Occurrence of Antirodies to Group Specific Chlamydia Antigen in Finnish Sheep,Cattle and Horse Sera

A serological survey on the occurrence of group-specific chlamydial antibodies in random sera of Finnish sheep, cattle and horses was performed. The whole material consisted of 1347 serum samples, including 432 ovine, 454 bovine and 461 equine sera. The sera were sent to the laboratory for various serological tests during 1968–1972. Of the ovine sera 9.5%, bovine 12.8 % and equine 7.1 % showed a titer ≥ 1:16 in the complement fixation test. No definite geographic differences could be found in the distribution of the herds which showed positive results. The ubiquity of chlamydial infections in domestic mammals and their role as a cause of clinical diseases is discussed.     

Alarming Proportions of Methicillin-Resistant Staphylococcus aureus (MRSA) in Wound Samples from Companion Animals,Germany 2010–2012

Staphylococcus (S.) aureus is an important cause of wound infections in companion animals, and infections with methicillin-resistant S. aureus (MRSA) are of particular concern due to limited treatment options and their zoonotic potential. However, comparable epidemiological data on MRSA infections in dogs, cats and horses is scarce, also limiting the knowledge about possible links to MRSA isolates from human populations. To gain more knowledge about the occurrence and genotypic variation of MRSA among wound swabs of companion animal origin in Germany we performed a survey (2010–2012) including 5,229 samples from 1,170 veterinary practices. S. aureus was identified in 201 (5.8%) canine, 140 (12.2%) feline and 138 (22.8%) equine swabs from a total of 3,479 canine, 1,146 feline and 604 equine wounds, respectively. High MRSA rates were identified with 62.7%, 46.4% and 41.3% in S. aureus of canine, feline and equine origin, respectively. Further genotyping including spa typing and multilocus sequence typing (MLST) revealed a comparable distribution of spa types among canine and feline MRSA with CC22 (47.6%; 49.2%) and CC5 (30.2%; 29.2%) as predominant lineages followed by CC398 (13.5%; 7.7%) and CC8 (4.0%; 9.2%). In contrast, the majority of equine MRSA belonged to CC398 (87.7%). Our data highlight the importance of S. aureus and MRSA as a cause of wound infections, particularly in cats and horses in Germany. While “human-associated” MRSA lineages were most common in dogs and cats, a remarkable number of CC398-MRSA was detected in horses, indicating a replacement of CC8-MRSA as the predominant lineage within horses in Germany. These data enforce further longitudinal epidemiological approaches to examine the diversity and temporal relatedness of MRSA populations in humans and animals to assess probable sources of MRSA infections. This would enable a sound risk assessment and establishment of intervention strategies to limit the additional spread of MRSA.     

Structural aspects of the plasminogen of various species

The N-terminal amino acid sequence of equine, ovine, canine, goat and rabbit plasminogen were determined and compared with those already known of the human, bovine, porcine and feline molecule. Furthermore, the kringle 4 domains of equine, ovine, canine and goat plasminogen, prepared by limited cleavage with elastase, were sequenced and compared with the known species of human, bovine, porcine and chicken plasminogen. Homology with the human kringle 4 ranges between 73% (chicken) and 90% (bovine). Comparison of sequences, fragmentation patterns with elastase and adsorption on lysine-Bio-Gel suggests the same structural and functional domains in the animal species as in human plasminogen.     

Prevalence of antibodies to Neospora sp. in horses from Alabama and characterisation of an isolate recovered from a naturally infected horse [corrected

An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of the 536 serum samples. Endpoint titres for the positive samples were 1:50 (35/6.5%), 1:100 (19/3.5%), 1:200 (7/1.3%) and 1:1600 (1/0.2%). Tachyzoites were first seen in cultured bovine turbinate cells 32 days after inoculation with spinal cord homogenates from the horse with CNS disease. Tachyzoites reacted with known N. caninum-positive serum from horses, cows, dogs and mice, but did not react with murine anti-Toxoplasma gondii or equine anti-Sarcocystis neurona serum. Ultrastructural features of tachyzoites and results of comparison of tachyzoite immunodominant proteins revealed that they were identical to those of N. hughesi, a species described recently from a naturally infected horse. The isolate recovered from the naturally infected horse in the present study (designated NA1) is thought to be an isolate of N. hughesi, although confirmation of this awaits additional molecular characterisation. These results provide some additional evidence that N. hughesi is a valid species and that Neospora infections in horses may occur in widely separated geographic regions of the United States.     

