Metagenomic analysis of rumen microbial population in dairy heifers fed a high grain diet supplemented with dicarboxylic acids or polyphenols.

BACKGROUND: The aim of this study was to investigate the effects of two feed supplements on rumen bacterial communities of heifers fed a high grain diet. Six Holstein-Friesian heifers received one of the following dietary treatments according to a Latin square design: no supplement (control, C), 60 g/day of fumarate-malate (organic acid, O) and 100 g/day of polyphenol-essential oil (P). Rumen fluid was analyzed to assess the microbial population using Illumina sequencing and quantitative real time PCR. RESULTS: The P treatment had the highest number of observed species (P < 0.10), Chao1 index (P < 0.05), abundance based coverage estimated (ACE) (P < 0.05), and Fisher's alpha diversity (P < 0.10). The O treatment had intermediate values between C and P treatments with the exception of the Chao1 index. The PCoA with unweighted Unifrac distance showed a separation among dietary treatments (P = 0.09), above all between the C and P (P = 0.05). The O and P treatments showed a significant increase of the family Christenenellaceae and a decline of Prevotella brevis compared to C. Additionally, the P treatment enhanced the abundance of many taxa belonging to Bacteroidetes, Firmicutes and Tenericutes phyla due to a potential antimicrobial activity of flavonoids that increased competition among bacteria. CONCLUSIONS: Organic acid and polyphenols significantly modified rumen bacterial populations during high-grain feeding in dairy heifers. In particular the polyphenol treatment increased the richness and diversity of rumen microbiota, which are usually high in conditions of physiological rumen pH and rumen function.

Use of dicarboxylic acids and polyphenols to attenuate reticular pH drop and acute phase response in dairy heifers fed a high grain diet.

BACKGROUND: The aim of this study was to determine the ability of two feed additives, a fumarate-malate (FM) and a polyphenol-essential oil mixture (PM), in attenuating the drop of ruminal pH and the metabolic and immune response resulting from an excessively high grain diet. Six heifers were used in a 3 × 3 Latin square experiment and fed a low starch (LS) diet for 14 d, followed by a high starch (HS) diet for 8 d (NDF 33.6%, starch 30.0% DM). In the last 5 days of each period, barley meal was added to decrease rumen pH. During HS feeding all animals were randomly assigned to one of the following three dietary treatments: no supplement/control (CT), a daily dose of 60 g/d of FM, or 100 g/d of PM. Reticular pH was continuously recorded using wireless boluses. On d 21 of each period, rumen fluid was collected by rumenocentesis (1400 h), together with blood (0800 h) and fecal samples (0800, 1400, and 2100 h). RESULTS: The correlation coefficient of pH values obtained using the boluses and rumenocentesis was 0.83. Compared with CT and PM, the FM treatment led to a lower DMI. Nadir pH was lowest during CT (5.40, 5.69, and 5.62 for CT, FM and PM, respectively), confirming the effectiveness of both supplements in reducing the pH drop caused by high grain feeding. This result was confirmed by the highest average time spent daily below 5.6 pH (199, 16 and 18 min/d) and by the highest acetate to propionate ratio of the CT fed heifers. The PM decreased the concentrations of neutrophils (2.9, 3.2, and 2.8 10(9)/L) and acute phase proteins: SAA (37.1, 28.6 and 20.1 µg/mL), LBP (4.1, 3.8, and 2.9 µg/mL), and Hp (675, 695 and 601 µg/mL). Free lipopolysaccharides (LPS) were detected in blood and feces, but their concentrations were not affected by treatments, as the remaining blood variables. CONCLUSIONS: Data suggest that both additives could be useful in attenuating the effects of excessive grain feeding on rumen pH, but the PM supplement was more effective than FM in reducing the inflammatory response compared to CT.

Effect of induced ruminal acidosis on blood variables in heifers.

