Detection of Bovine and Porcine Adenoviruses for Tracing the Source of Fecal Contamination

Abstract

In this study, a molecular procedure for the detection of adenoviruses of animal origin was developed to evaluate the level of excretion of these viruses by swine and cattle and to design a test to facilitate the tracing of specific sources of environmental viral contamination. Two sets of oligonucleotides were designed, one to detect porcine adenoviruses and the other to detect bovine and ovine adenoviruses. The specificity of the assays was assessed in 31 fecal samples and 12 sewage samples that were collected monthly during a 1-year period. The data also provided information on the environmental prevalence of animal adenoviruses. Porcine adenoviruses were detected in 17 of 24 (70%) pools of swine samples studied, with most isolates being closely related to serotype 3. Bovine adenoviruses were present in 6 of 8 (75%) pools studied, with strains belonging to the genera Mastadenovirus and Atadenovirus and being similar to bovine adenoviruses of types 2, 4, and 7. These sets of primers produced negative results in nested PCR assays when human adenovirus controls and urban-sewage samples were tested. Likewise, the sets of primers previously designed for detection of human adenovirus also produced negative results with animal adenoviruses. These results indicate the importance of further studies to evaluate the usefulness of these tests to trace the source of fecal contamination in water and food and for environmental studies.

Most vertebrates are infected by adenovirus species. The family Adenoviridae has been classically divided into two genera: Mastadenovirus (adenoviruses infecting mammals) and Aviadenovirus (adenoviruses infecting birds). Two new genera, Atadenovirus and Siadenovirus, have recently been approved (5). The genus Mastadenovirus presently contains, among others, 51 human adenovirus (HAd), 5 porcine adenovirus (PAV), and 6 ovine adenovirus (OAV) serotypes. Bovine adenoviruses (BAVs) have been separated into two subgroups mainly because of the lack of genus-specific complement antigen in the BAV subgroup II members (2). BAV subgroup I (BAV1 to BAV3 and BAV10) belongs to the genus Mastadenovirus, while BAV subgroup II (BAV4 to BAV8) has now been clustered in the genus Atadenovirus, which also includes atypical OAVs, such as isolate 287 (OAV287) and members previously classified within the Aviadenovirus genus, such as egg drop syndrome (EDS) virus (5, 7, 8, 15). Another viral genomic arrangement has been described and considered as a fourth adenoviral genus, suggesting that a great variety of adenoviruses could be widely distributed (9, 16).

In spite of the existence of reported cases of pneumoenteritis or encephalitis (19), PAVs do not normally produce clinically severe pathologies. Several serotypes of BAVs have been isolated from calves suffering from keratoconjunctivitis, acute febrile disease, or pneumoenteritis (19), and some, such as BAV10 and BAV subgroup II, have even been associated with acute and fatal episodes in cattle (28). Moreover, in vitro inoculations have resulted in viral recovery from swabs of nasal and conjunctival mucosa and from feces of all infected calves during the course of clinical observation, indicating that a large and continuous viral excretion follows transient viremia (19). Extended periods of excretion in feces have also been described in cases of HAd infection, including those involving strains producing respiratory diseases. The study of PAV, OAV, and BAV will thus provide new information about the molecular epidemiology of these viruses and the diversity and prevalence of adenoviruses infecting farm animals.

In a previous study, a methodology for the detection of HAd in environmental samples was developed and applied to the analysis of viral contamination in sewage, river water, seawater, and shellfish (25). HAds have been shown to be more prevalent and stable than enteroviruses in the environment and in shellfish in widely divergent geographical areas (13, 21, 24), and the nested PCR assay developed for their specific detection has been proposed as a molecular index for the presence of fecal contamination of human origin in the environment, water, and food (24). The sensitivity of this nested PCR test was previously established to be as high as one viral particle, and the same procedure has been applied to the detection of animal adenoviruses. In the present study, two sets of oligonucleotides were designed, one for the specific detection of BAV and OAV and the other for the specific detection of PAV, to evaluate the level of excretion of adenoviruses by farm animals and to devise a test to trace the specific source of viral contamination in the environment.

