Gastroenterology

Intestinal Microbes and Digestive System Disease in Dogs

Intestinal Microbes and Digestive System Disease in Dogs
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Jan S. Suchodolski, MedVet, DrVetMed, PhD, AGAF, DACVM
Texas A&M University

The intestinal microbiota comprises viruses, bacteria, fungi, and protozoa. In the past, the word microflora has been used to describe this complex ecosystem, but microbiota (from –bios, “living organisms”) is the more appropriate term. The microbiome is the collective genome of all these microbes. Most studies to date have focused on the bacterial microbiota, which is estimated to make up the vast majority of the intestinal microbiota.

An estimated 100 trillion microbial cells are present within the intestine, up to 10 times the number of mammalian cells in the whole body. Combined, microbial genes outnumber host genes by an estimated factor of 10.

This complex ecosystem of gut bacteria has a tremendous influence on host health. The interactions between bacteria and host are mediated through direct contact between microbes and the immune system and through various microbiota-derived metabolites. A physiologic microbiome modulates the immune system, protects against enteropathogens, and provides nutritional benefits to the host.

Conversely, changes in the intricate relationship between gut bacteria and host cells affect the host’s immune responses and metabolic status and may result in disease (Figure 1). Recent studies have described intestinal dysbiosis (ie, changes in intestinal microbiota composition and/or diversity) in various acute and chronic gastrointestinal (GI) disorders.1 Additionally, initial data in human and animal models have linked chronic dysbiosis, such as that caused by antibiotic exposure, to extraintestinal disorders such as diabetes and obesity.2,3 These findings highlight the importance of gut microbiota and dysbiosis in regulating host metabolism, with effects reaching far beyond the GI tract.

The dysbiosis patterns and metabolic signatures observed in acute and chronic GI diseases and the metabolic syndrome are only beginning to be described. Dysbiosis signatures and metabolic alterations are being evaluated for their diagnostic and therapeutic potential. This article provides an overview of the bacteria in the canine intestine and the role of dysbiosis in the etiology of GI diseases.

Figure 1. The interplay between the intestinal microbiota and the host immune system and metabolism. (A) In a healthy intestine, proper spatial arrangements exist between the highly abundant luminal microbiota, the epithelium, and immune cells (dendritic cells, lymphocytes, macrophages, neutrophils) that are present in low numbers. The microbiota provides immunomodulatory stimuli to the immune system and metabolizes and ferments complex carbohydrates into beneficial short-chain fatty acids (SCFA). These SCFA provide energy for endothelial cells, are anti-inflammatory, and regulate intestinal motility. The normal microbiota also converts primary bile acids to secondary bile acids, which are also anti-inflammatory, induce glucagon-like peptide 1 (which in turn increases insulin), and decrease, for example, sporulation of Clostridium difficile. (B) In a diseased state, regardless of the initiating cause, the decreased production of antimicrobial peptides and mucus leads to increased intestinal permeability and translocation of bacteria. Innate immune receptors, such as Toll-like receptors on macrophages and other immune cells, recognize specific pathogen-associated molecular patterns due to dysbiosis (eg, lipopolysaccharides in bacterial cell walls) and trigger inflammatory reactions. Macrophages phagocytize microbes, also triggering immune responses that can lead to oxidative stress. Oxidative stress, in turn, can cause intestinal dysbiosis.

RARE BREED

Every dog harbors a unique microbial community, with distinct differences in the proportions of these bacterial groups. Yet the bacterial gene content is conserved across individuals, suggesting that functional aspects of the microbiome are similar across animals. Individual differences in microbial species may cause individualized responses to different diets, fiber sources, and probiotics.

THE INTESTINAL MICROBIOTA IN HEALTH

Identification and Components

Until recently, identification of intestinal bacteria was almost exclusively achieved using traditional bacterial culture. Fecal culture may still be useful for the detection of specific enteropathogens such as Salmonella or Campylobacter jejuni, as this approach allows for antibiotic susceptibility testing of clinical specimens, but the vast majority of intestinal bacteria are strict anaerobes and undetectable using standard cultivation methods. Therefore, routine bacterial culture does not allow in-depth characterization of complex intestinal bacterial communities (Figure 2). A number of molecular-based methods are now used for the characterization of the intestinal microbiota.4

Traditional bacterial culture as well molecular approaches have revealed differences in the type and amount of bacteria along the GI tract. Bacterial numbers in the duodenum of healthy dogs range from 102 to 109 colony-forming units (cfu)/g.5 The colon harbors much higher numbers, with up to 1011 cfu/g.6

