The prevalence of selected vector-borne diseases in dromedary camels (Camelus dromedarius) in the United Arab Emirates.

Vector-borne diseases (VBDs) affecting dromedary camels (Camelus dromedarius) have considerable importance in the United Arab Emirates (UAE) because of the consequences associated with production decline and economic losses. Our study aimed to determine the prevalence of selected VBDs in camels in the UAE and identify risk factors. This research is currently affected by the low number of epidemiological molecular surveys addressing this issue. Blood samples were obtained from 425 dromedary camels from different locations across the UAE. Whole genomic DNA was isolated, and PCR screening was done to detect piroplasmids (Babesia/Theileria spp.), Trypanosoma spp., and Anaplasmataceae spp. (Anaplasma, Ehrlichia, Neorickettsia and Wolbachia spp.). Amplicons were sequenced, and phylogenetic trees were constructed. Trypanosoma sequences were identified as T. brucei evansi, whereas Anaplasmataceae sequences were identified as A. platys-like. All camels were negative for Babesia/Theileria spp. (0%); however, 18 camels were positive for T. b. evansi (4%) and 52 were positive for A. platys-like (12%). Mixed infection with T. b. evansi and A. platys-like was found in one camel. Statistical analyses revealed that camels with a brown coat colour were significantly more prone to acquire the A. platys-like strain compared with those having a clearer coat. A similar finding was observed when comparing urban moving camels with desert indoor and urban indoor camels. Continuous disease surveillance is required to ensure and maintain the good health status of the camels in the UAE. Nonetheless, the risk of disease outbreak remains if the misuse of drugs continues.

Question of agent of camel balantidiosis solved: Molecular identity, taxonomic solution and epidemiological considerations.

Domestic camels (Camelus bactrianus, the Bactrian camel; and Camelus dromedarius, the dromedary) are pseudo-ruminant herbivores kept as livestock in rural, inhospitable regions (cold deserts and dry steppes of Asia, arid to semi-arid regions of Africa, western and central Asia). Their close contact with humans makes them a potential reservoir for zoonotic parasite infections, as has been suggested for human balantidiasis. However, there is confusion about the ciliate species that infects camels: Infundibulorium cameli was originally described in dromedaries, but this name has almost never been used and most authors identified their findings as Balantioides coli and, to a lesser extent, Buxtonella sulcata, a cattle ciliate. To clarify the taxonomic status of the parasite and the corresponding zoonotic significance for camels, we performed morphological characterization of cysts and genetic analysis (SSU-rDNA and ITS markers) of B. coli-like isolates from Bactrian camels from Bulgaria and from dromedaries from Spain and the United Arab Emirates. Our results indicate that the camel ciliate is not B. coli, nor is it B. sulcata, but is a different species that should be placed in the same genus as the latter. Thus, camels are not a reservoir for human balantidiasis. Although the correct genus name would be Infundibulorium according to the principle of priority, this would lead to confusion since this name has almost fallen into disuse since its initial description, but Buxtonella is almost universally used by researchers and veterinarians for the cattle ciliate. We therefore propose to apply the reversal of precedence and use Buxtonella as the valid genus name. Consequently, we propose Buxtonella cameli n.comb. as the name for the camel ciliate.

Complement Activation-Independent Attenuation of SARS-CoV-2 Infection by C1q and C4b-Binding Protein.

