Application of a fast and cost-effective 'three-in-one' MMR ELISA as a tool for surveying anti-MMR humoral immunity: the Hungarian experience.

In Hungary, between February 2017 and July 2019, 70 confirmed measles cases were reported, raising questions about the adequacy of population-level immunity. Although the assumed vaccination coverage is ≥99%, in a recent study, we detected potential gaps in the anti-measles humoral immunity. In Hungary, according to a decree by the Ministry of Public Welfare, beginning from 2021, the healthcare provider should conduct a serosurvey of anti-measles protection levels of healthcare professionals. To facilitate the compliance with this requirement, we developed a quick 'three-in-one' or 'triple' MMR (measles, mumps and rubella) indirect ELISA (IgG); an assay format that is currently not available commercially. High throughput applicability of the 'three-in-one' ELISA was verified using 1736 sera from routine laboratory residual samples, using an automated platform (Siemens BEP 2000 Advance). Assay verification was performed by comparing the full antigen repertoire-based 'target' assay with in-house 'control' assays using recombinant viral antigen coatings, and by validated commercially available kits. Indirect immunofluorescence was used as an independent reference method. Data were analysed using OriginLab, IBM SPSS, RStudio and MedCalc. In case of measles, we combined our current results with previously published data (Ntotal measles = 3523). Evaluation of anti-mumps and anti-rubella humoral antibody levels was based on the measurement of 1736 samples. The lowest anti-measles seropositivity (79.3%) was detected in sera of individuals vaccinated between 1978 and 1987. Considering the antigen-specific seropositivity ratios of all samples measured, anti-measles, -mumps and -rubella IgG antibody titres were adequate in 89.84%, 91.82% and 92.28%, respectively. Based on the virus-specific herd immunity threshold (HIT) values (HITMeasles = 92-95%, HITMumps = 75-86%, HITRubella = 83-86), it can be stated that regarding anti-measles immunity, certain age clusters of the population may have inadequate levels of humoral immunity. Despite the potential gaps in herd immunity, the use of MMR vaccine remains an effective and low-cost approach for the prevention of measles, mumps and rubella infections.

Molecular characterization of the Israeli B. bigemina vaccine strain and field isolates.

The present study demonstrated the genetic character of the Israeli Babesia bigemina vaccine strain and field isolates, based on rap-1a and rap-1c gene sequences. The RAP-1a of blood-derived Israeli B. bigemina field isolates shared 100% amino acid sequence identity. However, comparison of RAP-1c from various Israeli B. bigemina field isolates revealed that the total sequence identity among the field isolates ranged from 98.2 to 100%. High identity was observed when RAP-1a sequences from the Israeli vaccine strain and field isolates were compared with RAP-1a from Egypt, Syria, Mexico and South Africa, while, the Israeli RAP-1c sequences showed the highest identity to the Mexican isolate JG-29 and to the PR isolate from Puerto-Rico. Based on sequence variations between the rap-1a of the vaccine strain and that of the field isolate, and between the rap-1c of the vaccine strain and that of the field isolates, nPCR-RFLP procedures were developed that enable, for the first time differentiation between the Israeli B. bigemina vaccine strain and field-infection isolates. These assays could serve as fast and sensitive methods for detection and differentiation between Israeli B. bigemina vaccine strains and field isolates, as well as for epidemiological investigations.

Differentiation between Israeli B. bovis vaccine strain and field isolates.

The present study demonstrated for the first time the ability to distinguish between the Israeli Babesia bovis vaccine strain and field isolates. The existence of an additional EcoRI restriction site in the rhoptry-associated protein-1 (rap-1) gene, which is unique to the Israeli vaccine strain, and the abolition of one of the HaeIII restriction sites in the rap-1 gene of the vaccine strain enabled distinction between the Israeli B. bovis vaccine strain and field isolates, and this was the basis for polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) development. ClustalW sequence alignment of RAP-1-deduced amino acids of the Israeli B. bovis strains and of field isolates showed that the total sequence identity among the RAP-1 amino acid sequences ranged from 97.5% to 100%. However, comparison between amino acids of RAP-1 of the Israeli vaccine strain and of field isolates, on the one hand, and B. bovis strains from Argentina, Mexico, Brazil, and USA, on the other hand, revealed 90% identity. The PCR-RFLP assay offered the great advantage of being able to distinguish between vaccine and field isolates in mixtures and provide new insight into the molecular epidemiology of B. bovis infections in Israel.