Molecular tools have allowed identification of previously uncultivable, and hence unknown, bacteria. While the duodenum harbors a mixture of aerobic and facultative anaerobic bacteria, the colon is colonized almost exclusively by strict anaerobes.7 The bacterial groups Clostridiaceae, Bacteroidaceae, Prevotellaceae, and Fusobacteriaceae predominate in the large intestine (Figure 2). Of note is that every dog harbors a unique microbial community, with distinct differences in the proportions of these bacterial groups. Yet the bacterial gene content is conserved across individuals, suggesting that functional aspects of the microbiomes are similar across animals.8 Nevertheless, individual differences in microbial species may cause individualized responses to different diets, fiber sources, and probiotics.

The GI tract is also home to a diverse population of viruses and fungi. One study described up to 40 fungal species in canine fecal samples; most were various Candida spp.9 Based on these findings, it is expected that fungal organisms would be observed on routine fecal smears. Their exact contribution to health and disease remains unclear, as no significant differences in the types of fungi were reported when healthy dogs were compared with dogs with acute diarrhea.

Figure 2. Predominant bacterial taxa in fecal samples of healthy dogs. Each bar represents one healthy dog. Note that the bacterial families present vary widely, anaerobic bacteria predominate, and the ratios of these bacterial groups differ in each healthy dog. Routine bacterial culture misses these anaerobic bacteria. Fecal smears that evaluate ratios of gram-positive to gram-negative bacteria are also not useful to assess dysbiosis, as each dog harbors a unique ratio of these groups.

Roles

A balanced microbiome is crucial for maintaining host health. The normal microbiota performs the following functions:

  • Modulates the immune system
  • Keeps invading enteropathogens in check
  • Provides nutrients to the host by metabolizing and fermenting various dietary components

Intestinal bacteria also contribute to the development of gut physiology. This has been demonstrated in studies with germ-free raised mice, which show altered epithelial architecture (eg, decreased number of lymphoid follicles) compared with mice that have been exposed to bacteria at birth.

Consistent crosstalk between the intestinal microbiota and host immune cells is mediated through a combination of microbial-derived metabolites (eg, short-chain fatty acids [SCFA], indole, secondary bile acids) as well as molecules on the surface of bacteria that activate receptors of the host innate immune system (eg, Toll-like receptors, dendritic cells).

Commensal bacteria, which prevent mucosal invasion of transient pathogens through competition for nutrients and epithelial adhesion sites, are an important part of the intestinal barrier. Furthermore, they establish a physiologically restrictive environment for nonresident bacterial species through secretion of antimicrobial compounds and modulation of luminal pH.

The major bacterial groups in the gut are strict or facultative anaerobes. The predominant bacterial families in the large intestine (Figure 2) ferment dietary carbohydrates (eg, starch, cellulose, pectin, inulin), resulting in the production of SCFA (eg, acetate, propionate, butyrate) and other metabolites. SCFA are an important source of energy and growth factors for intestinal epithelial cells and have a modulating effect on intestinal motility. Furthermore, SCFA are immunomodulatory. For example, butyrate induces immunoregulatory T cells and acetate beneficially modulates intestinal permeability.

An emerging area of research aims to better characterize the biologic functions of additional bacterial-derived metabolites that have been recently recognized as regulators of host health, such as indole and secondary bile acids. For example, dietary tryptophan is metabolized by bacteria into indole, which has been shown to decrease interleukin-8 expression, strengthen intestinal barrier function, and ameliorate nonsteroidal anti-inflammatory drug–induced enteropathy in mice.10 Some bacterial species in the colon convert primary bile acids into secondary bile acids (eg, lithocholic and deoxycholic acids). Therefore, secondary bile acids are present at much higher concentrations in the colon than primary bile acids. This high concentration of secondary bile acids in the colon is beneficial, as these acids are an important regulator of host homeostasis through activation of various receptors throughout the body. For example, the bile acid–specific membrane receptor TGR5 is expressed in the gallbladder, in bile duct epithelium, on monocytes and macrophages, and in muscle, kidney, pancreatic, and intestinal cells. The activation of these receptors, for which secondary bile acids have the highest affinity, downregulates the expression of proinflammatory cytokines and modulates insulin and glucose metabolism through activation of glucagon-like peptide 1.11

Secondary bile acids also inhibit germination of Clostridium difficile spores, whereas an increase in primary bile acids (an effect of dysbiosis) promotes the germination of bacterial spores. Intestinal dysbiosis leads to a decrease in bile-acid converting bacterial species and, therefore, is associated with bile acid dysmetabolism (decrease in secondary and increase in primary bile acids) and potentially systemic effects on host metabolism.12,13 Furthermore, an abnormal increase in primary bile acids may cause secretory diarrhea.

