodyweight, change in appetite and profuse diarrhea.

The symptoms of progressive CIBD are: The sick canine initially might only vomit or exhibit diarrhea once in a month,after a time it may increase in frequency to once in a week or once in a day, and in severe cases vomiting and passing diarrheic feces several times a day. The course of the disease may be an indefinite few weeks to several months.

Due to its undetermined etiology, CIBD could only depend on eliminating other causes to arrive at a diagnosis.

When the diseased canine show vomiting, flatulence, diarrhea, hematochezia or feces with sloughed epithelial membrane, partial or complete loss of appetite, the veterinarian would generally check for the presence of improper diet, food allergy, parasites, intestinal impaction, insufficiency of pancreatin or other digestive enzymes, bacterial or viral infection, intestinal tract lymphoma and so on; after addressing one by one these presumptive causes without any alleviation of the condition, a diagnosis of CIBD would be arrived at by this process of exclusion.

1 Object of study
This study was done on Canine Inflammatory Bowel Disease?CIBD?cases encountered in 1997 through 2003. CIBD is a chronic inflammatory disease of the intestines and, although discovered by veterinarians in the late 1970s, the etiological agent has yet to be determined. In spite of that fact that laboratory isolation had yielded many suspected pathogens?include viruses and bacteria)?artificial infection of laboratory animals with these failed to reproduce the disease. But in reality over 200 of the CIBD cases encountered by us resulted in fatal affliction. Furthermore, repeated PCR analysis of pathological material derived from dogs that died of CIBD failed to reveal virus strains pathogenic to canines. These evidences led us to believe that the majority of CIBD cases were caused by nonpathogenic viral agents, which in conjunction with secretion from certain bacteria or other organisms, were able to synergistically elicit disease just as if they were virulent viruses. With this end in view the authors fabricated a device in which bacteria and virus could be grown in one culture system?in an attempt to discover whether substances secreted by bacteria could effect a synergistic action on nonpathogenic viruses collected from various localities.

1.1 Laboratory materials
CIBD virus strains and original bacteria strains secreting trypsin-like enzyme or subtilisin-like enzyme were collected from kennels, veterinary clinics and hospitals located in various parts of the country. The virus strains were subjected to preliminary identification with electron microscopy, after which PCR amplification with known canine virus primers was carried out and those found positive were stored in liquid nitrogen pending use. Primers for PCR amplification of various canine viruses gene segments, as well as those for virus genes of the Canidae and Felidae were designed and maintained by the authors, the synthesis of the primers being entrusted to the Jikang Bioengineering Co., Ltd. of Dalian City. Molecular biology reagents, bacteria drug sensitivity test reagents and drug sensitivity paper disks were provided by Waisees Animal Hospital. Sterile bench, molecular virology and microbiological laboratory utensil were conventional items of this laboratory. The collector for bacteria-carrying dust was a self-made product. Bacteria and animal cell co-cultivation device was a patented invention of our laboratory.

1.2 Methods
1.2.1 Virus/bacteria synergism: Primer design for canine viruses, virus purification, virus nucleic acid extraction, PCR analysis and indirect ELISA, bacteria identification and antibiotic sensitivity test were done as reported previously (6-17). The original material of 80 CIBD specimens found to be canine virus positive by PCR detection was subjected to electron microscopy for virus morphological identification, and finally 15 representative virus strains were selected to undergo the following experiments.

1.2.1.1 Method used to assess the effect of concurrent bacterial infection on virus infection pathogenicity: Bacteria in the raw sample were isolated and cloned, then each of representative bacterial clones was co-cultivated in cell culture with the canine virus originating from the same raw sample, the cell culture procedure being as reported in (15).

Each of the isolated bacteria was inoculated into a culture chamber so that the bacteria were separated by a 0.22 micron pore size millipore membrane from the animal cells so that they could not get into direct contact with each other, the detailed procedure being:

(1)Each bacterial sample was streaked onto nutrient agar plate, incubated at 37 C for 12 hours, after which isolated colonies were picked for identification.