Genetic polymorphism of Babesia bovis merozoite surface antigens-2 (MSA-2) isolates from bovine blood and Rhipicephalus annulatus ticks in Israel.

This study demonstrated the genetic diversity among MSA-2c, MSA-2a1 and MSA-2b proteins of Babesia bovis isolates obtained from bovine blood and Rhipicephalus annulatus tick samples. The least identities that were observed among the deduced amino acid sequences of MSA-2c, MSA-2a1 and MSA-2b were 55, 63, and 71%, respectively. During the study four B. bovis calves, aged about 1 month, were found to be infected with virulent field strains and developed babesiosis. Probably, the calves had received insufficient antibodies, or the antibodies raised against the vaccine strain did not cross-protect against virulent field isolates. The complete msa-2 locus from the Israeli B. bovis vaccine strain and two field isolates were characterized. Similarly to the Australian strains and isolates, the msa-2 loci of the examined Israeli strain and isolates had only two msa-2 genes - msa-2c and msa-2a/b - located between msa-2c and orfB. Several of the examined samples, contained different MSA-2 genotypes concurrently. No obvious geographical relationships among isolates from various regions of Israel were established. Moreover, in the phylogenetic analyses, the Israeli deduced MSA-2 amino acid sequences of the three examined genes were clustered together with sequences derived from other countries, proving that the msa-2 gene sequences of B. bovis shared the same genetic characters worldwide. The present study clearly showed that the MSA-2 proteins of B. bovis isolates from Israel were genetically distinct from the vaccine strains. Thus, further research will be needed in order to understand the genetic diversity mechanisms of B. bovis, and the immunological responses of the infected animals.

Short communication: effect of pomegranate-residue supplement on Cryptosporidium parvum oocyst shedding in neonatal calves.

Cryptosporidium parvum is considered one of the most common enteropathogens, responsible for the high incidence of diarrhea and deleterious implications on immunity and health in neonatal calves. The pomegranate is well known for its health-promoting properties. Two experiments were designed to test the antiparasitical and antidiarrheal effects of concentrated pomegranate extract (CPE) supplement in milk in neonatal Holstein calves. Forty-one calves were randomly divided into control (n=20) and treatment (n=21) groups. For the first experiment, the treatment group was supplemented with 3.75% CPE in the daily milk ration, between 3 and 14 d of age, whereas the control group received only milk. Fecal samples were collected between d 5 and 13 to quantify Cryptosporidium oocysts, and the duration and intensity of diarrhea were evaluated. Reduced fecal oocyst count and diarrhea intensity and duration were revealed in the 3.75% CPE calves. No difference was noted in average daily gain between groups. In a second experiment, which was designed to test the effect of a lower CPE concentration (0.6% of daily milk allocation), no effects on fecal oocyst count and average daily gain were observed. However, compared with control, the lower CPE group was characterized by a shorter duration of diarrhea and higher weight gain among males at 14 d of age. These results suggest that the CPE supplement-to-calf milk ratio may potentially alleviate intestinal morbidity caused by Cryptosporidium.

Temporal association of ambient temperature and relative humidity with Spirocerca lupi infection of Onthophagus sellatus: a 14-year longitudinal study.