DYSBIOSIS

Intestinal dysbiosis is defined as differences in ratios of bacterial groups compared with those found in healthy dogs and is often accompanied by a reduction in species diversity. Intestinal dysbiosis has been reported in various acute and chronic GI disorders, but it can also be induced through use of broad-spectrum antibiotics.1

The importance of the gut microbiota in host immune regulation and metabolism (Figure 1) means that a dysbiotic microbiome may have negative consequences for the host. However, the extent of clinical signs varies between individuals. For example, administration of metronidazole to healthy dogs induced major changes in the gut microbiota, with reductions in commensal anaerobic bacteria and a concurrent increase in Escherichia coli; these changes were accompanied by extensive changes in metabolomic pathways in the gut lumen (eg, increase in oxidative stress, reductions in secondary bile acids).14 Nine of 16 dogs developed loose stools while on antibiotics, but the remaining dogs exhibited no clinical signs despite having similar microbial and biochemical changes. This suggests that clinical signs depend on the interplay of multiple microbial and host factors, some of which (eg, underlying genetic susceptibility of the host, dietary and environmental triggers) remain to be elucidated. Nevertheless, antibiotic-induced dysbiosis is an example of how changes in microbial composition and metabolism can affect host health, as antibiotic-induced dysbiosis in early childhood or repeated pulse therapy has been recognized as a risk factor for development of allergies, obesity, and inflammatory bowel disease in humans.3,15

The emerging epidemiologic data in humans and our evolving understanding of the immunomodulatory and metabolic properties of the gut microbiota suggest that proper diagnosis and correction of dysbiosis will be important goals in various diseases. A dysbiotic microbiome may cause harm through several mechanisms (Box 1), which are likely to be concurrent. Diarrhea can be due to bacterial enterotoxins that stimulate mucosal fluid secretions. Another recently recognized mechanism for diarrhea in humans is bile acid malabsorption due to the inability of the dysbiotic microbiota to convert primary to secondary bile acids.13 Initial studies suggest that such a mechanism may also occur in dogs and warrants further study.12,16

BOX 1

Effects of Dysbiosis

  • Overproduction and translocation of bacterial toxins
  • Inflammatory stimulation of the immune system
  • Reductions in anti-inflammatory metabolites (eg, SCFA, indoles, secondary bile acids)
  • Alterations in brush border enzymes
  • Damage to mucosal receptors
  • Competition for nutrients (eg, vitamin B12)
  • Increased intestinal permeability

Assessment of Dysbiosis

Because of the importance of the commensal microbiota to host homeostasis, it is important to diagnose dysbiosis. As noted, fecal bacterial culture cannot characterize the many anaerobes in the GI tract. It is estimated that only a very small percentage of intestinal bacteria are cultivable with standard laboratory techniques. The best way to fully characterize the microbiota is by high-throughput sequencing platforms that can provide an overview of the proportions of all bacterial groups within a sample; however, cost and long turnaround times limit the use of this method to research studies. Use of polymerase chain reaction (PCR) assays to target specific bacterial taxa that are consistently altered in dogs with chronic enteropathies (CE) can provide more rapid results.1

The results of these multiple PCR assays can be combined and expressed as a mathematical ratio, the Dysbiosis Index (DI; Figure 3). A negative DI (<0) indicates a normal microbiota, whereas a positive DI (>0) indicates dysbiosis associated with CE.17 The DI can then be used to monitor the microbiota’s response to therapy for CE. Initial long-term follow-up studies in dogs with CE suggest that the microbiome requires several months to normalize, even when dogs respond within a few weeks with a decrease in clinical activity scores.18

A recent small study evaluating 3 dogs with CE used the DI to monitor fecal microbial changes in response to fecal microbial transplantation.19 All 3 dogs initially showed an immediate reduction in the DI, but after 3 weeks dysbiosis returned in 1 dog (increase in DI above 2), which showed no improvement in clinical signs. In the remaining 2 dogs, a partial improvement in clinical signs was observed, and their DI stayed below 0 for most of the 8-week follow-up period. This initial small dataset suggests a potential for monitoring the microbiota over time in patients with CE and after fecal microbial transplantation, but more studies are needed to determine the accuracy and clinical utility of microbiota analysis.

