Eraldo Lourenso Zanella

DNA vaccination elicits protective immune responses against pandemic and classic swine influenza viruses in pigs.

ABSTRACT Swine influenza is a highly contagious viral infection in pigs that significantly impacts the pork industry due to weight loss and secondary infections. There is also the potential of a significant threat to public health, as was seen in 2009 when the pandemic H1N1 influenza virus strain emerged from reassortment events among avian, swine, and human influenza viruses within pigs. As classic and pandemic H1N1 strains now circulate in swine, an effective vaccine may be the best strategy to protect the pork industry and public health. Current inactivated-virus vaccines available for swine influenza protect only against viral strains closely related to the vaccine strain, and egg-based production of these vaccines is insufficient to respond to large outbreaks. DNA vaccines are a promising alternative since they can potentially induce broad-based protection with more efficient production methods. In this study we evaluated the potentials of monovalent and trivalent DNA vaccine constructs to (i) elicit both humoral and gamma interferon (IFN-γ) responses and (ii) protect pigs against viral shedding and lung disease after challenge with pandemic H1N1 or classic swine H1N1 influenza virus. We also compared the efficiency of a needle-free vaccine delivery method to that of a conventional needle/syringe injection. We report that DNA vaccination elicits robust serum antibody and cellular responses after three immunizations and confers significant protection against influenza virus challenge. Needle-free delivery elicited improved antibody responses with the same efficiency as conventional injection and should be considered for development as a practical alternative for vaccine administration.

Growth and carcass characteristics of pasture fed LHRH immunocastrated, castrated and intact Bos indicus bulls

The effectiveness of a luteinizing hormone-releasing hormone (LHRH) fusion protein vaccine or surgical castration, at two years of age, on growth and carcass characteristics of Bos indicus bulls was evaluated. Seventy Nelore-cross bulls were divided into three groups: (1) immunized, (2) castrated and (3) intact control. At slaughter (three years of age), intact bulls had higher body weights, ADG, carcass weights, and muscle percentage compared to immunized and surgically castrated animals. Both castrated and immunized animals had greater marbling and percent carcass fat than the intact bulls. Average tenderness scores were inferior for intact bulls compared to immunized and castrated animals, but these differences were not significant (P>0.05). Juiciness, flavor, thawing, nor cooking losses differed significantly among the three groups. Immunocastration was effective in producing carcass traits similar to that of surgical castration. Therefore, immunization with LHRH fusion proteins appears to have practical utility in the management and castration of grazing bulls.

Natural co-infection of torque teno virus and porcine circovirus 2 in the reproductive apparatus of swine

This work aimed to detect and study natural co-infection of Circoviridae torque teno virus (TTV) and porcine circovirus 2 (PCV2) in the swine reproductive apparatus. Semen and organs from 17 boars were tested by nested and real-time PCR. PCV2 was amplified from semen (47%), lymph nodes (84.6%) and testicles (35.3%). TTV2 was amplified from 16/17 testis and 13/13 lymph nodes. TTV1 DNA was detected in fewer testicle samples (2/17), which were also TTV2 positive. Analyzed ovaries, follicular fluid and uteri of 83 culled sows showed TTV2, TTV1 and PCV2 from 49.3%, 30.1% and 6.0% of the sows, respectively. Sperm analysis indicated insignificant differences between PCV2 and TTVs positive and negative boars. The most frequent pathologic lesion in sows was endometritis (28.9%), but this was unassociated with PCV2 or TTVs detection. These findings question the importance of PCV2 and TTV2 natural co-infection in the pathology of porcine reproductive failures.

