Efficacy of strain RB51 vaccine in protecting infection and vertical transmission against Brucella abortus in Sprague-Dawley rats

Immunizing animals in the wild against Brucella (B.) abortus is essential to control bovine brucellosis because cattle can get the disease through close contact with infected wildlife. The aim of this experiment was to evaluate the effectiveness of the B. abortus strain RB51 vaccine in protecting infection as well as vertical transmission in Sprague-Dawley (SD) rats against B. abortus biotype 1. Virgin female SD rats (n = 48) two months of age were divided into two groups: one group (n = 24) received RB51 vaccine intraperitoneally with 3 × 10¹⁰ colony forming units (CFU) and the other group (n = 24) was used as non-vaccinated control. Non-vaccinated and RB51-vaccinated rats were challenged with 1.5 × 10⁹ CFU of virulent B. abortus biotype 1 six weeks after vaccination. Three weeks after challenge, all rats were bred. Verification of RB51-vaccine induced protection in SD rats was determined by bacteriological, serological and molecular screening of maternal and fetal tissues at necropsy. The RB51 vaccine elicited 81.25% protection in SD rats against infection with B. abortus biotype 1. Offspring from rats vaccinated with RB51 had a decreased (p < 0.05) prevalence of vertical transmission of B. abortus biotype 1 compared to the offspring from non-vaccinated rats (20.23% and 87.50%, respectively). This is the first report of RB51 vaccination efficacy against the vertical transmission of B. abortus in the SD rat model.     

Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR

Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell’s serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n?=?5), aborted fetuses (n?=?13), and vaginal swabs (n?=?12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.     

Prevalence and molecular identification of Chlamydia abortus in commercial dairy goat farms in a hot region in Mexico

The aim of this study was to determine the seroprevalence and presence of Chlamydia abortus in Saanen breed female goats from commercial dairy goat farms under intensive production in the municipality of Guanajuato, Mexico. Sera were collected to determine the prevalence of anti-C. abortus IgG antibodies using recombinant enzyme-linked immunosorbent assay (rELISA) and cell culture. Polymerase chain reaction (PCR) was used to prove the presence of the pathogen in swab samples collected from the vagina and rectum of selected animals. Additionally, foetal tissue samples from a sudden abortion were collected. C. abortus prevalence in female goats of commercial milking farms sampled in Guanajuato, Mexico, was 4.87 % (n?=?246). Seropositive animals were found in six out of nine (66.6 %) dairy goat farms sampled, and prevalence among animals in individual farms ranged between 3.44 and 13.51 %. C. abortus was detected using PCR in spleen tissue from the aborted foetus. PCR-based detection, as well as isolation from vaginal and rectal swabs, was not possible in the present study. Isolation through cell culture was also unsuccessful from aborted foetal tissue samples. In conclusion, the results from rELISA and PCR show that C. abortus is present in dairy goat farms in the state of Guanajuato, Mexico.     

Seroprevalence,associated risk factors analysis and first molecular characterization of chlamydia abortus among Egyptian sheep

Chlamydia abortus is one of the most common abortive agents worldwide in sheep. Few studies have been reported C. abortus infection among sheep in Egypt but the available data is scarce. The objective of the present study was to determine the seroprevalence of C. abortus among sheep, the associated risk factors and its molecular characterization. The present study was conducted on 675 sheep in six Governorates at Northern Egypt. Data analysis confirmed the presence of antibodies against C. abortus in 93 out of 675 sheep. The logistic regression model was fitted to identify the associated risk factors with C. abortus infection. The results revealed that C. abortus increased significantly in ewes (OR = 4.04, 95 %CI: 1.44−11.28) during autumn season (OR = 3.6, 95 %CI: 1.64–8.28), in ewes with a history of abortion (OR = 1.4, 95 %CI: 0.87–2.50) and in farm where no lambing pen (OR = 2.2, 95 %CI: 1.30–3.94) or abscence of post abortion measures (OR = 1.96, 95%CI: 1.23-3.12). In addition, age, flock size and exchange of breeding ram had no significant effect on prevalence of chlamydiosis. Also, PCR assay was confirmed presence of C. abortus as accusative pathogen in aborted ewe and the genetic characterization of Egyptian C. abortus strain revealed 100 % identity with another strain from Iraq. A control program should be applied to reduce economic losses and risk of human infection.     