Analysis of fecal samples provides information only about changes in the luminal microbiota. The use of fluorescence in-situ hybridization (FISH) of intestinal biopsy specimens allows visualization of whether bacteria have translocated into the mucosal epithelium, as is observed in dogs with granulomatous colitis.20 A positive result indicates the need for antimicrobial therapy to clear the translocated bacteria. FISH requires special analysis and is only available in a few reference laboratories.

Although assessing the fecal microbiota is useful for recognizing dysbiosis in the large intestine, fecal samples probably do not accurately reflect the situation in the small intestine. Although the fecal samples of many dogs with small intestinal disease show dysbiosis, a subset of dogs may have exclusively small intestinal dysbiosis. Measurements of serum cobalamin and folate concentrations remain the most useful markers of small intestinal dysbiosis. Serum cobalamin may be decreased and serum folate concentrations may be increased in dogs with small intestinal dysbiosis; alteration of both parameters is highly suggestive of the condition.

Recent studies have evaluated links between dysbiosis and changes in various biochemical pathways (eg, abnormal metabolism of bile acids, amino acids, and tryptophan) that affect the host immune system and metabolism.18,21 Many novel metabolic biomarkers, such as concentrations of fecal bile acids, are being investigated for better assessment of the etiology and treatment of GI diseases, and these may soon become useful for routine practice.

Figure 3. The Dysbiosis Index (DI) is increased in dogs with chronic enteropathies (CE). The DI is a mathematical ratio that summarizes the abundances of various bacterial groups (eg, E coli, Faecalibacterium, Blautia, Fusobacterium) into a single number. A DI below 0 indicates a normal microbiota, and a DI above 0 indicates dysbiosis in the large intestine. The DI assay is commercially available through the Gastrointestinal Laboratory at Texas A&M University.

Therapeutic Considerations for Correction of Dysbiosis

The microbiota is an important player in host metabolism. Recent metabolomic studies have clearly linked dysbiosis with various diseases within and outside the GI tract. However, more work is needed to determine how to modulate the microbiome for best therapeutic success and to predict response to a specific therapy.

Diet and Antimicrobial Therapy

Dysbiosis is present in many dogs with CE and may be the cause of diarrhea in some patients, but dysbiosis may also be the consequence of GI inflammation in other patients. A gradient of various disease patterns across patients is likely, with host immune system and microbiome contributing to various degrees. Therefore, the presence of dysbiosis does not equate to an immediate need for antimicrobial therapy, as dogs with diet-responsive CE may also have a dysbiosis. Some animals with diarrhea respond favorably to antimicrobials, but antibiotics may induce diarrhea in others. Long-term antibiotic administration may induce dysbiosis patterns that could create a risk factor for various metabolic diseases, such as through induction of bile acid dysmetabolism.3 At this time, the best therapeutic approach to chronic GI disease remains empiric, with a sequential protocol of food trials, anti-inflammatory drugs, and/or antimicrobials.

Probiotics and Prebiotics

Because the microbiota is implicated in the pathophysiology of chronic GI disease, the addition of probiotic and prebiotic therapy may be appropriate. Probiotics are live microorganisms that, when administered in sufficient quantities, confer a health benefit to the host. Few studies have evaluated the benefits of probiotics in acute and chronic GI disease. Data suggest that probiotics have only a minor effect on the intestinal microbiota, but their beneficial effect in dogs with inflammatory bowel disease may be due in part to immune stimulation and/or enhancement of intestinal barrier function.22 Also, it appears that administration of higher doses and multiple strains leads to a higher probability that probiotic bacteria will be able to colonize the gut.

Prebiotics are fermentable and nonfermentable fibers that, after reaching the large intestine, are metabolized by intestinal bacteria to produce SCFA and other metabolites that may be immunomodulatory. Most commercial intestinal diets contain prebiotics.

Fecal Microbial Transplantation

Fecal microbial transplantation has garnered a lot of interest. Although it is a highly successful therapeutic approach in humans with recurrent C difficile, its use in CE of dogs requires further study because the pathophysiology between these diseases differs. Anecdotal evidence and small-scale studies suggest that fecal microbial transplantation may be promising in a subset of dogs with CE,19 but at this time proper patient selection is purely empiric and more studies are required.

