Mitigation of greenhouse gases in dairy cattle via genetic selection: 1. Genetic parameters of direct methane using noninvasive methods and proxies of methane.

Records of methane emissions from 1,501 cows on 14 commercial farms in 4 regions of Spain were collected from May 2018 to June 2019. Methane concentrations (MeC) were measured using a nondispersive infrared methane detector installed within the feed bin of the automatic milking system during 14- to 21-d periods. Rumination time (RT; min/d) was collected using collars with a tag that registered time (minutes) spent eating and ruminating. The means of MeC and methane production (MeP) were 1,254.28 ppm and 182.49 g/d, respectively; mean RT was 473.38 min/d. Variance components for MeC, MeP, and RT were estimated with REML using pedigree and genomic information in a single-step model. Heritabilities for MeC and MeP were 0.11 and 0.12, respectively. Rumination time showed a slightly larger heritability estimate (0.17). The genetic correlation between MeP and MeC was high (>0.95), suggesting that selection on either trait would lead to a positive correlated response on the other. Negative correlations were estimated between RT and MeC (-0.24 ± 0.38) and MeP (-0.43 ± 0.35). Methane concentration and MeP had slightly positive correlations with milk yield (0.17 ± 0.39 and 0.21 ± 0.36), protein percentage (0.08 ± 0.32 and 0.30 ± 0.45), protein yield (0.22 ± 0.41 and 0.31 ± 0.35), fat percentage (0.02 ± 0.40 and 0.27 ± 0.36), and fat yield (0.27 ± 0.28 and 0.29 ± 0.28) from bivariate analyses. Rumination time had positive correlations with milk yield (0.41 ± 0.75) and protein yield (0.26 ± 0.57) and negative correlations with fat yield (-0.45 ± 0.32), protein percentage (-0.15 ± 0.38), and fat percentage (-0.40 ± 0.47). A positive approximated genetic correlation was estimated between fertility and MeC (0.10 ± 0.05) and MeP (0.18 ± 0.05), resulting in slightly higher CH4 production when selecting for better fertility [days open estimated breeding values (EBV) are expressed with mean 100 and SD 10, inversely related to days from calving to conception; that is, greater days open EBV implies better fertility]. Positive correlations were also estimated for stature with MeC and MeP (0.30 ± 0.04 and 0.43 ± 0.04, respectively). Other type traits (chest width, udder depth, angularity, and capacity) were positively correlated with methane traits, possibly because of higher milk yield and higher feed intake from these animals. Rumination time showed positive EBV correlations with production traits and type traits, and negative correlations with somatic cell count and body condition score. Based on the genetic correlations and heritabilities estimated in this study, methane is measurable and heritable, and estimates of genetic correlations suggest no strong opposition to current breeding objectives in Spanish Holsteins.

Feeding broilers with dry whey powder and whey protein concentrate affected productive performance, ileal digestibility of nutrients and cecal microbiota community.

Dietary interventions are a common practice in the poultry industry to promote optimal performance and health of animals. Here, we aim at assessing the influence of supplementing broiler diets with dry whey powder (DWP) and whey protein concentrate (WPC) on nutrient coefficient of apparent ileal digestibility (CAID) and productive performance. Cecal microbiota composition was also determined using Illumina amplicon sequencing. Dietary treatments were control diet (no supplementation of DWP or WPC), 60-DWP (60 g/kg of DWP), and 80-WPC (80 g/kg of WPC). One-day-old male broilers were randomly assigned to one of three treatments, and housed in floor pens. In Trial 1, 90 1-day-old chicks were allocated to three pens/treatment, with 10 birds/pen, during 21 days for CAID evaluation. Diet 60-DWP increased Ca CAID (P=0.041), while diet 80-WPC improved Ca and P CAID (P<0.001 and 0.002, respectively) when compared with control diet. In Trial 2, 810 one-day-old chicks were allocated to nine pens/treatment, with 30 birds/pen, during 42 days. Feeding chickens with 60-DWP and 80-WPC increased their BW, average daily gain (ADG) and feed intake (FI) during the starter (P<0.001 for all variables) and grower-finisher periods (P<0.001 for BW and FI, and P=0.048 for ADG), and during the entire feeding period (P<0.05), when compared with control diet. Diets 60-DWP and 80-WPC reduced the feed conversion ratio of chickens during the starter period (P<0.001 and 0.003, respectively), while 60-DWP reduced this parameter during the entire feeding period (P=0.048), when compared to control diet. At day 42, cecal microbial communities of chickens that were fed with 60-DWP and 80-WPC differed from those fed with control diet (R=0.776, P=0.008; and R=0.740, P=0.008, respectively). The abundance of Bacteroides fragilis, Bacteroides spp., Escherichia coli/Shigella flexneri and Megamonas furniformis increased when 60-DWP and 80-WPC diets were offered, while the presence of Helicobacter pullorum decreased. Lactobacillus salivarius consistently increased in chickens with better feed conversion ratio, which were those fed with 60-DWP. The results obtained in the present study indicate that growth of chickens is improved by DWP and WPC supplementation because of a higher mineral digestibility, increased feed intake and modulation of cecal microbiota communities.