The complement system is a key component of the innate immune response to viruses and proinflammatory events. Exaggerated complement activation has been attributed to the induction of a cytokine storm in severe SARS-CoV-2 infection. However, there is also an argument for the protective role of complement proteins, given their local synthesis or activation at the site of viral infection. This study investigated the complement activation-independent role of C1q and C4b-binding protein (C4BP) against SARS-CoV-2 infection. The interactions of C1q, its recombinant globular heads, and C4BP with the SARS-CoV-2 spike and receptor binding domain (RBD) were examined using direct ELISA. In addition, RT-qPCR was used to evaluate the modulatory effect of these complement proteins on the SARS-CoV-2-mediated immune response. Cell binding and luciferase-based viral entry assays were utilised to assess the effects of C1q, its recombinant globular heads, and C4BP on SARS-CoV-2 cell entry. C1q and C4BP bound directly to SARS-CoV-2 pseudotype particles via the RBD domain of the spike protein. C1q via its globular heads and C4BP were found to reduce binding as well as viral transduction of SARS-CoV-2 spike protein expressing lentiviral pseudotypes into transfected A549 cells expressing human ACE2 and TMPRSS2. Furthermore, the treatment of the SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes with C1q, its recombinant globular heads, or C4BP triggered a reduction in mRNA levels of proinflammatory cytokines and chemokines such as IL-1ß, IL-8, IL-6, TNF-α, IFN-α, and RANTES (as well as NF-κB) in A549 cells expressing human ACE2 and TMPRSS2. In addition, C1q and C4BP treatment also reduced SARS-CoV-2 pseudotype infection-mediated NF-κB activation in A549 cells expressing human ACE2 and TMPRSS2. C1q and C4BP are synthesised primarily by hepatocytes; however, they are also produced by macrophages, and alveolar type II cells, respectively, locally at the pulmonary site. These findings support the notion that the locally produced C1q and C4BP can be protective against SARS-CoV-2 infection in a complement activation-independent manner, offering immune resistance by inhibiting virus binding to target host cells and attenuating the infection-associated inflammatory response.

Vertebrobasilar Contribution to Cerebral Arterial System of Dromedary Camels (Camelus dromedarius).

It is hypothesized that in the "more highly evolved" mammals, including the domesticated mammals, that the brainstem and the cerebellum receive arterial blood through the vertebrobasilar system whilst the internal carotid arteries primarily supply the forebrain. In camels, the arterial blood supply to the brain differs from that of ruminants since the internal carotid artery and the rostral epidural rete mirabile (RERM) are both present and the basilar artery contributes a significant proportion of cerebral afferent blood. In this study, we described the anatomical distribution of the vertebrobasilar system arterial supply in the dromedary. Secondly, we determined the direction of blood flow within the vertebral and basilar arteries using transcranial color doppler ultrasonography. Thirdly, we quantified the percentage arterial contributions of the carotid and vertebrobasilar systems to the dromedary brain. Fifty-five heads of freshly slaughtered male Omani dromedaries aged 2-6 years were dissected to determine the distribution and topography of the arterial distribution to the brain. Their anatomical orientation was assessed by casting techniques using epoxy resin, polyurethane resin and latex neoprene. The epoxy resin and polyurethane resin casts of the head and neck arteries were used to measure the diameter of vertebrobasilar arterial system and carotid arterial system at pre-determined locations. These arterial diameters were used to calculate the percentage of blood supplied by each arterial system. The vertebrobasilar system in dromedary camels consists of paired vertebral arteries that contribute to the ventral spinal artery and basilar artery at multiple locations. In most specimens the vertebral artery was the primary contributor to the basilar artery compared to that of the ventral spinal artery. In four specimens the ventral spinal arteries appear to be the dominant contributor to the basilar artery. Transcranial color doppler ultrasonography confirmed that the direction of blood flow within the vertebral and basilar arteries was toward the brain in animals examined in ventral recumbency and when standing. The vertebrobasilar system contributes 34% of the blood supply to the brain. The vertebrobasilar system is the exclusive supply to the medulla oblongata, pons and cerebellum.

Descriptive Analysis of Cerebral Arterial Vascular Architecture in Dromedary Camel (Camelus dromedarius).

The artiodactyl brain has multiple levels of vascular pooling and the rostral epidural rete mirabile (RERM) at its base. The current study is the first of its kind to precisely demonstrate the arterial vasculature of the dromedary brain, utilizing a new casting method with colored latex and epoxy paint. In total, 35 freshly slaughtered dromedary heads were injected with colored latex or colored epoxy paint prior to dissection in order to reveal cerebral vasculature; Ten processed heads were chemically digested with 5% potassium hydroxide to obtain hard casts of cerebral arteries and anastomosing structures. The outcomes of this study ascertain the distinct vascular features of dromedaries that set them apart from other artiodactyls. In addition to the RERM, the dromedary possesses a well-developed ophthalmic and chiasmatic rete. The dromedary is similar to giraffe, goat, cat and pig in the contribution of middle meningeal artery to the rete mirabile; however, dromedaries have several arteries emerging directly from the cerebral arterial circle that supply the choroid plexus and pineal gland. Additionally, dromedaries exhibit a dominant basilar system that dominates the blood supply to the medulla oblongata, pons, and cerebellum. In our study, we were able to graphically prove the lack of connection between the areas supplied by vertebrobasilar system and carotid system in the dromedary. Furthermore, the vertebral artery does not branch into the basilar artery; instead, it acts as a contributing vessel to the ventral spinal artery that later fuse to form the basilar artery. This study employed the new casting method to illustrate a new arterial source to RERM and the various anastomoses among arterial sources supplying the brain in the dromedary. These anastomoses play an important role in maintaining an uninterrupted cerebral blood supply, decreasing the vulnerability of the fragile brain against ischemia and stroke, as well as, play an important role in maintaining blood pressure and flow in long-necked dromedaries when they raise or lower their heads.

