Can you see my heart beating? A snake with a very big problem!

Snake transport box

I received an unusual phone call recently from the owner of an 18 month old False Water Cobra (Hydrodynastes gigas), a South American rear-fanged, mildly venomous species of snake (http://www.reptilesmagazine.com/Snake-Species/False-Water-Cobra/). The owner had noticed shortly after acquiring the snake that it had what was described as a wound on it’s underside through which you could see it’s insides! Naturally, I was quite worried so asked for the snake to be brought in for examination, and in the meantime they emailed me some photos and reassurance that the snake was acting perfectly normally, eating and defaecating fine and didn’t seem in any way bothered by the strange ‘wound’.

False Water Cobra Hospital Vivarium on AdmissionFalse Water Cobra Hospital Vivarium on Admission 1

Having examined the animal, it appeared that she had a defect in the body wall on her ventral surface or belly with a large hole visible in the ventral muscles. Bizarrely, this defect was at the level of the heart about a third of her length down her body. The only tissue separating her visibly beating heart from the outside world was a thin membranous layer of body cavity lining and some connective tissue which was quite transparent. I have seen some similar lesions in reptiles at the umbilicus or site where the blood vessels carry nutrients into the growing embryo from the egg or from maternal tissues in the case of live bearing species. Sometimes when these animals hatch or are born the hole in the body wall fails to close completely and an umbilical hernia or body wall defect is left behind. However, I have never seen or even heard of such a defect high up on the body overlying the heart! I concluded that this must be a congenital problem that the snake was born with, perhaps a fluke genetic mutation or a developmental insult during incubation of the egg which disrupted the normal growth of the embryo resulting in a physical abnormality at birth. Having spoken to a few reptile enthusiasts in the UK who keep this species of snake, it turns out there have been a few reported with the exact same defect in the exact same place, so it looks likely this is a genetic problem, most likely caused by inbreeding in certain lines due to relatively low genetic diversity in the UK population.

Snake membranous hernia before surgery

The risk of leaving the snake in this condition was that during the course of her life she may snag this fragile membrane protecting her heart and eviscerate herself or worse still puncture her heart. Although this risk was small, it would be safer to carry out a surgery now than always wonder and worry if she might do some damage and seriously injure herself, when pulling off her shed skin for example. I admitted her and set her up in our reptile ward in a heated vivarium to bring her body temperature up to preferred range. Once her temperature was correct I injected her with a combination of sedative drugs into the muscle in her back. I say I injected her, but actually I restrained her with a thick pair of gloves whilst my nurse Justyna gave her the injection. She was very feisty, strong, and objected to restraint, trying to bite the gloves. Obviously considering her potential to give a painful bite and possible envenomation we took great care at this stage. Rear fanged snakes are different to other venomous species in that they have modified salivary glands and teeth at the back of their mouth, with which they chew their prey in order to inject and subdue it. Therefore it would be difficult to get a dangerous bite  from a rear fanged species unless you allowed the snake to chew on you. Front fanged species can deliver a dry or wet bite from the front of the mouth with a rapid strike and minimal contact so are far more dangerous to work with and handle. After her injection we left her back in her hospital vivarium in the correct temperature to allow her to metabolise the drugs efficiently. Ectothermic or ‘cold-blooded’ animals such as reptiles rely on external temperature to control their biological functions and metabolic rate, so it is vital when treating ill specimens and indeed when undertaking anaesthesia and surgery to maintain their body temperature in the correct range so everything runs smoothly and drug dosages work effectively. After about a 20-30 minute induction period, now nice and relaxed after her sedation we removed her and placed her into an anaesthetic gas chamber so she fell further asleep and we could position and prepare her for surgery. She was maintained under anaesthesia by using a gas mask delivering a safe gaseous drug called Sevoflurane. Normally for a prolonged surgical procedure in a snake I would intubate the animal with a solid plastic or rubber tube placed into the windpipe or trachea for better control and access to her airway should she stop breathing for instance, but in the case of venomous species I prefer not to mess around in the mouth due to the risk of accidental envenomation. A correct sized mask with her head taped inside in place and sealing the entrance provides the next best option and is safer for all involved.