References

  1. Honneffer JB, Minamoto Y, Suchodolski JS. Microbiota alterations in acute and chronic gastrointestinal inflammation of cats and dogs. World J Gastroenterol 2014;20:16489-16497.
  2. Saari A, Virta LJ, Sankilampi U, et al. Antibiotic exposure in infancy and risk of being overweight in the first 24 months of life. Pediatrics 2015;135:617-626.
  3. Vrieze A, Out C, Fuentes S, et al. Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin sensitivity. J Hepatol 2014;60:824-831.
  4. Suchodolski JS. Diagnosis and interpretation of intestinal dysbiosis in dogs and cats. Vet J 2016; doi: 10.1016/j.tvjl.2016.04.011
  5. German AJ, Day MJ, Ruaux CG, et al. Comparison of direct and indirect tests for small intestinal bacterial overgrowth and antibiotic-responsive diarrhea in dogs. J Vet Intern Med 2003;17:33-43.
  6. Mentula S, Harmoinen J, Heikkilä M, et al. Comparison between cultured small-intestinal and fecal microbiotas in beagle dogs. Appl Environ Microbiol 2005;71:4169-4175.
  7. Suchodolski JS, Camacho J, Steiner JM. Analysis of bacterial diversity in the canine duodenum, jejunum, ileum, and colon by comparative 16S rRNA gene analysis. FEMS Microbiology Ecology 2008;66:567-578.
  8. Guard BC, Suchodolski JS. Horse Species Symposium—Canine intestinal microbiology and metagenomics: From phylogeny to function. J Anim Sci 2016;94:2247-2261.
  9. Foster ML, Dowd SE, Stephenson C, et al. Characterization of the fungal microbiome (mycobiome) in fecal samples from dogs. Vet Med Int 2013;2013:658373.
  10. Whitfield-Cargile CM, Cohen ND, Chapkin RS, et al. The microbiota-derived metabolite indole decreases mucosal inflammation and injury in a murine model of NSAID enteropathy. Gut Microbes 2016;7:246-261.
  11. Pavlidis P, Powell N, Vincent RP, et al. Systematic review: bile acids and intestinal inflammation-luminal aggressors or regulators of mucosal defence? Aliment Pharmacol Ther 2015;42:802-817.
  12. Honneffer J, Guard B, Steiner JM, et al. Mo1805 Untargeted metabolomics reveals disruption within bile acid, cholesterol, and tryptophan metabolic pathways in dogs with idiopathic inflammatory bowel disease. Gastroenterology 2015;148:S-715 (abstract).
  13. Duboc H, Rajca S, Rainteau D, et al. Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases. Gut 2013;62:531-539.
  14. Suchodolski JS, Olson E, Honneffer J, et al. Effects of a hydrolyzed protein diet and metronidazole on the fecal microbiome andmetabolome in healthy dogs. J Vet Intern Med 2016;30:1455 (abstract).
  15. Cox LM, Blaser MJ. Antibiotics in early life and obesity. Nat Rev Endocrinol 2015;11:182-190.
  16. Kent AC, Cross G, Taylor DR, et al. Measurement of serum 7alpha-hydroxy-4-cholesten-3-one as a marker of bile acid malabsorption in dogs with chronic diarrhoea: a pilot study. Vet Rec Open 2016;3:e000163.
  17. Alshawaqfeh M, Guard M, Minamoto Y, et al. A dysbiosis index to assess microbial changes in fecal samples of dogs with chronicenteropathy. J Vet Intern Med 2016;30:1536 (abstract).
  18. Minamoto Y, Otoni CC, Steelman SM, et al. Alteration of the fecal microbiota and serum metabolite profiles in dogs with idiopathic inflammatory bowel disease. Gut Microbes 2015;6:33-47.
  19. Gerbec Z. Evaluation of therapeutic potential of restoring gastrointestinal homeostasis by a fecal microbiota transplant in dogs. Master’s Thesis: University of Ljubljana, Slovenia, 2016.
  20. Simpson KW, Dogan B, Rishniw M, et al. Adherent and invasive Escherichia coli is associated with granulomatous colitis in boxer dogs. Infect Immun 2006;74:4778-4792.
  21. Guard BC, Barr JW, Reddivari L, et al. Characterization of microbial dysbiosis and metabolomic changes in dogs with acute diarrhea. PLoS ONE 2015; 10:e0127259.
  22. Rossi G, Pengo G, Caldin M, et al. Comparison of microbiological, histological, and immunomodulatory parameters in response to treatment with either combination therapy with prednisone and metronidazole or probiotic VSL#3 strains in dogs with idiopathic inflammatory bowel disease. Plos ONE 2014; 9:e94699.