BACKGROUND: Ruminal acidosis is responsible for the onset of different pathologies in dairy and feedlot cattle, but there are major difficulties in the diagnosis. This study modelled the data obtained from various blood variables to identify those that could indicate the severity of ruminal acidosis. Six heifers were fed three experimental rations throughout three periods. The diets were characterised by different starch levels: high starch (HS), medium starch (MS) and low starch, as the control diet (CT). Ruminal pH values were continuously measured using wireless sensors and compared with pH measurements obtained by rumenocentesis. Blood samples were analysed for complete blood count, biochemical profile, venous blood gas, blood lipopolysaccharide (LPS) and LPS-binding proteins (LBP). RESULTS: The regression coefficient comparing the ruminal pH values, obtained using the two methods, was 0.56 (P = 0.040). Feeding the CT, MS and HS led to differences in the time spent below the 5.8, 5.5 and 5.0 pH thresholds and in several variables, including dry matter intake (7.7 vs. 6.9 vs. 5.1 kg/d; P = 0.002), ruminal nadir pH (5.69 vs. 5.47 vs. 5.44; P = 0.042), mean ruminal pH (6.50 vs. 6.34 vs. 6.31; P = 0.012), haemoglobin level (11.1 vs. 10.9 vs. 11.4 g/dL; P = 0.010), platelet count (506 vs. 481 vs. 601; P = 0.008), HCO3(-) (31.8 vs. 31.3 vs. 30.6 mmol/L; P = 0.071) and LBP (5.9 vs. 9.5 vs. 10.5 µg/mL; P < 0.001). A canonical discriminant analysis (CDA) was used to classify the animals into four ruminal pH classes (normal, risk of acidosis, subacute ruminal acidosis and acute ruminal acidosis) using haemoglobin, mean platelet volume, ß-hydroxybutyrate, glucose and reduced haemoglobin. CONCLUSIONS: Although additional studies are necessary to confirm the reliability of these discriminant functions, the use of plasma variables in a multifactorial model appeared to be useful for the evaluation of ruminal acidosis severity.

Silent Infection of Highly Pathogenic Avian Influenza Virus (H5N1) Clade 2.3.4.4b in a Commercial Chicken Broiler Flock in Italy.

From October 2021 to January 2022, different incursions of clade 2.3.4.4b H5N1 HPAIV (Highly Pathogenic Avian Influenza Virus) occurred in several Italian regions with its main diffusion in Densely Poultry Populated Areas (DPPAs) of north-eastern Italy. Monitoring and control activities applied in the affected area clearly evidenced that turkeys and broilers were the most affected species, although several flocks of broilers at times resulted HPAIV H5N1 infected in absence of increased mortality and/or clinical signs. Thus, an approach based on sampling dead birds was adopted in the broiler sector to improve the early detection of infection; this protocol allowed us to confirm that 15 farms were HPAIV-infected with birds ready to be delivered to the slaughterhouse. The aim of this report is to describe the results of the diagnostic activities carried out in one HPAIV H5N1-infected broiler farm, three days after laboratory confirmation during the pre-movement testing without showing increased mortality or clinical signs. Thus, clinical signs, daily cumulative mortality rate (CMR), virus shedding, seroconversion, pathobiology of clade 2.3.4.4b H5N1 HPAIV as well as Avian Influenza Viruses (AIVs) environmental contamination were thoroughly examined in the infected holding. Such in-depth investigation demonstrated low infection prevalence in live birds, low environmental contamination, no seroconversion for AIVs, gross and microscopic findings compatible with systemic infection with peracute death in H5N1 HPAIV-infected birds.

Gene segment reassortment between Eurasian and American clades of avian influenza virus in Italy.

All genes of avian influenza A viruses are phylogenetically distinguished into two large clades, namely the American and Eurasian clade. Reassortments among the gene segments of influenza viruses belonging to the two distinct clades are rare events and have never been described in poultry in Europe and Asia before. This study presents the genetic characterization of two influenza viruses isolated from domestic mallards in Italy in 2004 and 2005. Phylogenetic analysis of the entire genome showed that these viruses contain mixed gene segments belonging to the American and Eurasian clades.

Genetic variation of Italian avian paramyxoviruses serotype 9.

The haemagglutinin-neuraminidase (HN) and fusion protein (F) gene of four avian paramyoviruses serotype 9 (APMV9) recently isolated from wild birds in Italy have been sequenced. A comparison between the sequences of these four isolates and the prototype virus PMV-9/domestic Duck/New York/22/78 revealed significant sequence variation that suggests that different lineages exist among APMV-9 viruses similar to that seen for APMV-1 (Newcastle disease).

