Infectious coryza in a grey crowned crane (Balearica regulorum) recovered from captivity.

We report Avibacterium paragallinarum and Klebsiella pneumoniae coinfection in a grey crowned crane (Balearica regulorum). The crane was recovered from illegal captivity and released at a grey crowned crane (GCC) rehabilitation facility located at Akagera National Park in Rwanda. One year after being transferred, the bird presented with clinical signs suggesting a respiratory disease. Those signs included severe dyspnoea with mouth breathing, sneezing and nasal discharge. The crane was put on a 3-day treatment with antibiotics (ceftiofur 200 mg/ml at 50 mg/kg intramuscularly) and anti-inflammatory drug (meloxicam, intramuscular injection at a dose of 2 mg/kg), after which the crane seemed to have recovered. A month later, the same crane presented similar clinical signs and was treated with enrofloxacin at 10 mg/kg intramuscularly. Despite the treatment, the crane died 19 h later. At necropsy, adhesive air sacculitis and hydroperitoneum were observed, and a reddish fluid in air sacs and in the abdominal cavity was found. Also, a marked hepatomegaly and splenomegaly were observed. Samples were collected for laboratory examination. Molecular tests done on the tracheal and cloacal swabs revealed A. paragallinarum and K. pneumoniae, respectively. This is the first case of A. paragallinarum and K. pneumoniae coinfection reported in a grey crowned crane. Our study contributes to knowledge on the ecological distribution of both these pathogens in wild birds. It provides an opportunity to investigate further the clinical significance of infectious coryza in Rwanda's wild and domestic birds.

INTRODUCTION

Gruidae is a highly threatened bird family in the world. In particular, the grey crowned crane (GCC, Balearica regulorum) is a recognised endangered species by the International Union for Conservation of Nature (IUCN). There has been a sharp population decline of up to 80% of the species over the last 3 decades (BirdLife, 2016; Smith et al., 2016). The rapid decline is largely caused by illegal trade, captivity and habitat destruction. These former two activities increase the risks of pathogen transmission between humans, livestock and wild birds.

Avibacterium paragallinarum, initially known as Haemophilus paragallinarum, is a gram‐negative, non‐motile bacterium known to cause infectious coryza. The bacterium is considered a significant challenge to the commercial poultry industry (Blackall et al., 2005). Infectious coryza is usually an upper respiratory disease affecting primarily domestic chicken (Wahyuni et al., 2018); however, it has also been reported in other bird species (Reece et al., 1981; Thenmozhi & Malmarugan, 2013).

Klebsiella pneumoniae is an important pathogen that causes infection in humans and animals. It is often associated with pneumonia, septicaemia and liver abscesses in infected individuals (Newire et al., 2013; Siu et al., 2011). K. pneumoniae is not uncommon in wild birds; thus, this poses a public health threat as a potential reservoir of multidrug‐resistant K. pneumoniae (Navon‐Venezia et al., 2017).

This case report describes a coinfection of A. paragallinarum and K. pneumoniae in a GCC recovered from illegal captivity in Rwanda and provides molecular insights into the isolated pathogens.

CASE PRESENTATION

Case history

In December 2015, a crane was recovered from illegal captivity and completed 60 days of quarantine before being released at a GCC rehabilitation facility located at Akagera National Park, Rwanda. The crane was able to fly and was free to fly in and out of the rehabilitation facility and liked to venture out of the National Park to nearby agricultural farms. In late November 2016, a guard at the GCC rehabilitation facility located in Akagera National Park reported that the crane was exhibiting abnormal behaviour. The crane was anorectic, listless, with a tendency to isolate itself. The crane was identified as ID 102. The veterinary clinician identified the crane as an adult weighing about 3 kg with a generally good body condition (body condition score of 4 on a scale of 1–5). The crane had severe dyspnoea with open mouth breathing, sneezing and nasal discharge. Also, it was lethargic and had a swollen abdomen. At auscultation, abnormal respiratory sounds were heard. Based on clinical signs, the examining veterinarian presumptively diagnosed infectious pneumonia. The crane was treated with an antibiotic (ceftiofur 200 mg/ml, intramuscular injection at 50 mg/kg, three injections at 3 days intervals) and an anti‐inflammatory drug (meloxicam, single intramuscular injection at a dose of 2 mg/kg). After that course of treatment, the GCC's health status improved, and the crane was returned to the rehabilitation facility.