Jan S. Suchodolski, MedVet, DrVetMed, PhD, AGAF, DACVM, earned his veterinary degree from the University of Veterinary Medicine in Vienna, Austria, and his PhD in veterinary microbiology from Texas A&M University. He is board certified in immunology by the American College of Veterinary Microbiologists (ACVM) and currently serves as associate professor and associate director for research at the Gastrointestinal Laboratory at Texas A&M. His research is focused on gastrointestinal function testing and intestinal microbial ecology, with an emphasis on probiotic and dietary interventions as therapeutic applications in animals with inflammatory bowel disease, obesity, and diabetes mellitus.

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for='input_6_10_1' id='input_6_10_1_label' >Street Address</label> </span><span class='ginput_full address_line_2' id='input_6_10_2_container' > <input type='text' name='input_10.2' id='input_6_10_2' value='' /> <label for='input_6_10_2' id='input_6_10_2_label' >Address Line 2</label> </span><span class='ginput_left address_city' id='input_6_10_3_container' > <input type='text' name='input_10.3' id='input_6_10_3' value='' aria-required="true"/> <label for='input_6_10_3' id='input_6_10_3_label' >City</label> </span><span class='ginput_right address_state' id='input_6_10_4_container' > <input type='text' name='input_10.4' id='input_6_10_4' value='' aria-required="true"/> <label for='input_6_10_4' id='input_6_10_4_label' >State / Province / Region</label> </span><span class='ginput_left address_zip' id='input_6_10_5_container' > <input type='text' name='input_10.5' id='input_6_10_5' value='' aria-required="true"/> <label for='input_6_10_5' id='input_6_10_5_label' >ZIP / Postal Code</label> </span><span class='ginput_right address_country' id='input_6_10_6_container' > <select name='input_10.6' id='input_6_10_6' aria-required="true"><option value='' selected='selected'></option><option value='Afghanistan' >Afghanistan</option><option value='Åland Islands' >Åland Islands</option><option value='Albania' >Albania</option><option value='Algeria' >Algeria</option><option value='American Samoa' >American Samoa</option><option value='Andorra' >Andorra</option><option value='Angola' >Angola</option><option value='Anguilla' >Anguilla</option><option value='Antarctica' >Antarctica</option><option value='Antigua and Barbuda' >Antigua and Barbuda</option><option value='Argentina' >Argentina</option><option value='Armenia' >Armenia</option><option value='Aruba' >Aruba</option><option value='Australia' >Australia</option><option value='Austria' >Austria</option><option value='Azerbaijan' >Azerbaijan</option><option value='Bahamas' >Bahamas</option><option value='Bahrain' >Bahrain</option><option value='Bangladesh' >Bangladesh</option><option value='Barbados' >Barbados</option><option value='Belarus' >Belarus</option><option value='Belgium' >Belgium</option><option value='Belize' >Belize</option><option value='Benin' >Benin</option><option value='Bermuda' >Bermuda</option><option value='Bhutan' >Bhutan</option><option value='Bolivia' >Bolivia</option><option value='Bonaire, Sint Eustatius and Saba' >Bonaire, Sint Eustatius and Saba</option><option value='Bosnia and Herzegovina' >Bosnia and Herzegovina</option><option value='Botswana' >Botswana</option><option value='Bouvet Island' >Bouvet Island</option><option value='Brazil' >Brazil</option><option value='British Indian Ocean Territory' >British Indian Ocean Territory</option><option value='Brunei Darussalam' >Brunei Darussalam</option><option value='Bulgaria' >Bulgaria</option><option value='Burkina Faso' >Burkina Faso</option><option value='Burundi' >Burundi</option><option value='Cambodia' >Cambodia</option><option value='Cameroon' >Cameroon</option><option value='Canada' >Canada</option><option value='Cape Verde' >Cape Verde</option><option value='Cayman Islands' >Cayman Islands</option><option value='Central African Republic' >Central African Republic</option><option value='Chad' >Chad</option><option value='Chile' >Chile</option><option value='China' >China</option><option value='Christmas Island' >Christmas Island</option><option value='Cocos Islands' >Cocos Islands</option><option value='Colombia' >Colombia</option><option value='Comoros' >Comoros</option><option value='Congo, Democratic Republic of the' >Congo, Democratic Republic of the</option><option value='Congo, Republic of the' >Congo, Republic of the</option><option value='Cook Islands' >Cook Islands</option><option value='Costa Rica' >Costa Rica</option><option