Carrier Animals Research Articles

Detection of theileriosis in cattle and buffaloes by polymerase chain reaction.

Bovine tropical theileriosis caused by Theileriaannulata is a tick-borne disease of great economic importance in tropical and subtropical regions of the world. The present study was undertaken to detect theilerosis in cattle and buffaloes by polymerase chain reaction (PCR). The diagnosis of theileriosis is usually carried out by blood smear staining technique, which is not sufficiently sensitive to detect the piroplasms in the carrier animals. In this study, a total of 116 samples were collected from infected as well as apparently healthy cattle and buffaloes. Screening of blood smears by Giemsa staining detected 15 samples (12.93%) positive for Theileria piroplasms out of 116 samples. However, the PCR based screening using the specific primers from the major merozoite-piroplasm surface antigen sequence of T.annulata (Tams1) gene detected 74 samples (63.79%) positive for T.annulata which included 59 samples found negative by Giemsa staining. Our study suggests that the PCR based screening is more sensitive and accurate method for diagnosis of tropical theileriosis in cattle and buffaloes.

Andreas Höfele Stage, Stake, and Scaffold: Humans and Animals in Shakespeare’s TheatreStage, Stake, and Scaffold: Humans and Animals in Shakespeare’s Theatre. Andreas Höfele. Oxford: Oxford University Press, 2011. Pp. xii+315.

<i>Andreas Höfele</i> Stage, Stake, and Scaffold: Humans and Animals in Shakespeare’s Theatre<i>Stage, Stake, and Scaffold: Humans and Animals in Shakespeare’s Theatre</i>. Andreas Höfele. Oxford: Oxford University Press, 2011. Pp. xii+315.

The molecular identification of Streptococcus equi subsp. equi strains isolated within New Zealand

AIMS: To identify Streptococcus equi subsp. equi (S. equi) by PCR analysis and obtain isolates by culture, in order to investigate the strains of S. equi infecting horses within New Zealand.METHODS: A diagnostic PCR, based on the amplification of the seeI gene for S. equi, was used on 168 samples submitted from horses with and without clinical signs of strangles. Samples were also processed and cultured on selective media for the isolation of β-haemolytic colonies. In addition, the hypervariable region of the seM gene of S. equi was amplified and then sequenced for strain typing purposes.RESULTS: Of the 168 samples, 35 tested positive for S. equi using PCR. Thirty-two confirmed samples were from horses with a clinical diagnosis of strangles and three were from horses where clinical information was unavailable. Only 22/35 (63%) confirmed S. equi samples were successfully isolated following culture. Strain typing demonstrated that two novel seM alleles of S. equi were found in New Zealand with SeM-99 strains being restricted to the North Island while SeM-100 strains were found in both North and South Islands.CONCLUSIONS: The application of PCR for the laboratory confirmation of strangles allowed for a rapid and sensitive identification of S. equi. Moreover, seM typing revealed that within the samples examined two strains of S. equi co-circulated within the North Island of New Zealand but only one strain in the South Island.CLINICAL RELEVANCE: PCR reduces the time required to obtain laboratory confirmation of strangles compared with culture methods. It also has greater sensitivity in detecting S. equi infections, which is of particular importance in the detection of carrier animals which normally shed low numbers of bacteria. Additionally, seM molecular typing can differentiate between bacterial strains, assisting in the monitoring of local strains of S. equi subsp. equi causing disease.

Campylobacter jejuni Colonization Is Associated with a Dysbiosis in the Cecal Microbiota of Mice in the Absence of Prominent Inflammation

