Staphylococcus aureus diversity and subclinical mastitis

This is the first study I’ve found that was interested in cataloging bacterial diversity among subclinical (or asymptomatic) infections. While they may be less threatening to the animal’s overall health, these infections have great significance in the world of animal agriculture, where they restrict growth (or in this case, milk production), and encourage the use of medicated feeds which in turn motivate people to purchase organic products. Identifying the risk factors and causes of these infections could therefore impact both the management of food animals, and any legislation defining how and when medications can be used. With that in mind, let’s jump back into mastitis, and everyone’s favorite gram-positive, S. aureus.

It’s because of my plasmids, people can’t help but stare.
Image from http://cellimagelibrary.org/

S. aureus is one of many bacteria that cause mastitis, however it is of additional importance as it often causes chronic or recurring cases of mastitis that result in unusable milk and discomfort of the animal. In this study, the authors investigated 11 dairy farms where they expected to find S. aureus, based on previous culture findings at each farm. They defined cows that they took milk samples from as having new or chronic infections based on somatic cell counts (SCC) in the milk. If values were >200,000 cells/mL for the month of collection the infections were considered new, whereas if cell counts were  >200,000 cells/mL for more than 2 months, those infections were considered chronic. They took a single milk sample from each teat of the infected cows, for a total of 1,354 mammary glands from 350 cows.

Pulse field electrophoresis was used to identify the different subspecies/serotypes/pulsotypes (pick your word), and to identify the genes coding for enterotoxin production that had been amplified by PCR. An ELISA test was used last to detect the presence of several enterotoxins.

As the majority of exposure to enterotoxins produced my S. aureus is through milk and dairy products, subclinical infections of S. aureus are very important as a food safety control point. Unlike cows with clinical cases that are removed from production, cows with subclinical infections continue to contribute milk that makes it to the consumer, provided that the SSC is <750,000 cells/mL. The authors were unable to detect a large amount of enterotoxin in their samples, but many of the pulsotypes contained the genes coding for their production. Other studies cited by the author report the common presence of these genes in S. aureus  samples, but expression rates are inconclusive or unexplored. This means that theoretically, subclinical cows could be introducing these bacterial toxins into consumer milk in small amounts.

It’s difficult to tell how significant these amounts might be. Toxic doses of one of the enterotoxins, “Toxic Shock Syndrome Toxin 1”, has been found to be as low as 100 micrograms/Kg in miniature pigs. The concentrations that may be introduced through contaminated milk, and the bioavailability when ingested, should be explored. Takeuchi et al. (1998) were able to detect the presence of TSST- 1 in bulk milk tanks, but no one has yet to quantify the amounts of TSST- 1 potentially present in pasteurized milk.

All that being said, what good is this new information? It can be argued that because these infections are chronic and/or subclinical that these strains of S. aureus aren’t very pathogenic, but they’re still causing inflammation. By identifying common serotypes and factors leading to the subclinical infection of a herd, perhaps there are simple management changes that can prevent infection. Milking is an almost sterile procedure, with sanitation of the teats both prior and following milking, wearing gloves, and forestripping; but there could be other tricks that would target risk factors related to the spread of subclinical pathogens, especially those that are specific to a location.

 

ResearchBlogging.org

Bulanda M, Zaleska M, Mandel L, Talafantova M, Travnicek J, Kunstmann G, Mauff G, Pulverer G, & Heczko PB (1989). Toxicity of staphylococcal toxic shock syndrome toxin 1 for germ-free and conventional piglets. Reviews of infectious diseases, 11 Suppl 1 PMID: 2928643

Oliveira L, Rodrigues AC, Hulland C, & Ruegg PL (2011). Enterotoxin production, enterotoxin gene distribution, and genetic diversity of Staphylococcus aureus recovered from milk of cows with subclinical mastitis. American journal of veterinary research, 72 (10), 1361-8 PMID: 21962279

Takeuchi, S., Ishiguro, K., Ikegami, M., Kaidoh, T., & Hayakawa, Y. (1998). Production of toxic shock syndrome toxin by Staphylococcus aureus isolated from mastitic cow’s milk and farm bulk milk Veterinary Microbiology, 59 (4), 251-258 DOI: 10.1016/S0378-1135(96)01253-9

Banned antibiotics in feather meal – A discussion with an author of the study

Following my recent post where I examined an article from Johns Hopkins that found multiple contaminants in commercial feather meal (including fluoroquinolones, a class of antibiotics that have been banned from use in poultry since 2005), I was honored to be contacted by one of the Authors, Dr. David Love. Dr. Love offered to continue the discussion with me, and was happy to answer my questions regarding the study, the media frenzy it has inspired, and some of the goals of the research conducted at the Center for a Livable Future. I immediately jumped at the chance, and was able to speak with him on the phone earlier this week.

