Rotaviruses A and C in dairy cattle in the state of Rio de Janeiro, Brazil

Abstract

Stool samples were collected from calves from nine family-based small dairy farms in the state of Rio de Janeiro, for detection and characterization of rotavirus (RV) species A, B, and C (RVA, RVB, and RVC, respectively) by reverse transcription polymerase chain reaction. Twenty-six samples (27.7%) were positive for at least one of the species: 22 (23.4%) samples were positive only for RVA, 3 (3.2%) were positive for RVC, and one sample (1.1%) had co-infection of RVA and RVC. RVB was not detected. Seven (21.9%; n = 32) animals with diarrhea and 19 (30.1% n = 62) asymptomatic animals were positive, with no significant difference in positivity (p = 0.3677). RV was detected in all properties studied, at rates between 14.3 and 80%, demonstrating the widespread circulation of RV in four of the seven geographic regions of the state of Rio de Janeiro. Infection was more prevalent among animals ≤ 6 months of age. Sequence analysis of a portion of the RVA VP6-encoding gene identified the I2 genotype. RVC was also detected; to our knowledge, this is the first description of this agent in cattle in Brazil. The data presented here should add knowledge regarding the importance and prevalence of RV in our national territory, and may facilitate the planning and implementation of control and prevention measures for bovine rotavirus infections in Brazil.

Introduction

Gastroenteritis is one of the most important diseases of cattle, primarily affecting young animals, thereby causing great economic losses for producers. Such economic losses are due to the expense of treating and managing sick animals, as well as developmental delays that postpone breeding, in addition to mortality [1, 2]. Rotaviruses (RVs) are important agents of gastroenteritis in intensively reared newborn calves. Disease usually occurs in young calves of 2 to 8 weeks of age. Susceptibility decreases with age, most likely due to physiologic changes and/or acquired immunity from previous exposures [1, 3, 4].

Cattle farming is one of the primary activities of Brazilian agribusiness, and has an essential role in the generation of foreign exchange [5]. Due to the importance of the meat and dairy industries to the Brazilian economy, monitoring of livestock is crucial for the detection of pathogenic viruses, for the understanding of microbial ecology, and for the establishment of prevention policies, thus leading to greater farm productivity. Although RVs are among the leading etiologies of gastroenteritis [6], there is still no monitoring of their enzootic circulation by federal agencies in Brazil; RV infection is also not included on the list of notifiable diseases that must be reported to the official veterinary service [7]. Vaccination, although available, is not mandatory [8].

RVs have zoonotic potential, demonstrated by human isolates that have genotypes of animal origin and that suggest interspecies transmission [9–12]. Transmission can occur through direct contact with infected animals and also through contaminated water, food, and surfaces, which together with gene reassortment can lead to vaccine failures [13–15]. In addition, vaccination itself can impose selective pressure that contributes to the emergence of novel immune escape variants [16]. Thus, the continuous surveillance of RV strains circulating in herds is critical.

Although the circulation of RV has been reported previously in Brazil, most studies have been focused on RV species A. Furthermore, there are no data available in the scientific literature regarding RV infections in the cattle herd in the state of Rio de Janeiro. Thus, this work will contribute to the monitoring of the circulation of RV species A, B, and C (RVA, RVB, and RVC, respectively) in cattle in Rio de Janeiro, expanding knowledge regarding the epidemiology of this virus in national cattle farming.

Materials and methods

Sample collection

This study was approved by the ethics committee on animal research of the Empresa de Pesquisa Agropecuária do Estado do Rio de Janeiro (PESAGRO, Rio de Janeiro, RJ, Brazil), process number 1/2014.