MATERIALS AND METHODS

Fecal samples.

Twenty-three swine fecal samples from seven different farms located within an area of 100 km² in Catalonia (Spain) were collected in two different periods, April 2001 and September 2002. The farms were selected so as to include samples from breeding and fattening animals, and the samples were pooled according to age and commercial purpose of the individuals. Samples from farms 1 and 2 were obtained from adult pigs of various ages, samples from farm 3 were obtained from fattening pigs (from 5 to 6 months old) and sows (up to 4 years old), and samples from farms 4 to 7 were obtained from adult fattening pigs (over 6 months old). Likewise, eight cattle fecal samples from two different farms were collected in April 2002. In this case, samples from farm 8 came from young fattening or breeding animals (from 3 to 18 months old), while samples from farm 9 came from calves and adult animals.

Sewage samples.

Twelve urban-sewage samples were collected monthly from the Barcelona sewage network between August 2000 and July 2001. The samples were collected at the entry of a depuration plant receiving 670,000 m³/day from the urban area of Barcelona, with a population of approximately 1,800,000 inhabitants, without a significant animal wastewater content. All samples were gathered in sterile 500-ml polyethylene containers and kept at 4°C for less than 8 h prior to analysis of viral-particle concentration in phosphate-buffered saline (PBS; pH 7.4). Samples were subsequently stored at −80°C.

Concentration of viral particles from fecal samples.

Pools of fecal samples from three, four, or five animals were prepared. One gram from each pool was eluted in 3.5 ml of 0.25 N glycine buffer (pH 9.5), kept in ice for 30 min, and then centrifuged (9,200 × g for 15 min) to separate any suspended solids. Finally, the supernatant was concentrated by ultracentrifugation (110,000 × g for 1 h at 4°C) and viral particles were resuspended in 100 μl of PBS and stored at −80°C.

Concentration of viral particles from sewage samples.

Recovery of viral particles was performed as described previously (25). Briefly, 40 ml of sewage was ultracentrifuged (110,000 × g for 1 h at 4°C) to pellet all the viral particles together with any suspended material. The pellet was eluted with 4 ml of 0.25 N glycine buffer (pH 9.5), and suspended solids were separated by centrifugation at 12,000 × g for 15 min. Viruses were finally concentrated by ultracentrifugation (110,000 × g for 1 h at 4°C), resuspended in 100 μl of PBS, and stored at −80°C.

Nucleic acid extraction.

The nucleic acids from the viral concentrates were extracted using a method selected in previous studies for its high sensitivity and efficiency in PCR inhibition. The procedure is based on the use of guanidinium isothiocyanate to denature viral capsid proteins and silica particles to bind viral nucleic acids (6). Finally, nucleic acids were eluted from the support in TE buffer (10 mM Tris-0.1 mM EDTA [pH 7.4]) and stored at −20°C.

Design of specific oligonucleotides.

Specific oligonucleotides for the detection of PAV and of BAV and OAV were designed based on alignments of all available sequences of the hexon gene present in the GenBank and EMBL (European Molecular Biology Laboratory) databases. Detailed sequences of the primer sets are presented in Table 1. Furthermore, two sets of oligonucleotides were designed to amplify a region of the protease gene of PAV and of the new genus Atadenovirus.

TABLE 1.