Desempenho e características de carcaça de bovinos de diferentes grupos genéticos

This experiment was carried out to evaluate the performance of steers of three genetic groups: Nellore (NN), ½ Guzerat × ½ Nellore (GN) and ½ Brahman × ½ Nellore (BN). Forty-one steers, 24 mo old, kept in pasture of Brachiaria grass. All animals were from the same herd and were raised under the same conditions and slaughtered at 3 years old. At the beginning of the experiment and at slaughter, Brahman-crossed animals were heavier than the animals from the other two groups. Means for body weight for the groups NN, GN and BN, were respectively, 324, 320 and 343 kg at the beginning of the experiment, and 474, 470 and 499 kg at slaughter. However, average daily gains were similar among the groups (0.388, 0.386 and 0.409 kg/d, respectively). Animals of BN group produced heavier hot carcasses (253 × 238 kg) than those obtained in the GN group, but these two groups did not differ from the NN group (242 kg). Percentages of muscle, fat and bones in the carcass were similar among the groups. Other carcass traits (dressing percentage, ribeye area, fat thickness and marbling) and meat tenderness, measured by a trained panel or by a texturometer were, also, similar among the three genetic groups. The crossing of other zebu breeds (Brahman or Guzerat) with Nellore did not improve quality characteristics of carcasses and meat; however, crossing with Brahman resulted in heavier animals with heavier carcasses.

Transmission of porcine circovirus 2 (PCV2) by semen and viral distribution in different piglet tissues

Porcine circovirus infections are caused by the porcine circovirus 2 (PCV2). Among six different clinical manifestations involving respiratory, enteric, nervous and reproductive signs, the postweaning multisystemic wasting syndrome (PMWS) is the most important and studied disease. However, reproductive failures associated with PCV2 have been increasingly reported. Some studies have shown the possible contamination of sows by semen of PCV2 positive boars. In order to investigate the transmission of PCV2 by contaminated semen and its ability to infect the sow and piglets, 20 PCV2 negative sows were inseminated, 10 with negative boar semen and 10 with previously nested-PCR tested positive boar semen. The sows were weekly monitored and blood samples were collected. Based on the results, 4 out 20 sows were selected (1 sow was PCR negative and inseminated with a negative semen, 2 sows were PCR negative and inseminated with a positive semen and 1 sow was PCR negative and inseminated with a positive semen, but became PCR positive around the 30 days of pregnancy). After weaning, 12 male piglets, 3 of each sow, were selected and maintained under isolation. In order to investigate which organs harbored the virus, the young pigs were necropsied around 9 months of age. Samples of serum collected monthly were tested by immunocitochemistry (ICC), and all 12 pigs serum converted. Samples of lymphoid, systemic and reproductive organs were analyzed by nested-PCR and immunohistochemistry (IHC). Evaluation of the samples by nested-PCR, revealed that several tissues were positive in 10 of 12 pigs, mainly the lymph nodes, bone marrow and spleen. Various samples were positive by IHC in 8 of 12 piglets, being the lymph nodes, tonsils and bulbourethral glands the most frequently positive. Thus, the results of testing different samples, in the 3 tests (ICC, nested-PCR and IHC) were complementary. These results show that PCV2 transmission through semen to the sows and piglets may occur and may also represent a potential risk for the herd.

Increased incidence of DNA amplification in follicular than in uterine and blood samples indicates possible tropism of Neospora caninum to the ovarian follicle

This study evaluated the presence of Neospora caninum in ovarian follicle aspirates and uterine flushes obtained from N. caninum seropositive dairy cows. Ninety-two cows that aborted within the previous 90 days were sampled to determine the presence of antibodies against N. caninum. Thirteen seropositive cows were chosen for collection of blood leukocytes, uterine flushes (UF; n=12) and follicular aspirates (OPU; n=13). Samples were centrifuged and the cellular sediment from the follicular fluid, uterine flushes and blood leukocytes were used for DNA extraction and PCR. Follicular aspirates had the highest frequency of DNA amplification for N. caninum (p<0.05, 92.3%; 12/13). Whereas uterine (4/12) and blood leukocyte (5/13) samples had similar (p>0.05) rate of positive results. Nonetheless, there was no agreement between blood leukocytes and follicular samples taken from the same animal (Cohen Kappa=-0.16). Similarly, blood leukocytes and uterine results had moderate agreement between them (Cohen Kappa=0.47). This study indicates that N. caninum is present in the ovarian follicle and uterus of seropositive cows, suggesting a possible risk of neosporosis transmission between females during oocyte and embryo collection and transfer. Hence, precautions should be taken to minimize the risk of N. caninum transmission. Furthermore, the high incidence of positive results in follicle samples, that exceeded that of their paired blood leukocytes, suggests a possible tropism of N. caninum for the ovarian follicle.