Seroprevalence of Chlamydophila abortus infection in yaks (Bos grunniens) in Qinghai,China

Chlamydophila abortus is an important amphixenosis which in a wide range of animals, associated with reproductive disorders in yaks. In order to assess the prevalence of this infection in yaks in Qinghai, China, a cross-sectional study was carried out, and a total of 674 serum samples were collected from June to October 2012 in six counties, and antibodies to C. abortus were examined by indirect hemagglutination (IHA) test. The overall seroprevalence of C. abortus in yaks was 17.66 % (119/674), and the seroprevalence of antibodies to C. abortus in yaks ranged from 11.82 to 28.43 % among the six different areas, and the difference was statistically significant (P?<?0.05). The seropositivity of C. abortus infection in different age groups varied from 16.33 to 18.49 %, and prevalence in yaks of ≥3 year (18.49 %) was slightly higher than that in yaks of <3 year, but the differences among the age groups were not statistically significant (P?>?0.05). The seroprevalence of C. abortus infection in male yak (16.8 %) was slightly lower than that in females (17.85 %), and the difference was not statistically significant (P?>?0.05). So far, this is the first systematic and comprehensive investigation of C. abortus infectionin in yaks in this area.     

Babesia bigemina infection in yak (Poephagus grunniens L.): Molecular detection and characterization

Yaks contribute significantly in the Himalayan high land economy. Specific information on prevalence of babesiosis in yaks is lacking. A fast and reliable PCR assay targeting Babesia bigemina small subunit ribosomal RNA sequence (SS rRNA) was laboratory standardized for molecular detection of B. bigemina in yaks. Restriction digestion of the PCR amplified 675 bp target sequence with Vsp I confirmed the prevalent species of Babesia as B. bigemina. Nucleotide sequencing and phylogenetic analysis of PCR amplified 675 bp SS rRNA sequence revealed a close genetic relationship with other bovine isolates of B. bigemina. A PCR based survey involving 94 blood samples of yak from the National Research Centre on Yak, Dirang, Arunachal Pradesh detected infection in 5.32% of yak blood samples, which was significantly higher in comparison to microscope based detection of infection in 2.13% blood smears. This is the first report on sensitive PCR based detection of B. bigemina infection in yaks and PCR-RFLP and nucleotide sequence analysis based molecular characterization of the B. bigemina isolated from yaks.     

Apparent seroprevalence,isolation and identification of risk factors for brucellosis among dairy cattle in Goa,India

Brucellosis is a highly contagious zoonotic infection affecting livestock and human beings. The disease has been reported worldwide except in few countries where it has been eradicated. The prevalence of brucellosis among cattle from 11 farms having a history of abortions was studied. A total of 481 samples comprising of blood, milk, vaginal swabs, vaginal discharges, placental tissues and fetal tissues were collected from 296 animals. Clinical samples were processed for the isolation of Brucella. Serum samples (n = 296) were tested by Rose Bengal Plate Test (RBPT) and indirect ELISA. A total of 90 (30.40%) and 123 (41.55%) samples were positive by RBPT and indirect ELISA, respectively. Also 27.02% samples were positive by both the tests. Brucella isolates (n = 8) were recovered from clinical samples using Brucella selective media. All the isolates demonstrated PCR amplification for the bcsp31 and IS711 genes. Amplification of Brucella abortus specific primer was demonstrated by all the isolates in AMOS PCR indicating isolates to be of either B. abortus biotype 1, 2 or 4. Risk factors for transmission of brucellosis among cattle population were studied by field surveys. It was observed that lack of awareness about brucellosis (OR = 8.739, P = 0.138) and inadequate floor space (OR = 0.278, P = 0.128) were crucial risk factors for transmission of bovine brucellosis.     

