Bacteriophage Therapy in Companion and Farm Animals.

Bacteriophages, which are viruses with restricted tropism for bacteria, have been employed for over a century as antimicrobial agents; they have been largely abandoned in Western countries but are constantly used in Eastern European countries with the advent of antibiotics. In recent decades, the growing spread of multidrug-resistant bacteria, which pose a serious threat to worldwide public health, imposed an urgent demand for alternative therapeutic approaches to antibiotics in animal and human fields. Based on this requirement, numerous studies have been published on developing and testing bacteriophage-based therapy. Overall, the literature largely supports the potential of this perspective but also highlights the need for additional research as the current standards are inadequate to receive approval from regulatory authorities. This review aims to update and critically revise the current knowledge on the application of bacteriophages to treat bacterial-derived infectious diseases in animals in order to provide topical perspectives and innovative advances.

Identification and characterisation of Gamma-herpesviruses in zoo artiodactyla.

BACKGROUND: Viruses within the γ-herpesviruses subfamily include the causative agents of Malignant Catarrhal Fever (MCF) in several species of the order Artiodactyla. MCF is a usually fatal lymphoproliferative disease affecting non-adapted host species. In adapted host species these viruses become latent and recrudesce and transmit during times of stress or immunosuppression. The undetected presence of MCF-causing viruses (MCFVs) is a risk to non-adapted hosts, especially within non-sympatric zoological collections. This study investigated the presence of MCFVs in six different zoological collections in the UK, to evaluate the presence of subclinical/latent MCFVs in carrier animals. METHODS: One-hundred and thirty eight samples belonging to 54 different species of Artiodactyla were tested by Consensus Pan-herpes PCR. The positive samples were sequenced and subjected to phylogenetic analyses to understand their own evolutionary relationships and those with their hosts. RESULTS: Twenty-five samples from 18 different species tested positive. All viruses but one clustered in the γ-herpesvirus family and within the Macavirus as well as the non-Macavirus groups (caprinae and alcelaphinae/hippotraginae clusters, respectively). A strong association between virus and host species was evident in the Macavirus group and clustering within the caprinae group indicated potential pathogenicity. CONCLUSION: This study shows the presence of pathogenic and non-pathogenic MCFVs, as well as other γ-herpesviruses, in Artiodactyla species of conservation importance and allowed the identification of new herpesviruses in some non-adapted species.

Prevalence of Serum Antibody Titers against Core Vaccine Antigens in Italian Cats.

Feline core vaccines strongly recommended for all cats are against Feline panleukopenia virus (FPV), Felid herpesvirus type 1 (FeHV-1), and Feline calicivirus (FCV), but cats can be classified as low- and high-risk based on their lifestyle. The aim of this study was to determine the actual seroprotection against FPV, FeHV-1, and FCV in a large cohort of Italian cats by using the VacciCheck test. A total of 740 cats (567 owned and 173 stray cats; 435 vaccinated and 305 unvaccinated) were analyzed for Protective Antibody Titers (PATs). Differences related to origin, sex, age, breed, FIV/FeLV status, health status, and time elapsed since last vaccination were evaluated. Less than half of the entire cohort (36.4%) had PATs for all three diseases simultaneously, increasing to 48.6% if weak positive values were also considered and 50.3% when considering only the 435 vaccinated cats. Particularly, antibodies were detected against FCV, FPV, and FeHV-1 at protective titers (PATs) in 78.6%, 68.1, and 49.1% of the cats, respectively. In general, owned, neutered, and adult FIV- and/or FeLV-negative cats were the most protected categories, even if not always for the three viruses. Most cats maintained high PATs for 3 years or longer after vaccination against FPV and FCV but not FeHV-1. Long-lasting protective immunity persisted for many years after the last vaccination (more than 18 years in the oldest cats). Nevertheless, since not all cats were protected after so many years and for all pathogens, checking protection via antibody titration could be the best choice to prevent immunity breakdowns. The discussion also focuses on the reliability of antibody titration for the two URTD (upper respiratory tract disease) viruses which, unlike for FPV, is not widely accepted as a valid index of protection.

Partial Replacement of Synthetic Vitamin E by Polyphenols in Post-Weaning Piglets.