Genome-wide association study of periweaning failure-to-thrive syndrome (PFTS) in pigs

Porcine periweaning-failure-to-thrive syndrome (PFTS) is a condition that affects newly weaned piglets. It is characterised by a progressive debilitation leading to death, in the absence of infectious, nutritional, management or environmental factors. In this study, we present the first report of PFTS in South America and the results of a genome-wide association study to identify the genetic markers associated with the appearance of this condition in a crossbred swine population. Four chromosomal regions were associated with PFTS predisposition, one located on SSCX, one on SSC8, and the two other regions on SSC14. Regions on SSC8 and SSC14 harbour important functional candidate genes involved in human depression and might have an important role in PFTS. Our findings contribute to the increasing knowledge about this syndrome, which has been investigated since 2007, and to the identification of the aetiology of this disease.

Identification of Genetic Regions Associated with Scrotal Hernias in a Commercial Swine Herd

In this paper, we have used two approaches to detect genetic associations with scrotal hernias in commercial pigs. Firstly, we have investigated the effects of runs of homozygosity (ROH) with the appearance of scrotal hernias, followed by a Genome Wide Association Study (GWAS). The phenotype classification was based on visual appearance of scrotal hernias. Each affected animal was matched to a healthy control from the same pen. In the total, 68 animals were genotyped using the Porcine SNP60 Beadchip, out of those, 41 animals had the presence of hernias and 27 were healthy animals. Fifteen animals were removed from the analysis due to differences in genetic background, leaving 18 healthy animals and 35 piglets with scrotal hernia. Further, the detection of extended haplotypes shared ROH were conducted for health (control) and affected (case) animals and a permutation test was used to test whether the ROH segments were more frequent in case/case pairs than non-case/case pairs. Using the ROH, we have identified an association (p = 0.019) on chromosome 2(SSC2) being segregated on animals with the presence of scrotal hernias. Using a GWAS, a region composed by 3 SNPs on the sexual chromosome X (SSCX) were associated with scrotal hernias (p < 1.6 × 10−5), this region harbors the Androgen Receptor Gene (AR).

Oxidative Status of Boar Semen during Storage

The intensification of the use of artificial insemination in the swine industry has increased the interest to develop proper conditions to store semen better for longer periods of time without affecting its fertility. Trolox is a vitamin E analog with antioxidant property to prevent oxidative stress. Therefore, the objective of this study was to evaluate the Trolox antioxidant capacity on boar semen during storage period (86 h) at 15°C. For that, semen samples from 5 different boars were collected and diluted with a Long-Term (LT) extender with and without the addition of Trolox in a concentration of 50 µM. The sperm motility, oxidative stress and antioxidant protection were measured in those samples during an 84 h storage time. Our results indicated that Trolox was able to minimize the loss of sperm motility after 24 h of storage. The storage time was positively correlated with TBARS production. However in the presence of Trolox, after 12 h, TBARS content were significantly lower than those of sperm cells stored with LT diluent only. The concentrations of Non-Protein SH groups (NPSH), after 36 h of storage, were lower in sperm cells stored only with LT diluent when compared to the group supplemented with Trolox. Catalase and Superoxide Dismutase activities were decreased after 48 h storage at 15°C in both groups (with or without Trolox). Spontaneous spermatozoa ROS formation was high in viable swine sperm during the LT storage period in samples without Trolox. In conclusion, LT diluent with the addition of antioxidant as Trolox can reduce the oxidative stress in sperm cells, which could be used to protect spermatozoa against oxidative damage, preventing motility losses in swine semen.

Identificação de paternidade para avaliação da contribuição da primeira e segunda doses inseminantes na composição da leitegada suína

A protocol of two inseminations (12h and 36h; time schedule) per estrous was applied in fifty sows in order to study the individual influence of the insemination order on the offspring composition. Analyses of microsatellites were used for identification of the paternity. The first and the second inseminations contributed respectively with 34.8% and 65.2% of all piglets that were born. Piglets were born from both inseminations (mixed litters) in 80% of the farrowings. Each of the first or the second insemination contributed to 10% of the litters. Although the first insemination was applied before the first half of the estrous, it contributed to the piglets that were born in 90% of the farrowings.