Recent studies have hypothesized that circulation of classical swine fever virus (CSFV) variants when the immunity induced by the vaccine is not sterilizing might favour viral persistence. Likewise, in addition to congenital viral persistence, CSFV has also been proven to generate postnatal viral persistence. Under experimental conditions, postnatal persistently infected pigs were unable to elicit a specific immune response to a CSFV live attenuated vaccine via the mechanism known as superinfection exclusion (SIE). Here, we study whether subclinical forms of classical swine fever (CSF) may be present in a conventional farm in an endemic country and evaluate vaccine efficacy under these types of infections in field conditions. Six litters born from CSF-vaccinated gilts were randomly chosen from a commercial Cuban farm at 33 days of age (weaning). At this time, the piglets were vaccinated with a lapinized live attenuated CSFV C-strain vaccine. Virological and immunological analyses were performed before and after vaccination. The piglets were clinically healthy at weaning; however, 82% were viraemic, and the rectal swabs in most of the remaining 18% were positive. Only five piglets from one litter showed a specific antibody response. The tonsils and rectal swabs of five sows were CSFV positive, and only one of the sows showed an antibody response. After vaccination, 98% of the piglets were unable to clear the virus and to seroconvert, and some of the piglets showed polyarthritis and wasting after 36 days post vaccination. The CSFV E2 glycoprotein sequences recovered from one pig per litter were the same. The amino acid positions 72(R), 20(L) and 195(N) of E2 were identified in silico as positions associated with adaptive advantage. Circulation of chronic and persistent CSF infections was demonstrated in field conditions under a vaccination programme. Persistent infection was predominant. Here, we provide evidence that, in field conditions, subclinical infections are not detected by clinical diagnosis and, despite being infected with CSFV, the animals are vaccinated, rather than diagnosed and eliminated. These animals are refractory to vaccination, likely due to the SIE phenomenon. Improvement of vaccination strategies and diagnosis of subclinical forms of CSF is imperative for CSF eradication.

Background: Canine monocytic ehrlichiosis (CME) is caused by the tick-borne pathogen Ehrlichia canis, an obligate intracellular Gram-negative bacterium of the family Anaplasmataceae with tropism for canine monocytes and macrophages. The trp36 gene, which encodes for the major immunoreactive protein TRP36 in E. canis, has been successfully used to characterize the genetic diversity of this pathogen in different regions of the world. Based on trp36 sequence analysis, four E. canis genogroups, United States (US), Taiwan (TWN), Brazil (BR) and Costa Rica (CR), have been identified. The aim of this study was to characterize the genetic diversity of E. canis in Cuba based on the trp36 gene. Methods: Whole blood samples (n = 8) were collected from dogs found to be infested with the tick vector Rhipicephalus sanguineus sensu lato (s.l.) and/or presenting clinical signs and symptoms of CME. Total DNA was extracted from the blood samples and trp36 fragments were amplified by PCR. Nucleotide and protein sequences were compared using alignments and phylogenetic analysis. Results: Four of the trp36 sequences obtained (n = 8) fall within the phylogenetic cluster grouping the US genogroup E. canis strains. The other E. canis trp36 sequences formed a separate and well-supported clade (94% bootstrap value) that is phylogenetically distant from the other major groups and thus represents a new genogroup, herein designated as the ‘Cuba (CUB) genogroup’. Notably, dogs infected with the CUB genogroup presented frequent hemorrhagic lesions. Conclusions: The results of this study suggest that genetic diversification of E. canis in Cuba is associated with the emergence of E. canis strains with increased virulence.

Classical swine fever virus (CSFV) induces trans-placental transmission and congenital viral persistence; however, the available information is not updated. Three groups of sows were infected at mid-gestation with either a high, moderate or low virulence CSFV strains. Foetuses from sows infected with high or low virulence strain were obtained before delivery and piglets from sows infected with the moderate virulence strain were studied for 32 days after birth. The low virulence strain generated lower CSFV RNA load and the lowest proportion of trans-placental transmission. Severe lesions and mummifications were observed in foetuses infected with the high virulence strain. Sows infected with the moderately virulence strain showed stillbirths and mummifications, one of them delivered live piglets, all CSFV persistently infected. Efficient trans-placental transmission was detected in sows infected with the high and moderate virulence strain. The trans-placental transmission occurred before the onset of antibody response, which started at 14 days after infection in these sows and was influenced by replication efficacy of the infecting strain. Fast and solid immunity after sow vaccination is required for prevention of congenital viral persistence. An increase in the CD8+ T-cell subset and IFN-alpha response was found in viremic foetuses, or in those that showed higher viral replication in tissue, showing the CSFV recognition capacity by the foetal immune system after trans-placental infection.