Vitamin E is an essential nutrient usually recommended in post-weaning piglets, when a decline in the serum vitamin E concentration is observed. Selected polyphenols have the potential to partially replace vitamin E in animal feed. The aim of this study was to investigate the effect of the dietary inclusion of some commercial polyphenol products (PPs) on the growth performance, antioxidant status and immunity of post-weaning piglets. A total of 300 piglets (BW 7.18 kg ± 1.18) were randomly assigned to six dietary groups: CON- (40 mg/kg vitamin E); CON+(175.8 mg/kg vitamin E); and PP1, PP2, PP3 and PP4, in which 50% vitamin E of CON+ was replaced with PP with equivalent vitamin E activity. The PP1 group exhibited lower performance (p < 0.05) than the other dietary groups, but a similar performance to that commonly registered in pig farms. Dietary polyphenols did not influence the IgG concentration or the IL-6, IL-10, IFN-γ and TNF-α cytokine concentrations. A lower IL-8 level was found in the PP4 group than in the other groups. The diets that affected the vitamin A content showed the highest value (p < 0.05) in the PP1 group, and a trend was noted for vitamin E with a higher content in PP4 and CON+. The polyphenols-enriched diets, especially the PP3 diet, maintained an antioxidant capacity (whole blood KRL) similar to the CON+ diet. In conclusion, the replacement of vitamin E with all PPs enables partial vitamin E substitution in post-weaning piglets.

Effect of Aging on the Immune Response to Core Vaccines in Senior and Geriatric Dogs.

Elderly dogs are steadily increasing worldwide as well as veterinarians' and owners' interest in their health and wellness. Aging is not a disease, but a combination of changes negatively affecting the organism in general and the immune system in particular, resulting in a decline in protection over time. The aim of this study was to measure the specific serum antibody titers against the main dangerous and widespread viral diseases preventable by core vaccinations in senior and geriatric dogs using the in-practice test VacciCheck. A cohort of three hundred fifty elderly dogs was analyzed for Protective Antibody Titers (PATs) against CPV-2, CDV and CAdV-1. The age ranged from 5 to 19 years, with two hundred fifty-eight seniors (73.7%) and ninety-two geriatrics (26.3%), and 97.4% of them were vaccinated at least once in their lives. More than half of the entire study population (52.9%) had PATs simultaneously for all three diseases, with 80.5% seniors and 19.5% geriatrics. Specific PATs were found in 88.6% of aging dogs for CPV-2, 82.3% for CadV-1 and 66.0% for CDV, demonstrating that unprotected aging dogs represent a minority. Unexpectedly, the larger elderly dogs resulted as more protected than smaller ones for CPV-2. Protection then decreases over time, with geriatric dogs less protected than senior ones. Veterinary practitioners should therefore always consider whether to maintain core vaccinations in aging dogs as in adults on a three-year basis or opt instead for closer boosters (every 1 or 2 years). PATs for core vaccines could then represent a good biomarker of protection and their titration could become a standard of care, especially in such a sensitive period of the dogs' life.

Therapeutic Applications of Botulinum Neurotoxins in Veterinary Medicine.

Botulinum neurotoxins (BoNTs) are emerging as multipurpose therapeutic compounds for the treatment of several different syndromes involving peripheral and central nervous systems, and muscular and musculoskeletal disorders both in human and veterinary medicine. Therefore, the study of BoNTs is rapidly developing and identifying newly produced BoNT variants. Efforts should be made to clarify the biological and pharmacological characteristics of these novel BoNTs as well as the natural ones. The high potential of BoNTs as a therapeutic compound for medical syndromes lies in its ability to reach a specific cell type while bypassing other cells, thus having mild or no side effects. In this paper the recent developments in BoNTs are reviewed with the aim of analyzing the current knowledge on BoNTs' biological mechanisms of action, immunogenicity, formulations, and therapeutic applications in the veterinary field, highlighting advantages and drawbacks and identifying the gaps to be filled in order to address research priorities.

Tobacco Seed-Based Oral Vaccination against Verocytotoxic O138 Escherichia coli as Alternative Approach to Antibiotics in Weaned Piglets.