As those who read the post last week have seen, my primary concern with the study was not to do with it’s results or conclusion, but in how the press release was worded. He didn’t feel that it was as misrepresentative as I initially interpreted it, and we quickly moved on discussing just why this article was picked up so quickly.

“I’m not sure how much more clear it could be, we specifically said feather meal, and the title of the study says ‘feather meal, a previously unrecognized route for reentry into the food supply’…I think on the whole we were careful, I don’t think we can come out of this paper with twelve samples and make sweeping generalizations, it’s important to point out that our big recommendation of the study in the last line was that more research should be done…It’s really at the intersection of the media and what they’re interested in, the consumer and what their interests are, and then our story as the authors. Consumers are so interested in what’s going on with their food. We say we did a study on chicken, there’s energy there, and if that’s what they want to talk about, it tells me that we need more transparency in packaging, labeling, and more consumer education. “

I agree, everyone is interested in what they eat, and he makes a great point that we shouldn’t ignore that interest as scientists or producers as it reflects consumer demand. Another point I wanted clarification on was the statement that self-regulation and our current FDA guidelines aren’t sufficient to keep contaminants out of food.

“From the looks of the latest FDA Guidance there’s a lot of strong language, but no teeth in the language. I think for the draft guidance for 213 we’re hopeful, as there will be a larger role for veterinarians in prescribing antibiotics. As for self regulation, I would be more willing to support it if there was more transparency. Many other countries go out of their way to report use, and we in the U.S. have trouble dividing up which antibiotics are used for growth promotion, prophylaxis, and therapy. It would be hard to go about but if we could get that, and reduce or cut growth promotion uses, we would be able to actually measure progress on how we’re reducing antibiotic use in animals.”

He made a very strong point, and following publishing my post last week I came upon  a commentary published by the authors discussing the issue created by unintentional overuse of antibiotics in feed. The article actually provided many of the citations supporting their arguments that I mockingly asked for last week, and I encourage anyone interested to check out the data behind the conclusions. In wrapping up our discussion, I asked where the authors planned to go next with follow-up research.

“A lot of people want to know. Well we found this stuff in feathers, now lets look at meat, at the consumer level with what you buy at the grocery store.”

A logical next step, and one that I’m sure will have even more interest than the findings from feather meal.

Out of our discussion, I discovered a different perspective of the research that I believe was reflected in the discussion, but was completely missed by the media and myself. While the source of the contaminants is obviously a big question, that wasn’t the purpose of the study. The authors were examining feather meal as a route to antibiotic introduction that could have implications in terms of creation of AB-resistant bacteria. Regardless of how it got there (like through contaminated groundwater, as I suggested), a small percentage of chicken producers use it as a feed supplement, thus introducing fluoroquinolones into our food supply through a previously unknown method, and thus not subject to withdrawal times that prevent meat contamination. Further exploration of this research goal will probably concern testing the meat of chickens being fed feather meal for the presence of fluoroquinolones, and seeing if they do allow a sufficient amount to reenter the food supply that may warrant a withdrawal period.

In reflecting on my first post on the subject, I believe that my own response to the press release provided an excellent example of the point I was making. As this case and my interpretation of it reveal, it’s extremely easy to think that your statements were clear and representative of the science at the time, but under outside scrutiny can still be misinterpreted whether in a press release or a blog post. I’m sure I’ll remember this article when I get to publish my first paper, and take a good, hard look at the press release before approving it.

 

 

I want to sincerely thank Dr. David Love for taking the time to speak with me about his research, food safety, and agriculture research in general. I greatly enjoyed our discussion and hope that I get to work with him again. Quotes used in this post are transcribed from my notes I took during our discussion, and are used with his prior review and permission.

If you are still interested in this topic I encourage you to read all you can about it, there’s no end to the depth of the science and social issues involved. I’ve linked to the original article several times, but you can also read the supplementary material here that includes some of the anecdotal evidence in support of the presence of some of the contaminants. You can also read the National Chicken Council’s response to the NY times opinion piece that first made this research so popular. Finally, here’s some research from Chile correlating concentrations of enrofloxacin (a fluoroquinolone)  in feathers with withdrawal times in chickens treated with the drug.

