Ornithobacterium rhinotracheale in Moroccan Poultry: Antimicrobial Susceptibility Profiles, Characterization of Recent Isolates, and Retrospective Study (2019–23) of Its Occurrence in Different Poultry Production Systems

Respiratory diseases are one of the main causes of economic losses in the poultry industry each year (1,2). Ornithobacterium rhinotracheale (ORT) is considered to be one of the most economically important pathogens in poultry (3,4,5). ORT is an infectious disease caused by a Gram-negative, rod shaped, nonmotile and pleomorphic bacterium that belongs to the family Flavobacteriaceae (4,6). It was recognized as a separate genus containing one species by Vandamme et al. (6). Recently, another species isolated from humans, named Ornithobacterium hominis, was added to the genus (7). ORT infection is associated with respiratory disease in both turkeys and chickens (8). Over the years, the host range of the bacterium has expanded and now includes pheasants, pigeons, partridges, gulls, ostriches, quails, guinea fowl, and rooks (9,10,11,12). There are currently 18 identified serotypes (A–R); however their correlation with the pathogenicity of the strains is still not demonstrated (4,5). Nonetheless, the pathogenicity of ORT infection has been reported to vary depending on environmental factors such as litter, ventilation, and density management in intensive poultry houses (1,4,5). In addition, co-infections with other pathogens were associated with exacerbation of disease duration and severity, including low pathogenic avian influenza subtype H9N2, avian metapneumovirus, infectious bronchitis virus, Avibacterium paragallinarum, and Escherichia coli (5,13,14,15,16,17). Furthermore, it was demonstrated that live infectious bronchitis virus vaccine can exacerbate ORT infection (18).

Riemerella anatipestifer (RA) is the closest related bird pathogen to ORT based on 16S ribosomal RNA (rRNA) sequence similarity (4,19,20). RA is also a Gram-negative, rod-shaped bacterium belonging to the family Flavobacteriaceae within the genus Riemerella (21). The first description of the exudative septicemia associated with RA in geese dates back to 1904 by Riemer (22). It is also the etiologic agent of infectious serositis, the most economically significant disease of domestic ducks. Other reports of similar pathologic findings have been reported in other species, including geese, turkeys, chickens, and wild birds (20,23,24). There are currently 21 reported serotypes that differ in virulence; additionally, a difference in the virulence between strains of the same serotype has been observed (25). Both ORT and RA are transmitted horizontally, but vertical transmission is possible but yet to be proven (5,24). Although RA has been isolated from chickens (Gallus gallus), the species has been recognized as refractory to the infection (26). In 1992, Mouahid (27) reported the isolation of an RA strain from chicken lungs; the strains were described as “Riemerella-like taxon 2.” However, the pathogenicity of the strain was not discussed (20,27). In recent years, several reports of the bacterium have been documented in turkeys and chickens (23,28,29,30).

Culture of ORT is notoriously fastidious, requiring 24 to 48 hr for growth and often being overgrown by other bacteria in mixed cultures. This challenging nature, combined with the significant economic losses associated with ORT infections in the poultry industry, has prompted extensive research into more rapid and sensitive diagnostic tools for ORT. In 1998, Van Empel described primers targeting specific 16S rRNA sequences of ORT, establishing a protocol that demonstrated high sensitivity and specificity (31). Building on these advancements, Abdelwhab et al. (32) developed a TaqMan quantitative polymerase chain reaction (qPCR) assay based on 16S rRNA for rapid detection of ORT directly from clinical samples or cultured isolates. This assay was reported to be 100 times more sensitive than conventional PCR and reduced detection time by half. Moreover, in order to enhance resolution in identifying and differentiating ORT isolates, multiple studies have employed sequencing of the 16S rRNA gene. This method effectively distinguishes Ornithobacterium species with sequence identities ranging from 85.1% to 100% (5,11). Furthermore, 16S rRNA sequencing has facilitated detailed phylogenetic mapping, separating RA onto distinct branches alongside other closely related bacteria, including ORT (19). However, comparative studies have shown that other molecular techniques offer higher discriminatory power. For instance, Chou et al. (10) evaluated random amplified polymorphic DNA (RAPD), single-enzyme amplified fragment length polymorphism (SE-AFLP), and 16S rRNA sequencing and concluded that RAPD and SE-AFLP were more effective at differentiating ORT isolates than 16S rRNA sequencing alone. In addition, Veiga et al. (12) reported that partial sequencing of the rpoB gene provides higher resolution for distinguishing ORT from Ornithobacterium-like bacteria. Similar findings were observed for rpoB gene sequencing in differentiating Riemerella species within the Flavobacteriaceae family (20).