First molecular identification and subtype distribution of Blastocystis sp. isolated from hooded crows (Corvus cornix) and pigeons (Columba livia) in Tehran Province,Iran

Blastocystis is a common intestinal parasite among humans and animals such as non-human primates, pigs, cattle, birds, amphibians, and less frequently, rats, reptiles and insects. Since Blastocystis is a widely transmissible parasite between humans and mammals or birds, it is prominent to determine whether newly secluded non-human isolates are zoonotic. There are no comprehensive studies in Iran assessing the prevalence and molecular identification of Blastocystis infection in birds, especially in pigeons and crows. So, the aim of this study was to identify Blastocystis subtypes (STs) in crows and pigeons in Tehran province, Iran, using Nested PCR-RFLP and sequencing. Overall, 300 Blastocystis isolates from birds (156 pigeons and 144 crows) were subtyped by PCR, and the homology among isolates was then confirmed by RFLP analysis of the 18S rRNA gene. The prevalence of Blastocystis infection was detected 42.9% in pigeons and 44.4% in crows. All positive pigeons were owned by ST13 (100%). Among crows, 46 samples (71.8%) like pigeons were ST13, and 13 samples (20.3%) were ST14. Five samples (7.9%) remained unknown. This study was the first report of ST13 and ST14 of Blastocystis from birds. In the present study, our data revealed a high prevalence of Blastocystis sp. in pigeon’s and crow’s samples and the isolates from these birds were classified into two genetically distinct STs. Therefore, birds appear to be infected with various STs. It is important to determine the phylogenetic relationships between unknown STs from these birds and the multiple STs of Blastocystis.     

Prevalence and risk factors associated with Chlamydophila abortus infection in dairy herds in Jordan

A cross-sectional study was carried out to determine seroprevalence and to identify risk factors associated with Chlamydophila abortus infection in 62 nonvaccinated dairy herds (671 cows) in Jordan between January and June 2007. Information regarding herd management was recorded through a personal interview with farmers. Antibodies against C. abortus were detected using an ELISA test kit. Chi-square analysis and multivariable logistic regression model were used to identify risk factors associated with C. abortus seropositivity. The true prevalence of antibodies against C. abortus in individual cows and cattle herds were 19.9?% and 66.3?%, respectively. Univariable Chi-square analysis revealed three variables with P????0.25 that were further offered to multivariable logistic regression analysis. Small-sized herds were identified as a risk factor for seropositivity to C. abortus, while sweeping followed by water hosing and using disinfectants were identified as protective factors. Cows in the age groups of >8 and ??10?years old and >2 and ??6?years old had the highest and lowest significant seroprevalence to C. abortus, respectively. Results of this study indicated that C. abortus is highly prevalent in Jordan's dairy herds and Chlamydophila infection could be controlled by applying strict biosecurity measures in the dairy farms.     

Investigation of bovine brucellosis in the Northeastern Turkey

Bovine brucellosis, caused by Brucella abortus, is a significant problem for both public and animal health in Turkey. This study was conducted on the calving seasons between 2001 and 2006. A total of 626 serum samples of cattle obtained from 27 herds with a history of abortions was examined for Brucella antibodies by RBPT, SAT and ELISA. Of the cattle sera analysed, 221 (35,30%) and 206 (32, 92%) and 247 (39,45%) were found to be positive by RBPT , SAT and ELISA, respectively. B. abortus was isolated from 48 (32,21%) of 149 lung samples and stomach contents of the aborted fetuses. Based on the biochemical tests and the agglutination tests with monospecific A and M antisera, only 3 of the isolates were found to be B. abortus biotype 1 and the remaining 45 were biotype 3. This study also revealed that the dominant biotype of B. abortus was biotype 3 in this region. The determination of the agents responsible for bovine brucellosis and serosurvey of this disease are expected to help better understanding of this zoonotic infection in this region and neighbouring countries.     