Additional government resources on AB-resistant bacteria statistics and USDA residue testing: FDA NARMS report and USDA Redbook.

Please feel free to leave comments on how you feel about the research, the media presentation, and my own interpretation! I know for a fact all you people from ResearchBlogging.org have opinions, I read them all the time!
ResearchBlogging.org

Love, D., Davis, M., Bassett, A., Gunther, A., & Nachman, K. (2010). Dose Imprecision and Resistance: Free-Choice Medicated Feeds in Industrial Food Animal Production in the United States Environmental Health Perspectives, 119 (3), 279-283 DOI: 10.1289/ehp.1002625

Love, D., Halden, R., Davis, M., & Nachman, K. (2012). Feather Meal: A Previously Unrecognized Route for Reentry into the Food Supply of Multiple Pharmaceuticals and Personal Care Products (PPCPs) Environmental Science & Technology, 46 (7), 3795-3802 DOI: 10.1021/es203970e

San Martín B, Cornejo J, Iragüen D, Hidalgo H, & Anadón A (2007). Depletion study of enrofloxacin and its metabolite ciprofloxacin in edible tissues and feathers of white leghorn hens by liquid chromatography coupled with tandem mass spectrometry. Journal of food protection, 70 (8), 1952-7 PMID: 17803156

Newsworthy: Veterinarians Find Infections Faster by Monitoring Nt-pCNP

"Stinger", a dog with sepsis from a bite wound. Image from Valley Center Veterinary Clinic, Valley Center, CA.

N-terminal portion of pro C-type natriuretic peptide. Try to say that one three times fast. ScienceDaily has a cool article detailing a couple new studies showing that this peptide (Nt-pCNP) could be a solid indicator of sepsis as opposed to a generalized inflammatory response. It could potentially be added to current serum chemistry analysis, or packaged as an ELISA snap for quick, in-house diagnostics for pertinent cases.

Overuse of antibiotics has been a long term problem that is being well addressed in human medicine, however they are used much more broadly and liberally in animals due to their non-prescription access (especially in large animal work), and their use as a diagnostic tool for patients who can’t speak and often can’t afford thorough diagnostics. A veterinarian who’s client is unwilling to pay for a culture will often send broad spectrum antibiotics home anyway as a less expensive option in the hopes that they will take care of the problem.

I was unable to find a chemistry profile of Nt-pCNP, but the journal article itself talks a bit about C-type natriuretic peptide. CNP is produced by vascular endothelial cells and immune system macrophages. It “inhibits microbial growth and modifies pathogenicity of microorganisms” (DeClue, 2011). The problem with looking for just CNP as an indicator of sepsis is that it has a very short half life, and tends to degrade even faster in removed serum. Therefore, the researchers decided to use Nt-pCNP as their target molecule.  Nt-pCNP is created in a 1:1 ratio with CNP as a byproduct, and is much more durable and long-lived in both the bloodstream and collected serum.

The results of the study support the hypothesis  that CNP is a good indicator of sepsis, however like anything else, it’s not ideal. CNP was shown to be a poor indicator of sepsis when the infection was peritoneal. This includes gastrointestinal perforations or other possible infections found within the peritoneum (the authors mention that using peritoneal fluid as opposed to serum from a distal point may yield better sensitivity). Taking these false negatives into account, the test had a 65.5% sensitivity, for all other origins of sepsis in the study, sensitivity was 92%. Unfortunately, there appears to be a large potential for ambiguous negatives when peritoneal infection is suspected, but it’s always important to remember to educate clients that medicine is rarely black and white. It’s nice that House is able to identify exactly what’s wrong with each of his patients every week, but most of the time, we’re just going to give them supportive care based on the most likely result. Some of the limitations of the study that the authors mentioned were the small sample size and uncontrolled natures of the ailments that may have influenced the blood chemistry (samples taken from bacterial vs. viral infections, condition as of admission, underlying secondary infection or ailment, etc.).