Snake anaesthesia preparing for surgery

 

Here’s a video link showing the heart visibly beating underneath the membranous layer of the abdominal wall defect:

Once Justyna prepped her with a surgical scrub and insulating layers to maintain her body temperature lying belly up on a surgical heat mat, I was ready to begin. I carefully dissected away the thin membrane covering her heart from the scales on either side of the deficit. I had to be extremely careful as this layer of tissue was so thin and the heart was literally beating against it directly underneath. Tiny blades and scissors were required, and I cut in time between the beats to avoid cutting at the moment the heart filled to maximum capacity with blood. Myself, Justyna and the veterinary student who is seeing practice with us this week to learn more about exotic pet medicine were all holding our breaths at times. Credit to the student Conor for taking the photographs while I operated by the way. Justyna found this anaesthetic very easy to monitor throughout as we were literally watching the heart beating throughout so she could record heart rate and strength with the naked eye for a change! Often we use sensitive probes to monitor heart rate through the body wall but not needed in this case.

And here is a video during the surgery when I had carefully dissected away the thin tissue revealing the heart beating underneath before I began to suture the defect closed:

Once the tissue had been separated away from the half sized scales lining either side of the lesion I set about suturing the hole back together, bringing the soft tissue of the internal body wall and the edges of the scales together to meet in the middle and form a tight seal which would heal and protect the heart and blood vessels underneath. I was quite happy with a neat and tidy job at the end. She was given a long acting pain medication and anti-inflammatory by injection, and a reversal drug for the sedatives given earlier and placed back in a clean dry cage with paper substrate to recover. We turned up the heat temporarily to boost her metabolic rate and aid her clearance of the anaesthetic drugs, and within 20 minutes she was groggy but wandering around her cage getting her bearings again. It always surprises me how tough these animals are. Reptiles have been around for millions of years and they really are quite resilient and remarkable in what they can withstand. Many of my clients fret and worry about anaesthetic risk in particular if their pet reptile needs a surgery for example. Although the general risk is higher than in dogs and cats I find them to be remarkably sturdy anaesthetic patients for the most part, so the risk is still quite low as long as you seek out an experienced and knowledgeable reptile vet.

Snake repair after closure

 

In terms of aftercare, she just needs to be kept clean and dry. No antibiotics were prescribed as it was a sterile op and good hygiene practices should prevent infection from here. Being a semi-aquatic species that likes to bathe in water, I’ve specified that she is not allowed a water dish in which she could submerge as the wound could act as an entry point for water and bacteria or other contamination into the body cavity if she submerged in the coming week or so. I will keep her in overnight and send her home tomorrow. So far she is making an excellent recovery. The sutures I placed are dissolvable over time and should slough off the next time she sheds her skin or possibly after that, so I just need to recheck her in a week to make sure the wound looks good, is healing well and is free of infection. Problem solved!

Snake in recovery

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Metabolic Bone Disease: Another Rubber Tortoise!

WARNING: THE VIDEO AT THE END OF THIS ARTICLE IS DISTRESSING AND FEATURES A DEAD ANIMAL IN A POOR PHYSICAL STATE. PLEASE WATCH AT YOUR DISCRETION.

Virtually every week I am presented with a pet reptile which has an advanced disease relating to poor owner preparation or knowledge on how to keep these specialist animals. Today I had a tortoise patient booked in as a health check, but knew instantly on taking it out of it’s container that I would be humanely putting it to sleep as it had been suffering for a long time from metabolic bone disease (MBD) relating to poor husbandry and nutrition. I am angry, frustrated and weary of having to destroy animals that can be kept very successfully in captivity with a little bit of preparation, initial outlay of financial investment, research and common sense. I am astounded that I am having the same conversations over and over again with reptile owners who really don;t know how to keep the animals in their care, but have taken someone else’s word for it without doing extensive research of their own. I will explain what led to this animal’s demise, and how it could have been prevented. I hope this post gets shared widely as the owner has kindly agreed I can share the story so that other owners may be educated and welfare improves for at least some animals as a result. I have to commend the owner of this tortoise for at least bringing the tortoise to the vet. I’m sure many don’t and their animal dies slowly at home. My role as a vet is to educate owners and safeguard animal welfare, often having to be quite diplomatic in the face of pretty distressing cases of animal suffering. I recognise the tone of this post comes across as an angry rant, but I need to drum home an important message in the hopes I help some new keepers revise the care they are providing to their pet reptile and correct any issues before they cause such suffering as I saw today. Firstly, before I get to the details of this case let me explain a little about metabolic bone disease (MBD).

