Serologic diagnosis of Oestrus ovis (Diptera: Oestridae) in naturally infested sheep

Sera from eighteen control sheep supposed to be free from parasitism by Oestrus ovis Linnaeus, 1761, and from 100 sheep raised in an enzootic area of O.ovis infestations were tested to detect anti-Oestrus antibodies by double immunodiffusion (DD) and indirect haemagglutination (IH) tests with somatic crude antigens from first (L1), second (L2) and third (L3) instar of O.ovis larvae. At necropsy, eighty-eight out of 100 sheep from the O.ovis infested area were found to be parasitized while the eighteen control ovines did not show Oestrus larvae. Examination of the sera from the parasitized sheep by DD showed positive results of 42% for L1, 59% for L2 and 18% for L3. Screening the sera with IH gave sensitivities of 100% for L1, 100% for L2 and 97.7% for L3. Sheep, naturally parasitized by gastrointestinal nematodes, presented no cross immune reactions in DD tests with the three larval stages of O.ovis or with L2 larvae in IH tests.     

The rabbit antibody response to immunization with Haemophilus ducreyi A comparative study for immunoglobulin detection using diffusion-in-gel enzyme-linked immunosorbent assay, countercurrent immunoelectrophoresis, and double-diffusion-in-gel

Abstract Haemophilus ducreyi was used for immunization of rabbits. The serum level of specific IgG, IgM, and IgA antibodies to the same agent was determined by diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) and precipitating antibodies were detected with countercurrent immunoelectrophoresis (CIE) and double-diffusion-in-gel (DD) techniques. Antibodies to H. ducreyi could be detected in all rabbits within two weeks with all three techniques used.     

Responsivenes of total content changes of magnesium and zinc status in patients infected with Giardia intestinalis

The aim of the study was to investigate the changes of total content of the essential elements of zinc and magnesium levels in patients infected with Giardia intestinalis. Zinc and magnesium concentrations were measured in 64 patients who were positive for the intestinal parasite G. intestinalis. Scores were obtained for the positives and their 60 age- and sex-matched G. intestinalis-negative healthy controls. The mean concentration of magnesium in blood was significantly lower in G. intestinalis-positive patients than in their controls both in females (p<0.05) and males (p<0.05). The average zinc concentration in G. intestinalis-positive female patients was 0.76±0.3 mg/L and it was 0.60±0.2 mg/L in controls (p>0.05). The mean values of the zinc in blood were 0.73±0.2 mg/L in G. intestinalis-positive male patients and 0.82±0.1 mg/L in controls (p>0.05). No correlation could be demonstrated between age and mean values of zinc and magnesium in both G. intestinalis-positive females/males and controls (p>0.05). No significant correlation could be found between blood zinc and magnesium levels in G. intestinalis-positive female/male patients and controls (p>0.05). Magnesium levels were found to be clearly decreased, whereas no change was observed in zinc level in the patients infected with G. intestinalis compared to controls.     

Genetic characterization of Pompeii and Herculaneum Equidae buried by Vesuvius in 79 AD

DNA extracted from the skeletons of five equids discovered in a Pompeii stable and of a horse found in Herculaneum was investigated. Amino acid racemization level was consistent with the presence of DNA. Post‐mortem base modifications were excluded by sequencing a 146 bp fragment of the 16S rRNA mitochondrial gene. Sequencing of a 370 bp fragment of mitochondrial (mt)DNA control region allowed the construction of a phylogenetic tree that, along with sequencing of nuclear genes (epsilon globin, gamma interferon, and p53) fragments, gave us the possibility to address some questions puzzling archaeologists. What animals—donkeys, horses, or crossbreeds—were they? And, given they had been evidently assigned to one specific job, were they all akin or were they animals with different mitochondrial haplotypes? The conclusions provided by molecular analysis show that the Pompeii remains are those of horses and mules. Furthermore one of the equids (CAV5) seems to belong to a haplotype, which is either not yet documented in the GenBank or has since disappeared. As its characteristics closely recall those of donkeys, which is the out group chosen to construct the tree, that appears to have evolved within the Equidae family much earlier than horses, this assumption seems to be nearer the truth. J. Cell. Physiol. 199: 200–205, 2004© 2003 Wiley‐Liss, Inc.     