value='Côte d&#039;Ivoire' >Côte d&#039;Ivoire</option><option value='Croatia' >Croatia</option><option value='Cuba' >Cuba</option><option value='Curaçao' >Curaçao</option><option value='Cyprus' >Cyprus</option><option value='Czech Republic' >Czech Republic</option><option value='Denmark' >Denmark</option><option value='Djibouti' >Djibouti</option><option value='Dominica' >Dominica</option><option value='Dominican Republic' >Dominican Republic</option><option value='Ecuador' >Ecuador</option><option value='Egypt' >Egypt</option><option value='El Salvador' >El Salvador</option><option value='Equatorial Guinea' >Equatorial Guinea</option><option value='Eritrea' >Eritrea</option><option value='Estonia' >Estonia</option><option value='Eswatini (Swaziland)' >Eswatini (Swaziland)</option><option value='Ethiopia' >Ethiopia</option><option value='Falkland Islands' >Falkland Islands</option><option value='Faroe Islands' >Faroe Islands</option><option value='Fiji' >Fiji</option><option value='Finland' >Finland</option><option value='France' >France</option><option value='French Guiana' >French Guiana</option><option value='French Polynesia' >French Polynesia</option><option value='French Southern Territories' >French Southern Territories</option><option value='Gabon' >Gabon</option><option value='Gambia' >Gambia</option><option value='Georgia' >Georgia</option><option value='Germany' >Germany</option><option value='Ghana' >Ghana</option><option value='Gibraltar' >Gibraltar</option><option value='Greece' >Greece</option><option value='Greenland' >Greenland</option><option value='Grenada' >Grenada</option><option value='Guadeloupe' >Guadeloupe</option><option value='Guam' >Guam</option><option value='Guatemala' >Guatemala</option><option value='Guernsey' >Guernsey</option><option value='Guinea' >Guinea</option><option value='Guinea-Bissau' >Guinea-Bissau</option><option value='Guyana' >Guyana</option><option value='Haiti' >Haiti</option><option value='Heard and McDonald Islands' >Heard and McDonald Islands</option><option value='Holy See' >Holy See</option><option value='Honduras' >Honduras</option><option value='Hong Kong' >Hong Kong</option><option value='Hungary' >Hungary</option><option value='Iceland' >Iceland</option><option value='India' >India</option><option value='Indonesia' >Indonesia</option><option value='Iran' >Iran</option><option value='Iraq' >Iraq</option><option value='Ireland' >Ireland</option><option value='Isle of Man' >Isle of Man</option><option value='Israel' >Israel</option><option value='Italy' >Italy</option><option value='Jamaica' >Jamaica</option><option value='Japan' >Japan</option><option value='Jersey' >Jersey</option><option value='Jordan' >Jordan</option><option value='Kazakhstan' >Kazakhstan</option><option value='Kenya' >Kenya</option><option value='Kiribati' >Kiribati</option><option value='Kuwait' >Kuwait</option><option value='Kyrgyzstan' >Kyrgyzstan</option><option value='Lao People&#039;s Democratic Republic' >Lao People&#039;s Democratic Republic</option><option value='Latvia' >Latvia</option><option value='Lebanon' >Lebanon</option><option value='Lesotho' >Lesotho</option><option value='Liberia' >Liberia</option><option value='Libya' >Libya</option><option value='Liechtenstein' >Liechtenstein</option><option value='Lithuania' >Lithuania</option><option value='Luxembourg' >Luxembourg</option><option value='Macau' >Macau</option><option value='Macedonia' >Macedonia</option><option value='Madagascar' >Madagascar</option><option value='Malawi' >Malawi</option><option value='Malaysia' >Malaysia</option><option value='Maldives' >Maldives</option><option value='Mali' >Mali</option><option value='Malta' >Malta</option><option value='Marshall Islands' >Marshall Islands</option><option value='Martinique' >Martinique</option><option value='Mauritania' >Mauritania</option><option value='Mauritius' >Mauritius</option><option value='Mayotte' >Mayotte</option><option value='Mexico' >Mexico</option><option value='Micronesia' >Micronesia</option><option value='Moldova' >Moldova</option><option value='Monaco' >Monaco</option><option value='Mongolia' >Mongolia</option><option value='Montenegro' >Montenegro</option><option value='Montserrat' >Montserrat</option><option value='Morocco' >Morocco</option><option value='Mozambique' >Mozambique</option><option value='Myanmar' >Myanmar</option><option value='Namibia' >Namibia</option><option value='Nauru' >Nauru</option><option value='Nepal' >Nepal</option><option value='Netherlands' >Netherlands</option><option value='New Caledonia' >New