Microscopic observation in live tissue, awesome! But don’t ignore the methods

It has been far too long since I wrote a blog post. Look out internet, I have a blogging itch that needs scratching, and it’ll probably cause a rash!

…I apologize for that mental image.

The NIH sent me an email this week (via the various government listservs I’m enrolled in) that was proudly declaring that the mysteries of the cell were being solved right now, so I took the clickbait. In it was a cool study where we were able to actively watch mitochondria oscillate inside a living animal.

Fig from the article
http://dx.doi.org/10.1016/j.celrep.2014.09.022

There are two rabbit holes to enter with this article. The first is the observation of interest, which was mitochondrial oscillation. While these slinky moves have been observed in cell cultures, the authors wanted to see if there were any differences in cells that were part of a living, breathing animal.

Movement isn’t a surprising thing. If you’ve ever drawn the ATP synthase lollipop (totally relatable experience for everyone, right?), you already know that some of the main membrane proteins in mitochondria are constantly rolling around attaching phosphates to create ATP. The cool thing though, is that since they’re synching up with each other, that means there could be cellular communication mechanisms that are helping coordinate mitochondrial efforts to produce energy as needed. Which makes sense if you’re generating ATP in response to a stress event on a cellular level.

But what if you’re an animal? A slice of muscle tissue is made up of many muscle fibers, all of which contain mitochondria. It seems like there would be a need to coordinate increased energy production if you were planning to use all those fibers in sync to move. When looking at the cellular tissue inside a living rat’s salivary gland epithelium (the covering layer of the gland), the authors observed that mitochondria oscillated in sync not only within individual cells, but in sync with other cells in the tissue. The authors describe it in their press release wonderfully:

“You look through the microscope, and it almost looks like a synchronized dance”

It’s always more fun looking at living cells and tissue, it reminds you that all of that stuff we can’t see is always buzzing around without our notice and crawling all over our skin and gut.

Hypochondriacs I apologize for that second image.

So since we like looking at living things, let’s get to the second cool part that the press release seems to gloss over, how the hell were we observing cell structures in a living animal!

The principal author, Roberto Weigert, was the first one to publish this technique, so I went to that article to better understand what’s going on. I don’t want to dig into the microscopy so much, as the technical information is a little overwhelming. I’ll just say that there are really cool microscopes that can use near-infrared light to penetrate deep into different tissues (segregated visually by the injection of fluorescent dyes). The article has some amazing images of mouse vasculature that are both easy to observe and understand. But that technology isn’t what this blog is about. What I want to know is, what did this study entail for the animals used?

For this procedure, the research rats were anesthetized and had their salivary glands “externalized”, meaning that they gained access to them presumably by opening/removing the skin, fat, and muscle layers and segregating the gland as far as they could for the procedure (you’d be surprised how far you can pull things out while they’re still attached). Then, they bathed/saturated the glands with various dyes and chemical/hormone baths depending on what they were observing in that particular instance.

Once the images were taken, presumably these brave rats were euthanized, I couldn’t find a reference in the procedure but ultimately it had no bearing on the ability to replicate the experiment and was not included.

Now imagining these experiments in vivo (in living animals) brings up nasty words like vivisection. However it’s always important that the authors of the study aren’t left to singly decide if the research is necessary or not, it’s up to the Animal Care and Use Committee to allow the use of animal subjects for research at the NIH.

In order to use and ultimately euthanize these animals, the authors had to prove that: the information learned from the study will benefit humans and/or animals, there is a rationale for using animals including why a surrogate (e.g. cell culture) would not work (the authors make a great statement in their press release by describing the observations as if you’re looking at a tree vs. the forest), and a description of how the authors have actively attempted to minimize pain and discomfort for the animals used.

Ultimately I chose to write about this article because the methods were cool, but also to acknowledge the animal use inherent, but understated, in this type of research. It’s important to remember that often new information comes at the cost of continuing to support animal research when justified, and to not hide the facts from ourselves.

In order to responsibly care for all of our domestic species, we need to remember that before they were beef, eggs, milk, nuggets, or a data point, they needed to be cared for and euthanized humanely.

ResearchBlogging.org

Natalie Porat-Shliom, Yun Chen, Muhibullah Tora, Akiko Shitara, Andrius Masedunskas, & Roberto Weigertemail (2014). In Vivo Tissue-wide Synchronization of Mitochondrial Metabolic Oscillations Cell Reports : http://dx.doi.org/10.1016/j.celrep.2014.09.022

Weigert R, Porat-Shliom N, & Amornphimoltham P (2013). Imaging cell biology in live animals: ready for prime time. The Journal of cell biology, 201 (7), 969-79 PMID: 23798727

The poultry microbiome, once again proving that culture-based ecology misleads us all

Shigella penetrating the intestinal wall. Source: cellimagelibrary.org

If the world was enriched and homogenized, we would actually have a very good idea of what the microbiological community within looks like. Fortunately, the world is much more complex than the miniature environments we culture in the lab, and high throughput sequencing (HTS) is allowing us to fully appreciate micro-biodiversity. As new information becomes available, many of our models for microbial communities continue to be challenged by the actual composition of species in natural environments.