Background Campylobacter jejuni causes enterocolitis in humans, but does not incite disease in asymptomatic carrier animals. To survive in the intestine, C. jejuni must successfully compete with the microbiota and overcome the host immune defense. Campylobacter jejuni colonization success varies considerably amongst individual mice, and we examined the degree to which the intestinal microbiota was affected in mice (i.e. a model carrier animal) colonized by C. jejuni at high relative to low densities.MethodsMice were inoculated with C. jejuni or buffer, and pathogen shedding and intestinal colonization were measured. Histopathologic scoring and quantification of mRNA expression for α-defensins, toll-like receptors, and cytokine genes were conducted. Mucosa-associated bacterial communities were characterized by two approaches: multiplexed barcoded pyrosequencing and terminal restriction fragment length polymorphism analysis.ResultsTwo C. jejuni treatments were established based on the degree of cecal and colonic colonization; C. jejuni Group A animals were colonized at high cell densities, and C. jejuni Group B animals were colonized at lower cell densities. Histological examination of cecal and colonic tissues indicated that C. jejuni did not incite visible pathologic changes. Although there was no significant difference among treatments in expression of mRNA for α-defensins, toll-like receptors, or cytokine genes, a trend for increased expression of toll-like receptors and cytokine genes was observed for C. jejuni Group A. The results of the two methods to characterize bacterial communities indicated that the composition of the cecal microbiota of C. jejuni Group A mice differed significantly from C. jejuni Group B and Control mice. This difference was due to a reduction in load, diversity and richness of bacteria associated with the cecal mucosa of C. jejuni Group A mice.ConclusionsHigh density colonization by C. jejuni is associated with a dysbiosis in the cecal microbiota independent of prominent inflammation.

Bovine Tick-borne Protozoan Diseases: Emerging Threats

©2013. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited. Tick-borne protozoan diseases, Theileriosis and Babesiosis, are major health and management problems of cattle, small ruminants and buffaloes in Africa, Asia and Latin America. Recently, tickborne diseases were ranked high in terms of their impact on poor farming communities in developing countries. Whereas the global economic importance of ticks is particularly high for livestock, there is also a relevant impact on public health in the northern hemisphere. Laboratory diagnosis of infection with piroplasms was traditionally based on microscopy of peripheral blood smears to find the parasites within erythrocytes as well as serological tests. Nevertheless, both methods have limitations in detecting carrier animals with low numbers of infected erythrocytes and discriminating pathogenic from non-pathogenic species, particularly in mixed infections, and cross-reactivity problems have been described for serology. To overcome these drawbacks, molecular tests based on PCR assays have been used for the sensitive and specific detection of several piroplasms. More recently, real-time PCR procedures have been developed for a more rapid detection of the infection with reduced contamination risk and generally with higher sensitivity. However, the multiplexing capacity of PCR assays is limited and does not allow for the simultaneous detection of the various Babesia and Theileria spp. that can infect a single host. Interestingly, the DNA microarray assays have proven to be useful for monitoring waterborne protozoan pathogens; however to date, it is not implicated in diagnosis of bovine tickborne protozoan diseases. The DNA microarray format may have the potential to differentiate among several clinically important tick-borne piroplasms within a few hours. The uniquely designed DNA microarray may eventually allow rapid differentiation of specific parasite genotypes and assist in diagnostic and epidemiological studies of veterinary parasites.

Serological study of Leptospirosis in central Nepal

Background: Leptospirosis, an infectious disease caused by spirochetes of the genus Leptospira, is the most widespread zoonosis in the world. Humans acquire infection through contact with the urine of infected or carrier animals, either directly or through contaminated water or soil. There are only few reports documenting the serological evidence of leptospirosis in Nepal. Thus, present study aims to determine the status of leptospirosis in central Nepal. Material and Methods: A descriptive cross sectional study was conducted in 1266 patients suspected of leptospirosis in Chitwan Medical College Teaching Hospital for a period of two years. Blood samples collected from the patients were processed for qualitative detection of leptospiral IgM antibody by ELISA (Enzyme linked immunosorbent assay). Results: Of the 1266 samples subjected to ELISA, seropositivity was seen in 61 samples (4.8%) with the highest rate observed in the autumn season (63.9%) followed by summer season (21.3%). Majority (39.3%) of the seropositive individuals were agricultural workers. Leptospirosis was more common in males and in the people of 16-30 years. Conclusion: Leptospirosis is common in central Nepal. Epidemiological surveillance and laboratory capacity across the region need strengthening to determine its true prevalence in the community.