First detection of anti-Besnoitia spp. specific antibodies in horses and donkeys in Italy

Among Apicomplexa protozoa infecting equids, Besnoitia spp., Toxoplasma gondii and Neospora spp. represent important issues from a sanitary and zootechnical viewpoint. However, only scarce epidemiological data are available on the spread of the infections in horses and donkeys in Europe. Therefore, a serosurvey was planned to estimate the prevalence of these Sarcocystidae species in Italian equids. Serum samples from 268 horses and 18 donkeys raised in Italy were collected and serologically analyzed to detect anti-Besnoitia spp., anti-T. gondii and anti-Neospora spp. antibodies: an approach based on an initial screening by in-house ELISA followed by a confirmatory WB was used. Two horses (0.7%) and four donkeys (22.2%), showed antibodies anti-Besnoitia spp. Ten horses (3.7%) resulted positive to T. gondii and one of these (0.4%) was seropositive also to Neospora spp. This is the first detection of anti-Besnoitia spp. specific antibodies in Italian horses and donkeys. The study confirmed the circulation of Besnoitia spp. among equids in Europe. Low prevalence of T. gondii and Neospora spp. in horses raised in Italy was reported. Nevertheless, it is noteworthy to consider that consumption of horse meat could represent a source for human toxoplasmosis.     

Counterimmunoelectrophoresis as a routine mycoserological procedure

Counterimmunoelectrophoresis (CIE) has been compared in a diagnostic laboratory with agar gel double diffusion (DD) as a routine procedure for detection of antibodies to pathogenic and allergenic fungi and actinomycetes. It was shown to be of particular value in detecting antibodies to Aspergillus fumigatus. Thus 72 of 106 sera in which precipitins were detected were positive by CIE alone. Some sera were positive only by CIE to antigens prepared from Histoplasma capsulatum, Allescheria boydii, Candida albicans and C. parapsilosis.     

Prevalence,Risk Factors and Multilocus Genotyping of Giardia intestinalis in Dairy Cattle,Northwest China

Giardia intestinalis is a cosmopolitan protozoan parasite that can infect a range of animals, including dairy cattle. As information regarding the prevalence and genotyping of G. intestinalis infection in dairy cattle in northwestern China is limited, 2,945 feces samples from 1,224 dairy cattle in Gansu Province and from 1,614 in Ningxia Hui Autonomous Region (NXHAR) were examined between December 2012 and March 2014. The overall prevalence of G. intestinalis was 3.63% (107/2,945), with 2.63% and 4.38% in Gansu and NXHAR, respectively. Logistic regression analysis showed region, age and season to be significant risk factors for G. intestinalis infection. Assemblage analysis identified 106 assemblage E and one assemblage A at the triose phosphate isomerase (tpi) locus in this study. Intravariations were also detected at tpi, glutamate dehydrogenase (gdh) and beta giardin (bg) loci within assemblage E, showing seven, three, and five new subtypes, respectively. Moreover, 13 new multilocus genotypes (E20‐E32) were observed in assemblage E. Effective strategies and measures should be taken to prevent and control giardiasis in Gansu and NXHAR.     

