Establishment of proliferative cell nuclear antigen gene as an internal reference gene for polymerase chain reaction of a wide range of archival and fresh mammalian tissues.

Polymerase chain reaction (PCR) from paraffin-embedded tissues provides a powerful tool to amplify DNA from a variety of recent and archival material. Because DNA from paraffin-embedded samples is more degraded than from fresh material, the amplification of reference genes is essential to exclude false-negative results. This study describes the use of the proliferative cell nuclear antigen (PCNA) gene as a reference gene in a range of animal species and in humans. The PCNA-PCR to amplify a fragment extending from exon 5 through exon 6 and including the intervening intron 6 gave a reproducible pattern, with a 280-base pair (bp) band from canine, equine, bovine, ovine, and caprine samples showing high sequence homology. Porcine, guinea pig, tiger, and lion samples, however, gave an additional fragment of approximately 197 bp. The whole intron 6 from these fragments is missing, possibly representing a pseudogene. In feline samples only the 197-bp fragment could be detected. This study shows that the PCNA gene is highly conserved across a broad range of animal species and is well suited as an internal control for PCR analysis in veterinary medicine.     

Reproduktionszyklen beim Weißen Alpenschaf: Effekt des Widders und der Schur

Inhalt: Bei weiblichen Tieren der Rase Weiβes Alpenschaf (WAS) wurden die Einflüsse des Kontaktes mit einem Widder und der Frühjahrsschur (als Temperaturstress) als mögliche Impulse für die Aufhebung des saisonalen Anöstrus untersucht. Die Geschlechts-aktivität der Tiere wurde mittels des wöchentlich gemessenen Progesteron-Plasmaspiegels beurteilt. Ein vasektomierter Widder hielt bei den meisten Schafen der untersuchten
Gruppe die Geschlechtszyklen während des ganzen Jahres aufrecht; kein deutlicher Anostrus konnte beobachtet werden. Die Schur vor oder nach dem Weidebeginn ver-bunden mit einem Wechsel von schlechtem zu gutem Futter und mit einer Klimaänderung induzierte hingegen keine Unterbrechung des Anöstrus .
Contents: Reproductive cycles in White Alpine Sheep: effect of ram and shearing
The ability to interrupt the seasonal anestrus in the presence of a ram (pheromone effect) and after an early shearing (temperature stress) has been investigated on ewes of the race "white alpine sheep". The occurence of estrus was assessed by measuring of plasma progesterone in weekly intervals. No clear anestric period was observed in a group of 10 animals accompanied by a vasectomized ram throughout the whole year. The reproductive cycles of most ewes were regular (18 days). On the other hand, the shearing before or after the beginning of the grazing period that is accompanied by the change of a poor to a rich feed and by a change of climatic conditions, did not systematically influence the anestrus in our experiments .     

Detection of chlamydiae in boar semen and genital tracts

Chlamydiae cause abortion and reproductive disorders in sows. Although organisms can infect the male genital tract, little is known about the disease situation in boars. Hence, we examined the prevalence of chlamydial infection in semen and genital tracts of boars. Samples collected from Swiss boars (group A: n=42), and boars from Germany (group B: n=39) were examined by bacteriology, LPS-ELISA, immunohistochemistry (IHC) and polymerase chain reaction (PCR). The latter methodology involved use of three PCR assays including 16Sig rDNA, IGS-S (intergenic spacer 16S/23S-Short) and IGS-L (intergenic spacer 16S/23S-Long) PCR for comparison methods. PCR sensitivity and the presence of potential PCR inhibitors were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. Detection limits of the 16Sig and IGS-S PCR were 10 templates, while the IGS-L PCR was less sensitive (100 templates). Of 25 semen samples that were collected from group A, one semen sample was positive for Cp. psittaci and two were positive for Chlamydia-like organisms by 16Sig PCR. Screening of sera from Swiss boars revealed three animals with positive reactions in the LPS-ELISA, although we failed to detect chlamydiae within organs of these or sera-negative animals by IHC or IGS-S PCR. In group B, 10 ejaculates were positive for Chlamydia (C.) suis and two were positive for Chlamydia-like organisms by 16S PCR. The identification of DNA from Chlamydia-like organisms in semen from both groups of boars was surprising and a role for these bacteria in reproductive diseases requires further assessment. In conclusion, the prevalence of chlamydial infection was low in group A animals indicating that venereal transmission may not be significant for Chlamydia-associated reproductive diseases in pigs, although rare cases may occur.     