Embryo transfer in the dog and cat

The practicality and feasibility of embryo transfer technology in dogs and cats is quickly becoming a clinical reality. Although progress has been slow, I anticipate that embryo transfer will be a practical and an economical technique in the near future. Most importantly, it is essential that the practical lessons learned with equine and bovine embryo transfer be integrated into the development of canine and feline programs.     

The development of PCR methodology for the identification of species of the tapeworm Moniezia from cattle, goats and sheep in central Vietnam

The aims of this study were to investigate the prevalence of Moniezia spp. in domestic ruminants in central Vietnam and to develop a polymerase chain reaction (PCR) technique to distinguish M. expansa from M. benedeni. Among 2040 examined domestic animals (540 cattle, 800 goats, 700 sheep) Moniezia was recovered from 5.4% of cattle, 16.4% of sheep and 20.6% of goats. A set of primers for PCR was designed to classify M. expansa and M. benedeni based on the amplification of DNA corresponding to the internal transcribed spacer of 5.8S rRNA. The 457 specimens (75 from cattle, 162 from goats, 150 from sheep, 30 from horses, 30 from chickens and 10 from dogs) were subjected to PCR for classification of Moniezia spp. PCR products with the expected sizes were amplified from bovine, ovine and caprine specimens. No specific PCR products were found for specimens from horses, chickens and dogs. Of the 75 specimens from cattle, nine were classified as M. expansa and 66 were M. benedeni. Among 162 caprine specimens, 138 were M. expansa and 24 were M. benedeni. The distribution of M. expansa and M. benedeni in 150 ovine specimens was 132 and 18, respectively. These results show that M. expansa is dominant in goats and sheep, whereas M. benedeni is more common in cattle; PCR can be used for classification of these two species.     

Validation of a heterologous fertilization assay and comparison of fertilization rates of equine oocytes using in vitro fertilization,perivitelline, and intracytoplasmic sperm injections

IVF in horses is rarely successful. One reason for this could be the failure of sperm to fully capacitate or exhibit hyperactive motility. We hypothesized that the zona pellucida (ZP) of equine oocytes prevents fertilization in vitro, and bypassing the ZP would increase fertilization rates. Limited availability of equine oocytes for research has necessitated the use of heterologous oocyte binding assays using bovine oocytes. We sought to validate an assay using bovine oocytes and equine sperm and then to demonstrate that bypassing the ZP using perivitelline sperm injections (PVIs) with equine sperm capacitated with dilauroyl phosphatidylcholine would result in higher fertilization rates than standard IVF in bovine and equine oocytes. In experiment 1, bovine oocytes were used for (1) IVF with bovine sperm, (2) IVF with equine sperm, and (3) intracytoplasmic sperm injections (ICSIs) with equine sperm. Presumptive zygotes were either stained with 4′,6-diamidino-2-phenylindole from 18 to 26 hours at 2-hour intervals or evaluated for cleavage at 56 hours after addition of sperm. Equine sperm fertilized bovine oocytes; however, pronuclei formation was delayed compared with bovine sperm after IVF. The delayed pronuclear formation was not seen after ICSI. In experiment 2, bovine oocytes were assigned to the following five groups: (1) cumulus oocyte complexes (COCs) coincubated with bovine sperm; (2) COC exposed to sucrose then coincubated with bovine sperm; (3) COC coincubated with equine sperm; (4) COC exposed to sucrose, and coincubated with equine sperm; and (5) oocytes exposed to sucrose, and 10 to 15 equine sperm injected into the perivitelline (PV) space. Equine sperm tended (P = 0.08) to fertilize more bovine oocytes when injected into the PV space than after IVF. In experiment 3, oocytes were assigned to the following four groups: (1) IVF, equine, and bovine COC coincubated with equine sperm; (2) PVI of equine and bovine oocytes; (3) PVI with equine oocytes pretreated with sucrose; and (4) ICSI of equine oocytes. Oocytes were examined at 24 hours for cleavage. No equine oocytes cleaved after IVF or PVI. However, ICSI conducted with equine sperm treated with dilauroyl phosphatidylcholine resulted in 85% of the oocytes cleaving. Sperm injected into the PV space of equine oocytes did not appear to enter the ooplasm. This study validated the use of bovine oocytes for equine sperm studies and indicates that failure of equine IVF is more than an inability of equine sperm to penetrate the ZP.     

