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1.
The prevalence of Cryptosporidium species in calves and heifers with relation to diarrhea from several herds was investigated in this study. Fecal samples were collected from 135 and 120 pre-weaned calves and 79 and 130 heifers raised in the Central Anatolia (CAR) and Mediterranean Regions (MR) of Turkey, respectively. A total of 86 post-weaned calves in CAR were also included in the study. For diagnostic comparison, all samples were examined by microscopic examination, SSU rRNA nested PCR and TaqMan real-time PCR for the presence of oocyst and Cryptosporidium DNA. In total, 102 (34.0 %) and 93 (37.2 %) of the examined samples from CAR and MR were found positive for Cryptosporidium DNA with both nested PCR and real-time PCR analyses, respectively with an overall prevalence of 35.5 %. The diagnostic sensitivity and specificity of microscopic examination were determined as 68.7 % and 100.0 % compared to molecular tools, respectively. RFLP and sequence analyses of the SSU rRNA from the PCR products revealed that 138 (70.8 %) out of 195 positive isolates were C. parvum further confirming the species-specific real-time PCR results. Among the remaining 57 (29.2 %) positive isolates, 30 (15.4 %) and 27 (13.8 %) were characterized as C. ryanae and C. bovis, respectively. C. parvum was the dominant species in pre-weaned calves especially with diarrhea while C. bovis and C. ryanae were mostly found in post-weaned calves and heifers. The sequence analyses of the gp60 gene of C. parvum isolates revealed two subtypes (IIaA13G2R1, IIaA14G1R1) belonging to zoonotic family IIa, with IIaA13G2R1 being the most common in diarrheic calves.  相似文献   

2.
Antibodies against Theileria sergenti and Babesia ovata were detected by indirect fluorescent antibody (IFA) and complement fixation (CF) methods. Both of the antibodies against T. sergenti and B. ovata could be detected with a single IFA testing procedure. Complement fixing antibody tests for T. sergenti in experimentally infected cattle were positive for about 70 days after the parasites could no longer be detected in the blood smear, and were negative thereafter. B. ovata CF antibody titres were negative on the 270th day post-infection, while the IFA tests against both parasites were positive for longer periods. B. ovata IFA-test titres remained relatively high until the 420th day after inoculation. In a survey of sera from naturally infected animals, the rate of detecting antibodies against both parasites was higher using the IFA test than using the CF test. The IFA method was more effective for diagnosis of Babesia infection than the CF technique.  相似文献   

3.
Cryptosporidium oocysts were found in 43 out of 77 calves from two farms in Iwate Prefecture and nine farms on Tanegashima Island, Kagoshima Prefecture, Japan. The DNA fragments of 18S ribosomal RNA (18S rRNA) gene were amplified by a nested PCR from 43 oocyst-positive as well as one oocyst-negative samples. All of them were precisely identified as C. parvum by analyzing the nucleotide sequences of the 18S rRNA gene. C. parvum oocyst-positive calves ranged in age from 6 to 13 days old and significantly have watery diarrhea (P<0.05). Sequences of the gene encoding the 60-kDa glycoprotein (GP60) in 43 Cryptosporidium oocyst-positive samples were identical to that of the zoonotic IIaA15G2R1 subtype. We therefore suggest that calves could be potential sources of C. parvum infections in humans.  相似文献   

4.
A BHV-4 specific nested PCR was used for the detection of viral DNA in serum samples of rabbits and calves. All animals were followed up for 62 days, blood samples were taken for PCR studies every second day. Maternal infection of calves resulted in the repeated regular reappearance (10-14 days) of the virus (DNA) in serum samples. When PCR positive five-day-old calves were infected with tissue culture adapted virus, the reappearance of the DNA in the serum was shown to be irregular, nevertheless, DNA peaks reappeared during the whole observation period. A PCR negative calf infected at the age of 60 days was found to possess viraemia until p.i.d. 32. In rabbits treated intravenously with BHV-4 the inoculum or a primary viraemia was detected at p.i.d. 2-3 and p.i.d. 14-16. Published data on human herpesviruses suggest, that the target cells might be a pluripotent stem cell population of the bone marrow and differentiated virus-infected cells destroyed by the immune system might be the source of viral DNA detected in the serum. Frequency of DNA reappearance was depended on the age of the infected animals but not on the inoculated amount of BHV-4. The described phenomenon might be part of BHV-4 infection of very young animals.  相似文献   