In the world of food safety, we rely on these models to set policy on a regulatory level, and to set critical limits down at the production level. Which tests we run on what products depend directly on what organisms (that cause food borne illness or spoilage) are supposed to be found on that type of food. The authors of this study that came out in PLOS ONE this February examined the microbiome associated with poultry products from farm to fork (meaning from clucking chicken to packaged poultry product) using HTS rather than culture/enrichment methods. The results indicate that there is an unappreciated amount of diversity between different stages of the poultry production process, and that we may not acknowledge the presence of some organisms as much as we should.

In the study, samples were taken from multiple steps in the poultry production process: wet and dry litter, fecal samples, fluid from carcasses collected during the cooling process following slaughter, and fluid from raw retail poultry products (legs, wings, and breasts). Other than the retail portion, all of the samples collected were from the same batch of birds from start to finish. The available RNA from viable cells in each sample was amplified and identified as belonging to specific species using a combination of Illumina sequencing and database referencing (blastn and usearch).

From this pile of data, lists of organisms were compiled to compare the ecosystem profile for each point in production.

The numbers refer the the number of unique taxa found in each group

The authors were very surprised by the amount of diversity between the two litter samples (wet and dry) and the fecal sample. They expected to see very similar profiles, as all of the predicted microbes in those groups would be inoculated from contact with fecal material (young chicks have no inherited microflora, and are coprophagous); however, all of the groups’ microbial communities had very little in common. As shown above, of the hundreds of unique species identified, only 52 were actually found at every stage from farm to fork.

In evaluating food safety, several results are of concern. The first was that the authors found significant amounts of Shigella spp., which have traditionally not been associated with poultry products and may not be a part of many sanitation programs. The second is that in one of their dry litter samples, the authors found a large amount of C. jejuni. It’s presence was interesting as previous studies have found it difficult to cultivate C. jejuni onto dry litter, suggesting that it will not grow in that environment. This discovery further shows that our attempts to cultivate bacteria are not indicative of their behavior in “the wild”. There may be nutrient gradients or a symbiont in play that allows C. jejuni to grow; therefore the possible contamination of dry litter has to be acknowledged in that facility’s Campylobacter monitoring program.

The last point of interest I’ll discuss here is the large amount of unique species that were found in samples following slaughter. This suggests that these species did not come from the farm, but rather were introduced during slaughter and processing. Interestingly, among Campylobacter spp., there was little to no abundance of C. jejuni in the samples, but differing amounts of other Campylobacter spp. This is revealing, as we have been predisposed to expect C. jejuni to be present due to our use of selective media.

Let’s fully appreciate the amount of diversity found within the processing facility, the authors collected two post-processing samples labeled carcass rinse and carcass weep. The rinse was composed of fluid shaken off of the carcass following its removal from the chlorinated chill tanks, and the weep was the drippings from the same carcass 48 hours later. 2/3 of the unique species found the weep samples were not found in the rinse. The authors interpret this as being due to the fact that the sterilization of carcasses is not the goal of poultry processing, and provide the example that viable Salmonella can be recovered from carcasses even after they are sent through the standard antimicrobial processes. The goal is to reduce enumeration, not sterilization.

Finally, in examining the retail samples, we get what we expect. Similar organisms as the weep, with some new faces, presumably because they persisted through processing at undetectable levels, and slowly grew as the product was stored in refrigeration.

The authors conclude by examining some potential symbionts that would allow C. jejuni to persist, but ultimately say that due to the high number of environments C. jejuni can occupy, attempting to exclude it in a universal way will not be very effective.

So all in all, a thorough example of the misdirection we receive from culture bias, and a startling look at how, given enough incubation time, properly processed meat can still support a huge amount of microbial diversity, including many food borne pathogens.

Appreciate this diversity, and make sure you cook your chicken to temperature.

 

ResearchBlogging.org

Oakley BB, Morales CA, Line J, Berrang ME, Meinersmann RJ, Tillman GE, Wise MG, Siragusa GR, Hiett KL, & Seal BS (2013). The Poultry-Associated Microbiome: Network Analysis and Farm-to-Fork Characterizations. PloS one, 8 (2) PMID: 23468931

Why rooster crowing isn’t that impressive, and chickens get jet lag like the rest of us.