Short communication: Signs of host genetic regulation in the microbiome composition in 2 dairy breeds: Holstein and Brown Swiss.

This study aimed to evaluate whether the host genotype exerts any genetic control on the microbiome composition of the rumen in cattle. Microbial DNA was extracted from 18 samples of ruminal content from 2 breeds (Holstein and Brown Swiss). Reads were processed using mothur (https://www.mothur.org/) in 16S and 18S rRNA gene-based analyses. Then, reads were classified at the genus clade, resulting in 3,579 operational taxonomic units (OTU) aligned against the 16S database and 184 OTU aligned against the 18S database. After filtering on relative abundance (>0.1%) and penetrance (95%), 25 OTU were selected for the analyses (17 bacteria, 1 archaea, and 7 ciliates). Association with the genetic background of the host animal based on the principal components of a genomic relationship matrix based on single nucleotide polymorphism markers was analyzed using Bayesian methods. Fifty percent of the bacteria and archaea genera were associated with the host genetic background, including Butyrivibrio, Prevotella, Paraprevotella, and Methanobrevibacter as main genera. Forty-three percent of the ciliates analyzed were also associated with the genetic background of the host. In total, 48% of microbes were associated with the host genetic background. The results in this study support the hypothesis and provide some evidence that there exists a host genetic component in cattle that can partially regulate the composition of the microbiome.

Changes in the dynamics of Coxiella burnetii infection in dairy cattle: an approach to match field data with the epidemiological cycle of C. burnetii in endemic herds.

This study aimed to evaluate changes in the epidemiological status of Coxiella burnetii in dairy cattle herds to better understand the epidemiology of the infection and to predict its evolution. Bulk-tank milk (BTM) and serum samples were collected from 94 dairy cattle herds and analyzed by ELISA (BTM and sera) and PCR (BTM) in study 1 (S1). Two years later (study 2; S2), the same farms were visited with a similar sampling approach. To estimate seroconversion during this period, blood samples were collected from the maximum possible number of animals surveyed in S1. Environmental samples were collected in S2 to identify active shedding. Farms were allocated into 3 different categories in each study according to PCR and ELISA results: category A, with BTM ELISA and PCR positive herds and at least 1 seropositive animal; category B, with BTM ELISA or PCR positive herds or individual sera positive; and category C, with all negative results among herds. Changes in herd category between S1 and S2 were grouped in 9 classes. Two statistical models, one to search for drives of within-herd changes in C. burnetii infection status and another to look for variables modulating individual changes in C. burnetii antibody level, were built. Several herds in category A in S1 remained in that category 2 yr later, indicating that C. burnetii can remain within a herd for a long time. Most of the herds with seroconversion and detection of the bacterium in the environment belonged to category A, suggesting active and recent infections. Changes in the epidemiological status of herds were driven by local densities of domestic ruminants, showing the implication of neighbor reservoirs; whereas individual changes in antibody levels were modulated by variation in the epidemiological status of herds. Observed changes in epidemiological status allowed depiction of the hypothesized life cycle of C. burnetii within dairy cattle herds, which should be tested by future long-term series studies on C. burnetii infection to help fitting control measures (e.g., vaccination) to within-herd C. burnetii status.

Short communication: investigation of Coxiella burnetii occurrence in dairy sheep flocks by bulk-tank milk analysis and antibody level determination.