Sero-surveillance of equine infectious anemia virus in equines in India during more than a decade (1999–2012)

Equine infectious anemia (EIA) is a retroviral infection of horses. Horses infected by EIA virus (EIAV) become inapparent carriers that remain asymptomatic for the remainder of their life span and serve as infection source to other horses. In this study, agar gel immunodiffusion test and ELISA were used to investigate the presence of antibodies to EIAV in equines. A total of 67,042 equine serum samples from 19 states and two union territories were tested during April 1999 to September 2012. The results revealed that none of the animals were positive for antibodies to EIAV from 1999 to December 2009. However, two EIAV sero-positive cases one each from indigenous and thoroughbred equines were detected in 2010 and 2012, respectively. Occurrence of EIA after a long gap of 11years is indicative of reemergence of EIA in India which warrants concerted efforts in nationwide surveillance and monitoring for detection and elimination of EIAV carrier animals to prevent EIA outbreak.

Leptospirosis in Fox Squirrels (Sciurus niger) of Larimer County, Colorado, USA

Leptospirosis is a zoonotic disease caused by the bacterium Leptospira interrogans. The organism is typically maintained within a geographic region by colonizing renal tubules of carrier animals and shed into the environment in urine. We assessed whether L. interrogans was present in fox squirrels (Sciurus niger) in Larimer County, Colorado, USA, and whether it is associated with disease. Twenty-two squirrels were trapped from 29 November 2011 to 15 December 2011 for use in an unrelated study. The squirrels were individually housed for 33-65 days and euthanized; no clinical disease was observed. On gross examination, significant renal lesions were observed in 6 of 22 animals (27%). Histologically, affected animals had severe neutrophilic tubulitis with interstitial nephritis. Immunohistochemistry was conducted on the kidneys of all animals and 10 of 22 (45%) were positive for L. interrogans, with varying severity of infection. The same 10 squirrels were serologically positive for antibodies specific to L. interrogans. These results suggest that L. interrogans is present in fox squirrels in Larimer County, Colorado, USA, and may be associated with varying degrees of renal disease. Further investigation into the role of wildlife in the ecology of leptospirosis within the region is warranted.

New insights into the melanophilin (MLPH) gene controlling coat color phenotypes in American mink

The mutation causing the Silverblue color type (pp) is one of the most used recessive mutations within American mink (Neovison vison) fur farming, since it is involved in some of the popular color types such as Violet and Saphire which originate from a combination of recessive mutations. In the present study, the genomic and mRNA sequences of the melanophilin (MLPH) gene were studied in Violet, Silverblue and wild-type (wt) mink animals. Although breeding schemes and previous literature indicates that the Violet (aammpp) phenotype is a triple recessive color type involving the same locus as the Silverblue (pp) color type, our findings indicate different genotypes at the MLPH locus. Upon comparison at genomic level, we identified two deletions of the entire intron 7 and of the 5′ end of intron 8 in the sequence of the Silverblue MLPH gene. When investigating the mRNA, the Silverblue animals completely lack exon 8, which encodes 65 residues, of which 47 define the Myosin Va (MYO5A) binding domain. This may cause the incorrect anchoring of the MLPH protein to MYO5A in Silverblue animals, resulting in an improper pigmentation as seen in diluted phenotypes. Additionally, in the MLPH mRNA of wt, Violet and Silverblue phenotypes, part of intron 8 is retained resulting in a truncated MLPH protein, which is 359 residues long in wt and Violet and 284 residues long in Silverblue. Subsequently, our findings point out that the missing actin-binding domain, in neither of the 3 analyzed phenotypes affects the transport of melanosomes or the consequent final pigmentation. Moreover, the loss of the major part of the MYO5A domain in the Silverblue MLPH protein seems to be the responsible for the dilute phenotype. Based on our genomic DNA data, genetic tests for selecting Silverblue and Violet carrier animals can be performed in American mink.