The first documented cases of ORT and RA infections in Moroccan turkey farms were reported by Croville et al. in 2018 (33). That study screened 178 tracheal swab samples collected from 17 turkey farms experiencing acute respiratory outbreaks in Morocco and France, using a nanofluidic PCR platform. ORT was prevalent, being detected in 6 out of 8 sampled farms, whereas RA was identified in only one sample. However, the role of RA in the observed clinical and pathologic findings was not elucidated, and the study did not report detailed pathologic lesions from the affected farms. Despite this initial report, a comprehensive investigation of ORT and RA infections in Moroccan poultry has yet to be conducted. The current study addressed this gap by investigating the genetic characteristics of Moroccan ORT isolates and evaluating their antimicrobial susceptibility profiles during the 2023 outbreaks. Furthermore, in response to the rising number of ORT cases observed in clinical submissions in recent years, we conducted a retrospective analysis to assess the frequency of these cases and analyze the epidemiologic trends of outbreaks over a 5-yr period (2019–23).

This study analyzed case files from Dr. Mouahid’s Veterinary Clinic (Temara, Morocco) spanning January 2019 to December 2023 (Supplemental Table S1). Data on submission dates, flock locations (administrative regions in Morocco), production system, bird age, and flock population sizes are recorded in Excel files. These files also include information on diseases diagnosed through necropsy and the associated pathogens involved identified through laboratory investigations.

For this study, all case files related to breeder turkeys, broiler breeders, laying hens, broiler turkeys, and broiler chickens from 2019 to 2023 from all ages and from different geographic locations were retrieved from the database. For the purpose of the analysis, these poultry types were separated in two categories: “layers” included breeder turkeys, broiler breeders, and laying hens, while “broilers” included both broiler chicken and turkeys. An “ORT case” was defined as the presence of ORT-suggestive lesions identified during necropsy in one or more diseased birds from a single case submission and confirmed through bacteriologic analysis and/or PCR. A case submission included up to eight birds for chickens and up to four turkeys.

For the temporal distribution analysis, these periods were considered: winter from December to February, spring from March to May, summer from June to August, and fall from September to November.

We used Fisher’s exact test to determine whether the number of ORT cases increased during the study period. Additionally, potential risk factors associated with ORT diagnosis were evaluated using a univariable logistic regression analysis. The factors tested included age, season, poultry type, and geographic location.

Subsequently, the statistically significant factors identified in the univariable analysis were included in a multivariable logistic regression analysis (34). The odds ratio (OR), 95% confidence interval (CI), and p values were estimated using maximum likelihood methods. All statistical analyses were performed using SPSS Statistics 29.0 (35).

For this study, all the cases submitted from January to December 2023 that were suspected of ORT infection on the basis of clinical signs and gross pathologic lesions were subjected to laboratory investigations including bacteriology for the culture and isolation of ORT, following which the antimicrobial susceptibility profile of the isolates was assessed; in addition, organ tissues were analyzed for histopathologic lesions and for the molecular confirmation of ORT using real-time qPCR in addition to conventional PCR followed by partial sequencing of the rpoB gene.

Necropsies were performed on all cases in this study. Each case submission comprised up to eight birds for chickens and up to four turkeys from the same flock. Gross pathologic lesions were recorded. Cases suspected of ORT infection were sampled for further laboratory investigations. For bacteriologic screening, samples of the liver, spleen, trachea, lungs, air sacs, ovarian follicles, and bone marrow were aseptically recovered during necropsy. Additionally, tracheal and lung swabs, as well as lung and tracheal tissue samples, were collected. Swabs were used for real-time qPCR, while lung and tracheal tissues were immediately placed in transport media (Difco™ Brain Heart Infusion Broth, Cat. No. 237500 [(2009]; Becton, Dickinson and Company, Sparks, MD) and transported to the Avian Pathology Unit laboratory at the Agronomy and Veterinary Hassan II Institute for further molecular analysis. Tissue samples were homogenized in Dulbecco’s nutrient broth, and the resulting suspensions were centrifuged at 8000 × g for 5 min at 4 C. Subsequently, a 500-μl aliquot of the supernatant was clarified at 12,000 × g for 5 min and analyzed immediately. Finally, lung samples were placed in 10% neutral buffered formalin for histopathologic analysis.