MBD is a condition whereby an animal lacking in calcium, ultraviolet (UV) light exposure or both eventually becomes so deficient that it starts to absorb calcium from it’s skeleton and in the case of turtles and tortoises, it’s shell, in order to maintain normal metabolic function. Calcium is required for a variety of bodily functions including skeletal growth, neurological activity, muscle contractions and hormonal control for example. Reptiles in captivity are particularly vulnerable to MBD as they are reliant on their owners to provide them with artificial UV lighting, replication of a varied and natural diet, as well as appropriate calcium and other vitamin/mineral supplementation. The absorption of UVB light from natural sunlight allows reptiles to synthesise Vitamin D3 in their skin, and vitamin D3 allows them then to absorb calcium from their diet in the gut. This delicate balance between sunlight, vitamin D and dietary calcium sources is reliant on environmental temperatures, and is hindered if reptiles are kept in conditions which are colder than their preferred optimal temperature zone (POTZ). So you can appreciate that there is a fine balance between several interlinked factors that could contribute to the development of MBD when we take reptiles into captivity and take control of their immediate environment. Temperature, lighting and diet are the three factors that lead to the development of MBD. Get any one of those factors incorrect and you are setting your animal up for ill health. For further reading on this topic I can highly recommend reading the following book from leading reptile lighting specialists Arcadia: (www.arcadia-reptile.com/mbd-book-reviews/).

The basis of keeping any reptile as a pet is very simple. I will share this basic ‘secret’ now. It is not rocket science. It is very simple and achievable. Although it is often inconvenient or more expensive than some people are prepared to pay. And it is not often achieved by taking shortcuts or following some pet shop’s or online forum’s advice. The simple rule of reptile keeping is this:

REPLICATE THEIR NATURAL CONDITIONS IN THE WILD!

Sounds easy, right? Well it is. But unfortunately there are a multitude of sources of information giving conflicting advice, a bewildering array of equipment and housing options commercially available, and an unfortunate trend in the reptile hobby of keeping animals in conditions which allow them to survive with minimal effort or expense. The aim of keeping reptiles as pets should be to provide them everything they need to allow them to thrive and flourish in as near natural conditions as possible, not just to survive with the minimum provisions they need. It is not up to the pet shop staff to give you the right equipment. It is not up to the guy on some forum who told you how to keep your pet alive. It is up to YOU to research the conditions your animal comes from in the wild, and how best to replicate them in captivity. There is no one perfect list or care sheet that does this work for you. Different keepers do things different ways. What you need to research is what temperature, humidity, diet and light your animal needs and then ensure you are providing that by monitoring the conditions in your particular set up. Just because someone does it their specific way and has had success, doesn’t mean you will achieve the same in your particular setup by following their lead. There are many variables that might affect your animal’s long term health.

I have lost count of the amount of times I ask someone the question: “What temperature is your vivarium?” and am met with the reply “I don’t know, it’s warm”. A thermometer so you can record and monitor the temperature, and a thermostat to control the temperature are two vital pieces of equipment for providing the correct environment. A thermometer costs very little, a thermostat significantly more. But without them how do you know you are providing the animal with the correct range of temperatures it needs to survive? YOU DON’T! So you could be providing sub optimal temperatures sometimes or overheating your animal at others, resulting in biological stress and impaired immunity or overall health. If you have no thermostatic control of your vivarium, then the temperatures will fluctuate depending on conditions outside the vivarium in the room for example. Hence why advice telling you a specific wattage of bulb is suitable for a certain species is far too vague, as it doesn’t take into account how cold or warm the room in which the vivarium is housed tends to be. Rather, good advice should tell you the correct temperature  range for your species, and it is up to you to find the correct bulb or other heating to achieve that temperature range that allows natural thermoregulation. UV lighting needs to be the correct intensity for the species of reptile being kept, needs to give enough exposure to UV rays by it’s spread and location in the enclosure and needs to be replaced frequently to maintain high enough levels in the enclosure. These bulbs also cost money. Diet needs to be researched. Convenient options often lead to long term problems. It takes time and significant effort, as well as significant expense, to provide a varied and natural diet. If you are not willing to invest this time, research, effort and expense into keeping a pet reptile, DO NOT BUY ONE!

