Molecular characterization of bovine rotavirus circulating in beef and dairy herds in Argentina during a 10-year period (1994-2003)

Group A bovine rotavirus (BRV) is one of the main causes of neonatal calf diarrhea. The present study reports the incidence of rotavirus diarrhea and the genotypes of BRV strains circulating in beef and dairy herds from Argentina, during a 10-year period (1994-2003). Group A BRV was detected in 62.5% (250/400) of the total studied cases of diarrhea. Positive cases were analyzed by heminested multiplex RT-PCR for P and G genotypes identification. Sixty percent of them were typed as P[5]G6, 4.4% P[11]G10, 4.4% P[11]G6 and 2.4% P[5]G10. Additionally, 9.2% of the cases were initially typed as G8 combined with P[5] or P[11], but sequence analysis revealed they belonged to genotype G6, lineage Hun4-like. Partial typing was assessed in 12.0% of the cases. One of the partially typed samples was closely related to genotype G15. BRV was detected in 71% and 58% of the outbreaks registered in beef and dairy farms, respectively. A clear differential distribution of G/P types was found according to the herd type. P[5]G6 was the prevalent strain in beef herds, while P[11] was the prevalent P-type in dairy herds (71%), associated in similar proportions with G6 and G10, These findings indicate that BRV genotypes included in the current commercially available rotavirus vaccines (G6, G10, P[5] and P[11]) should protect calves from most Argentinean field strains. Nevertheless, continuous surveillance is necessary to detect the emergence of new variants.     

Detection and analysis of bovine rotavirus strains circulating in Australian calves during 2004 and 2005

Bovine rotavirus (BRV) has been detected in both dairy and beef cattle herds worldwide. Stool samples collected from calves in the Gippsland region of Victoria, Australia were screened to determine the presence of BRV. A total of 100 faecal samples were collected from calves with and without diarrhoea across three farms during 2004 and 2005. Group A BRV was detected in 26% of faecal samples (22 from diarrheic calves and four from asymptomatic calves). Genotyping analysis of rotavirus positive samples indicated that G6P[5] was the most prevalent genotype (38.5%) followed by G6P[5 + 11] (15.4%). G10P[11] and G6 + G10P[5] were each detected at a rate of 7.7%, and G6 + G10P[11] was found in a single sample (3.8%). Seven samples (26.9%) could not be G and/or P typed. Thirty percent of the BRV positive samples were mixed infections, indicating that individual calves were co-infected with more than one strain of rotavirus. The G6P[5] strains exhibited high VP7 identity (>97% amino acid identity) with B-60, a G6 strain identified in Victorian calves during 1988. A G10P[11] isolate was closely related (>97% amino acid identity in VP7 and VP4 proteins) to a Victorian G10P[11] strain (B-11) also identified during 1988. This study demonstrates that BRV is a contributing pathogen to diarrhoeal disease in Victorian calves, with sequence analysis suggesting long-term conservation of the VP7 protein over a 16-year period.     

Molecular epidemiology of bovine toroviruses circulating in South Korea

The prevalence of the bovine torovirus (BToV) and its genetic characterization have been reported in North America, Europe and Japan. Therefore, this study examined the prevalence and genetic diversity of the BToV in a total of 645 diarrheic fecal samples from 629 Korean native beef calf herds using RT-PCR and nested PCR with the primer pairs specific to a part of the BToV membrane (M) gene. Overall, 19 (2.9%) out of 645 diarrheic samples from 19 herds (6.9%) tested positive for BToVs by either RT-PCR or nested PCR. A comparison of the nucleotide (nt) and amino acid (aa) sequences of a part of the BToV M gene (409bp) among the BToVs showed the Korean BToVs to have comparatively higher sequence homology to the Japanese and Dutch BToVs than to the American and Italian BToVs. Generally, the Korean BToV strains clustered with the Japanese and Dutch BToV strains. However, the American and Italian BToV strains clustered on a separate major branch, suggesting that these are more distantly related to other known BToV strains. These results suggest that the BToV infections are sporadic in diarrheic calves in South Korea, and the Korean BToV strains are more closely related to the Japanese and Dutch BToVs than to the American and Italian BToVs.     