Chlamydia-related abortions in cattle from Graubunden, Switzerland

In 2001, the first case of bovine chlamydial abortion was reported in canton Graubunden, Switzerland. In this region, Chlamydophila (Cp.) abortus is endemic in small ruminants. Hence, we aimed to investigate the incidence of chlamydia-related abortions in cattle from Graubunden. During breeding seasons of 2003-2004, formalin-fixed and paraffin-embedded placenta specimens (n = 235) from late-term abortions in cattle were analyzed by histopathology, immunohistochemistry with a Chlamydiaceae-specific monoclonal antibody against chlamydial lipopolysaccharide (LPS), and 2 different polymerase chain reaction (PCR) methods (16 S ribosomal ribonucleic acid [rRNA] PCR, intergenic spacer [IGS-S] PCR), followed by PCR product sequencing. In 149 of 235 cases (63.4%), histopathologic lesions such as purulent and/or necrotizing placentitis were observed. Chlamydial antigen was clearly demonstrated in immunohistochemistry in only 1 of 235 cases (0.4%). Cp. abortus or Cp. psittaci was found in 12 of 235 (5.1%) and 10 of 235 cases (4.2%) by 16 S rRNA PCR and IGS-S PCR, respectively. However, we detected, by 16 S rRNA PCR, 43 of 235 cases (18.3%) to be positive for chlamydia-like organisms. In contrast to the situation in small ruminants in the canton Graubunden, bovine abortion from Cp. abortus seems not to play an important role. Nevertheless, zoonotic potential should be taken into account when handling abortion material from cattle. The significance of chlamydia-like isolates other than Waddlia chondrophila remains an open question in abortion and needs further investigation.     

Demonstration of rotavirus antigen in trypsin-digested paraffin tissue sections by immunofluorescence

Trypsin-digested paraffin tissue sections were used to demonstrate rotavirus particles in small intestinal epithelial cells of dirrheic neonatal calves, using direct fluorescent-antibody assay. The results were compared with other techniques to demonstrate rotavirus particles in tissue sections by immunofluorescence. Enzyme treatment of deparaffinized tissue sections gave excellent results with distinct specific fluorescence and minimal background. Prior staining of the tissue section with Mayer's hematoxylin made simultaneous observation of the same tissue section by light and ultraviolet microscopies possible.     

Porcine colon explants in the study of innate immune response to Entamoeba histolytica

Human amebiasis is caused by the protozoan Entamoeba histolytica. This protozoan is responsible for muco-hemorrhagic diarrhoea and liver abscess in affected populations. E. histolytica can be asymptomatic commensally confined to the intestinal lumen or can result in invasion of the colonic mucosa leading to ulceration and/or liver abscesses. Recently, human colonic explants have been identified as valuable in the study of host-parasite interactions. Here we investigated the potential of porcine colonic explants as an alternative to human tissues which are far less available. Porcine colonic explants were cultured with two strains of E. histolytica, one virulent (HM1:IMSS) and one avirulent (Rahman). Results from histopathological and real-time PCR analysis showed that porcine explants cultured with virulent ameba trophozoites react similarly to their human counterparts with an invasion of the tissue by the trophozoites and the triggering of typical innate immune response against the parasite. On the contrary, explants cultured with avirulent ameba trophozoites were preserved. The study open the way to the use of porcine colonic explants in the study of the complex interactions between the parasite and the host.     

Efficacy of Botanical Insecticides in Control of Flea Beetle (Phyllotreta cruciferae) on Cabbage (Brassica oleracea Linn) on Field

An investigation of effect of Latana camara,Tephrosia vogelii and Zingiber officinale applied at 5%,10%and 25%concentrations on flea beetle of cabbage was conducted on the Teaching and Research Farm of the Ladoke Akintola University of Technology Ogbomoso in the early seasons of 2011 and 2012.The experiment consisted of 11 treatments laid in a Randomized Complete Block Design(RCBD)with three replications.Data were subjected to analysis of variance and significant means were compared using the Least Significant Difference(LSD)at p0.05.All the tested plant extracts exhibited moderate efficacy in the control of flea beetle.The treatment plant extracts performed as well as synthetic insecticides(0.76±0.05 and 0.76±0.05,respectively).It was discovered that 25%w/v of Z.officinale gave the best yield of 0.50 kg·hm~(-2).The result showed that extract of T.vogelii(0.71±0.00)and L.camara(0.96±0.23)could suppress flea beetle population at different hours of spraying,while Z.officinale showed an effect at 48 HAS(0.71±0.00)and 72 HAS(0.76±0.05)after spraying.     

Retrospective study of myocardial canine parvovirus infection by in situ hybridization.

The diagnosis of myocardial canine parvovirus (CPV) infection used to depend on the presence of pathognomonic intranuclear inclusion bodies. The in situ hybridization technique, however, allowed to detect CPV specific nucleic acid in myocardial tissue where no inclusion bodies were found. Hence, we applied this technique to check formalin-fixed paraffin-embedded myocardial tissue from puppies with heart lesions for the presence of CPV. The tissues had been collected between 1977 and 1989. A biotinylated probe was used for in situ hybridization. This way CPV specific nucleic acid was detected in 3 dogs where CPV myocarditis had not been diagnosed on routinely stained slides because of the lack of intranuclear inclusion bodies. However, in spite of the application of the in situ hybridization technique no further myocardial CPV infection was detected in puppies with heart lesions from after 1979, confirming that the number of puppies with myocardial CPV infection declined after that year.