Serological and molecular comparison of Mannheimia haemolytica and Pasteurella trehalosi strains isolated from wild and domestic ruminants in the French Alps

Over a period of 17 years, 84 bacterial isolates identified as Mannheimia haemolytica or M. glucosida, and 52 isolates identified as Pasteurella trehalosi were detected in the lungs of domestic and wild ruminants in the French Alps. The isolates were serotyped according to their surface capsular antigens, and those sharing common antigens were further characterized by pulsed field gel electrophoresis. The results showed that the bacterial isolates included in the study clustered according to the host species from which they were isolated. These findings indicate that the transmission of serotypes of M. haemolytica, M. glucosida or P. trehalosi from an animal host in which they are common to another species sharing the same geographical space may be a rare epidemiological event.     

Genotyping of Clostridium perfringens isolated from domestic and exotic ruminants and swine

Clostridium perfringens types A, B, C, D and E are known to cause severe enteritis/enterotoxaemia and diseases (especially caused by type A) belonging to the gas oedema complex in many species. Samples from the small intestine as well as faeces of domestic and exotic animals suffering from enterotoxaemic signs or having died within days after first occurance of toxaemia were submitted for typing C. perfringens toxovars by multiplex PCR. The following species have been investigated: domestic sheep (Ovis ammon; n = 10), domestic goat (Capra aegagrus hircus; n = 26), Japanese serow (Capricornis sumatraensis; n = 4), lechwe waterbuck (Hydrotragus leche; n = 1), blackbuck (Antilope cervicapra; n = 1), European reindeer (Rangifer tarandus tarandus; n = 4), domestic swine (Sus scrofa; n = 52), and collared peccary (Tayassu albirostris; n = 1). Interestingly, the predominant C. perfringens toxovar in domestic sheep was type A. This toxovar could also be diagnosed in all reindeer, in three Japanese serows, one lechwe waterbuck and most pigs (n = 47), the majority of those being at suckling age. Type D was the most prevalent toxovar (n = 18) in domestic goats, but also types A and E could be identified as pathogens in this species. Type C could only be found in domestic swine (n = 5) and in one case of clostridiosis in a Japanese serow. Two cases of enterotoxaemia in goats, one case in reindeer, and a single case in blackbuck and collared peccary were caused by C. perfringens type E. Genotyping of C. perfringens is recommended before starting vaccination programmes as it could be shown, that the importance of specific toxovars has been underestimated in specific species and/or age groups.     

Characterization of fluoroquinolone resistance in Escherichia coli strains from ruminants.

Thirty-seven fluoroquinolone-resistant Escherichia coli strains from ruminants (according to Clinical and Laboratory Standards Institute guidelines) were screened by molecular methods for mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes and for the presence of the qnrA gene. One of the strains studied was an enterohemorrhagic E. coli (EHEC) strain potentially pathogenic for humans. Three E. coli strains resistant to enrofloxacin (minimal inhibitory concentration [MIC] = 2 microg/ml) but not to ciprofloxacin (MIC = 1 microg/ml) presented single mutations in the gyrA and parC genes, while 34 strains resistant to both fluoroquinolones presented double and single mutations in gyrA and parC, respectively (31 strains), or double mutations in gyrA and parC (3 strains). The EHEC strain presented a double amino acid substitution in the GyrA protein (Ser-83-->Leu and Asp-87-->Gly) and a double amino acid substitution in the ParC protein (Gly-78-->Cys and Ser-80-->Arg), one of which has not been previously described. The present study shows that most of the mutations in the QRDR of the gyrA and parC genes of fluoroquinolone-resistant E. coli strains from ruminants are the same as those seen in E. coli strains from other animal species and humans and that there are no differences in mutation patterns in the QRDR of E. coli strains from healthy ruminants and those with diarrhea. No strains carried qnrA, which indicates that this gene does not play an important role in the selection of fluoroquinolone-resistant E. coli strains from ruminants.     

