Molecular Characterization of Fowl Adenovirus from Brazilian Poultry Farms

Abstract

Fowl adenovirus (FAdV) can cause different poultry diseases with economic losses in the broilers and layers commercial farms. FAdV is currently classified into five species and 12 serotypes, disseminated in poultry flocks worldwide. The present study aimed to identify FAdV species and serotypes in Brazilian poultry farms. A total of 678 chicken flocks from the main Brazilian poultry-producing regions were evaluated for FAdV infection between 2020 and 2023. FAdV was detected by a real-time PCR targeting 52K gene and further genotyped by partial sequencing of the hexon gene followed by phylogenetic analyses. The results demonstrated that FAdV was detected in 72 flocks (10.6%). In 46 of these samples, FAdV species and serotypes could be identified, including three main species: Aviadenovirus ventriculi (FAdV-A = 15), Aviadenovirus gallinae (FAdV-D = 15) and Aviadenovirus hepatitidis (FAdV-E = 16). Phylogenetic analysis based on 173 partial hexon sequences (including sequences from this study, 44 previously sequenced in Brazil, and 86 data from other countries) revealed five separate clades for FAdV species. All Brazilian FAdVs were classified into the same three species reported above (FAdV-A = 19, FAdV-D = 34, FAdV-E = 37), and also in well-supported subclades for each serotype: FAdV-A1 (n = 19), FAdV-D9 (n = 1), FAdV-D11 (n = 33), FAdV-E6 (n = 1), FAdV-E8a (n = 33), FAdV-E8b (n = 3). Amino acid substitutions in the hyper variable regions (1, 2 and 3) and conserved motifs of the Hexon protein were further analyzed, enabling discrimination between closely related serotypes. This study demonstrates the circulation of different FAdVs in Brazil, highlighting FAdV-A1, FAdV-D9, FAdV-D11, FAdV-E6, FAdV-E8a and FAdV-E8b. The findings reported here also indicate genetic and amino acid diversity in the Hexon protein of the FAdVs in Brazilian poultry farms, which are of importance for molecular surveillance and poultry diseases control strategies.

1. Introduction

Fowl adenoviruses (FAdVs) belong to the genus Aviadenovirus in the family Adenoviridae [1]. FAdVs harbor a genome of double-stranded DNA spanning approximately 43 to 46 kilobases (kb) and coding for 10 primary structural proteins and 11 nonstructural proteins. The viral capsid comprises major (Hexon, Penton base, and Fiber) and minor structural proteins (VI, VIII, IIIa) as well as core proteins (V, VII, X, TP). In addition, there are eleven nonstructural proteins: E1A, E1B, E3, E4, DNA polymerase (pol), DNA-binding protein (DBP, E2A), IVa2, 52/55K protein, early protein (EP), 100K protein, and 33K protein [2,3].

FAdVs are classified into five species (Aviadenovirus ventriculi, Aviadenovirus quintum, Aviadenovirus hydropericardii, Aviadenovirus gallinae and Aviadenovirus hepatitidis), formerly recognized by capital letters (A, B, C, D and E, respectively) [4,5,6]. They are also classified into 12 serotypes (1, 2, 3, 4, 5, 6, 7, 8a, 8b, 9, 10 and 11) according to cross-neutralization tests. Aviadenovirus ventriculi and Aviadenovirus quintum have only one serotype (FAdV-A1 and FAdV-B5, respectively), Aviadenovirus hydropericardii has two serotypes (FAdV-C4 and -C10), Aviadenovirus gallinae has four serotypes (FAdV-D2, -D3, -D9, and -D11), and Aviadenovirus hepatitidis has also four serotypes (FAdV-E6, -E7, -E8a and -8b) [4].

FAdV-associated diseases have been reported in broilers and layer hens, including inclusion body hepatitis (IBH), hepatitis hydropericardium syndrome (HHS), adenoviral gizzard erosions (AGE) and eventually runting stunting syndrome (RSS). IBH and HHS outbreaks have resulted in high avian mortality in commercial poultry flocks, leading to significant economic losses for the poultry industry worldwide. Both diseases are associated with sudden mortality, apathy, jaundice, ruffled feathers, lethargy, hydropericardium, ascites, and high mortality rates [2,4,7,8,9]. On the contrary, AGE has been associated with less severe clinical signs, such as apathy, poor weight gain, growth retardation, and flock heterogeneity, often accompanied by mild diarrhea.