Post-weaning diarrhoea and enterotoxaemia caused by Escherichia coli are serious threats in the pig (Sus scrofa domesticus) livestock industry and are responsible for economic losses related to mortality, morbidity and stunted growth. The aim of this study was to evaluate the effect of an engineered tobacco seeds-based edible vaccine in O138 Escherichia coli-challenged piglets throughout a multidisciplinary approach. Thirty-six weaned piglets were enrolled and randomly divided into two experimental groups, a control (C; n = 18) group and a tobacco edible vaccination group (T, n = 18), for 29 days of trial. At days 0, 1, 2, 5 and 14, piglets of the T group were fed with 10 g of the engineered tobacco seeds line expressing F18 and VT2eB antigens, while the C group received wild-type tobacco seeds. After 20 days, 6 piglets/group were orally challenged with the Escherichia coli O138 strain (creating four subgroups: UC = unchallenged control, CC = challenged control, UT = unchallenged tobacco, CT = challenged tobacco) and fed with a high protein diet for 3 consecutive days. Zootechnical, clinical, microbiological, histological and immunological parameters were assayed and registered during the 9 days of post-challenge follow up. At 29 days post-challenge, the CT group displayed a lower average of the sum of clinical scores compared to the CC group (p < 0.05), while the CC group showed a higher average sum of the faecal score (diarrhoea) (p < 0.05) than the CT group. A decreased number of days of shedding of the pathogenic strain was observed in the CT compared to the CC group (p < 0.05). Specific anti-F18 IgA molecules were significantly higher in the CT group compared to the CC group's faecal samples during the post-challenge period (p < 0.01). In conclusion, edible vaccination with engineered tobacco seeds showed a protective effect on clinical symptoms and diarrhoea incidence during the post-challenge period, characterized by a limited time of pathogenic strain shedding in faeces.

Can Chemotherapy Negatively Affect the Specific Antibody Response toward Core Vaccines in Canine Cancer Patients?

The life expectancy of our pets has been getting longer in recent years due to new therapeutic opportunities, better nutrition, and better diagnostic approaches. This positive effect, however, has been accompanied by a concomitant increase in neoplasms, particularly in canine patients. Therefore, veterinarians inevitably face new issues related to these diseases, poorly or never investigated in the past, such as the possible side effects resulting from chemotherapy. The aim of this study was to investigate whether and how chemotherapy influences the antibody response against CPV-2, CDV, and CAdV-1 in dogs vaccinated before starting chemotherapy. Twenty-one canine patients with different types of malignancies were sampled before, during, and after different chemotherapy protocols to determine their actual levels of seroprotection against CPV-2, CDV, and CadV-1 by using the in-practice test VacciCheck. Differences related to sex, breed size, type of tumor, and chemotherapy protocol were evaluated. No statistically significant changes in antibody protection emerged for any of the chemotherapy protocol used, suggesting that, contrary to expectation, chemotherapy does not have a marked immunosuppressive effect on the post-vaccine antibody response. These results, although preliminary, may be useful in improving the clinical approach to the canine cancer patient, helping veterinarians fully manage their patients, and enabling owners to feel more confident about their pets' quality of life.

Prevalence of Serum Antibody Titers against Core Vaccine Antigens in Italian Dogs.

Canine vaccination is the main tool for preventing dangerous and widespread diseases. The strongly recommended (core) dog vaccines are against Canine Parvovirus type 2 (CPV-2), Canine Distemper Virus (CDV), and Canine Adenovirus (CAdV-1), but vaccination protocols should be tailored to dog lifestyles. Vaccination guidelines suggest vaccinating adult dogs no more frequently than every 3 years using modified live (attenuated) vaccines (MLV), thus obtaining a long-lasting (sometimes throughout life) specific protection in many but not all animals. The aim of this study was to determine the actual levels of seroprotection against CPV-2, CDV and CAdV-1 in a cohort of Italian dogs by using the in-practice test VacciCheck. A total of 1,027 dogs (951 vaccinated and 76 unvaccinated) were analyzed for Protective Antibody Titers (PATs) against CPV-2, CDV, and CAdV-1. Differences related to sex, age, breed size, health status, and time elapsed since last vaccination were evaluated. Half of the entire canine cohort (50.6%) had PATs for all three viruses (68.5% considering only vaccinated dogs). In particular, 90.8% of dogs were protected against CPV-2, 68.6% against CDV, and 79.8% against CAdV-1. Most dogs remained protected for 3 years after vaccination or longer. Revaccination on a 3-year basis can then be recommended for core MLV vaccines without altering individual's seroprotection or even herd immunity.