To estimate the prevalence of Coxiella burnetii in the dairy sheep population from the Basque Country (northern Spain), a study was carried out combining molecular and serological techniques. First, bulk-tank milk samples from 154 flocks belonging to the Latxa Breed Farmers Association were analyzed by PCR, with 22% of flocks testing positive for C. burnetii. Then, a selection of 34 flocks (7 PCR positive and 17 negative) was investigated for the presence of serum antibodies by ELISA test on 1,011 ewes (approximately 30 ewes per flock). A total of 8.9% of the animals were seropositive, 67.6% of the flocks had at least one seropositive animal, but only in 14.7% of them was seroprevalence greater than 25%. Older ewes showed a significantly greater prevalence (17.5%) compared with yearlings (7.5%) or replacement lambs (1.5%). A marginally significant association was found between seroprevalence and PCR detection of C. burnetii in bulk-tank milk. The widespread distribution of C. burnetii in the region advocates for the implementation of Q fever control strategies and highlights the potential risk of sheep as a reservoir and infection source for other domestic and wildlife species and the human population.

Flock-prevalence of border disease virus infection in Basque dairy-sheep estimated by bulk-tank milk analysis.

Bulk-tank milk (BTM) samples from 154 sheep flocks were used to estimate BDV prevalence in the Basque Country in Spain using an ELISA and a RT-PCR test. The proportion of antibody-positive flocks was 68% but varied significantly between provinces and was 93% in Araba and 54-55% in Bizkaia and Gipuzkoa. Most ELISA-positive flocks had very low antibody inhibition percentage (AIP) indicating high seroprevalence and recent BDV exposure. However, only 9% flocks were PCR-positive suggesting few infected ewes were being milked at the time of sampling. Phylogenetic analysis of the 5' NCR sequences of BDV from seven infected flocks showed that all except one clustered within the group formed by BDV type C strains from a previous study in the region, whereas the remaining isolate was closest to BDV type A. These results suggest that BDV strains in most Basque flocks have a common origin and differences in prevalence between provinces are associated to recent events affecting BDV spread such as use of communal pastures and sheep trading. The widespread distribution of BDV in the region, advocates for the implementation of BDV control strategies and highlights the potential risk of sheep as a pestivirus reservoir for other species.

Pathological and aetiological studies of multifocal interstitial nephritis in wasted pigs at slaughter.

Multifocal interstitial nephritis in pigs has been associated with several infectious agents. The objective of the present study was to investigate several different potential infectious agents associated with "white-spotted" kidneys in pigs suffering from wasting at slaughter (aged 6-8 months). Twenty-nine case kidneys (with a "white-spotted" gross appearance) classified into 3 macroscopic lesional grades, and 15 control kidneys (lacking gross lesions), were obtained from a pig abattoir. Laboratory analyses to detect potential associations with the aforementioned pathological condition with Leptospira spp., porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), and bacteria, were carried out. Microscopically, interstitial nephritis with a lymphofollicular inflammatory pattern (follicular nephritis) was observed in both case and control kidneys, with a higher frequency seen in the former ones. No leptospires were identified, although antibodies to the Pomona and Bratislava serovars were detected. Some pyogenic bacteria were also isolated from both case and control kidneys. PCV2 nucleic acid was only detected in 1 case kidney. PRRSV antigen was not found in any tested sample. Some pigs were tested positive for PPV by serology. Apparently, none of the studied agents were specifically associated as being the potential cause of the renal lesions in the studied wasted pigs. The fact that these chronic lesions may have been the consequence of a previous infection with one of these studied microorganisms, or more, and eventually with other non-tested infectious agents during the growing-finishing period, cannot be ruled out.

Intrauterine Neospora caninum inoculation of heifers.

Here, we studied the potential of Neospora caninum tachyzoites to infect heifers when administered in utero by artificial insemination via contaminated semen. Eighteen primiparous cyclic heifers were hormonally synchronized and artificially inseminated. Nine of them, which were inseminated with semen containing 10(7) live N. caninum NC-1 isolate-tachyzoites, reacted with seroconversion and a specific IFN-gamma response. Moreover, N. caninum DNA was demonstrated by a nested-PCR in the blood of all nine heifers and in brain, lungs, liver and uterine horn of several of them. In contrast, nine heifers inseminated with tachyzoite-free semen developed no antibody or IFN-gamma responses, and no parasite DNA was detected in blood or organs. At necropsy, viable embryos were detected in one and six of the infected and non-infected heifers, respectively. No specific Neospora DNA was detected in any of the embryos. This study provides evidence that intrauterine inoculation via contaminated semen cause N. caninum infection in cattle.