Plasmodium ovale wallikeri in Western Lowland Gorillas and Humans, Central African Republic.

Human malaria parasites have rarely been reported from free-ranging great apes. Our study confirms the presence of the human malaria parasite Plasmodium ovale wallikeri in western lowland gorillas and humans in Dzanga Sangha Protected Areas, Central African Republic, and discusses implications for malaria epidemiology.

No impact of strongylid infections on the detection of Plasmodium spp. in faeces of western lowland gorillas and eastern chimpanzees.

BACKGROUND: Although a high genetic diversity of Plasmodium spp. circulating in great apes has been revealed recently due to non-invasive methods enabling detection in faecal samples, little is known about the actual mechanisms underlying the presence of Plasmodium DNA in faeces. Great apes are commonly infected by strongylid nematodes, including hookworms, which cause intestinal bleeding. The impact of strongylid infections on the detection of Plasmodium DNA in faeces was assessed in wild, western, lowland gorillas from Dzanga Sangha Protected Areas, Central African Republic and eastern chimpanzees from Kalinzu Forest Reserve, Uganda. METHODS: Fifty-one faecal samples from 22 habituated gorillas and 74 samples from 15 habituated chimpanzees were analysed using Cytochrome-b PCR assay and coprological methods. RESULTS: Overall, 26.4% of the analysed samples were positive for both Plasmodium spp. and strongylids. However, the results showed no significant impact of intensity of infections of strongylids on detection of Plasmodium DNA in gorilla and chimpanzee faeces. CONCLUSION: Bleeding caused by strongylid nematode Necator spp. cannot explain the presence of Plasmodium DNA in ape faeces.

A comparative molecular survey of malaria prevalence among Eastern chimpanzee populations in Issa Valley (Tanzania) and Kalinzu (Uganda).

BACKGROUND: Habitat types can affect vector and pathogen distribution and transmission dynamics. The prevalence and genetic diversity of Plasmodium spp. in two eastern chimpanzee populations-Kalinzu Forest Reserve, Uganda and Issa Valley, Tanzania-inhabiting different habitat types was investigated. As a follow up study the effect of host sex and age on infections patterns in Kalinzu Forest Reserve chimpanzees was determined. METHODS: Molecular methods were employed to detect Plasmodium DNA from faecal samples collected from savanna-woodland (Issa Valley) and forest (Kalinzu Forest Reserve) chimpanzee populations. RESULTS: Based on a Cytochrome-b PCR assay, 32 out of 160 Kalinzu chimpanzee faecal samples were positive for Plasmodium DNA, whilst no positive sample was detected in 171 Issa Valley chimpanzee faecal samples. Sequence analysis revealed that previously known Laverania species (Plasmodium reichenowi, Plasmodium billbrayi and Plasmodium billcollinsi) are circulating in the Kalinzu chimpanzees. A significantly higher proportion of young individuals were tested positive for infections, and switching of Plasmodium spp. was reported in one individual. Amongst the positive individuals sampled more than once, the success of amplification of Plasmodium DNA from faeces varied over sampling time. CONCLUSION: The study showed marked differences in the prevalence of malaria parasites among free ranging chimpanzee populations living in different habitats. In addition, a clear pattern of Plasmodium infections with respect to host age was found. The results presented in this study contribute to understanding the ecological aspects underlying the malaria infections in the wild. Nevertheless, integrative long-term studies on vector abundance, Plasmodium diversity during different seasons between sites would provide more insight on the occurrence, distribution and ecology of these pathogens.

Molecular detection of Anaplasma platys infection in free-roaming dogs and ticks from Kenya and Ivory Coast.