The reptile hobby as it stands is under increasing pressure from animal rights and welfare lobbyists arguing that these animals should not be kept in captivity at all due to the significant welfare problems they experience and the relatively high (often over exaggerated) mortality rates. I know many wonderful, knowledgeable and extremely capable reptile keepers as friends and as clients who provide amazing care to the animals they look after. However, there is a massive welfare problem with captive reptiles which cannot be ignored. I see it in my line of work very frequently that beginner or inexperienced keepers are making very costly husbandry or nutrition mistakes which are costing the lives of a huge number of animals up and down the country every day. As a hobby, we are defensive when criticised by animal rights groups, but to ignore that there are huge welfare problems in the hobby is actually doing ourselves a disservice.

Anyway, back to the tortoise I had to euthanise today. It was only a year and a half old and had been purchased from someone online. It had been kept in a vivarium with a single combined UV and heat lamp as the sole heat source. The owner didn’t know what temperatures were being provided. There was no thermostat. The pet shop staff told the owner that bulb was suitable for tortoises. The bulb had not been changed for well over a year. The tortoise was fed commercial tortoise pellets from a large plastic tub as it’s staple diet. It also ate lettuce and cucumber. No calcium, multivitamin or mineral supplements were provided. The owner noticed it’s shell was ‘looking weird’ around Christmas time. Today is the last day of March. It was brought in today because it had stopped eating. It had also stopped moving very much. It’s shell was very flat and very soft. TORTOISES SHOULD NOT HAVE SOFT SHELLS! IF THEY DO, YOU’VE BEEN DOING IT WRONG! Why are we still seeing cases where reptile owner’s are doing it so wrong?

Here’s my take on the above husbandry failings. You should research tortoise or any pet’s care long before you buy one. You should have read all available information, and asked as many questions as you can of experienced keepers until you are an expert on their care. Clear up any confusion you have before you purchase an animal. Don’t just listen to one point of view. Explore the theory, and examine the logic behind the varied advice you are given. Steer clear of prescriptive advice that tells you ‘X species needs Y type of bulb’. REPTILE HUSBANDRY IS NOT THAT SIMPLE!

Mediterranean tortoises do best in large open topped well ventilated enclosures such as ‘tortoise tables’. Vivaria can lead to problems with temperature and humidity for Mediterranean tortoises. A single combination UV and heat bulb may work for some keepers who have mastered the pretty simple task of knowing and controlling the temperature correctly using a thermometer and thermostat, selecting the correct wattage bulb for their set up and providing a nutritious, appropriate captive diet with correct supplementation. It does not work for everyone, and I would argue that pet shops should not be selling these bulbs for beginners to keeping tortoises as these are the owners who are likely to get other factors such as diet wrong too. A preferable option is to provide a heat lamp and a separate UVB fluorescent tube which covers the entire length of the enclosure. That way the tortoise will receive UV light throughout the entire enclosure, even when it is not basking directly under the heat lamp. Tortoises do not bask in the one spot for long periods, they roam. By choosing a combination bulb your tortoise may not receive the optimal level of UVB exposure over time and therefore if your temperatures and diet are also somewhat deficient your tortoise will not thrive. It may even develop metabolic bone disease. Remember the intricate relationship between temperature, lighting and dietary balance? Take any one of those factors out or skimp on their provision and you are setting yourself up for a case of entirely avoidable MBD, among other potential health problems. If you do not know the temperature you are keeping your reptile at, buy a thermometer and find out. Pretty simple concept. If the temperature fluctuates widely in your specific set up due to external factors then buy a thermostat and make sure it is controlled. Yes, that costs money. If you can’t afford one, maybe you can’t afford a reptile. If you can’t afford one for your newest reptile, maybe you have too many reptiles. NOT KNOWING WHAT TEMPERATURE YOU KEEP YOUR PET REPTILE AT IS A COMPLETE NEGLIGENCE AS A PET OWNER.