Conventional inactivated bivalent H5/H7 vaccine prevents viral localization in muscles of turkeys infected experimentally with low pathogenic avian influenza and highly pathogenic avian influenza H7N1 isolates.

Highly pathogenic avian influenza (HPAI) viruses cause viraemia and systemic infections with virus replication in internal organs and muscles; in contrast, low pathogenicity avian influenza (LPAI) viruses produce mild infections with low mortality rates and local virus replication. There is little available information on the ability of LPAI viruses to cause viraemia or on the presence of avian influenza viruses in general in the muscles of infected turkeys. The aim of the present study was to determine the ability of LPAI and HPAI H7N1 viruses to reach muscle tissues following experimental infection and to determine the efficacy of vaccination in preventing viraemia and meat localization. The potential of infective muscle tissue to act as a source of infection for susceptible turkeys by mimicking the practice of swill-feeding was also investigated. The HPAI virus was isolated from blood and muscle tissues of all unvaccinated turkeys; LPAI could be isolated only from blood of one bird and could be detected only by reverse transcriptase-polymerase chain reaction in muscles. In contrast, no viable virus or viral RNA could be detected in muscles of vaccinated/challenged turkeys, indicating that viral localization in muscle tissue is prevented in vaccinated birds.

Pathogenicity of a QX strain of infectious bronchitis virus in specific pathogen free and commercial broiler chickens, and evaluation of protection induced by a vaccination programme based on the Ma5 and 4/91 serotypes.

The aims of this study were firstly to evaluate the pathogenicity of an Italian isolate of the QX strain of infectious bronchitis (IB) virus using 1-day-old female specific pathogen free chicks (layer type) and 1-day-old female commercial broiler type chickens, and secondly to assess the level of protection induced in these birds by a vaccination programme including the IB Massachusetts and 4/91 serotype live attenuated vaccines. Unvaccinated birds showed clinical signs of varying severity, predominantly affecting the upper respiratory tract. Vaccinated birds appeared healthy, with the exception of a very mild conjunctivitis affecting a limited number of the broilers. Vaccination fully protected specific pathogen free birds, since no histopathological lesions were observed, nor was virus detected following challenge. In broilers, replication of the challenge virus was not prevented but was significantly reduced. This study confirms that vaccination at 1 day old and at 14 days of age using the Ma5 and 4/91 IB vaccines may be instrumental in reducing the economic impact of QX IB virus infections in layer and broiler farms.

Influenza virus surveillance in wild birds in Italy: results of laboratory investigations in 2003-2005.

Following the avian influenza (AI) epidemics occurring in different areas of the world, a surveillance program funded by the Italian Ministry of Health was implemented. In the framework of this program, an investigation of wild birds was carried out to assess the circulation of AI viruses in their natural reservoir. More than 3000 samples, mainly cloacal swabs, were collected from migratory wild birds belonging to the orders Anseriformes and Charadriiformes. Samples were screened by means of a real-time reverse transcriptase polymerase chain reaction (RRT-PCR), then processed for attempted virus isolation in embryonated fowl's specific pathogen-free eggs. Approximately 5% of the samples were positive for type A influenza viruses by RRT-PCR, and from 14 of those samples AI viruses were isolated and fully characterized. The isolates, belonging to 8 different avian influenza virus subtype combinations (H10N4, H1N1, H4N6, H7N7, H7N4, H5N1, H5N2, and H5N3), were obtained from migratory Anseriformes.

Lethal nephrotropism of an H10N1 avian influenza virus stands out as an atypical pathotype.