The first case of Demodex gatoi in Austria, detected with fecal flotation

Feline demodicosis is a rare parasitic condition caused by three different species of mites (Demodex cati, Demodex gatoi, and an unnamed species). D. gatoi inhabits the superficial skin layer (stratum corneum) and is easily transmitted between individual cats. A 2-year-old female spayed Cornish Rex was presented with alopecia and pruritus. The dermatological examination revealed bilateral alopecia and excoriations on trunk, limbs, and belly. The second cat in the household, a 3-year-old female spayed Thai, showed no clinical signs. Superficial and deep skin scrapings were performed and cellophane tapes applied, and living D. gatoi mites could be detected in both cats. Oral ivermectin (0.25 mg/kg every other day) was subscribed. Feces were collected from both cats and fecal flotation with sugar and zinc solutions performed. When compared to skin scrapings and cellophane tapes, D. gatoi was detected more frequently and in higher numbers in fecal samples. Our findings suggest that D. gatoi can be efficiently diagnosed with coproscopy, particularly in asymptomatic carrier animals. DNA was extracted from the flotation liquid, and a PCR protocol for the species verification was designed. A fragment targeting a 325-bp DNA fragment of the D. gatoi mitochondrial 16S rDNA gene was amplified with a 100% similarity to the D. gatoi entry in GenBank® (GI 421920216). We report the first finding of D. gatoi in Austria and propose fecal flotation as a valuable tool for mite detection. Fecal flotation liquid is suitable for DNA extraction and PCR-based species verification of D. gatoi.

Methicillin resistant staphylococci and broad-spectrum β-lactamase producing Enterobacteriaceae in horses

The use of β-lactam antibiotics results in the selection of bacteria showing resistance toward this class of antibiotics. The review focuses on the increasing importance of methicillin resistant staphylococci and broad-spectrum β-lactamase-producing Enterobacteriaceae in horses. Diagnostic protocols that optimize accurate identification of these bacteria from both clinical samples and samples obtained from putative carrier animals are described. In addition, the opportunities and pitfalls for preventive and curative measures are discussed.

Molecular detection and identification of Anaplasma species in sheep from Ahvaz, Iran

Summary Ovine anaplasmosis is a tick-borne rickettsial disease, widespread in tropical and subtropical areas. In the present study, a PCR-RFLP method based on major surface protein 4 (MSP4) gene, was utilized for the detection of Anaplasma infection in 119 sheep blood samples collected from different parts of Ahvaz in the southwest of Iran. PCR identified Anaplasma infections in 87.4% (104/119) of the samples in contrast to the routine blood smear examination, which revealed inclusion bodies in only 33.6% (40/119) of samples. RFLP assessment revealed that all PCR positive samples were A. ovis, while for the first time in Iran, a mixed infection with A. marginale was seen in 50% (52/104) of Anaplasma infected samples. These results suggest higher sensitivity of PCR method over the conventional microscopic technique for diagnosis of anaplasmosis, particularly in carrier animals. It also revealed that ovine anaplasmosis caused by A. ovis and A. marginale is present and highly prevalent in Ahvaz and appears to be the first report from this region.

Fine mapping for Weaver syndrome in Brown Swiss cattle and the identification of 41 concordant mutations across NRCAM, PNPLA8 and CTTNBP2.

Bovine Progressive Degenerative Myeloencephalopathy (Weaver Syndrome) is a recessive neurological disease that has been observed in the Brown Swiss cattle breed since the 1970’s in North America and Europe. Bilateral hind leg weakness and ataxia appear in afflicted animals at 6 to 18 months of age, and slowly progresses to total loss of hind limb control by 3 to 4 years of age. While Weaver has previously been mapped to Bos taurus autosome (BTA) 4∶46–56 Mb and a diagnostic test based on the 6 microsatellite (MS) markers is commercially available, neither the causative gene nor mutation has been identified; therefore misdiagnosis can occur due to recombination between the diagnostic MS markers and the causative mutation. Analysis of 34,980 BTA 4 SNPs genotypes derived from the Illumina BovineHD assay for 20 Brown Swiss Weaver carriers and 49 homozygous normal bulls refined the Weaver locus to 48–53 Mb. Genotyping of 153 SNPs, identified from whole genome sequencing of 10 normal and 10 carrier animals, across a validation set of 841 animals resulted in the identification of 41 diagnostic SNPs that were concordant with the disease. Except for one intergenic SNP all are associated with genes expressed in nervous tissues: 37 distal to NRCAM, one non-synonymous (serine to asparagine) in PNPLA8, one synonymous and one non-synonymous (lysine to glutamic acid) in CTTNBP2. Haplotype and imputation analyses of 7,458 Brown Swiss animals with Illumina BovineSNP50 data and the 41 diagnostic SNPs resulted in the identification of only one haplotype concordant with the Weaver phenotype. Use of this haplotype and the diagnostic SNPs more accurately identifies Weaver carriers in both Brown Swiss purebred and influenced herds.