Fecal specimens from 94 calves up to 10 months of age were collected between October 2013 and September 2014. The calves were from nine family-based small dairy farms located in the following municipalities within the state of Rio de Janeiro, Brazil: Northwest Aperibé and Itaocara, North Campos de Goytacazes, and Southeast Itaboraí and Sampaio Corrêa. As these are small rural properties, with a reduced number of animals in each herd, it is common to sell and/or exchange calves and/or heifers, as well as adult animals, between owners, depending on the time of year (season dry season or rainy season), the scarcity of pasture available on the property to feed the animals, and the financial need of the owner. Most of the properties did not have sanitary control and zootechnical data and for this reason it was not possible to verify the vaccination status of the herd. There is also the introduction of animals into herds acquired at auctions, as well as the movement of animals between properties during agricultural exhibitions, often without complying with the presentation of the GTA (Animal Transit Guide), and, generally, without the period of “quarantine” is completed, before its introduction into the herd, along with the other animals. This was an observational analytical study using convenience samples. In this way, the veterinary collaborators of the study visited the properties according to the previously scheduled visit calendar and collected fecal samples from the herd without the need for prior notification of cases of diarrhea. Between 4 and 15 samples were collected per farm, depending on the size of the herd. Samples were collected either directly from the rectum of animals gathered in handling corrals or with the aid of swabs when needed, and were stored at − 20 °C. Thirty-two samples were from diarrheal animals and 62 were obtained from asymptomatic animals.

Virus detection

Stool suspensions were prepared in 10% (w/v) phosphate-buffered saline (pH 7.2) and then centrifuged at 2,500 × g for 5 min. Nucleic acid was extracted from 300 μL of the supernatant using the guanidine isothiocyanate-phenol–chloroform method. Specimens were tested for the presence of RVA, RVB, and RVC by reverse-transcription PCR (RT-PCR) amplification of a target fragment of the VP6-encoding gene, using the protocol described previously [17, 18]. PCR products were separated by 1.2% (w/v) agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. A 100-bp DNA ladder (Ludwig Biotec) was used to determine molecular size.

PCR products from twelve RVA-positive samples were further analyzed by sequencing. Overlapping sequences were assembled and edited using SeqMan, EditSeq, and MegAlign in the Lasergene software package (DNASTAR). Phylogenetic analysis was performed with MEGA software (v.7.0.14; https://www.megasoftware.net/) [19]. A dendrogram was constructed using the maximum likelihood method based on the Kimura 2-parameter model. Statistical significance was estimated by bootstrap analysis with 1,000 pseudoreplicates. Sequences were compared to reference RVA strains from GenBank (https://www.ncbi.nlm.nih.gov/nucleotide/). Sequences generated in our study were deposited into GenBank under accessions OL441149-OL441160. Nucleotide identities were determined by using the MegAlign p-distance algorithm.

Statistical analysis

Statistical analyses were performed using the chi-square test. Statistical significance was defined by p-value < 0.05.

Results

RV was detected in the stool of 26 (27.7%) of the 94 calves tested. Twenty-two (23.4%) samples were positive only for RVA, 3 (3.2%) were positive for RVC, and 1 sample (1.1%) had co-infection of RVA and RVC. RVB was not detected. Seven (21.9%; n = 32) animals with diarrhea and 19 (30.1%; n = 62) of the asymptomatic animals were positive, with no significant difference in positivity (p = 0.3677). RV was detected in all studied properties, at frequencies between 14.3 and 80%, demonstrating the widespread circulation of the virus in four of the seven geographic regions of the state of Rio de Janeiro (North, Northwest, Metropolitan, and Lakes) (Table 1).

Table 1.

Distribution of RV among cattle in the state of Rio de Janeiro, Brazil

Region*	City	Samples tested	Positive samples n (%)
NO	Aperibé	23	5 (21,7)
	Itaocara	32	8 (25)
N	Campos de Goytacazes	14	2 (14,3)
M	Itaboraí	15	3 (20)
L	Sampaio Corrêa/Saquarema	10	8 (80)

^*NO northwest, N north, M metropolitan, L Lagos, RV rotavirus

When analyzing viral circulation in each separate municipality, we observed that (i) in the municipalities of the Northwest region, Aperibé and Itaocara, prevalence rates were 21.7% and 25%, respectively; (ii) the highest prevalence (80%) was observed in Sampaio Corrêa/Saquarema, Lakes region; (iii) 20% of the animals tested in the municipality of Itaboraí in the Metropolitan region were infected by RV; (iv) the municipality of Campos de Goytacazes, in the North region of the state presented a prevalence of 14.3%; (v) RVA was detected in all municipalities, while RVC was detected only in Itaboraí and Itaocara. A co-infection (RVA + RVC) was detected in Itaocara. The two RVC-positive samples detected in the municipality of Itaboraí were obtained in December 2013, while the two samples from Itaocara were obtained in August 2014, demonstrating the independent circulation of the virus in different herds.

