Verification by polymerase chain reaction of vertical transmission of Theileria sergenti in cows

To evaluate the transplacental transfer of Theileria sergenti infection in cattle, we used DNA probes to detect T. sergenti in 6 pregnant cows and their calves. All the animals were monitored by parasitologic, serologic, and polymerase chain reaction (PCR) assays for a predicted 875-base-pair (bp) DNA product and a 684-bp amplicon detected by nested PCR in the blood and spleens of aborted fetuses. An open reading frame (ORF) starting at nucleotide 170 and terminating at position 1021 was shown to code for a polypeptide of 283 amino acid residues. All 6 dams and 5 calves were positive for T. sergenti in all tests. One calf was positive only with nested PCR. We conclude that transplacental transmission of T. sergenti is a significant problem. The relevance of the data in the programmed introduction of new (especially pregnant) animals into established clean herds needs serious consideration with regard to control of theileriosis and other tickborne diseases.     

The detection of the meq gene in chicken infected with Marek's disease virus serotype 1

In the genome of strains of very virulent Marek's disease virus serotype 1(vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF, termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CV1988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.     

Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica)

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.     

Immunohistochemical analysis of expression patterns of tumor necrosis factor receptors on lymphoma cells in enzootic bovine leukosis

Tumor necrosis factor-alpha (TNF-alpha) has been reported to be associated with the progression of lymphoproliferative neoplastic diseases and retroviral infections. Hence we examined immunohistochemically the expression patterns of TNF-receptors (TNF-RI and RII) on lymphoma cells derived from the 29 cases of enzootic bovine leukosis (EBL). Lymphomas obtained in 29 animals with EBL were histopathologically classified into three types: diffuse mixed type (10 cases), diffuse large type (9 cases), and diffuse large cleaved type (10 cases). Immunohistochemically using a monoclonal antibody to a bovine lymphocyte surface antigen, the lymphomas were classified into three phenotypes: B-1a (CD5+/CD11b+), B-1b (CD5-/CD11b+) and B-2 (conventional B) (CD5-/CD11b-). Interestingly, the lymphoma cells in all animals expressed TNF-RII, but not TNF-RI. Although, in EBL, lymphoma cells of which the histopathological and immunological property differs has been formed, the expression patterns of TNF-Rs had the universality in all lymphoma cells. TNF-RII, which induces cell proliferation, was expressed but TNF-RI, which induces cell apoptosis was not expressed on all lymphoma cells, suggesting that TNF-Rs play an important role in the malignant proliferation of B cells and formation of lymphomas in EBL.     

Complete cDNA sequences and phylogenetic analyses of the Th1 and Th2 cytokines of the bactrian camel (Camelus bactrianus)

The complementary DNAs of the Th1 (IL-2, IL-12p35, and IFN-gamma) and Th2 (IL-4, IL-10 and IL-13) cytokine genes of the bactrian camel (Camelus bactrianus) were cloned, sequenced, and analyzed. IL-2, IL-4, IL-10, IL-12p35, IL-13, and IFN-gamma were found to have 465, 402, 537, 669, 411, and 501 bp length open reading frames with 154, 133, 178, 222, 136, and 166 amino acid encodings, respectively. The homology ranged from 58.8% to 100% between the nucleotide sequences of the camel cytokine genes and the published sequences of other mammalian genes, including the llama, pig, cow, horse, human, and mouse. The cDNA had highest homology with orders Artiodactyla (pigs and cattle) and Perissodactyla (horses), especially to the recently cloned llama sequences.     

Molecular detection of Anaplasma phagocytophilum in cattle and Ixodes persulcatus ticks

The tick-borne pathogen, Anaplasma phagocytophilum (A. phagocytophilum), the causative agent of human granulocytic anaplasmosis (HGA), is increasingly becoming a public health concern as an aetiological agent for emerging infectious disease. We found A. phagocytophilum infection in a pooled sample of field-collected Ixodes persulcatus (I. persulcatus) ticks from one district in Hokkaido, Japan. Thus, to further investigate the prevalence in field-collected ticks, we used PCR assays targeting the A. phagocytophilum gene encoding 44 kDa major outer membrane protein (p44) for screening of I. persulcatus ticks and samples from cattle from pastures. Out of the 281 I. persulcatus ticks, 20 (7.1%) were found to harbor A. phagocytophilum DNA. The infection rate for A. phagocytophilum in cattle was 3.4% (42/1251). In future studies, it will be necessary to investigate effects of the infection in order to understand its pathogenesis of A. phagocytophilum in domestic animals.     

Odorant receptor-derived cAMP signals direct axonal targeting

In mammals, odorant receptors (ORs) direct the axons of olfactory sensory neurons (OSNs) toward targets in the olfactory bulb. We show that cyclic adenosine monophosphate (cAMP) signals that regulate the expression of axon guidance molecules are essential for the OR-instructed axonal projection. Genetic manipulations of ORs, stimulatory G protein, cAMP-dependent protein kinase, and cAMP response element-binding protein shifted the axonal projection sites along the anteriorposterior axis in the olfactory bulb. Thus, it is the OR-derived cAMP signals, rather than direct action of OR molecules, that determine the target destinations of OSNs.     

Experimental transmission of Bovine leukemia virus in cattle via rectal palpation

We examined whether Bovine leukemia virus (BLV) was transmitted by rectal palpation using a common sleeve between a BLV-infected cow and BLV-negative steers. Three of four steers developed antibodies against BLV as determined by agar-gel immunodiffusion (AGID) test between 7 to 10 weeks after the first rectal palpation using common sleeves from BLV-infected cow. In the steers, BLV proviral DNA were detected by PCR 1 to 5 weeks earlier than detection of the antibodies by the AGID test. Our experiments demonstrated that rectal palpation is a potential cause of BLV spread in herds and that detection of BLV proviral DNA in cattle by PCR is useful screening test for early diagnosis of BLV infection.