Formalin-fixed lung samples were embedded in paraffin, and 5-μm-thick sections were prepared and subsequently stained with hematoxylin and eosin following standard histologic procedures.

To isolate ORT, lung and trachea samples were cultured on 5% sheep blood agar supplemented with 5 μg/ml of gentamicin. The plates were incubated at 37 C under micro-aerophilic conditions for 24–48 hr. From each plate, three to five colonies were selected based on their characteristic morphology. Small, pinpoint, circular, gray and opaque colonies were subjected to Gram staining; in addition, biochemical and enzymatic identification of the isolates was performed using oxidase and catalase tests, as well as the API 20NE system (BioMérieux SA, La Balme-les-Grottes, France), following the manufacturer’s instructions (36).

Comparative bacteriologic investigations were carried out on organs collected from affected flocks. For this purpose, samples were also cultured on tryptic soy agar, bile esculin azide agar, MacConkey agar, brilliant green agar, xylose lysine deoxycholate agar, eosin methylene blue (EMB) agar, Chapman agar, blood agar with nalidixic acid, and in selenite cystine broth. E. coli strains isolated during this study were subjected to serotyping by slide agglutination test using commercially available O-antisera O1, O2, and O78 (Ceva Biovac, Beaucouzé, France) according to the manufacturer’s instructions.

The disk diffusion method was used to determine the antimicrobial susceptibility profiles of Moroccan ORT isolates following the recommendations of the Clinical Laboratory Standards Institute (CLSI) for fastidious gram-negative organisms (37). In total, 17 antimicrobials authorized for use in poultry in Morocco (38) were tested. These included spiramycin (100 μg), erythromycin (10 μg), neomycin (30 μg), tetracycline (30 μg), doxycyline (30 μg), ampicillin (10 μg), amoxicillin (25 μg), enrofloxacin (5 μg), flumequine (30 μg), cefalotin, trimethoprim/sulfamethoxazole (TMPS; 1.25/23.75 μg), lincomycin (2 μg), colistin (10 μg), fosfomycin (50 μg), and florfenicol (30 μg). Additionally, susceptibility was also tested for streptomycin (300 μg) and gentamicin (10 μg). All the antimicrobial disks used were from Oxoid (Basingstoke, U.K.).

DNA extraction from tracheal and lung swabs was performed using the Kylt®DNA extraction Mix II kit (AniCon Labor GmbH, Hoeltinghausen, Germany) according to the manufacturer’s instructions. Amplification of ORT DNA was conducted with the Kylt®ORT kit (AniCon Labor GmbH, Hoeltinghausen, Germany). The final volume of each reaction was 20 μl, which contained 16 μl of the Reaction-Mix and 4 μl of the extracted sample. The real-time qPCR thermal profile was 95 C for 10 min, followed by 42 cycles at 95 C for 15 sec and 60 C for 1 min. All the reactions were carried out on the ABI 7500Fast thermocycler (Applied Biosystems, Foster City, CA). For comparative analysis, DNA extracted from swab samples was also tested for Mycoplasma gallisepticum and Mycoplasma synoviae using the Kylt® MGS triplex kit (AniCon Labor GmbH, Hoeltinghausen, Germany) in accordance with the manufacturer’s instructions.

Twelve positive samples analyzed by qPCR were selected for amplification and sequencing of the rpoB gene. The samples originated from nine flocks during 2023 outbreaks. The nine flocks consisted of five layer flocks of caged-reared laying hens from north-central Morocco (Fes-Meknes region) aged between 18 and 68 weeks reporting an elevated mortality rate (0.16% to 0.49% weekly mortality rates) with no repercussion on the production rate; two turkey flocks from the central region of Morocco (Gharb and Rabat-Salé-Kenitra regions) aged 73 and 85 days that presented with elevated mortalities (0.4% weekly rate) and respiratory clinical signs with ruffled feathers; and, lastly, two broiler chicken flocks from the region of Rabat reporting elevated mortalities with rales, head swelling, and ruffled feathers with lameness and weakness. The broiler chicken flocks were aged 29 and 32 days with a weekly mortality rate of 0.96% and 2.19%, respectively (Table 1).