Oligonucleotide primers used for PCR amplification and sequencing of HAd, PAV, and BAV

Region	Virus type	Positiona	Amplification reaction	Primer	Sequenceb	Annealing temp (°C)	Product size (bp)
Hexon	HAd2
	HAd40	18858-18883	First left	HL	5′-GCCGCAGTGGTCTTACATGCACATC-3′	55	301
	HAd41	19136-19158	First right	HR	5′-CAGCACGCCGCGGATGTCCAAAGT-3′
	HAd2	18937-18960	Nested left	NHL	5′-GCCACCGAGACGTACTTCAGCCTG-3′	55	143
	HAd2	19051-19079	Nested right	NHR	5′-TGTACGAGTACGCGGTATCCTCGCGGTC-3′
Hexon	PAV	20627-20647	First left	PALF	5′-GATGTCATGGAYAACGTCAAC-3′	54	612
	PAV	21217-21238	First right	PARF	5′-CACGGAGGAGTCRAACTGGATG-3′
	PAV	20711-20733	Nested left	PALN	5′-TACTGCMAGTTYCACATCCAGGT-3′	57	344
	PAV	21035-21054	Nested right	PARN	5′-GGAATGGAGATGGGCAGGTT-3′
Hexon	BAV	19235-19256	First left	BALF	5′-GRTGGTCIYTRGATRTRATGGA-3′	52	641
	BAV	19852-19872	First right	BARF	5′-AAGYCTRTCATCYCCDGGCCA-3′
	BAV	19347-19370	Nested left	BALN	5′-ATTCARGTWCCWCARAARTTTTTTGC-3′	51	430
	BAV	19747-19769	Nested right	BARN	5′-CCWGAATAHRIAAARTTKGGATC-3′
Protease	PAV	19106-19130	First left	Pr-PALF	5′-CACCAGCCRCACMGAGGCGTCATCG-3′	60	659
	PAV	19741-19716	First right	Pr-PARF	5′-GGCGGTGKGMGCGRAAGTASGIGGA-3′
	PAV	19239-19259	Nested left	Pr-PALN	5′-CTTCCTGGGMACMTTTGACAA-3′	51	356
	PAV	19571-19592	Nested right	Pr-PARN	5′-TGTSGGGSYAGYKGICAAAGG-3′
Protease	BAV II	17380-17404	First left	Pr-BALF	5′-TCAGCTGGTAGTGCWRCRACATGAG-3′	52	677
	BAV II	18032-18056	First right	Pr-BARF	5′-GAAGAAGATGAWGAAGAMGAYTAA-3′
	BAV II	17472-17494	Nested left	Pr-BALN	5′-ACTTTTGATTGYMGATTTCCAGG-3′	46	625
	BAV II	17973-17996	Nested right	Pr-BARN	5′-TTTTATTAADCCAAKYTTWGTATT-3′

^aThe sequence positions for the HAds, PAVs, BAVs, and BAV subgroup II viruses refer to the HAd2, PAV3, BAV3, and BAV4 complete genomes, respectively.

^bM = A + C, Y = C + T, R = A + G, D = G + A + T, W = A + T, K = G + T, S = G + C, I = inosine.

Enzymatic amplification.

For the detection of viral DNA genomes in fecal samples, a nested PCR technique was applied using the primers described in Table 1. For a typical one-step reaction, 10 μl or a 10-fold dilution of the extracted nucleic acids (corresponding to 4 and 0.4 ml of sewage and to 100 and 10 mg of feces) were analyzed in a 50-μl reaction mixture containing 1× PCR buffer [160 mM (NH₄)₂SO₄-670 mM Tris-HCl (pH 8.8) at 25°C], 1.5 mM MgCl₂, 0.025 mM concentrations of each deoxynucleoside triphosphate, 25 pmol of each external primer (HL/HL for HAd detection, BALF/BARF and Pr-BALF/Pr-BARF for BAV detection, and PALF/PARF and Pr-PALF/Pr-PARF for PAV detection), and 2 U of Taq DNA polymerase (Bioron GmbH, Ludwigshafen, Germany). Thermal cycling of the amplification mixture was performed in a programmable heat block (Gene Amp PCR system 2400; Applied Biosystems). In all PCR assays for detection of HAd, the first denaturation cycle was performed for 4 min at 94°C. The conditions for the 29-cycle amplification were as follows: denaturing at 92°C for 60 s, annealing at 55°C for 60 s, and extension at 72°C for 75 s. Amplifications were completed with a 7-min extension step at 72°C. For BAV and PAV detection, the amplification conditions were as described above, except that the annealing temperatures were modified as indicated in Table 1. The external primers were used in these first 30 cycles of amplification, and then 1 μl of first-round product was added to a new 50-μl PCR mixture containing 0.16 μM concentrations of each nested primer (NHR/NHL for HAd, BALN/BARN and Pr-BALN/BARN for BAV, and PARN/PALN and Pr-PALN/PARN for PAV detection) in a new 30-cycle amplification. In all cases, amplifications were completed with a 7-min extension step at 72°C. The results were analyzed by gel electrophoresis on a 3% agarose gel (Genotek) stained with ethidium bromide.