UV lighting needs to be changed on a regular basis. The bulbs give out light for long periods, but the output of the UV spectrum of light declines with time, generally over the course of 6-12 months depending on the manufacturer and quality of the bulb. Document when you buy new bulbs and when they need to be replaced. Young, actively growing reptiles in particular with high calcium demands cannot do well with a sub optimal level of UV light, even for short periods without suffering.  Commercial pelleted foods are not a staple diet, they are a supplement to a healthy diet. Their nutritional content declines with time. They may be convenient for the owner as they sit in a tub ready to be poured into a feeding bowl, but tortoises need a variety of fresh greens, weeds, grasses, flowers and small amount of vegetables to thrive. Dry pelleted food is not a substitute for a fresh diet. Diets also need to be supplemented with calcium, and other vitamins and minerals that may be lacking in the limited diet we invariably provide in captivity. Lettuce, cucumber and tomato is an extremely poor diet as it has a high water content but very few valuable nutrients. As an aside which is unrelated to this case, I hear from time time time about the act of feeding tortoises dog and cat food. NEVER FEED ANIMAL PROTEIN TO HERBIVOROUS MEDITERRANEAN TORTOISES. (Jam sandwiches or chips are also off the list. I’m not joking!).

If you notice your tortoise or other reptile pet acting or looking strange, then BRING IT TO THE VET! Don’t wait until it can’t move or no longer eats. Don’t wait until you’ve read a few articles and self diagnosed it, then tried to treat it at home. Don’t ask other hobbyists on five separate internet forums until you hear get the answer you want or whatever other DIY home treatment option you might try first. IF YOU CANNOT AFFORD VETERINARY FEES, YOU CANNOT AFFORD THE PET! Reptiles hide illness very well to avoid predation int he wild. If you think they are sick, they are probably sick. Seek a qualified and experienced reptile vet at the earliest opportunity and work with them. Don’t lie to them or withhold important information that could aid their diagnosis (this happens a lot too, though not in this case). A deformed or misshapen shell in tortoises and turtles is always indicative of a problem. If the shell is softening this problem is advanced.

This tortoise today had a softened, flattened shell like a rubber ball. This did not happen overnight. The owner noticed at Christmas that something was wrong. But only brought it in to my clinic when it had stopped eating and stopped moving. The tortoise was so deficient in calcium over a long period of time due to poor heating, poor lighting and poor diet that it had absorbed it’s shell and skeleton of calcium, thus softening the bones to the point they could no longer support it’s body weight. It was undoubtedly in immense pain. It was having to pull it’s forelimbs back and forth to try to keep breathing as it’s collapsed rubbery shell was compressing it’s lungs and causing respiratory distress. It was undoubtedly in multi-systemic organ failure at the tender age of 18 months. It had the potential to outlive it’s owner, it’s owners children and potentially grandchildren if a little more research and effort had gone into it’s care. Sadly, it was beyond help, and I have to take comfort in the fact I could relieve it’s suffering and put it out of it’s misery humanely. I am however, still angry, jaded, frustrated and tired of seeing this problem crop up over and over again whilst trying to maintain an upbeat, positive demeanour that will help educate the owner rather than turn them against the idea of veterinary care.

Below is the video of the tortoise which may be distressing to some people. Please view at your discretion. Please also note that I took this video AFTER the tortoise had died, so it is no longer suffering. It is explicit to show the extreme changes that can occur with MBD as a direct result of poor husbandry and nutrition. If it makes one person improve the care of their pet tortoise it will be worth posting, so please feel free to share this article.

Salmonella: Transmission from Pet Reptiles

Zoonoses or Zoonotic diseases are those that can be transmitted between humans and animals. Salmonella is a zoonosis from pet reptiles that I am asked about on a fairly regular basis, and usually relates to the risk associated with reptile pets and Salmonellosis in humans. Whilst reptiles, amphibians and many other exotic pets can and do transmit Salmonella infection from time to time, sensible hygiene precautions can generally prevent this occurrence. Other precautions are recommended such as refraining from buying wild caught animals which are more likely to be infected, but also may be stressed and debilitated leading to enhanced shedding of infective material. Similarly, buying pet reptiles from clean, professional outlets or breeders may also reduce the risk of acquiring an infected animal in the first place due to decreased chances of transmission between animals where hygiene and cleanliness are scrupulous. Many different Salmonella species and serovars have been isolated from reptiles commonly kept as pets, several of which are infectious to humans.

