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ANAEROBES IN HEALTH AND DISEASE OF ANIMALS
A.S. Upadhye and M.D. Venkatesha

Courtesy : Festschrift - Dr. S. Ramachandran

Introduction 

Anaerobes play an important role in nutrition, health and disease processes in animals. Nutrition is dependent to a considerable extent upon the microbial fermentations that occur in the gastro-intestinal tract (GI tract) of animals. This realization has called for extensive research into the nature and activities of the normal microbial population of the GI tract. Considerable scientific data have been built up to understand the nature of microbial population and their activities. However, there are many gaps still to be covered in our knowledge of this complex phenomenon.

The conditions of the GI tract are highly suitable for the development of vast and diverse microbial populations. The environment of GI tract is primarily anaerobic, there is plenty of food and nutrients available all the time for microbes; the pH and temperature are highly favourable and in addition, the inhibitory end products are gradually removed. Since the environment of the GI tract is anaerobic, the organisms of functional significance attracting most widespread interest are anaerobes. The colonization of the GI tract of the newborn animal occurs within a few days after the birth. Anaerobes are the predominant bacteria associated with the GI tract outnumbering aerobic organisms by as much as 102 to 104. High concentrations of bacterial population have been observed in the rumen, colon and caecum. 

Oxygen is inimical to anaerobic bacteria. It is speculated that oxygen toxicity in anaerobes is due to the absence of the protective enzymes, catalase, peroxidase and superoxide dismutase found in aerobes. Anaerobic bacteria have been divided into obligate anaerobes that do not form colonies on agar surface exposed to 0.5% or more oxygen and moderate anaerobes that are capable of growth at oxygen levels ranging from 2-8%. Strict obligate anaerobes are typically members of the normal GI tract flora, whereas diseases are produced by mostly moderate anaerobes. 

The indigenous microflora play many important roles. The GI microflora constitute a complex ecosystem and assist the animal in digestion of feed consumed and act as source of nutrients (microbial protein and vitamins). It has also been demonstrated that indigenous microflora by early colonization provide protection against infection by pathogenic microorganisms and play an essential role in providing optimal resistance to disease. 

Anaerobic bacteria are also responsible for a number of diseases in man and animals. Some of the well-known diseases such as enterotoxaemia, black-quarter, tetanus, botulism etc. are caused by clostridia, which originate exogenously from soil. However, some organisms, which form part of the indigenous flora of the intestinal, genital and respiratory tracts, may cause infections under suitable conditions. These opportunistic pathogens were completely overlooked and have come into prominence recently. The endogenous infections are reported to be ten times more common than the exogenous infections. 

Anaerobes are difficult to grow because of their sensitivity to oxygen. Studies on anaerobes were delayed till techniques for isolation of anaerobes were developed and also due to the difficult and laborious procedures involved. 

In the present paper, the role of nonpathogenic indigenous anaerobic flora in health and of pathogenic endogenous / exogenous anaerobes in diseases of animals will be discussed. 

Anaerobic bacteria in nutrition and health 

Rumen digestion 
The digestive anatomy and physiology of ruminants is markedly different from that of non-ruminants. The rumen is essentially a fermentor chamber containing 1010 bacterial cells and 105 -106 protozoal cells / gram of rumen contents, together with an unknown (probably 8% of the ruminal mass) number of anaerobic fungi. This considerable concentration of rumen microorganisms varies from time to time and exists in symbiotic relationship with the host. Many of the plant materials consumed by the ruminants are digested and fermented by the rumen microbes to form chiefly volatile fatty acids (VFA), carbon dioxide and methane. The rumen VFA are of considerable value to the host. Ruminants have evolved to utilize the VFA resulting from fermentation as their main source of energy, growth and lactation. In addition to aiding digestion through breakdown of higher carbohydrates, these microorganisms are also responsible for synthesis of certain essential amino acids and vitamins. The advantage of this foregut fermentation compared to hind gut fermentation is that the microbial cells formed as a result of the fermentation are available to the host as they pass down the tract to the ruminant gastric stomach, the abomasum. The microbial protein is the most important source of amino acids for absorption. 