Extenders for preservation of canine and equine spermatozoa at 5 degrees C

Two experiments were conducted to evaluate the effects of six extenders and three glycerol levels on the motility of sperm stored at 5 degrees C. Using a split-ejaculated design, semen from 10 dogs and 12 stallions was extended with egg-yolk-tris (EYT), egg-yolk-bicarbonate (EGB), Beltsville F-3 (BF-3), Cornell University (CUE), caprogen (CAP) and heated skim milk (SM) extenders. After cooling to 5 degrees C, additional extender containing 0% to 12% glycerol was added to provide a final concentration of 0%, 3% or 6% glycerol. Regardless of glycerol level, a higher (P<0.05) percentage of canine sperm retained their potential for progressive motility in CAP extender than in EYT, SM, CUE, EGB or BF-3 extenders. The SM extender was the best (P<0.05) for maintaining motility of equine sperm. The inclusion of 6% glycerol depressed (P<0.05) motility of canine sperm, but there was no effect (P>0.05) of glycerol concentration on the percentage of motile equine sperm. For both species, the interaction of glycerol level and extender was nonsignificant. CAP may be useful for storage of canine sperm at 5 degrees C and SM may be satisfactory for storage of equine sperm.     

Identification of a Pegivirus (GB Virus-Like Virus) That Infects Horses

The recent identification of nonprimate hepaciviruses in dogs and then in horses prompted us to look for pegiviruses (GB virus-like viruses) in these species. Although none were detected in canines, we found widespread natural infection of horses by a novel pegivirus. Unique genomic features and phylogenetic analyses confirmed that the tentatively named equine pegivirus (EPgV) represents a novel species within the Pegivirus genus. We also determined that EPgV causes persistent viremia whereas its clinical significance is undetermined.     

Comparative analysis of vertebrate EIF2AK2 (PKR) genes and assignment of the equine gene to ECA15q24–q25 and the bovine gene to BTA11q12–q15

The structures of the canine, rabbit, bovine and equine EIF2AK2 genes were determined. Each of these genes has a 5'' non-coding exon as well as 15 coding exons. All of the canine, bovine and equine EIF2AK2 introns have consensus donor and acceptor splice sites. In the equine EIF2AK2 gene, a unique single nucleotide polymorphism that encoded a Tyr329Cys substitution was detected. Regulatory elements predicted in the promoter region were conserved in ungulates, primates, rodents, Afrotheria (elephant) and Insectifora (shrew). Western clawed frog and fugu EIF2AK2 gene sequences were detected in the USCS Genome Browser and compared to those of other vertebrate EIF2AK2 genes. A comparison of EIF2AK2 protein domains in vertebrates indicates that the kinase catalytic domains were evolutionarily more conserved than the nucleic acid-binding motifs. Nucleotide substitution rates were uniform among the vertebrate sequences with the exception of the zebrafish and goldfish EIF2AK2 genes, which showed substitution rates about 20% higher than those of other vertebrates. FISH was used to physically assign the horse and cattle genes to chromosome locations, ECA15q24–q25 and BTA11q12–15, respectively. Comparative mapping data confirmed conservation of synteny between ungulates, humans and rodents.     

Non-Avian Animal Reservoirs Present a Source of Influenza A PB1-F2 Proteins with Novel Virulence-Enhancing Markers

PB1-F2 protein, expressed from an alternative reading frame of most influenza A virus (IAV) PB1 segments, may possess specific residues associated with enhanced inflammation (L62, R75, R79, and L82) and cytotoxicity (I68, L69, and V70). These residues were shown to increase the pathogenicity of primary viral and secondary bacterial infections in a mouse model. In contrast to human seasonal influenza strains, virulence-associated residues are present in PB1-F2 proteins from pandemic H1N1 1918, H2N2 1957, and H3N2 1968, and highly pathogenic H5N1 strains, suggesting their contribution to viruses'' pathogenic phenotypes. Non-human influenza strains may act as donors of virulent PB1-F2 proteins. Previously, avian influenza strains were identified as a potential source of inflammatory, but not cytotoxic, PB1-F2 residues. Here, we analyze the frequency of virulence-associated residues in PB1-F2 sequences from IAVs circulating in mammalian species in close contact with humans: pigs, horses, and dogs. All four inflammatory residues were found in PB1-F2 proteins from these viruses. Among cytotoxic residues, I68 was the most common and was especially prevalent in equine and canine IAVs. Historically, PB1-F2 from equine (about 75%) and canine (about 20%) IAVs were most likely to have combinations of the highest numbers of residues associated with inflammation and cytotoxicity, compared to about 7% of swine IAVs. Our analyses show that, in addition to birds, pigs, horses, and dogs are potentially important sources of pathogenic PB1-F2 variants. There is a need for surveillance of IAVs with genetic markers of virulence that may be emerging from these reservoirs in order to improve pandemic preparedness and response.     