Molecular characterization of a novel bovine group A rotavirus

In this study, by partial sequence analysis of the genome segments encoding VP5* and VP7, we characterized a novel bovine group A rotavirus, namely, Tak2, that was detected from adult cattle diarrhea in Tochigi Prefecture, Japan. The nucleotide (nt) and deduced amino acid (aa) sequences of the genome segments encoding VP5* and half of the amino terminal portion of VP7 of Tak2 revealed a low identity with those of group A rotaviruses carrying previously published P and G type specificities (VP5*: nt identity, 61.6%-67.6% and aa identity, 58.0%-71.4%; half of the amino terminal portion of VP7: nt identity, 57.8%-73.5% and aa identity, 61.2%-70.9%). Additionally, phylogenetic analysis of the nt sequences of the genome segments encoding VP5* and half of the amino terminal portion of VP7 revealed that Tak2 formed a branch separate from the established P and G types. These results suggested that Tak2 could possess novel P and G types yet not reported among group A rotaviruses.     

Molecular characterization of unusual bovine rotavirus A strains having high genetic relatedness with human rotavirus: evidence for zooanthroponotic transmission

We report here the genomic characterization of two rare rotavirus A (RVAs) G1P[11] and G9P[X] strains detected in cattle calves from two different geographical locations in India during routine rotavirus surveillance. These strains possessed unusual G types (VP7 gene) on a bovine/artiodactyl genotype constellation, G1‐P[11]‐I2‐Rx‐Cx‐Mx‐Ax‐N2‐T6‐E2‐H3 (HR‐B91) and G9‐P[X]‐I2‐Rx‐Cx‐Mx‐Ax‐N2‐T6‐E2‐H3 (WB‐H2). This is the first report on molecular characterization of G9 in cattle, and second report on G1 in cattle. The VP7 gene of HR‐B91 occupied lineage IIc within G1 while that of WB‐H2 occupied IIIb within G9 genotype. The latter was found to be very diverse from other RVA strains of G9 genotype, and this may emerge as a new genotype in due course. The study provides evidence of zooanthroponotic transmission of human G1 and G9 RVA genes to calves. Of note, the G9 genotype was found to serve as the ancestral genotype for G1. Phylogenetic analysis of remaining gene segments revealed close relatedness to artiodactyl or artiodactyl‐like human RVA strains. The findings of this study highlight the potential role of interspecies transmission and reassortment events in generating the rare rotavirus strains.     

Molecular characterization of canine coronavirus strains circulating in Brazil

To characterize canine coronavirus (CCoV) circulating in diarrheic puppies in Brazil, 250 fecal samples collected between 2006 and 2012 were tested. By using RT-PCR to partially amplify the M gene, CCoV RNA was detected in 30 samples. Sequence analysis of the M protein grouped eight strains with CCoV-I and another 19 with CCoV-II prototypes. To genotype/subtype the CCoV strains and assess the occurrence of single or multiple CCoV infections, RT-PCR of the S gene was performed, and 25/30 CCoV-positive strains amplified with one or two primer pairs. For 17/25 samples, single infections were detected as follows: six CCoV-I, nine CCoV-IIa and two CCoV-IIb. Eight samples were positive for more than one genotype/subtype as follows: seven CCoV-I/IIa and one CCoV-I/IIb. Sequence analysis revealed that the CCoV-I and IIa strains shared high genetic similarity to each other and to the prototypes. The Brazilian strains of CCoV-IIb displayed an aminoacid insertion that was also described in CCoV-IIb-UCD-1 and TGEV strains. Among the 25 CCoV-positive puppies, five had a fatal outcome, all but one of which were cases of mixed infection. The current study is the first reported molecular characterization of CCoV-I, IIa and IIb strains in Brazil.     

Spatial analysis of bovine spongiform encephalopathy in Galicia, Spain (2000-2005)

In Spain, the first bovine spongiform encephalopathy (BSE) case was detected in 2000 in a cow born in the Galicia region (Northwestern Spain). From then and until October 2005, 590 cases were detected, 223 of them in Galicia.

In 1994, meat and bone meal (MBM) was banned on ruminant feed and, in 1996, an EU decision mandating an overall change in MBM processing was implemented. This decision was gradually applied in the territory and not enforced before July 1998. The objective of this study was to explore clustering of BSE cases and estimate the standard incidence ratio (SIR) of BSE in Galicia. Our study was based on the BSE cases detected during the surveillance period 2000–2005 in the Galicia region. These cases were divided, based on birth date, into two periods: animals born from 1994 to July 1998, and those born after July 1998. We tested the role of cross-contamination on the geographical SIR distribution for both periods. Hierarchical Bayesian models were used to model the overdispersion and lack of independence of the SIR estimates. The geographical distribution of the standard incidence ratio of BSE between both periods was different. In the second period, the SIR was reduced in some areas. The reduction in these areas could be attributable to the changes in the processing of MBM. We did not find any statistical link between the poultry population and the standard incidence ratio, but pig population had a positive effect.     