5.
Onuma  M.  Kubota  S.  Kakuda  T.  Sako  Y.  Asada  M.  Kabeya  H.  Sugimoto  C. 《Tropical animal health and production》1997,29(4):119S-123S

Bovine piroplasmosis caused byTheileria sergenti is a major cause of economic loss in grazing cattle in Japan. Infected calves show chronic anaemia with intraerythrocytic piroplasms and occasionally die in severe cases. We found that parasite stocks and isolates consist of genetically and antigenically mixed populations. To differentiate parasite populations bearing 3 allelic forms of p32/34, an immunodominant piroplasm surface protein, 3 sets of oligonucleotide primers were designed to amplify either of 3 alleles by polymerase chain reaction (PCR). By using this allele-specific PCR, we found that the majority ofT. sergenti-infected calves in Japan harbored mixed parasite populations bearing C and I type parasites. To controlTheileria infection, we produced 2 vaccine candidates: recombinant baculovirus p32 and synthetic peptide containing Lys-Glu-Lys (KEK) motif. Immunization with either recombinant p32 or synthetic peptide containing KEK sequences with Freund’s complete adjuvant resulted in low parasitemia and reduced the clinical symptoms compared to control calves. Interestingly, the parasite with the p32 allelic form corresponding to the one used as the immunogen was suppressed.

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6.
The protective efficacy of Mycobacterium bovis BCG (1 × 106 single dose) was evaluated under field conditions. A total of 140 male Holstein Friesian calves, one to two week-old were selected. Two groups of 70 each were formed, one group was vaccinated and the other was injected with a placebo during their second week of age and followed until 12 months of age. The study considered a positive case of tuberculosis to be an animal that had a positive reaction to the three following tests in a row: tuberculin, IFNγ PPD-B and IFNγ ESAT6-CFP10 during the 12 months of exposure. The results showed a 59.4% efficacy (IC95%: 47.64-71.16). The non-vaccinated calves were 2.4 times more at risk of becoming infected (IC95%: 1.07-5.68) compared to vaccinated animals. As a complementary test a PCR test was performed using nasal exudates in some animals from both groups using a Mycobacterium complex detection kit. All the positive PCR reactions (5/44) were found in the non-vaccinated animals. These findings suggest that the use of the BCG vaccine, even though it is not capable of protecting 100%, does prevent TB vaccinated animals from excreting bacilli in their nasal secretions at their first year of age.  相似文献   

7.
The present study was carried out in a herd with concurrent infections of Mycoplasma wenyonii and 'Candidatus M. haemobos', to investigate if transplacental and/or vector-borne transmission is possible for one or both bovine haemoplasma species. For this purpose blood samples were collected from 38 mother animals and their newborn calves; as well as from 17 uninseminated cows twice three months apart. In addition, 311 mosquitoes and blood-sucking flies (Diptera: Culicidae, Tabanidae, Muscidae) were cought near the animals. DNA was extracted from all samples, followed by real-time PCR analysis. In 10.5% of neonate calves, that were born to cows harbouring both haemoplasmas, M. wenyonii and/or 'Candidatus M. haemobos' positivity was detected. Copy numbers in positive samples from cows and their calves indicated that - in comparison with M. wenyonii - 'Candidatus M. haemobos'-bacteraemia had usually lower levels. In samples of uninseminated cows the rate of infection with the latter species decreased. These findings may explain why M. wenyonii was significantly more frequently detected in blood-sucking flies, than 'Candidatus M. haemobos'. In conclusion, molecular evidence is provided for the first time on the transplacental transmission of bovine haemoplasmas. Regarding their spread by blood-sucking arthropods, new potential vectors were identified, i.e. the horn fly (Haematobia irritans), the stable fly (Stomoxys calcitrans) and two species of horse flies (Tabanus bovinus, T. bromius).  相似文献   