In the case of this test and many other lab tests, positives are very definitive and help us out, while negatives are ambiguous. This is true whether it’s a heartworm test, fine needle aspirate, fecal flotation, radiology, or any number of other diagnostic tests. Every one is a tool, and hopefully looking at Nt-pCNP levels will give us another way to confirm sepsis while our cultures are growing at the lab, or perhaps offering another faster or less expensive option that the situation necessitates.
This post was chosen as an Editor's Selection for ResearchBlogging.org
DeClue AE, Osterbur K, Bigio A, & Sharp CR (2011). Evaluation of serum NT-pCNP as a diagnostic and prognostic biomarker for sepsis in dogs. Journal of veterinary internal medicine / American College of Veterinary Internal Medicine, 25 (3), 453-9 PMID: 21457321

Article Review: Vaccine Reactions

I’m apparently still on this immunology kick, because I seem to be finding it everywhere. Heck, I recently learned that we’ve cured the allergic response to peanuts and eggs in lab mice. Check out the link, the author is hilarious and the material is interesting.

These two articles offer a great look at the overall prevalence and risk factors associated with vaccine-associated adverse events. The components within the vaccine that cause these events are the antigen itself, adjuvants, preservatives, stabilizers, and residues from the tissue culture used to grow the vaccine (Moore, 2005). Vaccine reactions are similar to any acute allergic reaction, and can present with a variety of mild to severe symptoms. The mild being lethargy, anorexia, fever, edema (generalized or local to the injection site), pruitis, uticaria (hives or wheals), and pain at the injection site; the severe being vomiting, dyspnea (labored or shortness of breath), and anaphylaxis. There’s a lot of information about when certain symptoms tended to occur at intervals after the vaccines were given, but any reaction that isn’t within the first 3 days is pretty much never going to be life threatening. If anaphylaxis is going to occur, it’s going to be immediately following vaccination.

The really useful information was the breakdown of risk factors that can be used for client communication. I’ve decided to discuss them here, broken down into cat and dog categories.

Image from meow-cats.com

First, let’s start with cats. I almost like these numbers more because you don’t have to take into account bias based on animal or breed size, as most cats fall into the <20 lbs category. Nonetheless you still have to remember that a 4 pound kitten does way only a fraction of that 5 year old chubby (BCS >5 on a 9 point scale) longhair it will grow to be.

So the first two risk factors require a little bit of thinking in context to explain the numbers. It seems that cats weighing 2-4 Kg (4.4-8.8 lbs) and approximately one year of age are most at risk compared to other weights and ages. The high numbers for these groups can be explained by the number of first encounter events that occur. If you’re recording vaccine reactions, you will record less in older age groups and higher weights (low weight under 10 lbs is going to be suggestive of a young age rather than a smaller cat), because if an adverse event occurred at a young age, either the animal is no longer vaccinated or steps are taken to reduce its risk (medication, strict scheduling, vaccine selection). That being said, just because the numbers are higher by circumstance, this information is very relevant in a clinical setting. Knowing the epidemiology of these events can help technicians at clinics determine when the discussion of vaccine reactions is “routine” or “protocol”, or when it really needs to be a time to educate the client. Vaccine reactions may need to be just a bullet point when Schrodinger is there for his 4th rabies booster and a discussion when he’s receiving his kitten series.

Sex and neuter status have a large impact on reaction risk as well. Intact males actually have a lower risk of adverse events than neutered males and spayed and intact females. Apparently, estrogen has an immune boosting effect, while testosterone has an immune suppressing effect. This benefits intact males when it comes to all allergic reactions (and possibly auto-immune disorders).

Here’s the big one, and the one clinics have the most control over. With each additional vaccine given in a single visit, the risk of an adverse event increases by 28% in cats. That’s huge. Any cases of severe anaphylaxis or death recorded in the study were preceded by the animals receiving 3 or more vaccines in one visit. So clearly the biggest thing any clinic can do to prevent adverse events (or at least severe ones) is to adopt a vaccination schedule that prevents multiple vaccinations from occurring within the same visit. This can be difficult as clients will not want to end up paying for multiple exams throughout the year, but with boosters outside of rabies, exams shouldn’t be necessary unless an annual or other scheduled exam is due. As far as specific vaccines being more prone to adverse events, the only suggestive evidence was when both FVRCP and FeLV were given within the same visit. This is explained by both having two concurrent vaccinations given, and also the theory that vaccines containing multiple antigens or covering multiple serovars (multivalent) are more likely to illicit reactions. Interestingly, while clients are often scared by the potential for vaccine caused neoplasia from the rabies vaccine, it was among the lowest reaction rates observed with the administration of a single vaccine.