DNA extraction was performed on lung and tracheal samples using the DNeasy Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The rpoB gene fragment (538 bp) was amplified with primers and protocols described by Veiga et al. (2019) (12). Briefly, the primers rpoB Fla-f (5′-TCAATTCGTTCTTTGGAAC-3′) and rpoB Fla-r (5′-GCATCATGTTAGATCCCAT-3′) were used to amplify the 538-bp region. The PCR reaction was carried out in a final volume of 25 μl, consisting of 5 μl of template DNA, 2.5 μl of 10X PCR reaction buffer (AmpliTaq Gold™ DNA Polymerase Kit, Cat. No. N8080241; Applied Biosystems), 1 μl of deoxynucleoside triphosphates, 1 μl of MgCl₂, 0.3 μl of Taq DNA polymerase, 13.2 μl of distilled water, and 1 μl of each primer at a concentration of 1 μM. The amplification protocol included an initial denaturation at 94 C for 3 min, followed by 30 cycles of denaturation at 94 C for 30 sec, annealing at 54 C for 30 sec, extension at 72 C for 45 sec, and a final extension at 72 C for 5 min. Conventional PCR amplifications were carried out using an Applied Biosystems Veriti™ Thermal Cycler (Thermo Fisher Scientific, Waltham, MA).

The rpoB PCR products were visualized on 1.5% agarose gels stained with purified ethidium bromide gel (Qiagen). The gel bands were then excised and purified using the Qiagen Gel Extraction Kit (Qiagen) following the manufacturer’s instructions. Sequencing was performed using the primers rpoBFla-f and rpoBFla-r− at Eurofins (Hamburg, Germany), using PeakTrace™ Basecaller and the PHRED 20 quality score. Sequencing reactions were carried out with the BigDye® Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific). After sequencing, the products were further purified using the BigDye™ X Terminator Purification Kit (Thermo Fisher Scientific) before sequence analysis.

Assembly and analysis of sequence data were carried out using the BioEdit Software version 5.0.9 (39). Phylogenetic analysis and tree construction for the rpoB glycoprotein were generated using the maximum likelihood method with 1000 bootstrap replicates with the MEGA software Version 5.05 program (40), and bootstrap values above 50 were labeled on major tree branches for reference. The identity of bacterial species was confirmed using the BLAST search in GenBank (41). The Moroccan ORT isolates were deposited in GenBank under accession numbers: PQ365702 to PQ365711. The two Moroccan RA isolates were deposited in GenBank are under accession numbers: PQ365712 and PQ365713.

In total, 2867 case files were reviewed during this 5-yr retrospective study, of which 185 were diagnosed as ORT-positive cases. Statistical analysis using Fisher’s exact test revealed a significant overall increase in ORT-positive cases over the study period (p < 0.001). Indeed, the test confirmed a significant difference in the number of ORT-positive cases between the earlier period (2019–21) and the later period (2022–23). This upward trend is clearly illustrated in Fig. 1, which depicts a consistent and marked rise in overall ORT cases over time. Fig. 2A further highlights this increase, stratified by production type. In terms of seasonal distribution, the majority of cases occurred during the period from June to November, corresponding to the summer and fall seasons, as shown in Fig. 2B. Univariable logistic regression analysis was used to explore potential risk factors, including age, poultry type, location, and season. All four variables were significantly associated with ORT diagnosis. Broiler-type productions (broiler turkeys and chickens) were found to be 3.63 times more likely to have ORT (p < 0.001), while poultry cases from the northwest part of Morocco were 3.11 times more likely to receive an ORT diagnosis (p < 0.001). Additionally, case submissions during the summer season were 1.66 times more likely to test positive for ORT (p = 0.0023). Birds aged less than 5 mo old were also significantly more likely to be diagnosed with ORT, with an odds ratio (OR) of 2.91 (p < 0.001). In contrast, case submissions during the winter season were significantly less likely to be associated with ORT infection (OR = 0.61; p < 0.05). Similarly, poultry cases originating from the north-central region of Morocco were significantly less likely to have an ORT diagnosis (OR = 0.29; p < 0.001; Table 2). All variables were subsequently included in a multivariable logistic regression model, which identified age as the sole significant factor with an adjusted OR of 4.87, a 95% confidence interval of 2.54–9.37, and p value <0.001.

In total, 84 ORT cases were identified during 2023 (Supplemental Table S1), out of which 10 flocks were confirmed to be positive for ORT by bacteriology. On the other hand, all the flocks were positive using real-time qPCR, and 12 samples belonging to nine flocks were selected for rpoB gene sequencing (Table 1).