Quality control of amplification method.

Standard precautions were applied in order to reduce the probability of sample contamination. Every sample was analyzed twice in independent experiments, and negative controls were added after every two samples. Positive amplifications were confirmed by sequencing of the amplified DNA.

Sequencing and analysis of amplicons.

Amplicons obtained after nested PCR were purified using a QIAquick purification kit (Qiagen, Inc.) according to the manufacturer's instructions. Sequencing reactions were prepared in both orientations with an ABI PRISM Big Dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase FS (Applied Biosystems) according to the manufacturer's instructions. Sequencing was performed using an ABI PRISM 377 analyzer (Applied Biosystems) by Serveis Científico-Tècnics of the University of Barcelona, and the sequences were compared with those in the GenBank database by using the BLAST program of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). Sequences were aligned using the ClustalX 1.8 program, and phylogenetic analysis was performed using the neighbor-joining program of the PHYLIP software package for comparison with representative sequences characterized previously.

Nucleotide sequence accession numbers.

The sequences reported in this paper have been deposited in the GenBank database under accession numbers AY288805 to AY288813 (porcine hexon sequences), AY288814 to AY288818 (bovine hexon sequences), and AY288819 to AY288821 (bovine protease sequences).

RESULTS

Swine fecal samples.

Twenty-three swine fecal samples were collected from seven different farms located within 100 km of each other in Catalonia (northeastern Spain). Five pools of samples from both the first and second farms were analyzed, and three pools from each farm were found to be positive for PAV. Likewise, from the third farm, five pools of samples, two from fattening pigs and three from sows of various ages, were tested. PAVs were detected in one of the two pools from fattening pigs and in two of the three pools from sows. We also analyzed two pools of samples taken from fattening pigs from each of the other four farms, and all of these were positive. Amplified regions within the hexon gene were 344 bp long. Nine of these regions were sequenced, and the nine strains of PAV were named Por1 to Por9.

The number of detectable viral particles in swine feces was estimated to be 10¹ to 10³ genome equivalents/g of pooled fecal sample by limiting decimal dilution analysis using the PAV hexon primers. No significant relationships were established between positive cases and the ages of animals or their commercial aim, even though excretion of PAV was detected in sows and especially in fattening pigs.

Cattle fecal samples.

In the two farms studied, three of the four bovine fecal samples were positive for the presence of BAVs. Both farms were located in the same area and were controlled by the same sanitary staff. The amplicons detected were 410 bp long and presented differences in their nucleotide sequences. The six identified strains were named BovA to BovF.

The number of viral particles was estimated to be 10¹ to 10⁴ genomic equivalents/g of pooled fecal sample by limit dilution analysis using the BAV hexon oligonucleotide set. Although more samples should be tested in order to establish reliable relationships, in this study half of the young-animal samples and all of the old-animal samples were positive for BAV.

Sewage samples.

Twelve sewage samples from the Barcelona sewage network were collected between August 2000 and July 2001. All samples were analyzed for PAVs, BAVs, and HAds by using the specific primers designed in the hexon region. As expected, none of the 12 samples were positive for PAV or BAV, whereas 11 samples were positive for HAd.

Sequence analysis of adenovirus excreted by swine.