Caledonia</option><option value='New Zealand' >New Zealand</option><option value='Nicaragua' >Nicaragua</option><option value='Niger' >Niger</option><option value='Nigeria' >Nigeria</option><option value='Niue' >Niue</option><option value='Norfolk Island' >Norfolk Island</option><option value='North Korea' >North Korea</option><option value='Northern Mariana Islands' >Northern Mariana Islands</option><option value='Norway' >Norway</option><option value='Oman' >Oman</option><option value='Pakistan' >Pakistan</option><option value='Palau' >Palau</option><option value='Palestine, State of' >Palestine, State of</option><option value='Panama' >Panama</option><option value='Papua New Guinea' >Papua New Guinea</option><option value='Paraguay' >Paraguay</option><option value='Peru' >Peru</option><option value='Philippines' >Philippines</option><option value='Pitcairn' >Pitcairn</option><option value='Poland' >Poland</option><option value='Portugal' >Portugal</option><option value='Puerto Rico' >Puerto Rico</option><option value='Qatar' >Qatar</option><option value='Réunion' >Réunion</option><option value='Romania' >Romania</option><option value='Russia' >Russia</option><option value='Rwanda' >Rwanda</option><option value='Saint Barthélemy' >Saint Barthélemy</option><option value='Saint Helena' >Saint Helena</option><option value='Saint Kitts and Nevis' >Saint Kitts and Nevis</option><option value='Saint Lucia' >Saint Lucia</option><option value='Saint Martin' >Saint Martin</option><option value='Saint Pierre and Miquelon' >Saint Pierre and Miquelon</option><option value='Saint Vincent and the Grenadines' >Saint Vincent and the Grenadines</option><option value='Samoa' >Samoa</option><option value='San Marino' >San Marino</option><option value='Sao Tome and Principe' >Sao Tome and Principe</option><option value='Saudi Arabia' >Saudi Arabia</option><option value='Senegal' >Senegal</option><option value='Serbia' >Serbia</option><option value='Seychelles' >Seychelles</option><option value='Sierra Leone' >Sierra Leone</option><option value='Singapore' >Singapore</option><option value='Sint Maarten' >Sint Maarten</option><option value='Slovakia' >Slovakia</option><option value='Slovenia' >Slovenia</option><option value='Solomon Islands' >Solomon Islands</option><option value='Somalia' >Somalia</option><option value='South Africa' >South Africa</option><option value='South Georgia' >South Georgia</option><option value='South Korea' >South Korea</option><option value='South Sudan' >South Sudan</option><option value='Spain' >Spain</option><option value='Sri Lanka' >Sri Lanka</option><option value='Sudan' >Sudan</option><option value='Suriname' >Suriname</option><option value='Svalbard and Jan Mayen Islands' >Svalbard and Jan Mayen Islands</option><option value='Sweden' >Sweden</option><option value='Switzerland' >Switzerland</option><option value='Syria' >Syria</option><option value='Taiwan' >Taiwan</option><option value='Tajikistan' >Tajikistan</option><option value='Tanzania' >Tanzania</option><option value='Thailand' >Thailand</option><option value='Timor-Leste' >Timor-Leste</option><option value='Togo' >Togo</option><option value='Tokelau' >Tokelau</option><option value='Tonga' >Tonga</option><option value='Trinidad and Tobago' >Trinidad and Tobago</option><option value='Tunisia' >Tunisia</option><option value='Turkey' >Turkey</option><option value='Turkmenistan' >Turkmenistan</option><option value='Turks and Caicos Islands' >Turks and Caicos Islands</option><option value='Tuvalu' >Tuvalu</option><option value='Uganda' >Uganda</option><option value='Ukraine' >Ukraine</option><option value='United Arab Emirates' >United Arab Emirates</option><option value='United Kingdom' >United Kingdom</option><option value='United States' >United States</option><option value='Uruguay' >Uruguay</option><option value='US Minor Outlying Islands' >US Minor Outlying Islands</option><option value='Uzbekistan' >Uzbekistan</option><option value='Vanuatu' >Vanuatu</option><option value='Venezuela' >Venezuela</option><option value='Vietnam' >Vietnam</option><option value='Virgin Islands, British' >Virgin Islands, British</option><option value='Virgin Islands, U.S.' >Virgin Islands, U.S.</option><option value='Wallis and Futuna' >Wallis and Futuna</option><option value='Western Sahara' >Western Sahara</option><option value='Yemen' >Yemen</option><option value='Zambia' >Zambia</option><option value='Zimbabwe' >Zimbabwe</option></select> <label for='input_6_10_6' id='input_6_10_6_label' 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