Circadian rhythms and jet lag.  There, cyclic crowing behavior explained.

Quite a lot of people are discussing this study from Japan examining the effect of light on the crowing behavior of roosters. The authors observed several birds in experimental conditions where light intensity and duration were controlled, taking observations with audio recorders and cameras. The scenarios presented were a daylight cycle of 12 hours of light and dim light respectively, and constant dim light. Observations were recorded for a period of 14 days, producing this graph.

So many reporters on the study have run with this, making declarations about what great timekeepers roosters are, and how cool it is that they don’t need the sun to know when dawn is.

Well, approximately when dawn is.

“Under dimLL conditions, a free-running rhythm of crowing was observed with a period of 23.7 ± 0.1 h (n = 4), but this free-running rhythmicity gradually damped out”

Interesting, so the sun is unnecessary until it’s been gone for a while, then we start to get some variation. This dampening effect is even more obvious when you place testosterone implants in the roosters.

Testosterone implant roosters calling out “Bro, do you even lift?”

Don’t get me wrong, the fact that Roosters have this accurate of a circadian clock is impressive! It’s very interesting biologically, but it’s not some infallible atomic clock. While many news sites are toting that Roosters are independent of the sun, the opposite is true. Circadian rhythms are directly calibrated primarily by light cycling, with temperature being another important environmental cue. To confirm the roosters knew what time it was, the authors examined the effect of light or recorded crowing sounds at different times of day. They found that there were fewer crowing behaviors at random dawn times than at the “correct time” of day.

This doesn’t mean the roosters know it’s 5pm, but their circadian rhythm is telling them that it isn’t dawn. However, the sun still “came up” so we witness some halfhearted crowing. Anyone who has ever traveled out of their timezone knows exactly how this feels: these roosters have jet lag. While the sun may be coming up, their circadian clocks are telling them that it feels like a different time of day, so they crow in response to the light, but with reluctance and confusion, much in the same way you sleepily get up on vacation when the Louvre opens, even though it feels like 5PM to you.

“But Austin,” you tell me, “aren’t you anthropomorphizing?” While I admit roosters may not empathize with trans-Atlantic vacations, we know that chickens are dependent on daylight to calibrate their biological rhythms because we do it all the time. We increase egg production by simulating summer lighting year round, and alter feed intake in broilers by changing their daylight cycle. We also use this trick to bring mares into heat.

The loss of rhythm observed in 24 hour dim light is likely to become more and more sporadic, and even more so if the roosters were housed singly (as there is some group consensus due to competitive crowing). I would propose that if you could keep the roosters on a light cycle that progressively moved forward an hour a day until dawn was at 2pm, the roosters would crow with the same strict rhythm independent of the actual sun. If the authors of the study choose to pursue this hypothesis, an easy test would be to simply progress their artificial sun’s rise and fall over time.

Alternatively, we could fly several roosters with us to Paris, and see if they wake us up before the Louvre opens.

ResearchBlogging.org

Shimmura, T., & Yoshimura, T. (2013). Circadian clock determines the timing of rooster crowing Current Biology, 23 (6) DOI: 10.1016/j.cub.2013.02.015

“Organizational silos,” and how they prevent effective zoonotic disease tracking

It appears that the agencies that we rely on to track disease outbreaks need to start tracking disease, not just their own jurisdiction.

An article in Sociology of Health and Illness piqued my interest this last week that reveals the amount of segregation different government agencies have when dealing with zoonotic disease. The understanding of the goals and connections between livestock, wildlife, and human health among these agencies are often apathetic at best, and antagonistic at worst.

The author of the article took it upon himself to interview several government agencies with different species and regional jurisdictions, and was able to reveal what he calls “organizational silos” that develop when the values and cultures of these different agencies prevent them from working with outside groups. When attempting to monitor emerging infectious disease (EID), identification of cross-species movement is critical to predicting and preventing pandemics. Unfortunately, while they may be able to acknowledge the geographical movement of EID’s, many organizations are blinded by their specific oversight of humans or animals.

Copied from the article: Diagram showing the crossover between domestic animals, wildlife, and human EID. Important emergence factors for each circle are listed on the outside.