8.
To obtain information about the occurrence and genotype distribution of G. intestinalis and C. parvum in Austrian cattle, faecal samples from diarrhoeic calves younger than 180 days of age originating from 70 farms were examined. Of the 177 faecal samples, 27.1% were positive for Giardia cysts (immunofluorescence microscopy) and 55.4% for Cryptosporidium oocysts (phase-contrast microscopy). Positive samples were characterized by nested PCR for Giardia, 83.3% (triosephosphate isomerase; tpi) and 89.6% (β-giardin; bg) were positive, while the Cryptosporidium nested PCR returned 92.5% (60-kDa glycoprotein) positive results. Sequence analysis revealed one assemblage A-positive sample and 30 (bg) respectively 29 (tpi) assemblage E-positive samples for G. intestinalis. For C. parvum four subtypes within the IIa family (IIaA15G2R1, n = 29; IIaA19G2R2, n = 3; IIaA21G2R1, n = 2; IIaA14G1R1, n = 1) could be differentiated. Validation of two immunochromatographic point-of-care tests resulted in a sensitivity of 29.2% and 77.6%; a specificity of 98.4% and 91.1% for the detection of Giardia intestinalis and Cryptosporidium parvum, respectively. Results confirm the widespread occurrence of both protozoa in diarrhoeic calves in Austria.  相似文献   

9.
The present study developed and validated a species-specific loop-mediated isothermal amplification (LAMP) assay for the rapid detection and discrimination of two benign bovine Theileria species – T. sergenti and T. sinensis. The LAMP assay is inexpensive and easy to perform and involves a rapid reaction-the amplification can be performed in 55 min or 50 min under isothermal conditions of 61 °C or 63 °C, respectively, by employing a set of four species-specific primer mixtures. The results can be checked using agarose gels. The optimal assay conditions, under which the assay exhibited with no cross-reaction with other closely related tick-borne parasites (T. annulata, Babesia bovis, B. bigemina, B. major, B. ovata, B. U. sp., Anaplasma marginale) or between the two Theileria species of interest, was established. The assay is approximately 10-fold more sensitive than the conventional specific PCR assay. The LAMP assay was validated using DNA from 6 standard stocks in the laboratory and was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infection cattle or yaks in China. These findings indicate that this Theileria species-specific LAMP assay may have potential clinical applications for the detection and differentiation of two benign bovine Theileria species – T. sergenti and T. sinensis, especially in endemic countries.  相似文献   

10.
Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.  相似文献   

11.
A total of 938 faecal samples (461 cattle calves, 264 buffalo calves, 55 lambs, 116 kids and 42 piglets) from different livestock farms and individual small holdings in six targeted states of India were collected and screened by modified Ziehl–Neelsen staining technique to determine the prevalence of Cryptosporidium spp. and its association with age, sex, season and faecal consistency in domesticated animals. Overall, 16.2 % of the animals were positive for Cryptosporidium infection with prevalence of 16.3, 24.2, 1.8, 3.5 and 19.1 % in cattle calves, buffalo calves, lambs, kids and piglets, respectively. The prevalence of infection was significantly higher (p?<?0.05) in bovines (19.3 % cattle and 33.7 % buffalo) below 1 month of age than in animals between 1 and 3 months of age. But in piglets, it was higher in the age group of 1 to 3 months (22.6 %) than in younger animals (9.1 %). Also, higher prevalence (p?>?0.05) was recorded in females than in males. Seasons had a significant effect (p?<?0.05) on the prevalence of infection in large ruminants, with the highest prevalence in monsoon (cattle 28.8 % and buffalo 36.6 %) followed by pre-monsoon and post-monsoon season. However, in case of sheep and goats, the prevalence was higher (p?>?0.05) in post-monsoon than in monsoon season. A high degree of association was noticed between Cryptosporidium infection and diarrhoea in ruminants screened during the present study. But, in case of pigs, the prevalence was higher in non-diarrhoeic than in diarrhoeic animals. Genotyping of Cryptosporidium spp. based on nested PCR amplification of partial 18S rRNA and its subsequent digestion with SspI, VspI and MboII restriction enzymes revealed prevalence of Cryptosporidium parvum in representative number of positive samples of cattle, buffalo and goats.  相似文献   