Among 56 animals for whom age was reported, 48 were ≤ 6 months old, and 33.3% (16/48) of these had RV infection, 3 of whom had diarrhea at the time of sample collection. Six of the 8 (75%) oldest animals (7 to 10 months of age) had subclinical infections. Among animals whose age was not reported, 15.8% (6/38) tested positive for RV and 4 had diarrhea. RVA was detected at all ages, whereas RVC was only detected in younger animals (≤ months of age). Co-infection of RVA and RVC was detected in a 3-month-old animal.

Sequence analysis identified the I2 genotype, forming a clade with bovine strains detected in Brazil, India, and Japan (Fig. 1). Interestingly, the strains in this clade showed greater nucleotide identity with the simian RV strain SA-11-H6 than with other bovine RVA strains (89.4–97.6%).

Fig. 1.

Dendrogram constructed from partial sequences of the VP6 of bovine RVA strains. The distances were corrected using the Kimura 2-parameter model and the dendrogram was constructed using the maximum likelihood method. Statistical support was provided by bootstrapping 1000 pseudoreplicates. Bootstrap values above 75% are given as branch nodes. Genbank reference strain accession numbers are shown next to the strain identification. Strains detected in this study are indicated by red diamonds. Strains previously identified in Brazil are indicated by black circles

Discussion

Although many studies have elucidated the epidemiology of RV among cattle, there are few reports regarding its prevalence among Brazilian cattle [20–27]. In this context, this study stands out as the first report of the detection of RV in cattle feces in the state of Rio de Janeiro.

The RV prevalence found in this study (27.7%) can be considered high if compared to the reported Brazilian national prevalence rates, which vary between 2.5 and 19.4% [20–27]. The difference in positivity possibly reflects the sensitivities of the methodologies employed. Some studies used the combination of enzyme immunoassay and polyacrylamide gel electrophoresis [20–23], while others used RT-PCR [24–27], which was used in the present study, and has greater sensitivity. Another factor that may have affected the positivity rate is the origin of the samples. Most of the aforementioned studies were carried out in the states of São Paulo, Goiás, Paraná, Mato Grosso, Mato Grosso do Sul, and Minas Gerais, with samples from dairy herds belonging to producers associated with a dairy cooperative in which the animals were managed with adequate nutritional and health practices [20–27]. In this study, samples came from herds belonging to small producers, which lack sanitary and zootechnical control.

An important finding was the detection of asymptomatic circulation in 30.1% (19/62) of the studied animals. Asymptomatic RV excretion by cattle has been reported previously [21, 22, 24, 28, 29]. The prevalence of asymptomatic infections is an important factor from an epidemiological point of view, as carrier animals can serve as a reservoir of infection. Another important observation was a 75% prevalence of asymptomatic carriage among older animals (> 6 months of age). This is a relevant finding, because the literature reports that older animals are the main source of infection in newborn animals [1, 3].

Among the RV species infecting cattle, RVA is the most frequently detected, whereas RVC is observed sporadically [3, 30–33]. In this study, RVA was detected in 84.6% (22/26) of positive samples; RVC was detected in 11.5% (3/26) and co-infection was detected in one sample (3.9%). Bovine RVA and RVC coinfection has been reported previously [31]. Perhaps the low prevalence of RVC in our study was due to the age of the studied animals, which were mostly young, while RVC infection is observed primarily in adult animals and is associated with decreased milk production [34]. Although RVC prevalence was relatively low, our finding is relevant because it is the first report of RVC infection in Brazilian cattle.

The absence of RVB was not surprising since this species has not yet been described in Brazilian cattle. However, the age of the studied animals may have been an important factor because infection by this agent is generally described in adult animals [35, 36], while most samples analyzed in our study came from young animals.