Method for obtaining ovine uterine secretions from unilaterally pregnant ewes

Development of the ovine conceptus was confined to the uterine horn ipsilateral to the corpus luteum (CL) by placing a ligature around that uterine horn at a point near the uterine body on day 5 of pregnancy. On day 140 of gestation, seven of 10 ewes were still pregnant and from 21 to 815 ml of uterine fluid (488 +/- 94 ml, X +/- SEM) were collected from the nongravid uterine horn. Total recoverable protein (X +/- SEM) was 13.4 +/- 3.4 grams. Polyacrylamide gel electrophoresis of the reduced proteins in presence of sodium dodecyl sulfate indicated that protein composition of uterine fluid was distinct from that of colostrum, serum, amniotic fluid, and allantoic fluid, and revealed the presence of two major polypeptides with molecular weights of about 57,000 and 58,500, respectively, plus numerous other minor components. Gel filtration on columns of Sephadex G-200 and Sepharose CL-6B suggested that these polypeptides formed a series of aggregates of high molecular weight when kept under nonreducing conditions. Glucose (.18 +/- .03 mg/ml), but not fructose, was present in uterine fluid. In addition, high levels of prostaglandin F (451.4 +/- 83.3 ng/ml) were present.     

Identification of Bartonella strains isolated from wild and domestic ruminants by a single-step PCR analysis of the 16S-23S intergenic spacer region

Of the 20 species or subspecies of Bartonella currently known, 7 cause various diseases in humans with many being zoonotic. However, some Bartonella species appear only to cause asymptomatic bacteraemia in their hosts. In ruminants, three Bartonella species (B. bovis, B. capreoli and B. schoenbuchensis) have recently been described. However, limited or no information has yet been published concerning their mode of transmission and their possible pathogenicity for domestic cattle. The phylogenetic relationship of these species with other bacteria of the Bartonella genus has only been recently investigated. It is therefore necessary to develop appropriate tools that will easily allow identification of these ruminant strains for epidemiological and clinical studies. A single-step PCR assay, based on the amplification of a fragment of the 16S-23S rRNA intergenic spacer (ITS), was evaluated for identification of Bartonella isolated from domestic cattle and from free-ranging or captive cervids. For each Bartonella species tested, the PCR assay led to a product that was unique either for its length or its sequence. All ruminant isolates tested could be easily differentiated among themselves and from the other Bartonella species. Furthermore, sequence analysis of the PCR products revealed a close relationship between all ruminant Bartonella strains. Therefore, ITS PCR testing appears to be a convenient tool for a quick diagnosis of ruminant Bartonella species.     

Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese

1. The aim of this study was to determine the antibiotic susceptibility of 93 Lactobacillus strains isolated from domestic geese raised on Polish farms. The minimal inhibitory concentration (MIC) of 13 antimicrobial substances was determined by the broth microdilution method.

2. All strains were sensitive to the cell wall inhibitors ampicillin and amoxicillin (MIC ≤ 8 μg/ml). Resistance to inhibitors of protein synthesis and to fluoroquinolone inhibitors of replication was found in 44.1% and 60.2% of isolates, respectively; 26.9% strains were resistant to neomycin (MIC ≥ 64 μg/ml), 23.6% to tetracycline (MIC ≥ 32 μg/ml), 15% to lincomycin (MIC ≥ 64 μg/ml), 18.3% to doxycycline (MIC ≥ 32 μg/ml), 9.7% to tylosin (MIC ≥ 32 μg/ml), 56% to flumequine (MIC ≥ 256 μg/ml) and 22.6% to enrofloxacin (MIC ≥ 64 μg/ml).

3. Bimodal distribution of MICs indicative of acquired resistance and unimodal distribution of the high MIC values indicative of intrinsic resistance were correlated with Lactobacillus species. Eleven (11.8%) strains displayed multiple resistance for at least three classes of antibiotics.

4. Data derived from this study can be used as a basis for reviewing current microbiological breakpoints for categorisation of susceptible and resistant strains of Lactobacillus genus and help to assess the hazards associated with the occurrence of drug resistance among natural intestinal microflora.

     

Overview of helminth problems in domestic ruminants in Switzerland

Potent anthelmintics were introduced into the Swiss market several decades ago. Despite this, gastrointestinal nematodes (GIN), lungworms and the large liver fluke (Fasciola hepatica) can successfully inhabit Swiss ruminant farms. This is mainly due to a high reproductive capacity as well as very efficient survival strategies. In addition some species readily develop anthelmintic resistance. GIN-infections in young cattle are under comparatively good control. However, prophylactic measures are compromised where adult stock is also affected due to incomplete development of immune protection. Under these circumstances control measures must include all age groups. This results in fewer helminths in refugia thus may accelerate the development of anthelmintic resistance. This review aims to present a synopsis of the significance of the major helminth infections obtained on pasture by large and small ruminants in Switzerland. Currently available strategies for strategic helminth control are summarized and an outlook is given on new developments which might expand the spectrum of control measures relevant for veterinary practice in the future.     