There are many telling comments contained in his interviews, and I encourage you to read the article to get the whole scope of the problem, but I’ve chosen to list a few of my favorites here:

From the Director of Animal Health Division at a state Department of Agriculture:

“‘We got a positive [flu result] on one of our routine surveillance tests’ of a poultry farm, Spencer complained, and ‘we were required to contact the USDA right away because of the pandemic Asian strain’. Spencer added, ‘It seems a little silly because there was no clinical illness on the property, and the strain came back something pretty common…’ In Spencer’s eyes, it was ‘hard to justify’ reporting the flu strain to the USDA… These days, Spencer said he passes on information about disease events to the state DOH and leaves it to them to tell local health boards. ‘If somebody screws up’, he shrugged, ‘at least we can blame the [Department of Health]’.”

Not an uncommon perspective for many organizations, or even coworkers! Let’s hear from another director at the USDA Animal and Plant Health Inspection Service (APHIS):

 “Clinton argued that the ‘single biggest threat for disease’ comes from ‘wildlife intermingling with domestic livestock’. He told me, ‘You can’t control the birds’ and he rightly pointed out that ducks are flu incubators. If the bird flu – which Clinton called the top priority of his agency – becomes pandemic in humans, he told me, it will come from waterfowl.”

Interesting, I might argue that we have much more interaction with domestic fowl (can’t remember the last time I handled a wild duck), but let’s see what others had to say about this viewpoint.

“Nina Marano, a zoonotic disease expert at the CDC, told me that ‘most of the outbreaks have occurred through interaction with domestic poultry’. Another example: though poultry farmers singled out wild birds called cattle egrets as the source of a 2004 flu outbreak in California, the egrets tested negative – it turned out that contaminated egg containers circulating between farms were the culprit (McNeil 2004).”

Finally, one last example of how a zoonotic disease often isn’t treated as such by human health agencies. From a Director of the Infectious Disease Bureau of a city Public Health Commission:

“When I asked Sanders to describe a zoonose that she responded to, she mentioned a recent outbreak of salmonella…and she believed that the pathogen came from two live poultry markets in Chinatown. What I found telling was that, in Sanders’ lengthy discussion of this outbreak, she did not mention any communication with veterinary medicine agencies.While the Disease Bureau’s response to salmonella followed protocol, it did not turn to the Department of Agriculture, the USDA, or any other agencies involved in animal health for help or information. Nor did it share information with them.”

Clearly here the city health board considered this a food safety issue, but payed no attention to the implications of getting meat from an approved source (a domain which definitely belongs to the USDA), or the fact that other agriculture agencies may be interested in a salmonella outbreak. There are many other telling quotes within these interviews, and I again encourage you to check out the article.

The author of the study concludes that the only examples we get of harmonious collaboration are for those diseases which are in the public eye such as rabies and influenza (H5N1 and H1N1), though we still have lines drawn even when the public is asking for action (“‘we have enough H1N1 to worry about without worrying about turkeys’. He
concluded that turkey infection is ‘a Department of Agriculture issue’”). The most shining example of the failure to communicate by these institutions in the article is the discovery of Bird Flu in the US.

The first human cases of H5N1 in the US were wrongly diagnosed with St. Louis encephalitis, resulting in the deaths of 3 patients. A veterinary pathologist at the Bronx zoo observed neurological symptoms in some of the zoo’s birds and suspected a link, however encephalitis would not have killed her birds. Both the CDC and local DOH would not accept new information from her, instead keeping the encephalitis diagnosis. She then sent specimens to a friend at an Army Medical Research Institute of Infectious Diseases, who revealed the etiology of the disease and I’m sure had a hilarious conversation with the CDC and DOH (could you please explain to us why this veterinarian is doing your job casually on the side, and doing it better?). By the time the CDC received/accepted this information, H5N1 was endemic in the area.

Nothing against the CDC, it’s a fantastic organization, but this highlights the closed lines of communication that exist between human and animal agencies the author discusses. In order to prevent the next EID crisis, rigorous epidemiology is critical. Refusing to acknowledge the importance of cross-species movement to the virulence and emergence of a disease that falls under your agency does not only prevent you from identifying the next source of infection, but leaves you with nothing but reactive measures catered to a epidemic that you refuse to fully appreciate.

ResearchBlogging.org

Jerolmack, C. (2012). Who’s worried about turkeys? How ‘organisational silos’ impede zoonotic disease surveillance Sociology of Health & Illness DOI: 10.1111/j.1467-9566.2012.01501.x

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

Adopter preferences in selecting shelter cats, what about coat color?

I’ve recently been collecting evidence that suggests that we could increase adoption rates by incorporating more group housing into shelters. In building my study, I’m now interested in what factors make cats less desirable at shelters so that potentially we could market less desirable cats by placing them in housing that will make them more desirable. Lepper et. al (2002) examined just that, in a study with over 4000 cats they examined multiple variables in order to determine certain predictors of adoption. They also examined dogs, but I’ll just be discussing the cats today. Check out the article for yourself if you are interested in the dog analysis.