The VFA produced by microbial fermentation are mainly acetic, propionic and butyric acids with smaller amounts of higher branched acids. The microbial decomposition of carbohydrates in the rumen varies according to the kind and number of microorganisms present, which in turn are under the influence of the character of the feed. The products of carbohydrate fermentation serve as sources of energy for body processes and for synthesis of body fat. Improvements in feed utilization by ruminants depend upon, in part, on advantageous manipulation of feeds and feeding practices to exploit the potential of rumen microbes. Studies on the nature of rumen microorganisms and their role in utilization of varieties of feed available would be of great value. Attempts to modify rumen microflora have also been made for better utilization of a variety of organic wastes as feed. Ruminants are able to utilize both protein and non-protein nitrogenous substances like urea for body maintenance and production as the microbial population of rumen synthesizes microbial protein of relatively high biological value which becomes available to the animal by the normal process of protein digestion and absorption in the abomasum. 

The rumen microflora 
The environment of the rumen is well adapted for the maintenance of a large and diverse microbial population, which consists of bacteria, protozoa and fungi. The predominant rumen bacteria and their salient characters are shown in Table 1. In animals predominantly on a forage ration, the majority of bacteria are Gram-negative and with high grain ration there is increased proportion of Gram-positive cells. Among the diverse groups, cellulose digesters and ureolytic species are of great importance because of their ability to digest cellulose and hydrolyze urea respectively. Significant differences in the numbers as well as types of microorganisms in the rumen of animals fed on different feeding schedules have been reported (1,2,3). 

Protozoa in the rumen comprise about 105 -106 cells / gram of ruminal contents. Higher number of protozoa is generally found in rumen when diets of high digestibility are fed. Frequent feeding also increases the concentration of protozoa.

Table 1 

Important rumen bacteria and their salient characters

Grinding and pelleting the feed, starvation or prolonged under-nutrition are known to drastically reduce the protozoa in the rumen due to increased acidity. The anaerobic fungi are the most recently recognized group of rumen microbes. When animals are fed a high forage diet, rumen fungi contribute up to 8% of the microbial mass. It is still unclear whether fungi are functionally significant. However, they have been shown to degrade cellulose and xylose indicating some role in fibre digestion. 

All the typical microorganisms of the rumen appear to be specific to that habitat. Direct contact between animals seems necessary for transfer of organisms from one animal to another. Roughage diet rather than a concentrate diet allows the most rapid development of a considerable variety of typical rumen microorganisms. It might probably be due to the influence of the low pH attained in the rumen of animals fed on concentrate diet.

Early attempts to isolate rumen bacteria met with little success because of the complex nutritional requirement of rumen organisms and their requirement of strict anaerobic conditions. Procedures of isolation and characterization of anaerobic rumen bacteria are cumbersome and time consuming. Starting from sample collection all the procedures have to be carried out under strict anaerobic environment, with no oxygen contact at any stage. Various anaerobic methods using anaerobic jars, gas pack system, anaerobic chambers or glove boxes and roller tube techniques have been developed. 

Due to their dependence on nutrition of microbial fermentation, ruminants are subjected to certain maladies not present in other animals. These are bloat, acute indigestion, nitrate poisoning, ammonia toxicity etc. which are related to the activities of these microorganisms in the rumen. 

Indigenous intestinal microflora
There is considerable evidence that when antibiotics are used to disrupt the intestinal flora, the experimental animals become very susceptible to colonization with pathogens, but when the indigenous flora is undisturbed they are quite resistant. The various mechanisms involved in prevention of colonization by pathogens may be due to competition between indigenous flora and pathogens for nutrients, toxic metabolites produced by indigenous flora and adverse environmental conditions and competition for association sites (4). 

The hypothesis that nutrient competition is responsible for exclusion of non-indigenous organisms from the intestinal tract is based on series of observations indicating that population control mechanisms in the intestine are consistent with chemostat theory. A fundamental concept of chemostat theory is that bacteria in mixtures compete for essential growth factors. Multiplication of E. coli, fusobacter and eubacteria was greatly suppressed when organisms were inoculated. A major factor limiting multiplication of the organisms was lack of source of utilizable carbohydrates. It is speculated that competition for utilizable carbohydrates is of overriding importance in the regulation of populations in the intestinal tract. 