12.
We examined the relationships between calf productivity, anemia and Theileria orientalis sergenti infection from records of 109 Japanese shorthorn calves during a grazing period and we quantified the relative importance of the T. orientalis sergenti infection‐resistance criteria used. A decrease in average parasitemia (AvePara) and an increase in average packed cell volume (AvePCV) would cause a decrease in the number of drug treatments. An increase in AvePCV would decrease the number of drug treatments more efficiently than a decrease in AvePara. An increase in AvePCV would cause an increase in dairy gain, while a decrease in AvePara would cause a decrease in dairy gain. Therefore, an improvement in the capacity to control anemia development would improve T. orientalis sergenti infection resistance more efficiently than an improvement in the capacity to control parasitemia development.  相似文献   

13.
Equine infectious anemia (EIA) is a highly infectious disease in members of the Equidae family, caused by equine infectious anemia virus (EIAV). The disease severity ranges from subclinical to acute or chronic, and causes significant economic losses in the equine industry worldwide. Serologic tests for detection of EIAV infection have some concerns given the prolonged seroconversion time. Therefore, molecular methods are needed to improve surveillance programs for this disease. We attempted detection of EIAV in 6 clinical and 42 non-clinical horses in Nuevo Leon State, Mexico, using the agar gel immunodiffusion (AGID) test for antibody detection, and nested and hemi-nested PCR for detection of proviral DNA. We found that 6 of 6, 5 of 6, and 6 of 6 clinical horses were positive by AGID, nested PCR, and hemi-nested PCR, respectively, whereas 0 of 42, 1 of 42, and 9 of 42 non-clinical horses were positive by these tests, respectively. BLAST analysis of the 203-bp 5′-LTR/tat segment of PCR product revealed 83–93% identity with EIAV isolates in GenBank and reference strains from other countries. By phylogenetic analysis, our Mexican samples were grouped in a different clade than other sequences reported worldwide, indicating that the LRT/tat region represents an important target for the detection of non-clinical horses.  相似文献   

14.
Given the fact that numerous microbial species can be detected in pregnant female dogs, the objective of this study was to assess the transplacental transmission of Brucella canis, Ehrlichia canis, Neospora caninum and Toxoplasma gondii in stillborn puppies. This study involved 41 stillborn puppies, 78.6% of which were positive for T. gondii, 52.4% for N. caninum and 59.5% for B. canis. E. canis was not detected in any of the analyzed puppies. Pregnancy is an important physiological condition for the transmission of infectious agents to puppies and transplacental transmission may be epidemiologically relevant in the spread of these opportunistic agents.  相似文献   

15.
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16.
Laboratory confirmation methods are important in bovine cysticerosis diagnosis as other pathologies can result in morphologically similar lesions resulting in false identifications. We developed a probe-based real-time PCR assay to identify Taenia saginata in suspect cysts encountered at meat inspection and compared its use with the traditional method of identification, histology, as well as a published nested PCR. The assay simultaneously detects T. saginata DNA and a bovine internal control using the cytochrome c oxidase subunit 1 gene of each species and shows specificity against parasites causing lesions morphologically similar to those of T. saginata. The assay was sufficiently sensitive to detect 1 fg (Ct 35.09 ± 0.95) of target DNA using serially-diluted plasmid DNA in reactions spiked with bovine DNA as well as in all viable and caseated positive control cysts. A loss in PCR sensitivity was observed with increasing cyst degeneration as seen in other molecular methods. In comparison to histology, the assay offered greater sensitivity and accuracy with 10/19 (53%) T. saginata positives detected by real-time PCR and none by histology. When the results were compared with the reference PCR, the assay was less sensitive but offered advantages of faster turnaround times and reduced contamination risk. Estimates of the assay's repeatability and reproducibility showed the assay is highly reliable with reliability coefficients greater than 0.94.  相似文献   