It has been shown that there is a clear correlation with fearful behaviors and euthanasia (Gourkow and Fraser, 2006), therefore we should be doing everything we can to reduce fearful behaviors. I’ve suggested that when we are forced to make euthanasia decisions based on space,  we need to take temperament into account to evaluate the potential for adoption.

Among other variables however, this study illuminates factors outside the control of behavior modification that may

Gidget is available for adoption at Heartland Humane Society (Corvallis)

influence adopt-ability. Comparing coat color, the authors chose to set tabby cats as the standard, and were able to determine that white, color point, and grey cats were the most likely to be adopted. Brown and Black were the least likely, a fact that any former shelter employee wouldn’t be surprised to hear.

Cats’ whose color or features could be attributed to a specific breed (such as Persians) were in high demand. This study found that coat length did not have an effect on adoption rates, but other researchers have found that more than half of adopters list coat length as an important factor in their selection. Interestingly, Siamese cats  had no higher rate of adoption than other cats in the study.

To me, this information says that we could easily be selecting animals that are potentially less desirable, and placing them in the front or lobby areas of shelters in group settings, and keeping kittens and more desirable animals in less prominent housing. This simple management change could potentially increase adoption rates overall, and of course reduce euthanasia rates, the goal of any shelter.

 

ResearchBlogging.org

Merry Lepper, Philip H. Kass, & Lynette A. Hart (2002). Prediction of Adoption Versus Euthanasia Among Dogs and Cats in a California Animal Shelter Journal of Applied Animal Welfare Science, 5, 29-42 DOI: 10.1207/S15327604JAWS0501_3

 

Factors affecting adoption rates in shelter cats, is welfare the immediate concern? What about marketing?

"Kipling" currently up for adoption at Heartland Humance Society (Corvallis)

As I continue my research trend concerning adoption rates in shelter cats, I came across this thesis by Nadine Gourkow, who is much more famous than I ever realized while reading the article. A brief google search of her name reveals that she is at the forefront of shelter cat welfare, and clearly I need to read more of her work as I continue to shape my study.

Her masters thesis explores the background for my intended study, in that it seeks to examine not only those factors inherent to the cats and their housing situation, but in what potential adopters are looking for. I’m hoping to collect as much of this information as possible to develop a hypothesis as to whether shelter layout could increase adoption rates by marketing less attractive cats in situations where they may be perceived as more adoptable.

I’ll admit firsthand that I didn’t read all 80 pages of the thesis, I was primarily interested in the methods, results, and discussion, as such I may have missed some of the finer points of Ms. Gourkow’s introduction. My personal interest was in the conclusions she drew based on the study design and how they could impact my own study design.

Four treatments were used in the study, which varied by complexity of cages (from a single animal in a barren cage to multiple cats in an enriched enclosure) and frequency/consistency of handling by shelter staff. I was primarily interested in the difference between singly housed cats compared with multiple cat housing.

I was pleased to see that this study only contained adult cats, as I’ve had concerns about the skewed adoption rate of kittens found more often in group housing in other studies. It was unfortunate that the handling effects could not be separated from the housing treatment in the analysis, however, it seems clear that either the consistent handling or cage enrichment had a large effect.

Where the cats in the single, barren, cage (standard) treatment experienced a 45% adoption rate, all of the other treatments had 74% or more adopted.

Did you read that? By adding a perch and hiding area (along with consistent handling) to a cat housed alone, they increased the number of cats adopted by 29%.

That’s an impressive change, however, I am more concerned with single vs. group housing, and thus was even more interested in what adopters listed as important criteria in their selection of a cat. The results clearly suggest that cats housed in groups have an advantage.

When it comes down to what adopters want to see in a cat, we can’t necessarily alter desirable traits such as “friendliness towards adopter,” “playfulness,” or “happy disposition.” But we can suppress other desirable traits by not providing opportunities for animals to exhibit them. The factors that I want to focus on are those that are enhanced or only provided through the use of group housing.

Being able to enter the cage with cats (74% of respondents)

Friendly with other cats (69%)

Able to view with other cats (52%)

Alternatively, while it may be essential for the cats’ mental well being, seeing perches and toys in cages was not very influential for adopters (38% or lower). These items may still play a large role in adoption rates due to other factors such as playfulness (86%) and reduction of fear behaviors (which dramatically increase euthanasia rates), but from a marketing and management standpoint, the easy change would be to house more cats in groups.

Arguments against housing cats in groups are primarily based on disease management. I recently read that roughly half of the cats in shelter environments become infected with contagious upper respiratory disease during their stay, and my own anecdotal experience supports that percentage. The article here, however, showed no difference in animals quarantined or euthanized due to illness between single and group treatments.