There is considerable evidence that toxic metabolites including hydrogen sulfide, free bile acids and short chain fatty acids are inhibitory to intestinal bacteria. Hydrogen sulfide contributes to the suppression of E. coli. Data are available on the participation of VFA in excluding pathogens from the intestinal tract. Toxic metabolites interfere with the ability of pathogens to associate with intestinal mucosal surfaces. The acids are involved in the exclusion of pathogens from the intestinal tract. Salmonella enteritidis is inhibited by suspensions of intestinal contents specifically from caecum and colon. There is inverse relationship between VFA concentration and enterobacteria population levels of intestine. Treatment with streptomycin causes an increase in the pH of caecal contents and decreases VFA concentration, which results in more hospitable environment for multiplication of intestinal pathogens. 

Disease processes depend on the persistence of the pathogens in the intestinal tract. The inhibitory activity of the flora, the doubling time of the pathogens in the ruminal contents often exceed the dilution rate and under these conditions the pathogens are washed out of the intestinal tract. To ensure survival in this ecosystem, the pathogen has to associate with the intestinal mucosa. Ability to associate with the intestinal mucosa, is an important determinant for the successful colonization of the intestinal tract by the pathogenic organisms. Data from several experiments indicate that flora components compete with the pathogens for mucosal association sites. The flora firmly attached to mucosa, block colonization by pathogens. Antibodies to some of the indigenous flora have been demonstrated. The role of these antibodies in giving partial protection against antigenically related pathogens is not known. 

Anaerobes in disease 
Many species of pathogenic anaerobic bacteria are involved in disease processes of animals. Anaerobic bacteria cause specific diseases as well as nonspecific diseases. These diseases may be grouped into two groups based on the characteristics of the causal agents. The first group has spore-forming anaerobes (clostridial group) responsible for causing specific diseases in animals and originates exogenously. The second group consists of non-spore forming anaerobes, which cause non-specific diseases and originates endogenously (5,6,7).

Spore forming anaerobes
Clostridia are widely distributed in nature and have their main habitat in soil and also occur as common inhabitants of the GI tract of man and animals. It is from the soil or from vegetation contaminated with faecal contents that the infection originates. The soil borne infections are most wide spread among domestic animals including poultry. Clostridial diseases affecting different species of animals and the pathogenesis of clostridial infection in general are shown in Table 2. Most of the clostridial infections are acute and fatal and the predisposing factors such as tissue / muscle damage, over feeding, liver fluke infestation, etc. play predominant role in outbreaks of diseases. Pathogenic clostridia are grouped into histotoxic (invasive) and toxigenic groups (non-invasive). Histotoxic clostridia produce a number of exotoxins and tissue destroying enzymes such as haemolysin, hyaluronidase, fibrinolysin, DNAse etc. with the help of which they are able to invade tissues and cause infection. Toxigenic clostridia produce highly potent toxins but are not able to invade tissues. The toxins produced outside the body or at local sites, get absorbed and cause disease (8,9,10).

Clostridia are Gram positive, motile / non-motile anaerobic spore forming bacilli. Oxygen tolerance varies among species. Some are strict anaerobes, others are oxygen tolerant. Clostridial spores are resistant to adverse environmental conditions and survive in soil for long periods. Clostridia are biochemically active and produce a number of toxins and enzymes. Some of the clostridia such as C. novyi, C. perfringens and C. botulinum are further typed based on toxins produced.