Specific FAdV species/serotypes have also been associated with different diseases, highlighting FAdV-D2 and FAdV-E8a, -E8b and -E11 with IBH [9,10,11,12], FAdV-C4 with HHS [7,13] and FAdV-A1 with AGE [9,14,15]. These FAdV diseases have been controlled in poultry farms using different vaccines, including inactivated and live-attenuated formulations (with FAdV-C4, FAdV-E8a, -E8b being most frequently used). Due to the serotype-specific nature of immunity and the high diversity of circulating strains, multivalent or specific autogenous vaccines are often employed to enhance protection in commercial poultry farms [9].

In South America, FAdV outbreaks have been reported over the past few decades. In Brazil, FAdV-E8a, -E8b, and -E11 were identified in broiler chickens showing signs such as hydropericardium, malabsorption, dwarfism, and stunted growth. These cases were associated with clinical outbreaks of IBH [8,11]. In Peru (2016), a significant IBH outbreak caused by FAdV-8b occurred in broiler chickens, resulting in reduced weight gain, 5–10% mortality, and necrotic livers [16]. In Ecuador (2019–2021), FAdV-D2, FAdV-E6, FAdV-E8a, and FAdV-E11 were detected in broilers, layer hens, and breeders, characterized by ruffled feathers, stunting, cloacal pasting, and diarrhea. These species/serotypes were also associated with enteric disease, IBH, suggesting vertical transmission in young birds [17]. In Chile (2024), the first reported case of IBH caused by FAdV-D11 occurred in farmed flocks with sudden mortality of approximately 10%, without prior clinical signs [18].

The diagnosis of diseases caused by FAdV includes the field observation of specific clinical signs in affected flocks, followed by laboratory histopathological analyses in necropsied tissues. Macroscopic characteristic lesions are hepatic necrosis for HHS and IBH and gizzard erosion for AGE [10]. Serological analysis (ELISA) may be used as a supportive tool to detect anti-FAdV antibodies in flocks; however, current assays have technical limitations and may not reliably cover the full diversity of FAdV species and serotypes. Virus neutralization tests are useful to detect FAdV and to identify the specific serotype. Virus isolation is rarely performed, but it can be necessary for diagnostic confirmation. Molecular biology techniques have become increasingly common to detect and characterize FAdV species, serotypes and strains directly from clinical or environmental samples [4,9,19,20].

The current primary method for the FAdV detection relies on conventional or real-time PCR targeting two main viral genes: the 52K gene and the hexon gene. Günes et al. [19,21] developed a real-time PCR protocol targeting a conserved region of the 52K gene. This method enables broad detection of FAdVs, and subsequent species classification (A to E) is achieved through Sanger sequencing of the PCR product. Alternatively, the hexon gene, especially the loop 1 (L1) region flanked by pedestal 1 (P1), has been widely used for both detection and differentiation of FAdVs [22,23]. L1 region contains several hypervariable regions (HVRs) responsible for FAdV antigenic diversity [10,23]. Structural studies have identified up to nine HVRs located at the top of the Hexon protein, which are important for immunogenicity and host interactions [24]. Additionally, real-time PCR assays targeting conserved regions of the hexon gene have been developed for rapid detection, although they are not used for typing [15].

In recent years, FAdV infection has been diagnosed across Brazil through molecular analysis of hundreds to thousands of clinical samples. This study aimed to identify FAdV species and serotypes circulating in commercial poultry farms in Brazil, from September 2020 to December 2023.