Presence of Anaplasma phagocytophilum Ecotype I in UK Ruminants and Associated Zoonotic Risk.

Anaplasma phagocytophilum is the causative agent of tick-borne fever in sheep, pasture fever in cattle, and granulocytic anaplasmosis in humans. The increasing prevalence and transboundary spread of A. phagocytophilum in livestock, ticks, and wildlife in the UK poses a potential zoonotic risk that has yet to be estimated. Several ecotypes of A. phagocytophilum show variable zoonotic potential. To evaluate the possible risk associated with the transmission of A. phagocytophilum from ruminants to humans, the ecotype was determined by sequencing the groEL gene from 71 positive blood and tissue samples from UK ruminants. Thirty-four groEL sequences were obtained, fourteen of which were identified in multiple samples. Of the 13 nucleotide polymorphisms identified through pairwise comparison, all corresponded to synonymous substitutions. The subsequent phylogenetic estimation of the relationship with other European/world isolates indicated that all the groEL sequences clustered with other ecotype I sequences. The presence of ecotype I closely reflects that observed in ruminants in continental Europe and suggests a lower risk of zoonotic transmission from this reservoir.

Protective effect of phytogenic plus short and medium-chain fatty acids-based additives in enterotoxigenic Escherichia coli challenged piglets.

Post Weaning Diarrhea (PWD) is the most important multifactorial gastroenteric disease of the weaning in pig livestock. Phytogenic (PHY) natural extracts are largely studied as alternatives to antibiotic treatments in combating the global concern of the antimicrobial resistance. The aim of this study was to evaluate the protective effect of innovative phytogenic premix with or without short and medium chain fatty acids (SCFA and MCFA) in O138 Escherichia coli challenged piglets. Twenty-seven weaned piglets were allotted into four groups fed different diets according to the following dietary treatments: CTRL (n = 13) group fed basal diet, PHY1 (n = 7) fed the basal diet supplemented with 0.2% of phytogenic premix, PHY2 (n = 7) fed the basal diet supplemented with 0.2% of phytogenic premix added with 2000 ppm of SCFA and MCFA. After 6 days of experimental diet feeding, animals were challenged (day 0) with 2 × 109 CFU of E. coli and CTRL group was divided at day 0 into positive (challenged CTRL + ; n = 6) and negative control group (unchallenged CTRL-; n = 7). Body weights were recorded at -14, -6, 0, 4 and 7 days and the feed intake was recorded daily. E. coli shedding was monitored for 4 days post-challenge by plate counting. Fecal consistency was registered daily by a four-point scale (0-3; diarrhea > 1) during the post-challenge period. Tissue samples were obtained for gene expression and histological evaluations at day 7 from four animals per group. Lower average feed intake was observed in CTRL + compared to PHY2 and CTRL during the post-challenge period. Infected groups showed higher E. coli shedding compared to CTRL- during the 4 days post-challenge (p < 0.01). PHY2 showed lower frequency of diarrhea compared to PHY1 and CTRL + from 5 to 7 days post-challenge. No significant alterations among groups were observed in histopathological evaluation. Duodenum expression of occludin tended to be lower in challenged groups compared to CTRL- at 7 days post-challenge (p = 0.066). In conclusion, dietary supplementation of PHY plus SCFA and MCFA revealed encouraging results for diarrhea prevention and growth performance in weaned piglets.

Molecular Tools to Identify and Characterize Malignant Catarrhal Fever Viruses (MCFV) of Ruminants and Captive Artiodactyla.