Prevalence and antibiotic resistance profiles ofSalmonella serotypes isolated from backyard poultry flocks in West Bengal,India

The present study was conducted to determine prevalence, virulence gene profile, serotyping, and antibiotic resistance patterns ofSalmonella in birds kept under the backyard system in West Bengal, India. The study also incorporated the detection ofSalmonella prevalence in their environment, including feed, drinking water, utensils, litter, dried manure under the house, soil, and eggs, which helped to formulate a biosecurity strategy. The study was conducted in 4 agro-climatic zones, such as the terai, new alluvial, red laterite, and coastal. Out of 360 samples, 22Salmonella isolates (6.1%) were identified.Salmonella were isolated from cloacal swabs of 6 birds (15%, n = 40), from 4 feed samples (10%, n = 40), 8 drinking water samples (20%, n = 40), and 4 eggs (10%, n = 40). Similar antigenic structure, nucleotide sequence (invA) ofSalmonella Enteritidis and Typhimurium, and randomly amplified polymorphic DNA banding patterns ofSalmonella Enteritidis were observed. It seems that the sameSalmonella isolate was present in feed sample, cloacal swabs, and eggs in the terai zone, whereas, it was found in drinking water, birds, and eggs in the new alluvial and in drinking water and birds in the coastal zone. A zone-specific biosecurity strategy was formulated based on the findings. The isolates were found to be resistant to chloramphenicol, ciprofloxacin, gentamicin, levofloxacin, norfloxacin, and oxytetracycline. None of the isolates possessed genes for major extended spectrum β-lactamases. Thus, the present study identified the source ofSalmonella contamination in the backyard chickens and their eggs in India with possible forms of biosecurity strategies. Our study was the first attempt in India to determine the prevalence, virulence gene profile, serotyping, and antibiotic resistance pattern ofSalmonella in backyard birds, including the environment and product.     

A one-year descriptive epidemiology of zoonotic abortifacient pathogen bacteria in farm animals in Turkey

This study aimed to investigate the epidemiology of 10 suspicious pathogenic bacteria in 250 stomach contents of aborted calf, lamb, and goat foetuses in 2019. The 155 positive samples obtained from PCR consisted of 53 (58.88 %) bacteria from 90 lamb samples, 10 (43.47 %) bacteria from 23 goat samples, and 92 (67.15 %) bacteria from 137 calf samples. The five most common bacteria associated with abortions were Brucella melitensis, 52 (20.9 %); B. abortus, 13 (5.2 %); Leptospira spp., 34 (13.6 %); Campylobacter fetus, 52 (20.9 %); and Coxiella burnetii, 4 (1.6 %). The highest rate of B. melitensis (65.4 %), B. abortus (69.2 %), Leptospira spp. (67.6 %), and C. fetus (50 %) was detected in the aborted calf samples. The highest individual rate was that of C. fetus (5.2 %). The flock-herd rates of B. melitensis, B. abortus, Leptospira spp., C. fetus, and C. burnetii infections in the 29 farms studied were 34.48 %, 20.69 %, 62.06 %, 82.75 %, and 3.44 %, respectively, with a confidence level and interval of 95 %. The frequency of abortions caused by Leptospira spp. and Campylobacter fetus may be related to increasing in B. melitensis. The rates of aborted calf, lamb, and goat foetuses among the various sampling periods and regions were significantly (P < 0.01) different. In conclusion, precautions should be applied to reduce the spread of these bacterial agents in high-risk areas and to eliminate the risk of harbouring these zoonotic infections in humans. Therefore, these results must be taken into account in the development of control and protection strategies against abortions in animals.     

High diversity of coagulase negative staphylococci species in wild boars,with low antimicrobial resistance rates but detection of relevant resistance genes

This work was focused to determine the prevalence and the species diversity of coagulase-negative staphylococci (CoNS) in wild boars, and to study their antimicrobial resistance phenotype and genotype. Nasal samples of 371 wild boars from six Spanish regions were collected for CoNS recovery. The identification was performed by MALDI-TOF mass-spectrometry. Antimicrobial susceptibility for eight antimicrobial agents was studied by disc-diffusion method and the presence of 31 antimicrobial resistance genes by PCR.CoNS were detected in nasal samples of 136/371 animals tested (36.6%), and 161 isolates were obtained (1–3/animal); a high diversity of species was found (n = 17), with predominance of S. sciuri (n = 64), S. xylosus (n = 21) and S. chromogenes (n = 17). Among CoNS isolates, 22.4% showed resistance to at least one antimicrobial tested. Tetracycline-resistance phenotype was the most frequently detected (10.5%), generally mediated by tet(K) gene [associated or not with tet(L)]. Other relevant resistance genes were identified including unusual ones [mecA, erm(B), erm(F), mphC, erm(43), msr(A)/msr(B), lnu(A), dfrG, fexA, and cat_pC221].This is the first study in which CoNS isolates from wild boars are analysed. The knowledge of antimicrobial phenotype and genotype of CoNS in natural ecosystems is highly important since these staphylococcal species can act as vectors of relevant antimicrobial resistance mechanisms.     