Upon necropsy, birds of the 84 ORT-positive cases exhibited fibrinous pleuropneumonia, a hallmark lesion suggestive of ORT infection (Fig. 3). Additionally, in the nine flocks subjected to rpoB gene sequencing, other lesions were observed, including fibrinous polyserositis (perihepatitis, pericarditis, and airsacculitis). Fibrinous oophoritis was consistently present in all layer cases (5/5). In turkeys, sternal bursitis was identified in one of two cases, while in broiler chickens, exudative arthritis was noted in one of two cases. These findings raised suspicion of ORT in all cases. Concurrently, M. gallisepticum and E. coli were considered for their potential role in the observed polyserositis.

Histopathology of the lungs showed similar lesions for the birds infected with ORT and RA (Fig. 4), severe extensive serositis with heterophilic and histiocytic infiltration, and accumulation of fibrin with intralesional bacteria. In the lung parenchyma, similar cellular infiltrates were found in the interlobular septa with blood vessel congestion and edema. Cellular necrotic debris admixed with fibrin and bacteria were found in the lumen of parabronchi, and, in certain cases, fibrinonecrotic debris replaced the entirety of the parabronchial wall.

During the year 2023, 84 cases from flocks suspected to be ORT-positive were submitted for bacteriology testing, but only 10 samples were isolated. The isolates were collected from three broiler chicken flocks aged 14, 36, and 43 days of age, five broiler turkey flocks aged 41, 90, 113, 117, and 136 days, and two egg-type layer flocks aged 13 and 27 wk of age. Growth was observed after 48 hr of incubation, and colonies were often overgrown by other bacteria including E. coli, Pseudomonas aeroginosa, and Proteus.

All isolates reacted positively to p-nitrophenyl-β-d-galactopyranoside and oxidase tests within 48 hr at 37 C using the API 20NE identification system, while all other tests remained negative after 72 hr of incubation at 37 C. One isolate was urease-positive, resulting in a biocode of 0-2-2-0-0-0-4, whereas the other nine isolates produced a biocode of 0-0-2-0-0-0-4.

The nine flocks for which samples were sequenced for the rpoB gene and confirmed as ORT and RA also tested negative for ORT using bacteriologic methods. However, E. coli serotypes O1:K1 and O78:K80 were detected in two layer chicken flocks, while a nontypable E. coli strain was isolated from both broiler cases; as for turkeys, one flock tested positive for Mannheimia haemolytica (Table 1).

All ORT isolates were fully resistant to cefalotin, colistin, cosfomycin, and all three aminoglycosides tested (neomycin, streptomycin, and gentamicin). In addition, 67% of isolates showed resistance to enrofloxacin. Moreover, 67% of isolates were resistant to flumequine, TMPS, and fosfomycin. For ampicillin, 50% of turkey and broiler isolates showed resistance.

On the other hand, the majority of isolates (67%) showed sensitivity to penicillins (ampicillin and amoxicillin), notably ORT isolates of laying hens (100% sensitivity), followed by broilers with 67% sensitivity for amoxicillin, while turkey strains showed only 50% sensitivity to amoxicillin. Additionally, turkey isolates showed 100% sensitivity towards tetracyclines (doxycycline and tetracycline), while in broilers, a 67% sensitivity rate was noted. However, in layers, only a 50% sensitivity rate for doxycycline was observed, while all the isolates were intermediate for tetracycline. Finally, broiler turkey and chicken isolates showed 100% sensitivity to florfenicol (Table 3).

All the nine investigated flocks in this study tested positive for ORT using real-time qPCR. In addition, both M. gallisepticum and M. synoviae were detected in 2/5 cases of layers, while all the other flocks tested negative for both M. gallisepticum and M. synoviae (Table 1).

Ten Moroccan ORT isolates exhibited nucleotide similarity ranging from 98% to 100% with previously reported ORT strains from chickens and turkeys. These included strains isolated in the U.K. in 1988 and 2013 (CP003283 and MH746638), Chile in 2009 (MH746633), China in 2012 (MH746637), Poland in 2024 (PP264577 and PP264580), and the United States in 2019 and 2022 (CP094845 and CP132522). In contrast, two rpoB gene sequences obtained from lung samples of a laying hen flock and a broiler turkey flock were found to be genetically similar to known Riemerella anatipestifer isolates based on partial rpoB sequences. The two Moroccan Riemerella anatipestifer isolates shared 98.79% nucleotide sequence identity with each other and 99.39% identity with the non-Moroccan RA strain IPDH 98/90 (FJ999744), which was isolated from chicken lung and liver in Germany in 1990 and classified within the “Riemerella-like taxon 2” (8,28). The deduced amino acid sequence identities of the Moroccan RA isolates were 89.4% when compared to each other and ranged from 83.10% (Moroccan isolate CVM_2387 [PQ365712] and RA strain WJ4 [CP041029] isolated from a duck in China in 2000) to 95.15% (Moroccan isolate CVM_2387 [PQ365712] and strain IPDH 98/90 [FJ999744] isolated from chicken lung and liver in Germany in 1990) when compared to non-Moroccan RA strains.

