Sequence analysis of the msp4 gene of Anaplasma ovis strains

Anaplasma ovis (Rickettsiales: Anaplasmataceae) is a tick-borne pathogen of sheep, goats and wild ruminants. The genetic diversity of A. ovis strains has not been well characterized due to the lack of sequence information. In this study, we evaluated bighorn sheep (Ovis canadensis) and mule deer (Odocoileus hemionus) from Montana for infection with A. ovis by serology and sequence analysis of the msp4 gene. Antibodies to Anaplasma spp. were detected in 37% and 39% of bighorn sheep and mule deer analyzed, respectively. Four new msp4 genotypes were identified. The A. ovismsp4 sequences identified herein were analyzed together with sequences reported previously for the characterization of the genetic diversity of A. ovis strains in comparison with other Anaplasma spp. The results of these studies demonstrated that although A. ovismsp4 genotypes may vary among geographic regions and between sheep and deer hosts, the variation observed was less than the variation observed between A. marginale and A. phagocytophilum strains. The results reported herein further confirm that A. ovis infection occurs in natural wild ruminant populations in Western United States and that bighorn sheep and mule deer may serve as wildlife reservoirs of A. ovis.     

Evaluation of endotoxaemia in the prognosis and treatment of scouring merino lambs

This study looked at measurement of endotoxaemia as a tool in determining prognosis and probable response to treatment in scouring lambs. One hundred eighty-three lambs in the first 15-20 days of life, from eight Merino sheep farms located in the region of La Serena, south-west Spain, were used in this experiment. Scouring and normal/control lambs were selected following a clinical examination, the scouring group was further divided into subgroups, specifically those that did or did not survive 72 h following treatment. At the time of the clinical examination, faecal and blood samples were taken. Faecal culture and commercial faecal antigen tests for detection of enteropathogens in faeces and serum endotoxin measurement using chromogenic lymulus amoebocyte lysate (LAL) were carried out. Scouring lambs received 0.07 mg/kg liveweight halofuginone once a day for 3 days, a single oral dose of 0.20 mg/kg liveweight of spectinomycin and oral rehydration fluid. The pathogens isolated were Cryptosporidium spp. and Escherichia coli. The case fatality rate was 51% in the scouring lambs. Postmortem findings were consistent with enterotoxigenic E. coli infection. The concentration of endotoxin was 0.18 +/- 0.12 ng/ml in the control group, 0.35 +/- 0.17 ng/ml in the surviving lambs and 0.46 +/- 0.14 ng/ml in the non-surviving lambs. Significant differences between groups were found. Case fatality rate of the scouring lambs with endotoxaemia below 0.30 ng/ml was 0%, while it was 100% above 0.50 ng/ml. These results may be utilized as a prognostic indicator in lambs affected by E. coli and Cryptosporidium that will help aid in decision-making as to whether to treat a lamb or not based on its chances of survival.     

Concurrent infection with Trichinella spiralis and other helminths in pigs

The aim of this study was to investigate possible influence of different helmintosis in the development of Trichinella spiralis in experimental infected pigs. Forty-two Iberian pigs were allocated to six groups. Three groups were single inoculated with Ascaris suum, Metastrongylus apri or T. spiralis, respectively. Two groups were co-infected with T. spiralis and A. suum or T. spiralis and M. apri, respectively, while the last group included uninfected control pigs. Clinical signs were only observed in pigs with single or concurrent M. apri infections, with more severe respiratory symptoms in pigs with mixed M. apri infection. The number of A. suum and M. apri lung larvae, intestinal larvae of A. suum and adult M. apri were reduced in pigs with mixed Trichinella infections compared to pigs with single infections. In contrast, the number of liver white spots was higher in pigs with mixed infections. While T. spiralis muscular larval burdens were increased in pigs concomitantly infected with M. apri, they were reduced in pigs concomitantly infected with A. suum, compared to pigs receiving single infections with either of these helminths. Pigs with single or mixed A. suum infections showed higher eosinophil levels compared to the remaining groups. IgGt, IgG1, IgG2 and IgM against T. spiralis antigen could not be detected in pigs with single Ascaris or Metastrongylus infections, indicating that no cross-antibodies were produced. IgGt, IgG1 and IgM antibodies were detected earlier and generally at higher levels in mixed T. spiralis infections compared to single T. spiralis infections. The results suggest that T. spiralis had a low synergistic interaction with M. apri in concomitantly infected pigs, and an antagonistic interaction in concurrent infection with A. suum.     