Molecular identification of chlamydial cause of abortion in small ruminants in Jordan

Chlamydophila abortus (Ch. abortus) is the etiological agent of ovine enzootic abortion (OEA) and one of the most common infectious agents of abortion in small ruminants worldwide. RFLP-PCR analysis of the outer membrane protein gene (OMP2 gene) was used for diagnosis and characterization of chlamydial causes of abortion in small ruminants in Jordan. Sixty-six placental tissues and 15 vaginal swabs were collected from aborted ewes and does to identify cause of abortion in Jordan. Thirty-eight placental samples (58 %) and 13 vaginal swabs (87 %) were positive for chlamydial DNA. Shedding of bacteria in vaginal swabs was detected within 7 days after abortion. The results of this study showed that chlamydiosis is one of the important causes of abortion in small ruminants in Jordan. In addition, vaginal swab is an excellent sample for molecular diagnosis of chlamydiosis. DNA sequencing and RFLP analysis of the OMP2 reveal that all chlamydial cause of abortion in small ruminants in Jordan are due to Ch. abortus. While, Ch. pecorum was not detected in any sample. OMP2 gene of the isolated Jordanian strain was identical (100 %) to Ch. abortus FAS strain. In conclusion, Ch. abortus is an important cause of abortion in Jordan; vaginal swab within 7 days of abortion can be used for molecular diagnosis of chlamydiosis in small ruminants.     

Antimicrobial resistance in fecal generic Escherichia coli and Salmonella spp. obtained from Ontario sheep flocks and associations between antimicrobial use and resistance

The purpose of this study was to examine the prevalence and patterns of antimicrobial resistance (AMR) in enteric bacteria obtained from Ontario sheep flocks, and associations between antimicrobial use (AMU) and AMR. Forty-nine sheep producers participated for a 1-year interval between 2006 and 2008. Two-hundred and eighty-three pooled fecal samples were collected from the flocks during initial and final visits. Up to 3 isolates of Salmonella spp. and generic E. coli per pooled fecal sample were tested for susceptibility to 15 antimicrobials. Resistance was infrequent among Salmonella (0%, n = 7 isolates) and low among E. coli (13.1%; n = 849) isolates. A small number of isolates were resistant to antimicrobials classified as being of very high importance to human health. Tetracycline resistance was most frequently observed (12.0%). Logistic regression was used to model potential AMU (qualitative and quantitative) risk factors for tetracycline resistance in generic E. coli from final visits. Qualitative analysis indicated that the use of injectable sulfonamides [including trimethoprim-sulfonamide combinations (TMS)] and tetracycline in the feed and water were significantly associated with tetracycline resistance (OR = 2.6, P = 0.01; and OR = 4.8, P ≤ 0.01, respectively). Quantitative analysis also indicated that TMS exposure rate was significantly associated with tetracycline resistance, which varied depending on the exposure rate. The exposure rate of tetracycline in the feed and water was only significant after the removal of one influential flock, warranting further research examining flocks with higher tetracycline exposure rates. Although the prevalence of AMR in participating flocks was relatively low, risk factors for resistance were identified.     