The phylogenetic relationships of the Moroccan ORT isolates were reconstructed based on partial rpoB gene sequences. This analysis revealed a close genetic relationship between the Moroccan isolates and previously reported ORT isolates from diverse geographic regions, as illustrated in Fig. 5. Similarly, the two Moroccan RA isolates exhibited high genetic similarity to the RA strains IPDH 98/90 (FJ999744) and WJ4 (CP041029), as depicted in Fig. 6.

ORT is an important respiratory pathogen that has been reported in several countries including South Africa, Egypt, Canada, and multiple European countries, including Germany, the U.K., and Austria (6,8,18,42). Yet, little is known about this infection in Moroccan poultry flocks. This study was designed to study the epidemiology of ORT in Moroccan poultry and to gain better insights into its genetic characteristics in combination with assessment of antimicrobial susceptibility profiles of Moroccan ORT isolates during 2023 outbreaks.

The 5-yr retrospective study (2019–23) revealed a significant increase in ORT cases (p < 0.001). This trend may be attributed to the rapid growth of the Moroccan poultry sector and the proliferation of intensive poultry farms in various regions of the country, which in turn increases the pathogen load in the environment, facilitating the spread of bacterial and viral infections between farms. This aligns with recent findings from Egypt, where Ellakany et al. (18) reported an increase in ORT incidence in broiler farms from 7.27% in 2010 to 11.66% in 2019, which was attributed to similar intensification of poultry production systems. Improved diagnostic capacity globally and locally in Morocco due to the adoption of molecular techniques such as PCR for the rapid diagnosis of poultry pathogens has also likely contributed to the observed increase in reported cases. These advancements enhance the detection and reporting of diseases, which may not have been accurately identified in the past. Another contributing factor could be the introduction of respiratory viruses, such as low pathogenic avian influenza subtype H9N2 into Moroccan poultry flocks. This virus compromises the immune system of birds, making them more susceptible to secondary bacterial infections, including ORT (43). Similar interactions between viral and bacterial pathogens have been widely documented, underscoring the importance of managing co-infections to control respiratory disease outbreaks effectively (5,13,14,15,16,17). Finally, environmental factors may also play a role in the increased incidence of ORT. To test this hypothesis, we analyzed the effect of two environmental variables, the geographic location and the season, on ORT incidence, in addition to poultry type and age as potential risk factors. Univariable logistic regression analysis identified the four variables as significant risk factors. Poultry cases from the northwest region of Morocco were 3.11 times more likely to receive an ORT diagnosis (p < 0.001). This region primarily includes case submissions from Rabat-Salé-Kénitra and Casablanca-Settat, two areas that host the country’s largest poultry production sites (44,45). The higher incidence of ORT in these regions may be attributed to the close proximity of different production types and the growing complexity of the motorway network, which facilitate the introduction and spread of avian pathogens. The transport network in these cities has been identified as a major contributor to disease introduction and dissemination. In fact, Farhi (46) conducted a cross-sectional epidemiologic study mapping vehicle movement between poultry farms within the Casablanca-Settat region. The study recorded 945 poultry transport trips across 126 communes, with live poultry transport accounting for over 44% of these trips. Notably, three central communes—Had Soualem, Hay Mohammadi, and Sahel Oulad H’Riz—were identified to be at higher risk areas for disease introduction and spread due to their transport networks.