2. Materials and Methods

2.1. Sampling

Clinical samples from 678 flocks of broilers or layers showing clinical signs of possible FAdV infection on Brazilian poultry farms were sent for routine diagnostic analysis between September 2020 and December 2023. Most of the farms were broiler production facilities, located in different poultry-producing regions across the country. The main clinical signs were the sudden onset of mortality with chicken presenting ruffled feathers, as well as enlarged and pale-yellow with multiple petechial hemorrhages in some liver samples. Pools of chicken tissues and organs (usually including trachea, lungs, kidneys, liver and cecal tonsils) from approximately nine birds per flock were separated and packed in plastic bags, transported in ice boxes and then maintained at −20 °C in a freezer until sent to the molecular biology laboratory (Simbios Biotecnologia, Cachoeirinha, Brazil).

2.2. Molecular Analysis

Cotton-tipped swabs were scraped into all the different chicken tissues and organs. Total nucleic acid was extracted from these samples with commercial reagents NewGene (Preamp and Prep) according to the supplier’s instructions (Simbios Biotecnologia, Cachoeirinha, Brazil). FAdV detection was performed using two molecular targets. First, samples were screened by qPCR targeting the 52K gene, following the protocol described by Günes et al. [21]. Next, a region of the hexon gene was amplified as previously described [22]. Amplified hexon fragments were purified and further sequenced for FAdV species/serotype identification. The resulting electropherograms were edited and analyzed using the Geneious v.2021.2.2 (Biomatters, Auckland, New Zealand; available at www.geneious.com). Consensus sequences were generated from the hexon gene fragments and then submitted to the BLASTn (Basic Local Alignment Search Tool) tool on the NCBI (National Center for Biotechnology Information) platform for preliminary identification of FAdV species and serotype, based on sequence similarity with publicly available reference genomes. FAdV’s partial hexon gene sequences, clearly assigned to species/serotypes (n = 42), were submitted to GenBank (accession numbers PX310701-PX310742).

2.3. FAdV Hexon Gene Dataset

FAdV hexon complete and partial gene sequences with collection date and country available in Genbank were downloaded and aligned with reference datasets previously constructed [8,25,26]. A total of 173 partial hexon gene sequences were included, representing 18 countries (Table S1). This dataset comprised 90 Brazilian sequences, including 46 obtained in this study and 44 previously deposited in GenBank. Target region extraction was performed based on samples sequenced in this study at nucleotide positions 343 to 870 of the hexon gene in the program Geneious v.2021.2.2 (Biomasters, Auckland, New Zealand; available at www.geneious.com).

2.4. Phylogenetic Analysis

FAdV sequences obtained in this study were included in a reference dataset of hexon gene sequences representing known FAdV species and serotypes. Multiple nucleotide sequence alignment was performed using MAFFT v.7 [27] with the default parameters under the L-INS-i algorithm, which is suitable for sequences with global homology. The alignment was manually inspected and edited, when necessary, to ensure proper codon alignment and to remove ambiguous or poorly aligned regions.

Recombination events were verified using the RDP5 v.5.34 [28] with the default settings using the algorithms RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan, 3Seq, and LARD. The RDP5 v.5.34 software also defined the beginning and end breakpoints of the potential recombinant sequences. Using four or more algorithms, putative recombinant events were considered significant when p ≤ 0.01 was observed for the same event.

After a nucleotide-based phylogenetic tree was reconstructed using the maximum-likelihood (ML) method implemented in the W-IQ-TREE web server [29], with the optimal nucleotide substitution model selected using ModelFinder [30], and 1000 replicates of the ultrafast bootstrap approximation [31]. BioEdit Sequence Alignment Editor v.7.2.5 generated the nucleotide sequence similarity matrix.

2.5. Amino Acid Substitution Analysis

Amino acid alignments corresponding to a specific segment of the Hexon peptide chain were generated using MAFFT v.7 [27]. This segment includes HVR1 (amino acids 137 to 200), HVR2 (amino acids 201 to 216), HVR3 (amino acids 232 to 262), and HVR4 (amino acids 281 to 286), with positions determined based on the reference sequence AC_000014 encoding a 942 amino acids residues Hexon protein [12]. These hypervariable regions were initially described by Crawford-Miksza and Schnurr [32] and have been subsequently used by other authors to study FAdV diversity [23,33,34]. The resulting alignments, including both the sequences generated in this study and publicly available reference sequences, were exported and manually curated in Geneious v.2021.2.2 (Biomatters, Auckland, New Zealand; available at www.geneious.com), where the main amino acid substitutions were visually inspected and compared across sequences to identify potential serotype- or intraspecies-specific markers.