Serological evidence of Leptospira interrogans serovar Bratislava infection and its association with abortions in cattle in northern Spain.

A cross-sectional study was carried out in the Basque Country of Spain to determine the seroprevalence of 10 Leptospira serovars in a population of dairy cattle with poor fertility, and a case-control study was carried out in another northern area to investigate the role of Leptospira interrogans serovar Bratislava in abortions. L. Bratislava was the most prevalent serovar in the cross-sectional study, with 25.4 per cent of the cows testing positive in the microagglutination test when a cut-off of 1:10 or higher was applied, followed by Leptospira Hardjo (8.2 per cent), Leptospira Pomona (7.7 per cent), Leptospira Autumnalis (0.7 per cent) and Leptospira Copenhageni (0.1 per cent). In the case-control study the seroprevalence of L. Bratislava was significantly higher among the cows which had aborted when a titre of 1:300 or more was used as a cut-off (9.7 per cent v 3.4 per cent, P=0.008); 69 per cent of the L. Bratislava-infected cows that had aborted apparently aborted as a result of the infection.

Age-specific seroprevalence of Border disease virus and presence of persistently infected sheep in Basque dairy-sheep flocks.

Using p125/p80 antibody and antigen-ELISA tests, age-specific seroprevalence and presence of persistently infected (PI) sheep were investigated in six commercial latxa dairy-flocks, housed for variable periods. The flocks all had a recent history of Border disease (BD). Every flock included seropositive sheep and seven 0.5-3-year-old PI sheep were detected in two of four flocks tested. Age-specific antibody patterns differed according to the presence or absence of PI sheep in the flock. In flocks free of PI sheep, seroprevalence was 6-13% in 1-year-old sheep and 42-93% in older sheep. In contrast, seroprevalence was 67-99% in sheep raised with PI sheep for at least 1 year and 29-33% in replacement 0.5-0.6-year-old sheep (including a PI sheep) indicating that Border disease virus (BDV) transmission in Basque dairy-flocks can be relatively slow. Moderate seroprevalence in young replacement sheep should not discourage further testing to detect PI sheep, and our results highlight the risk of failing to achieve "natural vaccination" prior to pregnancy by mixing PI sheep with BDV-unexposed ewes.

Neospora caninum infection in breeder bulls: seroprevalence and comparison of serological methods used for diagnosis.

AIMS: To investigate the seroprevalence of Neospora caninum infection in breeder bulls. In addition, the level of agreement of serological methods used was evaluated. METHODS: A total of 285 bulls of different ages and breeds raised for reproductive purposes in Spain were sampled for serum antibody activity to N. caninum. Sera were tested using three validated assays: the indirect fluorescent antibody test (IFAT) and two commercially available enzyme-linked immunosorbent assays (ELISA). RESULTS: Antibodies to N. caninum were found in 13.7% of bulls sampled by at least one of the three validated serological assays. The seroprevalence of neosporosis oscillated between 11.2 and 13.3% depending on the serological technique used. No significant associations (P > 0.05) were observed between N. caninum infection and bull breed or age. The serum antibody levels were predominantly low and close to threshold levels in all positive samples. A very good agreement was found among the serological methods used. CONCLUSIONS: This is the first seroprevalence study of bovine neosporosis in breeder bulls and results showed a moderate presence of N. caninum chronic infections.

Bovine abortion associated with Neospora caninum in northern Spain.

During 1996, aborted bovine fetuses from dairy herds in norther Spain were examined by histopathological, immunohistochemical and serological methods for evidence of infection by Neospora caninum. Microscopical brain changes consistent with those of neosporosis were detected in 36 of 81 fetuses and specific antibodies (IFAT titres > or = 1/16) in 32 of 63. Eight fetuses with neurological lesions were seronegative, and eight without lesions were seropositive. In 25 of 34 fetal brains with lesions consistent with published descriptions of neosporosis, immunohistochemical analysis revealed the presence of N caninum antigen. Depending on the diagnostic criteria, the prevalence of neosporosis related to bovine abortion was between 32 and 57 per cent of the fetuses submitted and between 33 and 58 per cent of the herds involved. Data on the geographical, seasonal and fetal age distribution of the infection are given for the fetuses examined and the value of the different diagnostic methods is discussed.