( 2)WCK cell line was seeded into 40 Koch's flasks using 1640 cell culture medium, and when the monolayer showed 80% confluence, 20 CIBD virus isolates were separately inoculated into the culture flasks.
Group A?Ten specially fashioned bacterial culture chambers inoculated separately with each of ten bacterial clones, was placed in the culture medium of 15 flasks containing WCK cell monolayers, allowed to continue incubating at 37 C before removing the chambers, after which the WCK cells were further incubated for 36 hours.

Group B: Fifteen CIBD virus isolates were separately inoculated into 15 cell culture flasks?and allowed to continue incubation at 37 C for 48 hours.

Group C: Fifteen bacterial culture chambers each inoculated with a bacterial clone were transferred into separate cell culture flasks, allowed to incubate at 37 C for 12 hours before removing the chambers, then continuing incubation for another 36 hours.

Group D:
1.2.2 Bacteria drug sensitivity test was done as previously reported (18).
1.3 Assessment
Based on the appearance of CPE in cell culture inoculated with low pathogenic canine viruses, the following conclusion could be arrived at.
A. Should only cell cultures inoculated with bacteria secreting substilin-like protease show CPE, whereas those inoculated with bacteria secreting trypsin-like protease did not show CPE, it would indicate that the disease and mortality in the CIBD case under study was caused by concurrent infection of substilin-like secreting bacteria.
B. Should only cell cultures inoculated with bacteria secreting trypsin-like protease show CPE, whereas those inoculated with bacteria secreting trypsin-trypsinlike protease did not show CPE, it would indicate that the disease and mortality in the CIBD case under study was caused by concurrent infection of trypsin-like enzyme secreting bacteria;
C. Should only cell cultures inoculated with bacteria secreting substilin-like and trypsin-like protease show CPE, whereas the non-inoculated ones did not show CPE, it would indicate that the high mortality in the CIBD case under study could have been caused by concurrent infection of substilin-like or trypsin-like protease secreting bacteria.
D. Should the cell cultures that were only inoculated with CIBD virus show CPE?it would indicate that the high mortality in the CIBD cases under study was caused by highly pathogenic CIBD viruses.

2. Results
On the basis of preliminary morphological identification with electron microscopy and PCR testing done in this study, it could be concluded that two viruses and one infective agent constituted the viral pathogens?see electron micrographs fig. 1, 2, 3). Proposed nomenclature for these 3 virus pathogens: (1) Synergistic Canine Reovirus. The disease produced by this virus acting synergistically with enzymes secreted by bacteria listed in 2.2 is tentatively called Canine Synergistic Reovirus Inflammatory Bowel Disease. (2) Canine Synergistic Orthomyxovirus. The disease produced by this virus acting synergistically with enzymes secreted by bacteria listed in 2.2 is tentatively called Canine Synergistic Orthomyxovirus Inflammatory Bowel Disease. (3) Canine Synergistic Sub-parvovirus. The disease produced by this virus acting synergistically with enzymes secreted by bacteria listed in 3.3 is tentatively called Canine Synergistic Sub-parvovirus Inflammatory Bowel Disease.

2.2 The tested bacteria could be divided into 3 categories:?1?trypsin-like enzyme producing bacteria. (2?substilin-like enzyme producing bacteria. ?3?bacteria producing unidentified protease?table 1?.
Table 1
Bacteria strain TSB SSB UPB Antibiotic
1-34 yes sensitive
35-46 yes resistant
47-54 yes sensitive
55-58 yes resistant
59-73 yes sensitive
74-80 yes resistant

N.B. * TSB = Trypsin-like secreting bacteria
** SSB = Substilin-like secreting bacteria
*** UPB = Unidentified protease-secreting bacteria
2.2
Most of the bacterial strains whether secreting trypsin-like or substilin-like proteases were found sensitive to antibiotics (table 2)
Table 2

2.3
Group A All 15 WCK cell culture flasks, in the presence culture chambers containing either trypsin-like secreting or substilin-like secreting bacteria, developed CPE.
Group B The 15 WCK cell culture flasks, which had only been inoculated with CIBD virus, did not show CPE.
Group C The 15 WCK cell culture flasks, in which only bacteria culture chambers containing purified isolates had been placed, did not exhibit CPE.
Group D The 5 WCK cell culture flasks, in which neither virus nor bacteria was inoculated, did not show any CPE.