Foot-and-mouth disease virus carrier status in Bos grunniens yaks

BackgroundThe carrier status of foot-and-mouth disease virus (FMDV) is complicated, and the role of carrier animals in virus transmission is controversial. To investigate the carrier status of FMDV in animals that live in high altitude, Bos grunniens yaks were infected experimentally with FMDV O/Akesu/58.ResultsAll of the yaks showed clinical signs of foot-and-mouth disease (FMD). Total antibody levels against FMDV measured by liquid-phase blocking enzyme-linked immunosorbent assay (LPB-ELISA) and antibody levels against nonstructural proteins (NSP) showed dynamic changes. Three of the five yaks were indentified as carrier animals by RT-PCR method, and the OP fluids from carrier yaks can cause cytopathic effect (CPE) on BHK-21 cells. At last, five persistent infection strains were isolated. Nucleotide mutations of VP1 gene were analyzed.ConclusionsAfter infected with FMDV, all of the yaks showed typical clinical signs. Yaks can keep carrier status for at least 8 months. Total antibody levels against FMDV measured by LPB-ELISA and antibody levels against NSP were at high level for carrier yaks. Sequence alignment of the five isolated strains showed obvious gene and protein mutations.

Identification of bovine leukocyte adhesion deficiency (BLAD) and bovine citrullinaemia (BC) alleles in Holstein cows reared in Antalya region

Bovine leukocyte adhesion deficiency (BLAD) and bovine citrullinaemia (BC) are autosomal recessive genetic diseases in Holstein cattle breeds and both result in death of homozygous animals. The aim of this study was to investigate the presence of BLAD and BC alleles in Holstein cows reared in the Antalya region of Turkey. In this study, blood samples were collected from a total of 504 Holstein breed randomly selected from the populations raised in different parts of Antalya. The polymerase chain reaction–restriction fragment length polymorphism analysis was used to identify individuals with these diseases. To cut the target regions TaqI and AvaII restriction enzymes were used to investigate the BLAD and BC alleles, respectively. In this study, from a total of 504 samples examined, homozygous recessive (mutant) BLAD and BC alleles were not determined. In addition there was no BC carrier animal in the populations from the Antalya region but the frequency of BLAD carriers (heterozygous) were found to be 2% in the same populations.

Human Puumala and Dobrava Hantavirus Infections in the Black Sea Region of Turkey: A Cross-Sectional Study

This study was carried out to better understand the epidemiology of hantaviruses in a province of Turkey (Giresun) where human hantavirus disease has recently been detected. In this cross-sectional study, a total of 626 blood samples from healthy people aged 15 and 84 years old were collected both in urban and rural areas in 2009. The sera were tested by enzyme-linked immunosorbent assay (ELISA), immunoblotting assay, and the focus reduction neutralization test (FRNT). We screened the samples by an ELISA and found that 65/626 samples reacted positively for the presence of hantavirus-reactive immunoglobulin G (IgG). Twenty of the 65 ELISA-positive samples could be confirmed by an immunobloting assay, and the overall seroprevalence was thereby calculated to 3.2% (20/626). The seroprevalence of the people living in wood areas or adobe houses 9/17 (52.9%) was significantly higher than among people living in concrete houses 10/47 (21.3%) (p=0.014). Finally, 3 of the 20 immunoblot-positive sera were confirmed as specific for the Puumala hantavirus serotype by FRNT, 1 serum was confirmed as Dobrava virus-specific, whereas 1 serum was found to be equally reactive to Dobrava and Saaremaa viruses. We will now focus on further investigations of the ecology and epidemiology of hantaviruses in humans and their carrier animals in Turkey, studies that have already been started and will be further intensified.

Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA to monitor changes in mouse gut bacterial communities during Salmonella enterica serovar Enteritidis latent infection

Changes in intestinal microbial flora during a 4-week period of Salmonella enterica serovar Enteritidis colonization in resistant mice (latent carrier animals) were evaluated using a culture independent method involving denaturing gradient gel electrophoresis. The contents of the ileocecal portion of the intestines produced 26 bands. Fifty-seven percent of the bands were expressed in more than 80% of the samples. Forty percent of the bands present in the negative control were common to all samples, and 60% differed from those obtained 12 h and 1, 5, 10, and 28 days post-inoculation (PI). A dendrogram distinguished the negative control as the external group, and 2 clusters were formed with 76% similarity, separating the 12-h PI and 3-day PI time points from the others. These groupings were also revealed through multivariate analysis in a principal component analysis and the Venn diagram. The production of interferon γ 12 h and 3 days PI may explain this brief imbalance in microbiota that was quickly reversed in the subsequent days. These findings demonstrate that S. enterica serovar Enteritidis can colonize the gut and persist in balance with the microbiota of resistant hosts.

Leptospirosis in the Republic of Korea: Historical Perspectives, Current Status and Future Challenges

Leptospirosis is an important public health problem in the Republic of Korea (ROK), occurring sporadically or in outbreaks during the autumn season. Wild rodents, Apodemus agrarius, have been mainly involved in human leptospirosis. The majority of carrier animals are infected with Leptospira interrogans serovar lai. The characteristic pulmonary involvement or hemorrhage may increase the clinical severity or result in fatal outcomes, and these aspects continue to be a threat to people in endemic areas. While the disease incidence has been relatively low in recent years, there have been newer findings of livestock (zoo animals and racing horses) and rats (Rattus norvegicus) captured in urban environments as potential animal carriers. Many avenues of research are still open to define current changes in ecology, epidemiology, and the disease burden in both humans and animals in the ROK, together with global warming and climate change issues. In addition, national policy regarding the weighted wildlife monitoring system and the enhanced disease surveillance program is required to facilitate better monitoring and understanding of this disease.

Cytogenetic Elaboration of a Novel Reciprocal Translocation in Sheep

Reciprocal translocations represent one of the most common structural chromosomal rearrangements observed in both humans and domestic animals. In these translocations, the balanced forms are most frequent but may remain undetected because the carriers show a normal phenotype. For this reason, routine cytogenetic analysis of domestic animals should necessarily rely on banded karyotypes. In fact, during a screening analysis, carried out on phenotypically normal young sheep (Ovis aries, OAR, 2n = 54) from Laticauda-Comisana hybrids, a new structural rearrangement was detected. Two abnormal acrocentric chromosomes (the smallest and the largest one) were found in all metaphases of this carrier animal, suggesting the presence of a reciprocal translocation (rcp). CBA and RBA banding were performed in order to characterize the translocation, and FISH with chromosome-specific BAC probes and telomere probes was applied to confirm the cytogenetic data. The translocation was classified as rcp(4q;12q)(q13;q25).

PCR based diagnosis of trypanosomiasis exploring invariant surface glycoprotein (ISG) 75 gene

The invariant surface glycoprotein (ISG-75) gene of Trypanosoma evansi buffalo isolate from Karnataka state in India was sequenced and analyzed to elucidate its relationship with other isolates/species. The sequenced ISG-75 gene was also explored to device a polymerase chain reaction (PCR) strategy for the diagnosis of trypanosomiasis in carrier animals. The six cloned ISG gene sequences revealed the open reading frame (ORF) of 1572 and 1527 nucleotide (nt) encoding a polypeptide of 523 and 508 amino acids (aa) respectively and belongs ISG-75 gene family. Sequence analysis revealed 91–100% and 65–99% similarity at nt and aa levels, respectively with other isolates/species and belongs to the RoTat 1.2 strain. The diagnostic PCR based on ISG-75 sequence amplifies a 407bp product specifically from the different T. evansi isolates and could detect 0.04pg and 1.2ng of DNA from purified trypanosomes and T. evansi infected rat blood samples respectively. Subsequently the PCR detected 0.02 and 0.27trypanosomesml−1 respectively, from purified trypanosomes and T. evansi (buffalo isolate) infected rat blood. By the developed PCR assay trypanosomal nucleic acid was detected in experimental rats and buffalo on 24h post infection (p.i.) and 3rd day post infection (d.p.i.), respectively. The developed ISG-75 gene based PCR assay could be useful in detection of carrier status of trypanosomiasis in animals.