New insights into the epidemiology of bovine piroplasmoses in Italy

Few studies have been published on bovine piroplasmoses in Italy, and therefore a clear picture of the epidemiology of these infections is difficult to obtain. Vertebrate and invertebrate hosts in Central and Northern Regions of Italy were investigated in 2005 and 2006, when microscopy, molecular tools and serological tests were applied to 468 blood samples drawn from cattle in order to evaluate the presence of these protozoa and identify possible risk factors. Ticks were also collected, identified and analyzed by molecular techniques.Microscopy identified 6.5% of the animals as positive, whereas PCR detected piroplasm DNA in 21.6%. BLAST analysis showed 67 amplicons (17.0%) referable to the Theileria sergenti/buffeli/orientalis group, 17 (4.3%) to Theileria annae, and 1 to Babesia divergens. Serology evidenced a prevalence of 45.4% for Babesia bovis, 17.4% for Babesia bigemina, and 34.9% for B. divergens. The 127 collected ticks were identified as belonging to 5 species, mostly represented by Rhipicephalus bursa, Hyalomma marginatum and Ixodes ricinus. Molecular analyses evidenced the presence of B. bovis and B. bigemina, in 3 and 5 ticks, respectively.Our findings suggest that different species of piroplasms are circulating in bovine populations in Central and Northern Italy, and provide new insights into the complex epidemiology of bovine piroplasmoses in Italy.     

Genotypic characteristics of bovine viral diarrhea virus 2 strains isolated in northern Italy

Two strains of Bovine viral diarrhea virus 2 (BVDV-2) were isolated from calves in northern Italy. Variations in the 5'-untranslated region (UTR) of the genome were studied by primary structure alignment and neighbor-joining method based phylogenetic tree analyses and by palindromic nucleotide substitutions at the three variable loci in the 5'-UTR. Genetic analysis indicated their appurtenance to genovar BVDV-2a. Nucleotide sequence at the 5'-UTR of strain BS-95-II, one of the Italian isolates from healthy calves, showed 98% homology to that of the Japanese isolate OY89, a cytopathic strain derived from cattle with mucosal disease.     

Detection and molecular characterization of calf diarrhoea bovine coronaviruses circulating in South Korea during 2004-2005

Although the widespread occurrence of calf diarrhoea (CD) bovine coronavirus (BCoV) infections have been reported in most cattle producing countries, only the genetic differences in the BCoVs from American and Canadian isolates and/or strains have been identified and compared. Hence, it is unclear if the BCoVs circulating in the other countries have distinct genetic characteristics. The aim of this study was to determine the prevalence and genetic diversity of CD BCoVs based on the deduced amino acid (aa) sequences of the spike (S) and haemagglutinin/esterase (HE) proteins in South Korea. RT-PCR and nested PCR using the primer pairs specific to the nucleocapsid gene, BCoVs detected the BCoVs in 56 (15.6%) of 359 diarrhoeic faecal samples. Phylogenetic analysis of the entire S gene indicated that 10 Korean CD BCoV strains clustered with other Korean BCoV strains with different clinical forms but were different from the American and Canadian BCoV strains. Moreover, the phylogenetic data of the aa sequences of the HE gene revealed all the Korean CD strains to be distinct from the other Korean BCoV strains with different clinical forms. These results suggest that the Korean BCoVs cause endemic infections in diarrhoeic calves in Jeonnam province and have taken a different evolutionary pathway from the BCoVs in other countries. Moreover, the different BCoV strains are circulating in the different clinical forms in South Korea. These results also suggest that vaccines against the BCoVs can be developed with each Korean BCoV in different clinical forms.     