Another argument suggests that strange cats experience more stress when placed together, but Kessler and Turner along with Ms. Gourkow both reveal that space per cat plays a larger role in mitigating that stress, and that after a certain period stress levels become similar across all groups.

The picture that I’m beginning to piece together from these papers is that cats that are housed in groups are more desirable and consistently enjoy higher adoption rates. To use this information, I would hope that shelters would incorporate more group housing, and select animals for limited space based on adoptable attributes. I’ll continue exploring group housing’s influence on adoption rates, but now I’m interested in finding other traits that could make animals less desirable so that we can make placement decisions based on an animals marketable traits. The goal of this line of thinking being: if we can increase adoption rates for our less adoptable animals, can we reduce euthanasia rates overall?

Edit: I found the published version of the study featured in this thesis, and added the citation below.

ResearchBlogging.org

Nadine Gourkow (2001). FACTORS AFFECTING THE WELFARE AND ADOPTION RATE OF CATS IN AN ANIMAL SHELTER University of British Columbia

N Gourkow, & D Fraser (2006). The effect of housing and handling practices on the welfare, behavior and selection of domestic cats (Felis sylvestris catus) by adopters in an animal shelter Animal Welfare, 15, 371-377

Do cats in shelters acclimate faster if given a bunkmate?

Lucy is currently up for adoption at Heartland Humane Society (Corvallis) and is housed singly.

This article is one of many that I’m currently reviewing to build the introduction for the original research I plan to complete this summer/fall. This is the first of several posts discussing shelter cats to come in the next several weeks.

This study by Kessler and Turner (1997) took a look at the stress levels of cats introduced to a shelter/boarding facility-type environment over the first two weeks of their stay, and cross-examined those housed alone, in pairs, and in groups. 45 homeless animals that had already been at the facilities for some time were selected as a control, and 140 animals staying for temporary boarding were observed for the first two weeks of their stay.

Overall, the authors were able to conclude that in a two week stay, two-thirds of the cats acclimated very well, and after two weeks their stress levels, while still higher, were very comparable to the control. They suggest that other options be explored by the owners of the other third, with a special emphasis on the 4% of the cats who were extremely stressed even after a two week stay.

I endorse this wholeheartedly as I often watched animals for owners as a job when I was much younger. While they may be given more brief human contact, working out a deal with a house-sitter or neighbor to take care of your pets while you are away can be much less stressful for them than if they are placed in an unfamiliar environment with strange people and animals. You reduce their risk of exposure to disease, and help some young lad save money for college (I did…though some of it bought movie popcorn).

The more surprising conclusion was that housing the cats singly, pairs, or groups appeared to have no influence on the stress levels of the animals. There appears to be a slightly faster decline in stress for group housed cats on the authors’ graph, however it isn’t addressed, and the difference is minimal.

The conclusions have merit, but I have several problems with the selection of control animals in this study, namely, the fact that they aren’t representative of the experimental group. The biggest problem is that only homeless shelter cats were used for the control, and only boarding cats for the experimental group, where  either all homeless shelter cats or all boarding cats should have been included. The second large flaw I see is that all of the control animals were housed in groups of 6 to eight, effectively ruining any comparisons you may want to make when looking at the other housing situations. In a study named Stress and Adaptation of Cats (Felis silvestris catus) Housed Singly, in Pairs, and in Groups, you would think the control would use all of those situations.

The authors briefly mentioned the stress caused by cats that may be less social, or housed with familiar animals versus strangers, but were unable to control those factors with the way the data was collected and prepared. When it comes down to it, the control group just wasn’t…controlled. They were unable to fully examine how quickly single or pair cats acclimated to the boarding facility because you couldn’t compare them to a control cat in the same situation.

Kessler and Turner have another study (1999) examining stress levels of shelter cats in terms of animal density and cage size. Interestingly enough, they found that group density was “highly correlated with the stress level of animals housed in groups”, indicating that we should have seen some differences from the study above as well. That research was done two years after the 1997 study, so perhaps the authors also thought that those questions remained unanswered from the original study. I’ll be looking out for some newer research on the subject and may chime in on it again soon.

 

ResearchBlogging.org

M R Kessler, & D C Turner (1997). Stress and Adaptation of Cats (Felis Silvestris Catus) Housed Singly, in Pairs and in Groups in Boarding Catteries Animal Welfare, 6, 243-254

M R Kessler, & D C Turner (1999). Effects of Density and Cage Size on Stress in Domestic Cats (Felis Silvestris Catus) Housed in Animal Shelters and Boarding Catteries Animal Welfare, 8, 259-267

 

If you’re interested in the cat featured in this post, head on over to Heatland Humane Society to meet him!