BACKGROUND: Anaplasma platys is a bacterium parasitic in the canine platelets, representing the causative agent of canine cyclic thrombocytopenia, with a worldwide distribution, mainly in tropical countries. The agent has zoonotic potential, being reported in several human clinical cases. The suspected vector is the tick Rhipicephalus sanguineus (sensu lato), widely distributed in animals and humans in the tropical regions of South America, Africa, Asia and Australia, but also in southern Europe. Only few scattered data concerning the epidemiology of A. platys in sub-Saharan Africa are available. The aim of our study was to evaluate the epidemiological status of A. platys in dogs and cats from rural communities in eastern and western Africa, where dogs and their ticks live in close contact with humans. METHODS: A total of 216 canine and 22 feline blood samples and ticks feeding on them were collected in 2013 and 2014 from eight localities in Ivory Coast and four localities in Kenya. PCR was performed using specific primers targeting a portion of the 16S rRNA gene, followed by sequencing. RESULTS: The main results highlight the high prevalence of A. platys infection in dogs in both Eastern and Western Africa and report it for the first time in Eastern Africa and in Rhipicephalus camicasi. CONCLUSIONS: The presence of A. platys DNA in R. camicasi feeding on negative hosts together with the relatively high prevalence of A. platys, correlated with the absence of the probable vector Rhipicephalus sanguineus (s.l.) in Kenyan Island suggest the involvement of other tick species in the transmission of A. platys.

Cytauxzoon Infections in Wild Felids from Carpathian-Danubian-Pontic Space: Further Evidence for a Different Cytauxzoon Species in European Felids.

Parasitic protists of the genus Cytauxzoon are detected in a wide range of wild and domestic felids. Bobcats are a confirmed reservoir of Cytauxzoon felis in North America while domestic cats are susceptible hosts suffering from severe or fatal illness. Cytauxzoon infections are mainly reported from American felids and, recently, several sub-clinical and clinical findings were reported from European, Asian, and African felids. In 2014, the collection of organs of 4 Eurasian lynx and 12 wild cats from 11 Romanian localities was carried out to determine the prevalence and genetic diversity of Cytauxzoon spp. We detected an overall high prevalence of 62.5% in both species of wild felids; 50% in wild cats and 100% in Eurasian lynx. The phylogenetic analysis indicates 2 distinct clades of Cytauxzoon in felids, with all of our sequences clustering with sequences of Cytauxzoon sp./Cytauxzoon manul from Palaearctic felids. Further studies, development of new genetic markers, and experimental transmission studies are required for clarifying the taxonomy and life cycle of feline Cytauxzoon in the Old World.

Phylogeny and Morphological Variability of Trypanosomes from African Pelomedusid Turtles with Redescription of Trypanosoma mocambicum Pienaar, 1962.

Little is known about host specificity, genetic diversity and phylogenetic relationships of African turtle trypanosomes. Using PCR targeting the SSU rRNA gene, we detected trypanosomes in 24 of 134 (17.9%) wild caught African pelomedusid turtles: Pelusios upembae (n=14), P. bechuanicus (n=1), P. rhodesianus (n=3) and P. subniger (n=6). Mixed infection of Trypanosoma species was confirmed by PCR in three specimens of P. upembae, and in one specimen each of P. bechuanicus, P. rhodesianus, and P. subniger. Microscopic examination of stained blood smears revealed two distinct forms (broad and slender) of trypomastigotes. The broad form coincided in morphology with T. mocambicumPienaar, 1962. Accordingly, we have designated this form as the neotype of T. mocambicum. In phylogenetic analysis of the SSU rRNA gene, all the new turtle trypanosome sequences grouped in a single clade within the strongly supported "aquatic" clade of Trypanosoma species. The turtle trypanosome clade was further subdivided into two subclades, which did not correlate with host turtle species or trypanosome morphology. This study provides the first sequence data of Trypanosoma species isolated from freshwater turtles from tropical Africa and extends knowledge on diversity of trypanosomes in the Afrotropical zoogeographical realm.

Ecology of malaria infections in western lowland gorillas inhabiting Dzanga Sangha Protected Areas, Central African Republic.