Image from peteducation.com

Dogs had much more biased data within the age and breed groups because there is an obvious relationship between body mass and the potential for reaction. When looking at the dog population, a chihuahua can be as little as 6% of the weight of a bullmastiff, yet they receive the same 1ml dose of vaccine. This means that an 8 lb Chihuahua is going to receive proportionally 15 times more vaccine than a 120 lb bullmastiff. Not surprisingly, this causes a bit of inflation in the number of reactions in groups that are smaller in size, such as toy breeds and puppies. The highest risk group in size was 0-10Kg (0-22lbs) and the highest risk age was approximately 2 years of age (with higher rates for <2 than the rates of >2).

Just like I mentioned before when talking about cats, the greatest risk factor for reactions in dogs was the amount of vaccines given in one visit. The difference though, is how the large weight distribution in dogs makes this even more important. Small dogs (<10Kg) are similar to cats in that their risk increases by 24% with every additional vaccine administered that visit, while large  dogs (10-45Kg) increase their risk by 12 percent. All 3 dogs in the study that suffered fatal reactions received 4 or more vaccines at once.

Breed dispositions were difficult to pinpoint, as the suspected breeds are all small breeds which suffer a higher rate of reaction already due to their size. There is suspicion that dachshunds may be predisposed to allergic reactions in general, but so far the evidence is inconclusive concerning vaccines. Only the Lyme vaccine appeared to carry a higher risk than any other, showing again that, with the exception of neoplasia concerns, rabies does not carry with it any additional risk. Spayed and neutered animals, as in cats, are more susceptible to reactions; however the difference between intact and spayed females is much larger in dogs than in cats (where they are nearly identical). Dogs do seem to display an interesting trend where vaccine reactions are more likely to occur on the 3rd booster in a series, likely catching clinicians and clients off guard as they have received the first two without incident. This just states again that the puppy and kitten periods (and new patients) are of much more relevance when discussing vaccine reactions with clients.

The articles are both great, and contain an excellent statistical analysis of millions of animals. They provide a great overall picture of the epidemiology of vaccine-associated adverse events, and are definitely worth a read for both veterinary doctors and staff. Knowing a couple of the more important statistics can reassure the client and lend credibility to technicians that are responsible for discussing these issues.

ResearchBlogging.orgMoore, G., DeSantis-Kerr, A., Guptill, L., Glickman, N., Lewis, H., & Glickman, L. (2007). Adverse events after vaccine administration in cats: 2,560 cases (2002–2005) Journal of the American Veterinary Medical Association, 231 (1), 94-100 DOI: 10.2460/javma.231.1.94

Moore GE, Guptill LF, Ward MP, Glickman NW, Faunt KK, Lewis HB, & Glickman LT (2005). Adverse events diagnosed within three days of vaccine administration in dogs. Journal of the American Veterinary Medical Association, 227 (7), 1102-8 PMID: 16220670

Newsworthy: Vaccine linked to “bleeding calf syndrome”

When I first started working in an actual clinic, I was blown away with the education I received in vaccine administration. Before at the shelter my instruction included solely how to administer them and not to be bitten while doing so. Spending a minute to educate clients on vaccine reactions, the steps we took to prevent them from happening, and the importance of the proper scheduling of a series were all new to me, and considering how seriously we took all these things, it vastly contrasted with my training at the shelter. How vaccines work has always been interesting to me, and the immunology involved isn’t terribly complicated on the surface. Even if the mechanisms escape me, I can still visualize the flowchart (something I wish I could consistently do with G-proteins, a crucial topic but one I constantly have to review).

Image from Veterinary Laboratories Agency

Anyway, the point is I was really interested in this article. Bleeding calf syndrome is technically called bovine neonatal pancytopenia (BNP), but is probably still a frightening thing to see. It actually only emerged in 2007. The characteristic bleeding is caused by thrombocytopenia after the calf’s bone marrow becomes compromised. The lack of platelets causes the appearance of bleeding through the skin the name refers to. A group of doctors in Germany were able to determine the etiology of the condition (which has a calf mortality rate of 90%). Based on another study, they knew that the colostrum given to affected calves could also induce the symptoms in other unrelated calves, and after not finding evidence from pathogenic or genetic causes looked at an “immune mediated process” (Deutskens, 2011).