The parasitic nematode Spirocerca lupi causes major morbidity and mortality in dogs. The scarab beetle Onthophagus sellatus is its major intermediate host in Israel. We investigated the prevalence of beetle infection by S. lupi in different years between 1994 and 2008. The average monthly maximum and minimum relative humidity (RH) and ambient temperature (AT) throughout the study period were calculated based on daily meteorological data. The infection prevalence decreased over the study period, possibly due to a chronological change resulting from increased preventive treatment of dogs against S. lupi, or climate change. Multivariate analysis was performed for these two hypotheses. Under the first hypothesis, chronological change was forced into the model, and environmental variables were inserted stepwise. The final model included beetle-collection date, minimum RH (RH min) during the month preceding beetle collection, its interaction with maximal AT (AT max) during that same month, and the interaction of maximal RH (RH max) and AT max, during the month of beetle collection. Under the second hypothesis, chronological change was not forced. The final model included RH max during the month of beetle collection, average RH (RHave) during the month preceding beetle collection, and its interaction with AT max during the latter month. The results suggest that under both hypotheses, RH and AT during the month preceding beetle collection influence S. lupi's ability to develop and survive in O. sellatus, and may be used to predict the risk to dogs of S. lupi infection.

Polyphenols from Pistacia lentiscus and Phillyrea latifolia impair the exsheathment of gastro-intestinal nematode larvae.

The infection of grazing ruminants with gastro-intestinal nematodes (GINs) is a severe problem in the Middle East. However, goats that graze the south-western slopes of the Carmel Heights in Israel have very low faecal egg counts, despite high grazing density. We hypothesized that polyphenols from Pistacia lentiscus L. and/or Phillyrea latifolia L. - both prevalent woody species of the region that are consumed by goats - have anthelmintic bioactivity. We tested this hypothesis by using the larval exsheathment inhibition assay (LEIA). Extracts were prepared from leaves of either plant species using 70% ethanol (E70), 100% ethanol (E100), or boiling water (W). Larvae were incubated in a phosphate-buffered saline solution with or without plant extract (1200µg/ml) and then exposed to an exsheathment solution expected to elicit 100% exsheathment after one hour. All extraction methods of P. lentiscus were highly effective at inhibiting larval exsheathment, but higher potency was found for the E70 than for E100 extraction method, while W was intermediate. Only the E70 extract of P. latifolia was highly effective relative to the control. The E70 extract of P. lentiscus had more than 7 times the potency of the E70 extract of P. latifolia. Irrespective of solvent and tannin-equivalent used, P. lentiscus contained more than double the quantity of total polyphenols than P. latifolia. The polyphenols of P. lentiscus consisted mainly of galloyl derivatives (63.6%), flavonol glucosides (28.6%), and catechin (7.8%). In P. latifolia, oleuropein and its derivative tyrosol accounted for 49.3 and 23.1% of phenolics, respectively, the remainder being flavones (luteolin and quercetin) and their glucoside derivatives. Results of the LEIA test suggest that extracts of tannin-rich plants interfere with the very early stage of host invasion and that high concentration of galloylated derivatives may explain anthelmintic activity.

Consumption of Pistacia lentiscus foliage alleviates coccidiosis in young goats.

Coccidiosis near weaning is a major cause of diarrhea, ill-thrift, and impaired performance in small ruminants. A recent survey showed that in villages of the Samaria Hills, Israel, shepherds treat young, weaned goat kids afflicted with diarrhea by cutting and feeding them the foliage of Pistacia lentiscus L. (lentisk) or by tethering them close to lentisk bushes which they browse. The aim of the present study was to assess whether lentisk leaves do indeed have anti-coccidial value, and, if positive, to ascertain the role of tannins in this effect. We monitored for 24 (Experiment 1) and 30 (Experiment 2) days the effect of lentisk feeding on the development of naturally occurring coccidiosis in weaned kids artificially infected with parasitic nematodes. In Experiment 1, kids were infected with nematodes and fed lentisk foliage (PIS) or cereal hay (HAY). Coccidiosis developed at the early stage of the nematode infection, when dietary treatments were initiated. Kids in the PIS group had a lower (P<0.02) concentration of oocysts per gram feces (opg). In Experiment 2, aimed at verifying if tannins are the active component in lentisk foliage, coccidiosis occurred at the peak of the nematode infection, before experimental diets were initiated. Dietary treatments were: cereal hay (HAY), or lentisk foliage consumed without (PIS) or with (PISPEG) a 20-g daily supplement of polyethylene glycol (PEG; MW 4000), a molecule that impairs tannin-bonding with proteins. Goats fed the PIS diet had lower fecal opg counts than counterparts of the HAY (P<0.001) and PISPEG (P<0.002) treatments. Fecal opg counts for the HAY and PISPEG treatments did not differ, suggesting that the anti-coccidial moiety in lentisk was indeed tannins. Our results strongly suggest that: (i) in agreement with the ethno-veterinary anecdotal evidence, exposure of young, weaned goat kids to lentisk foliage alleviates coccidiosis; and (ii) this positive effect is associated with tannins. As coccidiosis is a major affliction of kids, providing them with tannin-rich browse near weaning could be an environmentally friendly way of improving their welfare and health status, in particular under bio-organic farm management.