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The difficulty in assessing infection risk from reptile pets lies in the fact that certain Salmonella spp. can be commensal organisms in the gastrointestinal tract of pet reptiles, and also in that they are often intermittently shed so even if screening faecal samples it can be difficult to ensure a pet reptile is Salmonella free. Pets can appear perfectly healthy with no outward signs of infection yet carry a pathogenic strain that could result in severe illness in humans. Transmission to humans is usually via the faecal oral route, and as such any contact with faecal material should be minimised. Hygiene precautions such as washing hands after handling animals, wearing gloves for cleaning reptile housing and for maintenance of aquatic environments and filtration systems are the best defence against infection. Salmonella bacterial cells have been demonstrated to survive and remain infective in the environment for long periods of time, up to 6 months in dried reptile faeces and up to 6 weeks in contaminated aquarium water. Therefore, indirect transmission from the environment to oral ingestion can also be a significant route of infection in homes with reptile pets, particularly where cleanliness and hygiene practices are poor.

Crypto-gecko1-250x250

Signs of infection in humans include diarrhoea, vomiting, lethargy, abdominal cramps/pain, fever and in severe cases dehydration or even septicaemia and death. The most serious risk for infection is in young children, the elderly and immunocompromised individuals. One of the issues which comes up from time to time in the media, is whether reptiles and amphibians are suitable pets for children in the first place. This debate raged most notably in the 1980’s and early 1990’s when thousands of baby terrapins were being sold to young children in the UK and USA during the teenage mutant ninja turtles craze, resulting in several high profile cases of Salmonellosis in children. Several more recent high profile outbreaks in the USA have linked Salmonella outbreaks in children to aquatic frogs sold for home aquaria.

Transmission can be increased in young children for a variety of reasons, mainly due to not washing hands after handling pets or putting their hands in faecal contaminated water and then touching their mouths. Immunity in young children is also underdeveloped compared with healthy adults so transmission risk is again increased. As such, it is prudent to advise against aquatic pets such as turtles and frogs for young children for safety reasons as much as for the fact that these are specialist pets requiring detailed care and environmental conditions. In a household with very young children that would not understand these precautions, it may be better to limit access to such pets or consider waiting until children are a bit older to allow interaction with them. Similarly, it may be worth re-homing pet reptiles or amphibians if there is a member of the household who is on immunosuppressive medications like steroids or chemotherapy agents, or those with immunocompromising conditions. The alternatives would be rigorous and regular testing to screen for Salmonella spp, recognising that several tests are needs to rule out the disease and also that strict biosecurity will have to ensure infection is not introduced. This could potentially be very difficult to achieve especially considering some outbreaks of pathogenic Salmonella have been traced to rodent or insect food items required for many captive reptile diets.

If keeping reptiles or amphibians in the home with children it is crucial to teach them the importance of good hand washing after handling any animal, not just exotic species. Supervised handling if at all should be encouraged. Cleaning of reptile housing and equipment should never be carried out in areas where food is to be prepared, or drinking water is sourced or even in showers and baths used by the household. Preferably these tasks should be carried out outside the home or in a dedicated sink or washing facility. Cages or tanks housing exotic pets should be cleaned and disinfected with an appropriate disinfectant at the correct concentration on a regular basis. This is especially important for aquatic species who obviously occupy faecal contaminated water and therefore often have a higher potentially pathogenic load of gastrointestinal micro-organisms than other species.

In summary, perhaps reptile and amphibian pets are too great a risk for certain households depending on what practical arrangements can be made to limit risk of infection to vulnerable individuals, particularly young children. It is important to be aware however that with sensible hygiene precautions such pets pose a very low risk and incidence of disease transmission from pets to owners is greatly exaggerated by the media when occasional cases occur. Any signs of gastrointestinal illness in households containing such pets should be investigated by a medical doctor, especially in the vulnerable groups discussed. It is important to also mention that gastroenteritis caused by Campylobacter spp is also sometimes transmitted by captive reptiles and amphibians, and can cause similar signs.

Bibliography:

http://www.arav.org/wp-content/uploads/2013/03/Salmonella-Information-for-Veterinarians.pdf

http://www.apsu.edu/files/iacuc/Zoonoses-fish-reptiles-amphibians.pdf

http://www.cfsph.iastate.edu/Factsheets/pdfs/reptile_associated_salmonellosis.pdf

http://www.eurosurveillance.org/images/dynamic/EE/V15N22/art19581.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1297927/pdf/11285792.pdf