3. Results

3.1. FAdV Detection and Serotype Identification

FAdV was detected in 72 (10.6%) samples from different poultry flocks by qPCR targeting the 52K gene. DNA from these positive samples was then used to amplify the hexon gene by nested PCR and sequence it. A total of 46 samples could be effectively genotyped using the hexon gene sequences obtained (there were samples with negative nested PCR results, as well as samples with positive results without valid sequencing data). Only sequences with conclusive nucleotide data allowing species/serotype identification were included in the subsequent analyses.

In a first BLASTn analysis of the DNA sequences, three species (A, D and E) and five serotypes could be detected: FAdV-A1 (15; 32.6%), FAdV-E8a (15; 32.6%), FAdV-D11 (14; 30.4%), FAdV-D9 (1; 2.2%) and FAdV-E6 (1; 2.2%).

3.2. Recombination and Phylogenetic Analysis

The recombination analysis performed with the 173 partial hexon gene sequences, including both publicly available and the 46 newly obtained sequences, demonstrated two possible recombination events in the partial region of the hexon gene here evaluated (nucleotides 343 to 870). The first event (Event 1) was identified in sequence SB033_D_9_Brazil_2021, one of the samples sequenced in this study. It showed recombination with KY229183_D_11_Brazil_2016 as the minor parent, while the major parent could not be determined. This event was supported by six out of seven detection methods (RDP, GENECONV, MaxChi, Chimaera, SiScan, and 3Seq). The second event (Event 2) involved sequence AF339916_B_5, which recombined with KP274034_B_IvoryCoast_2012 as the major parent, while the minor parent remained unidentified. This event was also supported by six detection methods (RDP, GENECONV, MaxChi, SiScan, and 3Seq) (Table S2).

Phylogenetic analysis demonstrated that the 173 partial hexon sequences clearly clustered into separate clades according the five FAdV main species: FAdV-A (n = 26), FAdV-B (n = 39), FAdV-C (n = 18), FAdV-D (n = 43) and FAdV-E (n = 47). FAdVs from Brazil grouped into three clades: FAdV-A (n = 19, including 15 from this study), FAdV-D (n = 34, including 15 from this study), and FAdV-E (n = 37, including 16 from this study) (Figure 1).

Regarding serotype, FAdV-A species clade had all 26 strains from serotype 1 (26/26; 100%); FAdV-B species clade had all 39 strains from serotype 5 (39/39; 100%); FAdV-C species clade had 17 strains from serotype 4 (17/18; 94.5%) and one from serotype 10 (1/18; 5.5%); FAdV-D species clade had one strain from serotype 3 (1/43; 2.3%), two from serotype 9 (2/43; 4.7%), three from serotype 2 (3/43; 7%) and 37 from serotype 11 (37/43; 86%); and FAdV-E species clade included one strain from serotype 7 (1/47; 2.1%), three from serotype 6 (3/47; 6.4%), eight from serotype 8b (8/47; 17%), 35 from serotype 8a sequences (35/47; 74.5%) (Figure 1).

Among the sequences of the present study (n = 46), five serotypes could be identified within the three FAdV species (A, D and E): FAdV-A had one serotype (A1), FAdV D two serotypes (D9 and D11) and FAdV-E two serotypes (E6 and E8a). The clustering into serotypes specific clades was supported by bootstrap values of >70% (Figure 1). Serotypes identified in this study could be further separated into subclades, which were also supported by high bootstrap values.

In addition, a nucleotide similarity analysis of the partial sequenced hexon gene region (nucleotide positions 343 to 870) was performed among all here sequenced FAdVs (n = 46) and GenBank reference sequences (n = 55). Sequences from this study classified as FAdV-A1, D9, D11, E6, and E8a shared nucleotide identities ranging from 97 to 100% (FAdV-A1), 98% (FAdV-D9), 79 to 100% (FAdV-D11), 99% (FAdV-E6), and 54 to 100% (FAdV-E8a) (Table S3).