The family Herpesviridae includes viruses identified in mammals, birds and reptiles. All herpesviruses share a similar structure, consisting of a large linear double-stranded DNA genome surrounded by a proteic icosahedral capsid further contained within a lipidic bilayer envelope. The continuous rise of genetic variability and the evolutionary selective pressure underlie the appearance and consolidation of novel viral strains. This applies also to several gamma(γ)-herpesviruses, whose role as primary pathogen has been often neglected and, among these to newly emerged viruses or virus variants responsible for the development of Malignant Catarrhal Fever (MCF) or MCF-like disease. The identification of γ-herpesviruses adapted to new zoological hosts requires specific molecular tools for detection and characterization. These viruses can cause MCF in livestock and wild animals, a disease generally sporadic but with serious welfare implications and which, in many cases, leads to death within a few days from the appearance of the clinical signs. In the absence of a vaccine, the first step to improve disease control is based on the improvement of molecular tools to identify and characterize these viruses, their phylogenetic relationships and evolutionary interaction with the host species. A Panherpes PCR-specific test, based on the conserved DNA polymerase gene, employing consensus/degenerate and deoxyinosine-substituted primers followed by sequencing, is still the preferred diagnostic test to confirm and characterize herpesviral infections. The drawback of this test is the amplification of a relatively short sequence, which makes phylogenetic analysis less stringent. Based on these diagnostic requirements, and with a specific focus on γ-herpesviruses, the present review aims to critically analyze the currently available methods to identify and characterize novel MCFV strains, to highlight advantages and drawbacks and to identify the gaps to be filled in order to address research priorities. Possible approaches for improving or further developing these molecular tools are also suggested.

SARS CoV-2 infections in animals, two years into the pandemic.

In December 2019, several cases of pneumonia caused by a novel coronavirus, later identified as SARS-CoV-2, were detected in the Chinese city of Wuhan. Due to its rapid worldwide spread, on 11 March 2020 the World Health Organization declared a pandemic state. Since this new virus is genetically similar to the coronaviruses of bats, SARS-CoV-2 was hypothesized to have a zoonotic origin. Within a year of the appearance of SARS-CoV-2, several cases of infection were also reported in animals, suggesting human-to-animal and animal-to-animal transmission among mammals. Natural infection has been found in companion animals as well as captive animals such as lions, tigers, and gorillas. Among farm animals, so far, minks have been found to be susceptible to SARS-CoV-2 infection, whereas not all the relevant studies agree on the susceptibility of pigs. Experimental infections have documented the susceptibility to SARS-CoV-2 of further animal species, including mice, hamsters, cats, dogs, ferrets, raccoon dogs, cattle, and non-human primates. Experimental infections have proven crucial for clarifying the role of animals in transmission and developing models for viral pathogenesis and immunotherapy. On the whole, this review aims to update and critically revise the current information on natural and experimental SARS-CoV-2 infections in animals.

Recent advances and public health implications for environmental exposure to Chlamydia abortus: from enzootic to zoonotic disease.

Environmental transmission of Chlamydia abortus as a result of enzootic disease or disease outbreaks and the threats posed by this pathogen has been previously reported, however a state-of-the-science review of these reports and the identification of future research priorities in this area is still lacking. This study provides an overview of the current knowledge of host-pathogen-environment interactions, addressing public health risks and identifying critical questions and research gaps. We performed a systematic PubMed and Web of Science search for publications related to Chlamydia abortus in the past four decades, and we reviewed and combined the evidence critically discussing and commenting the results. A total of 182 studies, 5 chapters of specific books and the "OIE terrestrial manual" were included in this review. There were substantial variations between the studies in topic addressed and experimental design. Overall, the literature largely supports the crucial role played by environmental exposure on the acquisition of zoonotic disease caused by Chlamydia abortus. We also identify the paucity of information related to interspecies transmission and pathogen adaptation in relation to environmental dissemination and zoonotic risk. This analysis further highlights the need for additional research given that environmental transmission represents a serious risk not only to susceptible patients (pregnant women and immunocompromised individuals), but also for other species including wildlife.

IL-1R8 as Pathoimmunological Marker for Severity of Canine Chronic Enteropathy.

Chronic enteropathy (CE) is a severe multifactorial gastrointestinal disease that affects dogs and is driven by poorly characterized inflammatory pathways. Imbalance of pro-inflammatory response regulators, including IL-1R8, may be due to different factors, among which the infection with Helicobacteraceae is known to lead to a vicious circle in which excessive pro-inflammatory signaling and gastrointestinal injury reinforce each other and boost the disease. We investigated the expression of IL-1R8 in large intestine biopsies of dogs with or without clinical signs of CE and with previously assessed enterohepatic Helicobacter spp. colonization status by mean of quantitative real-time PCR. Our study revealed that IL-1R8 is downregulated in both acutely (p = 0.0074) and chronically (p = 0.0159) CE affected dogs compared to healthy controls. The data also showed that IL-1R8 expression tends to decrease with colonization by Helicobacter spp. Interestingly, a negative correlation was detected between the level of expression of IL-1R8 and the severity of macroscopic lesions identified by endoscopy and the crypt hyperplasia score. We further compared the expression levels between males and females and found no statistically significant difference between the two groups. No significant difference was observed in IL-1R8 expression profiles with the age of the animals either. Interestingly, an association was uncovered between IL-1R8 expression level and dog breed. Together, our data advance knowledge on gastrointestinal pathoimmunology in dogs and highlight the potential utilization of IL-1R8 as a diagnostic, prognostic and therapeutic biomarker for canine chronic enteropathy.