Many clinical signs shown by the animals such as diarrhoea, acute death, necrotic lesions and wounds lead to suspicion of clostridial infection. Direct microscopic examination of the lesion shows the presence of a large number of characteristic spore forming clostridia. Suitable samples from the affected tissues may be inoculated into enriched and selective media for isolation of the causative agents following standard anaerobic techniques. Spore selection technique of heating the sample at 800C for 10 minutes and then inoculating will be helpful. Characterization 

Table 2

 Clostridia causing infections of animals

Clostridium : Histotoxic

Clostridium : Toxigenic

of the organism may be made by specific biochemical tests. Toxin-antitoxin neutralization tests may be carried out in mice. Several commercial kits are being marketed for rapid identification of anaerobes in general. Fluorescent antibody reagents for C. septicum, C. chauvoei, C. novyi, and C. sordelli from Wellcome research Labs permit rapid identification of species. A recently marketed rapid glutamic acid decarboxylase microdilution test has been found to aid in the differentiation of commonly isolated clostridia. Diagnostic clostridial antiserum is also available commercially. 

Clostridial diseases are preventable. Because the course of the clostridial diseases is acute and often fatal, prophylactic vaccination of animals that are at risk is the method of choice. Vaccines prepared from C. septicum, C. chauvoei, C. novyi, C. sordelli, C. haemolyticum are available commercially. Typically, the vaccines consist of inactivated broth cultures of the bacteria, so that antibodies develop to both bacterial surface antigens and also the toxic products. The vaccines are prepared of individual bacterial agents or combinations of agents. The pathogens that are prevalent in the area determine the choice. Anti-tetanus vaccinations are done with adjuvanted tetanus toxoid. Botulinum toxoids have been used as vaccines to prevent botulism in animals including poultry and are currently used for other therapeutic measures in certain neurological conditions. 

Penicillin / ampicillin have excellent inhibitory activity against most clostridia. Chloramphenicol, piperacillin, metronidazole and imipenem are also active against all clostridia. Clostridia have shown resistance to cephalosporins, tetracycline, aminoglycosides and ciprofloxacin. 

Non-sporeforming anaerobes
Most of the infections caused by non-spore forming anaerobes are endogenous in origin since they are part of the normal bacterial flora of the mucosal surfaces of the alimentary / respiratory tracts. The infections originating from non-sporeforming anaerobes are shown in Table 3. The non-sporeforming anaerobes are strict obligatory anaerobes and being associated with intestinal, genital and respiratory tracts, they may cause uterine and pelvic infections, endometritis, pyometra, septic abortions, pulmonary infections, abdominal infections, liver abscesses etc. A variety of superficial soft tissue infections are also caused by these organisms, such as sinuses, abscesses, skin and wound infections. These endogenous infections are nonspecific, less acute in their onset and do not produce severe toxaemia. The organisms are comparatively of low pathogenicity and do not produce primary infection when inoculated singly into experimental animals. They cause disease at sites that have been debilitated or are seats of some preceeding pathological change. The Gram-negative non-sporeforming bacilli and cocci together are the commonest cause of anaerobic bacterial infections whose significance in pathogenesis is often overlooked. Understanding their role in disease is hampered by difficulties in isolation and identification of the large number of species involved and the complexity of their interaction with the host and each other. 

Table 3 

Non-clostridial anaerobes causing infections of animals

Bacteriological investigations carried out on clinical specimens from different species of animals with uterine and pelvic infections, pyometra, necrotic pneumonia, liver abscesses, intra-abdominal infections have revealed the presence of obligatory anaerobic species belonging to different genera. Relative incidence of anaerobic bacteria in various infections are intra-abdominal 90-95%, liver abscess 50%, female genital tract 55%, septic abortions 73%, soft tissue infections 100%, urinary tract infections 1%. Generally anaerobic cocci are more prevalent in the upper respiratory tract but sparse in the intestinal tract. 

Virulence factors associated with these organisms are responsible for enhanced pathogenicity. Capsule with its antiphagocytic property promotes abscess formation. Production of the cytotoxins and enzymes such as protease, lipase promotes destruction of tissue and spread. The tissue destruction also provides nutrition and anaerobic environment for the growth. 

The conditions necessary for the development of many of these endogenous infections are not clearly understood. Probably, when the local resistance of the tissues is lowered by inadequate diet or by unrelated infections, rapidly progressive infections may result. The endogenous infections also originate as secondary complications of surgical intervention of intestinal, respiratory and genital tracts. 