3.3. Amino Acid Substitution in the Hexon Protein

Amino acid substitutions in a partial segment of the Hexon protein (amino acid positions 115 to 286) were analyzed to identify hotspots and detect molecular signatures specific to serotypes and lineages. This analysis included 127 sequences retrieved from GenBank and 37 sequences obtained in this study (Table S4). The results are summarized in

Table 1. Amino acid variations in the HVR1 region of the hexon gene among different FAdV serotypes. Positions are numbered according to the alignment of the hexon gene region (148–158 aa and 193–194 aa). Only representative substitutions are shown; dots (.) indicate identity with the reference (FAdV-1). The complete alignment of the hexon region (115–286 aa) is available in Table S5.

Serotypes	148	149	150	151	152	153	154	155	156	157	158	193	194
FAdV-1	N	V	V	G	Q	M	T	N	V	Y	T	S	R
FAdV-2	V	I	T	.	.	.	.	T	P	.	E	D	E
FAdV-3	V	I	T	.	L	.	.	.	P	.	Q	D	D
FAdV-4	S	A	S	.	.	L	S	.	.	.	.	R	R/Q
FAdV-5	V	I	T	.	.	.	.	H	P	.	A	A	T
FAdV-6	I	I	T	.	.	.	.	.	P	.	E	.	S
FAdV-7	S	I	T	.	.	.	.	.	P	.	S	T	T
FAdV-8a	T	I	T	.	.	.	S	.	P	.	E	.	T
FAdV-8b	T	I	T	.	.	.	.	.	P	.	K	T	P
FAdV-9	V	I	T	.	L	.	.	T	P	.	R	D	D
FAdV-10	T	A	S	.	.	L	S	.	.	.	.	R	Q
FAdV-11	V	I	T	.	.	.	.	T	P	.	E	D	A

and

Table 2. Amino acid variations in the HVR1, HRV2, HRV3 and conserved motifs of the Hexon protein among different FAdV intra-species. Positions are numbered according to the alignment of the Hexon peptide chain (142–273 aa). Only representative substitutions are shown; dots (.) indicate identity with the reference (FAdV-A1). The complete alignment of the sequenced hexon gene region (115–286 aa) is available in Table S6.

Species	142	143	145	146	148	154	155	162	164	165	166	167	171	178	198	213	215	235	241	243	244	245	253	259	273
FAdV-A1	S	T	P	Q	N	T	N	R	D	K	T	A	Q	V	D	G	K	Q	M	N	G	G	A	Q	T
FAdV-C4	T	A	G	N	S	S	.	S	T/S	.	D	T	.	.	A	Q	N	.	.	.	A/T	.	.	N	E
FAdV-C10	T	A	G	N	T	S	.	.	S	.	A	T	.	.	.	R	.	.	.	.	A	.	.	N	D
FAdV-D2	T	G/E	N/S	K	V	.	T	Q	A	.	D	K	I	.	./V	N	R	P	.	S	S/T	A/.	G	A	.
FAdV-D3	T	D	N	K	V	.	.	Q	N	N	.	N	I	.	.	N	R	P	.	.	T	.	G	T	S
FAdV-D9	.	E	N	K	V	.	T	Q	N	.	.	N	V	.	.	N	R	P	.	K	T	D	.	T	S
FAdV-D11	T	E	S	K	V	.	T	Q	A	.	D	K	I	.	A	N	R	P	.	S	N	A	G	A	.
FAdV-E6	D	E	N	K	I	.	.	Q	S	P	.	.	A	T	T	N	R	A	L	S	T	S	G	.	S
FAdV-E7	E	D	D	K	S	.	.	Q	T	P	.	.	A	S	A	N	R	A	L	S	T	S	G	.	S
FAdV-E8a	E	D	N	K	T	S	.	Q	T	A	A	E	V	S	A	N	R	A	.	S	.	S	G	D	L/Y/I
FAdV-E8b	D	D	N	N	T	.	.	Q	T	A	.	.	A	S	A	N	R	T	L	T	T	A	G	.	S

, Tables S5 and S6.