The nucleotide sequence alignment of detected strains (Por1 to Por9) showed that all of the strains were closely related to the IAF strain of PAV type 3 (accession no. AJ237815) (Fig. 1). Sequences shared 93 to 98% similarity. This classification was confirmed by amplification of a 356-bp fragment of the protease gene, which showed 98% similarity to the PAV type 3 IAF strain.

FIG. 1.

Sequence alignment of the hexon regions of nine PAV-positive samples in reference to PAV3 strain IAF. Dots indicate sequence identities, and dashes indicate lack of nucleotide. M = A + C, Y = C + T, and W = A + T.

Sequence analysis of adenovirus excreted by cattle.

The detected sequences of the amplified region of the hexon protein were compared with the sequences available in GenBank (Fig. 2). Isolates BovD and BovE were identical (100%), but the highest similarity in comparison to reference sequences was only 82%, which was seen with BAV type 2 subtype A (accession no. AF252854). The detected strains BovA, BovC, and BovF showed a high similarity to the THT/62 strain of BAV type 4 (93 to 98%) (accession no. NC_002685). Similarity among them was approximately 92%. The BovB strain was identical (100%) to the Fukuroi strain of BAV type 7 (accession no. AF238232). Thus, these strains were closer to BAVs of subgroup II and therefore to the Atadenovirus genus.

FIG. 2.

Sequence alignment of the hexon regions of six BAV-positive samples. Dots indicate sequence identities, and dashes indicate lack of nucleotide. M = A + C, Y = C + T, and W = A + T.

To confirm the classification of BovB, BovC, and BovF in this new genus, we amplified a region of the protease gene in these strains, producing 525-bp amplicons. The sequences detected in BovA and BovC were identical (100%) to the BAV4 THT/62 strain, whereas BovB shared 98% similarity to the BAV7 Fukuroi strain (see Fig. 4 for the phylogenetic analysis of the detected isolates).

FIG. 4.

Phylogenetic reconstruction of BAV strains analyzed in this study. Reference strains of BAV2, BAV3, BAV4, and BAV7 (accession no. AF252854, AF030154, NC_002685, and AF238232, respectively), EDS (accession no. Y09598), FAV1 (fowl adenovirus type 1; accession no. U46933), HAd2, HAd12, HAd17, and HAd40 (accession no. J01917, X73487, AF108105, and L19433, respectively) are represented. The neighbor-joining tree was constructed using 437 nucleotides of a region of the protease gene of BAV genomes.

Oligonucleotide specificity.

The specificity of the designed oligonucleotides was initially tested by analysis of the sequences available in the databases. All of the strains detected, as well as HAd serotypes 2, 5, 12, and 41 at concentrations of 10⁷ viral particles/ml, were analyzed using the three sets of primers developed. The assays developed for the specific detection of HAd, PAV, and BAV produced positive results only when the corresponding strain was tested (Fig. 3). Even though some bands appeared in some cases, none of them showed the expected size, so they represented nonspecific amplifications. Moreover, 11 of the 12 urban-sewage samples that contained fecal contamination of human origin tested positive for HAds but were negative for amplicons with the porcine and bovine sets of primers.

FIG. 3.

Specificity assays performed with designed sets of oligonucleotides. Each adenovirus strain detected was tested with human (H), porcine (P), and bovine (B) oligonucleotides. The molecular sizes expected of the amplicons were 344 bp for swine, 410 bp for cattle, and 123 bp for human fragments. Lane M, molecular size marker (φX174 cleaved with HaeIII).

DISCUSSION

The PAV isolates found in this study were highly similar to the previously described PAV3 IAF strain (26). This is a strain derived from PAV3 strain 6618 and adapted to pig kidney cells and which has been isolated in several countries (10, 14). Nonetheless, levels of identity among the nine isolates were slightly greater than those between the isolates and the reference strain, and all of the isolates even showed a deletion of the same amino acid. Although confirmation through further studies is necessary, the similarity among these isolates suggests the existence of a stable variant of porcine serotype 3 in the area studied.