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: Banned antibiotics found in feather meal at Johns Hopkins – How did they get there, and why does your press release contain different conclusions?

An article recently published in Environmental Science and Technology details a study conducted at Johns Hopkins where when examining samples of commercially available feather meal (used as a protein supplement feed or fertilizer) they found trace amounts of fluoroquinolones, a class of antibiotics that have been banned for use in animal feed for 6 years. This is an interesting find, and definitely warrants further research before any broad sweeping conclusions can be made. You know…unless you publish a press release condemning the entire industry for breaking the law and trying to kill us all (question, if all the chicken consumers are dead, how does that lead to higher profits?).

The article itself is very well done. The authors collected samples of feather meal from several states and Canada and tested them for various pharmaceuticals. They also autoclaved the samples to see how the heating processes involved in creation of the product affected degradation/digestion of any of the compounds, and exposed E. Coli cultures to the meal to see if the presence of one or more of the compounds was enough to select for antibiotic resistant populations (they were).

The researchers examined each of the compounds of interest and proposed mechanisms for their presence. Some of them are used at various levels legally within the industry, and the presence of many others (such as caffeine) can be explained by their introduction through various feedstuffs (such as coffee pulp and green tea powder) (Love et al., 2012).

Obviously of most interest to the researchers were the levels of fluoroquinolones, and they hesitantly proposed possible mechanisms for their introduction into feather meal.

“Fluoroquinolones (enrofloxacin, norofloxacin, or ofloxacin) were detected in 6 of 10 U.S. samples, which was not expected because fluoroquinolone use in U.S. poultry production has been banned since 2005. These findings may suggest that the ban is not being adequately enforced or that other pathways, for example, through use of commodity feed products from livestock industries not covered by the ban, may inadvertently contaminate poultry feed with fluoroquinolones…To better interpret our findings, corroborating evidence in the form of antimicrobial usage practices and dosing amounts would be needed.” (Love et al., 2012)

Clearly we’re not ruling out the possibility of these antibiotics being fed, but there is no cause and effect relationship here. They also make note that feather’s contain antibiotics in higher concentrations than meat or other tissues, even after legally defined withdrawal times to remove them from edible tissue. The conclusions here are justly cautious, and place no blame or accusations upon the industry.

The other interesting find was that the feather meal tested would select for antibiotic resistant strains of E. Coli when exposed to cultures. However, this was only testable with autoclaved samples of the meal. And did we mention that the only samples tested here were sourced from China (who according to the article use many more antibiotics than we do in poultry production)?

“These initial results suggest, but cannot prove, that the inhibiting substance may be an antibiotic/bacteriostatic. Autoclaving may have attenuated the quantity and bioavailability of antimicrobial drugs originally present.” (Love et al., 2012)

Again, a cautious observation and hardly conclusive. Comparisons were made from standard cultures exposed to low levels of relevant antibiotics to see if the same strains were removed, but this data cannot be correctly compared as the feather meal was not controlled enough to isolate those compounds. The authors finish their discussion with an appreciation for the novel information they found and an invitation for others to verify, replicate, and build upon their results.

“We have previously described risks related to administration of medicated feed to food animals, which may promote selection for antimicrobial resistance. The presence of antimicrobials in feather meal, as determined in this study, is a previously unrecognized source of these drugs in animal feed. Because this is the first study of PPCPs in feather meal, we invite independent verification of our results by others. More work is needed to determine whether the detected levels of PPCPs in feather meal have an impact on the quality of food animal products and the safety of consumers.” (Love et al., 2012)

And there we have it, an exciting new study that presents a lot of questions to be used for follow-up research. So we’ll publish it and make sure to put out a press release so that not only those keeping up with the journals can read and understand what we’re currently researching.

Well…it seems like the authors like to be scientific when submitting journal articles, but prefer big headlines and sensationalizing when trying to popularize their research. In their official press release, these researchers quickly turn from cautious scientists to industry whistleblowers.

“The discovery of certain antibiotics in feather meal strongly suggests the continued use of these drugs, despite the ban put in place in 2005 by the FDA…The public health community has long been frustrated with the unwillingness of FDA to effectively address what antibiotics are fed to food animals.” – David Love, PhD

Comments from Keeve Nachman, PhD, show a level of conviction that I had no idea he possessed in the original article.