Importantly, FAdV HVR1 into Hexon protein exhibited one small segment with 11 (148 to 158) and another with two (193 to 194) amino acid substitutions among FAdVs serotypes. The first segment could differentiate serotypes 1, 3, 4, 5, 6, 7, 8a, 8b, 9 and 10 since it presented the following and specific amino acids sequences: NVVGQMTNVYT to serotype 1, VITGQMTHPYA to serotype 5, TASGQLSNVYT to serotype 10, SASGQLSNVYT to serotype 4, VITGLMTNPYQ to serotype 3, VITGLMTTPYR to serotype 9, IITGQMTNPYE to serotype 6, SITGQMTNPYS to serotype 7, TITGQMTNPYK to serotype 8b and TITGQMSTPYE to serotype 8a. The second segment was capable of differentiating serotypes 1, 5, 2, 11, 6, 7, 8b, 8a, having the following amino acids: SR to serotype 1, AT to serotype 5, DE to serotype 2, DA to serotype 11, SS to serotype 6, TT to serotype 7, TP to serotype 8b and ST to serotype 8a (Table 1 and Table S5).

Furthermore, some additional amino acids specific positions allowed intra-species differentiation of FAdV serotypes and strains. FAdV-C serotypes 10 and 4 exhibited seven amino acid substitutions, while FAdV-D serotypes 2, 9, 11 and 3 had five main changes. FAdV-E serotypes 6, 7, 8b and 8a present an even higher amino acid substitution frequency, totaling 16 hotspots (Table 2 and Table S6).

4. Discussion

FAdVs have been reported in several countries, resulting in significant economic losses to the poultry industry and being associated with a range of diseases. These include IBH, HHS, and AGE, as well as respiratory and enteric disorders [9,35]. In this study, FAdV was detected in different tissues, sites of active viral replication, through PCR amplification of the 52K gene [21]. In addition, partial sequencing of the hexon gene was performed to identify FAdV species, serotypes, and even lineages. Among the Brazilian sequences analyzed in the present study, FAdV-A1 and FAdV-E8a were predominant. The predominance of FAdV-D and FAdV-E species in poultry production in Brazil has already been reported by other authors [8,36,37,38]. HHS is mainly caused by FAdV-D and FAdV-E [9]. On the contrary, FAdV-A1 has been associated with AGE as previously reported [39].

Phylogenetic analysis based on the partial nucleotide sequence of the hexon gene classified the sequences into five distinct clades (one for each species) and confirmed the occurrence of the species FAdV-A (serotype 1), FAdV-D (serotypes 11 and 9), and FAdV-E (serotypes 8a, 8b, and 6) in Brazilian poultry farms. In addition, sequences from this study and downloaded from the GenBank also revealed that local FAdV viral strains clustered with sequences from other countries. FAdV-A1 Brazilian sequences clustered with reference strains from China and Japan, FAdV-D11 with strains from China and Ecuador, FAdV-D9 with strains from Canada and Japan, FAdV-E6 with strains from Japan and Ecuador, and FAdV-E8a with strains from China and Japan. These results emphasize the worldwide distribution of these FAdV species.

Noteworthy, an increasing number of FAdV clinical cases have been described globally. In Europe, FAdV-E (serotypes 8b and 8a) and FAdV-D (serotypes 11 and 2) have been more frequent in Spain [40]. In Asia, FAdV-C (serotype 4) and FAdV-E (serotypes 8a and 8b) seem to predominate in China [41], FAdV-D and FAdV-E (serotypes 11 and 8b, respectively) in India [42]. In Korea, a study shows that FAdV-E (serotype 8b) is replacing FAdV-C (serotype 4), possibly due to selective pressure for the use of vaccines [43,44]. In Africa, three species (FAdV-A, FAdV-D and FAdV-E) have been associated with poultry diseases in Egypt [34]. In North America, FAdV-C4 was demonstrated to be the most prevalent in Mexico [7]. In South America, different FAdVs species were associated with poultry problems in Ecuador and Chile [17,18].