The BAV isolates detected showed a much greater variability, including sequences related to the two BAV subgroups described to date. The BAV2-like strain is a member of BAV subgroup I. This type has been divided into two subspecies (A and B) based on hemagglutination properties and restriction enzyme cleavage patterns (3). A noticeable variation in this serotype could thus be detected. Further studies are being performed to characterize this isolate. The BAV4-like and BAV7-like strains belong to BAV subgroup II and therefore to the Atadenovirus genus. Experimental infections associate serotype 4 with weak bronchopneumonia. Serotype 7 was first described in blood samples from a cow that suffered an acute feverish episode (17), suggesting that a temporary viremia is a pathogenic process common to BAV infections. Thus, the divergence was high, indicating that several serotypes may coexist in a reduced number of individuals. Diverse adenoviral distribution has already been discussed for BAVs in relation to the proposal that they evolved exclusively with their hosts (classical Mastadenovirus members) or may have extended their spectrum of hosts to related species, such as sheep (1, 4), deer (20), and even moose (27).

The phylogenetic study based on protease sequences clearly generated two clusters (Fig. 4). One cluster contains classical HAd, PAV, and BAV subgroup I, whereas the other includes atypical OAV and avian adenovirus as well as BAV subgroup II. Hexon analysis had already provided evidence to support this arrangement (data not shown). The protease gene has been widely used in phylogenetic analysis, leading to the conclusion that BAV subgroup II, together with EDS virus and OAV287, belong to a new genus named Atadenovirus because of the viruses' high content of adenines and thymines (5, 29). All of the bovine and porcine fragments studied shared the same active residues, His₅₁-Glu₇₁-Cys₁₂₂. This catalytic triad has been described in every adenovirus protease with the exception of HAd2 and HAd5, in which aspartic acid (Asp[D]) is found instead of glutamic acid (Glu[E]) (11). The Cys₁₀₃ residue, determined to be a binding site of the cellular activator pVI peptide (22), was also conserved. Nevertheless, the amino acid Pro₁₃₇, believed to be necessary for intracellular trafficking, was found in porcine strains but not in any of the bovine strains analyzed. This residue has been detected in all Mastadenovirus strains but not in Aviadenovirus or Atadenovirus (15).

Animal feces may contaminate the environment and water via runoff, drainage, or direct deposition or by contamination through slaughterhouse sewage. Although the analysis of a larger number of samples is necessary, this study reports a high PAV and BAV prevalence in animal feces, as has been described for HAd in humans. This finding as well as adenoviral features such as their DNA genome, uniform geographical and seasonal distribution (24), and stability under a wide range of environmental conditions (12, 18, 23) lead us to regard this viral family as a suitable indicator of viral contamination. The methodology reported in this work is reliable and specific. The sets of oligonucleotides used in the developed tests were designed for the hexon gene after consideration of all of the sequences available in the nucleotide databases, including those of Atadenovirus and Mastadenovirus. The hexon protein is composed of a variable triangular surface and two highly β-barrel regions called P1 and P2. Our amplified fragment is located within a P1-P2 shared region. Experimentally, neither the BAV nor the PAV primer sets used in the nested PCR assays produced nonspecific amplifications in the fecal samples and urban-sewage samples tested. Furthermore, the set of primers described previously for the detection of HAds produced negative results with any of the samples containing the detected animal adenovirus strains. In a previous study, the specificity of HAd oligonucleotides had already been evaluated using nine slaughterhouse sewage samples containing only fecal contamination of animal origin, such as cattle, swine, and sheep (24). Similarly, the primers designed for detection of animal adenoviruses produced negative results when the HAd strains Ad2, Ad5, Ad12, and Ad41 were tested at concentrations of 10⁷ viral particles/ml. These results suggest that further analysis of animal adenoviruses and evaluation of the potential application of these tests to identify PAVs and BAVs in addition to HAds may lead to a useful tool for tracing the source of fecal contamination in water, food, and the environment.