“In recent years, we’ve seen the rate of fluoroquinolone resistance slow, but not drop…With such a ban, you would expect a decline in resistance to these drugs. The continued use of fluoroquinolones and unintended antibiotic contamination of poultry feed may help explain why high rates of fluoroquinolone-resistant Campylobacter continue to be found on commercial poultry meat products over half a decade after the ban.” (I guess we know for sure they’re still being used in the US, I must have missed that citation in your introduction)

“A high enough concentration was found in one of the samples to select for bacteria that are resistant to drugs important to treat infections in humans” (Note: the concentration comment is true, however, your study stated that there was not controlled enough testing to prove that any specific compound present caused the bacterial selection)

“We strongly believe that the FDA should monitor what drugs are going into animal feed…Based on what we’ve learned, I’m concerned that the new FDA guidance documents, which call for voluntary action from industry, will be ineffectual. By looking into feather meal, and uncovering a drug banned nearly 6 years ago, we have very little confidence that the food animal production industry can be left to regulate itself.”

Wow, this press release from the actual authors of the article must be legit, after all, they wrote it, and they wouldn’t write/act completely differently so as to both dangle a carrot to the media and still get their research published in a peer-reviewed journal right?

I knew the Colonel was lying to me. Source: kfc.com

Well, the press release did its job, and Nachman can be found interviewing left and right about how he was “floored” by the result, and how that the more he learns, “the more [he is] drawn to Organic”. There’s a severe lack of integrity here, and the misrepresentation of your data and analysis to cater to anti-big-Ag sentiments is irresponsible and unethical.

So, future scientific corespondents for the Daily Mail aside, what are some other proposed mechanisms for the presence of these banned antibiotics? After all, I’m suggesting that they aren’t being fed routinely as the authors apparently really believe. Let me rephrase that, I’m not ignoring multiple unproven mechanisms for the purpose of shock science.

I spoke with James Hermes PhD, a professor and Poultry Science Extension Specialist here at Oregon State University, about the article. He had obviously heard of it and shared with me some discussions he had had with his colleagues. Their proposed mechanism for the introduction of the pharmaceuticals was through groundwater.

“Feather meal is boiled at the rendering plant, it’s processed with a lot of water, so anything in the groundwater can end up in the meal…Just recently near [Corvallis] they did some testing and found nearly everything we use at home. For years they told us to flush our excess medications…It’s always been there, we’ve just only recently been able to look at such small concentrations 1 parts per billion, trillion, or even possibly quadrillion.” – James Hermes, PhD

So in addition to whatever chickens could be exposed to via drinking the water and concentrating pollutants in their feathers, additional water and pollutants are introduced during the rendering of the product. He encouraged me to find some research showing that ground water contains any and all of the things discovered in the feather meal, and I found it.

So is this a possible mechanism for the introduction of these contaminants? I suggest the authors of the study follow their own advice in the article and explore this mechanism. Perhaps see if the levels of the pharmaceuticals change in the feathers both prior to and after rendering/boiling, explore if they are present in organically produced feather meal, and find out if there are still large concentrations of fluoroquinolones in the groundwater of the areas processing feather meal. And hey, if they want to keep on trucking with scare tactics, I think finding this stuff in the water supply will be much more frightening to the public given that we can’t buy organic water (don’t start).

Finally, in evaluating consumer exposure to the present antibiotics in feather meal, we should keep in mind the steps necessary in the shortest route to the consumer. First, there have to be high concentrations of antibiotics in feather meal; second, that feather meal must be fed to an animal used for food, third; those antibiotics must be retained and remain active until slaughter of that animal at a high enough concentration; fourth, they have to survive gastric juices and be absorbed into the small intestine of the person eating the meat/milk. Nevermind that along the way, the authors of the relevant study note that at any point the vector for the antibiotics is heated most of them will degrade.

Obviously, if the feather meal is used as fertilizer instead of a by product feed, then there are a few more steps that need to take place to get those pollutants onto the plate.

Now if you’ll excuse me, I need to finish this compelling article on how those fat cats selling nautral almond extract are trying to murder me .

 

ResearchBlogging.org
D.C. Love, R.U. Halden, M.F. Davis, & K.E. Nachman (2012). Feather Meal: A Previously Unrecognized Route for Reentry into the Food Supply of Multiple Pharmaceuticals and Personal Care Products (PPCPs) Environmental Science and Technology, 46, 3795-3802

Kolpin, D., Furlong, E., Meyer, M., Thurman, E., Zaugg, S., Barber, L., & Buxton, H. (2002). Pharmaceuticals, Hormones, and Other Organic Wastewater Contaminants in U.S. Streams, 1999−2000: A National Reconnaissance Environmental Science & Technology, 36 (6), 1202-1211 DOI: 10.1021/es011055j

 

 

Update: following this post I had a chance to talk with one of the Authors of the article, read about our discussion here.