Bloodmeal analysis in Culicoides midges collected near horses,donkeys and zebras in the Eastern Cape,South Africa

An upsurge in African horse sickness (AHS) in the Eastern Cape, South Africa, from 2006 led to an epidemiological reassessment of the disease there. Light trapping surveys carried out near horses, donkeys and zebras in 2014–2016 collected 39 species of Culicoides midge (Diptera: Ceratopogonidae) that are potential vectors of AHS. To establish if these midges fed on equids, DNA sequences were obtained from the gut contents of 52 female midges (35 freshly blood‐fed, 13 gravid and four parous), representing 11 species collected across 11 sites. Culicoides leucostictus fed on all three equids. Culicoides bolitinos, Culicoides imicola and Culicoides magnus fed on both horses and donkeys. Culicoides onderstepoortensis fed on donkeys, and Culicoides similis and Culicoides pycnostictus fed on zebras. Bloodmeals from cows, pigs, warthogs, impalas and a domestic dog were also identified in various species, but none of the midges tested had fed on birds. These results contribute to knowledge of the vectorial capacity of several species of Culicoides with regard to AHS in the Eastern Cape and point to potential reservoir hosts, of which donkeys, zebras and domestic dogs have previously been found to harbour AHS. Blood‐fed midges were also obtained throughout winter, indicating the potential for endemic AHS in the province.     

Dot-immunobinding assay in the detection of IgG antibodies against farmer's lung antigens

Circulating antibodies against Faenia rectivirgula, Thermoactinomyces candidus, T. vulgaris and Aspergillus fumigatus were studied in the sera of 14 clinically proven farmer's lung patients and 10 normal controls using three immunological methods. These methods were agar gel double diffusion (DD), biotin-avidin-linked immunosorbent assay (BALISA) and dot-immunobinding assay (DIBA). Agar gel diffusion, the least sensitive of the three methods, failed to detect antibodies in some of the patients, while BALISA detected antibodies even in the normal controls. However, the sensitivity of dot-immunobinding assay was in between DD and BALISA while the specificity was comparable to DD to all the antibodies except against A. fumigatus antigens. Dot-immunobinding assay gave faster results than DD and the blots can be stored as record for longer periods of time without fading.     

The albinism of the feral Asinara white donkeys (Equus asinus) is determined by a missense mutation in a highly conserved position of the tyrosinase (TYR) gene deduced protein

A feral donkey population (Equus asinus), living in the Asinara National Park (an island north‐west of Sardinia, Italy), includes a unique white albino donkey subpopulation or colour morph that is a major attraction of this park. Disrupting mutations in the tyrosinase (TYR) gene are known to cause recessive albinisms in humans (oculocutaneous albinism Type 1; OCA1) and other species. In this study, we analysed the donkey TYR gene as a strong candidate to identify the causative mutation of the albinism of these donkeys. The TYR gene was sequenced from 13 donkeys (seven Asinara white albino and six coloured animals). Seven single nucleotide polymorphisms were identified. A missense mutation (c.604C>G; p.His202Asp) in a highly conserved amino acid position (even across kingdoms), which disrupts the first copper‐binding site (CuA) of functional protein, was identified in the homozygous condition (G/G or D/D) in all Asinara white albino donkeys and in the albino son of a trio (the grey parents had genotype C/G or H/D), supporting the recessive mode of inheritance of this mutation. Genotyping 82 donkeys confirmed that Asinara albino donkeys had genotype G/G whereas all other coloured donkeys had genotype C/C or C/G. Across‐population association between the c.604C>G genotypes and the albino coat colour was highly significant (P = 6.17E?18). The identification of the causative mutation of the albinism in the Asinara white donkeys might open new perspectives to study the dynamics of this putative deleterious allele in a feral population and to manage this interesting animal genetic resource.     

Two variants in the KIT gene as candidate causative mutations for a dominant white and a white spotting phenotype in the donkey