Epidemiological investigation of infectious salmon anaemia (ISA) outbreaks in Norway 2003-2005

Epidemiological information was summarized from 32 outbreaks of infectious salmon anaemia (ISA) on salmon farming sites in Norway in 2003–2005. Virus isolates from the outbreak sites were genotyped, and the genotyping was used to assess possible associations between outbreak sites due to adjacent location, sharing fish farming authorisation, sharing smolt suppliers or sharing broodfish origin of the fish. The ISA outbreaks were distributed along most of the Norwegian coast and showed a variable clinical picture. The virus genotypes clustered into three genogroups. Pairs of outbreak sites matched for adjacent location or registered under the same authorisation, all shared genogroup, which was a significantly higher number of corresponding genogroups than expected by chance. For outbreak sites sharing smolt suppliers, corresponding genogroups appeared in 7 out of 12 matched pairs, which was not significant. An evaluation of broodfish origin associated with genogroups did not support transmission linked to broodfish origin. In conclusion, genotyping of virus isolates from ISA outbreaks supports associations between adjacent outbreaks. This is consistent with horizontal transmission. The present study failed to find evidence for vertical transmission (patterns of genogroups related to smolt suppliers or broodfish companies were not identified).     

A serologic survey of bovine rotavirus infection in Italy

Serum neutralization tests conducted by microtiter procedure on 2970 serum samples from calves, heifers and adult cows from representative regions of Italy indicate that rotaviruses, and the infection which they cause, existed in Italy long before the virus was isolated. At the present time the infection is distributed fairly uniformly throughout the cattle population with both sexes and all age groups being equally susceptible.     

Observed prevalence of tick-borne pathogens in domestic animals in Sicily, Italy during 2003-2005

The objective of this study was to characterize the observed prevalence of tick-borne pathogens (TBP) in domestic animals in Sicily, Italy during 2003-2005. Serological (competitive ELISA and indirect immunofluorescence antibody, n = 3299) and DNA tests (polymerase chain reaction and reverse line blot, n = 2565) were conducted on horse, donkey, cattle, sheep, goat, pig and dog samples. Pathogens analysed included Anaplasma, Ehrlichia, Rickettsia, Babesia and Theileria species, and Coxiella burnetii. The most prevalent TBP were Anaplasma and Babesia species. The results reported herein suggested that cattle could serve as the major reservoir for Babesia and Theileria spp. while for Anaplasma spp. cattle, dogs, sheep and goats may be the most important reservoir species. These results expanded our knowledge about the prevalence of TBP in Sicily and provided information to understand the epidemiology of tick-borne diseases and may help to implement measures to diagnose, treat and control transmission to humans and animals in this region.     

Molecular characterization of a Korean bovine parainfluenza virus type 3 isolate

Bovine parainfluenza virus type 3 (BPIV-3) was isolated from Korean native cattle that presented clinical signs of mild pneumonia. The complete genome of a representative isolate (12Q061) was sequenced. The newly identified strain, which was found to be distinct from the previously reported genotypes A (BPIV-3a) and B (BPIV-3b) and closely related to the Chinese strain SD0835, was tentatively classified as genotype C (BPIV-3c). Our results suggest a relationship between BPIV-3 genetic variation and the geographic location of its isolation. Identification of these new BPIV-3 genotypes may facilitate the development of improved diagnostic methods and vaccines. This is to our knowledge the first report of the identification and molecular characterization of BPIV-3 in Korea.     

Detection of group a rotavirus strains circulating in calves in Tunisia

Faecal samples were collected from 89 dairy calves to determine the prevalence of rotavirus infection in Tunisia and the genomic diversity of bovine rotavirus strains. After screening of all faecal samples by enzyme-linked immunosorbent assay, rotavirus strains were analysed by RNA polyacrylamide gel electrophoresis and characterized antigenically by monoclonal antibodies to the VP6 subgroup. The VP7 genotype was determined by nested RT-PCR. Of the 89 calves tested, 27 (30%) were positive for rotavirus antigen. Four different long electrophoretypes were identified. All VP6 typeable strains carried the subgroup I specificity. G8 genotype was the most prevalent, but G6 and mixed strains G(6 + 8) were also detected.     

Molecular characterization of avian infectious bronchitis virus strains isolated in Colombia during 2003

Sixteen infectious bronchitis virus (IBV) isolates were recovered from broilers and layers from five geographic poultry regions in Colombia. The viruses were isolated from tracheas, lungs, and cecal tonsils of birds, previously vaccinated with the Massachusetts strain, that were showing respiratory signs. Further analysis of the IBV isolates was achieved by phylogenetic analysis comparing their deduced amino acid sequences in the hypervariable region 1 of the S1 gene with reference strains. Four unique genotype clusters containing isolates with indigenous genotypes were observed. One isolate was found to be the Connecticut genotype and three isolates were found to be the Massachusetts genotype.