Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.     

Identification of polymorphisms and association analysis with meat quality traits in the porcine KIAA1717 and HUMMLC2B genes

Skeletal muscle genes are potential candidates for production and meat quality. Screening a subtracted cDNA library constructed with mRNA obtained from longissimus dorsi muscles of F1 hybrids Landrace × Yorkshire and their female parents Yorkshire, we isolated two partial sequences coding for the H3-K4-specific methyltransferase (KIAA1717) and skeletal muscle myosin regulatory light chain (HUMMLC2B) genes. Database search revealed KIAA1717 and HUMMLC2B encoded proteins with SET domain and EF-hand calcium binding motif, respectively. In the present work we identified their partial polymorphisms and two SNPs, one (C1354T) at the 3′ untranslated region (UTR) of KIAA1717 and one (A345G) at the SINE (PRE-1) element of HUMMLC2B, both created/disrupted a restriction site for endonuclease Msp I. The selected pigs were genotyped at the KIAA1717 C1354T and HUMMLC2B A345G sites by means of a PCR-RFLP protocol. Significant associations were observed for the KIAA1717 C1354T polymorphic site with meat marbling (longissimus doris (p < 0.05), biceps femoris (p < 0.01)) and intramuscular fat (p < 0.01). HUMMLC2B A345G were significantly associated with meat pH (longissimus doris (p < 0.05), biceps femoris (p < 0.01)), drip loss (p < 0.01), water holding capacity (p < 0.01) and meat color value (longissimus doris (p < 0.01), biceps femoris (p < 0.05)). Further studies are needed to confirm these preliminary results.     

Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen

Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs.     

Inflammation-associated responses in piglets induced with post-weaning colibacillosis are influenced by dietary protein level

Forty piglets (average body weight = 5.32 kg) were used to investigate the effect of dietary crude protein (CP) content on immunological responses following a challenge with enterotoxigenic Escherichia coli (ETEC) K88. Pigs, housed 4 per pen, were randomly allotted to 2 diets: 1) a high, 225 g/kg CP diet (HCP) or 2) a low, 176 g/kg CP diet (LCP) supplemented with crystalline amino acids. Pigs were orally challenged with 6 mL of an ETEC K88 suspension containing 10¹⁰ cfu/mL on d 8 after weaning. Blood samples were collected from 10 pigs (1 pig/pen) on d 7 (at weaning), −24 h, 8 h, 72 h and 7 d after the challenge for determination of plasma urea N (PUN) and serum concentrations of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) and haptoglobin (Hp). Tumor necrosis factor alpha, IL-1β and Hp were measured as indicators of inflammatory responses. The concentrations of serum TNF-α at 8 h, 72 h and 7 d after challenge were similar to the level observed at 24 h before challenge but higher (P < 0.05) than the weaning level. Pigs fed the LCP diet had lower (P = 0.032) concentrations of IL-1β (72 vs. 116 pg/mL) at 8 h post-challenge compared with those fed the HCP diet. Likewise, pigs fed the LCP diet tended to have lower (P = 0.088) concentration of Hp (9 vs. 25 mg/dL) compared with those fed the HCP diet at 8 h post-challenge. Compared with the weaning concentration, PUN concentration at 72 h after ETEC challenge was higher (P < 0.05) in pigs fed the HCP diet. The results indicate that the LCP diet supplemented with crystalline amino acids reduced inflammatory responses, as indicated by serum IL-1β, in piglets infected with ETEC K88.     

Clinical findings and outcomes of ulcerative keratomycosis in 30 horses in the mid‐Atlantic United States (2006–2007)

The purpose of this study was to determine the clinical course and outcome associated with keratomycosis in horses in the mid‐Atlantic USA. Records of horses diagnosed with keratomycosis at New Bolton Center from November 2006 to November 2007 with positive fungal culture were retrospectively studied. Neither horses with ulcerative keratitis and a negative fungal culture nor those with stromal abscesses were included. Subject details, history, clinical findings, therapy and outcome were recorded. Thirty horses fitted both inclusion criteria (diagnosis of keratomycosis and positive corneal fungal culture). Fourteen of 30 cases occurred during summer. Aspergillus was the most commonly cultured fungal genus (17/30, or 57%) followed by Alternaria (4/30). Seventeen horses had positive bacterial and fungal cultures. Fifteen of 30 horses were treated surgically by a keratectomy and amnion (8) or conjunctival (7) graft. Itraconazole was the most common topical anti‐fungal therapy and was utilised in 25/30 horses. Globe survival was 97% (29/30). All surviving globes had a positive menace response and were visual at the last examination. It was concluded that in the mid‐Atlantic USA, fungal keratitis is common, has the highest incidence in summer, and is usually associated with a positive outcome. Aspergillus may be a relatively more common corneal pathogen in this region than elsewhere in the USA. Surgical cases were more likely to have fungal hyphae identified on cytology and tended to be hospitalised longer than medical cases. There was no apparent association between surgical disease and all other patient, organism and treatment variables.