White spotting phenotypes have been intensively studied in horses, and although similar phenotypes occur in the donkey, little is known about the molecular genetics underlying these patterns in donkeys. White spotting in donkeys can range from only a few white areas to almost complete depigmentation and is characterised by a loss of pigmentation usually progressing from a white spot in the hip area. Completely white‐born donkeys are rare, and the phenotype is characterised by the complete absence of pigment resulting in pink skin and a white coat. A dominant mode of inheritance has been demonstrated for spotting in donkeys. Although the mode of inheritance for the completely white phenotype in donkeys is not clear, the phenotype shows similarities to dominant white in horses. As variants in the KIT gene are known to cause a range of white phenotypes in the horse, we investigated the KIT gene as a potential candidate gene for two phenotypes in the donkey, white spotting and white. A mutation analysis of all 21 KIT exons identified a missense variant in exon 4 (c.662A>C; p.Tyr221Ser) present only in a white‐born donkey. A second variant affecting a splice donor site (c.1978+2T>A) was found exclusively in donkeys with white spotting. Both variants were absent in 24 solid‐coloured controls. To the authors’ knowledge, this is the first study investigating genetic mechanisms underlying white phenotypes in donkeys. Our results suggest that two independent KIT alleles are probably responsible for white spotting and white in donkeys.     

Horse haemoglobin phenotyping by agarose gel isoelectric focusing comparison of Thoroughbreds with other Equidae*

By using isoelectric focusing in thin agarose slab gels 1049 Thoroughbred, 82 Nooit-gedachter, 45 Percheron and 244 horses of other breeds were examined. The numbers of other Equidae tested were 107 donkeys, 50 mules, 4 common zebras (Equus burchelli boehmi) and 8 mountain zebras (Equus zebra hartmannae). Phenotypic data are presented for all tested animals and gene frequencies are calculated for the horses.     

<Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> and <Emphasis Type="Italic">Giardia intestinalis</Emphasis> in Calf Diarrhoea in Sweden

The objective of this study conducted in 75 herds was to investigate the presence and significance of Cryptosporidium parvum and Giardia intestinalis in Swedish dairy calves in comparison with rotavirus, coronavirus and Escherichia coli K99+. The farmers were asked to collect faecal samples from each heifer calf that had diarrhoea between birth and 90 days of age, and also from a healthy calf of the same age. In total, 270 samples were collected and analysed. C. parvum, either alone or together with G. intestinalis and/or rotavirus, was detected in 16 (11%) and 6 (5%) of the samples from diarrhoeic and healthy calves, respectively. Even though a higher proportion of diarrhoeic calves shed C. parvum, the difference between the groups was not statistically significant (p = 0.067), possibly due to the low number of positive samples. G. intestinalis was found in 42 (29%) of the diarrhoea samples and in 29 (23%) of the samples from healthy calves. Rotavirus and coronavirus were demonstrated in 24% and 3% of the diarrhoea samples, respectively, whereas E. coli K99+ was only found in samples from 2 healthy calves. C. parvum and G. intestinalis were found in samples from calves 7 to 84 days of age and during all seasons. The results confirm that C. parvum is present in Swedish dairy herds and might have clinical significance. G. intestinalis was the most common agent found but the importance of this parasite remains unclear. Both parasites have suggested zoonotic potential and thus warrant further attention. In addition, rotavirus is a major pathogen in neonatal enteritis in Sweden, whereas coronavirus and E. coli K99+ seem to be of less importance.     

Relationships between body condition score and ultrasound skin-associated subcutaneous fat depth in equids

Background

In equids, health and welfare depend on body composition. A growing number of equids are now used as leisure and companion animals, and often found overfeed. The need for a close monitoring of body fatness led to the search for tools allowing a rapid and non-invasive estimation of fatness. This study intends to assess real-time ultrasonography (RTU) usefulness in establishing a relationship between ultrasound measures of subcutaneous fat–plus–skin thickness (SF-Skin) and body condition score (BCS) in horses and donkeys. Forty-three healthy animals (16 donkeys and 27 horses) were used in this study to generate 95 records (RTU and BCS pairs), in multiple RTU sessions for 2 years. Using visual appraisal and palpation, BCS was graded in a 1–9 points scale. Real-time ultrasonography images were taken using a 7.5 MHz linear transducer, placed perpendicular to the backbone, over the 3rd lumbar vertebra. ImageJ was used to measure the SF-Skin on RTU images. The relation between BCS and SF-Skin measurements was tested by linear and polynomial regression analysis.