African great apes are susceptible to infections with several species of Plasmodium, including the predecessor of Plasmodium falciparum. Little is known about the ecology of these pathogens in gorillas. A total of 131 gorilla fecal samples were collected from Dzanga-Sangha Protected Areas to study the diversity and prevalence of Plasmodium species. The effects of sex and age as factors influencing levels of infection with Plasmodium in habituated gorilla groups were assessed. Ninety-five human blood samples from the same locality were also analysed to test for cross-transmission between humans and gorillas. According to a cytB PCR assay 32% of gorilla's fecal samples and 43·1% human individuals were infected with Plasmodium spp. All Laverania species, Plasmodium vivax, and for the first time Plasmodium ovale were identified from gorilla samples. Plasmodium praefalciparum was present only from habituated individuals and P. falciparum was detected from human samples. Although few P. vivax and P. ovale sequences were obtained from gorillas, the evidence for cross-species transmission between humans and gorillas requires more in depth analysis. No association was found between malaria infection and sex, however, younger individuals aged ≤6 years were more susceptible. Switching between two different Plasmodium spp. was observed in three individuals. Prolonged monitoring of Plasmodium infection during various seasons and recording behavioural data is necessary to draw a precise picture about the infection dynamics.

Piroplasms in feral and domestic equines in rural areas of the Danube Delta, Romania, with survey of dogs as a possible reservoir.

Rural areas of Romania, particularly the localities covering Danube Delta, are still not sufficiently explored in terms of epidemiological aspects, despite the large density of domestic animals living in close contact with people and natural environment of the Danube Delta Biosphere Reserve. Between 2010 and 2012, a survey on equine piroplasmids species was performed in this area, using a total of 178 horses, 15 donkeys and 177 dogs from 18 localities inside and outside the Danube Delta. None of the sampled hosts showed any clinical symptoms typical for equine piroplasmoses. A 25.4% overall prevalence for both equine species of piroplasmids was detected by PCR. Detection by "catch-all" primers followed by multiplex PCR revealed 20.3% prevalence of T. equi, beside 2.2% of B. caballi and 3.0% of mixed infections for all examined animals. Based on sequencing of 67 PCR amplicons of 18S rRNA gene of T. equi, four genotypes (A, C, D and E) were detected, whereas four B. caballi sequences clustered within a single genotype (A). A single dog infected with T. equi was found and confirmed by sequencing, clustering within genotype D. Our study further proved limited host specificity of piroplasmid. Higher prevalence was revealed inside the Danube delta, presumably caused by different environmental conditions, different tick densities and lower availability of veterinary services.

Infections by Babesia caballi and Theileria equi in Jordanian equids: epidemiology and genetic diversity.

Microscopic diagnosis of equine piroplasmoses, caused by Theileria equi and Babesia caballi, is hindered by low parasitaemia during the latent phase of the infections. However, this constraint can be overcome by the application of PCR followed by sequencing. Out of 288 animals examined, the piroplasmid DNA was detected in 78 (27·1%). Multiplex PCR indicated that T. equi (18·8%) was more prevalent than B. caballi (7·3%), while mixed infections were conspicuously absent. Sequences of 69 PCR amplicons obtained by the 'catch-all' PCR were in concordance with those amplified by the multiplex strategy. Computed minimal adequate model analyses for both equine piroplasmid species separately showed a significant effect of host species and age in the case of T. equi, while in the B. caballi infections only the correlation with host sex was significant. Phylogenetic analyses inferred the occurrence of three genotypes of T. equi and B. caballi. Moreover, a novel genotype C of B. caballi was identified. The dendrogram based on obtained sequences of T. equi revealed possible speciation events. The infections with T. equi and B. caballi are enzootic in all ecozones of Jordan and different genotypes circulate wherever dense horse population exists.

Stray dogs of northern Jordan as reservoirs of ticks and tick-borne hemopathogens.