Endogenic anaerobic infections share many of the clinical features that are common to most types of pyogenic sepsis. There are, however, features that may help to distinguish these infections from other types of bacterial infections. The proximity of the infection to mucosal surfaces and the conditions that interfere with the integrity of the mucosal surface reflect the portal of entry of these endogenously derived organisms and the copious foul smelling discharges, which are more characteristic of these infections. 

Most suitable specimens for cultural examination of the infections are pus or other exudate and pieces of tissue from infective lesions of the gastrointestinal, genital or respiratory tract. The clinical specimens for anaerobic culture must be collected by methods that avoid contamination with the normal bacterial flora. A variety of specimen transport systems have been developed to protect bacteriological specimens from oxygen. The specimen needs to be processed fast in the laboratory by following standard anaerobic techniques. 

Copious purulent material often with formation of large abscesses is very common with these infections for which treatment of first importance is surgical drainage. Anti-microbial therapy alone without surgical drainage of pus in the tissues is not of much use. Ampicillin, clindamycin, vancomycin and metronidazole have been found to be effective. There are no prophylactic vaccines developed for endogenous infections. Autogenous vaccines have been tried (11,12,13,14)

Conclusions 
Anaerobic bacteria play a significant role in nutrition, health and disease processes of animals. Even though intensive studies have been carried out on the type, nature and activities of indigenous non-pathogenic microflora of the GIT, much needs to be done particularly on the utilization of unutilizable feed wastes, use of non-protein nitrogenous sources for feeding animals for higher and economic production of livestock. Use of selective microbes and their adaptation to the rumen environment needs to be done for efficient utilization of feeds and synthesis of vitamins. Also, the role of indigenous anaerobic flora in providing protection against infection by pathogens and their role in modulating immune response is receiving greater attention. 

Animal diseases due to pathogenic anaerobes continue to be important in the livestock economy. As these bacteria have soil and animal gut as their natural habitat they probably cannot be eliminated from causing infections of the animals. However, predisposing factors such as tissue damage, liver fluke infestation, sudden changes in the gut environment can be reduced. Since clostridia and their products (toxins) are highly immunogenic, many of the clostridial infections can be prevented by prophylactic vaccinations. There is a need for development of improved diagnostics and vaccines such as subunit, synthetic and recombinant vaccines for protection against clostridial infections. Development of prophylactic vaccines and other control measures to reduce the infections caused by non-sporeforming microbes need to be undertaken.

References

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3. Methods in Microbiology Vol. 3 B (1969). Academic Press, London.
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5. Willis, A.T. (1969). Clostridia of wound infection. Butterworths, London.
6. Smith, L.D. and Williams, B.L. (1984). The pathogenic anaerobic bacteria. Charles C. Thomas, Springfield, Illinois.
7. Hatheway, C.L. (1990). Clin. Microbiol. Rev., 3: 66.
8. Betty A. Forbes et al. (1998). “Diagnostic Microbiology”. p. 686. Mosby Inc., St. Louis. Missouri.
9. Mackie and McCartiney. (1996). Practical medical Microbiology. p. 501. Eds. J.G. Collee et al. Churchill, Livingstone, Edinburgh.
10. Carlton, L.G. (1993). Pathogenesis of bacterial infections in animals. p. 273. Ed. Charles O.Thoen. Iowa State University Press, Ames.
11. Shapton, D.A. and Board, R.G. (1971). Isolation of anaerobes. Academic Press, London.
12. Holdman, L.V. et al. (1977). Anaerobic Laboratory Manual, VPI Institute and State University, Blacksburg.
13. Anaerobic Bacteria - Role in disease (1974). p. 89. Eds. Albert Balows et al. Charles C. Thomas, Springfield, Illinois.
14. Manual of Clinical Microbiology (1991). p. 488. American Society for Microbiology.

Authors Corresponding address: 

Dr. A.S. Upadhye
Former Professor and Head,
Veterinary Microbiology, Veterinary College, University of Agricultural Sciences, Hebbal, Bangalore - 560 024, India.

Dr. M.D. Venkatesha
Scientist,
Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore - 560 024, India

The views expressed in this article are of the author(s), and any clarifications can be obtained from the author(s).