Anthelmintic activity of Pistacia lentiscus foliage in two Middle Eastern breeds of goats differing in their propensity to consume tannin-rich browse.

The Damascus and Mamber breeds of goats thrive in Middle Eastern Mediterranean regions where the tannin-rich (20% of polyethylene glycol-binding tannins) brush species Pistacia lentiscus L. (lentisk) is ubiquitous. In light of the increasing recognition of the anthelmintic activity of plant tannins, we examined the effect of offering lentisk foliage for 24 days on fecal egg excretion in 5.5-month-old Damascus and Mamber kid goats (n=28) following infection with 10,000 L3 larvae of mixed gastro-intestinal nematodes (GIN). Lentisk foliage was consumed with or without a daily supplement of 20 g polyethylene glycol (PEG; MW 4000). Lentisk tannins showed a strong protein-depletive effect that was totally reversed by the addition of PEG. At the peak of infection, kids of the two breeds lost weight unless they were fed with lentisk without PEG. Fecal egg counts (FEC) were lowest - and did not differ from 0 - in kids fed lentisk without PEG, highest in the controls fed hay as roughage, and intermediate in kids fed lentisk and PEG (241, 1293, and 705 eggs per gram, respectively, SEM 180; P<0.001); therefore, the anthelmintic activity of lentisk was only partly attributable to tannins. The suppressive effect of lentisk on FEC ceased when feeding was discontinued, suggesting that female parasites were not killed but their fertility was reversibly impaired. Damascus kids showed lower FEC than their Mamber counterparts, inferring that the effect of foraging on tannin-rich species is only additive to genetic differences between goat breeds in their sensitivity to GIN infection. On the basis of our results we would expect yearlong lentisk grazing to result in no or very low GIN infection, and Damascus goats to have some advantage over Mamber goats where chemical control of GIN is unfeasible. There appears to be a trade-off between the benefits of lentisk tannin as drug and its side-effects (protein depletion) when given at high level; how goats balance this trade-off requires further elucidation.

Impaired nocifensive behaviours and mechanical hyperalgesia, but enhanced thermal allodynia in pituitary adenylate cyclase-activating polypeptide deficient mice.

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) and its receptors (PAC1 and VPAC) have been shown in the spinal dorsal horn, dorsal root ganglia and sensory nerve terminals. Data concerning the role of PACAP in central pain transmission are controversial and we have recently published its divergent peripheral effects on nociceptive processes. The aim of the present study was to investigate acute somatic and visceral nocifensive behaviours, partial sciatic nerve ligation-evoked chronic neuropathic, as well as resiniferatoxin-induced inflammatory thermal and mechanical hyperalgesia in PACAP deficient (PACAP(-/-)) mice to elucidate its overall function in pain transmission. Neuronal activation was investigated with c-Fos immunohistochemistry. Paw lickings in the early (0-5 min) and late (20-45 min) phases of the formalin test were markedly reduced in PACAP(-/-) mice. Acetic acid-evoked abdominal contractions referring to acute visceral chemonociception was also significantly attenuated in PACAP knockout animals. In both models, the excitatory role of PACAP was supported by markedly greater c-Fos expression in the periaqueductal grey and the somatosensory cortex. In PACAP-deficient animals neuropathic mechanical hyperalgesia was absent, while c-Fos immunopositivity 20 days after the operation was significantly higher. In this chronic model, these neurons are likely to indicate the activation of secondary inhibitory pathways. Intraplantarly injected resiniferatoxin-evoked mechanical hyperalgesia involving both peripheral and central processes was decreased, but thermal allodynia mediated by only peripheral mechanisms was increased in PACAP(-/-) mice. These data clearly demonstrate an overall excitatory role of PACAP in pain transmission originating from both exteroceptive and interoceptive areas, it is also involved in central sensitization. This can be explained by the signal transduction mechanisms of its identified receptors, both PAC1 and VPAC activation leads to neuronal excitation. In contrast, it is an inhibitory mediator at the level of the peripheral sensory nerve endings and decreases their sensitization to heat with presently unknown mechanisms.