17.
The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.  相似文献   

18.
North Korea is located on the northern part of the Korean Peninsula in East Asia. While tick-borne pathogens of medical and veterinary importance have been reported from China and South Korea, they have not been reported from North Korea. To screen for zoonotic tick-borne pathogens in North Korea, ticks were collected from domestic goats. A total of 292 (27 nymph, 26 male, 239 female) Haemaphysalis (H.) longicornis were collected and assayed individually for selected tick-borne pathogens. A total of 77 (26.4%) were positive for Anaplasma bovis, followed by Bartonella (B.) grahamii (15, 5.1%), Anaplasma phagocytophilum (12, 4.1%), Bartonella henselae (10, 3.4%), and Borrelia spp. (3, 1.0%) based on 16S ribosomal RNA and ITS species-specific nested polymerase chain reaction. Using the groEL-based nested PCR, a total of 6 and 1 H. longicornis were positive for B. grahamii and B. henselae, respectively. All products were sequenced and demonstrated 100% identity and homology with previously reported sequences from other countries in GenBank. This is the first report of the detection of tick-borne pathogens in the North Korea and suggests that farm animals may act as reservoirs for zoonotic tick-borne pathogens.  相似文献   

19.
To evaluate transplacental transmission of Toxoplasma gondii in naturally infected ewes, blood samples were collected from 55 pregnant ewes and their offspring, before ingestion of colostrum. From 16 offspring of positive ewes and nine offspring from negative ewes, blood samples were obtained after 48 h and 7, 14, 21, 28, 35, 42, 49 and 56 days after birth. T. gondii antibodies were detected in serum samples using the indirect fluorescence antibody test (IFAT ≥ 64). Four of the 30 positive ewes (13.3 %) had offspring positive for T. gondii before ingesting colostrum (vertical transmission). The colostrum antibody titers decreased every week, and only 20 % (2/10) of the lambs in continued to present detectable antibody titers until day 56 after birth. Therefore, vertical transmission of T. gondii in lambs was indication of occur and is an important route for transferring and maintaining the agent in sheep herds in the Brazilian semiarid region.  相似文献   

20.
Babesia spp. infections were investigated in Bos taurus x Bos indicus dairy cows and calves and in Boophilus microplus engorged female ticks and eggs. Blood samples and engorged female ticks were collected from 25 cows and 27 calves. Babesia spp. was detected in ticks by microscopic examination of hemolymph of engorged female and by squashes of egg samples. Cattle infection was investigated in blood thin smears and by DNA amplification methods (PCR and nested PCR), using specific primers for Babesia bovis and Babesia bigemina. Merozoites of B. bovis (3 animals) and B. bigemina (12 animals) were detected exclusively in blood smears of calves. DNA amplification methods revealed that the frequency of B. bigemina infection in calves (92.6%) and in cows (84%) and of B. bovis in calves (85.2%) and in cows (100%) did not differ significantly (P > 0.05). Babesia spp. infection was more frequent in female ticks and eggs collected from calves (P < 0.01) than from cows, especially in those which had patent parasitemia. Hatching rates of B. microplus larvae were assessed according to the origin of engorged females, parasitemia of the vertebrate host, frequency and intensity of infection in engorged female tick, and frequency of egg infection. Hatching rate was lower in samples collected from calves (P < 0.01) than from cows, and in those in which Babesia spp. was detected in egg samples (P < 0.01).  相似文献   

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