A nephrotropic H10N1 avian influenza virus (AIV) with an intravenous pathogenicity index (IVPI) of 1.9 and a haemagglutinin monobasic amino acid cleavage site motif, was genetically and phenotypically characterized. Specific pathogen free chickens of 3 or 6 weeks of age were challenged with a 10(6)EID50/0.1mL dose by either oro-nasal or intravenous route, to study the distribution, tissue tropism and virulence of the virus. Direct transmission was tested by introducing sentinel birds on day 4 post infection. Virus shedding and viremia were investigated by means of type A influenza real-time RT-PCR. Dead birds were necropsied and selected organs were collected for histology, immunohistochemistry, and to detect and re-isolate the virus. Serological analyses were carried out to evaluate seroconversion, three weeks from challenge. The oro-nasal challenge of the 6-week-old birds elicited 47% mortality as a result of viremia and massive replication of the virus in the kidneys. Unexpectedly, among birds of 3 weeks of age the same challenge caused 5% mortality and few clinical signs. Surprisingly the intravenous administration of the virus in the 3-week-old birds recorded an IVPI of 2.4. A full genome characterization of the virus could not identify any molecular determinant underlying the observed phenotype. Our findings describe the complex pathobiology of an AIV of the H10 subtype that stands out for its peculiar pathogenicity and tissue tropism in chickens.

Evaluation of the genetic diversity of avian paramyxovirus type 4.

Avian paramyxoviruses (APMVs) belong to the genus Avulavirus in the family Paramyxoviridae and include at least nine serotypes, APMV-1 to -9, as well as two additional provisional serotypes. Newcastle disease virus (NDV), which comprises APMV-1, is the most extensively studied APMV because it is an important poultry pathogen. A moderate level of antigenic and genetic diversity is recognized for APMV-1 isolates, but our knowledge of the antigenic and genetic diversity of the other APMV serotypes is limited. APMV-4 is frequently isolated from waterfowl around the world. To date complete genome sequences of APMV-4 are available for only strains, which were isolated from ducks in Hong Kong, Korea and Belgium over a period of 37 years. We have carried out genome sequencing from the nucleocapsid (N) gene-end signal to the polymerase (L) gene-start signal of five APMV-4 strains recently isolated from Italy. Each of the eight APMV-4 strains has the same F protein cleavage site, DIQPR↓F. They also share a high level of nucleotide and amino acid sequence identity: for example, the F and HN glycoproteins have greater than 97% sequence identity between the various strains. Thus, comparison of these eight strains of APMV-4 did not provide evidence of substantial diversity, in contrast to similar studies with APMV-2, -3, and -6, in which the F and HN glycoproteins exhibited up to 20-30% amino acid sequence variation within a subgroup. Reciprocal cross-HI assay using post infection chicken sera also failed to detect significant antigenic variation among the available APMV-4 strains.

Usutu virus in Italy: an emergence or a silent infection?

A two year study (2008-2009) was carried out to monitor the Usutu virus (USUV) circulation in Italy. Sentinel horses and chickens, wild birds and mosquitoes were sampled and tested for the presence of USUV and USUV antibodies within the WND National Surveillance plan. Seroconversion evidenced in sentinel animals proved that in these two years the virus has circulated in Tuscany, Emilia Romagna, Veneto and Friuli Venezia Giulia regions. In Veneto USUV caused a severe blackbird die-off disease involving at least a thousand birds. Eleven viral strains were detected in organs of 9 blackbirds (52.9%) and two magpies (0.5%) originating from Veneto and Emilia Romagna regions. USUV was also detected in a pool of Culex pipiens caught in Tuscany. According to the alignment of the NS5 partial sequences, no differences between the Italian USUV strains isolated from Veneto, Friuli and Emilia Romagna regions were observed. The Italian North Eastern strain sequences were identical to those of the strain detected in the brain of a human patient and shared a high similarity with the isolates from Vienna and Budapest. Conversely, there were few differences between the Italian strains which circulated in the North Eastern regions and the USUV strain detected in a pool of C. pipiens caught in Tuscany. A high degree of similarity at both nucleotide and amino acid level was also found when the full genome sequence of the Italian North Eastern isolate was compared with that of the strains circulating in Europe. The North Eastern Italian strain sequence exhibited 97% identity to the South African reference strain SAAR-1776. The deduced amino acid sequences of the Italian strain differed by 10 and 11 amino-acids from the Budapest and Vienna strains, respectively, and by 28 from the SAAR-1776 strain. According to this study two strains of USUVs are likely to have circulated in Italy between 2008 and 2009. They have developed strategies of adaptation and evolution to spread into new areas and to become established.