Distribution of the lingual lymphoid tissue in domestic ruminants

The distribution and organisation of the intralingual lymphoid tissue was studied in sheep, goat and cattle. For each species, the tongues of two animals were harvested and divided in sample blocks extending over the total surface of the tongue. With 2.5 mm intervals, ten serial histological sections were made for conventional histological staining (haematoxylin-eosin, Van Gieson, Masson's trichrome) and immunohistochemical staining of lymphoid cells (anti-CD3, anti-CD21, anti-CD45). Lymphocytes were scattered in the subepithelial propria-submucosa and in the connective tissue cores of the lingual papillae. The connective tissue cores of fungiform papillae, including those located on the lingual apex, and vallate papillae showed relatively more lymphocytes than the propria-submucosa. Lymphoid cell aggregations were even more abundant beneath the grooves surrounding the vallate papillae in small ruminants. In cattle, a well-organised lingual tonsil was additionally found at the root of the tongue. CD3-positive lymphocytes were observed in all species examined. CD21-positive lymphocytes were numerous in the lymphoid nodules of the bovine lingual tonsil but very scarce in the ovine and caprine tongues. Therefore, the lymphoid cell aggregations in the tongues of small ruminants should not be referred to by the term 'lingual tonsil'.     

Assessment of three methods for multilocus fragment typing of Cryptosporidium parvum from domestic ruminants in north west Spain

The performance of three different methods, capillary electrophoresis (CE), high resolution slab-gel electrophoresis and sequencing, for PCR fragment size analysis of two Cryptosporidium parvum microsatellite regions, ML1 and ML2, was investigated by analysing 27 isolates from calves and 14 from lambs. To assess genetic variability of this protozoan in domestic ruminants in north west Spain, results were combined with sequence analysis of the 60 kDa glycoprotein (GP60) gene creating a multilocus type and analysed by farm and host species. CE showed greater overall typability (T), discriminatory power and ease of use than slab-gel electrophoresis and sequencing which were both affected by PCR stutter, especially at ML2. CE fragment sizes were consistently 4 bp longer compared to sequencing which is considered the gold standard for allele sizing but which gave the lowest typability; CE sizes were therefore adjusted. Only three alleles were identified at the ML1 locus (ML1-238, ML1-229 and ML1-226). The ML2 locus was more polymorphic and eight alleles were found (ML2-235, ML2-233, ML2-231, ML2-229, ML2-227, ML2-225, ML2-201 and ML2-176). Adjusted ML1 and ML2 CE fragment sizes were combined with GP60 subtype for 37 of the 41 C. parvum isolates which were typable at all three loci (T=0.90): nine multilocus types (MLTs) were identified. The discriminatory power of the 3-locus typing method was 0.83. Greater genetic variability was observed in calf isolates (7 MLTs) than in those from lambs (4 MLTs) although more calf isolates were studied. The most common MLT in cattle was MLT1 (ML1-238, ML2-231, GP60 subtype IIaA15G2R1), while MLT3 (ML1-238, ML2-227, GP60 IIaA16G3R1) was predominant in lambs. Our findings demonstrate that high discrimination can be achieved by means of multilocus typing. CE appears to be an economic and rapid option for performing microsatellite fragment size analysis offering good typability, discrimination and ease of use but may require calibration to sequenced standards.     

Host-parasite checklist of nematodes of domestic ruminants in New Zealand

Extract

Nematodes of domestic ruminants are abundant and widespread throughout New Zealand, and, although the economic importance of some of them is well-recognized, the literature regarding the occurrence, incidence, and distribution of individual species is sparse and very incomplete. A partial explanation of this may be that in diagnostic laboratories in this country, as elsewhere, it is usual for the purposes of routine diagnostic worm counts to identify the parasites at the generic level only. Thus, little information is gained regarding the geographical and seasonal distribution of individual species where more than one species of a genus may occur in the same location in any particular host.