Spirocerca lupi in dogs: prophylactic effect of doramectin.

Spirocerca lupi is primarily a parasite of dogs, which typically causes oesophageal nodules, aortic aneurysms, and spondylitis. This study investigated the efficacy of doramectin as a prophylactic agent for canine spirocercosis. Five beagle dogs were injected subcutaneously with doramectin (400 microg/kg on 3 occasions 30 days apart q30d), while 5 other beagle dogs served as untreated controls. All dogs were inoculated with 40 infectious S. lupi larvae (L3) one month after the last doramectin treatment. All control dogs and 4/5 treated dogs became infected. Two control dogs died of ruptured aortic aneurysms, while no deaths occurred in treated dogs. Oesophageal nodules appeared 40-103 day later in treated as compared to control dogs, and eggs appeared in the faeces 49-106 day later in treated as compared to control dogs. The mean faecal egg count on day 223 in the treatment group was reduced by 99.77%. All control dogs had thoracic radiographic changes during the study, while only 2/5 study dogs showed radiographic changes. This study shows that although doramectin did not entirely prevent canine spirocercosis it reduced the clinical signs associated with infection and delayed and reduced egg output.

Controlling the onset of natural cryptosporidiosis in calves with paromomycin sulphate.

A prospective, controlled-blind field trial was conducted to evaluate the efficacy and safety of paromomycin sulphate, given for 10 days from birth, in preventing natural cryptosporidiosis in calves. In the untreated control group, oocyst shedding and diarrhoea followed the pattern that had occurred on the farm before the trial, consistent with a perinatal infection, whereas in the treated group, the prepatent period was significantly longer than in the control group (P < 0.01) and oocyst shedding and diarrhoea started only after the drug was withdrawn. However, the regimen did not reduce the incidence of disease in the treated group.

Disseminated eosinophilic disease resembling idiopathic hypereosinophilic syndrome in a dog.

True idiopathic hypereosinophilic syndrome has been described in human beings and cats, but not in dogs. The syndrome is characterised by prolonged unexplained peripheral mature eosinophilia, the infiltration of many organs by eosinophils, organ dysfunction and a fatal outcome. This paper describes an idiopathic disseminated eosinophilic disease in a dog involving various organs, manly the heart and the lungs, accompanied by a leukemoid eosinophilic response, and a fatal outcome. The histopathological findings included the infiltration of the myocardium, lung parenchyma, liver, spleen, lymph nodes and skeletal muscles with eosiniphils.

Antibodies to selected canine pathogens and infestation with intestinal helminths in golden jackals (Canis aureus) in Israel.

Blood and fecal samples, collected from 46 healthy adult free-ranging golden jackals captured in two different locations in Israel, were examined. A serological Study was conducted to investigate the prevalence of circulating antibodies reacting with four common canine pathogens: canine distemper virus (CDV), canine parvovirus (CPV), Ehrlichia canis and Leishmania infantum. Faecal flotation and haematological tests were also performed. The seroprevalence of CPV, E. canis, CDV, and L. infantum were 72.3% (34/47), 54.3% (25/46), 52.2% (24/46), and 6.5% (3/46) respectively. Faecal flotation tests revealed a high prevalence of Ancylostoma caninum (13/17, 76%) and a low prevalence of Dipilidium caninum infestation. Examination of blood smears revealed Hepatazoon canis gamonts in one jackal. Golden jackals are among the most common free-ranging carnivores in Israel and neighboring countries. Their habitats are in proximity to densely populated areas and they bear close phylogenic relation to the domestic dog. These facts, combined with the high prevalence of the jackals' exposure to the major canine pathogens demonstrated in this study, suggest that they may serve as a reservoir for the transmission of certain diseases to domestic dogs.