3种四环素耐药基因型肉牛肠道大肠杆菌对四环素类抗生素的耐药性分析

以低于治疗水平的氯四环素(CT)及低于治疗水平的氯四环素和治疗水平的氧四环素组合(CT-OX)两种方式分别对肉牛进行抗生素处理,研究其对肠道大肠杆菌耐药基因型的影响。从粪便样品分离大肠杆菌,并通过抗菌药物纸片法和稀释法敏感性试验测试分离出的大肠杆菌对四环素、氧四环素和氯四环素的敏感性。利用针对耐药基因tet(A)、tet(B)和tet(C)的引物对176个四环素耐药或中介的细菌样品进行多重PCR试验,结果发现所有样品均携带一种或两种耐药基因,tet(A)在两组样品中的流行基本相同,但CT组中tet(B)的流行比例显著小于CT-OX组(P<0.05),而tet(C)的流行比例则显著CT-OX组(P<0.05)。同时,在对四环素表现为中介的52个样品检测结果中,发现其中92.3%携带tet(C)基因。另外,最小抑菌浓度值(MICs)结果表明,药物敏感性同时取决于四环素类别和耐药基因型两方面。利用real-time PCR在转录水平上对tet(C)基因进行分析,发现耐药型与中介型并非上游调控造成。对tet(C)基因的测序分析结果发现,耐药型的第1063位碱基由T突变为G。由上述数据可知,对肉牛的四环素饲喂种类可以影响到大肠杆菌的耐药基因流行。     

Prevalence of intestinal parasites and genotyping of Giardia intestinalis in pet shop puppies in east Japan

The current study examined the prevalence of intestinal parasites and genotypes of Giardia intestinalis in puppies from nine pet shops in east Japan. Fresh fecal samples from 1794 puppies (≦3 months old) were collected on one occasion. Giardia spp. was examined for specific coproantigen using ELISA kit (SNAP^®Giardia, IDEXX Laboratories, Inc., USA). Other intestinal parasites were detected microscopically using the formalin-ethyl acetate sedimentation technique. Genotyping was determined for the random 29 stool samples identified as Giardia spp. positive using PCR and direct sequencing of the glutamate dehydrogenase (gdh) gene. Overall prevalence of protozoan Giardia spp. and Cystoisospora spp. revealed 23.4% and 11.3%, respectively. Prevalence of ascarids, Strongyloides spp. and hookworms were recorded 1.8%, 1.1% and 0.1%, respectively. Protozoan Giardia spp. and Cystoisospora spp., thus, represent important pathogens among pet shop puppies. All genotyped G. intestinalis isolates were belonged to assemblage C or D, identified as dog-specific genotypes. Zoonotic assemblage A and B were not demonstrated. The result suggests that the risk of zoonotic transmission of G. intestinalis from pet shops puppies to humans may be quite low in Japan.     

Prevalence and molecular analysis of Sarcocystis infections in cattle in Northwest Iran and the first global report of S. gigantea in cattle

Cattle are intermediate host for several species of Sarcocystis, including S. cruzi, S. hirsuta, and S. hominis with high prevalence worldwide. The present study aimed to determine the prevalence of Sarcocystis infection, species identification, and phylogenetic analysis of the parasite in cattle in Northwest Iran. The samples of diaphragm and esophagus from 290 cattle were collected from slaughterhouses in Northwest Iran and subjected to macroscopic, microscopic, and histopathology examinations, PCR-RFLP, sequencing and phylogenetic analyses. Tissue cysts of Sarcocystis spp. were detected in 92% of cattle by digestion and microscopic tests. Based on the PCR–RFLP and specific PCR, 87.9% and 1.03% of isolates were identified as S. cruzi, and S. hominis, respectively. Macrocyst was seen in a single sample that was identified as S. gigantea. The haplotype network exhibited the extension of the various haplotypes of S. cruzi between neighboring provinces in Northwest Iran. Heterogeneity analysis of S. cruzi 18S-rRNA sequences indicated genetic diversity among S. cruzi isolates (Haplotype diversity: 0.733-0.854) consisting 16 haplotypes; however, the nucleotide differences showed low diversity (0.01481 to 0.03351). Pair wise sequence distance matrix amongst S. cruzi sequences indicated an intra-species divergence of 0%-7.8% and identity of 92.6%-100%. Sarcocystis infection is highly prevalent in cattle in Northwest Iran, with the predominance of S. cruzi, and genetic variants of this species are unequivocally distributing in Northwest provinces. First global detection of S. gigantea in cattle reflects new insights of transmission dynamic and biology of this parasite in Iran.     

Prevalence and genotyping of bovine Cryptosporidium species in the Mediterranean and Central Anatolia Region of Turkey

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.