Sarcocystis fayeri sp. n. from the horse.

Hearts, diaphragms, esophagi, and spinal cords from 266 horses were obtained at slaughter in Creston, Ohio. Tissues were examined microscopically for Sarcocystis in sections, digested in trypsin to obtain bradyzoites, and fed to 10 dogs and 10 cats. Intramuscular cysts were found in selections of two hearts from 57 horses and four esophagi from 107 horses. The cysts were up to 900 micron long and up to 70 micron wide. The cyst wall was 1 to 2 micron thick and cross-striated. The enclosed bradyzoites were banana-shaped, 15 to 20 by 20 to 3 micron, and contained several PAS-positive granules. Bradyzoites were found in trypsin digests of seven of 57 (13%) equine tissues (heart, diaphragm, esophagus but not spinal cord) in one experiment and 10 of 47 (21%) esophagi, eight of 47 (17%) diaphragms but none of 47 hearts and spinal cords in another experiment. All of 10 dogs shed sporulated sporocysts or oocysts in feces 12 to 15 days (12 in one, 13 in eight, and 15 days in one) after digesting tissues from 169 horses. The sporocysts were 11 to 13 (12.0 +/- 0.5) by 7 to 8.5 (7.9 +/- 0.5) micron. In histologic sections of canine small intestine the sporocysts were located in the lamina propria near the tips of the villi. The 10 cats fed tissues from 266 horses did not shed Sarcocystis. A new name, S. fayeri, is proposed for this organism. Sarcocystis fayeri sporocysts (12 by 8 micron) are shorter than those of S. betrami (15 by 10 micron), the other species of Sarcocystis from the horse. The prepatent period is 12 to 15 days for S. fayeri and 8 days for S. bertrami (synonym S. equicanis Rommel and Geisel 1975).     

Characterization and differentiation of equine umbilical cord-derived matrix cells

Stem cells are being evaluated in numerous human clinical trials and are commercially used in veterinary medicine to treat horses and dogs. Stem cell differentiation, homing to disease sites, growth and cytokine factor modulation, and low antigenicity contribute to their therapeutic success. Bone marrow and adipose tissue are the two most common sources of adult-derived stem cells in animals. We report on the existence of an alternative source of primitive, multipotent stem cells from the equine umbilical cord cellular matrix (Wharton's jelly). Equine umbilical cord matrix (EUCM) cells can be cultured, cryogenically preserved, and differentiated into osteo-, adipo-, chondrogenic, and neuronal cell lineages. These results identify a source of stem cells that can be non-invasively collected at birth and stored for future use in that horse or used as donor cells for treating unrelated horses.     

Species specificity of lupus-like anticoagulants

Mixing studies using activated partial thromboplastin time (APTT) technique were performed on 14 patients with lupus-like anticoagulants (LLACs) using human, equine, bovine, porcine and canine plasma. Eleven patients significantly prolonged the APTT of normal human plasma (patient/control ratio = greater than 1.15) but no patient inhibited bovine plasma. However, with one exception, equine APTT ratios were concordant with human ratios. Seven of fourteen patients also inhibited porcine plasma but in each case porcine APTT ratios were lower than their human or equine counterparts. None of five patients tested inhibited canine plasma. Collectively, these results suggest heterogeneity among LLACs at least with regard to species specificity.     

The Use of Naturally Occurring Cancer in Domestic Animals for Research into Human Cancer: General Considerations and a Review of Canine Skeletal Osteosarcoma

For many years, research into human cancer has concentrated on human patients and on artificially induced neoplasms in inbred murine hosts. Cancer, however, affects a great variety of mammals, particularly those that have been domesticated. Suchf naturally occurring neoplasms are common in dogs, cats, cattle, horses, etc., and offer fertile ground for studies relating to epidemiologyf, etiology, immunobiology, and therapy. Canine osteosarcoma is described in detail. The clinicopathologic features of this canine tumor closely approximate that of human osteosarcoma and thus make canine osteosarcoma an invaluable comparative model. Canine osteosarcoma and other naturally occurring tumors lie intermediate between the mouse models and human cancer. The use of these veterinary models in the future fabric of cancer research will broaden its base and will influence our conceptual approach to research and clinical options.     