What they found was that there was a correlation between cows vaccinated for Bovine Viral Diarrhea Virus (BVDV) and calves suffering from BNP. After a lot of spectroscopy and protein identification, they found that the vaccine actually was the cause. What the issue was, is that the BVDV vaccine is made using kidney cells from cows, instead of another species (for example many human vaccines are cultured in pig cells). Here’s how that works: there is a specific protein “map” coating all nucleated cells that the immune system uses to identify which team they play for. Anything with a different protein coat is assumed to be foreign, and is attacked. This is the major reason why donated organs are rejected, because the donor has a slightly different “map” than you and your immune system assumes it’s trying to hurt you. With the vaccine grown in bovine cells, remnants or copies of this Major Histocompatability Complex (MHC, the “map”) are introduced to the mother, who has an immune response to them.

This is where it gets interesting, the antibodies the mother makes to attack this are called alloantibodies, and they don’t hurt the mother. They just become another antibody she reserves along with the ones that attacked the rest of the BVDV vaccine. None of her cells have that foreign “map”, so none of her cells need to worry about that extra antibody she created. However, the alloantibody gets stowed away in the colostrum along with all the others just before partuition, still ready to attack the MHC the vaccine was made with. If by coincidence the MHC of the calf is the same as the one in the vaccine cultures, then those alloantibodies given to the calf through the dam’s colostrum will attack cells within the calf, starting with blood cells and moving to the bone marrow. Making it technically not an auto-immune response, because it comes from a foreign immune system, but still a case of friendly fire. Those alloantibodies in the colostrum treat every calf cell featuring that MHC as if it’s an infection.

It should be noted that other vaccines for BVDV that are grown using non-bovine cultures do not cause these problems. This is because if the mother creates antibodies for the MHC of another species, there is no way that the calf can be affected by them. The authors of both the news article and the journal article mention that this serves as an example why same-species vaccine cultures and formation of alloantibodies should be avoided.

Check out the journal article for yourself, the introduction and discussion are well written and interesting. There’s also a similar alloantibody caused disease in humans called Neonatal Alloimmune Thrombocytopenia that’s interesting, the major difference being that the alloantibodies are introduced through the placenta instead of ingested through colostrum.

ResearchBlogging.orgDeutskens F, Lamp B, Riedel CM, Wentz E, Lochnit G, Doll K, Thiel HJ, & Rumenapf T (2011). Vaccine-induced antibodies linked to Bovine Neonatal Pancytopenia (BNP) recognize cattle Major Histocompatibility Complex class I (MHC I). Veterinary research, 42 (1) PMID: 21878124

Article Review: Leptospira and Leptospirosis

In my latest ScienceDirect purge, I came across this article covering Leptospirosis. I had no idea it would be such a dense read, figuring it would be a simple review of the disease with emphasis on new discoveries. I ended up using a lot of immunology references and Google searches. This article isn’t just an entry from the Merck manual.

The article does a very good job of covering Lepto microbiology, but I was especially impressed with the point they made to identify everything we don’t know. Indeed that was the emphasis of the article, that lepto contains so many pathways unique to it as a bacterium that we don’t know nearly as much about it as we do something like E. Coli. Additionally, Lepto is extremely hard to culture, as you end up with non-virulent colonies. They identify and isolate the virulent daughters by inoculating lab animals.

You might assume that immunity to Lepto is a simple thing, given how prevalent Lepto vaccination is due to the zoonotic risk. However the article makes the point multiple times that immunity to one Lepto serovar does not grant immunity to others, though occasionally it can help grant passive immunity or resistance across different species. While the exchange of genetic material between parent and daughter lepto colonies is not well understood, it appears to be slow mutating, which is interesting given how unique the antigens between serovars seem to be.

There’s a lot of complicated immunology discussed in the article that I don’t feel qualified to comment on, but it’s very interesting, and I recommend glancing through. The more microbiology I learn the more I understand that 99% of the workings of the cell happen on membranes (a statement that probably produces a loud “duh” from any student, biologist, or doctor). For a more clinical discussion of Lepto, a simpler reference like Merck or Blackwells will help, as well as several peer-reviewed sources that the article itself recommends for information on clinical presentations.

Adler, B., & de la Peña Moctezuma, A. (2010). Leptospira and leptospirosis Veterinary Microbiology, 140 (3-4), 287-296 DOI: 10.1016/j.vetmic.2009.03.012