The gut microbiome correlates with conspecific aggression in a small population of rescued dogs (Canis familiaris)

Aggression is a serious behavioral disorder in domestic dogs that endangers both dogs and humans. The underlying causes of canine aggression are poorly resolved and require illumination to ensure effective therapy. Recent research links the compositional diversity of the gut microbiome to behavioral and psychological regulation in other mammals, such as mice and humans. Given these observations, we hypothesized that the composition of the canine gut microbiome could associate with aggression. We analyzed fecal microbiome samples collected from a small population of pit bull type dogs seized from a dogfighting organization. This population included 21 dogs that displayed conspecific aggressive behaviors and 10 that did not. Beta-diversity analyses support an association between gut microbiome structure and dog aggression. Additionally, we used a phylogenetic approach to resolve specific clades of gut bacteria that stratify aggressive and non-aggressive dogs, including clades within Lactobacillus, Dorea, Blautia, Turicibacter, and Bacteroides. Several of these taxa have been implicated in modulating mammalian behavior as well as gastrointestinal disease states. Although sample size limits this study, our findings indicate that gut microorganisms are linked to dog aggression and point to an aggression-associated physiological state that interacts with the gut microbiome. These results also indicate that the gut microbiome may be useful for diagnosing aggressive behaviors prior to their manifestation and potentially discerning cryptic etiologies of aggression.

leading to discomfort or pain that could also contribute to irritability or aggression. 103 Here, we conducted an exploratory analysis of fecal samples originating from a small shelter-104 housed population of pit bull type dogs seized from organized dogfighting to determine if canine 105 aggression could be predicted based on the composition of the gut microbiome.

Materials and Methods
108 Sample Collection 109 A single fecal sample was collected from the kennel of each of thirty-one pit bull type 110 dogs residing at a temporary shelter while in protective custody. This population served as the 111 focus of this pilot study because it enabled control over as many factors as possible, including 112 breed type, environment, diet, and medical care, while providing access to a population with a 113 relatively more frequent aggressive phenotype compared to typical populations. Upon intake into 114 the shelter and prior to the initiation of this study, an animal welfare agency catalogued various 115 parameters of each individual, which were used in this study's analysis as covariate data (Table   116 S1). Animal welfare employees collected feces using aseptic technique within an hour of 117 defecation and immediately froze them at -18C to -20°C to fix bacterial growth and preserve the 118 DNA content. Fecal samples were shipped to Oregon State University and stored at -20°C. 119 Thirty of the dogs were on a diet of Iams Proactive Health minichunks adult kibble (chicken-120 based formula) and one dog was on a diet of Iams Puppy. Fourteen males and seventeen females 121 were sampled from. Each dog received a behavior evaluation conducted by the animal welfare 122 agency shortly after intake that categorized these dogs as intraspecifically aggressive (n = 21) or 123 non-aggressive (n = 10) based on exhibited aggression towards unfamiliar dogs. Data from these 124 evaluations were sent to Oregon State University along with the stool samples for analysis. With 125 the exception of the collection and processing of fecal material, this study did not involve any 126 manipulation of, measurement of, or contact with dogs that had not already occurred.
No animal subjects, animal handling or study specific animal interactions were required 130 for the purpose of this study. Dog fecal samples were collected from shelter kennels after natural 131 deposit. Previously collected behavioral data from the animal welfare agency's records were 132 used in analysis. Therefore, this study was determined to be exempt from institutional animal 133 care and use review by Oregon State University's ethical review board.   The QIIME (v1.8.0) bioinformatics pipeline was used to quality control raw sequences as 147 well as quantify the diversity of microorganisms isolated from the fecal samples. The Illumina-153 core_diversity_analysis.py script, samples were subject to rarefaction through random sub-154 sampling of sequences at a depth of 40,000 reads, which corresponded to the lowest sequencing 155 depth obtained across samples. The BIOM table generated from the core_diversity_analysis.py 156 script was imported into R and potentially spurious OTUs were filtered by removing those that 157 (1) were found in fewer than three samples and (2) were observed fewer than 20 times across all 158 samples from all subsequent analyses. The resulting OTU matrix was subsequently processed 159 using the beta_diversity.py script to calculate the weighted and unweighted UniFrac distances 160 between all pairs of samples (Lozupone & Knight, 2005). Alpha diversity was calculated in R 161 (v3.2.3) using the diversity function in the vegan package (v2.3-3).

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The bacterial phylotypes that were observed across the dog fecal samples were compared 190 between behavioral groups to resolve those phylotypes that vary in association with aggression 191 (Fig. 2). Firmicutes, Fusobacteria, Bacteroidetes, and Proteobacteria were the dominant phyla in 192 all fecal samples, which is consistent with dominant bacterial phyla discovered in previous  Of the 578 clades that are prevalent in either aggressive or non-aggressive dogs, 96 219 significantly differ in abundance between the two populations (q < 0.2). Of these clades, 39 have 220 a mean relative abundance that is significantly higher in the gut microbiomes of aggressive dogs, 221 while 57 have a higher relative abundance in non-aggressive dog microbiomes. A complete list 222 of clades that associate with behavior can be found in Table S4. Of particular note is our finding 223 that nine clades with the genus Bacteroides are elevated in the gut microbiomes of non-224 aggressive dogs compared to aggressive dogs. This finding indicates that the relative abundance 225 of these lineages within Bacteroides may predict aggression status and that their depletion may 226 contribute to aggression. We also find that the genus Lactobacillus contains 25 clades that are    Our investigation finds that the composition of the gut microbiome differs between 254 aggressive and non-aggressive dogs in the population that we studied. The rescued, shelter-255 housed dogs included in this investigation proved useful for this study because they included 256 aggressive and non-aggressive individuals and were taken into the shelter at the same time, 257 maintained in the same facility, exposed to the same diet, and generally of consistent breed type. 258 Despite our attempt to homogenize the sources of variation amongst these dogs, we observed 259 extensive variation in the composition of the gut microbiome within each behavioral cohort. This 260 intra-cohort variation indicates that the stool samples we studied are subject to cryptic factors 261 that associate with microbiome composition (e.g., age of host (Conley et al., 2016a)). This is 262 unsurprising given that individuals living outside of a laboratory setting (including pet and 263 shelter dogs, as well as humans) are subject to genetic and environmental diversity that cannot 264 fully be controlled for. That said, the identification of significant differences between these 265 populations under naturalistic conditions heightens the applied value of these findings. Future 267 clarify the relationship between microbiome composition and the gut microbiome.

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Several taxa significantly differ in their relative abundance between aggressive and 269 nonaggressive dogs. For example, we find that that lineages within the genus Bacteroides are 270 elevated in non-aggressive dogs, which might be expected given that species within this genus,  The abundance of monophyletic clades within phylotypes stratify aggressive and nonaggressive dogs.
(A) Illustrating a subtree within the Bacteroides phylotype containing node 874 (red branches), which is a monophyletic clade that is both common to and relatively more abundant amongst the non-aggressive individuals than the aggressive individuals. The heat map adjacent to this subtree illustrates the log10 relative abundance of each lineage in this subtree across the individuals subject to our investigation. The red rectangle highlights the relative abundance of the lineages within node 874. The vertical blue line separates aggressive and non-aggressive individuals. (B) illustrating a similar subtree, but in this case, it has been extracted from within the Lactobacillus phylotypes and highlights a monophyletic clade (node 3489) that is common to and relatively more abundant amongst the aggressive dogs.