On the morning of the 19 December 2016, the same crane's health status had deteriorated. It was again isolated and treated with intramuscular injection of enrofloxacine at 10 mg/kg. Despite the care provided, it died the following day.

Necropsy findings

The crane was submitted for necropsy, which was conducted on the same day by a veterinary pathologist. Macroscopically, there was severe congestion of the subcutaneous tissue, pectoral and abdominal muscles (Figure 1). Also, the trachea was severely congested. The pericardium and air sacs were filled with about 100 ml of blood‐tinged fluid. There was also severe pleuropneumonia with air sacs adhered to the parietal pleura. In the abdominal cavity, splenomegaly and hepatomegaly were observed. Moreover, there were green strikes on the liver surface. The intestine had some remains of ingested food, and six intestinal roundworms were recovered from the large intestine (data not shown). Other organs had no significant macroscopic lesion.

FIGURE 1

Severely congested trachea with a shiny mucosal surface

Laboratory findings

Prior to commencing treatment, cloacal and tracheal swabs were collected on FTA cards and sent to Molecular Diagnostic Services (Pty) Ltd, (a private laboratory based in Durban, South Africa). The DNA and total RNA were extracted using an in‐house automated magnetic beads protocol. Both cloacal and tracheal swabs tested negative for newcastle disease virus (NDV) and avian influenza virus (AIV) by PCR. 16S rRNA PCR and sequencing were also performed. The sequences were then submitted to BLAST/n at http://www.ncbi.nlm.nih.gov/BLAST to confirm the sequence identities (Altschul et al., 1990). Tracheal and cloacal swabs were PCR positive for A. paragallinarum and K. pneumoniae, respectively. Each sequence was aligned with its homologous sequences from A. paragallinarum and K. pneumoniae, using a strain of Escherichia coli as an outgroup. The alignment and phylogenetic tree for the nucleotide sequences were generated using the Neighbour‐joining distance algorithm, and the Kimura 2‐parameter (K2P) model (Kimura, 1980) with 10,000 bootstrap replicates using CLC sequence viewer software (version 8; CLC Bio Inc., Massachusetts, USA) (Wen et al., 2018). The nucleotide sequences determined in this study were submitted to GenBank under accession numbers MK747257 and MK733284 for K. pneumoniae and A. paragallinarum, respectively.

The chicken A. paragallinarum isolates retrieved from the database formed distinct clusters phylogenetically distant from the isolate of this study (Figure 2), suggesting that the latter may be genetically different from genotypes causing infectious coryza in poultry. However, the probability of transmitting these pathogens between chickens and wild birds cannot be ruled out because these isolates are from different geographical locations. Moreover, only one isolate from the GCC was used.

FIGURE 2

Neighbour‐joining method and the Kimura 2‐parameter model were used to construct a phylogenetic tree showing the relationships between Klebsiella pneumoniae 16S rRNA gene sequences retrieved from GenBank and one isolate from this study (K. pneumoniae MK747257). The numbers displayed are bootstrap values (multiplied by 100) based on 10000 replicates (Dopazo, 1994). Escherichia coli strain ATCC 35218 was used as an outgroup. The scale bar represents the number of substitutions per nucleotide

On the other hand, the K. pneumoniae isolate from the GCC were indistinguishable from K. pneumoniae isolates from human clinical cases, implying more genetic similarity between them and the possibility of cross‐infection between various hosts (Figure 3).

FIGURE 3

Neighbour‐joining method and the Kimura 2‐parameter model were used to construct a phylogenetic tree showing the relationships between Avibacterium paragallinarum 16S rRNA gene sequences retrieved from GenBank and one isolate from this study (A. paragallinarum MK733284). The numbers displayed are bootstrap values (multiplied by 100) based on 10,000 replicates (Dopazo, 1994). The scale bar represents the number of substitutions per nucleotide, and Escherichia coli strain ATCC 35218 was used as an outgroup