Cross-sectional prospective survey to study indication-based usage of antimicrobials in animals: Results of use in cattle

Background

Indication-based data on the use of antimicrobials in animals were collected using a prospective cross-sectional survey, similarly as for surveys carried out in human medicine, but adapting the questionnaire to include veterinary-specific issues. The participating veterinarians were randomly selected from a sample population of practising veterinarians. The sampling was stratified to take into account the proportions of different types of veterinary practice in the country. All patients consulting the veterinary practice during a 1-week period were included in the study and veterinarians returned a completed questionnaire for each patient receiving antimicrobial treatment. As cattle received most of the treatments, results from the survey are given using cattle as an example species.

Results

The survey was sent to 681 veterinarians, of whom 262 (39%) responded. In total 2850 questionnaires were completed. The largest quantities of antimicrobials, measured in kilograms, were used for cattle, followed by pigs, dogs and horses. The species that were treated most were cattle (n = 1308), dogs (n = 989) and cats (n = 311). For cattle, the most common reason for treatment was acute mastitis (52%), followed by dry-cow therapy (21%), subclinical mastitis (6%) and treatment for acute enteritis (4%). The remaining treatments covered 17% of cattle patients and 15 different indications. For acute mastitis, parenteral or intramammary treatment was used in 36% and 34% of the cases, respectively. The remaining 30% received both treatments simultaneously. Of the parenteral treatments (n = 459), benzyl penicillin was used in 83% of the treated animals (n = 379), while fluoroquinolones were used in 49 cases (11%). Of the 433 cows receiving intramammary treatment, ampicillin combined with cloxacillin was most commonly used (n = 157; 36%), followed by cephalexin+streptomycin (n = 113; 26%).

Conclusion

This cross-sectional prospective survey provided a useful method for the collection of information on the indication-based use of antimicrobials in different animal species. Cattle were the most commonly treated animal species during the study period. The most common indication for antimicrobial use in cows was mastitis. Benzyl penicillin was the drug most frequently used for the treatment of mastitis, which seems appropriate according to the national guidelines on the use of antimicrobials in cattle in Finland.

     

Detection of sex chromosome aneuploidy in dog spermatozoa by triple color fluorescence in situ hybridization

With the development of a direct visualization of sex chromosome in a single sperm by fluorescence in situ hybridization (FISH) technique, the frequency of aberration (aneuploidy) in spermatozoa in several mammals has been investigated. However, there is no report in the incidence of X-Y aneuploidy in the sperm population of dogs. Therefore, in this study, the aneuploidy in dog spermatozoa was examined by multicolor FISH using specific molecular probes for canine sex chromosomes and autosome. Semen from eight male Labrador retrievers was used as specimen. For decondensation of sperm nuclei, the specimen was treated with 1 M NaOH for 4 minutes at room temperature. Probes for chromosomes X, Y, and 1, labeled with SpectrumGreen, Cy3 and Cy5, respectively, were hybridized with decondensed spermatozoa. Fluorescence in situ hybridization signals in sperm heads were clearly detected in each specimen, regardless of the sperm donor. The FISH signal of at least one of the three probes was detected in all sperm heads examined. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X and Y chromosomes in spermatozoa of all the eight dogs. Mean percentage of sex chromosome aneuploidy was 0.127% (ranged between 0% and 0.316%). This percentage of canine sex chromosome aneuploidy was lower than the one reported in cattle, horses, river buffalo, and goats sperm, but higher than that observed in mice and sheep.     

The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pairs in closely related species

The high degree of polymorphism displayed by DNA microsatellites makes them useful as DNA markers in linkage studies. A search of the DNA sequence databases revealed that the locations of dinucleotide microsatellites are often conserved among mammalian species, enabling the prediction of the presence of DNA microsatellites using comparative genetic data. In closely related species such as cattle and sheep, this conservation was close enough to allow PCR primers designed for use in one species to be used to analyze microsatellite length polymorphism in the other. A total of 48 sets of primer pairs, flanking bovine microsatellites and giving polymorphic PCR products in that species, were tested with template DNA from sheep, horses, and humans. Specific products were obtained in 27 cases (56%) with ovine DNA, 20 of which (42%) showed polymorphisms. With equine DNA, 3 (6.2%) gave specific but monomorphic products, while no specific products were obtained using human DNA. The ability to use heterologous PCR primers, coupled with comparative mapping information will facilitate the use of DNA microsatellites in gene mapping studies in closely related species such as cattle and sheep, rat and mouse, or primates.