Results

The BCS values were similar in horses (5.50; from 3 to 8 points) and donkeys (5.14; from 3 to 7 points). The SF-Skin measures show a similar trend (a mean of 7.1 and 7.7 mm in horses and donkeys, respectively). A polynomial regression among BCS and SF-Skin explained 92 and 77 % of the variation in donkeys and horses respectively. The coefficient of determination was considerably higher for the regression developed for donkeys compared with that of horses (R2 = 0.92 vs. 0.77, respectively), which reduced the accuracy of the method in horses. Both the linear and polynomial models tested show a strong relationship among BCS and SF-Skin for donkeys (R2 > 0.91; P < 0.01) and horses (R2 > 0.74; P < 0.01), despite that the extremes for BCS did not existed in our sample.

Conclusions

Our results showed the potential RTU usefulness to monitor body fat in equids. Using a high-frequency transducer and RTU together with image analysis allowed the identification of small SF-skin variations. This report will support further studies on the relationships between SF-Skin and BCS, particularly in extreme BCS scores.

     

Prevalence of non-strongyle gastrointestinal parasites of horses in Riyadh region of Saudi Arabia

This study aimed to provide recent data on the occurrence of non-strongyle intestinal parasite infestation in horses in the Riyadh region of Saudi Arabia as a basis for developing parasite control strategies. We conducted necropsy for 45 horses from September 2006 to November 2007 in the Riyadh region, Saudi Arabia. 39 out of 45 horses were infected with intestinal parasites with an infestation rate of 86.6%. Infestations with seven nematode species and two species of Gasterophilus larva were found. The most prevalent parasites were Strongyloides westeri (64.4%) and Parascaris equorum (28.8%) followed by Habronema muscae (22.2%). Trichostrongylus axei and Oxyuris equi were less common at (11.1%) and (8.8%), respectively. Habronema megastoma and Setaria equine were found in two horses only (4.4%). Gasterophilus intestinalis larvae were recovered from 39 horses (86.6%) and Gasterophilus nasalis larvae were found in 17 horses (37.7%). Season had a significant effect on the prevalence of P. equorum and G. nasalis, while age of horses had a significant effect only on the prevalence of P. equorum. The husbandry in Saudi Arabia appears to be conductive to parasites transmitted in stables or by insects rather than in pasture.     

Characterization of a red blood cell antigen in donkeys and mules associated with neonatal isoerythrolysis

A red cell antigen of donkeys and mules was identified using antibodies in serum from a mare which produced a mule foal affected with neonatal isoerythrolysis (NI). Subsequently antibodies with similar activity were identified in the sera of other mares which had produced mule foals and were produced by immunization of horses with blood from donkeys. The antigen detected by these antibodies does not correspond to any recognized horse red cell alloantigen. This may be a xenoantigen since all donkeys (and mules) tested have shared this antigen and all horses tested have lacked the antigen. The results suggest that all mule pregnancies (donkey sire x horse dam) are incompatible with regard to this factor and a potential for neonatal isoerythrolysis exists in all cases.     

The proteomic response of sea squirts (genus Ciona) to acute heat stress: a global perspective on the thermal stability of proteins

Congeners belonging to the genus Ciona have disparate distributions limited by temperature. Ciona intestinalis is more widespread with a cosmopolitan distribution ranging from tropical to sub-arctic zones, while Ciona savignyi is limited to temperate-latitudes of the northern Pacific Ocean. To compare the heat stress response between congeners, we quantified changes in protein expression using proteomics. Animals were exposed to 22 °C, 25 °C, and 28 °C for 6 h, then recovered at a control temperature (13 °C) for 16 h (high heat stress experiment). In a second experiment we exposed animals to lower levels of heat stress at 18 °C, 20 °C, and 23 °C, with a 16 °C control. A quantitative analysis, using 2D gel electrophoresis and MALDI-TOF/TOF mass spectrometry (with a 69% and 93% identification rate for Ciona intestinalis and Ciona savignyi, respectively), showed changes in a number of protein functional groups, including molecular chaperones, extracellular matrix proteins, calcium-binding proteins, cytoskeletal proteins and proteins involved in energy metabolism. Our results indicate that C. intestinalis maintains higher constitutive levels of molecular chaperones than C. savignyi, suggesting that it is prepared to respond faster to thermal stress. Systematic discrepancies between estimated versus predicted molecular masses of identified proteins differed between protein families and were more pronounced under high heat conditions, suggesting that thermal sensitivities are lower for cytoskeletal proteins and ATP-synthase than for any other protein group represented on 2D gels.     