We also found that broiler-type productions, including turkeys and broiler chickens, were 3.63 times more likely to receive an ORT diagnosis (p < 0.001). Turkeys, in particular, are known to be highly susceptible to ORT infection (5), which explains the significant association. Age was another significant risk factor, with birds aged less than 5 mo being 2.91 times more likely to be diagnosed with ORT (p < 0.001). Multivariable logistic regression analysis further identified age as the sole significant factor among the four variables tested. To enhance the accuracy of the statistical analysis, we categorized bird ages into two groups: <5 mo and >5 mo. This classification was chosen to ensure an adequate sample size and to distinguish laying birds based on the onset of egg-laying. Birds under 5 mo of age primarily include all broiler-type productions (both turkeys and chickens), which likely accounts for the significant results. This classification may not be ideal for analyzing ORT epidemiology. A more detailed approach, with small age intervals and separate analyses for each production type, is recommended to better understand the role of age as an epidemiologic risk factor for ORT infection. Interestingly, we found that case submissions during the winter season were significantly less likely to be associated with ORT infection (p < 0.05), while cases presented during the summer season were 1.66 times more likely to test positive for ORT (p = 0.0023). These findings correlate in part with those of Hauck et al. (16), which reported a higher concentration of cases from August to January. Conversely, it was previously established that low temperatures enhance the survival of the pathogen in the environment, which in turn increases the incidence of ORT infection during winter (4). These conflicting findings highlight the complexity of ORT epidemiology and the need for further studies to clarify seasonal influences on disease occurrence.

We also report in this study the results of ORT outbreak investigations during 2023, where the isolation and characterization of Moroccan ORT isolates during outbreaks of the year 2023 were carried out, and the antimicrobial susceptibility profiles of isolates were determined. Birds were submitted for respiratory clinical signs and elevated mortality rates, and during the necropsy examination, they presented with an exudative pleuropneumonia, more often unilateral, which is strongly correlated with ORT infection in poultry (5,47). Less commonly, we found congestion and hepatization of the lung, unilateral or bilateral.

During this study, we successfully isolated 10 ORT samples from 84 ORT-suspected and PCR-confirmed cases in 2023. This low isolation rate can be attributed to the difficulty in culturing ORT but also because most of the flocks examined had already received antibiotic treatment, which can further hinder the isolation process (18,48,49).

Moreover, many cases presented for examination involved complex infections, where ORT was likely in advanced stage; such mixed infections, as previously demonstrated for RA, can interfere with bacterial isolation (50). Furthermore, while gentamicin, to which most ORT isolates are resistant, was added to the growth medium to suppress other bacteria, it did not fully prevent the overgrowth of resistant bacteria, further contributing to the low isolation rate. Similar findings were reported by Canal et al. (51).

The antimicrobial susceptibility results revealed full resistance of Moroccan ORT isolates to the aminoglycosides gentamicin and neomycin. Interestingly, one turkey isolate exhibited intermediate resistance to streptomycin. This finding could be attributed to the use of the disk diffusion method, which is less precise compared to microdilution. Furthermore, the absence of ORT-specific breakpoints necessitates reliance on CLSI guidelines for fastidious gram-negative organisms (5), potentially affecting the interpretation of results. The fastidious growth requirements of ORT further complicate the accurate measurement of inhibition zones in laboratory settings. Streptomycin, notably, is not authorized for use in poultry in Morocco. The lack of authorized use and the presumed absence of exposure to this antibiotic in Moroccan poultry may contribute to the observed reduced resistance in Moroccan ORT strains. While significant genetic changes in Moroccan isolates are unlikely, this finding underscores the need for further investigations focusing on genetic resistance factors or whole-genome sequencing, which would be valuable to confirm this observation.

The antimicrobial susceptibility results also revealed full resistance to colistin and cefalotin, with 67% of isolates showing resistance to enrofloxacin, flumequine, and the combination TMPS. These findings are consistent with previous studies (42,52,53,54). In contrast, Marien et al. (2) demonstrated in an experimental triple infection (ORT, E. coli, and avian metapneumovirus) that enrofloxacin was the most effective treatment, significantly reducing bacterial recovery in respiratory organs. FLORFENICOL ranked second in efficacy, while amoxicillin had no significant impact on bacterial load or recovery from the respiratory tract. Hess et al. (42) also reported 100% sensitivity to TMPS in Austrian isolates, with 89% of isolates exhibiting intermediate resistance to enrofloxacin. These results differ from our findings, potentially due to the widespread and heavy use of antibiotics in Moroccan poultry for prophylactic and therapeutic purposes. A survey of antimicrobial consumption in broiler production in Morocco by Rahmatallah et al. (55) identified enrofloxacin, colistin, and TMPS as the most heavily used antimicrobials, followed by tetracycline, florfenicol, and amoxicillin. Frequent exposure to these drugs is a strong potential driver of antimicrobial resistance. In the United States, Nagaraja and Thachil (56) demonstrated similar trends, reporting increased resistance in Minnesota ORT isolates from 1996 to 2010, with resistance to enrofloxacin rising from 4% to 80% and resistance to tetracyclines rising from 2% to 50%. Furthermore, Rahmatallah et al. (55) highlighted critical issues with dosing practices in Morocco, including underdosing of TMPS and amoxicillin and overdosing of tetracycline, enrofloxacin, and colistin. These practices further enhance the development of antimicrobial resistance. Our findings corroborate these concerns, with 50% resistance to amoxicillin observed in turkeys and broiler chickens, likely driven by its frequent use to control enteric problems in broiler production systems (55). For cefalotin, Umali et al. (54) reported 50% resistance in addition to 25% intermediate resistance in Japanese isolates. No comparable data currently exist for Moroccan isolates, underscoring the need for further local studies to evaluate ORT antimicrobial resistance patterns.