Tranquillizing Effect of Passiflora incarnata Extract: Outcome on Behavioral and Physiological Indicators in Weaning Pigs with Intact Tails.

Tail docking has been used in the pig industry to decrease the occurrence of tail biting behavior. This abnormal behavior has a multifactorial origin since it is a response to simultaneous environmental, nutritional and management changes. Given the calming properties of Passiflora incarnata, we hypothesized that dietary supplementation with the extract in weaned pigs could result in a modification of behavior and physiologic indicators linked to stress. Weaned piglets (n = 120, mean body weight 9.07 ± 2.30 kg) were randomly allocated to one of two dietary treatments: control diet (CON) and CON supplemented with 1 kg/t of P. incarnata (PAS). The trial was 28 days long. The presence of skin lesions was assessed at d-1, d-10, d-19, and d-28, and saliva samples were collected for IgA and cortisol determinations at the same sampling times. Results showed the PAS group was characterized by equal growth performance as the CON group, fewer ear lesions (p < 0.05), less aggressive behavior (p < 0.001), higher enrichment exploration (p < 0.001) and lower cortisol levels (p < 0.01). Time effect was observed for tail lesions (p < 0.001) and behavioral observations (p < 0.001). Additional research is required to determine the effect of P. incarnata extract using a larger number of animals and longer period of supplementation when risks associated with tail biting are uncontrolled.

Translational Approach to Induce and Evaluate Verocytotoxic E. coli O138 Based Disease in Piglets.

Pig livestock was influenced by several global concerns that imposed a re-thinking of the farming system, which included the reduction in chemical dependency and the development of antimicrobial alternatives. Post-weaning diarrhea and enterotoxaemia caused by Escherichia coli, are serious threats that are responsible for the economic losses related to mortality, morbidity and stunted growth in weaning piglets. The aim of the study was to set up experimental conditions to simulate the simultaneous outbreak of post-weaning diarrhea and enterotoxaemia in weaned piglets, through verocytotoxic O138 Escherichia coli challenge, with a multidisciplinary approach. Eighteen piglets susceptible to F18 VTEC infection were selected by polymerase chain reaction for polymorphism on the fucosyltransferase 1 gene and randomly divided in two experimental groups, non-infected controls (C; n = 6) and infected ones (I; n = 12) and housed into individual pens at the same environmental conditions for 29 days. At day 20, I pigs were orally inoculated with Escherichia coli O138 and fed a high protein ration for 3 days. Zootechnical, clinical, microbiological, histological and immunological parameters were evaluated along the follow up (3 and 9 days). Experimental infection, confirmed by bacteria faecal shedding of the I group, significantly affected the clinical status. The I group showed significantly higher total scores, corresponding to medians of the sum of daily scores from days 1 to 3 (Σ3) and 1 to 9 (Σ9) post infection, epiphora, vitality, hair irregularity, oedema and depression. Histological examination showed evident inflammatory infiltrate of lymphocytes, and follicular hyperplasia in I pigs; in the same group, the immunohistochemical and immunological assays revealed an increase in IgG in the intestinal crypts and CD3-positive T cells in intestinal epithelium. The experimental Escherichia coli infection in controlled conditions is crucial for both the evaluation of innovative compounds and the elucidation of the mechanisms associated with the persistence of antibacterial resistant strains. In conclusion, the adopted infection model, carried out on receptor-mediated susceptible piglets, allowed us to identify a discriminative panel of clinical symptoms related to Escherichia coli O138 infection, and could be used to assess the protective effect of antibiotic alternatives.

What we have lost: Mastitis resistance in Holstein Friesians and in a local cattle breed.