Dogs are competent reservoir hosts of several hemopathogens including zoonotic agents and can serve as readily available source of nutrition for many blood-feeding arthropods. Three hemopathogens had been detected for the first time in Jordan. The PCR prevalence of Anaplasma phagocytophilum, Hepatozoon canis, and piroplasmid DNA were 39.5%, 28.9%, and 7.9% (n=38) respectively. Sequencing of amplicons of PCR with universal primers targeting the 18S rRNA gene of piroplasmids shows the highest similarity to equine piroplasmids species Theileria equi from two dogs and Babesia caballi from a single dog. Ticks of two genera Rhipicephalus and Haemaphysalis, were detected in this study (n=268). The brown dog tick, Rhipicephalus sanguineus was the most abundant species (95.1%, n=255), followed by Haemaphysalis erinacei (3%, n=8) and Haemaphysalis parva (1.9%, n=5). The two Haemaphysalis species were detected for the first time from dogs in Jordan. Regarding its high prevalence, we expect R. sanguineus being a possible vector of detected pathogens.

Quest for the piroplasms in camels: identification of Theileria equi and Babesia caballi in Jordanian dromedaries by PCR.

DNA of two species of piroplasmids was detected in dromedaries during a survey of blood protozoans in Jordan between 2007 and 2009. Ten clinically healthy camels (10%) originating from three Jordanian districts were found, using a PCR assay, to harbor Theileria or Babesia species in their blood and no mix infection was determined. Analysis of the partial 18S rRNA gene sequences of these parasites allowed their unambiguous identification as equine piroplasmids Babesia caballi (n=6) and Theileria equi (n=4). In case of latter species, a novel genotype was found in horses. This first molecular-based species determination of piroplasmids from camels further contributes to the growing evidence of low host specificity of piroplasmids.

Genetic diversity of the class II major histocompatibility DRA locus in European, Asiatic and African domestic donkeys.

The major histocompatibility complex (MHC) genes coding for antigen presenting molecules are the most polymorphic genes in vertebrate genome. The MHC class II DRA gene shows only small variation in many mammalian species, but it exhibits relatively high level of polymorphism in Equidae, especially in donkeys. This extraordinary degree of polymorphism together with signatures of selection in specific amino acids sites makes the donkey DRA gene a suitable model for population diversity studies. The objective of this study was to investigate the DRA gene diversity in three different populations of donkeys under infectious pressure of protozoan parasites, Theileria equi and Babesia caballi. Three populations of domestic donkeys from Italy (N = 68), Jordan (N = 43), and Kenya (N = 78) were studied. A method of the donkey MHC DRA genotyping based on PCR-RFLP and sequencing was designed. In addition to the DRA gene, 12 polymorphic microsatellite loci were genotyped. The presence of Theileria equi and Babesia caballi parasites in peripheral blood was investigated by PCR. Allele and genotype frequencies, observed and expected heterozygosities and F(IS) values were computed as parameters of genetic diversity for all loci genotyped. Genetic distances between the three populations were estimated based on F(ST) values. Statistical associations between parasite infection and genetic polymorphisms were sought. Extensive DRA locus variation characteristic for Equids was found. The results showed differences between populations both in terms of numbers of alleles and their frequencies as well as variation in expected heterozygosity values. Based on comparisons with neutral microsatellite loci, population sub-structure characteristics and association analysis, convincing evidence of pathogen-driven selection at the population level was not provided. It seems that genetic diversity observed in the three populations reflects mostly effects of selective breeding and their different genetic origins.

A survey for piroplasmids in horses and Bactrian camels in North-Eastern Mongolia.

Equine piroplasmosis caused by Babesia caballi and Theileria equi is widespread in Asia. The presence of these haemozoans in Mongolia was previously confirmed in domestic as well as in reintroduced Przewalski horses in which they cause significant pathology. The data on occurrence of piroplasms from Bactrian camels in Asia is lacking. A total of 192 horses, 70 Bactrian camels, and additional 16 shepherd dogs from the Hentiy province were included in our study. No clinical signs typical for piroplasmid infection were observed during the field survey. Microscopic examination revealed the presence of T. equi in blood smears from 67% of examined horses, with camels and dogs being negative. A two step PCR approach was used to detect piroplasms in peripheral blood. In the first "catch all" PCR reaction, amplification of the 496 bp-long fragment of the SSU rRNA gene enabled the detection of Babesia and Theileria spp. Second round multiplex PCR reaction used for species discrimination allowed the amplification of T. equi- and B. caballi-specific 340 bp and 650 bp-long regions of the SSU rRNA, respectively. This assay detected T. equi in 92.7% of horses, while the infections with B. caballi and dual infections were rare. In both PCR setups, camels and dogs were negative indicating that in the studied region, these hosts do not share piroplasms with horses.