Complete genome sequences of avian paramyxovirus serotype 6 prototype strain Hong Kong and a recent novel strain from Italy: evidence for the existence of subgroups within the serotype.

Complete genome sequences were determined for two strains of avian paramyxovirus serotype 6 (APMV-6): the prototype Hong Kong (HK) strain and a more recent isolate from Italy (IT4524-2). The genome length of strain HK is 16236 nucleotide (nt), which is the same as for the other two APMV-6 strains (FE and TW) that have been reported to date, whereas that of strain IT4524-2 is 16230 nt. The length difference in strain IT4524-2 is due to a 6-nt deletion in the downstream untranslated region of the F gene. All of these viruses follow the "rule of six". Each genome consists of seven genes in the order of 3'N-P-M-F-SH-HN-L5', which differs from other APMV serotypes in containing an additional gene encoding the small hydrophobic (SH) protein. Sequence comparisons revealed that strain IT4524-2 shares an unexpectedly low level of genome nt sequence identity (70%) and aggregate predicted amino acid (aa) sequence identity (79%) with other three strains, which in contrast are more closely related to each other with nt sequence 94-98% nt identity and 90-100% aggregate aa identity. Sequence analysis of the F-SH-HN genome region of two other recent Italian isolates showed that they fall in the HK/FE/TW group. The predicted signal peptide of IT4524-2 F protein lacks the N-terminal first 10 aa that are present in the other five strains. Also, the F protein cleavage site of strain IT4524-2, REPR downward arrow L, has two dibasic aa (arginine, R) compared to the monobasic F protein cleavage site of PEPR downward arrow L in the other strains. Reciprocal cross-hemagglutination inhibition (HI) assays using post-infection chicken sera indicated that strain IT4524-2 is antigenically related to the other APMV-6 strains, but with 4- to 8-fold lower HI tiers for the test sera between strain IT4524-2 and the other APMV-6 strains. Taken together, our results indicated that the APMV-6 strains represents a single serotype with two subgroups that differ substantially based on nt and aa sequences and can be distinguished by HI assay.

A conventional, inactivated oil emulsion vaccine suppresses shedding and prevents viral meat colonisation in commercial (Pekin) ducks challenged with HPAI H5N1.

The ongoing H5N1 epidemic is currently affecting a number of avian species, including waterfowl. These birds appear to have an important role as reservoirs of infection and comprehensive data on the efficacy of vaccination is currently lacking. The present paper reports the effect of a two dose vaccination programme with a conventional inactivated product on infection, lateral spread, shedding and presence of virus in commodities such as meat and viscera of Pekin ducks. Vaccination of this species appears to be efficacious in suppressing viral shedding, and preventing viraemia and lateral spread of infection to unvaccinated and vaccinated Pekin ducks.

Active surveillance for avian influenza viruses in wild birds and backyard flocks in Northern Italy during 2004 to 2006.

Following the avian influenza epidemics that occurred in Italy between 1997 and 2003, the Italian Ministry of Health in collaboration with veterinary authorities promoted, funded and implemented a national surveillance programme. The main objectives of the surveillance effort were to identify avian influenza viruses circulating in wild birds and to investigate the role of backyard poultry flocks in the dynamics of infection in a densely populated poultry area. Over 2 years (2004 to 2006), 164 backyard flocks and 4083 wild birds (mainly migratory Anseriformes and Charadriiformes) were sampled in three regions in the North of Italy. Samples collected were screened by means of real-time reverse transcriptase-polymerase chain reaction and the positive samples were processed for attempted virus isolation in embryonated fowl's specific pathogen free eggs. At the end of the study period, 27 low-pathogenic avian influenza viruses had been isolated from backyard flocks and 49 strains obtained from wild birds. Of these, 26 belonged to the H5 or H7 subtype and were closely related to contemporary low-pathogenic strains of Eurasian lineage. The findings confirm that backyard free-range farming is at high risk for avian influenza virus introduction, and confirm the role of wild waterfowl in the introduction and perpetuation of low-pathogenic avian influenza viruses during the winter season in Southern Europe.