Paramyxoviruses are well-known pathogens of the central nervous and respiratory systems of many host species. In the last few decades, many novel paramyxoviruses have emerged causing devastating illnesses in different aquatic and terrestrial animals, including in some cases a species jump to humans [1]. Parainfluenza virus type 3 (PIV3) is an enveloped, single-stranded negative sense RNA virus within the Respirovirus genus of the Paramyxoviridae family [2, 3]. The Respirovirus genus includes human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), Sendai virus (murine PIV1) and bovine parainfluenza virus type 3 (BPIV3) [2]. In some instances when animals are subjected to high stressful conditions, infection with BPIV3 can contribute to tissue damage and immunosuppression, resulting in severe bronchopneumonia from secondary bacterial infections [4]. The resulting disease is part of the bovine respiratory disease complex (BRDC) and is considered as the most significant illness associated with feedlot cattle in the USA [5], and possibly worldwide. Other respiratory viruses such as bovine herpesvirus 1, bovine viral diarrhea virus (BVDV) and bovine respiratory syncytial virus (BRSV) have also been associated with BRDC development in feedlot cattle [6]. The clinical presentation of bovine infections with BPIV3 can vary considerably, ranging from asymptomatic infections to severe respiratory illness. In most cases where BPIV3 is implicated in disease, usual clinical signs include coughing, fever and nasal discharge [6].

Three genotypes, A (BPIV3a), B (BPIV3b) and C (BPIV3c) have been described, based on genetic and phylogenetic analysis [7]. The BPIV3b genotype could hypothetically be a lineage from a strain that recently crossed from another host species into cattle [8]. PIV3 infections were found in a wide variety of mammals including cattle, humans, sheep [9], goats [10], bison [11], guinea pigs [12], black and white rhinoceros [13], moose [14], bighorn sheep [15] and camels [16]. Cross-species infections have been reported in numerous instances, including HPIV-3 in guinea pigs [12], BPIV3 in a human, BPIV3 in sheep, and ovine PIV3 in cattle [17]. In Argentina, serological studies conducted in 1980 and 1984 showed a high incidence of antibodies against BPIV3 (77%) in cattle from the main livestock breeding regions [18, 19]. In addition, positive serology was reported in domestic and wild South American camelids [20–22] and the virus was isolated from cattle and sheep [23]. However, little is known about the circulation of BPIV3 in cattle and other ruminants in Argentina, as well as about the genotypes that are present in this Southern American country.

Production systems have evolved to mixed managements, where alternative production species, such as the water buffalo (Bubalus bubalis), coexist in the same habitat as with cattle. Water buffalo breeding represents an important economic alternative in Argentina, which allows access to national and international markets. This species is susceptible to several viral infections [20, 24, 25] including BPIV3 as reported in a very early in 1966 in Egypt [26]. In addition to its potential relevance with respect to water buffalo health, this finding bears epidemiological significance due to the risk of transmission of the virus to cattle. The aim of this study was to characterize antigenically and genetically BPIV3 isolates from a respiratory and reproductive syndrome outbreak in dairy buffaloes and compare the buffalo isolate with contemporaneous BPIV3 circulating in cattle.

In the present study, we describe the isolation of BPIV3 from two water buffaloes with respiratory and reproductive signs. The virus was isolated from nasal and vaginal swabs from two out of twelve animals with reproductive and respiratory signs studied. To our knowledge little is known about the pathogenesis of this virus in buffalo. Should there be a syndrome similar to that present in cattle where the virus is transiently excreted [27], the unsuccessful virus isolation from the then remaining symptomatic animals could be related to the time point of sampling, which could have taken place at the end of the acute infection.

The IF assay showed strong and specific binding of anti-BPIV3 antibodies to cells infected with these three positive clinical isolates. Sera from buffaloes were able to neutralize the activity of the 7 N, 7 V and 2 V isolates and that of the reference bovine virus (BPIV3 strain SF), but the neutralization titers against the latter were lower than against buffalo isolates, suggesting antigenic differences.

The sequence of the M amplified fragment showed 94% nucleotide identity between buffalo isolates and BPIV3b strain Q5592 from Australia [8]. This is the first report of molecular characterization of a BPIV3 isolated from buffaloes. In order to determine homology between PIV3 isolates from Argentinean bovine and buffaloes, we characterized retrospectively six isolates collected from bovine respiratory outbreaks that occurred in 2009 and 2011. These isolates showed high percentages of nucleotide and amino acid identity with A and C genotypes (Table 1). There are only few reports about BPIV3 circulation in Argentina, Epstein et al described the isolation, virological and physicochemical characterization of PIV3 from Argentinean cattle and sheep in 1974 [23]. However, phylogenetic studies of these isolates were not carried out. Phylogenetic reconstructions based on the alignment of the M-gene nucleotide sequences demonstrated that the 7 N, 7 V and 2 V isolates clustered in the B genotype group. So far, this genotype has only been previously reported in Australia [8]. The phylogenetic position of the Argentinean buffalo isolates might be correlated with the antigenic differences observed by cross-neutralization studies, where the reference strain belonging to the BPIV3a group was lesser neutralized than the buffalo viral isolates by sera of buffaloes. Therefore the serological and molecular characterization of buffalo BPVI3 isolates show that these viruses are more similar to B than to A (SF reference strain) and C BPVI3 genotypes (SD0835 reference strain). Interestingly, the genotype B isolated from buffaloes was not found among the bovine samples, analyzed in this work, that clustered with genotypes A and C. Genotype B was until now observed only in Australia with the hypothesis of a recent crossing from another species into cattle [8]. This evidence of genotype B in buffaloes should need further investigation to know if this species could be one of the host species of this genotype. Therefore analysis of a larger number of isolates from cattle and buffaloes is needed to determine whether the three genotypes circulate in both species, or if the distribution observed is representative. Importantly, this is the first report of bovine B and C genotype circulation out of Australia and China, respectively. The circulation of genotype C only in China was attributed to a geographic restriction [7]. This hypothesis can be ruled out by our results and this observation was most likely due to the relative frequencies of these genotypes with respect to the samples analyzed.

To our knowledge this is the first report of the isolation of a parainfluenza virus type 3 from vaginal secretions. However, it is important to highlight that the presence of the virus in the secretions could be due to oro-genital contact between animals. Evidence of viral replication in the genital mucosa needs new investigations.

The isolation and characterization of BPIV3 from water buffaloes were reported. We identified the isolates as bovine parainfluenza virus type 3 genotype B that differs from genotypes A and C circulating in Argentinean cattle herds reported so far. Further studies would be required to determine if A and C genotypes also circulate in buffalo herds, as well as to elucidate the role of buffalo as reservoirs, especially of the B genotype, and in the transmission of this virus to cattle.