Counterimmunoelectrophoresis and opportunistic fungal infections

Sera from 35 apparently normal humans, 37 compromised human patients, 30 hedgehogs and 30 sheep, were examined for precipitating antibodies to four opportunistic fungi — Absidia corymbifera, Aspergillus fumigatus, Candida albicans and Rhizopus arrhizus — using Counterimmunoelectrophoresis (CIE).Precipitins to A. fumigatus were almost exclusively confined to specimens obtained from the compromised human group (51% of those examined) while Candida precipitating antibodies were detected in the sera of both normal (26%) and compromised (49%) humans and in 10% of the hedgehog specimens. Serum precipitins against the two phycomycetes included in the investigations were rare.Because of the complexity of most fungal antigen extracts, it appears essential that sera be tested against a number of different antigen concentrations if CIE is to be used with confidence in fungal serology.     

Rapid enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies to Candida albicans

A rapid enzyme-linked immunosorbent assay (ELISA) where the performance time was shortened to 4h was compared with counter-immunoelectrophoresis (CIE) and a standard ELISA procedure for the detection of IgG antibodies to Candida albicans in 61 patients with suspected invasive candidosis. Using a C. albicans cytoplasmic antigen the rapid ELISA compared well with CIE and the standard ELISA. Seventeen sera that reacted with two concentrations of C. albicans antigen in CIE were also positive in both forms of ELISA. Four sera that were CIE-negative were positive in the standard ELISA and three were also positive in the rapid ELISA. The rapid ELISA provides a sensitive and reproducible test for routine serological investigation of different forms of candidosis.     

Evaluation of Rv0220, Rv2958c,Rv2994 and Rv3347c of Mycobacterium tuberculosis for serodiagnosis of tuberculosis

Tuberculosis (TB), the leading cause of death among infectious diseases worldwide, is caused by Mycobacterium tuberculosis (M. tuberculosis). Early accurate diagnosis means earlier prevention, treatment and control of TB. To confirm efficient diagnostic antigens for M. tuberculosis, the serodiagnosis value of four recombinant proteins including Rv0220, Rv2958c, Rv2994 and Rv3347c was evaluated in this study. The specificities and sensitivities of four recombinant proteins were determined based on enzyme‐linked immunosorbent assay (ELISA) by screening sera from smear‐positive pulmonary TB patients (n = 92), uninfected individuals (n = 60) and patients with Mycoplasma pneumoniae (n = 32) that potentially cross‐react with M. tuberculosis. The ELISAs showed that Rv0220, Rv2958c, Rv2994 and Rv3347c exhibited high specificities and sensitivities in detecting immunoglobulin G (IgG) antibody, with 98.3/91.3%, 91.7/85.9%, 93.3/89.1% and 93.3/80.4% respectively. According to the receiver‐operating characteristic (ROC) analysis, the area under the ROC of the target proteins was 0.988, 0.969, 0.929 and 0.945 respectively. Western blot was established to evaluate the immunoreactivities of target proteins to mice and human sera. Results demonstrated that Rv0220, Rv2958c, Rv2994 and Rv3347c could specifically recognize TB‐positive sera and the sera of mice immunized with the corresponding protein. Thus, Rv0220, Rv2958c, Rv2994 and Rv3347c were valuable potential diagnostic antigens for M. tuberculosis.