DISCUSSION

We have presented data confirming infectious coryza caused by A. paragallinarum infection in grey crowned crane (B. regulorum). This report highlights the coinfection of a grey crowned crane by A. paragallinarum and K. pneumoniae supporting the severity of infectious coryza. Coinfection of A. paragallinarum with other opportunistic pathogens has been reported in the past. Naturally and experimentally infected birds display a severe clinical picture characterised by a respiratory distress syndrome (Kishida et al., 2004; Morales‐Erasto et al., 2016). The clinical signs described are in line with previous upper respiratory distress reports in other avian species (Blackall, 1999). The severe, extensive inflammatory lesions potentially resulted from complications due to Avibacterium spp. coinfection with other bacteria as previously reported (Morales‐Erasto et al., 2016; Paudel et al., 2017). K. pneumoniae is generally reported as opportunistic bacteria that can occasionally cause pneumonia and a wide range of extra intestinal lesions such as liver abscesses in humans and animals (Brisse & Duijkeren, 2005; Davis & Price, 2016; Du et al., 2014). This opportunistic pathogen is reported in Rwanda as increasingly resistant to commonly used antibiotics in humans (Carroll et al., 2016; Ntirenganya et al., 2015). Animals are known possible reservoirs of resistant K. pneumoniae (Navon‐Venezia et al., 2017) that can cross‐infect humans in various ways, including close contact.

Infectious coryza is a well‐known upper‐respiratory tract disease of domestic chicken. The pathological condition has a significant economic impact on the poultry farming industry. It can lead to a substantial drop in egg production that can range between 14% and 41% and can cause mortality in chickens of 0.7%–10% (Thitisak et al., 1988), thus impeding farmers’ economic development. Infectious coryza has not been reported in Rwanda, but it is highly prevalent in neighbouring countries (Byarugaba et al., 2007; Wambura, 2010).

To the best of our knowledge, this is the first report of A. paragallinarum and K. pneumoniae coinfection in a bird in Rwanda. These findings call for the need for further investigation of occurrence of these pathogens in various hosts in Rwanda. Evidence abounds elsewhere of wild birds transmitting pathogens to humans and livestock and has been well described (Bonnedahl & Järhult, 2014; Boseret et al., 2013; Shyaka et al., 2015). Moreover, K. pneumoniae is of clinical significance because it is a zoonotic pathogen and has been reported as increasingly prevalent and resistant to most antimicrobials in clinical settings in Rwanda (Ntirenganya et al., 2015; Umuhoza & Barton, 2014). These two identified pathogens were isolated from a GCC recovered from illegal captivity; thus, there is a possibility that these pathogens were acquired from, or spread to, other animal species such as poultry (A. paragallinarum) and/or humans (K. pneumoniae). Also, we cannot rule out the possibility that one or both pathogens infected the crane after recovery from captivity. The two hypotheses emphasise that keeping wild birds as pets could potentially be a source of pathogen spillover events between wild birds, humans and other livestock species.

Therefore, there is a need to study the broad epidemiology of public health and “One Health” pathogens, most importantly those threatening the livestock industry such as K. pneumoniae and A. paragallinarum. In this regard, it is imperative to adopt a holistic approach that would focus on generating data such as genome sequences of isolates from various hosts, including humans, livestock, and the environment. Such data could be used to better understand the epidemiology of various pathogens in humans, animals and the environment in Rwanda. Moreover, this could shed some light on the possible overlap between genotypes, implying a circulation of the identical genotypes between various hosts. This would be the first important step towards understanding the role of wild birds in the transmission cycle of various pathogens to humans and other animal species.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ETHICS STATEMENT

This study did not require ethical approval as it is linked to a clinical case. Animal care and all procedures employed adhered to World Organisation for Animal Health (OIE) Terrestrial Animal Health Code 2012 (use of animals in research and education). We obtained written consent from Rwanda Wildlife Conservation Association (RWCA) to publish this study.

AUTHOR CONTRIBUTIONS

Conceptualisation, funding acquisition, investigation, methodology, project administration and writing—review and editing: Olivier Nsengimana. Investigation, methodology, resources, writing – original draft and writing—review and editing: Gervais Habarugira. Investigation, methodology and writing—review and editing: Lonzy Ojok. Investigation and writing—review and editing: Deo Ruhagazi. Investigation and writing—review and editing: Albert Kayitare. Conceptualisation, data curation, formal analysis, investigation, methodology, resources, software, validation, visualisation, writing—original draft and writing—review and editing: Anselme Shyaka.

PEER REVIEW

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.766.