All 12 samples collected from the nine flocks reported in our study tested positive for ORT by real-time qPCR. However, sequencing of the rpoB gene revealed that two sequences were genetically similar to RA. This discrepancy may result from sequencing being performed directly on DNA extracted from clinical samples, potentially amplifying rpoB sequences from RA present in mixed bacterial populations. As such, the amplification of rpoB sequences from RA present in the samples cannot be ruled out. This limitation underscores the challenges of accurately identifying ORT in clinical samples containing diverse bacterial populations, potentially leading to co-amplification of nontarget DNA. While rpoB sequencing showed a comparable resolution to multilocus sequence typing in differentiating ORT and closely related species (12), its application in clinical samples may not always provide definitive results, particularly when mixed infections are present. Advanced techniques such as whole genome sequencing or multilocus sequence typing could overcome these challenges by providing higher resolution (11), enabling more precise differentiation among these species and offering a more comprehensive understanding of the genetic relationships among isolates.

Mortality rates in these nine flocks ranged from 0.16% up to 2.19% per week. The severity of clinical signs and mortality in ORT infection was reported to depend on various factors including co-infections (4,5). In fact, the lowest mortality rate recorded in our study was in two layer flocks (0.16%–021%) that did not experience any coinfections; the two other layer flocks with M. gallisepticum/M. synoviae, ORT, and E. coli quadruple infection showed higher weekly mortality rates of 0.32% and 0.49%. The highest mortality rate was recorded in two broiler flocks. A 0.9% rate was recorded in a flock with a double ORT/E.coli infection, and another flock recorded a 2.19% mortality rate with a quadruple co-infection with low pathogenic avian influenza subtype H9N2, infectious bursitis disease virus, ORT, and E. coli. ORT infection rate in turkeys was higher as compared to chickens; this is in line with the results of Hauck et al. (16), which found a higher percentage of affected turkeys as compared to chickens, and the reports of differences in the severity of ORT infection depending on species (5,57). Furthermore, our results are in line with previous reports of ORT detection as primary cause of respiratory disease in poultry or implicated in complex diseases as a complicating pathogen, with the latter being more common in the field (5,47).

In conclusion, ORT infection is widespread in Moroccan poultry flocks and is significantly implicated in respiratory diseases in poultry. The incidence of ORT infection notably increased over the 5-yr period from 2019 to 2023, with relapse rates reaching up to 40%. This highlights the need for a better understanding of the infection and close monitoring of the antimicrobial susceptibility profiles of Moroccan isolates to manage outbreaks effectively and mitigate antibiotic resistance. Additionally, RA was detected in broiler turkeys and layer chickens, necessitating further research to elucidate its pathogenic role in respiratory disease outbreaks.

Supplemental data associated with this article can be found at https://doi.org/10.1637/aviandiseases-D-24-00078.s1.

The authors would like to thank Dr. Arbani Oumayma and Soumaya Chaikhi for helping with sequence analysis and submission. The authors are also grateful to all the staff of Mouahid’s Veterinary Clinic, Morocco, as well as the Avian Pathology Unit at Agronomy and Veterinary Institute Hassan II, Morocco, for their support and technical assistance.

Abbreviations:

CI =

confidence interval;

CLSI =

Clinical Laboratory Standards Institute;

OR =

odds ratio;

ORT =

Ornithobacterium rhinotracheale;

qPCR =

quantitative polymerase chain reaction;

RA =

Riemerella anatipestifer;

RAPD =

random amplified polymorphic DNA;

rRNA =

ribosomal RNA;

SE-AFLP =

single-enzyme amplified fragment length polymorphism;

TMPS =

trimethoprim/sulfamethoxazole