Furthermore, an analysis based on the amino acid sequences of the Hexon proteins of FAdVs was performed. The alignment of the different FAdV serotypes revealed important differences between species, serotypes and also strains. Specific amino acid residues were identified in this protein, highlighting some hyper variable segments, consistent with previous reports [12,34]. Some of these regions produce antigenic epitopes that play a critical role in the immunogenicity of the virus [34,45]. Previous studies suggested that pathogenic FAdV-C4 possess arginine (R), while non-pathogenic FAdVs of this same species/serotype possess isoleucine (I) at position 188 [46]. Here, amino acid substitution analysis was performed to complement the nucleotide-based phylogenetic analysis and to provide additional resolution for the differentiation of serotypes and lineages. This analysis allowed the identification of molecular signatures that differentiate lineages and enable further intraspecific evaluation of FAdVs.

In addition, some FAdV sequencing data obtained in this study have suggested the occurrence of some mixed infections with more than one FAdV species/serotype in the poultry flocks. PCR-based methods combined with restriction enzyme analysis have already demonstrated that co-infections with different FAdV serotypes occur frequently in broiler chickens [13,22]. Mixed infections with two or more adenovirus serotypes have also been frequently reported worldwide [13,22,47,48]. In Brazil, De La Torre et al. [8] identified the presence of serotypes FAdV-8b and FadV-11 co-circulating in chickens. However, it remains unclear whether such mixed infections influence disease severity or involve synergistic interactions between serotypes. The detection of multiple serotypes in the same poultry flock may suggest either additive pathogenic effects (where one serotype enhances the impact of another) or potential cross-protection among them [13,49,50]. These findings reinforce the importance of robust molecular epidemiology for effective field surveillance, ensuring accurate detection of all circulating types. This approach is essential for guiding the formulation of effective multivalent autogenous vaccines and for avoiding misinterpretation of co-infections as the emergence of new serotypes or species.

As presented above, this study provided an overview of the diversity of FAdV species/serotypes on commercial poultry farms in Brazil. This raises curiosity about more specific epidemiological questions about FAdV. Unfortunately, the study samples are from a third-party laboratory that performs diagnostics for other companies, lacking access to more detailed epidemiological data, such as the precise geographic distribution of farms (Brazilian states and regions), the types of birds (broilers, layers, breeders), and detailed clinical symptoms in the sampled flocks. Furthermore, some organs collected for analysis may not have been optimal for identifying some of the more specific clinical manifestations of FAdV in poultry. Further studies are needed to better understand and correlate the FAdVs circulating in the country, associating them with clinical cases and outbreaks of diseases related to this virus.

To prevent and control viral poultry diseases in endemic areas, it is recommended that affected flocks be properly immunized. Different vaccines have been used in commercial poultry, including inactivated, live-attenuated, subunit, and combined strains. Some FAdV species specific vaccines have been most commonly used in the large-scale production of commercial vaccines [9,35,51,52,53]. However, recent serological and molecular data on many FAdV species/serotypes in specific production regions/farms have encouraged the production and use of inactivated autogenous vaccines [35]. On some farms where more than one FAdV serotype has been detected, different FAdV strains should be included in the vaccine formulation, offering the best chance of inducing protection, which is usually serotype-specific [9]. The use of autogenous vaccines against specific FAdVs has proven very effective in controlling outbreaks [40]. The development and adequate production of these vaccines, as well as the implementation of a successful vaccination program, first require adequate molecular characterization of the prevalent FAdV species/serotype. Therefore, continuous molecular epidemiological surveillance of species and serotypes should be performed in commercial poultry production flocks suspected of being infected with pathogenic FAdVs.

5. Conclusions

This study identified the main FAdV species and serotypes (FAdV-A1, FAdV-D11, FAdV-D9, FAdV-E8a, and FAdV-E6) circulating in commercial poultry farms in Brazil from 2020 to 2023. This knowledge will be important for more effective prevention/control of diseases caused by FAdV, which have been frequently observed in many commercial poultry farms (layers and broilers) in Brazil.