In Holstein Friesian dairy cows, selective pressure for increased milk production has led to a higher propensity to disease, including mastitis, when compared to less selected and lower producing dairy breeds. The biology underpinning the higher resistance to disease of such "local breeds" is not fully understood. With the aim of investigating the factors associated to this phenomenon, we applied a multidisciplinary approach to compare innate immune response patterns, metabolic parameters, milk protein profiles and the milk microbiota in Holstein Friesian and Rendena cows reared in the same farm and under the same management conditions. Quarter milk samples and blood plasma were collected from all cows at dry-off, 1day after calving, 7-10days after calving and 30days after calving. Quarter milk samples were subjected to bacteriological culture, characterization of the milk microbiota by 16S metagenomics, milk protein profiling by electrophoresis and densitometry, somatic cell counting, measurement of the inflammation marker cathelicidin and assessment of different innate immune-related mediators such as lysozyme, CD45, IL-1ß, TNF-α, PTX3, IL-1R8. In parallel, the main inflammometabolic parameters were measured in blood plasma samples. Despite having relatively few animals (6 moderate-yielding Holstein Friesian and 4 low-yielding Rendena) some important differences were apparent. Holstein Friesian cows showed a more severe fat mobilization and systemic inflammatory response postpartum in comparison with Rendena cows, which had a greater postpartum muscle mass and an increased amino acid mobilization compared to Holstein Friesians. Upon bacteriological analysis, contagious bacteria such as Staphylococcus aureus and Streptococcus agalactiae were absent, but significant differences were seen in the general composition of the milk microbiota of the two breeds. Concerning the milk protein abundance profile, pronounced differences were seen in colostrum, with significantly higher amounts of immunoglobulins and other immune-related proteins in Rendena. Added to this, the expression of innate immune related genes such as PTX-3, IL-1ß, TNF-α, and KRT5 expression in milk epithelial and leukocyte cell components, respectively, was lower in Holstein Friesian colostrum compared with Rendena. In conclusion, several differences were observed in the two breeds, in spite of the same farming conditions. The observations reported in this work present numerous pointers to the factors that may provide autochthonous, more rustic breeds with a higher resistance to disease.

In vitro replication activity of bovine viral diarrhea virus in an epithelial cell line and in bovine peripheral blood mononuclear cells.

The present study focused on the in vitro infection of Madin-Darby bovine kidney (MDBK) cells and bovine peripheral blood mononuclear cells (PBMCs) from naÏve animals with non-cytopathic (ncp, BVDV-1b NY-1) and cytopathic (cp, BVDV-1a NADL) strains. Infections with 0.1 and 1 multiplicity of infections (MOI) and incubation times of 18 and 36 hr were compared. Twelve BVDV naÏve heifers were enrolled to collect PBMCs. The viral loads in MDBK cells and in PBMCs after in vitro infections were measured by real-time polymerase chain reaction (PCR) assays. The highest viral loads were measured at 1 MOI and 36 hr post infection in both cell systems and the lowest at 0.1 MOI and 18 hr with the exception of the cp strain NADL in PBMCs, for which the highest viral load was observed at 0.1 MOI and 36 hr. Viral load mean values were higher for the cp strain than the ncp strain irrespective of the extent of the infection period and MOI. The models of infection studied uncovered different replication activities respectively according to the biotype of virus, the cell substrate and the duration of infection. Replication tends to be higher in PBMCs, particularly at low MOIs and for the ncp strain.

In vitro permissivity of bovine peripheral blood mononuclear cells to bovine viral diarrhoea virus is dependent on the animal specific immune status.

The in vitro permissivity to infection with homologous and heterologous bovine viral diarrhoea virus (BVDV) strains of bovine peripheral blood mononuclear cells (PBMCs) from eight naïve and eight BVDV-1b immune animals was studied. Four reference strains (BVDV-1a NADL, BVDV-1b NY-1, BVDV-2 125 and BVDV-2 890) were selected, based on genotype, prevalence and biotype. Virus neutralizing antibody titres were determined at bleeding and the viral loads were measured in PBMCs by end point titration in cell culture and by real-time PCR. PBMCs from both naïve and immune animals became infected by all BVDV strains tested, although virus titres were lower for immune heifers than naïve ones; the differences were significant for NADL (P<0.05) and 890 (P<0.001) strains. The in vitro model used in this study showed that PBMCs from immune animals are susceptible to re-infection with both homologous and heterologous BVDV strains, albeit at a lower extent than naïve cattle.