Sequencing analysis of RVA demonstrated the spread of these viruses within the Rio de Janeiro herd, since strains detected in 3 distinct regions (Lakes, Metropolitan and Northwest) regions exhibited nucleotide identity of > 98%. A previous molecular characterization of the VP6-encoding gene demonstrated the circulation of genotypes I1, I2, and I5 among bovine RVA strains [37]. The phylogenetic analysis of the strains detected in this study identified the I2 genotype, which is the most common among cattle. Other studies have also described the circulation of the I2 bovine genotype in Brazil [16, 38].

Previous studies have reported the similarity between bovine and simian RVA strains [39, 40] that included Brazil isolates [16]. Our dendrogram disclosed that the analyzed strains formed a clade with other bovine RVA I2 strains from Brazil (Y136), India (RUBV51 and RUBV117), and Japan (Azuk-1 and Dai-10), and with the simian RVA SA-11-H96. Recently, an analysis of multiple RVA strains from different host species revealed three distinct clades within the I2 genotype [41]. Bovine strains group into the three clades; however, strains belonging to clade 3 include bovine, human, and deer viruses, and the simian strain SA-11-H96. A bovine strain containing VP7 of simian origin has also been described [42]. These findings suggest that these strains may have emerged from gene reassortment in these host species.

Failures of vaccine protection have been attributed to several factors, including antigenic differences between vaccine and circulating RVA strains [1]. Several studies conducted in Brazil confirm this assessment, and reinforce the need for continuous monitoring of circulating strains and analysis of their genetic and antigenic composition in comparison with vaccine strains [23, 25, 38, 43].

A striking feature of the RV genome is its propensity for genetic reassortment [13, 14, 44]. This has been considered the main evolutionary mechanism of RV, facilitating the crossing of the interspecies barrier and allowing the transmission of heterologous strains and their adaptation to new host species [9–12, 14, 15]. Several studies have demonstrated gene reassortment between bovine RV strains and strains originating from other hosts, including humans, resulting in symptomatic infections [10–12, 15, 45–50]. In fact, the human infections caused by strains containing genomic segments of bovine origin have also been described in Brazilian children with diarrhea [51–54]. This finding emphasizes the urgent necessity of creating and maintaining a veterinary surveillance program similar to that of the human public health system. Sharing information between these two surveillance systems in a One Health paradigm would probably facilitate the understanding of the complex evolution of these viruses, in addition to guiding prevention and control programs for human and animal rotavirus infections.

Conclusions

This study demonstrated the circulation of RV among dairy cattle in all municipalities surveyed, covering 4 of the 7 regions of the state of Rio de Janeiro. The high RV prevalence found in our study suggests that this pathogen may be widely disseminated in the state’s cattle herd. The presence of RVC was also demonstrated, which, to our knowledge, is the first description of this agent in Brazilian cattle. Thus, the data presented here are relevant and should improve knowledge regarding the importance and dissemination of bovine RV in our national territory, and assist in the planning and implementation of control and prevention measures for bovine rotavirus in Brazil.

Author contribution

A. R. M. M. was responsible for sample analysis and data interpretation, and drafted the manuscript. G. S. M. contributed to methodology, sample analysis, and the reviewing and editing of the manuscript. N. S. was responsible for the conception and design of the study, contributed substantially to data analysis and interpretation, and critically revised the manuscript for important intellectual content. The final manuscript has been reviewed and approved by all the authors.

Funding

This study was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq grants 404984/2018–5 and 301469/2018–0), and the Fundação Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro, Brazil (FAPERJ, grant E-26/202.909/2017).

Declarations

Ethical approval.

The study was approved by the ethics committee on animal research of the Empresa de Pesquisa Agropecuária do Estado do Rio de Janeiro (PESAGRO, Rio de Janeiro, RJ, Brazil), process number 1/2014.

Conflict of interest

The authors declare no conflict of interest.

Disclaimer.

The funders were not involved in the study design, data collection, data interpretation, or the decision to submit the work for publication.