3. Discussion
Nowadays when veterinarians confront the diagnosis of CIBD?they chiefly adopt deductive elimination of likely pathogens and artificial infection of laboratory animals. However, the method of excluding likely pathogens is not effective when dealing with a pathogen that cannot induce disease by itself. No international veterinary organization has been able up to reproduce CIBD in experimental animals by inoculation of isolated pathogen alone up to date.

As mentioned in the preface of this paper, veterinarians used to suspect that bacterial infection was the cause of CIBD. Since some CIBD cases could be healed with antibiotics?bacteria were considered the pathogen?however, up to the present nobody has succeeded in reproducing the disease by introducing isolated organisms into the intestine of experimental dogs, and so there is no proof to verify this hypothesis. The present CIBD study revealed that all the bacterial isolates were not capable of inducing disease by themselves alone?nor could the virus isolates do so alone. They must in conjunction exert synergistic action before disease could be produced. From table 1 and table 2 it can be gathered that the majority of synergistic bacteria are sensitive to antibiotics?and after their being compromised by antibiotics, synergistic action with CIBD viruses cease. This is a rational explanation why antibiotics can be used to heal, yet using isolated bacteria cannot induce CIBD by introducing them into the canine intestinal tract.

A dog is constantly getting in contact with various antigens during its life, which will elicit the production of corresponding antibodies, and this is the reason why serum therapy can have curative effect in CIBD.
Of the CIBD cases in this study?Alsatian, Sharpei and Rottweiler dogs born locally but not in large kennel groups, are not prone to contract CIBD. In contrast, imported purebred Alsatian, Sharpei and Rottweiler appear more susceptible to the disease. That purebred dogs born locally, but not collectively raised in kennels, should have stronger resistance to local pathogens conforms to pathological principles. Imported purebred dogs initially exposed to synergistic pathogens in a new environment become sick rather easily. Incidentally, no evidence for genetic susceptibility was found in this study. Consequently?the authors cannot agree to hypotheses regarding CIBD pathogenesis put forward by certain scholars abroad.

Many cases of CIBD have been encountered all over the world since the early 1970s. Since the canine population lacked antibodies to evolving new serotypes, one would anticipate a pandemic within a short space of time, but that did not materialize, why? This can be attributed to the inclination for clinicians in general to use wide spectrum antibiotics. Under the effect of broad-spectrum antibiotics the complicating bacteria secreting trypsin-like or substilin-like proteases are suppressed, and so CIBD progeny viruses lacking the enzymes for infectivity cannot replicate, with the result that the range of infected cells becomes greatly restricted. Table 1 and Table 2 show several unclassified antibiotic-tolerant bacteria, which can, however, produce enzymes able to produce a synergistic effect. One may speculate that receptors on the surface of the viruses may need modification by bacterial proteases to effect synergism in infection. Of more concern is the fact that some of these synergistic bacteria are antibiotic resistant, which may greatly reduce the efficacy of supportive therapy. This will be a research area of great value. In this study?15 cloned bacteria each inside a bacterial culture chamber was placed into each of the group A and group C cell culture flask, incubated at 37 C for 12 hours, after which the chamber was removed, this procedure being adopted to ensure that enough bacterial enzyme would be secreted while not depriving the cells of nutrients.

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Appendix: bacteria and cell culture co-cultivation device
This is an in vitro co-cultivation device?featuring an mammalian cell culture bottle (1) plus a bacterial culture chamber (3) with the latter placed inside the former. The bacterial culture chamber is an enclosed structure?its wall fitted with a millipore membrane allowing passage of medium constituents not larger than 0.22 micron in size.

According to patent required description of the device (4) the bacterial culture chamber (3) features an "a" wall (32) and a "b" wall (32) squeezing on a ring-shaped seal (33) kept in position by screws (35) to make an enclosed structure. On the "b" wall is a small opening (36) closed with a 0.22 micron pore size millipore membrane (34), which prevents bacteria from getting out the chamber, while allowing free passage of bacterial secretion and culture medium.

According to patent required description of the device (5), the "a" wall and "b" wall as well as positioning screws can be made of stainless steel or heat-stabile plastics, and the ring-shaped seal can be of heat-stabile non-toxic rubber or plastics.
Procedures for nested PCR of purified virus (see references 2, 5, 6, 7, 8, 9, and 10)

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