Leishmania-specific isotype levels and their relationship with specific cell-mediated immunity parameters in canine leishmaniasis

In the current retrospective study, Leishmania infantum-specific IgG, IgA and IgM levels were determined by ELISA in 106 untreated dogs with clinically-patent leishmaniasis (Sx) and in 171 clinically healthy dogs (Asx) from Spain to investigate the relationship between these Ig isotypes and clinical status. In addition, we studied if different Leishmania-specific humoral pattern exists between Asx dogs with and without cellular mediated immunity (CMI). Fifty-six dogs were assessed by means of lymphoproliferation assay (LPA), interferon production (IFN) and leishmanin skin test (LST), 71 dogs by means of both LPA and IFN and 44 only by LST. Both Sx and Asx dogs produced Leishmania-specific IgG, IgA and IgM antibodies, however the levels and proportion of positive dogs for each Ig isotype were significantly higher in Sx than in Asx ones (P<0.001). Analysis of immunological profiles determined for each cellular technique (positive and negative cellular response for each technique combined with positive or negative specific humoral response) showed that Asx dogs constituted a high heterogeneous group. No correlations were observed between CMI tests and specific IgG or IgM levels. However, a significant inverse correlation was demonstrated between specific IgA levels and LPA response (Spearman's r=-0.220; P=0.035). In general, the low correlation detected between CMI tests and isotype levels might indicate that the immune response is not strongly polarized in the majority of Asx dogs. Additionally, this study suggests that dogs developing T-cell response are probably able to avoid the dissemination of the parasite at least to mucosal surfaces and, as a consequence, to produce low or background specific IgA levels. Further studies are needed to investigate the relationship between specific IgA and parasite load, especially at mucosal site.     

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.     

Epidemiologic investigation of an outbreak of goose parvovirus infection in Sweden

An outbreak of goose parvovirus (GPV) infection on a Swedish goose farm in the spring of 2004 increased the mortality rates from 2% in the early unaffected hatches to 90% and 99% respectively in the two hatches following virus introduction and 40% in goslings hatched later in the same breeding season. In this paper we describe the clinical observations, diagnostic procedures, and epidemiologic investigation carried out to elucidate the source of the infection. The diagnosis was confirmed by serology, virus isolation, and sequence analysis of a 493-bp-long fragment of the VP1 gene. Phylogenetically the causative virus was closely related to pathogenic GPV strains isolated in 2003 and 2004 from Poland and the United Kingdom, respectively. The Swedish isolate exhibited less homology with pathogenic strains from Hungary and Asia and with attenuated vaccine strains. The epidemiologic investigation showed that the virus was first introduced to a contract farm (farm A) and then was transferred with newly hatched goslings to the farm that had submitted the birds for necropsy (index farm). The exact time and source of the virus introduction to farm A could not be determined with absolute certainty. Possible sources of the infection included backyard goose eggs that had been delivered to farm A for subcontract incubation and hatching, wild geese that frequented the flock of breeding geese on pasture on farm A, and a clutch of Canada goose eggs (Branta canadensis) that had been produced by wild geese and was hatched in the same machine as the eggs produced by farm A.     

Effects of transcutaneous electrical nerve stimulation on the fatty acid profile of rabbit longissimus dorsi muscle (preliminary report)

This study was designed to investigate whether transcutaneous electrical nerve stimulation (TENS) of the longissimus dorsi muscle (MLD) of rabbits induces specific proportional changes in the muscle fatty acid composition. Ten 4-week-old Pannon White rabbits were exposed to TENS treatment two times a day, with the following settings: 30 Hz, 20 micros impulse length, 10 mA, 2 x 20 min. After a treatment period of 50 days rabbits were slaughtered and the fatty acid composition of the MLD was determined by gas chromatography. The TENS treatment increased the proportions of linoleic (C18:2 n-6), linolenic (C18:3 n-3) and gondoic acids (C20:1 n-9), compared with the control group. The level of palmitic (C16:0), stearic (C18:0), oleic (C18:1 n-9) and eicosapentaenoic (C20:5 n-3) acids significantly decreased. The proportion of total unsaturated fatty acids significantly increased. On the basis of the results obtained, TENS may have similar effects on the muscle fatty acid profile like physical training. Based on the supposal that the composition of membrane structure was also affected, the electrical stimulation of muscles may have further consequences, e.g. on membrane properties.     

In vitro and in vivo efficacy of alpha-cyclodextrin for treatment of experimental cryptosporidiosis

The efficacy of alpha-cyclodextrin against infection by Cryptosporidium parvum was evaluated using in vitro and in vivo models. Cyclodextrins are water-soluble cyclic hexamers of glucose units with hydrophobic cavities capable of solubilizing lipophiles and are widely used as drug excipients in the pharmaceutical industry. The viability of purified C. parvum oocysts, exposed for 30, 60, 90, 120 min and 24h to different concentrations of alpha-cyclodextrin (2.5, 5, 7.5, 10, 12.5 and 15%), was evaluated by inclusion or exclusion of two fluorogenic vital dyes and by an excystation technique. Preventive and curative efficacies against cryptosporidial infections, at different doses (2.5 and 5%) and regimes of administration of alpha-cyclodextrin, were determined in an experimental neonatal mice model. Results of the viability assay showed a decrease in oocyst viability that was associated with an increase in exposure time, for each of the concentrations used. Moreover, a high proportion of nonviable oocysts (81%) was observed when C. parvum oocysts were exposed to alpha-cyclodextrin (2.5%) for 24h. The intensity of infection, determined 7 days post-inoculation by examination of intestinal homogenates, was significantly lower (P<0.05) than in the control litters, for all the assays carried out with alpha-cyclodextrin. Only 38.8% of the animals became infected when the alpha-cyclodextrin solution (5%) was administered 2h before inoculated oocysts, and every 24h at 1 and 2 days post-inoculation.