The Importance of Next-Generation Sequencing in Avian Veterinary Medicine

Introduction

Effective treatment of diseases requires accurate diagnoses. Infectious diseases in avian medicine present special diagnostic challenges. Globalization, climate change, and the rising popularity in avian pets, especially psittacines, has increased the emergence and spread of novel pathogens that are of particular concern to veterinarians. Moreover, for every antibiotic that has been developed to treat psittacine birds, pathogens have been isolated that are resistant to treatment.¹⁵ Culture and susceptibility remain the standard diagnostic tools for detection, diagnosis, and treatment of avian pathogens and their antibiotic resistance. However, this approach is centuries-old and new technologies have been developed recently that provide a more complete picture of what may be causing infections.

Based on novel molecular-based DNA technologies, we now know more than 10²⁰ microbes from three different domains of life inhabit this world together with us. These microbes affect metabolism, health, and can cause infectious diseases. Only about 1% of these microbes are culturable in the lab.³ The other 99% go unrecognized by standard culture methods and are often missed upon analysis of infectious disease cases. This applies especially for anaerobic bacteria, or fungi, which can take weeks to grow, or those organisms that currently cannot be cultured in the lab for various reasons. The results are recurring and chronic infections of psittacine birds with unidentified etiology.

Molecular diagnostic tools, such as DNA sequencing, including whole genome sequencing, are available to aid the urgent medical need to detect and identify all culprits that cause infections.

What is next-generation sequencing?

Next-generation DNA sequencing (NGS) offers the ability to diagnose infections using microbial DNA as the analyte, thus bypassing culture testing along with its deficiencies. The “no-growth” result often associated with culturing is no longer an issue, as no clinical sample is sterile; even urine from healthy pets typically harbors bacteria and fungi.4,8

For NGS diagnostics, microbial, genomic DNA or RNA (converted to cDNA) is extracted from clinical samples, such as urine, feces, blood, or skin/ear/nose/throat swabs, then purified and tagged. The resulting DNA molecules are copied thousands of times and massively sequenced in parallel. Thus, millions of DNA sequence reads from all microbes in a sample are able to be examined at once. These DNA reads are then trimmed, host DNA is filtered out using special software, overlapping reads are assembled into long sequences and the genes are compared to genomic databases to assign the microbial species identity and associated pathogenicity and predict antimicrobial activity based on resistance genes. This process provides a complete picture of which microbes are present and allows the quantification of each microbial species at the time and sight of sample collection from the patient. Next-generation sequencing-based diagnostic tools are increasingly used in veterinary practices to further improve infection diagnostics, while also aiding good antibiotic stewardship by selecting the most useful antibiotics. These types of tests are offered by specialty labs, like the MiDOG labs pet microbiome test (Irvine, CA) for example.

Applications to avian medicine

A case study of a 29-year-old female Congo African grey parrot (Psittacus erithacus) with soft, foul-smelling stool highlights how NGS can show a more comprehensive picture of infection than culturing.¹⁴ In this case, culture identified one Enterococcus sp., while the pet microbiome NGS test showed two species:  Enterococcus faecium and an Anaerosporobacter species, specifically Clostridium baratii. Metronidazole therapy cleared the Enterococcus infection, but clinical signs correlated to the presence and numbers of the Anaerosporobacter species persisted. This species, however, was never detected in repeated cultures as it is an anaerobic organism with special growth requirements. This report demonstrates the first diagnosis of Anaerosporobacter sp. from the feces of an avian species using microbiome DNA sequencing as compared to traditional culture techniques.¹⁴

Another example of the applicability of NGS in psittacine diagnostics is its utility for diagnosing avian chlamydiosis, which is caused by several strains of the Gram-negative, intracellular organism Chlamydia psittaci.^1,10 Diagnosis of avian chlamydiosis is challenging, particularly in the asymptomatic bird.⁵ Culture and serology are of limited utility in diagnostic settings, as positive antibody titers do not necessarily indicate an active infection and delayed culture results may lead to fatal outcomes. Consequently, contemporary veterinary diagnostics has increasingly used polymerase chain reaction (PCR) assay testing as the gold standard for avian chlamydiosis clinical diagnostics.⁵ Next-generation sequencing can be even  more useful, particularly in subclinical presentation of avian chlamydiosis. Results from a study detecting and characterizing C. psittaci in captive birds supports the clinical applicability of using genomic sequencing to identify, analyze, and treat psittacines more effectively.⁷