Yeasts in the gastrointestinal tract of preweaned calves and possible involvement of Candida glabrata in neonatal calf diarrhea.

To examine the possibility of a mycotic involvement in neonatal calf diarrhea (NCD) the presence of fungi was assessed in (a) the intestinal contents of dead calves and fecal samples submitted for routine laboratory examination, (b) fecal specimens, sampled once in winter and once in summer, of calves raised on 2 farms with different management systems, and (c) mucosal scrapings of various segments of the digestive tract of a diarrheic calf, massively shedding Candida glabrata. C. glabrata was the most prevalent fungal species isolated from the routine samples. It was the only fungus which was shed by the calves on the 2 farms, for continuous, more or less prolonged periods, but exclusively in the winter months. Diarrhea and C. glabrata shedding seemed to be associated. C. glabrata colonized the abomasum (the functional equivalent of the monogastric stomach) but not the other segments of the digestive tract of the euthanized calf. Based on the findings of this study it seems that while some yeast species may be considered as commensals of the digestive tract of calves, and consequently their isolation from intestinal contents or fecal samples has no clinical significance, others, such as C. glabrata may be involved in enteric pathogenic processes. Moreover, characteristics of the culture, previous chemotherapeutic treatments, the animal's age and possibly climatic conditions should be taken into account before deciding on the fungal isolate's clinical relevance. Determination of mycotic involvement in NCD by routine mycological examination of intestinal contents and fecal samples of diarrheic calves may be useful to avoid unnecessary and potentially harmful antibacterial therapy.

Sudden death due to Paragonimus kellicotti infection in a dog.

A sudden death due to Paragonimus kellicotti infection in a dog that had had no previous clinical signs of illness until the day of admission to the veterinary hospital is documented. The clinical, haematological and biochemical abnormalities, as well as postmortem findings, are presented. This report represents the first case of canine paragonimiasis in Israel, and discusses the possibility of this fluke becoming established in the Middle East.

The influence of aestivation in land snails on the larval development of Muellerius cf. capillaris (Metastrongyloidea: Protostrongylidae).

The larval development of Muellerius cf. capillaris in aestivating Trochoidea seetzenii and Theba pisana was delayed: in the first snail 82% of the parasites remained as second-stage larvae (L2) after as much as 90 days, and in the second snail 60% remained as L2 after 50 days. Reactivation of T. seetzenii after 59 days of aestivation caused the larvae to develop to the third stage (L3). The number of recovered larvae among T. seetzenii was consistently higher in active vs aestivating snails (P < 0.05). Such differences were not evident among T. pisana (P > 0.05). In active T. pisana, larval development was faster than in active T. seetzenii, whereas there were no such differences between aestivating snails of these 2 species. Aestivating infected T. seetzenii had lower body weights than same-size active non-infected, as well as infected snails. Aestivating infected T. pisana were not weighed, but they too exhibited poor body condition.

Spirocerca lupi infection in the dog: aberrant migration.

The nematode Spirocerca lupi (S. lupi), a parasite of dogs and other carnivores, affects mainly the esophagus and the aorta leading to gastrointestinal, respiratory, and circulatory signs. Aberrant migration of the worm to unusual anatomical structures, especially the thoracic cavity, resulting in atypical clinical signs is being reported more frequently. Aberrant migration of S. lupi is reviewed, and two such cases (i.e., migration to the heart, causing an aortico-pulmonary "window-like" opening, and to a subcutaneous abscess in the caudal thoracic region) are presented.