Pathology Team Discovers Novel Species of Bacteria

A pathologist is not a good pathologist if they are not curious. In these cases, the bacteria were inside macrophages, which is not something we commonly see, and immediately raised suspicion that we might be dealing with an unusual organism. That prompted us to investigate further, rather than stopping at a diagnosis. Many discoveries in pathology happen this way, when a clinical case does not quite follow the textbook and someone decides to look deeper. Having residents who are willing to pursue those questions is essential for advancing the field.

—Dr. Francisco Conrado, associate professor in the Department of Comparative Pathobiology at Cummings School

A Labrador Retriever and a Rottweiler cross arrived within weeks of each other last fall to the emergency room at Henry and Lois Foster Hospital for Small Animals (FHSA) at Cummings School of Veterinary Medicine at Tufts University. Both were in acute respiratory distress and succumbed to their symptoms the next day. FHSA’s Pathology team set out to identify the bacteria present in both dogs and, in doing so, uncovered a new genomic species and the pathogenic potential of bacteria not previously considered infectious. 

Featured in the Journal of Applied Microbiology this past February, “First report of Castellaniella spp. infection in dogs and genomic evidence of a novel species” details the first two documented cases of dogs infected by the Castellaniella bacteria and characterizes the novel species found in one dog.

“These two cases were striking because of how rapidly they progressed and how severe the outcomes were,” says Conrado (he/him), co-author of the report. “Bacteria in the genus Castellaniella are not typically associated with infections in mammals, and certainly not in dogs. At this stage, we do not know whether this represents an emerging pathogen with broader relevance, possibly even zoonotic implications, or whether it is a rare presentation of an organism that will remain uncommon. The important step was documenting and publishing the finding. Once a report like this exists, other pathologists and institutions know to keep it on their radar.”

First author of the case study is Dr. Lina Crespo Bilhalva (she/her), clinical pathology resident in the Department of Comparative Pathobiology at Cummings School. Incidentally, Crespo also discovered the erythrocytic iridovirus in a Brazilian butter frog while conducting research for her Ph.D.

Standard cultures could not identify bacteria in the pleural effusion (fluid between the lungs and chest wall) sampled from both dogs. Under the microscope, Crespo observed bacteria almost exclusively inside macrophages, which—aside from a few exceptions—are not typically the primary cells responsible for phagocytosing bacteria. 

“When you're a pathologist, you always want to get the most accurate and specific diagnosis. The dogs died fast, with acute clinical signs. We then suspected something different based on the similar bacterial morphology, the fact that they were located inside macrophages, and the inconclusive culture results in both samples,” says Crespo.

Dr. Joyce Knoll (she/her), professor in the Department of Comparative Pathobiology at Cummings School and co-author of the study, says, “Lina brought this unique bacteria to everyone’s attention. We see many samples containing bacteria, but these bacteria were unusual. There are a limited number of organisms that will hang out in macrophages, but this didn’t look like something any of us recognized.” 

Crespo sent the samples to former colleagues at Purdue University for polymerase chain reaction (PCR) testing, which identified the bacteria’s genus in both cases as Castellaniella. The species could not be differentiated. 

“I had never heard of Castellaniella before. I got curious about it,” says Crespo. 

Crespo reached out to MiDOG, a company that provides next-generation sequencing to determine the species. MiDOG conducted metagenomic analysis of the samples and collaborated on the case report. This analysis identified two species of Castellaniella. Notably, the Rottweiler cross was infected with an unclassified Castellaniella species not previously described—a novel genomic species.

“I was not expecting this case to be a novel bacterial species,” says Crespo. “If you think about wildlife, there's a lot to be discovered—for example, if you collect a sample for next-generation sequencing from a random tree frog species from Panama, you might find something new. In this case, we were working with hospitalized dogs.”

Castellaniella are classified as nonpathogenic environmental bacteria, most often found in contaminated soil and groundwater, agricultural systems, and wastewater. Castellaniella thrive in these complicated environments. Their genes are quite adaptive, and many are associated with multidrug resistance.

Crespo researched cases of Castellaniella causing infection and found only three reports: a deceased porpoise and mass mortalities of Daurian pikas (a species related to rabbits) at a zoo and among broiler breeder chickens. The two dogs are the first cases of domestic mammals.

The case report challenges the classification of Castellaniella as nonpathogenic and warns that the bacteria could potentially threaten human and animal health. It sets an alert that Castellaniella can be pathogenic and currently detectable only by next-generation sequencing diagnostics.

“In addition to knowledge about a range of infectious diseases, Lina also came to us with some expertise in molecular medicine—knowledge in how to use the genetic material of an organism to help identify it. That was very important in this case, since routine methods for culturing the sample didn’t work,” says Knoll. 

Dr. Emily Brinker (she/her), assistant professor in the Department of Comparative Pathobiology at Cummings School and co-author of the case study, was the primary anatomic pathologist on one case. “These case reports help expand our current knowledge base to help other dogs. For instance, if future cases with a similar appearance reveal Castellaniella, the microbiologist will know this isn't a contaminant, but something to pay attention to and report out. Although the dogs in our cases did not survive, this hopefully gives the foundation for someone else's dog to be diagnosed before death and successfully treated.” 

Co-authors of the case report also include the FHSA Emergency Medicine and Critical Care residents who treated the patients, Drs. Sabrina Manley and Paola Mayorga Guasch, and Dr. Gisela Martinez-Romero, assistant clinical professor in the Department of Comparative Pathobiology at Cummings School, in addition to partners at Purdue and MiDOG.

“This project was truly a collaborative effort,” says Conrado. “Much of modern pathology depends on these kinds of networks. Because we work across species and organ systems, collaboration with clinicians, researchers, and external partners is essential for turning unusual clinical observations into meaningful scientific discoveries.”