Benefits of next-generation sequencing 

NGS versus PCR

Unlike targeted PCR panels, NGS is an untargeted and de-novo approach to identifying microbial pathogens, so no prior knowledge about potential infectious organisms is needed. The capacity of PCR panels is also limited to eight to 38 targets, and oftentimes veterinarians are forced to use heuristics to determine which pathogen should be targeted on a panel for diagnosis. Pet birds in particular are prone to common infections and may be more susceptible to rarer opportunistic bacteria and fungi.¹³ NGS technology has been widely adapted to study the “microbiome” or “microbial profile” in all kinds of different sample types from humans and animals (see below). The utility of this technology to not only detect all microbes present but also the ability to identify novel pathogens, as well as antibiotic resistances has warranted its application in psittacine clinical diagnostics to achieve better health outcomes for patients.

NGS versus culture

The results from research and case studies underscore the clinical applicability of using genomic sequencing to identify, analyze, and eventually treat infections more effectively. Additionally, the fast turnaround time with NGS when compared to culture-based diagnostics is particularly promising for acute infections that progress rapidly in psittacine birds.

The indications for a veterinarian to run NGS testing are the same as for culture and susceptibility. Testing should only be performed when there is clinical justification, not routinely for lifestyle monitoring. The price of NGS testing may vary between labs, however, the cost is comparable to routine cultures. In some cases, the cost may be even cheaper to the client as fungal species as well as aerobic and anaerobic bacteria may be included in one sample test. Some specialty clinics have even replaced their culture or PCR testing with NGS-based tests for up to 90% of their samples.

Diagnosis of psittacine bacterial and fungal infections can be complex, given that standard culture-based diagnostics are unable to recreate the natural systems in which microorganisms develop. Considerations when developing culture media include nutrient distribution, pH, osmotic conditions, and temperature. Standard culture temperature does not mimic avian body temperature, which is several degrees higher than in mammals. Even when multidimensional matrices are employed to allow for several environmental combinations, these attempts are resource intensive and also fail to consider biofilm.¹² Biofilms remain completely undetected by most modern culture methods. In contrast to the planktonic bacteria that is cultured, biofilm has a physical outer layer that limits access to nutrients but encourages quorum sensing and environmental plasticity.⁹ These adaptations complicate microbial diagnostics, as several studies have found either no-growth culture results or false positives from free-floating planktonic bacteria.¹¹

NGS uncovers the avian microbiome

In terms of our current understanding of the psittacine microbiome, “normal” bacterial flora includes Lactobacillus, Corynebacterium, nonhemolytic Streptococcus, Micrococcus spp., and Staphylococcus epidermidis.¹¹ Culture-based diagnostics report common Gram-negative bacteria, such as Klebsiella, Pseudomonas, Aeromonas, Enterobacter, Proteus, Citrobacter spp., Escherichia coli, and Serratia marcescens.⁵ Common Gram-positive bacterial pathogens include Staphylococcus aureus, Staphylococcus intermedius, Clostridium, Enterococcus, Streptococcus, and other Staphylococcus spp.⁵ Other pathogens such as Mycoplasma spp., Salmonella spp., and Pasteurella spp. are particularly difficult to culture, but are highly associated with various infectious diseases in psittacines.

The avian gut microbiome has been increasingly studied in the last couple of years and provides a particularly useful insight into the health status of psittacine birds.¹⁶ The core gut microbiome of psittacine birds is composed primarily of three bacterial phyla:  the Firmicutes, Proteobacteria, and Actinobacteria.¹⁷ These bacteria are integral to various host functions, such as nutrition metabolism, vitamin synthesis, and the maturation of the gut immune system.² Veterinarians have increasingly begun to use NGS for clinical diagnostics due to the quicker turn-around time and more accurate characterization of the avian microbiome. For example, one study aiming to detect and characterize M. avium subspecies using NGS techniques reaffirmed the importance of NGS approaches in avian diagnostics.⁶

Outlook

The world of microbes impacts animal health dramatically. The use of new molecular technology to determine exactly which microbial commensals and pathogens are present in a patient’s sample, and how they may be susceptible to  treatment, will undoubtedly assist in saving lives and enable a better diagnosis for guided treatment decisions.

References